Identification of halosalicylamide derivatives as a novel class of allosteric inhibitors of HCV NS5B polymerase

Article abstract of DOI:10.1016/j.bmcl.2008.04.068

Halosalicylamide derivatives were identified from high-throughput screening as potent inhibitors of HCV NS5B polymerase. The subsequent structure and activity relationship revealed the absolute requirement of the salicylamide moiety for optimum activity. Methylation of either the hydroxyl group or the amide group of the salicylamide moiety abolished the activity while the substitutions on both phenyl rings are acceptable. The halosalicylamide derivatives were shown to be non-competitive with respect to elongation nucleotide and demonstrated broad genotype activity against genotype 1-3 HCV NS5B polymerases. Inhibitor competition studies indicated an additive binding mode to the initiation pocket that is occupied by the thiadiazine class of compounds and an additive binding mode to the elongation pocket that is occupied by diketoacids, but a mutually exclusive binding mode with respect to the allosteric thumb pocket that is occupied by the benzimidazole class of inhibitors. Therefore, halosalicylamides represent a novel class of allosteric inhibitors of HCV NS5B polymerase.

Full text of DOI:10.1016/j.bmcl.2008.04.068

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 3173–3177
Identification of halosalicylamide derivatives as a novel class of
allosteric inhibitors of HCV NS5B polymerase
Yaya Liu,* Pamela L. Donner, John K. Pratt, Wen W. Jiang, Teresa Ng, Vijaya Gracias,
Steve Baumeister, Paul E. Wiedeman, Linda Traphagen, Usha Warrior,
Clarence Maring, Warren M. Kati, Stevan W. Djuric and Akhteruzzaman Molla
Antiviral Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL 60064, USA
Received 26 February 2008; revised 24 April 2008; accepted 28 April 2008
Available online 1 May 2008
Abstract—Halosalicylamide derivatives were identified from high-throughput screening as potent inhibitors of HCV NS5B polymer-
ase. The subsequent structure and activity relationship revealed the absolute requirement of the salicylamide moiety for optimum
activity. Methylation of either the hydroxyl group or the amide group of the salicylamide moiety abolished the activity while the
substitutions on both phenyl rings are acceptable. The halosalicylamide derivatives were shown to be non-competitive with respect
to elongation nucleotide and demonstrated broad genotype activity against genotype 1–3 HCV NS5B polymerases. Inhibitor com-
petition studies indicated an additive binding mode to the initiation pocket that is occupied by the thiadiazine class of compounds
and an additive binding mode to the elongation pocket that is occupied by diketoacids, but a mutually exclusive binding mode with
respect to the allosteric thumb pocket that is occupied by the benzimidazole class of inhibitors. Therefore, halosalicylamides repre-
sent a novel class of allosteric inhibitors of HCV NS5B polymerase.
Ó 2008 Elsevier Ltd. All rights reserved.
Hepatitis C virus is the major etiological agent of non-
A, non-B hepatitis. The disease is a major heath prob-
lem with an estimated 170 million people infected world-
wide. HCV infection results in mild and acute liver
disease, but chronic infections are common and may
eventually develop into liver cirrhosis or hepatocellular
carcinoma.1–5 The current standard of care for treating
HCV is a combination therapy of pegylated interferon-
a (PEG-IFN-a) plus ribavirin. Response rates for
HCV patients having genotypes 2 or 3 on a 24-week
treatment regimen of PEG-IFN-a/ribavirin show a sus-
tained virological response approaching 80%. Patients
infected with genotype 1 HCV do not respond as well
to this combination therapy demonstrating sustained
virological response rates of <50% even after treatment
therapies of 48 weeks in duration.6,7
both the 50 and 30 ends. The HCV NS5B gene encodes
an RNA-dependent RNA polymerase whose enzymatic
activity is critical to the replication of viral RNA gen-
ome. Because of its demonstrated and vital role in viral
replication, HCV NS5B polymerase has been the most
studied viral protein target for small molecule HCV
therapy.
Various groups have reported several classes of small
molecule inhibitors of hepatitis C virus NS5B polymer-
ase (see Fig. 1). Among the numerous non-nucleoside
inhibitors, compounds with either thiophene, phenylala-
nine, and dihydropyranone scaffolds have been shown
to bind to HCV polymerase in the lower thumb alloste-
ric pocket by X-ray crystal structures.8,9 A series of com-
pounds based on a benzimidazole scaffold have been
reported.10–13 Recently, the X-ray crystal structures of
the bound benzimidazole scaffold have also been solved
and revealed these inhibitors bind to a site on the surface
Hepatitis C virus is a positive-sense single-stranded
RNA virus. The HCV genomic RNA is 9.5 kb in length
and consists of a long open reading frame, which is
flanked by highly conserved untranslated regions at
˚
of the upper thumb domain that is 14 A away from the
lower thumb allosteric pocket that is occupied by com-
pounds of either thiophene or phenylalanine scaffolds.14
A series of benzothiadiazine derivatives have also been
reported as initiation specific inhibitors.15–19 More re-
cently, acylpyrrolidine,20–22 proline sulfonamide,23
phenoxyphenyl acrylic acid,24,25 thiazolidine26 have also
Keywords: Hepatitis C; HCV; HCV NS5B polymerase; Antiviral.
*
Corresponding author. Tel.: +1 847 937 8626; fax: +1 847 938
2756; e-mail: yaya.liu@abbott.com
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.04.068

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Y. Liu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3173–3177
Table 1. IC50 values against genotype 1b polymerase: effects of various
R groups on the inhibition potency
InniittiiaattioionnYY444848PoPcokcetket
ThhuummbbPP449595PoPcokcekt et
H
R
O
O
B
I
N
H
A
ThhuummbbMM42432P3oPcokcekt et
I
Compound
R
IC50 (lM)
1
2
–H
46
37
30
54
46
33
11
10
5.6
1.9
–CH3
–NH2
–OH
–OCH3
–F
3
4
InniittiiaattioionnRR202000PoPcokcetket
Elloonnggaatitoionnnunculecoletiodteidpeocpkoectket
5
6
Figure 1. Ribbon diagram of the HCV RNA-dependent RNA
polymerase. The fingers, palm, and thumb subdomains are colored
in blue, red, and green, respectively. The catalytic site lies within the
center of the polymerase domains. The b-loop is colored in orange.
PDB accession code: 1GX6. Four target sites for antiviral molecules
have been identified to date, including the elongation nucleotide pocket
and initiation nucleotide inhibitor pocket, and allosteric thumb P495
and thumb M423 pockets (reproduced with permission).
7
–Cl
–Br
8
9
10
–OPh
–NHPh
was purchased from an outside vendor (Chembridge
Corp.).
been shown to bind to this initiation pocket A. The ben-
zofuran carboxamide27,,28 and anthranilic acid29 have
been shown to bind to initiation pocket B that overlaps
with initiation pocket A. The diketoacid derivatives
have been reported as product mimics that possibly
interact with the catalytic metal ions in the elongation
nucleotide pocket of the enzyme active site.30,31
We found bulky hydrophobic groups such as phenyl
ether (9) and phenyl amine (10) have the best activity
as summarized in Table 1 for the diiodo substituent ser-
ies. We then decided to evaluate the importance of the
phenol group of the salicylamide moiety. As shown in
Table 2, four pairs of compounds (11–18) with variation
of R1 as either hydroxyl group or amino group are com-
pared. The anilines (12, 14, 16, and 18) were about 12- to
19-fold weaker than the phenols (11, 13, 15 and 17).
Substitutions on the B ring did not change activity sig-
nificantly. The amide linkage in 19 can be replaced with
the imine in 20 without loss of activity.
Halosalicylamide derivatives were identified from high-
throughput screening as potent inhibitors of HCV
NS5B polymerase. The initial structure–activity rela-
tionship of the screening hits suggested that 4,6-diiodo
substitution on the phenol ring is better than dibromo
and dichloro substitution. We carried out further SAR
analysis on the effect of various R groups on all three
substituents. The synthesis of these analogs was accom-
plished by amide formation from commercially avail-
able acids and anilines as illustrated in Scheme 1. The
acid chlorides were formed from the halo salicylic acids
and then condensed with the appropriate aniline to give
the final analogs. The O-acetyl compound, 23, was syn-
thesized by first treating the salicylic acid with acetyl
chloride, followed by amide formation. Compound 20
The effects methylation of the phenol hydroxyl group
and the amide group of the salicylamide moiety are also
investigated in Table 3. The methylation of the amide
group yielded a compound (22) that is more than 40-fold
weaker than the parent compound (21). A similar loss
was observed for compound (25), with methylation of
amide compound (24). Methylamine of the phenol
group in 23, 26, and 27 completely abolished the activity
(Scheme 2).
Our structure–activity relationship studies of halosali-
cylamide derivatives revealed the absolute requirement
of the salicylamide moiety for optimum activity. Meth-
ylation of either the phenol group or the amide group
of the salicylamide moiety abolished activity while sub-
stitutions on both phenyl rings were generally accept-
able. The carboxyamide can be replaced with imine
without loss of activity.
Y1
Y2
Y3
B
R1
A
O
Y1
Y2
Y3
R1
A
O
HN
R2
X
B
OH
X
N
R2
b, c
X
X
X = Cl, Br, I
R1 = OH
Y= H, OH, Cl, Br, F, CH3, NHPh, OPh,
OCH3, NH2
a
The specific binding of the halosalicylamide derivatives
to HCV NS5B polymerase was confirmed by affinity
selection and NMR. We then decided to characterize
the mechanism of inhibition of this class of com-
pounds. We titrated a soluble analog compound 21
with increasing concentrations of the uridine triphos-
phate substrate.32 The halosalicylamide demonstrated
OC(O)CH3
OMe
NH2
R2 = H, CH3
Scheme 1. Reagents and conditions: (a) acetyl chloride, TEA, DCM,
rt, 4 h; (b) SOCl2, cat DMF, toluene 90 °C 1.5 h; (c) substituted
aniline; dioxane 85 °C, 2 h.

Y. Liu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3173–3177
3175
Table 2. IC50 values against genotype 1b polymerase: effects of amine replacement of the phenol hydroxyl group and the amide linkage replacement
of the salicylamide moiety
Y1
Linker
Y2
Y3
R1
B
X
A
X
Compound
X
Y1
Y2
Y3
R1
Linker
IC50 (lM)
11
12
13
14
15
16
17
18
19
20
–Cl
–Cl
–Cl
–Cl
–Br
–Br
–Br
–Br
–I
–H
–H
–H
–H
–H
–H
–H
–H
–OH
–Cl
–Cl
–Cl
–Cl
–Cl
–Br
–Br
–H
–Cl
–Cl
–OH
–NH2
–OH
–NH2
–OH
–NH2
–OH
–NH2
–OH
–OH
–CONH
–CONH
–CONH
–CONH
–CONH
–CONH
–CONH
–CONH
–CONH
–CHN
8.6
102
9.0
108
6.7
98
–CH3
–CH3
–H
–H
–Br
–Br
–H
6.9
131
8.6
8.8
–H
–Cl
–Cl
–I
–H
Table 3. IC50 values against genotype 1b polymerase: effects of methylation of phenol group and the amide group of the salicylamide moiety
Y1
Y2
Y3
R1
O
B
X
N
R2
A
X
Compound
X
Y1
Y2
Y3
R1
R2
IC50 (lM)
21
22
23
24
25
26
27
–I
–H
–H
–Cl
–Cl
–Cl
–Br
–Cl
–Br
–H
–Cl
–Cl
–Cl
–H
–H
–H
–H
–OH
–OH
–H
4.7
–I
–CH3
–H
–H
>200
>200
6.7
–I
–H
–H
–OAc
–OH
–OH
–Br
–Br
–Cl
–Cl
–H
–H
–CH3
–H
–H
135
>200
>200
–OCH3
–OCH3
–OCH3
non-competitive inhibition with respect to nucleotide
substrates. Competition studies using inhibitors of
known binding sites indicated an additive binding
mode for halosalicylamides to both the thiadiazine ini-
tiation nucleotide pocket and the diketoacid elongation
nucleotide pocket, but a mutually exclusive binding
mode with respect to the benzimidazole thumb alloste-
ric pocket as demonstrated in Figure 2.32 More impor-
tantly, this class of compounds demonstrated broad
genotype activity and replicon cell culture activity as
shown in Table 4. However, the poor solubility of this
class of compounds poses challenges for the structural
biology work. Further improvement in activity and
physical properties for this class of compounds is
desired.
OH is crucial for activity
Carboxyamide can be replaced with imine
OH
O
B
R
The high HCV copy number and the significant diver-
sity of the quasispecies that are present in HCV in-
fected patients coupled with the high potential for
the development of resistance suggest that multiple
drugs will likely be required to effectively treat this
disease. In our high-throughput screening campaign
effort, we identified halosalicylamide derivatives as a
new class of allosteric inhibitors that could be used
in combination with palm initiation inhibitors or
Cl < I = Br
N
H
A
Br = I > Cl
Scheme 2. SAR summary of halosalicylamides.

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Y. Liu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3173–3177
Compound 21 with thiadiazine Compound 21 with Diketoacid
Compound 21 with Benzimidazole
0.003
0.003
0.0025
0.002
0.0015
0.001
0.0005
0
0.005
0.004
0.003
0.002
0.001
0
0.002
0.001
0
-0.2
0
0.2 0.4 0.6 0.8
1
-0.5
0
0.5
1
-20 -10
0
10
20
30
40
Diketoacid (μM)
Benzimidazole (μM)
Thiadiazine (μM)
Figure 2. Inhibitor/inhibitor competition study using inhibitors of known binding sites. Various concentrations of inhibitors of known binding sites
are shown on X-axis. Varying concentrations of compound 21 are shown as (h, D, }, and s). The inverse of initial velocities are shown on Y-axis.
Table 4. Biochemical IC50s against various genotypes of polymerase, replicon cell culture EC50 and TD50 of compound 21
1a IC50
BK IC50
2a IC50
2b IC50
3a IC50
SEAP EC50
MTT TD50
21
5.7 lM
8.6 lM
16.2 lM
17.3 lM
14.3 lM
2.0 lM
21.8 lM
9. Love, R. A.; Parge, H. E.; Yu, X.; Hickey, M. J.; Diehl,
W.; Gao, J.; Wriggers, H.; Ekker, A.; Wang, L.; Thomson,
J. A.; Dragovich, P. S.; Fuhrman, S. A. J. Virol. 2003, 77,
7575.
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Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2008.04.068.
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