Hexahydro-pyrrolo- and hexahydro-1H-pyrido[1,2-b]pyridazin-2-ones as potent inhibitors of HCV NS5B polymerase

Article abstract of DOI:10.1016/j.bmcl.2008.08.017

Hexahydro-pyrrolo- and hexahydro-1H-pyrido[1,2-b]pyridazin-2-one analogs were discovered as a novel class of inhibitors of genotype 1 HCV NS5B polymerase. Among these, compound 4c displayed potent inhibitory activities in biochemical and replicon assays (IC₅₀ (1b) <10 nM; EC₅₀ (1b) = 34 nM) as well as good stability towards human liver microsomes (HLM t_1/2 = 59 min).

Full text of DOI:10.1016/j.bmcl.2008.08.017

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5002–5005
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Hexahydro-pyrrolo- and hexahydro-1H-pyrido[1,2-b]pyridazin-2-ones
as potent inhibitors of HCV NS5B polymerase
*
Frank Ruebsam , Zhongxiang Sun, Benjamin K. Ayida, Stephen E. Webber, Yuefen Zhou, Qiang Zhao,
Charles R. Kissinger, Richard E. Showalter, Amit M. Shah, Mei Tsan, Rupal Patel, Laurie A. LeBrun,
Ruhi Kamran, Maria V. Sergeeva, Darian M. Bartkowski, Thomas G. Nolan, Daniel A. Norris, 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
Article history:
Hexahydro-pyrrolo- and hexahydro-1H-pyrido[1,2-b]pyridazin-2-one analogs were discovered as a novel
class of inhibitors of genotype 1 HCV NS5B polymerase. Among these, compound 4c displayed potent
inhibitory activities in biochemical and replicon assays (IC50 (1b) <10 nM; EC50 (1b) = 34 nM) as well
as good stability towards human liver microsomes (HLM t1/2 = 59 min).
Received 12 June 2008
Revised 5 August 2008
Accepted 6 August 2008
Available online 9 August 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Hepatitis C Virus (HCV)
NS5B polymerase
Small-molecule, non-nucleoside NS5B
inhibitor
Structure-based design
Hexahydro-pyrrolo[1,2-b]pyridazin-2-one,
Hexahydro-1H-pyrido[1,2-b]pyridazin-2-
one
Hepatitis C virus (HCV), a blood-borne pathogen belonging to
the Flaviviridae family of viruses,1 is a major cause of acute hepati-
tis and chronic liver disease, including cirrhosis and liver cancer.
The disease affects an estimated 170 million individuals worldwide
and 4 million people in the US, with 3–4 million people newly
infected each year.2 Despite major efforts by research groups in
academia and the pharmaceutical industry, there is currently no
vaccine available to prevent hepatitis C, nor a HCV-specific antivi-
ral agent approved for treatment of chronic hepatitis C. The current
standard of care is a combination of pegylated interferon (IFN)
with ribavirin.3
However, the current HCV therapy suffers from inadequate sus-
tained viral response rates, in particular for patients infected with
genotype 1 HCV, along with significant side-effects, resulting in a
continuing medical need for improved treatments.4 Our research
has been focused on identifying novel non-nucleoside inhibitors
of the HCV NS5B protein, a virally encoded RNA-dependent RNA
polymerase (RdRp), the activity of which is critical for the replica-
tion of the virus.5 We focused our attention on the palm binding
site, one of several inhibitor binding pockets distinct from the
active site.6 Several series of NS5B inhibitors have been reported
to bind at the palm binding site.7 More specifically, we have previ-
ously reported that compounds containing the pyridazin-2-one
motif, as exemplified by compound 1 (Fig. 18) exhibit potent inhib-
itory activity against NS5B with IC50 (1b) values of <0.01 l
M.9
However, many of these compounds displayed limited oral bio-
availability in animals that was likely due to poor intestinal
absorption. We believe that this is related to the high polar surface
area (PSA) of these compounds, which is outside the normal range
typically correlated with good absorption (<140 Å2).9c,9d,10 Accord-
ingly, we explored other palm-binding NS5B inhibitors with
reduced PSA values relative to compound 1 (e.g., 211 and 312) in
the hope of improving both intestinal permeability and oral bio-
availability. Unfortunately, these alternate inhibitor series did not
display significant improvements in these two biological
parameters.
Here we disclose a new series of palm-binding NS5B inhibitors
that also possess lower PSA values relative to compound 1. The
described compounds (4, Fig. 1) incorporate hexahydro-pyrrolo-
and hexahydro-1H-pyrido[1,2-b]pyridazin-2-ones into their de-
sign, the former of which are formally derived from 3 by saturation
of the fused pyrrole ring. Our analysis of the co-crystal structure of
3b bound to the NS5B protein12 suggested that the saturated, and
* Corresponding author. Tel.: +1 858 530 3708; fax: +1 858 530 3644.
E-mail address: fruebsam@anadyspharma.com (F. Ruebsam).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.08.017

F. Ruebsam et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5002–5005
5003
R3
N
O
O
O
O
O
O
O
O
H
N
H
N
H
N
S
S
S
S
S
OH
N
N
OH
N
S
OH
N
OH
S
N
S
O O
4a
O O
O O
O O
R1
S
S
N
H
N
H
N
H
N
H
N
N
N
N
O
O
N
R2
O
N
R2
O
n
n = 1, 2
3a R2 = 4-F-Bn
IC50 1b < 0.01 μM
4
2
1
IC50 1b < 0.01 μM
EC50 1b = 0.011 μM
IC50 1b = < 0.01 μM
EC50 1b = 0.005 μM
PSA = 203 Å2
EC50 1b = 0.012 μM
PSA = 167 Å2
PSA = 190 Å2
3b R2 = CH2CH2CH(CH3)2
Figure 1. HCV NS5B polymerase inhibitors.
thus probably slightly puckered, ring system could be accommo-
dated in the palm binding site.
cules of general structure 3.12 While incorporating the 4-fluoro-
benzyl and isoamyl groups in R2 previously led to excellent repli-
con potencies (see Fig. 1), the activities of such derivatives in the
present series were disappointing (4a and 4b). Interestingly, the
replicon activities could be improved by introducing a tert-butyl-
ethyl group into the inhibitor design (4c) leading to a compound
that was roughly equipotent to 3a. Our comparison of the co-crys-
tal structures from this (Fig. 2) with the previously reported series
did not reveal any significant structural changes in the R2 binding
pocket that could explain these differences.
In our earlier work, we observed that the methanesulfonamide
group appended to the benzothiadiazine ring was crucial for
achieving potent activities in the biochemical and replicon as-
says.9c,9d,12 As such, it came as no surprise that N-methylation of
this moiety (R3 = Me) resulted in substantial increases of both
IC50 and EC50 values as seen in 4d.
We also explored variations on the left-hand ring system
including introduction of a methyl group at the C-4a bridgehead
(4e). The additional methyl group had a detrimental effect on the
potencies as well as on the compound’s stability toward HLM.
Expanding the ring size from 5- to 6-membered led to the hexahy-
dro-1H-pyrido[1,2-b]pyridazin-2-one analogs 4f and 4g, which
were well tolerated in the enzymatic assay, but led to diminished
activities against the replicon, the reasons for which are currently
not understood. Overall, with the exception of compound 4e, all
compounds under study exhibited good stability toward HLM
(see Table 1).
Compounds 4 were synthesized following the route shown in
Scheme 1. Commercially available cyclic
a-amino esters 5 were
N-aminated using freshly prepared 1-oxa-2-aza-spiro[2.5]octane13
and the intermediates were directly treated with aldehydes to
yield the imines 6. Reduction with sodium cyanoborohydride affor-
ded the corresponding N-substituted hydrazines 7, which were
coupled with acid 814 and subsequently cyclized in the presence
of sodium ethoxide to give the desired compounds 4. These com-
pounds could be further treated with iodomethane to yield analogs
that were N-methylated at the R2 sulfonamide moiety as shown,
for example 4d.
Table
1 details the structure–activity relationships (SAR)
obtained for compounds 4, focusing on their biochemical potencies
against HCV genotype 1b, activities against the HCV genotype 1b
subgenomic replicon in tissue culture, cytotoxicity, and stability
against human liver microsomes (HLM).
We began our SAR exploration by synthesizing 4a as the direct
analog of compound 3a. Compound 4a was a potent inhibitor in
the enzymatic assay thus confirming our hypothesis that a satu-
rated ring system would be tolerated in the palm binding site.
Since the activity of 4a in the replicon assay was relatively modest,
we went on to explore further structural variations in R2 with the
goal of improving this parameter. However, we were surprised to
note a different trend for the SAR around the R2 moiety as com-
pared to that observed for the corresponding unsaturated mole-
O
O
O
R1
R1
R1
b
a
OR
OR
OR
NH
N
N
n
N
R2
NH
R2
n
n
7
6
5
O
O
H
N
S
O
N
S
O
O
c
HO
N
H
8
Me
O
O
O
O
H
N
S
N
S
OH
N
S
OH
N
S
O O
d
R1
O O
R1
N
H
N
H
N
N
N
O
n
N
R2
O
n
R2
4d
4a-g
Scheme 1. Reagents and conditions: (a) i—1-oxa-2-aza-spiro[2.5]octane, toluene, 80 °C, 16 h; ii—RCHO, MeOH, 45 °C (39–97% over two steps); (b) NaCNBH3, MeOH, 25 °C, 2–
22 h (68–89%); (c) i—8, DCC, DCM/DMF, 25 °C, 2–16 h or EDC, NMM, DMF, 25 °C, 2–16 h; ii—NaOEt, EtOH, 60 °C, 3–16 h (8–81% over two steps); (d) MeI, K2CO3, DMF, 25 °C,
18 h (38%).

5004
F. Ruebsam et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5002–5005
Table 1
SAR of hexahydro-pyrrolo- and hexahydro-1H-pyrido[1,2-b]pyridazin-2-one analogs 4
Compounda
n
R1
R2
R3
IC50 (1b) [
l
M]b
EC50 (1b) [
l
M]b
CC50 (GAPDH) [
l
M]b
HLM t1/2 [min]b,c
3a
4a
4b
4c
4d
4e
4f
Figure 1
H
H
H
H
H
4a-Me
H
H
4-F-Bn
4-F-Bn
H
H
H
H
Me
H
H
<0.01
<0.01
0.012
<0.01
0.16
0.18
0.041
0.016
0.012
0.19
0.16
0.034
3.6
1.1
0.11
0.3
>1
>1
>1
>1
>33
>33
>33
>1
>60 (86%)
>60 (87%)
>60 (65%)
59
>60
4.9
1
1
1
1
1
2
2
CH2CH2CH(CH3)2
CH2CH2C(CH3)3
CH2CH2C(CH3)3
CH2CH2C(CH3)3
CH2CH2C(CH3)3
4-F-Bn
52
4g
H
>60 (102%)
a
All compounds were obtained from racemic starting materials.
See Ref. 9a for assay conditions.
For values >60 min, % remaining at 60 min is given in parentheses. All compounds were tested at 1 lM.
b
c
rupting this network of interactions in the compounds under study
leads to a loss of activity as evident in compound 4d, where the
methyl group presumably prevents the formation of some of these
H-bonds with the NS5B protein.
In addition to defining the above protein–ligand interactions, we
also wished to determine the impact (if any) that introduction of the
sp3 hybridized center at C-4a had on inhibitor binding to NS5B.
An overlay of the co-crystal structures obtained for compound 4c
and the related pyrrolo-[1,2-b]pyridazin-2-one compound 3b12
bound to the NS5B protein illustrates the different binding geome-
tries of the two series (Fig. 4). While the majority of the compounds’
atoms overlay very well, the saturated ring in 4c (adopting a puck-
ered conformation) clearly protrudes above and below the plane de-
fined by the pyrrolo-[1,2-b]pyridazin-2-one ring of 3b. Since the R2
substituents in these two compounds are slightly different (isoamyl
vs. tert-butylethyl), it is difficult to compare the binding conforma-
tion in the R2 sub-pocket. To the best of our knowledge, this is the
first structural analysis of a benzothiadiazine-containing NS5B
inhibitor bearing a sp3 hybridized center at the bridgehead carbon
(here at C-4a).
Table 2 shows the calculated physicochemical parameters and
in vitro DMPK data for a selected number of compounds. Formal
saturation of the fused pyrrole ring system present in compounds
3 did not alter polar surface area (PSA) which we had concluded to
be one of the parameters causing poor permeability in the previ-
ously reported series. With the exception of the N-methylated ana-
log 4d where PSA was reduced to 156 Å2, the PSA values associated
with all other compounds were calculated as 165 Å.2
Figure 2. Co-crystal structure of compound 4c bound to the NS5B protein (2.3 Å).
Figure 2 shows the co-crystal structure of 4c bound to the palm
binding site of NS5B.15 Figure 3 schematically details the interac-
tions between compound 4c and the NS5B protein as observed in
the co-crystal structure. As previously reported,9c,12 the sulfon-
amide substituent on the benzothiadiazine ring forms several H-
bonds with the NS5B protein.
These include an interaction between the sulfonamide NH and
the side chain of Asp318, as well as a H-bond between one sulfon-
amide oxygen and the side chain of Asn291. The other sulfonamide
oxygen forms a H-bond with a key structural water molecule. Inter-
Ser 556
Gly 449
HN
Ser 288
N
H
O
H
O
O
O
H
N
O
H
Tyr 448
Gln 446
O
H
N
O
H
H
Asn 291
N
H
O
H
S
N
H
O
O
O
S
H
O
N
H
O
N
H
Gly 410
Asn 411
Asp 318
N
HO
O
N
Met 414
Tyr 415
Leu 384
Ser 368
Cys 366
Pro 197
Tyr 448
Arg 200
Figure 3. Schematic representation of the interactions between compound 4c and
the NS5B protein. Hydrogen bonds are represented as dashed lines, and the residues
which make up the enzyme binding sub-sites are shown.
Figure 4. Overlay of co-crystal structures of compound 4c (orange, 2.3 Å) with
compound 3b (gray, 2.1 Å) bound to the NS5B protein.

F. Ruebsam et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5002–5005
5005
Table 2
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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;
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4. Hoofnagle, J. H.; Seeff, L. B. N. Eng. J. Med. 2007, 355, 2444.
5. Kolykhalov, A. A.; Agapov, E. V.; Blight, K. J.; Mihalik, K.; Feinstone, S. M.; Rice,
C. M. Science 1997, 277, 570.
Correlation of calculated physicochemical parameters and in vitro DMPK data of
selected analogs 4
b,d
Compound
clogPa
MLM t1/2 [min]b,c
Papp [(cm/s) Â 10À6
]
3a
4a
4b
4c
4d
4e
4f
0.66
0.53
0.57
0.92
0.99
1.4
>60 (90%)
>60 (94%)
63
7.5
17
10
18
0.10
0.20
0.20
0.44
2.0
0.60
0.31
0.10
6. Koch, U.; Narjes, F. Curr. Top. Med. Chem. 2007, 7, 1302.
7. (a) Slater, M. J.; Amphlett, E. M.; Andrews, D. M.; Bravi, G.; Burton, G.; Cheasty,
A. G.; Corfield, J. A.; Ellis, M. 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.;
Thommes, P.; Wilkinson, C.; Wonacott, A. J. Med. Chem. 2007, 50, 897; (b)
Dhanak, D.; Duffy, K. J.; Johnston, V. K.; Lin-Goerke, J.; Darcy, M.; Shaw, A. N.;
Gu, B.; Silverman, C.; Gates, A. T.; Nonnemacher, M. R.; 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, H.-Y.; Chan, G.; Khandekar,
S.; Keenan, R. M.; Sarisky, R. T. J. Biol. Chem. 2002, 277, 38322; (c) Evans, K. A.;
Chai, D.; Graybill, T. L.; Burton, G.; Sarisky, R. T.; Lin-Goerke, J.; Johnston, V. K.;
Rivero, R. A. Bioorg. Med. Chem. Lett. 2006, 16, 2205; d Blake, J. F.; Fell, J. B.;
Fischer, J. P.; Hendricks, R. T.; Spencer, S. R.; Stengel, P. J. WO2006117306,
2006.; (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.;
1.49
1.09
4g
>60 (100%)
a
Calculated using ACD/Labs, version 10.0, Advanced Chemistry Development,
Inc., Toronto ON, Canada, www.acdlabs.com, 2006.
b
See Ref. 9c for assay conditions.
For MLM (monkey liver microsome) half-lives >60 min, % remaining at 60 min
c
is given in parentheses. All compounds were tested at 1 lM.
d
Controls:
Papp
Atenolol
(low) = 0.4 (cm/s) Â 10À6
,
Papp
Propranolol
(high) = 10 (cm/s) Â 10À6
.
Kempf, D. J.; Kohlbrenner, W. Bioorg. Med. Chem. Lett. 2005, 15, 1577;
f
Hutchinson, D. K. et al. U.S. Patent US2005107364, 2005.; (g) 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.
While the calculated logP values indicated an increase in lipo-
philicity, in particular for the hexahydro-1H-pyrido[1,2-b]pyrida-
zin-2-one analogs 4f and 4g, the gain did not translate into
improved intestinal permeability as judged by the Caco-2 data.
Consistent with our previous conclusions, only the weakly active
N-methylated analog 4d, which has a lower PSA value relative to the
other inhibitors, displayed any significant permeability improve-
ment. Overall, all compounds under study displayed good solubility
8. All structures are arbitrarily drawn as one of several possible tautomers.
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.; 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; (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, 18, 3421.
in a rapid biochemicalassessment(>100 lM) but manyshowed only
low to modest stability toward monkey liver microsomes (MLM).
Compound 4c, the only inhibitor that combined high potencies in
the biochemical and replicon assays with good HLM half-life, was
evaluated in vivo in cynomolgus monkeys in which we found the
corresponding oral bioavailability to be poor (Fpo = 7%; AUCinf (PO/
IV) = 344/5178 ng/h/mL).16 Although the compound was very
unstable toward MLM, the experimental fraction absorbed for this
inhibitor (FApo = 8%, calculated based on the measured oral bioavail-
ability (Fpo%) and clearance of the compound in cynomolgus mon-
keys)17 suggested that poor intestinal permeability, rather than
extensive metabolism, was the likely cause of the low in vivo
exposures.
In summary, we describe a novel series of non-nucleoside inhib-
itors of genotype 1b HCV NS5B polymerase (4) that were formally
derived via saturation of the fused left-hand ring system previously
described (3). While this modification led to a number of very potent
compounds in the biochemical and replicon assays, the data also
indicated that permeability is still too low for the compounds in this
series to be effectively absorbed. Our ongoing efforts to further im-
prove the PK properties of the benzothiadiazine-containing NS5B
inhibitors will be reported in a future communication.
10. (a) Veber, D. F.; Johnson, S. R.; Cheng, H.-Y.; Smith, B. R.; Ward, K. W.; Kopple, K.
D. J. Med. Chem. 2002, 45, 2615; (b) Palm, K.; Stenberg, P.; Luthman, K.;
Artursson, P. Pharm. Res. 1997, 14, 568.
11. Ellis, D. A.; Blazel, J. K.; Webber, S. E.; Tran, C. V.; Dragovich, P. S.; Sun, Z.;
Ruebsam, F.; McGuire, H. M.; Xiang, A. X.; Zhao, J.; Li, L.-S.; Zhou, Y.; Han, Q.;
Kissinger, C. R.; Showalter, R. E.; Lardy, M.; Shah, A. M.; Tsan, M.; Patel, R.;
LeBrun, L. A.; Kamran, R.; Bartkowski, D. M.; Nolan, T. G.; Norris, D. A.;
Sergeeva, M. V.; Kirkovsky, L. Bioorg. Med. Chem. Lett. 2008, 18, 4628.
12. Ruebsam, F.; Webber, S. E.; Tran, M. T.; Tran, C. V.; Murphy, D. E.; Zhao, J.;
Dragovich, P. S.; Kim, S. H.; Li, L.-S.; Zhou, Y.; Han, Q.; Kissinger, C. R.;
Showalter, R. E.; Lardy, M.; Shah, A. M.; Tsan, M.; Patel, R.; LeBrun, L. A.;
Kamran, R.; Sergeeva, M. V.; Bartkowski, D. M.; Nolan, T. G.; Norris, D. A.;
Kirkovsky, L. Bioorg. Med. Chem. Lett. 2008, 18, 3616.
13. Schmitz, E.; Ohme, R.; Schramm, S.; Striegler, H.; Heyne, H.-U.; Rusche, J. J.
Prakt. Chem. 1977, 319, 195.
14. 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.
15. 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 = 86 Å, b = 106 Å, c = 126 Å and two protein molecules in the
asymmetric unit. Protein/inhibitor complexes were prepared by soaking these
crystals for 3 to 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
2.3 Å for compound 4c. The crystal structure discussed in this paper has been
deposited in the Protein Databank (www.rcsb.org) with entry code: 3CVK. Full
structure determination details are provided in the PDB entry.
Acknowledgments
The authors thank Drs. Peter Dragovich, Devron Averett and
Steve Worland for their support and helpful discussions during
the course of this work.
References and notes
16. 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.
17. Davies, B.; Morris, T. Pharm. Res. 1993, 10, 193. FApo = Fpo/(1-CL/Q) where
Q = 44 mL/min/kg (hepatic blood flow in cynomolgus monkey) and
CL = 3.3 mL/min/kg (clearance in cynomolgus monkey after IV administration).
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