Pyrrolo[1,2-b]pyridazin-2-ones as potent inhibitors of HCV NS5B polymerase

Article abstract of DOI:10.1016/j.bmcl.2008.04.066

Pyrrolo[1,2-b]pyridazin-2-one analogs were discovered as a novel class of inhibitors of genotype 1 HCV NS5B polymerase. Structure-based design led to the discovery of compound 3k, which displayed potent inhibitory activities in biochemical and replicon as

Full text of DOI:10.1016/j.bmcl.2008.04.066

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 3616–3621
Pyrrolo[1,2-b]pyridazin-2-ones as potent inhibitors
of HCV NS5B polymerase
Frank Ruebsam,* Stephen E. Webber, Martin T. Tran, Chinh V. Tran,
Douglas E. Murphy, Jingjing Zhao, Peter S. Dragovich, Sun Hee Kim, Lian-Sheng Li,
Yuefen Zhou, Qing Han, Charles R. Kissinger, Richard E. Showalter, Matthew Lardy,
Amit M. Shah, Mei Tsan, Rupal Patel, Laurie A. LeBrun, Ruhi Kamran,
Maria V. Sergeeva, Darian M. Bartkowski, Thomas G. Nolan,
Daniel A. Norris and Leo Kirkovsky
Anadys Pharmaceuticals, Inc., 3115 Merryfield Row, San Diego, CA 92121, USA
Received 28 March 2008; revised 23 April 2008; accepted 28 April 2008
Available online 1 May 2008
Abstract—Pyrrolo[1,2-b]pyridazin-2-one analogs were discovered as a novel class of inhibitors of genotype 1 HCV NS5B polymer-
ase. Structure-based design led to the discovery of compound 3k, which displayed potent inhibitory activities in biochemical
and replicon assays (IC50 (1b) < 10 nM; EC50 (1b) = 12 nM) as well as good stability towards human liver microsomes (HLM
t1/2 > 60 min).
ꢀ 2008 Elsevier Ltd. All rights reserved.
Hepatitis C virus (HCV) is a major cause of acute hep-
atitis and chronic liver disease, including cirrhosis and
liver cancer. Globally, an estimated 170 million individ-
uals, 3% of the world’s population, are chronically in-
fected with HCV and 3–4 million people are newly
infected each year.1 Currently, there is no vaccine avail-
able to prevent hepatitis C, nor a HCV-specific antiviral
agent approved for treatment of chronic hepatitis C.
The current standard of care is a combination of pegy-
lated interferon (IFN) with ribavirin.2 Low response
rates, in particular for patients infected with genotype
1 HCV, along with significant side-effects of current
HCV therapy result in a continuing medical need for im-
proved treatments.3
Most small molecule, non-nucleoside inhibitors of NS5B
bind to one of three binding pockets, distinct from the
active site.5 Among these, we focused our attention on
the palm binding site, which, based on our analysis, is
highly conserved across various HCV genotype 1
sequences.
Several series of NS5B inhibitors have been reported to
bind at the palm binding site.6 More specifically, com-
pound 1 (Fig. 17), containing the benzo[1,2,4]thiadi-
azine-1,1-dioxide motif, has been reported to exhibit
potent inhibitory activity against NS5B with an IC50
(1b) of 0.032–0.20 lM.6c,8 As previously reported, we
discovered that compounds containing 5-hydroxy-
3(2H)-pyridazinones, as exemplified by compound 2,
can also function as potent NS5B inhibitors.9 However,
for many of these compounds we found it challenging to
overcome their limited oral bioavailability. This was
probably due to poor cell permeability likely caused
by their high polar surface area (PSA), which is outside
the normal range typically correlated with good absorp-
tion.9c,d,10 Here we describe a related series of pyrrol-
o[1,2-b]pyridazin-2-one derivatives (3), which are
derived from (2) by fusing C6 and N1 of the pyridazi-
none ring. We hypothesized that the resulting reduction
in PSA combined with the increased lipophilicity might
Our research has been focused on identifying novel
inhibitors of the HCV NS5B protein, a virally encoded
RNA-dependent RNA polymerase (RdRp), the activity
of which is critical for the replication of the virus.4
Keywords: Hepatitis C virus (HCV); NS5B polymerase; Small mole-
cule; Non-nucleoside NS5B inhibitor; Structure-based design; Pyrrol-
o[1,2-b]pyridazin-2-one.
*
Corresponding author. Tel.: +1 858 530 3708; fax: +1 858 530
3644; e-mail: fruebsam@anadyspharma.com
0960-894X/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.04.066

F. Ruebsam et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3616–3621
3617
O
O
O
O
O
O
H
N
O
S
R3
7'
S
S
OH
N
N
OH
N
N
S
OH
N
O
6
N
H
S
6
N
H
N
H
R1
3
1
N
R2
1 N
N
O
O
O
2
3
1
IC50 1b = 0.032-0.20 μM 6c,8
IC50 1b < 0.01 μM
EC50 1b = 0.005 μM
PSA = 203 Å2
Papp = 0.02 (cm/sec) x 10-6
F = 2 % (monkey)
Figure 1. HCV NS5B polymerase inhibitors.
provide improved permeability properties and thereby
afford a beneficial effect on the in vivo PK properties
compared with analogs of compound 2.
ple, N-methylation of the R3 sulfonamide moiety pres-
ent in 3c led to a >11-fold loss in potency in the
biochemical assay (3f), while replacing the R3 sulfon-
amide with a methoxy group (3b) greatly diminished
the biological activity.
Table 1 details the structure–activity relationships
(SAR) obtained for compounds 3, focusing on their bio-
chemical potencies against HCV genotype 1b, activities
against the HCV genotype 1b subgenomic replicon in
tissue culture, cytotoxicity, and stability against human
liver microsomes (HLM).
As evident in the co-crystal structure of 3c bound to
NS5B11 (Fig. 2) and as previously reported,9c the R3 sul-
fonamide forms several H-bonds with the NS5B protein.
These include an interaction between the sulfonamide
NH and the sidechain of Asp318, as well as a H-bond
between one sulfonamide oxygen and a key structural
water molecule (Fig. 3). The other R3 sulfonamide oxy-
gen forms a H-bond with the sidechain of Asn291. These
favorable interactions may explain the good activity of
3c compared to 3a–b, 3f, 3i–j, and 3m–n, which
presumably lack some of these H-bonds with the
NS5B protein.
Initially, we prepared analog 3a as a direct comparison
with 1. This compound displayed low micromolar
NS5B inhibition properties and was also active in the
replicon cell-based assay. Encouraged by these results,
we introduced a sulfonamide R3 substituent known
from our previous studies to afford potent NS5B inhib-
itory properties.9c This modification quickly led to com-
pound 3c, which displayed excellent activity in both
biochemical and replicon assays. In line with our previ-
ous findings, the R3 substituent was critical for activity
and very sensitive to structural changes.9a,c For exam-
Somewhat surprisingly, introduction of a R3 cyclopro-
pylsulfonamide moiety into the pyrrolopyridazinone
inhibitor design (compound 3g) led to a considerable
Table 1. SAR of pyrrolo[1,2-b]pyridazin-2-one analogs 3
Compound Route
R1
R2
R3
IC50
EC50
CC50
HLM
(1b)a (lM) (1b)a (lM) (GAPDH)a (lM) t1/2 (min)
a
2
Ref. 9c and d Figure 1 CH2CH2CH(CH3)2 NHSO2Me
<0.01
0.98
2.2
0.005
5.3
>33
>100
>100
>1
>60 (100%)c
14
3a
3b
3c
3d
3e
3f
B,D
B,D
B,D
B,C
B,C
A
H
H
H
F
CH2CH2CH(CH3)2
CH2CH2CH(CH3)2 OMe
H
17
NDb
42
CH2CH2CH(CH3)2 NHSO2Me
CH2CH2CH(CH3)2 NHSO2Me
CH2CH2CH(CH3)2 NHSO2Me
CH2CH2CH(CH3)2 NMeSO2Me
CH2CH2CH(CH3)2 NHSO2cPr
<0.01
0.027
0.32
0.0085
0.019
NDb
0.19
>1
>60 (78%)c
NDb
>60 (55%)c
59
10
CN
H
H
H
H
H
H
H
H
NDb
>33
>33
>1
0.11
0.16
3g
3h
3i
B,D
A
0.096
0.005
0.12
NDb
0.012
0.022
0.33
CH2CH2C(CH3)3
CH2CH2C(CH3)3
4-F-Bn
NHSO2Me
NMeSO2Me
H
<0.01
0.06
0.85
A
>33
NDb
>1
49
3j
A
45
3k
3l
A
4-F-Bn
3-Cl,4-F-Bn
4-F-Bn
NHSO2Me
NHSO2Me
NMeSO2Me
O
<0.01
0.025
0.13
>60 (86%)c
>60 (102%)c
>60 (90%)c
A
>1
>10
3m
A
O
S
3nd
A
H
4-F-Bn
0.13
0.38
>33
>60 (100%)c
a See Ref. 9a for assay conditions.
b ND, not determined.
c For values >60 min, % remaining at 60 min is given in parentheses.
d Racemic.

3618
F. Ruebsam et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3616–3621
and 3m vs 3k). We went on to explore changes in the
R1 substituents by installing a nitrile group at the 6-po-
sition of the pyrrolopyridazinone ring. This modifica-
tion resulted in a substantial loss in activity (3e
compared to 3c), suggesting that the nitrile group may
be either sterically too encumbered or too polar in nat-
ure to fit well in the shallow hydrophobic R1 binding
pocket. Installation of the smaller and more lipophilic
fluoro moiety (3d) was better tolerated but still led to
a P3-fold loss in potency in the biochemical assay (3d
vs 3c).
We tested the stability of the above compounds toward
human liver microsomes, indicated as their HLM t1/2 in
Table 1. The majority of compounds exhibited moderate
(>30 min) to long half-lives (>60 min). Compound 3a
lacking the R3 substituent was among the least stable
compounds tested. While the most potent compound
3c had a reasonable half-life (42 min), introduction of
a R1 fluorine atom (3d) led to an improvement in stabil-
ity (t1/2 > 60 min, 78% remaining at 60 min). This result
suggested that the fluorine may reduce potential metab-
olism occurring on the pyrrole ring. When comparing 3h
with 3c, a significant loss in stability was observed, pre-
sumably because the tert-butyl ethyl moiety at R2 repre-
sents a better substrate for interactions with CYPs as
compared with the isoamyl group. Interestingly, the sta-
bility could be restored by N-methylation of the R3 sul-
fonamide but unfortunately at the cost of potency as
shown in compound 3i. Compound 3k proved to be
the optimal compound exhibiting a favorable combina-
tion of long HLM half-life and potent activities in bio-
chemical and replicon assays. Collectively, our results
suggest that modifications in the R2 and R3 regions
can be utilized to successfully overcome potential meta-
bolic liabilities in this series of NS5B inhibitors.
Figure 2. Co-crystal structure of compound 3c bound to the NS5B
˚
protein (2.1 A).
Ser 556
Gly 449
HN
Ser 288
N
H
O
O
O
O
H
N
O
H
Tyr 448
H2O
N
H2O
Asn 291
N
H
O
H
S
N
H
O
O
O
S
H
O
N
N
H
O
Gln 446
Asn 411
N
H
Asp 318
N
HO
O
Met 414
Tyr 415
Leu 384
Ser 368
Tyr 448
Pro 197
Arg 200
Cys 366
Figure 3. Schematic representation of the interactions between com-
pound 3c and the NS5B protein. Hydrogen bonds are represented as
dashed lines, and the residues which make up the enzyme binding
subsites are shown.
Table 2 details the results obtained from our in vivo PK
assessment of a selected number of compounds. To our
disappointment, compounds 3c and 3k, which were ini-
tially identified to be the most promising compounds in
this series, showed only low exposure levels after oral
dosing in cynomolgus monkeys. These compounds dis-
played good stability towards monkey liver microsomes
as well as reasonable solubility in our biochemical as-
says. However, as observed previously, MLM stability
did not correlate well with the corresponding clearance
data suggesting this process may not be primarily med-
iated via biotransformation.9d We therefore assumed
that their poor permeability, as indicated by their low
Papp values in the Caco-2 assay, was responsible for
the unsatisfactory PK properties. Accordingly, we rea-
soned that lowering the PSA and/or increasing the lipo-
philicity of the compounds under study might improve
their intestinal permeability and oral bioavailability.
N-Alkylation of the sulfonamides led to an increase in
lipophilicity as predicted by their clogP values (compare
3f and 3m with 3c). Importantly, these changes also re-
sulted in lower PSA values compared to the unsubstitut-
ed sulfonamide 3c, which we believe to be beneficial for
achieving good cell permeability. Indeed, in these cases
Caco-2 permeability was increased but only translated
into similar or slightly improved exposure levels for
loss in activity in the enzymatic assay. Similar introduc-
tion of this fragment into the substituted pyridazinone
inhibitors we previously studied (e.g., 2) led only to a
3- to 4-fold reduction in NS5B inhibitory potencies.9c
While the former result is not completely understood,
it appears likely that this modification may cause a
change in the overall geometry of the pyrrolopyridazi-
none structure in the binding pocket, resulting in the ob-
served loss in activity.
Changes in the R2 moiety were generally well tolerated,
with the tert-butyl ethyl analog 3h showing comparable
activities to 3c. Also, the 4-fluorobenzyl analog 3k re-
tained good enzymatic and antiviral potencies, while
introduction of an additional chlorine atom on the R2
benzyl ring led to a slight loss of activity (3l vs 3k). This
result may indicate a sterically and/or electronically
unfavorable interaction. Again, N-methylation of the
R3 sulfonamide for the analog containing a tert-butyl
ethyl R2 moiety also resulted in loss of activities (3i vs
3h). However, this change was not as pronounced as
that observed for compounds containing isoamyl and
4-fluorobenzyl fragments in the R2 position (3f vs 3c

F. Ruebsam et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3616–3621
3619
Table 2. Correlation of calculated physicochemical parameters, in vitro DMPK data and oral bioavailabilities of selected pyrrolo[1,2-b]pyridazin-2-
one analogs 3
PSAa
clogPa
MLM
b
t1/2 (min)
Solubility
limitd (lM)
Papp
[(cm/s) · 10ꢀ6
F (%)f
AUCinf
(ng/h/mL) po/iv
CL (iv)f
(mL/min/kg)
b,e
f
Compound
]
2
203
112
167
158
167
167
167
158
ꢀ0.1
1.99
0.48
1.23
0.85
0.83
0.66
0.73
>60 (82%)c
11
>100
>200
>100
>100
>100
>100
>100
>60
0.03
11
2
63
NDg
7
30/1334
12,887/20,478
83/NDg
79/1063
141/3774
25/1209
6/539
14
49
NDg
16
5
3a
3c
3f
>60 (66%)c
60
0.2
4.3
0.8
0.7
0.1
0.9
3g
3h
3k
3m
>60 (87%)c
14
>60 (90%)c
>60 (90%)c
4
2
14
31
9
1
6
107/1776
a Calculated using ACD/Labs, version 10.0, Advanced Chemistry Development, Inc., Toronto ON, Canada. Available from www.acdlabs.com, 2006.
b See Ref. 9c for assay conditions.
c For values >60 min, % remaining at 60 min is given in parentheses. Compound 2 was tested at 5 lM, all other compounds were tested at 1 lM.
d Determined by UV absorption (2% DMSO in 20 mM Tris–HCl, pH 7.5, 20 mM NaCl, 5 mM MgCl2, 5 mM dithiothreitol, 0.1 g/L bovine serum
albumin, and 100 U/mL RNAse inhibitor).
e Controls: Papp atenolol (low) = 0.4 · 10ꢀ6 (cm/s), Papp propranolol (high) = 10 · 10ꢀ6 (cm/s).
f 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.
g ND, not determined.
O
O
O
S
R3
Route A
O
N
OR
R1
+
N
HO
N
H
NH
R2
a, b, c
O
O
S
R3
5a: R3 = I
5b: R3 = NHSO2Me
4
OH
N
N
H
Route B
R1
N
N
R2
O
OH
O
O
O O
d
S
R3
OR
H2N
3
R1
+
N
N
R2
H2N
7a: R3 = H
6
7b: R3 = OMe
7c: R3 = NHSO2Me
Scheme 1. Reagents and conditions: (a) DCC, DCM or EDC, DMAP, DMF, rt, 12 h; (b) NaOEt, EtOH, 60–80 ꢁC, 8–12 h (39–82% over two steps);
(c) when R3 = I: NHRSO2R0, CuI, sarcosine, K3PO4, DMF, 100 ꢁC, 4–16 h, (15–65%); (d) i—pyridine, 120 ꢁC, 3 h; ii—DBU (2 equiv), 120 ꢁC, 16 h
(7–10% over two steps); or i—neat, 180 ꢁC, 20 min; ii—aq KOH, 110 ꢁC, 20 h (47–52% over two steps).
Route D
R1
O
O
O
c
b
OR
a
O
OR
OR
R1
R1
R1
N
N
N
NH
N
NH
R2
NH2
OR
RO
R2
d
8
9
4
10
O
O
e, f
Route C
O
OH
O
O
g
OEt
R1
R1
N
N
N
R2
O
N
R2
O
11
6
Scheme 2. Reagents and conditions: (a) i—NaH, DMF, rt, 1 h; ii—NH2Cl, Et2O; or NH2Cl, MTBE, Aliquatꢂ 336, rt, 1.5 h (62–75%); (b) RCHO,
NaCNBH3, MeOH, 16 h (37–71%); (c) EtOOCCH2COCl, 1,4-dioxane, 100 ꢁC, 1 h, (quant.); (d) NaOEt, EtOH, 40–60 ꢁC (25–82%); (e) Pd(PPh3)4,
NH2OBnÆHCl, DCM, 40 ꢁC, 16 h (82%); (f) COCl2, toluene, K2CO3, H2O, 0 ꢁC to rt, 16 h (57%); (g) CH2(CO2Et)2, NaH, DMA, 120 ꢁC, 16 h (55%).

3620
F. Ruebsam et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3616–3621
compounds 3f and 3m. Although introduction of a ter-
minal cyclopropyl group into the sulfonamide (3g) led
to an increase in lipophilicity, the polar surface area re-
mained unchanged. Consistent with our hypothesis,
only modest improvements in cell permeability and
exposure after oral dosing were observed compared to
3c. While some improvements were achieved, the calcu-
lated logP and polar surface area values for the majority
of the compounds in Table 2 are still outside the range
exhibited low nanomolar activity in both biochemical
and replicon assays as well as good stability toward
HLM. However, PK studies indicated that the introduc-
tion of the polar R3 sulfonamide moiety contained in 3k
significantly reduced oral bioavailability, presumably
due to an increase in PSA that resulted in poor absorp-
tion. Results from additional modifications to this inhib-
itor series will be reported in the future.
2
10
˚
of most known orally bioavailable drugs (<140 A ).
Consistent with this hypothesis, when the polar R3 sul-
fonamide group was removed (3a), a significantly lower
PSA combined with increased lipophilicity was
achieved, resulting in good Caco-2 permeability and oral
bioavailability. While the compounds described in this
work, with the exception of compound 3a, suffer from
high polarity that prevents them from being absorbed
effectively, these results provided us with additional
direction to further improve the PK properties of the
benzothiadiazine-containing NS5B inhibitors while
retaining potent biological activity.
Acknowledgments
The authors thank Drs. Devron Averett and Steve Wor-
land for their support and helpful discussions during the
course of this work.
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.;
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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, p 237; (c) De Clercq, E. Adv. Virus Res.
1993, 42, 1.
Compounds 3 were synthesized following Routes A or B
as shown in Scheme 1. In Route A, aminoesters 4 were
coupled with acids 512 in the presence of DCC or
EDC to form the corresponding amide intermediates.
Treatment with NaOEt afforded the desired compounds
3. Alternatively, in Route B, the esters 6 were condensed
with 2-aminobenzensulfonamides 713 by heating in pyr-
idine to furnish the corresponding amide intermediates,
which were then cyclized in the presence of DBU to yield
the desired compounds 3. When intermediate 5a, bear-
ing an iodo group at the 7-position, was employed, the
corresponding sulfonamides 3 could be accessed via
Cu-mediated displacement of the iodo moiety.14
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. Koch, U.; Narjes, F. Curr. Top. Med. Chem. 2007, 7, 1302.
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R.; Fenwick, R. H.; Fernandes, S.; Guidetti, R.; Haigh,
D.; Hartley, C. D.; Howes, P. D.; Jackson, D. L.; Jarvest,
R. L.; Lovegrove, V. L. H.; Medhurst, K. J.; Parry, N. R.;
Price, H.; Shah, P.; Singh, O. M. P.; Stocker, R.;
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K.; Lin-Goerke, J.; Darcy, M.; Shaw, A. N.; Gu, B.;
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Earnshaw, D. L.; Casper, D. J.; Kaura, A.; Baker, A.;
Greenwood, C.; Gutshall, L. L.; Maley, D.; DelVecchio,
A.; Macarron, R.; Hofmann, G. A.; Alnoah, Z.; Cheng,
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J. WO2006117306, 2006; (e) Pratt, J. K.; Donner, P.;
McDaniel, K. F.; Maring, C. J.; Kati, W. M.; Mo, H.;
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Montgomery, D.; Molla, A.; Kempf, D. J.; Kohlbrenner,
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inson, D. K. et al. U.S. Patent US2005107364, 2005.
7. All structures are arbitrarily drawn as one of several
possible tautomers.
The synthesis of key intermediates 4 and 6 is illustrated
in Scheme 2. Pyrrole 2-allyl esters 8 (with the exception
of 8e, where the methyl ester was used) were prepared
according to literature procedures.15 N-Amination,
using freshly or in situ prepared monochloramine,16 fol-
lowed by reductive alkylation in the presence of NaC-
NBH3, provided key intermediates 4. These could be
further elaborated into 6 via different routes as shown
in Scheme 2. Initially, we investigated accessing 6 via
formation of cyclic anhydrides 11, which upon treat-
ment with diethylmalonate in the presence of NaH affor-
ded the desired products 6 (Route C). However, this
route required hydrolysis of the esters prior to treatment
with phosgene, which initially led to decomposition of
the starting materials 4 when simple alkyl esters (e.g.,
methyl esters) were employed. While employing an allyl
ester, which was later removed in the presence of
Pd(PPh3)4, effectively solved that issue, we later focused
on the reaction sequence shown as Route D in Scheme 2.
In this route, compounds 4 were treated with ethyl mal-
onyl chloride to form the intermediates 10, which were
then converted to 6 in the presence of NaOEt.
8. Tedesco, R.; Shaw, A. N.; Bambal, R.; Chai, D.; Concha,
N. O.; Darcy, M. G.; Dhanak, D.; Fitch, D. M.; Gates,
A.; Gerhardt, W. G.; Halegoua, D. L.; Han, C.; Hofmann,
G. A.; Johnston, V. K.; Kaura, A. C.; Liu, N.; Keenan, R.
M.; Goerke, J. L.; Sarisky, R. T.; Wiggall, K. J.;
In summary, we have synthesized a novel class of pyrrol-
o[1,2-b]pyridazin-2-ones as potent inhibitors of geno-
type 1 HCV NS5B polymerase. Our optimization
efforts led to the discovery of compound 3k, which

F. Ruebsam et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3616–3621
3621
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Zimmerman, M. N.; Duffy, K. J. J. Med. Chem. 2006, 49,
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were collected to a resolution of 2.1 A for compound 3c.
The crystal structure discussed in this paper has been
deposited in the Protein Databank (www.rcsb.org) with
entry code: 3CO9. Full structure determination details are
provided in the PDB entry.
9. (a) Zhou, Y.; Webber, S. E.; Murphy, D. E.; Li, L.-S.;
Dragovich, P. S.; Tran, C. V.; Sun, Z.; Ruebsam, F.; Shah,
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Han, Q.; Zhao, Q.; Hermann, T.; Kissinger, C.; LeBrun,
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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.
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13. Intermediate 7a was prepared using modified literature
procedures as described in (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.;
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2289; (b) Hutchinson, D. K. et al. United States Patent
Application #: 2005/0107364; (c) Goldfarb, A. R.; Berk, B.
J. Am. Chem. Soc. 1943, 65, 738; intermediate 7b was
prepared using modified literature procedures as described
in (d) Fitch, M. D.; Evans, K. A.; Chai, D.; Duffy, K. J. Org.
Lett. 2005, 7, 5521; (e) Kovalenko, S. N.; Chernykh, V. P.;
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A. Chem. Heterocycl. Compd. (Engl. Transl.) 1999, 34, 791;
(f) Intermediate 7c was prepared as described in Dragovich,
P. S.; Murphy, D. E.; Tran, C. V.; Ruebsam, F. Synth.
Commun.
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2008.
Available
from
doi:10.1080/
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11. 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 4K,
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 A,
˚
˚
b = 106 A, c = 127 A and two protein molecules in the
asymmetric unit. Protein/inhibitor complexes were pre-
pared by soaking these crystals for 3–24 h in solutions
containing 15–20% DMSO, 20% glycerol, 20% PEG 4K,
0.1 M Hepes and 10 mM MgCl2 at pH 7.6 and an
inhibitor concentration of 2–10 mM. Diffraction data