Inhibitors of HCV NS5B polymerase. Part 1: Evaluation of the southern region of (2Z)-2-(benzoylamino)-3-(5-phenyl-2-furyl)acrylic acid

Article abstract of DOI:10.1016/j.bmcl.2005.03.066

A novel series of nonnucleoside HCV NS5B polymerase inhibitors were prepared from (2Z)-2-(benzoylamino)-3-(5-phenyl-2-furyl)acrylic acid, a high throughput screening lead. SAR studies combined with structure based drug design focusing on the southern heterobiaryl region of the template led to the synthesis of several potent and orally bioavailable lead compounds. X-ray crystallography studies were also performed to understand the interaction of these inhibitors with HCV NS5B polymerase.

Full text of DOI:10.1016/j.bmcl.2005.03.066

Bioorganic & Medicinal Chemistry Letters 15 (2005) 2481–2486
Inhibitors of HCV NS5B polymerase. Part 1: Evaluation of
the southern region of (2Z)-2-(benzoylamino)-3-(5-phenyl-2-
furyl)acrylic acid
Jeffrey A. Pfefferkorn,a,* Meredith L. Greene,b Richard A. Nugent,c Rebecca J. Gross,b
Mark A. Mitchell,b Barry C. Finzel,a Melissa S. Harris,a Peter A. Wells,d John A. Shelly,a
Robert A. Anstadt,b Robert E. Kilkuskie,b Laurice A. Koptab and Francis J. Schwendea
aPfizer Global Research and Development, Michigan Laboratories, 2800 Plymouth Road, Ann Arbor, MI 48105, USA
bPfizer Global Research and Development, Michigan Laboratories, 301 Henrietta Street, Kalamazoo, MI 49007, USA
cPfizer Global Research and Development, Research Technology Center, 620 Memorial Drive, Cambridge, MA 02139, USA
dPfizer Global Research and Development, La Jolla Laboratories, 10628 Science Center Dr., San Diego, CA 92121, USA
Received 21 January 2005; revised 16 March 2005; accepted 17 March 2005
Available online 12 April 2005
Abstract—A novel series of nonnucleoside HCV NS5B polymerase inhibitors were prepared from (2Z)-2-(benzoylamino)-3-(5-phen-
yl-2-furyl)acrylic acid, a high throughput screening lead. SAR studies combined with structure based drug design focusing on the
southern heterobiaryl region of the template led to the synthesis of several potent and orally bioavailable lead compounds. X-ray
crystallography studies were also performed to understand the interaction of these inhibitors with HCV NS5B polymerase.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
ical need exists for new, orally administered, anti-HCV
agents with good efficacy, spectrum, and tolerability.
Hepatitis C virus (HCV) is the major causative agent of
blood-borne non-A, non-B hepatitis, and it is estimated
to infect over 170 million people worldwide. Chronic
HCV infections have been associated with liver fibrosis,
liver cirrhosis, and heptacellular carcinoma.1,2 Current
HCV therapy consists of injectable a-interferon used
alone or in combination with ribavirin.2 Although effec-
tive in certain patient populations, interferon therapy
has several limitations including high cost and signifi-
cant side effects that often require dose adjustment or
discontinuation of therapy. Moreover, while not well
understood, interferon therapy has been shown to be
most effective against genotype 2 and 3 HCV infections
and least effective against genotype 1 infections. Unfor-
tunately, since 60% of patients in North America are in-
fected with the genotype 1 strains, a significant portion
of HCV positive patients will not benefit from interferon
therapy.1 As a consequence of these limitations, a med-
From a therapeutic perspective, hepatitis C virus offers a
number of potential targets for small molecule interven-
tion.3,4 The viral genome encodes for several nonstruc-
tural (NS) proteins including a protease (NS3/NS4a), a
helicase (NS3) and an RNA-dependent polymerase
(NS5B).4 In this report, we describe our efforts to devel-
op nonnucleoside inhibitors of the NS5B RNA-depen-
dent RNA polymerase.5 Given the essential role of this
enzyme in viral replication, it is anticipated that agents
capable of disrupting its function will prove efficacious
in the treatment of HCV infections.
O
O
O
H
O
H
NH
NH
HO
HO
R1
R2
O
A
Y
B
R3
Keywords: HCV Polymerase; Inhibitors.
*
PNU-248809 (1)
(2)
Corresponding author. Tel.: +1 734 622 1471; fax: +1 734 622
3171; e-mail: jeffrey.a.pfefferkorn@pfizer.com
Figure 1. Lead compound (1) and SAR strategy (2).
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.03.066

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J. A. Pfefferkorn et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2481–2486
High throughput screening of our internal compound
collection against HCV polymerase led to the identifica-
tion of PNU-248809 (1, Fig. 1) as a modest inhibitor of
CD21 NS5B polymerase6 (IC50 = 6.7 lM). Herein we de-
scribe our SAR strategy (2, Fig. 1) to investigate the
structural requirements of the A- and B-ring regions of
this template. In the following report, we describe modi-
fications to the northern region of the template.
to afford the final products. This sequence was used to
prepare a series of substituted B-ring analogs.
In addition to the furan and thiophene A-rings de-
scribed in Scheme 1, several other heterocyclic and aryl
A-rings were also prepared. Since preliminary SAR data
(vide infra) suggested that a 2-chloro substituent on the
B-ring of the template afforded optimal inhibitory activ-
ity against HCV NS5B polymerase, all subsequent
A-ring analogs were synthesized with this optimal sub-
stitution pattern. As illustrated in Scheme 2, a thiazole
A-ring analog was synthesized via initial a-bromination
of 2-chloroacetophenone 11 to provide bromide 12. Bro-
moketone 12 was then reacted with the modified Gabriel
2. Chemical synthesis
Initial synthesis of PNU-248809 (1) established a conve-
nient route for the parallel synthesis of analogs as out-
lined in Scheme 1. Commercially available N-(Cbz)-a-
phosphonoglycine trimethyl ester (3, Scheme 1) was first
deprotected by hydrogenolysis7 to afford an amine that
was acylated with benzoyl chloride in the presence of tri-
ethylamine to provide, after recrystallization, N-(Bz)-a-
phosphonoglycine trimethyl ester 4 in 50% yield over
two steps. Intermediate 4 was then condensed with var-
ious substituted 5-phenyl-2-furaldehydes (7, X = O) or
5-phenyl-2-thiophenecarboxaldehydes (7, X = S) to af-
ford an enamide product [8 (X = O) or 9 (X = S)] as a
single Z-stereoisomer.8 The aldehydes (i.e., 7) required
for this condensation were either purchased commer-
cially or prepared via palladium-mediated coupling of
a substituted phenyl boronic acids 5 with 5-bromo-2-
furanaldehyde (6, X = O) or 5-bromo-2-thiophenecarb-
oxaldehyde (6, X = S).9 The synthesis was completed
by saponification of the methyl ester of intermediate [8
(X = O) or 9 (X = S)] with aqueous LiOH in dioxane
10
reagent NaN(CHO)2 to afford, after acid catalyzed
deformylation, an amine intermediate that was acylated
with ethyl chlorooxacetate to provide amide 13. Treat-
ment of intermediate 13 with P2S5 at elevated tempera-
ture provided thiazole 14 in modest yield.11 The ester
of thiazole 14 was then converted to the corresponding
aldehyde 15 using a two-step reduction–oxidation se-
quence. The resulting aldehyde 15 was condensed with
N-(Bz)-a-phosphonoglycine trimethyl ester 4 in the pres-
ence of DBU to give, after hydrolysis of the intermediate
methyl ester, thiazole 16.
As illustrated in Scheme 3, several analogs containing
six-membered aryl and heteroaryl A-rings were also pre-
pared. In this general sequence, various aryl bromides
(17–19) were coupled with 2-chlorophenyl boronic acid
in the presence of a Pd(PPh3)4 to afford biaryls (20,
21, 23). Subsequently, the esters of heterobiaryls (21
and 23) were converted to the corresponding aldehydes
(22 and 24) via DIBAL reduction. Finally aldehydes
(20, 22, 24) were subjected to condensation with N-
(Bz)-a-phosphonoglycine trimethyl ester (4) and hydro-
lysis generating compounds 25–27.
O
O
a,b
NHCBz
NHBz
MeO
MeO
P(O)(OMe)2
P(O)(OMe)2
Finally an analog possessing a phenyl ether A-ring was
prepared as outlined in Scheme 4. Initial aromatic nucleo-
3
4
R1
R2
Cl
O
Cl
O
H
N
b,c,d
R2
R
CO2Et
(HO)2B
Br
R3
R1
c
R3
O
13
5
+
X
CHO
11: R = H
12: R = Br
a
X
CHO
7
e
6
O
O
H
N
Cl
O
h,i
d
O
H
NH
R
HO
S
NH
R4O
R1
R2
S
N
X
A
14: R = CO2Me
15: R = CHO
R3
B
f,g
Cl
16
R4 = Me, R1-3 = H, X = O
8:
Scheme 2. Reagents and conditions: (a) Br2, AlCl3, Et2O, 0 °C, 8 h,
40%; (b) NaN(CHO)2, MeCN, 25 ! 70 °C, 2 h, 65%; (c) 5% HCl/
EtOH, 25 °C, 24 h, 85%; (d) ethyl chlorooxacetate, Et3N, CH2Cl2,
25 °C, 2 h, 34%; (e) P2S5, CHCl3, 60 °C, 16 h, 95%; (f) DIBAL,
CH2Cl2, ꢀ78 °C, 3 h, 79%; (g) Dess Martin reagent, NaHCO3,
CH2Cl2, 0 ! 25 °C, 4 h, 82%; (h) N-(Bz)-a-phosphonoglycine tri-
methyl ester (4), DBU, CH2Cl2, 2 h, 65%; (i) LiOH (aq), 1,4-dioxane,
25 °C, 2 h, 91%.
9: R4 = Me, R1-3 = H, X = S
1: R1-4 = H, X = O
e
e
10: R1-4 = H, X = S
Scheme 1. Reagents and conditions: (a) 10% Pd–C, H2, MeOH, 25 °C,
8 h; (b) Bz–Cl, Et3N, CH2Cl2, 0 ! 25 °C, 50% over two steps; (c)
TBAB, K2CO3, Pd(OAc)2, H2O, 25 °C, 16 h; (d) DBU, CH2Cl2, 25 °C,
2 h; (e) LiOH (aq), 1,4-dioxane, 25 °C, 2 h.

J. A. Pfefferkorn et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2481–2486
2483
Table 1. Activity of compounds 1, 10, and 32–57 against HCV NS5B
polymerase
Z
X
O
Cl
Br
X
Z
a
R
R
O
O
O
17: X= CH, Z = CH, R = H
18: X= N, Z = CH, R = OEt
19: X= CH, Z = N, R = OEt
20: X= CH, Z = CH, R = H
21: X= N, Z = CH, R = OEt
22: X= N, Z = CH, R = H
23: X= CH, Z = N, R = OEt
24: X= CH, Z = N, R = H
NH
HO
b
b
R1
R2
X
A
H
R3
B
Compound
X
R1
R2
R3
NS5b IC50 (lM)
O
c,d
O
H
1
O
S
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
F
6.7
1.5
0.66
1.3
1.4
3.0
2.6
3.1
3.9
4.0
4.6
5.6
6.0
6.9
8.7
12
NH Cl
X
HO
25: X= CH, Z = CH
26: X= N, Z = CH
27: X= CH, Z = N
10
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
H
H
S
Cl
Cl
Me
Me
F
H
O
S
H
Z
H
O
S
H
Scheme 3. Reagents and conditions: (a) 2-ClC6H4B(OH)2, Pd(Ph3P)4,
Na2CO3, 80 °C, 12 h; (b) DIBAL, CH2Cl2, ꢀ78 °C, 3 h; (c) N-(Bz)-a-
phosphonoglycine trimethyl ester (4), DBU, CH2Cl2, 25 °C, 2 h; (d)
LiOH (aq), 1,4-dioxane, 25 °C, 2 h.
H
O
O
O
O
O
O
O
O
O
O
O
S
F
H
H
F
OCF3
H
H
H
CF3
H
H
Cl
H
Cl
CHO
Cl
NO2
H
O
a
OH
CF3
H
+
H
CHO
H
H
CF3
H
H
13
20
30
F
29
OMe
CH2OH
H
H
28
H
20
22
b,c
O
O
O
O
O
O
O
O
O
H
OCF3
H
O
H
NO2
H
25
27
O
H
SMe
H
NH
HO
H
OCF3
H
66
H
NO2
H
>100
>100
>100
>100
>100
H
H
CH2OH
H
O
H
CH2OH
H
SO2NH2
31
Cl
H
SO2NH2
H
H
Scheme 4. Reagents and conditions: (a) K2CO3, DMA, 150 °C, 12 h,
79%; (b) N-(Bz)-a-phosphonoglycine trimethyl ester (4), DBU,
CH2Cl2, 25 °C, 2 h, 71%; (c) LiOH (aq), 1,4-dioxane, 25 °C, 2 h, 89%.
(IC50 = 0.66 lM). In general, increasing size and/or
polarity of a substituent at the R1 position beyond Cl
or Me led to decreased activity as did substitution at
R2 and R3. These observations suggested that the B-ring
binding pocket was shallow and hydrophobic.
philic displacement reaction between 4-fluorobenzalde-
hyde (28) and 2-chlorophenol (29) in the presence of
K2CO3 at 150 °C to afforded 4-(2-chlorophenoxy)benz-
aldehyde (30). This aldehyde was condensed with N-
(Bz)-a-phosphonoglycine trimethyl ester (4) in the pres-
ence of DBU and the resulting intermediate saponified
to provide compound 31.
Evaluation of several alternative A-rings (Table 2) re-
vealed that the majority were less potent than the thio-
phene ring (e.g., compound 10). Surprisingly however,
phenyl ether 31 proved to be more potent
(IC50 = 0.20 lM). As a follow-up, the 2- and 3-isomers
(not shown) of compound 31 (4-isomer) were prepared.
3. Results and discussion
Our SAR strategy provided for initial screening of new
analogs against HCV polymerase in a continuous
read PicogreenÒ assay using a genotype 1b CD21
NS5B polymerase enzyme construct.12 Evaluation of
the compounds from Table 1 (B-ring substitution with
A-rings selected from either furan or thiophene)
revealed several interesting trends. First, A-ring thioph-
enes had preferred inhibitory activity compared with
A-ring furans as illustrated by several compound pairs
(10 > 1), (32 > 33), and (34 > 35). Second, small hydro-
phobic substituents were preferred at the R1-position
of the B-ring as highlighted by compound 32
Table 2. Activity of A-ring analogs against HCV NS5B polymerase
Compound
A-ring
NS5b IC50 (lM)
33 (Table 1)
Furan
1.3
0.20
0.66
1.3
31 (Scheme 4)
10 (Scheme 1)
25 (Scheme 3)
16 (Scheme 2)
26 (Scheme 3)
27 (Scheme 3)
Phenyl ether
Thiophene
Phenyl
Thiazole
Pyridine
Pyridine
5.2
11
>100

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J. A. Pfefferkorn et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2481–2486
The 2-isomer was inactive while the 3-isomer was two-
fold less potent than 31.
In order to further improve the activity of compound 31,
additional analogs were prepared (according to the
method of Scheme 4) and evaluated. Consistent with
the previous results of Table 1, preliminary data in this
phenyl ether series suggested that substitution at the B-
ring R1 position was necessary to achieve optimal activ-
ity while substitution at B-ring meta- or para-positions
was generally deleterious to activity (data not shown).
Table 3 highlights that for mono-substituted analogs
(R2 = H) halogens were preferred in the R1 position with
activity positively correlated with increasing halogen
size (I > Br > Cl > F).
˚
Figure 2. X-ray crystal structure (2.5 A resolution) of compound 59
To better understand the interaction of these inhibitors
with HCV polymerase, a representative compound, 59,
was soaked into crystalline CD21 NS5B enzyme and
the structure was solved (Fig. 2) revealing that the inhib-
itor interacts at a unique binding site on the polymer-
ase.13–15 The site is comprised of a deep hydrophobic
pocket located adjacent to the sequence motif (residues
363–369) that forms a b hairpin linking the palm domain
to the thumb domain of the polymerase. In models of
template-primer binding to RNA-dependent DNA poly-
merases, the backbone of this hairpin interacts with the
phosphate backbone of the substrate primer strand, so
we have denoted this binding site as the primer grip
site.16
(Table 3) bound to the primer grip site of CD21 NS5B polymerase.
Interaction of a small molecule at this primer grip site of
could be expected to inhibit with the activity of NS5B by
interfering with initiation and/or elongation. Examina-
tion of the inhibition kinetics of compound 59 in fact re-
vealed both effects. The NS5B polymerase assay used to
evaluate these analogs was run under conditions for de
novo initiation. The ssRNA template consisted of 32
mer heterpolymer RNA with no secondary structure
and the sequence CCC at the 30 terminus. Ribonucleo-
side triphosphates, rATP, rCTP, and rUTP were present
at 2 lM; rGTP was present at 50 lM for de novo initi-
ation opposite CC at the 30 terminus.17 Under these con-
ditions, 59 and related analogs bound at the primer grip
site decreased both the rate of de novo initiation and the
processive polymerase reaction (elongation of nascent
RNA strand).
Table 3. Activity of compounds 58–77 against HCV NS5B polymerase
O
O
Bresannelli et al. have modeled the de novo initiation
complex from X-ray crystallography of HCV NS5B
with ribonucleoside triphosphates (rNTP) and divalent
cations bound to the enzyme.18 The structural data indi-
cates three rNTP are bound at the C (catalytic) site, P
(primer) site, and I (interrogation) site of NS5B.
Ser367, Arg386, and Arg394, which hydrogen bond with
a and b phosphates of rNTP at the P site,18 contribute to
the formation of the primer grip site described in this re-
port. Distortion of the P site by inhibitor bound at the
primer grip site appears to decrease the rate of de novo
initiation. Inhibition by compound 59 was dependent
on rGTP concentration suggesting partial competition
between compound 59 and rGTP for binding to the en-
zyme. Binding of inhibitors at the primer grip site could
affect elongation of the nascent RNA strand by (i) low-
ering the affinity of the P site for primer strand or (ii)
distorting the adjacent C site where incoming rNTP
are added to the primer strand.
NH
HO
R1
H
A
B
O
R2
Compound
R1
R2
NS5b IC50 (lM)
58
59
31
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
I
Br
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
0.04
0.10
0.20
0.58
1.3
Cl
Me
CF3
SMe
F
1.8
6.9
Et
OMe
H
9.4
15
17
28
i-Pr
Ph
48
44
O-i-Pr
CN
NO2
t-Bu
Br
47
73
From a structure-based design perspective, the crystal
structure of bound 59 revealed several key inhibitor–en-
zyme interactions including a water-mediated hydrogen
bond between the carboxylic acid and Ser556. Addition-
ally, the carbonyl of the benzamide functionality accepts
a hydrogen bond from the backbone amide of the
Tyr448 residue. Interestingly, the A- and B-rings of the
>100
0.03
0.07
0.20
0.31
5.5
Cl
Cl
F
Me
Cl
Cl
NH2
NO2

J. A. Pfefferkorn et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2481–2486
Table 5. Pharmacokinetic properties of compounds 31 and 59
2485
inhibitor extend into a deep, lipophilic pocket within the
enzyme (formed by residues Pro197, Leu384, Met414,
Tyr415, and Tyr448) affording substantial hydrophobic
interactions. Moreover, there appears to be an electron-
ically favorable interaction between the B-ring 2-Br sub-
stituent and Tyr448, which is positioned at the bottom
of this pocket. Further examination of the crystal struc-
ture revealed that a second tyrosine residue (Tyr415) as
well as a serine residue (Ser368) were in fact in close
proximity to the B-ring opposite the 2-Br substituent.
The location of these residues suggested that a suitable
substituent occupying the 6-position might favorable en-
gage one (or both) of these residues. The preferred activ-
ity of selected 2,6-disubstituted compounds (73 and 74,
Table 3) suggests that such a binding mode might in fact
be operative.
Compound
%F
Cmax (lM)
t1/2 (h)
CL (L/h/kg)
31
59
42
21
22
9
1.9
1.5
0.31
0.20
200-fold improvement in activity for selected com-
pounds relative to the original screening lead. Moreover,
crystallography studies have suggested that these com-
pounds bind to the polymerase at a unique site, which
we have denoted as the primer grip site. Leading analogs
demonstrated only modest cellular activity, but exhib-
ited reasonable PK properties. The results of additional
SAR studies on this template are reported in the follow-
ing article.
Acknowledgements
In order to further evaluate the potential antiviral
efficacy of this series of NS5B polymerase inhibitors,
several leading analogs were evaluated in a cellular
sub-genomic replicon assay.19 As illustrated in Table 4,
analogs 31, 58, 59, and 73 had superior cellular activity
relative to the original screening lead (1), and all of the
compounds proved to be nontoxic at the doses evalu-
ated. It is notable however, that while compounds 31,
58, 59, and 73 proved to be very potent against isolated
NS5B polymerase, their activities in the cellular replicon
system were rather modest. We have rationalized that
this differential in enzyme versus cellular activity might
be attributable to a number of potential factors,20
including: (a) low membrane permeability due to the
presence of a carboxylic acid; (b) high levels of serum
protein binding; and/or (c) the inability of compounds
bound at this site of NS5B to effectively block replica-
tion in a functional replication system. In the following
manuscript, we attempt to address several of these po-
tential issues through modification of the northern re-
gion of this template.
We would like to thank Stephen Secreast for formula-
tion of selected compounds for pharmacokinetic studies.
We would also like to acknowledge all members of the
former Pharmacia HCV team for helpful discussions.
Finally, we would like to thank our former collabora-
tors at Chiron Corporation for providing us with
CD21 HCV NS5B.
References and notes
1. For a review of HCV epidemiology, pathogenesis, and
clinical treatment, see: (a) Lauer, G. M.; Walker, B. D. N.
Engl. J. Med. 2001, 345, 41; (b) Di Bisceglie, A. M. Lancet
1998, 351, 351.
2. For a clinical overview of HCV, see: Strader, D. B.; Seeff,
L. B. ILAR J. 2001, 42, 107.
3. Bartenschlager, R.; Lohmann, V. BailliereÕs Clin. Gastr.
2000, 14, 241.
4. Pockros, P. J. Expert Opin. Invest. Drugs 2002, 11, 515.
5. For selected examples of nonnucleoside NS5B polymerase
inhibitors. see: (a) Stansfield, I.; Avolio, S.; Colarusso, S.;
Gennari, N.; Narjes, F.; Pacini, B.; Ponzi, S.; Harper, S.
Bioorg. Med. Chem. Lett. 2004, 14, 5085; (b) Summa, V.;
Petrocchi, A.; Matassa, V. G.; Taliani, M.; Laufer, R.; De
Francesco, R.; Altamura, S.; Pace, P. J. Med. Chem. 2004,
47, 5336–5339; (c) Beaulieu, P. L.; Boes, M.; Bousquet, Y.;
DeRoy, P.; Fazal, G.; Gauthier, J.; Gillard, J.; Goulet, S.;
McKercher, G.; Poupart, M.-A.; Valois, S.; Kukoji, G.
Bioorg. Med. Chem. Lett. 2004, 14, 967; (d) Reddy, T. J.;
Chan, L.; Turcotte, N.; Proulx, M.; Pereira, O. Z.; Das, S.
K.; Siddiqui, A.; Wang, W.; Poisson, C.; Yannopoulos, C.
G.; Bilimoria, D.; LÕHeureux, L.; Alaoui, H. M. A.;
Nguyen-Ba, N. Bioorg. Med. Chem. Lett. 2003, 13, 3341;
(e) Gopalsamy, A.; Lim, K.; Ellingboe, J. W.; Krishna-
murthy, G.; Orlowski, M.; Feld, B.; van Zeijl, M.; Howe,
A. Y. M. Bioorg. Med. Chem. Lett. 2004, 14, 4221; (f)
Shipps, G. W.; Deng, Y.; Wang, T.; Popovici-Muller, J.;
Curran, P. J.; Rosner, K. E.; Cooper, A. B.; Girijavallab-
ham, V.; Butkiewicz, N.; Cable, M. Bioorg. Med. Chem.
Lett. 2005, 15, 115, and references cited therein.
Finally, several of the leading analogs from this series
were selected for pharamacokinetic profiling to evaluate
the overall potential of this series of inhibitors to deliver
an orally administered anti-HCV agent. As shown in
Table 5, compounds 31 and 59 were dosed to rats (Spra-
gue-Dawley) at 5 mpk and had oral bioavailabilities of
42% and 21%, respectively. At this dose, the compounds
achieved Cmax levels of 22 lM (31) and 9 lM (59) with
half-lives under 2 h and relatively low levels of
clearance.
In conclusion, we have reported the preparation of a no-
vel series of nonnucleoside inhibitors of HCV NS5B
polymerase. Structure–activity studies have afforded a
Table 4. Activity of selected compounds in sub-genomic relicon
assay19
Compound
IC50 (lM)
ED50 (lM)
CC50 (lM)
6. In its native form, NS5B HCV polymerase is a membrane-
associated enzyme with the full length enzyme possessing a
transmembrane anchor domain. However, in the absence
of cellular membranes (e.g., during crystallography and
biochemistry studies) this membrane anchor domain
induces complications, and as a result, a 21-amino acid
1
6.7
>100
>100
>100
>100
>100
>100
31
59
58
73
0.20
0.10
0.04
0.03
60
28
29
50
9
7
4
7

2486
J. A. Pfefferkorn et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2481–2486
truncated version of the enzyme, denoted CD21, is
15. For crystal structures of inhibitors bound to other sites on
HCV NS5B polymerase, see: (a) Love, R. A.; Parge, H. E.;
Yu, H.; Hickey, M. J.; Diehl, W.; Gao, W.; Wriggers, H.;
Ekker, A.; Wang, L.; Thomson, J. A.; Dragovich, P. S.;
Fuhrman, S. A. J. Virol. 2003, 77, 7575; (b) Wang, M.;
Ng, K. K.-S.; Cherney, M. M.; Chan, L.; Yannopoulos, C.
G.; Bedard, J.; Morin, N.; Nguyen-Ba, N.; Alaoui-Ismaili,
M. H.; Bethell, R. C.; James, M. N. G. J. Biol. Chem.
2003, 278, 9489.
16. (a) Jacob-Molina, A.; Ding, J.; Nanni, R. G.; Clark, A.
D.; Lu, X.; Tantillo, C.; Williams, R. L.; Kamer, G.;
Ferris, A. L.; Clark, P.; Hizi, A.; Hughes, S. H.; Arnold,
E. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 6320; (b) Huang,
H.; Chopra, R.; Verdine, G. L.; Harrison, S. C. Science
1998, 282, 1669.
17. (a) Luo, G.; Hamatake, R. K.; Mathis, D. M.; Racela, J.;
Rigat, K. L.; Lemm, J.; Colonno, R. J. J. Virol. 2000, 74,
851; (b) Zhong, W.; Uss, A. S.; Ferrari, E.; Lau, J. Y. N.;
Hong, Z. J. Virol. 2000, 74, 2017; (c) Zhong, W.; Ferrari,
E.; Lesburg, C. A.; Maag, D.; Ghosh, S. K. B.; Cameron,
C. E.; Lau, J. Y. N.; Hong, Z. J. Virol. 2000, 74, 9134.
18. Bressanelli, S.; Tomei, L.; Rey, F. A.; De Francesco, R. J.
Virol. 2002, 76, 3482.
19. Cellular replicon assay was performed in Huh-7 cells in
10% fetal calf serum following general procedure: Loh-
mann, V.; Ko¨rner, F.; Koch, J.-O.; Herian, U.; Theil-
mann, L.; Bartenschlager, R. Science 1999, 285, 110. EC50
values are reported as arithmetic mean along with
standard deviation for 3–10 independent measurements.
20. For a relevant discussion, see: Harper, S.; Pacini, B.;
Avolio, S.; Di Filippo, M.; Migliaccio, G.; Laufer, R.; De
Francesco, R.; Rowley, M.; Narjes, F. J. Med. Chem.
2005, 48, 1314–1317.
employed in crystallography and preliminary biochemistry
work. All enzyme inhibition data presented herein is from
a CD21 genotype 1b.
7. Horenstein, B. A.; Nakanishi, K. J. Am. Chem. Soc. 1989,
111, 6242.
8. Schmidt, U.; Griesser, H.; Leitenberger, V.; Lieberknecht,
A.; Mangold, R.; Meyer, R.; Riedl, B. Synthesis 1992, 487.
9. Bussolari, J. C.; Rehborn, D. C. Org. Lett. 1999, 1, 965.
10. Yinglin, H.; Hongwen, H. Synthesis 1990, 122.
11. Tanaka, C.; Nasu, K.; Yamamoto, N.; Shibata, M. Chem.
Pharm. Bull. 1982, 30, 4195.
12. All biological data was generated on analytically pure (1H
NMR, MS, and CHN) samples. IC50 values are reported
as the arithmetic mean for two to five independent
measurements. For complete assay details, see: Yagi, Y.;
Sheets, M.; Wells, P.; Shelly, J. A.; Poorman, R. A.; Epps,
D. E. PCT Int. Appl. WO 2004044228, 2004.
13. For crystallization conditions, see: Finzel, B. C. PCT Int.
Appl. WO 2005001417, 2005. Coordinates for structure
have been deposited at Protein Data Bank (www.rcsb.org)
under file name 1QXK.
14. To the best of our knowledge this represents the first
disclosure of a co-crystal structure of an inhibitor bound
at this site on NS5B polymerase. Previously, others have
disclosed a resistance mutation (M414 ! T414) at this
site, see: Nguyen, T. T.; Gates, A. D.; Gutshall, L. L.;
Johnston, V. K.; Gu, B.; Duffy, K. J.; Sarisky, R. T.
Antimicrob. Agents Chemother. 2003, 47, 3525; Addition-
ally, a previous analysis of NS5B revealed the presence of
a hydrophobic pocket containing elements of this site, see:
Leveque, V. J.-P.; Johnson, R. B.; Parsons, S.; Ren, J.;
Xie, C.; Zhang, F.; Wang, Q. M. J. Virol. 2003, 77, 9020.