5-Benzothiazole substituted pyrimidine derivatives as HCV replication (replicase) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.03.036

Based on a previously identified HCV replication (replicase) inhibitor 1, SAR efforts were conducted around the pyrimidine core to improve the potency and pharmacokinetic profile of the inhibitors. A benzothiazole moiety was found to be the optimal substituent at the pyrimidine 5-position. Due to potential reactivity concern, the 4-chloro residue was replaced by a methyl group with some loss in potency and enhanced rat in vivo profile. Extensive investigations at the C-2 position resulted in identification of compound 16 that demonstrated very good replicon potency, selectivity and rodent plasma/target organ concentration. Inhibitor 16 also demonstrated good plasma levels and oral bioavailability in dogs, while monkey exposure was rather low. Chemistry optimization towards a practical route to install the benzothiazole moiety resulted in an efficient direct C-H arylation protocol.

Full text of DOI:10.1016/j.bmcl.2012.03.036

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3229–3234
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
5-Benzothiazole substituted pyrimidine derivatives as HCV replication
(replicase) inhibitors
Ashok Arasappan a, , Frank Bennett a, Vinay Girijavallabhan a, Yuhua Huang a, Regina Huelgas a,
⇑
Carmen Alvarez a, Lei Chen a, Stephen Gavalas 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, Jeremy L. Clark b, Anita T. Fowler b, Feng Geng b, Hollis S. Kezar III 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
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:
Based on a previously identified HCV replication (replicase) inhibitor 1, SAR efforts were conducted
around the pyrimidine core to improve the potency and pharmacokinetic profile of the inhibitors. A ben-
zothiazole moiety was found to be the optimal substituent at the pyrimidine 5-position. Due to potential
reactivity concern, the 4-chloro residue was replaced by a methyl group with some loss in potency and
enhanced rat in vivo profile. Extensive investigations at the C-2 position resulted in identification of com-
pound 16 that demonstrated very good replicon potency, selectivity and rodent plasma/target organ con-
centration. Inhibitor 16 also demonstrated good plasma levels and oral bioavailability in dogs, while
monkey exposure was rather low. Chemistry optimization towards a practical route to install the benzo-
thiazole moiety resulted in an efficient direct C–H arylation protocol.
Received 27 January 2012
Accepted 7 March 2012
Available online 13 March 2012
Keywords:
HCV Replication inhibitor
HCV Replicase inhibitor
Carbanucleoside-like
5-Aryl pyrimidine
5-Benzothiazolyl pyrimidine
C–H arylation
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
DAAs (NS3 protease inhibitors, NS5B nucleoside and non-nucleo-
side inhibitors, and NS5A inhibitors) are in various stages of clinical
An estimated 3% of the human population is infected with hep-
atitis C virus (HCV).1 It is a global health burden and the leading
cause of liver cirrhosis, hepatocellular carcinoma and liver failure
in humans. HCV is a positive stranded RNA virus belonging to
trials.5 Due to the high mutation rate and genetic variability of HCV
(at least six genotypes, with numerous subtypes), novel small mol-
ecules with distinct resistance profile that will likely become part
of a combination regimen are highly sought-after.
the Flaviviridae family. Pegylated
a
-interferon and ribavirin combi-
Our previous efforts6 towards identification of novel small
molecule inhibitors of hepatitis C virus replication resulted in car-
banucleoside-like compound 1 with modest potency in the HCV
genotype 1b subgenomic replicon assay,7 and low rat plasma expo-
sure. Earlier target engagement studies suggested that compounds
of type 1 could possibly inhibit the HCV replication complex (rep-
licase). SAR studies around the aforementioned core demonstrated
the importance of an aryl substituted alkynyl linkage at the 5-
position of the pyrimidine ring. In an attempt to probe additional
tolerated functionality at the pyrimidine 5-position that could also
improve the potency/pharmacokinetic profile of the inhibitors, we
replaced the 5-alkynyl linkage with an aryl moiety. Herein we de-
scribe our investigations toward structures of type 2 (Fig. 1) that
resulted in several compounds with improved replicon potency.
nation therapy, the standard-of-care (SOC) until recently, does not
target the virus specifically.2 It is effective in less than 50% of geno-
type 1 patients, a population more prevalent in North America,
Europe and Japan. Furthermore, the undesirable side effects associ-
ated with the SOC makes it less than ideal for patient compliance.3
The past decade saw an explosion of growth towards discovering
novel small molecule direct-acting antivirals (DAA) that inhibit
HCV replication. These efforts resulted in the recent regulatory ap-
proval of NS3 protease inhibitors, boceprevir and telaprevir that
improved the cure rates when added to the SOC.4 Several other
⇑
Corresponding author. Tel.: +1 732 594 2607; fax: +1 732 594 1185.
E-mail address: ashok.arasappan@merck.com (A. Arasappan).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.03.036

3230
A. Arasappan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3229–3234
N
N
S
a
b
R
R
7
7
NH
2
Cl
X
S
Ar
8
9
N
N
5
2
N
1
NH
N
NHR
HN
OH
HN
2
Cl
HO
HO
I
N
1
2
HO
HO
OH
c
N
NH
d
HN
2
6
HO
EC = 0.2 µM; CC = 15 µM
50
50
9
Rat AUC
(PO) = 1 µM.hr
0-6h
10
O
O
Figure 1. Design of inhibitors based on lead 1.
Scheme 2. Reagents and conditions. (a) t-amylnitrite, THF, reflux, 30 min; (b) (i)
nBuLi, nBu3SnCl, THF, À78 °C, 3 h (used as crude); (ii) 6, PdCl2(Ph3P)2, CuI, Et3N,
DMF, microwave 120 °C, 15 min (<20%); (c) 2,2-dimethoxypropane, MsOH, acetone,
0–25 °C, 16 h (quant.); (d) (i) 9, Pd(Ph3P)4, CuI, Cs2CO3, DMF, microwave 120 °C,
15 min (5–13%); (ii) Aq. 1 N HCl, MeOH, 25 °C, 12 h (quant.).
Further in vivo evaluations provided compounds with enhanced
rat plasma exposure and high levels of target organ coverage.
Synthetic chemistry routes for the preparation of 5-aryl target
compounds are described in Schemes 1–4. Iodination of commer-
cially available 2-amino-4,6-dichloro pyrimidine 3 resulted in 4.
Treatment of 4 with carbasugar 5 provided the key intermediate
6 in good yield. A variety of aryl groups were installed at the 5-po-
sition of the pyrimidine ring via Suzuki or Stille reaction of iodide 6
with readily available aryl boronic acids or aryl tri-n-butyl stann-
anes, respectively.8 It was found that the required targets 7 could
be obtained in adequate yield using microwave conditions as
shown in Scheme 1.
Cl
Cl
N
Cl
I
a
b
N
N
N
Cl
N
Cl
Cl
NH
CF
Cl
N
NH
CF
11
12
13
3
3
Target compounds 21–32 (Table 1) containing unsubstituted
aromatic group at the 5-position of the pyrimidine moiety were
prepared using the route shown in Scheme 1. Installation of the
substituted benzothiazole moiety at the 5-position of the pyrimi-
dine ring was accomplished using the synthetic sequence shown
in Scheme 2. The substituted benzothiazole precursors 9 were
obtained from the corresponding 2-amino benzothiazole com-
pounds 8 via diazotization chemistry. Lithiation of 9 followed by
quenching with tri-n-butylstannyl chloride provided 2-stannyl
benzothiazoles that subsequently underwent the previously de-
scribed Stille protocol with iodide 6 to afford some of the required
targets 7. Alternatively, certain targets of type 7 were obtained via
direct C–H arylation of benzothiazoles 9 with diol-protected iodide
10. It was found that both palladium and copper catalysts were
required for the direct arylation reaction.9 Diol protection, as the
corresponding acetonide, was also crucial for the success of the
reaction. It is worth noting that the direct arylation reaction pro-
ceeded successfully with a fully functionalized pyrimidine motif
under microwave conditions, albeit in low yield.
Cl
N
N
X
N
I
c
d
N
S
N
NH
CF
HN
OH
NH
CF
HN
HO
HO
3
3
14
HO
HO
OH
e
15
: X = Cl
16: X = Me
Scheme 3. Reagents and conditions. (a) CF3CH2NH2, Et3N, THF (16%); (b) ICl, gl.
AcOH (quant.); (c) 5, EtOH, Et3N, reflux, 16 h (87%); (d) tri-n-butylstannyl
benzothiazole, Pd(Ph3P)4, CuI, Et3N, dioxane, 100 °C, ꢀ1 h (57%); (e) MeB(OH)2,
PdCl2(Ph3P)2, K2CO3, dioxane/water, 100 °C, 2 h (43%).
Me
I
N
Me
Scheme 3 depicts the representative synthesis of some of the
inhibitors with C-2 and C-4 modified pyrimidine core. Treatment
a
N
I
SMe
HN
O
b
N
HO
or
c or d
Cl
N
SMe
O
17
18
Cl
Cl
I
a
b
N
N
NH2. HCl
N
Me
Cl
N
NH
Cl
N
NH
HO
2
2
N
Me
S
HO
OH
N
3
4
S
N
5
e
N
SMe
HN
R
Cl
Cl
N
N
H
HN
HO
HO
I
Ar
N
N
19
(HCl salt)
c or d
O
O
HO
OH
N
NH
N
NH
2
HN
HN
2
20
HO
HO
Scheme 4. Reagents and conditions. (a) (i) 5, EtOH, Et3N, reflux, 16 h; (ii) 2,2-
dimethoxypropane, MsOH, acetone, 0–25 °C (79% for two steps); (b) tri-n-butyl-
stannyl benzothiazole, Pd(Ph3P)4, CuI, Et3N, dioxane, 100 °C, 16 h (80%); (c)
benzothiazole, Pd(Ph3P)4, CuI, Cs2CO3, DMF, 100 °C, 2 h (45%); (d) benzothiazole,
Pd(dppf)2Cl2, Ag2CO3, Ph3P, MeCN, 75 °C, 24 h (70%); (e) (i) mCPBA, CH2Cl2, 0–25 °C;
(ii) RNH2, MeCN, 80–100 °C, 12–16 h; (iii) Aq. HCl, dioxane, MeOH (60–80% for
three steps).
6
7
HO
OH
HO
OH
Scheme 1. Reagents and conditions. (a) ICl, gl. AcOH, 5 h (79%); (b) 5, EtOH, Et3N,
reflux, 16 h (80%); (c) ArB(OH)2, PdCl2(dppf)2, K2CO3, DME/water, microwave
120 °C, 5–15 min (20–42%); (d) nBu3SnAr, PdCl2(Ph3P)2, CuI, Et3N, DMF, microwave
120 °C, 15 min (29–50%).

A. Arasappan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3229–3234
3231
Table 1
Evaluation of 5-aryl series
of trichloropyrimidine 11 with a suitable amine, trifluoroethyl
amine, provided 2-amino substituted pyrimidine 12.10 A substan-
tial amount (ꢀ50–60%) of the 4-regioisomeric byproduct (structure
not shown) was easily separated by silica chromatography. Iodin-
ation and subsequent reaction with carbasugar 5 followed earlier
described conditions to afford intermediate 14. Stille reaction of
14 with tri-n-butylstannyl benzothiazole using palladium and
copper catalysts provided 15 in moderate yields. The methyl group
at the 4-position was installed via Suzuki reaction with methyl
boronic acid to provide the required target compound 16.
Cl
N
Ar
N
NH
2
HN
HO
HO
OH
While the synthetic sequence shown in Scheme 3 was suitable
for the initial series target compounds, it was not practical for SAR
development. Since our intention was to probe the requirement for
pyrimidine C-2 substitution, a more robust route that was amena-
ble for late stage C-2 amino installation was necessary. Such a
route is shown in Scheme 4. Thus, carbasugar 5 was treated with
known 2-methylthio pyrimidine derivative 17,11 followed by diol
protection to give the 5-iodo intermediate 18. Employing the Stille
protocol described above, 18 was smoothly converted to the key 5-
benzothiazole intermediate 19. At this juncture, due to several rea-
sons ((a) limited availability of stannyl reagent from commercial
sources, and (b) requirement of large quantities of environmentally
unfriendly stannyl reagent for multigram quantities of 19), we ex-
plored other methods of 5-benzothiazole installation. We were
pleased to find that the direct C–H arylation of benzothiazole using
conditions described earlier worked well to provide 19 in moderate
yield. Further investigations resulted in an optimized C–H aryla-
tion protocol that employed palladium catalyst, triphenyl phos-
phine and silver carbonate, and provided the key intermediate 19
in 70% isolated yield. Intermediate 19 was then processed in three
steps to targets of type 20 as shown in Scheme 4.
Compd #
Ar
EC50
(
lM)
MTS/GAPDH CC50
>25/–
(lM)
21
>25
>25
22
23
>25/>25
>25/—
N
N
>25
N
24
25
4
>25/>25
—/>25
O
S
>25
N
26
27
>25
3
>25/>25
>25/>25
O
N
S
28
7
25/—
N
H
Previously we had identified compound 1 as a HCV replication
(replicase) inhibitor with modest replicon potency, and low rat
plasma exposure. Our earlier work revealed an alkenyl, or more
importantly, an alkynyl moiety as the preferred substituent at
the 5-position of the pyrimidine ring. As a logical way forward in
developing the SAR around structure 1, and possibly to improve
the potency/pharmacokinetic profile of the inhibitors, we designed
targets of type 2 with an aryl ring as the pyrimidine 5-substituent.
These designed compounds were synthesized as shown in Schemes
1 and 2, and the potency data are shown in Table 1. The initial set
of targets prepared, monocyclic aryl rings at the pyrimidine
5-position (21–27), were largely inactive. Only the furanyl (24)
and thiazolyl (27) substituent showed marginal levels of replicon
potency, albeit less potent compared to 1. Interestingly, the ben-
zofuranyl compound 29 restored the replicon potency while
improving the selectivity index (EC50/CC50), compared to 1. Instal-
lation of benzoxazolyl (31) or benzothiazolyl (32) moiety onto the
pyrimidine motif had a profound impact on potency, with replicon
29
30
31
32
33
34
35
36
37
0.3
>25/—
—/>25
15/12
O
S
>25
0.05
0.02
0.18
0.65
N
O
N
18/14
S
N
S
F
>25/>25
>25/16
>25/>25
25/>25
16/6
N
Cl
S
N
MeO
EtO
0.045
0.03
0.05
S
EC50 equal to 0.05 or 0.02 lM, respectively. Replicon data for com-
pounds 28 and 30 indicated that only limited aryl substituents
were tolerated at the 5-position of pyrimidine ring. Having estab-
lished the optimum group at 5-position, we then probed the effect
of introducing additional functionality on the benzothiazolyl moi-
ety. While halo substituents (33 and 34) resulted in potency loss,
alkoxy groups (35 and 36) retained potency while improving the
selectivity. Introduction of a methylamino group at the 5- or 6-po-
sition on the benzothiazolyl ring (37 and 38) provided equipotent
inhibitors. However, a dimethylamino functionality was accommo-
dated only at the 5-position (40) and not at the 6-position (39) of
the benzothiazolyl moiety.
Since a naked benzothiazolyl moiety at the 5-position of the
pyrimidine ring provided a potent inhibitor with acceptable selec-
tivity, we decided to carry out C-2 modifications on substrate of
type 32 (Table 2). Before we embarked on such an investigation,
we were concerned with the possible reactivity of 4-chloro group
N
S
N
MeHN
MeHN
S
N
38
39
40
0.02
1
14/20
S
N
Me2N
Me2N
>25/>25
21/25
S
N
0.035
S

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A. Arasappan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3229–3234
Table 2
Evaluation of C-2 amino substituent.
N
Me
4
S
N
R
2
N
N
H
HN
HO
HO
OH
Compd #
R
EC50
(lM)
MTS/GAPDH CC50
(lM)
AUCa
(lM.h)
Liverb C6h (ng.g)
32
41
42
Hc
Hd
H
0.02
0.2
0.13
18/14
>25/>25
25/4
0.08
0.4
4.7
<100
250
—
Me
43
44
45
0.05
0.1
>25/>25
>25/12
2.7
6.2
—
75
515
—
0.31
>25/>25
46
47
0.16
0.09
14/13
13/12
19.6
5
3320
1245
48
49
50
0.06
0.05
0.25
10/10
8/8
2.3
3.2
0.5
510
405
150
>25/>25
51
52
NHR = NMe2
NHR = NMe2
0.14
13
16/18
>25/>25
11e
—
1030
—
f
OMe
53
54
55
0.035
0.035
0.020
15/21
18/18
7/8
5.9
2.4
0.9
875
400
130
OEt
OiPr
H
N
OEt
56
57
0.04
0.9
19/12
0.3
—
63
—
O
H
N
NHt Bu
>25/>25
O
H
N
S
58
59
0.26
0.04
>25/>25
25/25
—
—
O
O
O
0.2
370
N
16
0.03
0.8
17/25
7e
8415g
—
CF3
60h
61i
>25/>25
>25/>25
—
CF3
CF3
0.7
12.8
12765
62
63
0.14
0.1
>25/>25
19/21
13
—
6570
—
CF3
CF3
CF3
CF3
64
65
0.03
0.03
9/9
4/3
5.3
2.1
1075
2230
66
67
0.28
0.33
>25/>25
7/7
1.6
—
970
—
N
N
68
0.77
13/15
—
—
S
N
69
70
4.5
>25/>25
7/9
—
—
O
0.03
2.2
430

A. Arasappan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3229–3234
3233
Table 2 (continued)
Compd #
R
EC50
(lM)
MTS/GAPDH CC50
(lM)
AUCa
0.5
(lM.h)
Liverb C6h (ng.g)
71
72
0.006
0.015
5/6
4/6
180
75
S
0.2
S
N
N
S
73
0.03
0.03
21/23
18/13
0.5
3.4
150
125
74
75
N
0.015
0.03
11/5
9/7
0.02
0.1
10
N
76
100
a
AUC0–6 h, po (10 mpk), vehicle—0.4% mc.
Liver conc at 6 h.
Pyrimidine C-4 = Cl.
b
c
d
e
f
Pyrimidine C-4 = H.
AUC0–24h
.
Pyrimidine C-4 = Et.
Liver conc at 4 h.
Enantiomer of 16.
g
h
i
Pyrimidine C-4 = cyclopropyl.
was then studied extensively using the 4-methyl substituted target
42. Installation of a series of alkyl residues on the 2-amino moiety
was investigated first. Compared to 42, the C-2 N-methyl deriva-
tive 43 displayed improved potency, good selectivity and rat plas-
ma exposure. However, the target organ concentration was poor
for 43. The series of alkyl substituents studied (44–50, straight
chain, branched, small cyclics) were equipotent or less potent than
43. The rat pharmacokinetic profiles for the C-2 alkyl amino com-
N
Me
S
N
N
N
H
CF
3
HN
HO
16
(HCl salt)
HO
EC = 0.03 µM
OH
pounds were essentially similar, with the a-branched ones, 46 and
50
MTS/GAPDH CC = 17/25 µM
50
µ
47, exhibiting enhanced AUC and liver concentration. Introduction
of an N,N-dimethylamino residue at C-2 position (51) resulted in
potency loss, albeit with significantly improved rat AUC. The dras-
tic loss of potency observed for inhibitor 52, an analog of 51, with a
C-4 ethyl group demonstrated the SAR limitation for this position;
only a methyl group was tolerated at the C-4 pyrimidine position.
To expand the scope of the C-2 amino alkyl substituent, we
introduced heteroatoms and additional functionality on the alkyl
chain. Inhibitors with ether-containing side chains (53–55) were
very potent, with 53 showing good rat exposure and target organ
concentration. Inhibitors with amino-derived functionality on the
alkyl chain displayed mixed results; while the carbamoyl (56)
and morpholino (59) containing targets showed very good potency,
the urea (57) and sulfonamido (58) targets were less potent. Unfor-
tunately, the potent compounds in this series (56 and 59) did not
show appreciable rat plasma exposure. Introduction of a trifluoro-
ethyl amino moiety at the C-2 position had a profound impact on
the inhibitor profile. Thus, compound 16 exhibited very good
replicon potency and selectivity. Moreover, 16 displayed excellent
DNA CC = 19
M
50
Selectivity index (CC /EC ) >550
50
50
Pharmacokinetic data
Rat: IV (2 mpk, 40%HPBCD); PO (10 mpk, 0.4% MC)
AUC = 7 µM.h; F = 82%
0-24h
Liver Conc (PO) = 8415 ng/g
4h
Monkey: IV (1 mpk, 40% HPBCD); PO (3 mpk, 0.4% HPMC)
µ
= 0.3 M.h; F = 8%
AUC
0-24h
Dog: IV (1 mpk, 40% HPBCD); PO (3 mpk, 0.4% HPMC)
AUC = 7.8 µM.h; F = 35%
0-24h
Figure 2. Profile of lead compound 16.
on the pyrimidine ring. Hence we decided to remove or replace the
4-chloro functionality. Targets shown in Table 2 are the results of
efforts directed toward both C-4, and more extensively, C-2 modi-
fications. These compounds were prepared using routes shown in
Scheme 3 or Scheme 4. After initial evaluation in the replicon
assay, most of the compounds were subjected to rat in vivo studies.
Inhibitors were dosed orally (10 mpk, n = 2 rats for each com-
pound, formulation vehicle—0.4% mc) and plasma samples were
collected at various timepoints till 6 h post-dose. Rat plasma expo-
sure is reported as AUC0–6 h. The animals were sacrificed at 6 h;
liver was harvested and processed to measure the compound con-
centration (liver C6h). The results are depicted in Table 2.
Removal of the 4-chloro moiety of 32 provided inhibitor 41
(prepared via hydrogenation), which displayed a 10-fold loss in
replicon potency, albeit with some improvement in rat plasma
exposure. Replacement of the 4-chloro residue of 32 with a methyl
group (42) resulted in some loss in potency. Interestingly, there
was a significant increase in rat AUC for 42. Hence, introduction
of different functionalities on the pyrimidine 2-amino moiety
rat oral pharmacokinetic profile (AUC = 7 lM h) and target organ
exposure (liver C4h = 8415 ng/g). Several modifications were then
carried out around compound 16. The corresponding enantiomer,
60 (prepared from commercial carbasugar 5-ent—structure not
shown) resulted in substantial erosion of potency. Significant loss
in potency for 61, with a C-4 cyclopropyl group, (prepared using
cyclopropylboronic acid employing Suzuki conditions—see
Scheme 3) once again confirmed the requirement of only a small
methyl group at the C-4 position. Interestingly, compound 61 dis-
played some of the best rat in vivo profiles seen for this class of
compounds. Introduction of an (S) or (R)-methyl group on the
trifluoroethyl side chain, 62 or 63 respectively, resulted in a few
fold loss in potency, while retaining the PK characteristics (for

3234
A. Arasappan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3229–3234
62). Increasing the chain length between the C-2 amino moiety and
the terminal trifluoromethyl group (compounds 64 and 65) pro-
vided equipotent analogs, albeit with progressively diminishing
selectivity and in vivo profile. Installation of aryl or heteroaryl
group on the C-2 amino moiety was investigated next (66–69),
which resulted in loss of replicon potency. However, an aryl or het-
eroaryl residue with a methylene spacer (70–76) on the C-2 amino
moiety proved to be highly potent. For example, inhibitor 71
Acknowledgment
We thank the Structural Chemistry group for NMR and MS anal-
ysis for all new compounds.
References and notes
1. Lavanchy, D. Liver, International 2009, 29(s1), 74.
2. Feld, J. J.; Hoofnagle, J. H. Nature 2005, 436, 967.
displayed excellent potency with an EC50 = 0.006 lM. Unfortu-
3. Birerdinc, A.; Younossi, Z. M. Expert Opin. Emerging Drugs 2010, 15(4), 535.
4. (a) Boceprevir: (i) FDA press release dated May 13, 2011, http://www.fda.gov/
NewsEvents/Newsroom/PressAnnouncements/ucm255390.htm (ii) Venkatram
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2011, http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/
ucm256299.htm (ii) 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.;
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5. (a) Flisiak, R.; Parfieniuk, A. Expert Opin. Investig. Drugs 2010, 19(1), 63; (b)
Meanwell, N. A.; Kadow, J. F.; Scola, P. M. Annu. Rep. Med. Chem. 2009, 44, 397.
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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.
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7. (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.
nately, all compounds containing this substitution pattern (70–
76) demonstrated poor oral AUC and/or liver concentration.
Having extensively explored the requirement for appropriate
functionality on the C-2 amino moiety, and based on the potency,
selectivity, rat oral and target organ exposure, we decided to ad-
vance inhibitor 16 for further evaluations in higher species. The full
profile of 16 is shown in Figure 2. Thus, inhibitor 16 displayed very
good potency and a high selectivity index. In rat in vivo studies, 16
exhibited high oral exposures, both in plasma and more impor-
tantly, the liver, which is the primary reservoir of HCV. Target 16
also demonstrated excellent rat oral bioavailability. While monkey
plasma exposure was low, in dogs 16 exhibited good plasma con-
centration and oral bioavailability. Inhibitor 16 had no issues with
CYP inhibition (3A4, 2D6, 2C9 >20
lM), and was clean in an in-
house kinase panel counterscreen (22 kinases, IC50 >30
lM).
Our studies directed towards modifying the previously de-
scribed carbanucleoside-like structure 1 resulted in benzothiazole
moiety as an appropriate 5-substituent on the pyrimidine ring
with improved HCV replicon potency. However, the parent C-2
amino analog 32 displayed poor rat plasma exposure. Replacement
of the potentially reactive pyrimidine C-4 chloro residue with a
methyl group provided enhanced rodent plasma exposure (analog
42). Modulation of replicon potency and rat pharmacokinetic pro-
file was carried out via introduction of a mono-substituent on the
C-2 amino functionality. Different synthetic routes were employed
to access the target compounds. While the initial Stille conditions
proved effective in installing the 5-benzothiazole moiety, signifi-
cant chemistry optimization towards a practical route resulted in
a direct C–H arylation protocol on a fully substituted pyrimidine
core, that was equally efficient and devoid of environmentally
unfriendly tin reagent. Through extensive screening of various
groups on the C-2 amino residue, inhibitor 16 was identified with
the best overall profile (potency, selectivity, rat oral and target
organ exposure). Inhibitor 16 was studied in higher species, where
it displayed good plasma exposure and oral bioavailability in dogs.
Thus, our lead optimization efforts to discover HCV replicase inhib-
itors from the novel carbanucleoside-like pharmacophore resulted
in inhibitor 16 with desirable characteristics that warrant further
investigations.
8. (a) Torborg, C.; Beller, M. Adv. Synth. Catal. 2009, 351, 3027; (b) Farina, V.;
Krishnamurthy, V.; Scott, W. J. Org. React. 1997, 50, 1.
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MacCoss, M. Tetrahedron Lett. 2010, 51, 2797; (b) Pivsa-Art, S.; Satoh, T.;
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E. Bioorg. Med. Chem. 2011, 19, 836.
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