Novel HCV NS5B polymerase inhibitors derived from 4-(1′,1′-dioxo-1′,4′-dihydro-1′λ6-benzo[1′,2′,4′]thiadiazin-3′-yl)-5-hydroxy-2H-pyridazin-3-ones. Part 5: Exploration of pyridazinones containing 6-amino-substituents

Article abstract of DOI:10.1016/j.bmcl.2008.08.094

The synthesis of 4-(1′,1′-dioxo-1′,4′-dihydro-1′λ⁶-benzo[1′,2′,4′]thiadiazin-3′-yl)-5-hydroxy-2H-pyridazin-3-ones bearing 6-amino substituents 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 μM). In vitro DMPK data (microsome t_1/2, Caco-2 P_app) for many of the compounds are also disclosed, and a crystal structure of a representative inhibitor complexed with the NS5B protein is discussed.

Full text of DOI:10.1016/j.bmcl.2008.08.094

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5635–5639
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel HCV NS5B polymerase inhibitors derived from 4-(10,10-dioxo-10,40-
dihydro-10k6-benzo[10,20,40]thiadiazin-30-yl)-5-hydroxy-2H-pyridazin-3-ones.
Part 5: Exploration of pyridazinones containing 6-amino-substituents
*
Peter S. Dragovich , Julie K. Blazel, David A. Ellis, Qing Han, Ruhi Kamran, Charles R. Kissinger,
Laurie A. LeBrun, Lian-Sheng Li, Douglas E. Murphy, Michael Noble, Rupal A. Patel, Frank Ruebsam,
Maria V. Sergeeva, Amit M. Shah, Richard E. Showalter, Chinh V. Tran, Mei Tsan, Stephen E. Webber,
Leo Kirkovsky, Yuefen Zhou
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 synthesis of 4-(10,10-dioxo-10,40-dihydro-10k6-benzo[10,20,40]thiadiazin-30-yl)-5-hydroxy-2H-pyrida-
zin-3-ones bearing 6-amino substituents as potent inhibitors of the HCV RNA-dependent RNA polymer-
ase (NS5B) is described. Several of these agents also display potent antiviral activity in cell culture
experiments (EC50 < 0.10 lM). In vitro DMPK data (microsome t1/2, Caco-2 Papp) for many of the com-
pounds are also disclosed, and a crystal structure of a representative inhibitor complexed with the
Article history:
Received 20 July 2008
Revised 24 August 2008
Accepted 26 August 2008
Available online 29 August 2008
NS5B protein is discussed.
Keywords:
Ó 2008 Elsevier Ltd. All rights reserved.
Pyridazinones
5-Hydroxy-3(2H)-pyridazinone derivatives
Hepatitis C virus (HCV)
RNA-dependent RNA polymerase (RdRp)
Non-nucleoside NS5B inhibitors
Chronic hepatitis C virus (HCV) infection afflicts more than 170
million people worldwide and 3–4 million individuals are esti-
mated to become newly infected each year.1 Current HCV therapy
is comprised of combinations of pegylated interferon (peg-IFN) and
the nucleoside analog ribavirin (RBV). Unfortunately, response
rates to this therapy are sub-optimal and are particularly low in
patients infected with genotype 1 HCV.2 In addition, treatment
with peg-IFN/RBV is often associated with significant side effects
including flu-like symptoms, depression, and anemia.2,3 The rela-
tively low response rates and the significant side effect burden of
current HCV therapies necessitate the identification of more effec-
tive anti-HCV agents, especially for treatment of patients infected
with genotype 1 HCV.
The HCV RNA-dependent RNA polymerase (RdRp, NS5B) is an
attractive target for the development of novel HCV treatments.4
The activity of this virally encoded enzyme is essential for HCV rep-
lication5 and many nucleoside and non-nucleoside NS5B inhibitors
have been described in the literature.6,7 Encouragingly, several
such molecules have also exhibited antiviral activity in HCV in-
fected patients.8 Most of the non-nucleoside NS5B inhibitors bind
to one of three locations on the enzyme’s surface that are distinct
from the active site.7 Among these, the Anadys NS5B inhibitor
development efforts have focused on compounds which bind to
the ‘palm’ site of the NS5B protein.
We previously disclosed the identification of a novel series of
non-nucleoside NS5B inhibitors containing a 1,1-dioxo-1,4-dihy-
dro-1k6-benzo[1,2,4]thiadiazine moiety linked to a substituted 5-
hydroxy-3(2H)-pyridazinone (exemplified by structure 1,
Fig. 1).9–11 Many such compounds exhibit potent NS5B inhibition
activities and often display sub-micromolar antiviral properties
when tested against an HCV replicon in cell culture. Unfortunately,
these molecules also frequently exhibit poor pharmacokinetic
properties following oral administration to animals.9c,9d In an effort
to improve these undesirable pharmacokinetic characteristics, we
sought to diversify the 5-hydroxy-3(2H)-pyridazinone pharmaco-
phore by the inclusion of a 6-amino-substituent into the inhibitor
design (structure 2, Fig. 1).12 Below, we describe the preparation of
such compounds along with an initial assessment of their biologi-
cal properties and structure–activity relationships.
The desired 6-aminopyridazinones (2) were prepared using two
related synthetic methods (Scheme 1).13 Each of these methods in-
volved derivatization of an appropriately substituted 6-amino-5-
hydroxy-3(2H)-pyridazinone intermediate bearing an ethyl ester
* Corresponding author. Tel.: +1 858 530 3648; fax: +1 858 527 1540.
E-mail address: pdragovich@anadyspharma.com (P.S. Dragovich).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.08.094

5636
P. S. Dragovich et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5635–5639
O
O
O
O
H
H
S
N
CH3
S
N
CH3
OH
5
N
S
R1
N
OH
N
S
O
O
O
O
R2
N
H
S
N
H
3
N
N
1
N
O
N
R3
O
1
2
IC50 (1b) = <10 nM
EC50 (1b) = 5 nM
HLM T1/2 = >60min
Papp = 0.02 x 10-6 cm/sec
Figure 1. 5-Hydroxy-3(2H)-pyridazinone derivatives as HCV NS5B polymerase inhibitors.
R1
N
OH
O
O
O
H
N
Method A
a
S
CH3
R2
OEt
H2N
S
2
+
+
O
O
N
H2N
N
R3
O
3
4
b
O
O
S
I
Method B
R1
N
OH
N
O
O
S
I
a
H2N
R2
N
H
N
H2N
N
O
R3
5
6
Scheme 1. General methods for the synthesis of aminopyridazinone inhibitors (2). (a) Pyridine, 110 °C, 2–8 h; then add 2 equiv DBU, 110 °C, 2–12 h, 10–30%. (b) 0.05 equiv
CuI, 0.20 equiv sarcosine, 2.5 equiv K3PO4, 1.2 equiv H2NSO2CH3, DMF, 100 °C, 20–36 h, 10–20% (step b).
moiety at the 4-position (structure 3).14 The most straightforward
synthesis of 2 (Method A) entailed thermal condensation of 3 with
2-amino-5-methanesulfonylaminobenzenesulfonamide (4)15 in
pyridine to form an amide intermediate (not shown), followed by
addition of DBU and continued heating to effect benzothiadiazine
formation. For reasons that we do not currently understand, this
direct synthesis failed to provide significant quantities of 2 when
applied to several aminopyridazinone substrates 3. Accordingly,
we developed an alternate synthesis of compounds 2 that was em-
ployed for these problematic cases.
This alternate preparation (Method B, Scheme 1) involved ther-
mal condensation of iodo compound 515c,16 in the benzothiadi-
azine-forming derivatization of 3 to give 6. Copper-mediated
coupling of the iodide present in 6 with methanesulfonamide then
provided 2 in moderate overall yield.17
The structure–activity relationships associated with the amino-
pyridazinone-containing NS5B inhibitors under study are depicted
in Table 1. Many of these molecules incorporate an isoamyl
(CH2CH2iPr) substituent at the pyridazinone 2-position (R3 in
structure 2) that was known from our earlier studies to impart po-
tent NS5B inhibition properties to pyridazinone-containing com-
pounds.9 For example, a molecule that contained such an R3
fragment and a diethylamino moiety at the 6-position on the
pyridazinone ring displayed sub-micromolar levels of both NS5B
inhibition activity and antiviral potency (2a). Several related com-
pounds bearing N-methyl-N-alkyl-aminopyridazinone 6-substitu-
ents were also examined, and the corresponding NS5B inhibition
properties were observed to improve somewhat for those contain-
ing bulkier alkyl groups (cf. 2b with 2c and 2d). An even more dra-
matic improvement in both anti-NS5B and antiviral activity was
realized by replacing the diethylamino moiety present in the lead
molecule with a pyrrolidine ring (cf. 2e with 2a). However, incor-
porating a piperidine moiety at this location did not afford similar
enhancements (cf. 2f with 2a). These results parallel the SAR trends
previously observed during our variation of alkyl and aryl pyridaz-
inone 6-substituents in which compounds bearing 5-membered
cyclic structures were more potent NS5B inhibitors than analogs
containing 6-membered moieties.9
Interestingly, addition of a methyl group adjacent to the ring
nitrogen in compounds 2e and 2f had opposite effects on inhibitor
potency. Derivatization of the former compound in this manner re-
sulted in a worsening of anti-NS5B and antiviral properties (cf. 2e
with 2g), while similar modification of the latter molecule slightly
improved NS5B inhibition activity (cf. 2f with 2h). Incorporation of
a morpholine moiety in lieu of the piperidine ring in the inhibitor
design provided a considerable improvement in anti-NS5B proper-
ties (cf. 2i with 2f). However, this alteration was detrimental to
antiviral activity in cell culture, as 2i was considerably weaker in
the replicon assay than several other compounds with equivalent
NS5B inhibition properties (cf. 2i with 2e, 2j, and 2k). The large
EC50/IC50 ratio exhibited by 2i is likely due to the compound’s poor
cell membrane permeability properties. Consistent with this
hypothesis, 2i exhibited a Caco-2 Papp value that was significantly
lower (less permeable) than all of the other 6-aminopyridazinones
examined in this work (Table 1).
Several additional NS5B inhibitors were prepared which com-
bined optimal 6-amino substituents with non-isoamyl R3 moieties
that were also known from our previous SAR studies to impart po-
tent NS5B inhibition properties to the compounds that contained
them.9 For example, a molecule incorporating an R3 tert-butyl ethyl

P. S. Dragovich et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5635–5639
5637
Table 1
Structure–activity relationships of aminopyridazinone NS5B inhibitors (2)
Compounda
Synth.
method
R1
R2
R3
1a IC50
M)
1b IC50
M)
EC50
M)
CC50
M)
HLM t1/2
(min)
Caco-2 Papp
b
c
d
d
e
f
(
l
(
l
(
l
(
l
(10ꢀ6 cm/s)
2a
2b
2c
2d
2e
2f
A
B
B
A
A
A
A
Et
Pr
iPr
tBu
Pyrrolidine
Piperidine
2-Me-
Et
CH2CH2iPr
CH2CH2iPr
CH2CH2iPr
CH2CH2iPr
CH2CH2iPr
CH2CH2iPr
CH2CH2iPr
NDg
0.50
0.47
ND
<0.025
ND
0.43
0.14
0.038
0.047
0.014
0.35
2.2
>33
13
8
4
0.30
0.12
0.77
ND
0.32
ND
Me
Me
Me
0.58
0.65
0.50
0.017
1.0
> 1.0
> 1.0
>10
>1.0
>33
4
10
21
9
2gh
ND
0.047
0.10
>10
ND
Pyrrolidine
2-Me-
2hh
A
CH2CH2iPr
ND
0.17
1.1
>10
5
ND
Piperidine
Morpholine
Pyrrolidine
Pyrrolidine
tBu
2i
2j
2k
2l
2m
B
A
A
A
A
CH2CH2iPr
CH2CH2tBu
CH2CH2CyPr
CH2CH2tBu
Ph
0.073
0.086
0.038
ND
0.015
0.029
0.035
0.20
0.62
0.011
0.19
0.20
>10
> 33
> 10
>1.0
>33
>60 (69)
14
9
6
0.021
0.37
0.22
ND
Me
Pyrrolidine
ND
0.11
>10
>60 (90)
ND
a
See Ref. 13.
b
c
Genotype 1a NS5B polymerase. See Ref. 9b for assay method and experimental error.
Genotype 1b NS5B polymerase. See Ref. 9a for assay method and experimental error.
Genotype 1b HCV replicon activity and associated cytotoxicity. See Ref. 9a for assay method and experimental error. CC50 limit determined at the highest concentration of
d
the compound tested in the assay.
e
Half-life in presence of human liver microsomes. For values P60 min, % remaining at 60 min is given in parentheses. All compounds were tested at 1 lM. See Ref. 9a for
assay method and experimental error.
f
See Ref. 9c for assay method and experimental error. Controls: Papp Atenolol (low) = 0.3 (cm/sec) ꢁ 10ꢀ6, Papp Propranolol (high) = 10 (cm/s) ꢁ 10ꢀ6
.
g
ND, not determined.
Racemic.
h
(CH2CH2tBu) fragment and a pyrrolidine substituent at the 2-posi-
even poorer activity in the replicon cell-based assay. These obser-
vations highlight the difficulty of improving the HLM stability of
this inhibitor series while simultaneously retaining good potency,
permeability, and/or absorption characteristics.
tion of the pyridazinone ring displayed excellent NS5B inhibition
activity and potent antiviral properties in cell culture (2j, Table
1). Similarly potent anti-NS5B characteristics were noted for a
compound which contained an R3 cyclopropyl ethyl moiety
(CH2CH2CyPr) in lieu of the tert-butyl ethyl fragment (2k). How-
ever, the antiviral activity exhibited by the latter molecule was sig-
nificantly worse than that displayed by the former inhibitor. This
difference may be due to differing cell membrane permeability
properties of the compounds in question, although the correspond-
ing Caco-2 Papp values were similar for both molecules. Somewhat
surprisingly, combination of the tert-butyl ethyl R3 fragment with a
N-methyl-N-tert-butyl-aminopyridazinone 6-substituent resulted
in somewhat poorer NS5B inhibition activity relative to the corre-
sponding R3 isoamyl analog (cf. 2l with 2d). However, the two
compounds displayed similar antiviral properties in the cell-based
assay. While the precise reasons for these activity trends are not
currently understood, these results (along with those observed
above for compounds 2g and 2h) suggest that NS5B recognition
by this class of inhibitors is highly sensitive to subtle structural
variations within both the R3 moiety and the 6-aminopyridazinone
substituent. Accordingly, a molecule incorporating a phenyl R3
fragment (2m) displayed reduced NS5B inhibition activity and
drastically attenuated antiviral properties relative to the most po-
tent compounds described in this work.
The X-ray crystal structure of compound 2e bound to the NS5B
protein is shown in Figure 2.18 A schematic diagram depicting en-
zyme residues near the inhibitor as well as protein–ligand hydro-
gen bonding interactions is also provided (Fig. 3). As was
observed in previous pyridazinone-NS5B crystal structures,9a–c
the R3 isoamyl fragment of 2e 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 pyrrolidine ring
present in 2e bound to a much more shallow cleft near the surface
of the enzyme that was made up of residues Gly-410, Met-414, and
Gln-446. The 5-hydroxy group on the pyridazinone ring formed
hydrogen bonds with the backbone amide NH of Tyr-448 and a
structural water molecule that interacted with the backbone NH
of Gly-449. In addition, one of the oxygen atoms present in the
benzothiadiazine ring system of 2e and one of the methylsulfona-
mide oxygen atoms each formed a hydrogen bond with a second
structural water molecule (itself hydrogen-bonded to the side
The stability of the aminopyridazinone-containing compounds
toward human liver microsomes (HLM) was also assessed to better
predict their potential for metabolic transformation in vivo. As
shown in Table 1, the majority of the molecules tested displayed
relatively short HLM half-lives (<25 min) that were considerably
attenuated relative to the long half-life exhibited by compound 1
(Fig. 1). These results suggest that incorporation of 6-amino-sub-
stituents into the pyridazinone-containing NS5B inhibitor series
introduces at least one metabolically labile site into the inhibitor
design. One exception to this trend was compound 2i which dis-
played a long half-life in the HLM assay. As described above, how-
ever, this molecule also exhibited reduced Caco-2 permeability and
antiviral properties relative to the other aminopyridazinone-con-
taining inhibitors. The other exception (compound 2m) displayed
Figure 2. Co-crystal X-ray structure of compound 2e bound to the NS5B protein
(1.9 Å).18

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P. S. Dragovich et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5635–5639
SER 288
TYR 448
O
OH
GLY 449
O
ASN 291
O
N
H
H2O
NH
H2N
O
H2O
O
S
O
O
OH
GLN 446
GLY 410
S
NH
OH
N
N
O
N
N
H
ASP 318
N
MET 414
MET 414
TYR 415
O
CYS 366
ARG 200
PRO 197 TYR 448
LEU 384
Figure 3. Schematic diagram of compound 2e bound in the NS5B palm site. Hydrogen bonds are represented as dashed lines, and the residues which make up the enzyme-
binding subsites are depicted.19
7. For reviews describing non-nucleoside NS5B inhibitors, see: (a) Beaulieu, P. L.
chain of Ser-288). Hydrogen bonds were also noted between the
other methylsulfonamide oxygen atom and the side chain of Asn-
Curr. Opin. Invest. New Drugs 2007, 8, 614; (b) Ref. 6a.; (c) Koch, U.; Narjes, F.
Infect. Disord. Drug Targets 2006, 6, 31.
291 as well as between the methylsulfonamide NH moiety and
the side chain of Asp-318. Similar hydrogen bonding interactions
were observed in co-crystal structures of other benzothiadiazine-
containing NS5B inhibitors with NS5B,9a–c and these likely contrib-
ute to the potent polymerase inhibition properties exhibited by
many of the compounds described in this work.
8. (a) Sorbera, L. A.; Castaner, J.; Leeson, P. A. Drugs Future 2006, 31, 320; (b)
Villano, S.; Chandra, P.; Raible, D., et al., Digestive Disease Week 2006, Los
Angeles, CA, May 20–25, 2006; (c) Schmeltz A.; Dunne, M.; 47th ICAAC,
September 17–20, 2007; (d) Pharmasset press release, January 7, 2008.
9. (a) Zhou, Y.; Webber, S. E.; Murphy, D. E.; Li, L.-S.; Dragovich, P. S.; Tran, C. V.;
Sun, Z.; Ruebsam, F.; Shah, A. M.; Tsan, M.; Showalter, R. E.; Patel, R.; Li, B.;
Zhao, Q.; Han, Q.; Hermann, T.; Kissinger, C. R.; 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.
M.; Tsan, M.; Averill, A.; Showalter, R. E.; Patel, R.; Han, Q.; Zhao, Q.; Hermann,
T.; Kissinger, C. R.; 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, 3446; (d) Sergeeva, M. V.;
Zhou, Y.; Bartkowski, D. M.; Nolan, T. G.; Norris, D. A.; Okamoto, E.; Kirkovsky,
L.; Kamran, R.; LeBrun, L. A.; Tsan, M.; Patel, R.; Shah, A. M.; Lardy, M.; Gobbi,
A.; Li, L.-S.; Zhao, J.; Bertolini, T.; Stankovic, N.; Sun, Z.; Murphy, D. E.; Webber,
S. E.; Dragovich, P. S. Bioorg. Med. Chem. Lett. 2008, 3421.
In summary, we synthesized a new class of 4-(10,10-dioxo-10,40-
dihydro-10k6-benzo[10,20,40]thiadiazin-30-yl)-5-hydroxy-2H-pyrida-
zin-3-ones bearing 6-amino substituents as potent inhibitors of the
HCV RNA-dependent RNA polymerase (NS5B). Many of these
agents also display antiviral activity in cell culture experiments.
However, in vitro DMPK data suggest that it may be difficult to
combine potent antiviral activity, good metabolic stability, and
favorable permeability/absorption characteristics in this class of
NS5B inhibitors. Further optimization of the benzothiadiazine-con-
taining compounds will be described in future communications.
10. The structures of all compounds described in this work are arbitrarily drawn in
a single tautomeric form.
11. For other examples of benzothiadiazine-containing NS5B inhibitors, see: (a)
Hutchinson, D. K.; Rosenberg, T.; Klein, L. L.; Bosse, T. D.; Larson, D. P.; He, W.;
Jiang, W. W.; Kati, W. M.; Kohlbrenner, W. E.; Liu, Y.; Masse, S. V.; Middleton,
T.; Molla, A.; Montgomery, D. A.; Beno, D. W. A.; Stewart, K. D.; Stoll, V. S.;
Kempf, D. J. Bioorg. Med. Chem. Lett. 2008, 18, 3887; (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; (c) 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; (d) 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.; Zimmerman, M. N.; Duffy, K. J. J. Med. Chem. 2006, 49, 971; (e)
Pratt, J. K.; Donner, P.; McDaniel, K. F.; Maring, C. J.; Kati, W. M.; Mo, H.;
Middleton, T.; Liu, Y.; Ng, T.; Xie, Q.; Zhang, R.; Montgomery, D.; Molla, A.;
Kempf, D. J.; Kohlbrenner, W. Bioorg. Med. Chem. Lett. 2005, 15, 1577.
12. The research described in this work was conducted prior to our obtaining data
suggesting that pyridazinone PK improvements could be achieved through the
reduction of compound polar surface area. See Refs. 9c and 9d for additional
details regarding this analysis.
Acknowledgments
The authors thank Drs. Devron Averett and Steve Worland for
their support and helpful discussions during the course of this
work.
References and notes
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Drug Targets 2006, 6, 17.
13. The structures of all new compounds described in this work were consistent
with 1H NMR and LC–MS analysis data (P95% HPLC purity).
14. For the synthesis of the 6-amino-5-hydroxy-3(2H)-pyridazinone intermediates
required to prepare the compounds described in this work, see: Dragovich, P.
S.; Blazel, J. K.; Dao, K.; Ellis, D. A.; Li, L.-S.; Murphy, D. E.; Ruebsam, F.; Tran, C.
V.; Zhou, Y. Synthesis 2008, 610.

P. S. Dragovich et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5635–5639
5639
15. (a) Dragovich, P. S.; Murphy, D. E.; Tran, C. V.; Ruebsam, F. Synth. Commun.
2008, 38, 1909; (b) Ref. 11c; (c) Hutchinson, D. K., et al., United States Patent
Application #: 2005/0107364; (d) Goldfarb, A. R.; Berk, B. J. Am. Chem. Soc.
1943, 65, 738.
16. Sun, Z.; Li, L.-S.; Webber S. E.; Ruebsam, F.; Tran, M.T.; Dragovich P. S.; Kucera,
D.J.; Murphy, D.E.; Tran CV WO2007150001, 2007.
dimensions, a = 85 Å, b = 106 Å, c = 127 Å containing two protein molecules in
the asymmetric unit. Protein–inhibitor complexes were prepared by soaking
these NS5B crystals for 3–24 h in solutions containing 15–20% DMSO, 20%
glycerol, 20% PEG 4 K, 0.1 M HEPES, 10 mM MgCl2 at pH 7.6 and inhibitors at
concentrations of 2–10 mM. Diffraction data were collected to a resolution of
1.9 Å for compound 2e. This crystal structure has been deposited in the Protein
Databank (www.rcsb.org) with entry code: 3E51. Full details of the structure
determination are given in the PDB entry.
17. Deng, W.; Liu, L.; Zhang, C.; Liu, M.; Guo, Q.-X. Tetrahedron Lett. 2005, 46, 7295.
18. 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
19. The O atom at the 5-position of the pyridazinone in Figure 3 was arbitrarily
drawn in the protonated form. The precise extent of the protonation was not
determined.