Discovery of novel potent HCV NS5B polymerase non-nucleoside inhibitors bearing a fused benzofuran scaffold

Article abstract of DOI:10.1016/j.bmcl.2018.01.029

This letter describes the discovery of a fused benzofuran scaffold viable for preparing a series of novel potent HCV NS5B polymerase non-nucleoside inhibitors. Designed on the basis of the functionalized benzofuran derivative nesbuvir (HCV-796), these com

Full text of DOI:10.1016/j.bmcl.2018.01.029

Accepted Manuscript
Discovery of novel potent HCV NS5B polymerase non-nucleoside inhibitors
bearing a fused benzofuran scaffold
Min Zhong, Eric Peng, Ningwu Huang, Qi Huang, Anja Huq, Meiyen Lau,
Richard Colonno, Leping Li
PII:
S0960-894X(18)30039-8
https://doi.org/10.1016/j.bmcl.2018.01.029
BMCL 25555
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 September 2017
12 January 2018
17 January 2018
Please cite this article as: Zhong, M., Peng, E., Huang, N., Huang, Q., Huq, A., Lau, M., Colonno, R., Li, L.,
Discovery of novel potent HCV NS5B polymerase non-nucleoside inhibitors bearing a fused benzofuran scaffold,
Bioorganic & Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.01.029
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Discovery of novel potent HCV NS5B polymerase non-
nucleoside inhibitors bearing a fused benzofuran scaffold
Min Zhong*, Eric Peng, Ningwu Huang, Qi Huang, Anja Huq, Meiyen Lau,
Richard Colonno and Leping Li*
Presidio Pharmaceuticals, Inc., 1700 Owens Street, Suite 184, San Francisco, CA 94158, USA
This is where the receipt/accepted dates will go; Received Month XX, 2000; Accepted Month XX, 2000 [BMCL RECEIPT]
Abstract— This letter describes the discovery of a fused benzofuran scaffold viable for preparing a series of novel potent HCV NS5B
polymerase non-nucleoside inhibitors. Designed on the basis of the functionalized benzofuran derivative nesbuvir (HCV-796), these
compounds presumably bind similarly to the allosteric binding site in the “palm” domain of HCV NS5B protein. SAR of each potential
hydrogen-bonding interaction site of this novel scaffold is discussed along with some preliminary genotypic profile and PK data of
several advanced compounds. [BMCL ABSTRACT] ©2000 Elsevier Science Ltd. All rights reserved.
It was estimated that more than 170 million people
worldwide have been suffering from chronic infections
with various genotypes of Hepatitis C virus (HCV).
Chronic HCV infection has been regarded as one of the
main causes for chronic hepatitis, liver cirrhosis,
hepatocellular carcinoma, and the related liver
transplant and death.1-4 Following an extensive effort in
HCV drug discovery over the past decade, a number of
efficacious all-oral combination regimens5-12 have
recently been approved by the US FDA to cure HCV
infections.
which could be used in combination with an HCV
NS5A inhibitor (e.g. ravidasvir)14 and an HCV
NS3/4A protease inhibitor as a new all-oral combination
regimen.
Among the various classes of HCV drugs utilized in
highly effective combination regimens are HCV NS5B
polymerase non-nucleoside inhibitors.13 For example,
Viekira Pak® is composed of co-formulated ombitasvir
(HCV NS5A inhibitor, 12.5 mg/tablet), paritaprevir
(HCV NS3/4A protease inhibitor, 75 mg/tablet) and
ritonavir (CYP3A inhibitor, 50 mg/tablet) (two tablets
and once daily), and dasabuvir (HCV NS5B polymerase
non-nucleoside inhibitor, 250 mg/tablet, single tablet,
and twice daily), which is prescribed for treating
patients with genotype 1 HCV infection.8a Recently,
Viekira XRTM, a once-daily extended-release co-
formulation of the active ingredients in Viekira Pak®,
was approved by the US FDA.8c
Figure 1. Chemical structures of nesbuvir (HCV-796) (1) and a fused
benzofuran scaffold
Nesbuvir (HCV-796) (1, Figure 1) is a potent HCV
NS5B polymerase non-nucleoside inhibitor15 that
advanced to a Phase II human clinical trial in
combination with pegylated interferon alfa-2b (pegINF
2b) in the presence of ribavirin. This combination
regimen demonstrated good efficacy in genotype 1a (gt-
1a) infected subjects; however, its further development
was suspended due to unexpected elevations of liver
enzymes.16
As part of our HCV drug discovery effort, we were
interested in discovering and developing a novel potent
HCV NS5B polymerase non-nucleoside inhibitor,

On the basis of the reported co-crystal structure,17
nesbuvir binds to the allosteric binding site in the
“palm” domain of the HCV NS5B polymerase protein,
where the –NH– and the –C(O)– of the amide moiety at
the C3 position show H-bond interactions with Ser365
and Arg200, respectively. Also, the –C(O)– has H-bond
interaction with Cys316, the –S(O)2– shows H-bond
interaction with Arg200 and Met414, and the
cyclopropyl residue displays hydrophobic interaction
with the protein. The –OH seems not to exhibit direct
interaction with the protein. Presumably, it helps
improve the intrinsic solubility.
protein and the –CH2CH2OH moiety presumably serves
as a solubilizing group, we thought that linking the –N–
of the sulfonamide residue to the C5 in central benzene
ring with an aliphatic ring could retain hydrophobic
interaction to achieve the desired potency. As shown in
Table 1, when a five-member ring is employed, the
resulting compound (2a, entry 2) showed an EC50 of
0.304 M in the gt-1b replicon. The corresponding six-
member analog (2b, entry 3) showed slightly weaker
potency. Interestingly, with the increase of the ring size
from five-member to seven-, eight- and nine-member,
the resulting analogs (2c-e, entries 4-6) showed 10 to
60-fold increase in potency relative to 2a in the gt-1b
replicon. Considering the importance of lipophilicity to
the physicochemical property of a compound, the
seven-member ring was chosen as the backbone for
further chemical modifications.
With the understanding of the co-crystal structural
information, we employed a macrocyclization drug
design strategy18 (Figure 1) to the structure of nesbuvir
(1), which led to the discovery of a series of novel
potent HCV NS5B polymerase non-nucleoside
inhibitors bearing a fused benzofuran scaffold. Herein,
we report the SAR of each potential H-bonding
interaction region of this new scaffold and some
preliminary genotypic profile and rat PK data of several
advanced compounds.
Table 2. SAR of R2 in the fused benzofuran analogs (2c and 2f-q)
Entry Compd. R2
HCV inhibition EC50 (M)a
All of the fused benzofuran derivatives were prepared
by following our previously disclosed protocols.19
A
gt-1a
gt-1b
panel of wild-type (gt-1a and gt-1b) and gt-1a variant
(L31V+Y93H) HCV replicons were employed as the
primary cell-based assays to evaluate all of the
1
0.024
0.024b
0.005c
0.083
0.012
1
0.009c
0.017
2
3
4
-C(O)NHMe
-C(O)NH2
2c
2f
compounds.20 These replicon stable cell lines were
generated in naïve Huh-7 cells. As expected, these HCV
NS5B polymerase non-nucleoside inhibitors exhibit
strong potency against the gt-1a variant (L31V+Y93H)
replicon, in which the potency of HCV NS5A inhibitors
bearing a dimeric bis-imidazole motif are completely
diminished, demonstrating the complementary effect of
these two classes of HCV inhibitors.
d
>2.0
>2.0
>2.0b
-
-C(O)NMe2
-
2g
5
6
-C(O)NHEt
-C(O)NHcPr
-
-
2h
2i
>2.0
7
-C(O)NHCN
>2.0
0.090
>2.0
>2.0
>2.0
-
2j
8
-C(O)NHOMe
-C(O)NHS(O)2Me
-C(O)N(OH)Me
0.031
2k
2l
9
-
Table 1. SAR of the ring size in the fused benzofuran analogs (2a-e)
10
11
0.298
-
2m
2n
12
13
14
0.059
0.005
0.850
-
2o
2p
2q
0.033b
Entry
1
Compd.
n
CLogPa
HCV gt-1b
EC50 (M)
0.012
0.009b
0.304
>2.0
2.16
1
>2.0
2
3
4
0
1
2
2.52
2.93
3.49
2a
2b
2c
>2.0b
0.451
0.017
aThe data was generated in gt-1a (L31V+Y93H)_LucNeo Luciferase and
gt-1b (wild-type)_LucNeo Luciferase HCV replicons, respectively.
bThe data was generated in gt-1a (wild-type) HCV replicon. The potency
of an HCV NS5B inhibitor in gt-1a (wild-type) and gt-1a
(L31V+Y93H)_LucNeo Luciferase HCV replicons is generally
comparable to each other.
5
6
3
4
4.05
4.61
0.005
0.026
2d
2e
aCalculated values using ChemBioOffice 2014 developed by
CambridgeSoft. CLogP refers to the calculated logarithm of a
compound’s partition coefficient between n-octanol and water.
bReported value, see ref. 15a
cReported value, see ref. 15a
dNot determined
To understand the SAR in the R2 region (–C(O)NHMe
as R2 in 2c), a number of functional groups bearing
In view that the cyclopropyl moiety in nesbuvir (1)
(Figure 1) mainly has hydrophobic interaction with the

potential H-bond donor (e.g. –NH– or –OH) and
acceptor (e.g. –C=O or –C=N–) were employed. As
outlined in Table 2, neither primary (2f, entry 3) nor
tertiary (2g, entry 4) amide is tolerated. Replacement of
the –C(O)NHMe group with bulkier –C(O)NH-alkyls
(2h-i, entries 5-6), –C(O)NHCN (2j, entry 7), or –
C(O)NHS(O)2Me (2l, entry 9) is detrimental to the
potency. Also, the –C(O)NHMe cannot be switched to
an –C(O)N(OH)Me residue (2m, entry 10).
Interestingly, substitution of the –C(O)NHMe with –
C(O)NHOMe afforded potent 2k (entry 8) comparable
to 2c. Among the tested five-member heterocycles,
including 4,5-dihydro-1H-imidazole (2n, entry 11),
imidazole (2o, entry 12), [1,2,4]-triazole (2p, entry 13),
and tetrazole (2q, entry 14), that retain a potential pair
of H-bond donor and acceptor similar to –C(O)NHMe,
2o demonstrated fairly improved potency in both the gt-
1a and gt-1b replicons; however, lower intrinsic
solubility associated with 2o (CLogP = 4.37) relative to
2c (CLogP = 3.49) was observed.
olefinic analog 3b (entry 3) seems to be more potent.
Saturation of the double bond led to a more potent
analog (±)-3c (entry 4) with an EC50 of 10 nM and 3 nM
in the gt-1a and gt-1b replicons, respectively. Between
those two enantiomers, (5R)-3c is more potent than
(5S)-3c.21 Neither a carbonyl (3d, entry 5) nor hydroxyl
((±)-3e, entry 6) at the C5 position was well tolerated.
Introduction of a cyclopropyl residue at the C5 position
reasonably maintained good potency (3f, entry 7). Also,
improved potency was observed when the cyclopropyl
residue was fused to the C5 and C6 carbons ((±)-3g,
entry 8) and (5R,6R)-3g is more active than (5S,6S)-3g
in the gt-1a and gt-1b replicons.
To reduce the lipophilicity of (±)-3c (CLogP = 4.01)
(entry 4), an oxygen atom was inserted at the C6
position, giving (±)-3h (CLogP = 2.38) (entry 9) with
four-fold increase of the potency in the gt-1a replicon.
As expected, the corresponding S isomer ((5S)-3h, entry
4) is the more potent enantiomer with an EC50 of 9 nM
and 3 nM in the gt-1a and gt-1b replicons, respectively.
Moreover, the metabolic stability of (5S)-3h (entry 3,
Table 5) in rat and human liver microsomes (LMs) was
improved as compared to (±)-3c (entry 2, Table 5).
Replacement of the –O– in (±)-3h with –NH– ((±)-3i,
entry 10) resulted in a significant loss of the potency,
but the potency of (±)-3i was fairly recovered by
methylating the –NH– residue ((±)-3j, entry 11).
Interestingly, the lactam analog of (±)-3i (3k, entry 12)
demonstrated comparable potency to (±)-3h (entry 9) in
both the gt-1a and gt-1b replicons, while the
As shown in Table 3, in the R3 region (–S(O)2Me group
as R3 in 2c) (entry 1), significant decrease of the
potency in the gt-1a replicon was observed when the -
Me group was substituted with –NH2 (2p, entry 2), -
NMe2 (2q, entry 3), or –cPr (2r, entry 4), respectively.
Moreover, replacement of the –S(O)2Me group with –
C(O)Me (2s, entry 5) or –C(O)OMe (2t, entry 6) was
not tolerated.
Table 3. SAR of R3 in the fused benzofuran analogs (2c and 2p-t)
corresponding N-methylated analog (±)-3l (entry 13) is
less potent. Replacement of the lactam residue in (±)-3k
or (±)-3l with five-member heterocycles gave (±)-3m
(entry 14), (±)-3n (entry 15), and (±)-3o (entry 16),
respectively, with good potency. However, decreased
intrinsic solubility of (±)-3k-o relative to (±)-3i-j was
noted, possibly due to the increase of planarity of the
molecules. Other functional groups can also be attached
to the C7 position in (±)-3h. For example, when a
methyl group was introduced, the resulting (±)-3p (entry
17) showed comparable potency; while a cyclopropyl
moiety slightly reduced the potency ((±)-3q, entry 18).
Entry
Compd. R3
HCV inhibition EC50 (M)a
gt-1a
gt-1b
1
2
-S(O)2Me
0.083
0.017
2c
c
-S(O)2NH2
-S(O)2NMe2
-S(O)2cPr
-C(O)Me
0.719
0.605b
0.518
0.480b
0.423
0.335b
>2.0
-
2p
3
4
5
6
-
2q
2r
2s
2t
0.073
-
>2.0b
0.722
-C(O)OMe
0.164
Considering that (±)-3h has poor metabolic stability in
rat and human LMs, further reduction of the
aThe data was generated in gt-1a (L31V+Y93H)_LucNeo Luciferase and
gt-1b (wild-type)_LucNeo Luciferase HCV replicons, respectively.
bThe data was generated in gt-1a (wild-type) HCV replicon. The potency
values of an HCV NS5B inhibitor in gt-1a (wild-type) and gt-1a
(L31V+Y93H)_LucNeo Luciferase HCV replicons is generally
comparable to each other.
lipophilicity was considered to improve it. When an
HOCH2– moiety was introduced to the C7 position, the
resulting compound ((±)-3r, entry 19) exhibited an EC50
of 52 nM and 12 nM in the gt-1a and gt-1b replicons,
respectively; and more importantly, significant
cNot determined
improvement of the metabolic stability in rat and human
LMs was achieved (entries 4-5, Table 5). Among those
four diastereomers of (±)-3r,22 (5S,7S)-3r is the most
potent one with an EC50 of 9 nM and 3 nM in the gt-1a
and gt-1b replicons, respectively, while (5S, 7R)-3r is
Our main effort has been expended on the modifications
of the seven-member ring to understand the SAR. As
shown in Table 4, a terminal vinyl residue at C5 is
tolerated (3a, entry 2) and the corresponding internal

Table 4. SAR of the substituted 7-member ring moiety in the fused benzofuran analogs (2c and 3a-u)
HCV inhibition EC50 (M)a
HCV inhibition EC50 (M)a
Entry Compd.
Fused ring
moiety
Entry Compd.
Fused ring
moiety
gt-1a
gt-1b
0.017
gt-1a
gt-1b
0.015
1
2
3
4
5
0.083
12
13
14
15
16
(±)-3k
0.014
2c
3a
3b
0.019b
0.011b
1.795b
(5S)-3k
(5R)-3k
0.005
0.586
0.106
0.070
0.026
0.016
(±)-3l
(5S)-3l
(5R)-3l
0.180b
0.102b
0.688b
0.017
0.013
0.059
(±)-3m
0.026b
0.074
(±)-3c
(5R)-3c
0.010
0.009
0.010b
0.089
0.003
0.004
(±)-3n
(5S)-3n
(5R)-3n
0.054b
0.020b
0.617b
0.006
0.003
0.038
(5S)-3c
0.046
0.078
0.318
(±)-3o
(±)-3p
0.100b
0.011
3d
6
(±)-3e
0.730
0.118
0.107
17
0.033b
0.008
7
8
0.020
0.009
18
19
(±)-3q
(5S)-3q
(5R)-3q
0.130b
0.093b
>2.0b
0.019
0.013
0.666
3f
(±)-3g
0.044
0.035b
0.025
>2.0
(±)-3r
0.052b
0.009b
>2.0b
0.012
0.003
1.473
0.069
0.013
0.007
0.003
1.208
0.024
0.013
0.033
(5S,7S)-3r
(5R,7R)-3r
(5R,7S)-3r
(5S,7R)-3r
(±)-3s
(5R,6R)-3g
(5S,6S)-3g
0.007
>2.0
0.554b
0.064b
0.009b
0.004b
>2.0b
9
(±)-3h
0.021
0.028b
0.009
0.624
0.011
20
(5S,7S)-3s
(5R,7R)-3s
(5R,7S)-3s
(5S,7R)-3s
(±)-3t
(5S)-3h
(5R)-3h
0.003
0.686
0.100b
0.057b
0.040b
10
11
(±)-3i
0.265
0.043
21
22
(±)-3j
0.050
0.014
(±)-3u
0.016b
0.019
aThe data was generated in gt-1a (L31V+Y93H)_LucNeo Luciferase and gt-1b (wild-type)_LucNeo Luciferase HCV replicons, respectively.
bThe data was generated in gt-1a (wild-type) HCV replicon. The potency of an HCV NS5B inhibitor in gt-1a (wild-type) and gt-1a (L31V+Y93H)_LucNeo
Luciferase HCV replicons is generally comparable to each other.
reasonably potent with an EC50 of 64 nM and 13 nM in
the gt-1a and gt-1b replicons, respectively. As expected,
the corresponding (5R) isomers (i.e. (5R,7R)- 3r and
(5R,7S)-3r) do not possess comparable potency.
the C7 position, giving (±)-3s (entry 20) with improved
potency relative to (±)-3h. As expected, the metabolic
stability in rat and human LMs was significantly
improved (entries 6-7, Table 5) and (5S,7S)-3s is the
most potent diastereomer with an EC50 of 4 nM and 3
Moreover, a MeS(O)2CH2– moiety can be attached to

nM in the gt-1a and gt-1b replicons, respectively.
Furthermore, the C8 position in (±)-3h can be
substituted with either HOCH2– or MeS(O)2CH2– to
give (±)-3t (entry 21) or (±)-3u (entry 22), which has
comparable potency to (±)-3r or (±)-3s in the gt-1a and
gt-1b replicons, respectively.
replaced with 2-F-phenyl ((±)-3v, entry 2), the gt-1a
potency was retained but there was a 2-fold loss of the
gt-1b potency. As expected, the 2,4-diF-phenyl analog
((±)-3w, entry 3) showed comparable potency to (±)-3v.
Modulation of the conformation between R1 and the
central benzofuran moiety by substituting the 2-F in (±)-
3w with a bulkier 2-OMe yielded a much weaker
compound ((±)-3x, entry 4). Interestingly, the 4-F in
(±)-3c could be replaced with a 4-F-phenoxyl residue
((±)-3y, entry 5) without significantly reducing the
potency.
Table 5. Metabolic stability of representative compounds in rat and
human liver microsomes
Entry Compd.
% remaining of the parent CLogPa tPSAa
at 60 min
RLMb
HLMb
1
1
84.0
53.3
2.16
95.94
Table 7. SAR of the central substituted [6,5]-heterocycles
2
3
(±)-3c
0.8
5.0
0.8
4.01
2.38
75.71
84.94
(5S)-3h
32.6
4
5
6
7
(±)-3r
68.9
60.0
99.7
100
85.9
100
100
100
2.30
2.30
1.30
1.30
105.17
105.17
119.08
119.08
(5S,7S)-3r
(±)-3s
HCV inhibition EC50 (M)a
Entry Compd. Central substituted
[6,5]-heterocycle
(5S,7S)-3s
gt-1a
gt-1b
aCalculated values using ChemBioOffice 2014 developed by
CambridgeSoft. CLogP refers to the calculated logarithm of a
1
2
(±)-3c
(±)-4
0.010
0.003
0.801
0.523
>2.0b
0.182
compound’s partition coefficient between n-octanol and water. tPSA
referes to total polar surface area.
bRLM refers to rat liver microsome and HLM refers to human liver
microsome.
3
4
(±)-5
(±)-6
0.109
c
-
It is worth noting that the cytotoxicity of these potent
HCV NS5B analogs in Huh-7 naïve cells is reasonably
low. For example, the CC50 of (5S,7S)-3r is > 3 M and
that of (5S,7S)-3s is > 10 M, which was determined by
incubating a compound with the cells at rt for 24 h.
aThe data was generated in gt-1a (L31V+Y93H)_LucNeo Luciferase and
gt-1b (wild-type)_LucNeo Luciferase HCV replicons, respectively.
bThe data was generated in gt-1a (wild-type) HCV replicon. The potency
of an HCV NS5B inhibitor in gt-1a (wild-type) and gt-1a
(L31V+Y93H)_LucNeo Luciferase HCV replicons is generally
comparable to each other.
Table 6. SAR of R1 in the fused benzofuran analogs ((±)-3c and (±)-3v-y)
cNot determined
Moreover, several fused [6,5]-hetereocyclic systems
were also tested to compare with the central benzofuran
scaffold. As shown in Table 7, a compound bearing a
substituted imidazo[1,2-a]pyridine ((±)-4, entry 2),
pyrazolo[1,5-a]pyridine ((±)-5, entry 3), or 2H-indazole
((±)-6, entry 4) central core is much less potent than the
corresponding benzofuran analog ((±)-3c, entry 1) in the
gt-1a and gt-1b replicons, respectively.
Entry Compd.
R1
HCV inhibition EC50 (M)a
gt-1a
gt-1b
1
2
(±)-3c
(±)-3v
0.010
0.003
0.011
0.015
>2.0
0.007
0.008
0.056
3
4
(±)-3w
(±)-3x
In addition to testing these compounds in the wide-type
gt-1a and gt-1b HCV replicons, their preliminary
genotypic profiles were evaluated with wide-type gt-2a,
gt-3a and gt-4a HCV replicons, respectively. These
replicon stable cell lines were also generated in naïve
Huh-7 cells. As summarized in Table 8, compounds
(5S,7S)-3r and (5S,7S)-3s demonstrated superior overall
virological profiles to nesbuvir (1). Although (±)-3t and
(±)-3u have comparable gt-1a and -1b potency to (±)-3r
and (±)-3s, respectively, they exhibited less attractive
overall genotypic profiles.
5
(±)-3y
0.071
0.014
aThe data was generated in gt-1a (L31V+Y93H)_LucNeo Luciferase and
gt-1b (wild-type)_LucNeo Luciferase HCV replicons, respectively.
Meanwhile, a set of analogs of (±)-3c, in which the 4-F-
phenyl group was replaced with varying R1, was
prepared to establish the preliminary SAR. As outlined
in Table 6, when the 4-F-phenyl in (±)-3c (entry 1) was
To evaluate the pharmacokinetic of this novel series of
potent HCV NS5B polymerase non-nucleoside

inhibitors, a number of compounds along with nesbuvir
(1) were dosed at 1 mg/kg and 5 mg/kg in rat iv and po
PK in a solution formulation of 10% NMP/10%
solutol/40% PEG-400/40% saline, respectively. As
shown in Table 9, nesbuvir (1) showed good PK profile
with a half-life (t1/2) of 2.73 h, clearance (CL) of 10.4
mL/min/kg, steady state volume distribution (Vss) of
1.52 L/kg, and area under curve (AUCINF) of 1610
hr*ng/mL in rat iv PK, and bioavailability (F%) of 73%
in rat po PK (entry 1). As compared to nesbuvir (1),
(5R)-3c, (5R)-3h, and (5S)-3k exhibited relatively
higher clearance, shorter half life, lower exposure and
poorer bioavailability (entries 2-4). Both (5S)-3n and
(5S,7S)-3r performed comparably to nesbuvir (1)
(entries 5-6). Although (±)-3s showed similar iv PK
profile to nesbuvir (1), its bioavailability was much
lower, presumably due to the much decreased
showed reasonably good iv and po PK profile in rats.
Further work will be focused on in vitro virological
assessment of representative compounds in combination
with other direct acting agents (DAAs) and the
understanding of safety profile of this series of
compounds as compared to nesbuvir (1), which will be
discussed in due course.
Corresponding Author. *MyoKardia, Inc., 333
Allerton Avenue, South San Francisco, CA 94080; Tel:
(650) 636-7328; Fax: (650) 741-0901; E-mail:
mzhong@myokardia.com (MZ) or Assembly
Biosciences, Inc., 409 Illinois Street, San Francisco, CA
94158; Tel: (650) 521-2034; Fax: (415) 968-2864; E-
mail: lli@assemblybio.com (LL).
Acknowledgment. The authors thank Dr. Jason Xiang
and his chemistry team at Shanghai ChemPartner for
their excellent chemistry CRO services.
lipophilicity (entry 7).
Table 8. Genotypic profile of HCV NS5B polymerase non-nucleoside
inhibitors bearing a fused benzofuran scaffold relative to nesbuvir (1)
Supplementary material. Synthetic procedure to the
preparation of (5S,7S)-3r and the corresponding
spectroscopic data, including 1H NMR, COSY,
NOESY, and chiral HPLC data (PDF)
HCV inhibition EC50 (M)a
Compd.
gt-1a
gt-1b
gt-2a
gt-3a
gt-4a
0.024
0.012
0.072
0.019
0.021
1
References and notes
(±)-3r
0.052
0.012
0.003
0.062
0.012
0.020
0.006
0.032
0.009
(5S,7S)-3r 0.009
(±)-3s 0.009
(5S,7S)-3s 0.004
1. (a). Choo, Q. L.; Kuo, G.; Weiner, A. J.; Overby, L.
R.; Bradley, D. W.; Houghton, M. Science 1989, 244, 359–
362. (b). Kuo, G.; Choo, Q. L.; Alter, H. J.; Gitnick, G. L.;
Redeker, A. G.; Purcell, R. H.; Miyamura, T.; Dienstag, J.
L.; Alter, M. J.; Stevens, C. E.; Tegtmeier, G. E.; Bonino,
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0.007
0.003
0.033
0.019
0.064
0.018
0.141
0.134
0.017
0.010
0.053
0.070
0.017
0.007
0.117
0.051
(±)-3t
(±)-3u
0.040
0.016
aThe data was generated in wide-type gt-1a, gt-1b, gt-2a, gt-3a and gt-
4a_LucNeo Luciferase HCV replicons.
Table 9. Rat iv and po PK of representative compounds
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Hepatol. 1999, 6, 35–47.
3. Lauer, G. M.; Walker, B. D. N. Engl. J. Med. 2001,
entry Compd.a
t1/2
CL
Vss
AUCINF
F
(h)b
(mg/ml/kg)b (L/kg)b (hr*ng/mL)b (%)c
1
2
3
4
5
6
7
2.73
0.96
0.34
0.68
2.43
10.4
38.2
78.0
44.5
12.8
22.0
16.0
1.52
2.36
1.79
1.74
1.41
2.25
1.51
1610
439
73
22
16
14
57
73
30
1
345, 41–52.
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2441.
(5R)-3c
(5R)-3h
(5S)-3k
(5S)-3n
234
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379
1301
759
(5S,7S)-3r 1.32
(±)-3s 3.62
1059
aA solution formulation of 10% NMP/10% solutol/40% PEG-400/40%
saline was employed in the rat iv and po PK experiments.
bRat iv PK was dosed at 1 mg/kg in Sprague-Dawley rats (n=3).
cRat po PK was dosed at 5 mg/kg in Sprague-Dawley rats (n=3).
In summary, we have successfully discovered a series of
novel potent HCV NS5B polymerase non-nucleoside
inhibitors bearing a fused benzofuran scaffold. As
compared to nesbuvir (1), some of the compounds, such
as (5S,7S)-3r and (5S,7S)-3s, demonstrated better
potency in the gt-1a and gt-1b replicons and superior
genotypic profiles. Also, (5S,7S)-3r and (5S,7S)-3s
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co-formulated tablet of the HCV NS5A inhibitor
ledipasvir (90 mg/tablet) and the HCV NS5B polymerase
nucleoside inhibitor sofosbuvir (400 mg/tablet) (one tablet
and once daily) developed by Gilead Sciences, Inc., please
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A. R.; Borgia, G. Rev. Recent Clin. Trials 2014, 9, 115-
123. (c). The US FDA approved Viekira XRTM, a once-
daily, extended-release co-formulation of the active
ingredients in Viekira Pak® (ombitasvir, paritaprevir and
ritonavir tablets; dasabuvir tablets) on July 22, 2016. It is
prescribed for treating patients with chronic genotype 1
(gt-1) HCV infection, including those with compensated
cirrhosis (Child-Pugh A). Also, it is not used for people
with decompensated cirrhosis.
9. (a). The announcement of FDA approval of the HCV
NS5A inhibitor DaklinzaTM developed by Bristol-Myers
Squibb, Inc. in combination of the HCV NS5B polymerase
nucleoside inhibitor sofosbuvir for treating patients
infected with genotype 3 HCV, please see "FDA approves
new treatment for chronic hepatitis C genotype 3
infections". FDA News Release on July 24, 2015. (b). Gao,
M.; Nettles, R. E.; Belema, M.; Snyder, L. B.; Nguyen, V.
N.; Fridell, R. A.; Serrano-Wu, M. H.; Langley, D. R.;
Sun, J.-H.; O’Boyle, D. R., II; Lemm, J. A.; Wang, C.;
Knipe, J. O.; Chien, C.; Colonno, R. J.; Grasela, D. M.;
Meanwell, N. A.; Hamann, L. D. Nature 2010, 465, 96–
100.
13. (a). Eltahla, A.A.; Luciani, F.; White, P. A.; Lloyd, A.
R.; Bull, R. A. Viruses 2015, 7, 5206-5224. (b). Zhao, C.;
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15. (a). Kneteman, N. M.; Howe, A. Y. M.; Gao, T.;
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2009, 49, 745–752. (b). Howe, A. Y.; Cheng, H.; Johann,
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18. Macrocyclization strategy has been successfully
employed in the discovery of HCV NS3/4A protease
inhibitors with improved potency, resistance and PK
profile. See examples: (a). Rosenquist, Å.; Samuelsson, B.;
Johansson, P.-O.; Cummings, M. D.; Lenz, O.; Raboisson,
P.; Simmen, K.; Vendeville, S.; de Kock, H.; Nilsson, M.;
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Woodard, B. T.; Lemieux, C.; Do, M. G.; Zhang, H.;
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Josey, J. A.; Beigelman, L.; Blatt, L.; Seiwert, S. D. J.
Med. Chem. 2014, 57, 1753–1769. (c). Neelamkavil, S. F.;
Agrawal, S.; Bara, T.; Bennett, C.; Bhat, S.; Biswas, D.;
Brockunier, L.; Buist, N.; Burnette, D.; Cartwright, M.;
Chackalamannil, S.; Chase, R.; Chelliah, M.; Chen, A.;
Clasby, M.; Colandrea, V. J.; Davies, I. W.; Eagen, K.;
Guo, Z.; Han, Y.; Howe, J.; Jayne, C.; Josien, H.;
Kargman, S.; Marcantonio, K.; Miao, S.; Miller, R.;
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10. (a). The announcement of FDA approval of
TechnivieTM, a fixed-dose combination of the HCV NS5A
inhibitor ombitasvir, the HCV NS3/4A protease inhibitor
paritaprevir, and the CYP3A4 inhibitor ritonavir,
developed by AbbVie, Inc., please see " FDA approves
Technivie for treatment of chronic hepatitis C genotype 4".
FDA News Release on July 24, 2015. (b). Hézode, C.;
Asselah, T.; Reddy, K. R.; Hassanein, T.; Berenguer, M.;
Fleischer-Stepniewska, K.; Marcellin, P.; Hall, C.; Schnell,
G.; Pilot-Matias, T.; Mobashery, N.; Redman, R.; Vilchez,
R. A.; Pol, S. Lancet 2015, 385, 2502–2509.
11. The announcement of FDA approval of ZepatierTM, a
co-formulated tablet of the HCV NS5A inhibitor elbasvir
(50 mg/tablet) and the HCV NS3/NS4A protease inhibitor
grazoprevir (100 mg/tablet) developed by Merck, Inc.,
please see "FDA approves Zepatier for treatment of
chronic hepatitis C genotypes 1 and 4". FDA News Release
on January 28, 2016.

19. (a). Zhong, M.; Li, L. WIPO Patent Appl.
WO2012/058125. (b). Zhong, M.; Li, L. WIPO Patent
Appl. WO2013/163466.
replicon, while exhibiting comparable potency against both
the gt-1a (wide-type) and gt-1a (L31V+Y93H) replicons.
21. The stereochemistry of the chial center was elucidated
on the basis of the method reported in Harada, N.;
Watanabe, M.; Kuwahara, S.; Sugio, A.; Kasai. Y.;
Ichikawa, A. Tetrahedron: Asymmetry 2000, 11, 1249 and
Taji, H.; Kasai, Y.; Sugio, A.; Kuwahara, S.; Watanabe,
M.; Harada, N.; Ichikawa, A. Chirality 2002, 14, 81. The
detailed experimental procedure was described in reference
19b.
20. At the early stage of our HCV NS5B program, we
encountered chemical contamination issues of low double-
digit nM IC50 HCV NS5B polymerase non-nucleoside
inhibitors with low single-digit pM IC50 HCV NS5A
inhibitors when synthesizing these two classes of
molecules in the same chemistry lab without much
precautions. Therefore, the gt-1a HCV replicon bearing
double mutants (e.g. L31V+Y93H) in the region of HCV
NS5A protein was utilized as a surrogate, in which HCV
NS5A inhibitors show significantly diminished potency as
compared to that observed in the corresponding wide-type
gt-1a relpicon. For potent HCV NS5B inhibitors, they
demonstrate good potency in the gt-1b (wide-type)
22. See the supplemental material for detailed
experimental procedure and stereochemistry determination
on the basis of the known stereochemistry of the chiral
starting material and the spectroscopic data, including 1H
NMR, COSY, NOSEY, and chiral HPLC data.
Graphical abstract
Discovery of novel potent HCV NS5B polymerase
non-nucleoside inhibitors bearing a fused
benzofuran scaffold
Min Zhong*, Eric Peng, Ningwu Huang, Qi Huang,
Anja Huq, Meiyen Lau, Richard Colonno and
Leping Li*
Presidio Pharmaceuticals, Inc., 1700 Owens Street, Suite
184, San Francisco, CA 94158, USA
This letter describes the discovery of a fused benzofuran scaffold
viable for preparing a series of novel potent HCV NS5B non-
nucleoside inhibitors. Designed on the basis of the functionalized
benzofuran derivative Nesbuvir (HCV-796), these compounds
presumably bind similarly to the allosteric binding site in the
“palm” domain of HCV NS5B protein. SAR of each potential
hydrogen-bonding interaction site of this novel scaffold is
discussed along with some preliminary genotypic profile and PK
data of several advanced compounds.