5,5′- and 6,6′-Dialkyl-5,6-dihydro-1H-pyridin-2-ones as potent inhibitors of HCV NS5B polymerase

Article abstract of DOI:10.1016/j.bmcl.2009.09.051

The discovery of 5,5′- and 6,6′-dialkyl-5,6-dihydro-1H-pyridin-2-ones as potent inhibitors of the HCV RNA-dependent RNA polymerase (NS5B) is described. Several of these agents also display potent antiviral activity in cell culture experiments (EC₅₀

Full text of DOI:10.1016/j.bmcl.2009.09.051

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6047–6052
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
5,50- and 6,60-Dialkyl-5,6-dihydro-1H-pyridin-2-ones as potent inhibitors
of HCV NS5B polymerase
*
David A. Ellis, Julie K. Blazel, Chinh V. Tran, Frank Ruebsam, Douglas E. Murphy , Lian-Sheng Li,
Jingjing Zhao, Yuefen Zhou, Helen M. McGuire, Alan X. Xiang, Stephen E. Webber, Qiang Zhao,
Qing Han, Charles R. Kissinger, Matthew Lardy, Alberto Gobbi, Richard E. Showalter, Amit M. Shah,
Mei Tsan, Rupal A. Patel, Laurie A. LeBrun, Ruhi Kamran, Darian M. Bartkowski, Thomas G. Nolan,
Daniel A. Norris, Maria V. Sergeeva, Leo Kirkovsky
Anadys Pharmaceuticals, Inc., 3115 Merryfield Row, San Diego, CA 92121, USA
a r t i c l e i n f o
a b s t r a c t
The discovery of 5,50- and 6,60-dialkyl-5,6-dihydro-1H-pyridin-2-ones as potent inhibitors of the HCV
RNA-dependent RNA polymerase (NS5B) is described. Several of these agents also display potent antiviral
activity in cell culture experiments (EC50 <0.10 lM). In vitro DMPK data for selected compounds as well
as crystal structures of representative inhibitors complexed with the NS5B protein are also disclosed.
Article history:
Received 24 July 2009
Revised 9 September 2009
Accepted 14 September 2009
Available online 17 September 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Hepatitis C virus (HCV)
NS5B polymerase
Small-molecule
Non-nucleoside NS5B inhibitor
5,50- and 6,60-Dialkyl-5,6-dihydro-1H-
pyridin-2-ones
Hepatitis C virus (HCV) is a major cause of acute hepatitis and
chronic liver disease, including cirrhosis and liver cancer.
Globally, an estimated 180 million individuals, 3% of the world’s
population, are chronically infected with HCV and 3–4 million
people are newly infected each year.1 Currently, there is no vac-
cine available to prevent hepatitis C, nor an HCV-specific antiviral
agent approved for treatment of chronic hepatitis C. The current
standard of care is comprised of a combination of pegylated
interferon (peg-IFN) with the nucleoside analog ribavirin (RBV).2
Low response rates, in particular for patients infected with
genotype 1 HCV, along with significant side-effects of peg-IFN/
RBV therapies necessitate the identification of more effective
anti-HCV agents.3
The activity of the virally encoded HCV NS5B polymerase is
essential for HCV replication making it an attractive target for
the development of novel HCV treatments.4 A number of nucleo-
side and non-nucleoside NS5B inhibitors have been described in
the literature. Most small molecule, non-nucleoside inhibitors of
NS5B bind to a number of pockets that are distinct from the active
site.5,6 Among these, the NS5B inhibitor development efforts at
Anadys have focused on compounds which bind to the ‘palm’ site
of the NS5B protein.
We recently reported that molecules containing a 1,1-dioxo-
1,4-dihydro-1k6-benzo[1,2,4]thiadiazine moiety linked to a bicy-
clic 5,6-dihydro-1H-pyridin-2-one (e.g., structure 1, Fig. 1), are po-
tent inhibitors of the NS5B polymerase with significantly improved
oral bioavailability relative to our previously reported NS5B inhib-
itors.7 Additionally, monocyclic pyridinones (e.g., structure 2) have
been reported as potent NS5B polymerase inhibitors.8 Encouraged
by the favorable biological profile of 1, we wished to explore re-
lated monocyclic 5,6-dihydro-1H-pyridin-2-ones (e.g., structure
3) in the hope of identifying additional potent antiviral agents with
favorable pharmacokinetic (PK) properties. The results of these ef-
forts are described below.
Synthetic routes to produce 5-mono-alkylated and 5,50-di-alkyl-
ated pyridinones are shown in Scheme 1. In cases where disubsti-
tuted dialkyl malonates 4 were not commercially available, we
began our synthesis from a variety of mono substituted malonates.
Anion formation of mono substituted malonates using sodium hy-
dride followed by treatment with alkyl halides led to the di-substi-
tuted intermediates 4. De-symmetrization of the di-substituted
malonates was achieved by treatment with diisobutylaluminium
* Corresponding author. Tel.: +1 858 530 3652; fax: +1 858 527 1540.
E-mail address: dmurphy@anadyspharma.com (D.E. Murphy).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.09.051

6048
D. A. Ellis et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6047–6052
O
O
O
O
O
O
H
N
H
N
H
N
S
S
S
OH
N
N
S
O
OH
N
N
S
N
S
OH
R1
R2
R3
R4
H
H
O
O
O
O
O
O
5
N
H
N
H
N
H
6
O
O
N
R5
O
IC50 1a = 0.008 µM
IC50 1b = 0.023 µM
EC50 1b = 0.003 µM
IC50 1a < 0.025 µM
IC50 1b < 0.01 µM
EC50 1b = 0.016 µM
F = 21%
F
1
2
3
Figure 1. HCV polymerase inhibitors.
O
O
O
O
O
O
a
b
RO
OR
RO
OR
H
OR
R2 R1
R2 R1
R1
4
5
c
O
O
H
S
N
R: Me or Et
N
S
OH
R1
R2
O
O
O
R1
R2
O
O
H
N
N
H
f
S
OR
O
N
S
+
O
O
10
N
R5
O
NH
R5
6
HO
N
H
9
e
O
O
R1
R1
OR
d
OR
NH2
NH
R5
7
8
R1: H
Scheme 1. Reagents and conditions: (a) (i) NaH, THF, 25 °C, 20 min, (ii) R2X, 25 °C, 3 h, 76–98%; (b) DIBAL (2 equiv), CH2Cl2, ꢀ78 °C, 4 h, 8–89%; (c) (i) R5NH2, MeOH or EtOH,
(ii) NaCNBH3 or NaBH4, AcOH, 25–60 °C, 4–16 h, 33–90%; (d) (i) aldehydes or ketones, MeOH, (ii) NaCNBH3, NaOAc, 25 °C, 4–16 h, 10–79%; (e) (i) KHMDS, THF, ꢀ78 °C, 15 min,
(ii) R2X, ꢀ78 °C for 6 h then warm to 25 °C, 36–40%; (f) (i) EDCI, NMM, DMF, 25 °C, 1 h, (ii) NaOEt, EtOH, 70 °C, 1 h, 30–70%.
hydride (DIBAL) to afford the racemic b-formylesters 5. Reductive
amination of intermediates 5 with primary amines afforded the de-
sired 2,2-disubstituted b-aminoesters 6. Alternatively, reductive
alkylation of commercially available b-amino esters 7 with alde-
hydes or ketones followed by potassium hexamethyldisilazane
(KHMDS) mediated alkylation at the 2-position afforded the race-
mic mono-alkylated-b-aminoesters 6. Coupling of intermediates
6 to acid 99 using standard conditions afforded the corresponding
amide intermediates, which, upon treatment with sodium ethox-
ide, cyclized to yield the desired final products 10.
Synthetic routes to produce 6-mono-alkylated pyridinones are
shown in Scheme 2. Esterification of a variety of commercially
available b-substituted-b-amino acids 11, in either racemic or
optically pure form (ee >95%), followed by reductive alkylation
with 4-fluoro-benzaldehyde yielded the desired 3-alkyl-N-4-flu-
oro-benzyl-b-amino esters 13.10 Alternatively, treatment of alde-
hydes with diazo-ethylacetate 14 in the presence of stannous
chloride afforded b-ketoesters 15.11 Reductive amination of 15
with 4-fluoro-benzylamine led to the racemic 3,30-dialkyl-N-4-
fluoro-benzyl-b-amino esters 13. Coupling of intermediates 13
to acid 9, followed by cyclization, proceeded as described above
to afford the final products 16.
Table 1 details the structure activity relationships (SAR) ob-
tained for compounds with substitutions at the 5- and 6-posi-
tions of the pyridinone ring system bearing a 4-fluoro-benzyl
R5 substituent. The major conclusions of this study are summa-
rized below. Mono-substitution at the 5-position led to active,
yet relatively weak NS5B inhibitors compared to the bicyclic ana-
log 1. Addition of a methyl group to produce the 5,50-di-substi-
tuted pyridinones caused little change in potency (cf. 20 with
24). Similarly, 5-spirocyclic derivatives (26–28) also offered no
obvious advantage. Substitution at the 6-position afforded several
compounds with improved NS5B inhibition properties relative to
the 5-alkyl substituted analogs. Substitution of the 6-position
with ethyl and isopropyl groups afforded the most potent inhib-
itors in Table 1. Interestingly, compound 29 exhibited >4-fold
improvement in biochemical potency (1b) and 10-fold improve-
ment in antiviral potency as compared to the corresponding
enantiomer (30).
The stability of these compounds was assayed using human li-
ver microsomes (HLM). The HLM T1/2 results are summarized in
Table 1. The majority of compounds described above exhibited
long HLM half-lives (>60 min); except when an isoamyl group
was incorporated at either the 5- or 6-position. Similarly short

D. A. Ellis et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6047–6052
6049
O
O
H
N
S
OH
N
N
S
O
O
O
O
O
N
H
a
b
c
R3
R4
OH
NH2
11
R3: H, R4: Alkyl or R3: Alkyl, R4: H
OR
NH
OMe
NH2
12
R3
R4
R3
R4
R3
R4
16
O
13
R: Me or Et
F
F
e
O
O
d
R3 CHO
+
OEt
OEt
N2
R3
O
14
15
Scheme 2. Reagents and conditions: (a) TMSCHN2, 1:1 MeOH/benzene, 0–25 °C, 1 h, 90–98%; (b) (i) 4-fluoro-benzaldehyde, MeOH or EtOH; (ii) NaCNBH3 or NaBH4, AcOH,
25–60 °C, 4–18 h, 50%; (c) (i) compound 9, EDCI, NMM, DMF, 25 °C, 1 h, (ii) NaOEt, EtOH, 70 °C, 1 h, 22–36%; (d) SnCl2, CH2Cl2, 25 °C, 1 h, 10–55%; (e) (i) 4-fluoro-benzylamine,
MeOH or EtOH, (ii) NaCNBH3 or NaBH4, AcOH, 25–60 °C, 4–16 h, 47–55%.
Table 1
Exploration of various mono and di-alkylated pyridinones
O
O
H
N
S
N
S
OH
N
R1
O
O
5
R2
R3
R4
N
H
6
O
F
Compda
R1
R2
R3
R4
IC50 1ab
(l
M)
IC50 1bc
(lM)
EC50 1bc
(
lM)
CC50 (lM)
HLM T1/2 (min)
c
c
1
Figure 1
<0.025
0.18
NDd
1.4
<0.01
0.2
0.26
0.13
0.22
0.067
0.032
0.099
0.24
0.016
3.9
3.6
0.67
0.49
1.3
0.16
0.2
0.77
ND
0.55
0.77
2.1
0.083
0.97
0.045
0.81
2.9
>100
>33
>33
>33
>33
>33
>1
>33
>33
ND
>1
>33
>33
>1
ND
>1
>10
ND
ND
>1
>10
>10
>33
>60
>60
>60
6.9
17
18
19
20e
21
22
23
24
25
26
27
28
29f
30f
31f
32f
33
34
35
36f
37
38
H
H
H
H
CH3
CH3
CH3
CH3
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH2–(4-F)Ph
CH2CH@C(CH3)2
CH2CH2CH(CH3)2
CH3
CH2CH3
CH2-cy-Propyl
CH2CH2CH(CH3)2
Ph
ND
6.5
0.085
0.045
0.1
ND
ND
0.037
0.12
0.22
<0.025
0.042
0.026
0.042
ND
>60
>60
>60
5.7
ND
>60
>60
28
>60
>60
>60
>60
>60
7.6
1.1
–CH2CH2–
–CH2CH2CH2–
–CH2CH2CH2CH2–
0.025
0.042
0.091
<0.010
0.043
<0.010
0.063
0.016
0.15
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH2CH3
H
CH(CH3)2
C(CH3)3
H
H
H
Ph
H
CH2CH3
H
H
CH2CH(CH3)2
CH2CH2CH(CH3)2
CH(CH2CH3)2
H
(4-F)Ph
–CH2CH2CH2CH2–
ND
5.5
0.061
0.17
0.1
0.07
0.078
0.09
0.31
0.66
0.52
0.84
59
>60
>60
>60
0.15
0.028
a
b
c
d
E
f
All compounds were prepared in racemic form unless otherwise indicated.
See Ref. 12b for assay conditions and error.
See Ref. 12a for assay conditions and error.
ND = not determined.
Synthesized via hydrogenation of compound 19.
Single enantiomer.

6050
D. A. Ellis et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6047–6052
scribed series of 1-hydroxy-4,4-dialkyl-3-oxo-3,4-dihydronaph-
thalenes (structure 39, Fig. 4)14 could be achieved. Such
modifications would effectively ‘flip’ the orientation of the dihyd-
ropyridinone ring relative to that established for the 4-fluoro-ben-
zyl containing molecules of Table 1 (e.g., structure 40, Figure 4).
Accordingly, we designed a second series of 5,50-di-alkylated pyrid-
inones containing R1 and R2 substituents known from the 1-hydro-
xy-4,4-dialkyl-3-oxo-3,4-dihydronaphthalene series to afford
potent NS5B inhibitors.14 In order to further ‘force’ the molecules
into a flipped conformation, we also incorporated cyclic aliphatic
R5 substituents that we had previously shown to lead to less favor-
able interactions in the deep hydrophobic pocket occupied by the
4-fluoro-benzyl group of 29.7 Table 2 summarizes the SAR obtained
for this second series of 5,50-di-alkylated pyridinones.
Overall, the ‘flipped’ series strategy led to the synthesis of ac-
tive, yet only moderately potent NS5B inhibitors relative to com-
pound 1. Of the compounds listed in Table 2, inhibitors
containing an isoamyl R2 moiety exhibited the best enzymatic
and antiviral potency compared to all others shown. Interestingly,
tert-butyl-ethyl R2 groups led to a severe loss in anti-NS5B potency
relative to the isoamyl analogs (cf. 49 with 54). Increasing the R5
cyclic aliphatic ring size from cyclopropyl to cyclohexyl had rela-
tively little impact on the NS5B inhibition properties as shown
by compounds 41, 44, 49 and 57, where the R2 substituent was
held fixed as an isoamyl moiety. Replacement of the cyclohexyl
R5 substituent with a phenyl group exhibited a dramatic loss in
enzymatic activity (cf. 57 with 60). Removal of the R1 methyl (cf.
49 with 51) had only minimal effect on overall NS5B activity
whereas increasing the size of the R1 substituent to an ethyl group
(52) led to significantly reduced antiviral potency. All inhibitors
shown in Table 2 containing a 4-fluoro-benzyl R2 substituent dis-
played severe loss in potency. These results are consistent with
our hypothesis that molecules depicted in Table 2 adopt an alter-
nate NS5B binding conformation relative to those in Table 1.
Stability towards human liver microsomes (HLM) was also as-
sessed for the inhibitors listed in Table 2. Interestingly, after expo-
sure of the racemic analogs to HLM, analysis by chiral HPLC/LC–MS
indicated one enantiomer to be much more stable than the other
(44, 49, 57 and 60).15 No attempt was made to assign the absolute
stereochemistry of the more stable enantiomer. Chiral analysis was
not performed during the HLM assessment of 45, 46, 47, 48, 52, 53,
54, and 58. However, a measurement of % compound remaining vs.
time after exposure of these racemic analogs to HLM indicated a
sharp drop in concentration followed by a gradual decline. This
data again suggested the possibility of one enantiomer being much
more stable than the other. Double experimental decay curve fit-
ting was applied to both the rapid and gradual decay phases of
the plot. We interpreted the resulting half-lives as the actual
half-lives corresponding to each enantiomer and the results are re-
ported in Table 2.
Figure 2. Co-crystal X-ray structure of compound 29 bound to the NS5B protein.
O
O
H
N
S
OH
N
N
S
O
O
N
H
GLY 410
ARG 386
O
CYS 366
ARG 200
MET 414
F
TYR 448
PRO 197
TYR 415
LEU 384
Figure 3. Schematic representation of the interactions between compound 29 and
the NS5B protein.
half-lives were also observed for the alkene analog (19) and the
cyclobutyl spirocyclic derivative (28).
The X-ray crystal structure of compound 29 bound to the NS5B
protein is shown in Figure 2.13 A schematic diagram depicting en-
zyme residues near the inhibitor is also provided (Fig. 3). The 4-flu-
oro-benzyl fragment bound in
a deep hydrophobic pocket
comprised of NS5B residues Pro-197, Arg-200, Leu-384, Cys-366,
Met-414, Tyr-415 and Tyr-448. In contrast, the R4 ethyl substituent
bound in a shallow cleft comprised of Gly-410 and Met-414. Based
on these observations, we assumed a similar binding mode for all
of the 4-fluoro-benzyl containing inhibitors in Table 1.
Given the expected rotational freedom along the bond connect-
ing the benzothiadiazine and dihydropyridinone moieties of 3, we
hypothesized that depending on the substitution pattern chosen,
an NS5B binding mode similar to that exhibited by a previously de-
To further confirm our earlier hypothesis that the compounds
described in Table 2 would likely adopt a ‘flipped’ conformation
O
O
H
O
O
S
N
H
OH
N
S
S
N
N
S
O
O
O
O
O
N
H
R5
N
H
N
O
5
OH
R2 R1
39
40
Figure 4. HCV polymerase inhibitors containing a quaternary carbon center.

D. A. Ellis et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6047–6052
6051
Table 2
Exploration of the ‘flipped’ series of NS5B inhibitors
O
O
H
N
S
N
S
O
O
O
R5
N
H
N
OH
R2 R1
R5
R1
R2
IC50 1aa
(l
M)
IC50 1bb
(
lM)
EC50 1bb
(lM)
CC50 (lM)
HLM T1/2 (min)
b
b
Compd
1
Figure 1
cy-Propyl
cy-Propyl
cy-Propyl
cy-Butyl
cy-Butyl
cy-Butyl
cy-Butyl
cy-Butyl
cy-Pentyl
cy-Pentyl
cy-Pentyl
cy-Pentyl
cy-Pentyl
cy-Pentyl
cy-Pentyl
cy-Pentyl
cy-Hexyl
cy-Hexyl
cy-Hexyl
Ph
<0.025
0.068
0.075
NDc
<0.01
0.073
0.15
0.39
0.023
0.028
0.041
0.063
0.03
0.047
0.11
0.056
0.084
0.1
0.25
0.96
1
0.02
0.91
2
0.016
0.097
0.33
2.1
0.014
0.15
0.46
8.5
>100
>33
>10
>33
>10
>1
>1
ND
ND
>33
>33
>33
>1
>10
>10
>33
>33
>33
>10
ND
>60
5.5
10
ND
41
42
43
44
45
46
47
48
49
50
51h
52
53
54
55
56
57
58
59
60
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
CH2CH2CH(CH3)2
CH2CH2C(CH3)3
CH2–(4-F)Ph
CH2CH2CH(CH3)2
0.044
<0.025
0.17
0.11
0.049
0.061
0.65
0.12
0.048
0.057
ND
ND
ND
0.17
ND
ND
>60d/2.2e
>60f/5.1g
>60f/3g
>60f/2.8g
>60f/3g
>60d/2.6e
35
CH2CH2-cy-Propyl
CH2CH2C(CH3)3
CH2CH2CH3
CH2CH2CH2CH3
CH2CH2CH(CH3)2
CH2CH@C(CH3)2
CH2CH2CH(CH3)2
CH2CH2CH(CH3)2
CH2CH2-cy-Propyl
CH2CH2C(CH3)3
CH2C„CH
>10
0.045
0.24
0.043
0.3
0.17
0.31
2.9
14
0.025
1.4
H
32
CH2CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
>60f/3g
>60f/3.7g
>60f/1.6g
43
CH2–(4-F)Ph
ND
CH2CH2CH(CH3)2
CH2CH2C(CH3)3
CH2–(4-F)Ph
>60d/3.6e
>60f/1.4g
ND
ND
ND
CH2CH2CH(CH3)2
ND
3.4
ND
>60d/2.3e
a
b
c
See Ref. 12b for assay conditions and error.
See Ref. 12a for assay conditions and error.
ND = not determined.
Chiral analysis: peak 1.
Chiral analysis: peak 2.
Double experimental decay curve fitting—gradual decay phase.
Double experimental decay curve fitting—rapid decay phase.
Synthesized via hydrogenation of compound 50.
d
e
f
g
h
with respect to those in Table 1, we obtained a co-crystal structure
of compound 49 bound to the NS5B protein (Fig. 5). A schematic
diagram depicting enzyme residues is also provided in Figure 6.
As predicted, the pyridinone ring of 49 was observed to be
‘‘flipped” relative to the orientation noted for compound 29 with
the R2 isoamyl fragment of the former molecule occupying the
same deep hydrophobic pocket that was filled by the latter’s 4-flu-
oro-benzyl substituent.
ILE 447
O
O
H
N
S
O
GLN 446
GLY 410
S
O
N
O
N
N
H
OH
MET 414
TYR 415
CYS 366
ARG 200
TYR 448
LEU 384
PRO 197
Figure 6. Schematic representation of the interactions between compound 49 and
the NS5B protein.
Table 3 details the in vitro and in vivo DMPK parameters for
compound 44 compared to those observed for inhibitor 1. As ob-
served in the HLM stability studies, chiral analysis after treatment
of 44 with cynomolgus monkey liver microsomes (MLM) indicated
greater stability of one enantiomer over the other. Although oral
bioavailability was determined to be 16% for compound 44, a much
faster clearance rate relative to 1 was observed (Fig. 7),7 and as a
result, 44 was no longer detectable 12 h post oral administration.
In summary, we have identified a novel series of HCV inhibitors
derived from 5,50 and 6,60-dialkylated dihydro-pyridinone ring sys-
tems linked to a 1,1-dioxo-1,4-dihydro-1k6-benzo[1,2,4]thiadiazine
Figure 5. Co-crystal X-ray structure of compound 49 bound to the NS5B protein.

6052
D. A. Ellis et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6047–6052
Table 3
In vitro and in vivo DMPK parameters for selected compounds
Papp ((cm/s) ꢁ 10ꢀ6
)
FPO (%)
AUCinf (ng/h/mL) PO/IV
CL (IV)c (mL/min/kg)
C12 h (PO)/EC50
a
a,b
c
c
d
Compd
MLM T1/2 (min)
1
>60
55f
1.6
2.9
21
16
6041/29086
556/3563
0.63
4.7
10.49
0h
44e
13g
a
b
c
See Ref. 12c for assay conditions and error.
Controls: Papp atenolol (low) = (0.2–0.6) ꢁ 10ꢀ6 (cm/s), Papp propranolol (high) = (10–15) ꢁ 10ꢀ6 (cm/s).
Cynomolgus monkeys; dose: 1 mg/kg; formulation (for both po and iv administration): 1% DMSO, 9.9% cremophor EL in 50 mM PBS, pH 7.4.
C12 h (PO)/EC50 = plasma concentration 12 h after oral administration divided by EC50 (1b) value.
Dosed in racemic form.
Chiral analysis: peak 1.
Chiral analysis: peak 2.
Signal below lower limit of quantification.
d
e
f
g
h
7. Ruebsam, F.; Tran, C. V.; Li, L.-S.; Kim, S. H.; Xiang, A. X.; Zhou, Y.; Blazel, J. K.;
Sun, Z.; Dragovich, P. S.; Zhao, J.; McGuire, H.; Murphy, D. E.; Tran, M. T.;
Stankovic, N.; Ellis, D. A.; Gobbi, A.; Showalter, R. E.; Webber, S. E.; Shah, A. M.;
Tsan, M.; Patel, R. A.; LeBrun, L. A.; Hou, H. J.; Kamran, R.; Sergeeva, M. V.;
Bartkowski, D. M.; Nolan, T. G.; Norris, D. A.; Kirkovsky, L. Bioorg. Med. Chem.
Lett. 2009, 19, 451.
8. Donner, P. L.; Xie, Q.; Pratt, J. K.; Maring, C. J.; Kati, W.; Jiang, W.; Liu, Y.; Koev,
G.; Masse, S.; Montgomery, D.; Molla, A.; Kempf, D. J. Bioorg. Med. Chem. Lett.
2008, 18, 2735.
10000
1000
100
10
PO
IV
9. Ruebsam, F.; Tran, M. T.; Webber, S. E.; Dragovich, P. S.; Li, L.-S.; Murphy, D. E.;
Kucera, D. J.; Sun, Z.; Tran, C. V. PCT International patent application 2007, WO
2007150001 A1.
10. Although not rigorously tested, it was assumed that the chirality of the
starting materials was preserved throughout the synthesis to the final
products.
1
11. Holmquist, C. R.; Roskamp, E. J. J. Org. Chem. 1989, 54, 3258.
12. (a) Zhou, Y.; Webber, S. E.; Murphy, D. E.; Li, L.-S.; Dragovich, P. S.; Tran, C. V.;
Sun, Z.; Ruebsam, F.; Shah, A.; Tsan, M.; Showalter, R.; Patel, R.; Li, B.; Zhao, Q.;
Han, Q.; Hermann, T.; Kissinger, C.; LeBrun, L.; Sergeeva, M. V.; Kirkovsky, L.
Bioorg. Med. Chem. Lett. 2008, 18, 1413; (b) Zhou, Y.; Li, L.-S.; Dragovich, P. S.;
Murphy, D. E.; Tran, C. V.; Ruebsam, F.; Webber, S. E.; Shah, A.; Tsan, M.; Averill,
A.; Showalter, R.; Patel, R.; Han, Q.; Zhao, Q.; Hermann, T.; Kissinger, C.; LeBrun,
L.; Sergeeva, M. V. Bioorg. Med. Chem. Lett. 2008, 18, 1419; (c) Li, L.-S.; Zhou, Y.;
Murphy, D. E.; Stankovic, N.; Zhao, J.; Dragovich, P. S.; Bertolini, T.; Sun, Z.;
Ayida, B.; Tran, C. V.; Ruebsam, F.; Webber, S. E.; Shah, A. M.; Tsan, M.;
Showalter, R. E.; Patel, R.; LeBrun, L. A.; Bartkowski, D. M.; Nolan, T. G.; Norris,
D. A.; Kamran, R.; Brooks, J.; Sergeeva, M. V.; Kirkovsky, L.; Zhao, Q.; Kissinger,
C. R. Bioorg. Med. Chem. Lett. 2008, 18, 3446.
0
2
4
6
Time (h)
Figure 7. Average plasma concentrations of compound 44 in cynomolgus monkeys
at various times after iv and po administration.
moiety. SAR studies identified a number of potent compounds in
both biochemical and cellular HCV assays. Oral bioavailability was
determined for compound 44 to be 16%, however fast in vivo clear-
ance led to undetectable amounts of 44 at 12 h post dosing. Our
ongoingefforts tofurtherimprove potencyandPKpropertiesaround
this class of NS5B polymerase inhibitors will be reported in a future
Letter.
13. Crystals of HCV NS5B polymerase (genotype 1b, strain BK,
D21) were grown by
the hanging drop method at room temperature using a well buffer of 20% PEG
4 K, 50 mM ammonium sulfate, 100 mM sodium acetate, pH 4.7 with 5 mM
DTT. The crystals formed in space group P212121 with approximate cell
dimensions, a = 85 Å, b = 106 Å, c = 127 Å and two protein molecules in the
asymmetric unit. Protein/inhibitor complexes were prepared by soaking these
crystals for 3–24 h in solutions containing 15–20% DMSO, 20% glycerol, 20%
PEG 4 K, 0.1 M Hepes and 10 mM MgCl2 at pH 7.6 and an inhibitor
concentration of 2–10 mM. Diffraction data were collected to a resolution of
2.6 Å for compound 29 and 2.15 Å for compound 49. The crystal structures
discussed in this Letter has been deposited in the Protein Databank
(www.rcsb.org) with entry codes: 3IGV and 3GYN, respectively. Full
structure determination details are provided in the PDB entry.
Acknowledgments
The authors thank Drs. Peter Dragovich, James Appleman, Dev-
ron Averett and Steve Worland for their support and helpful dis-
cussions during the course of this work.
14. (a) Krueger, A. C.; Madigan, D. L.; Green, B. E.; Hutchinson, D. K.; Jiang, W. W.;
Kati, W. M.; Liu, Y.; Maring, C. J.; Masse, S. V.; McDaniel, K. F.; Middleton, T. R.;
Mo, H.; Molla, A.; Montgomery, D. A.; Ng, T. I.; Kempf, D. J. Bioorg. Med. Chem.
Lett. 2007, 17, 2289; (b) Bosse, T. D.; Larson, D. P.; Wagner, R.; Hutchinson, D.
K.; Rockway, T. W.; Kati, W. M.; Liu, Y.; Masse, S.; Middleton, T.; Mo, H.;
Montgomery, D.; Jiang, W.; Koev, G.; Kempf, D. J.; Molla, A. Bioorg. Med. Chem.
Lett. 2008, 18, 568.
15. Chiral analysis was performed on the racemic mixtures initially to verify
separation of the enantiomers and to ensure that concentrations of each were
approximately equal. After exposure of the racemic mixture to HLM, chiral
analysis was performed again. In all cases, after exposure, the area under one
peak (as determined by chiral HPLC) was substantially lower than the other.
Chiral HPLC-analysis was performed using the Chiralpak (Chiral Technologies
References and notes
1. (a) Kim, W. R. Hepatology 2002, 36, 30; (b) Alter, M. J.; Kruszon-Moran, D.; Nainan,
O. V.; McQuillan, G. M.; Gao, F.; Moyer, L. A.; Kaslow, R. A.; Margolis, H. S. N. Engl. J.
Med. 1999, 341, 556; (c) Alberti, A.; Benvegnu, L. J. Hepatol. 2003, 38, S104.
2. (a) Sidwell, R. W.; Huffman, J. H.; Khare, G. P.; Allen, L. B.; Witkowski, J. T.;
Robins, R. K. Science 1972, 177, 705; (b) Smith, R. A.; Kirkpatrick, W.. In Ribavirin
a Broad Spectrum Antiviral Agent; Academic Press: New York, 1980; Vol. xiii; (c)
De Clercq, E. Adv. Virus Res. 1993, 42, 1.
3. Hoofnagle, J. H.; Seeff, L. B. N. Eng. J. Med. 2007, 355, 2444.
4. Kolykhalov, A. A.; Agapov, E. V.; Blight, K. J.; Mihalik, K.; Feinstone, S. M.; Rice,
C. M. Science 1997, 277, 570.
5. For reviews describing nucleoside NS5B inhibitors, see: (a) Koch, U.; Narjes, F.
Curr. Top. Med. Chem. 2007, 7, 1302; (b) Carroll, S. S.; Olsen, D. B. Infect. Disord.
Drug Targets 2006, 6, 17.
6. For reviews describing non-nucleoside NS5B inhibitors, see: (a) Beaulieu, P. L.
Curr. Opin. Invest. New Drugs 2007, 8, 614; (b) Koch, U.; Narjes, F. Infect. Disord.
Drug Targets 2006, 6, 31.
Inc.) column AS-RH, 2.1 ꢁ 150 mm, 5
gradient conditions: solvent A: 0.1% formic acid in water, solvent B: 0.1%
formic acid in acetonitrile. Injected 10 of sample dissolved in 50%
lm, k = 312 nm with the following binary
lL
methanol–50% water (0.1 mg/mL). 55–95%B in 5 min followed by 0.5 min at
95%B (flow rate 0.3 mL/min). Compound mass was verified by analyzing the
eluent by either (+)-ES or APCI (+) LC–MS.