Design and evaluation of novel tetracyclic benzofurans as palm site allosteric inhibitors of HCV NS5B polymerase

Article abstract of DOI:10.1016/j.bmcl.2018.10.045

Hepatitis C virus (HCV) NS5B polymerase is a prime target for the development of direct-acting antiviral drugs for the treatment of chronic HCV infection. Several novel and potent HCV NS5B non-nucleoside inhibitors with unique tetracyclic bezonfuran-based structures were prepared and evaluated. Similar to clinical developmental compound MK-8876, N-linked (compounds 1 and 2) and C-linked (compounds 3 and 4) tetracyclic structures maintained broad spectrum anti-replicon potency profiles and demonstrated moderate to excellent oral bioavailability and pharmacokinetic parameters across the three preclinical animal species. To better understand the importance of tetracyclic structures related to pan genotypic potency profiles especially against clinically relevant GT1a variants, the teracycles with different ring size were prepared and in vitro evaluations suggested compounds with six number ring have better overall potency profiles.

Full text of DOI:10.1016/j.bmcl.2018.10.045

Accepted Manuscript
Design and evaluation of novel tetracyclic benzofurans as palm site allosteric
inhibitors of HCV NS5B polymerase
Hong Liu, Xing Dai, Shuwen He, Linda Brockunier, Karen Marcantonio, Steven
W. Ludmerer, Fangbiao Li, Kung-I Feng, Ravi P. Nargund, Anandan Palani
PII:
S0960-894X(18)30847-3
https://doi.org/10.1016/j.bmcl.2018.10.045
BMCL 26104
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
19 June 2018
23 October 2018
26 October 2018
Please cite this article as: Liu, H., Dai, X., He, S., Brockunier, L., Marcantonio, K., Ludmerer, S.W., Li, F., Feng,
K-I., Nargund, R.P., Palani, A., Design and evaluation of novel tetracyclic benzofurans as palm site allosteric
inhibitors of HCV NS5B polymerase, Bioorganic & Medicinal Chemistry Letters (2018), doi: https://doi.org/
10.1016/j.bmcl.2018.10.045
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Design and evaluation of novel tetracyclic benzofurans as palm
site allosteric inhibitors of HCV NS5B polymerase
Hong Liu, Xing Dai, Shuwen He, Linda Brockunier, Karen Marcantonio, Steven W. Ludmerer, Fangbiao Li, Kung-I Feng, Ravi P. Nargund and Anandan
Palani
Leave this area blank for abstract info.
The Hepatitis C virus (HCV) NS5B polymerase is a prime target for the development of direct-acting
H
N
O
N
O
NH
NH
antiviral drugs for the treatment of chronic HCV infection. Several novel and potent HCV NS5B non-
nucleoside inhibitors with unique tetracyclic bezonfuran-based structures were prepared and evaluated.
Similar to clinical developmental compound MK-8876, N-linked (compounds 1 and 2) and C-linked
(compounds 3 and 4) tetracyclic structures maintained broad spectrum anti-replicon potency profiles
and demonstrated moderate to excellent oral bioavailability and pharmacokinetic parameters across the
three preclinical animal species. To better understand the importance of tetracyclic structures related to
pan genotypic potency profiles especially against clinically relevant GT1a variants, the teracycles with
different ring size were prepared and in vitro evaluations suggested compounds with six-number ring
have better overall potency profiles.
N
N
N
N
F
F
O
O
N
S
N
S
O
O
F
F
O
2
1
O
O
O
NH
NH
N
N
N
N
F
F
O
O
N
S
N
S
O
O
F
F
O
O
4
3
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Design and evaluation of novel tetracyclic benzofurans as palm site allosteric
inhibitors of HCV NS5B polymerase
Hong Liu *, Xing Dai, Shuwen He, Linda Brockunier, Karen Marcantonio, Steven W. Ludmerer, Fangbiao
Li, Kung-I Feng, Ravi P. Nargund and Anandan Palani
Merck & Co., Inc., 2000 Galloping Hill Road, Kenilworth, New Jersey 07033, United States
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Hepatitis C virus (HCV) NS5B polymerase is a prime target for the development of direct-acting
antiviral drugs for the treatment of chronic HCV infection. Several novel and potent HCV NS5B
non-nucleoside inhibitors with unique tetracyclic bezonfuran-based structures were prepared and
evaluated. Similar to clinical developmental compound MK-8876, N-linked (compounds 1 and
2) and C-linked (compounds 3 and 4) tetracyclic structures maintained broad spectrum anti-
replicon potency profiles and demonstrated moderate to excellent oral bioavailability and
pharmacokinetic parameters across the three preclinical animal species. To better understand the
importance of tetracyclic structures related to pan genotypic potency profiles especially against
clinically relevant GT1a variants, the teracycles with different ring size were prepared and in
vitro evaluations suggested compounds with six number ring have better overall potency
profiles.
Keywords:
Hepatitis C virus
NS5B polymerase
Non-nucleoside inhibitors (NNI)
MK-8876
Tetracyclic structure
2018 Elsevier Ltd. All rights reserved.
Hepatitis C virus (HCV) infection has emerged as one of the most significant disease and there are estimates that more than 200
million people worldwide are infected.1 HCV is an enveloped and positive-strand RNA virus of the Flaviviridae family with a 9.6 kb
genome which encodes structural (Core, E1, E2 and p7) and nonstructural proteins (NS2, NS3 / 4A, NS4B, NS5A and NS5B).2 These
proteins, as well as the viral translation process using the internal ribosomal entry site and a range of host factors, are candidate targets
for therapeutic intervention.
The HCV non-structural protein 5B (NS5B), an RNA-dependent RNA polymerase (RdRp) is the catalytic component of the HCV
replication complex which is responsible for the transcription of HCV genomic RNA.3,4Similar to other polymerases, the crystal
———
 Corresponding author. Tel.: 908-740-2451.
E-mail: hong.liu@merck.com

structures of NS5B revealed a classical right-hand like structure where the active site in the top region of the palm subdomain is fully
,
encircled by an extensive interaction between the fingers and thumb subdomain.5 6Since human cells lack a RNA-dependent RNA
polymerase equivalent, targeting HCV NS5B with small molecule inhibitors is especially significant from the drug discovery point of
view. Molecules that bind to and inhibit the HCV RdRp fall into two main categories: nucleoside and non-nucleoside inhibitors.
Nucleoside Inhibitors (NIs) target the highly conserved active site and Non-nucleoside inhibitors (NNIs) target one of five allosteric
sites on the RdRp
referred as the thumb I, thumb II, palm I, palm II7 and more recently identified palm-β.8
To date Dasabuvir 9(palm I binder) and Beclatasvir10 (thumb I binder combined with a protease inhibitor) are only HCV NNIs to be
approved for the treatment of patients with GT1 infection. In MSD, an extensive investigation of derivatives of first clinical NNI
Nesbuvir11 led to the discovery of MK-8876,12 a non-nucleoside inhibitor with a unique tetracyclic structure binding simultaneously to
both palm I and palm II sites, exhibiting an excellent inhibitory activity against a broad-spectrum of HCV genotypes and especially
maintaining its potency in the low single digit nanomolar range against clinically relevant mutants.
O
O
O
NH
NH
N
N
F
F
HO
O
O
N
S
N
S
O
O
F
O
O
Nesbuvir
MK-8876
Figure 1. Discovery of MK-8876
However, MK-8876 also showed the key liabilities that we wanted to address: relatively low passive permeability (Papp= 7.9x10-6
cm/sec) and poor crystalline aqueous solubility (1.2 µg/ml) which resulted in high dose number13 (Do = 1900) and required an enabled
spray dried formulation for dosing. All these posed potential risk for clinical formulation due to uncertainty
in human dose and PK variability. As part of our continued efforts towards development of better anti-HCV agents suitable for
emerging pan-genotypic therapeutic strategies, the back-up to MK-8876 largely focused on improving PK and dose number by
increasing the permeability and solubility while maintaining the favorable potency profile and limited off-target concerns. An extensive
SAR exploration around central benzonfuran core as well as benzonfuran replacement was carried out as summarized in Figure 2.
N-link and C-link
ring size
change
MK-8876
and heteoaryl
R
O
O
NH
C2
5
N
2
N
F
6
N
S
O
O
O
indole substitions
core
F
C6
sulfonamide modification
bezonfuran alternatives
Figure 2. MK-8876 back-up strategies
Herein we report our initial efforts about modification of the tetracyclic core ring structure. We recognized the importance of the
tetracyclic flat structure to warrant broad-spectrum HCV virus resistant potencies especially against some mutants, but the linkage for
the oxazine ring between indole and pyridine remained unexplored. Since the concept of bioisosterism has been widely applied in lead
optimization program to achieve improved target inhibition as well as a desired physicochemical profile, we sought to optimize the
heteroatom linkage between indole and pyridine rings through bioisoteric replacements. Towards this goal, we prepared analogs
replacing O-linkage with its classical isosteres –NH- (compound 2) and –CH2- (compound 4) as well as corresponding unsaturated
isosters (compounds 1 and 3) for evaluation.
H
N
O
N
O
NH
NH
N
N
N
N
F
F
O
O
N
S
N
S
O
O
F
F
2
O
1
O
O
O
NH
NH
N
N
N
N
F
F
O
O
N
S
O
N
S
O
O
O
F
F
4
3
Figure 3. N-linked and O-linked MK-8876 analog
The synthesis of 1 and 2 was carried out using the approach detailed in Scheme 1. Starting with commercially available 4-
fluoroindole-2-boronic acid pinacol ester 5, selective replacement of the iodine of 6-chloro-2-iodopyridin-3-amine provided
intermediate 6 by Suzuki cross coupling. Under acidic conditions, the intermediate 6 was treated with triethyl orthoformate to generate

the cyclic 7. Another Suzuki cross coupling with the intermediate 8 afforded compound 1 which can easily be reduced at room
temperature by NaBH4, to give compound 2. The key intermediate 8 was prepared as described previously.12
H2N
N
OEt
H
H2N
Cl
H
N
N
N
O
O
N
Cl
EtO OEt
N
I
N
Cl
B
(i)
(ii)
7
6
F
5
F
F
O
N
O
O
NH
NH
B
O
F
N
O
N
N
S
O
O
F
8
O
N
S
O
(iv)
(iii)
O
F
1
H
N
O
NH
N
N
F
O
N
S
O
O
F
2
Scheme 1. Reagents and conditions: (i) PdCl2(dtbpf)), Cs2CO3, 1,4-Dioxane/water, 70 °C, 16 h, 92% (ii) 4.0 M HCl in 1,4-Dioxane,triethyl orthoformate, 1,4-
Dioxane, reflux, 16 h, 85% (iii) PdCl2(dtbpf)), Cs2CO3, 1,4-Dioxane/water, 70 °C, 16 h, 48% (iv) NaBH4, THF/MeOH, rt, 20min, 75%.
To prepare compounds 3 and 4, the optimized synthesis is shown in Scheme 2. Nicotinaldehyde 9 was converted into methoxyvinyl
pyridine 10 by Wittig olefination. Selective Suzuki cross coupling with intermediate 8 replaced the Br to yield 11 and second Suzuki
coupling replaced Cl to afford 12, followed by simultaneous hydrolysis and cyclization under acidic conditions to generate compound 3.
Subsequent hydrogenation using 5% Pd/C as catalyst gave compound 4.
OMe
O
O
B
NH
OMe
O
O
O
F
NH
O
N
SO2
8
Cl
N
F
(ii)
(i)
Cl
N
Br
O
Cl
N
Br
N
11
SHO2
9
10
OMe
O
O
O
H
N
NH
B
H
N
5
N
F
F
(iv)
O
N
(iii)
SO2
12
F
O
O
NH
NH
N
N
N
N
F
(v)
F
O
O
N
S
O
N
S
O
O
O
3
F
4
F
Scheme 2. Reagents and conditions: (i) Ph3P+CH2OMeCl-, t-BuOK, THF, 0 °C to rt, 92% (ii) Pd(dppf)Cl2, K2CO3, 1,4-Dioxane/water, 70 °C, 16 h, 94% (iii)
PdCl2(dtbpf)), Cs2CO3, 1,4-Dioxane/water, 85 °C, 16 h, 95% (iv) HCl in 1,4-dioxane, 70 °C, 2h, 44% (v) Pd/C, H2, THF/MeOH, 50 °C, 95%
The tetracyclic compounds 1, 2, 3, 4 and benchmark MK-8876 were then tested in an initial potency screen against GT1a, GT2a or
GT1b replicon cell lines using a luciferase-based reporter assay (Table 1).14 Similar to MK-8876, replicon activities of all compounds
against genotypes 1a, 2a and 1b were all under 10 nM in the presence of 10% FBS (fetal bovine serum). However the genotype 1b
replicon activities showed 3 to 9-fold protein shift in the presence of 50% NHS (normal human serum) compared to 10-fold activity
shift for MK-8876.
Based on above encouraging results, compounds 1, 2, 3 and 4 were further evaluated against the panel of genotype or mutant
replicon cell lines using a TaqMan based assay (Table 2).15
Table 1. Anti-HCV replicon activity of MK-8876 analog
[a]
EC50 (nM)
GT1a
(10%FBS)
[a] Data are the mean of n ≥ 2 determinations unless otherwise noted. [b] n=1.
Table 2. Anti-HCV TaqMan based assay of MK-8876 analog
Cmpd
GT2a
(10%FBS)
GT1b
(10%FBS)
GT1b
(50%NHS)
MK-8876
2.4 ± 0.52
1.6 ± 0.88
2.7 ± 0.16
27 ± 2.4
1
2
3
4
2.0 ± 0.11
3.3 ± 0.25
3.2 ± 0.11
16 ± 1.8
5.3 [b]
6.4 [b]
5.3 [b]
46 [b]
2.9 ± 1.4
6.3 ± 1.8
5.5 ± 2.5
30 ± 22
3.0 ± 2.2
5.7 ± 2.1
3.3 ± 1.2
13 ± 7.6

EC50 (nM) [a]
[a] Data are the mean of n ≥ 2 determinations.
Cmpd
1A
1A
1A
1B
2A
2B
3A
4A
As shown in Table 2, all compounds showed very good potency
(<10 nM) against all genotypes including well-characterized non-
nucleoside inhibitor mutations (C316Y and S365A) in the GT1a
background except compound 3 (11 nM) against GT1a mutant
C316Y. Across all genotype and mutants, the potency profiles for all
compounds with N-linked and C-linked tetracyclic structures were
very similar to the potency profile of MK-8876 confirming our
initial assumption of tetracyclic conformation yielding a pan-
genotypic potency profile.
C316Y
S365A
MK-8876
1.6
2.2
7.3
3.9
1.8
1.2
2.7
1.1
± 1.2
± 0.7
± 5.2
± 2.1
± 1.1
± 0.7
± 2.2
± 0.3
1
2
3
4
0.68
1.4
3.4
2.4
1.0
0.63
3.1
1.0
± 0.2
± 0.4
± 1.5
± 0.8
± 0.5
± 0.3
± 1.0
± 1.0
0.90
1.8
5.7
2.9
1.0
1.1
7.6
3.1
± 0.6
± 1.4
± 4.6
± 2.3
± 0.4
± 0.9
± 6.7
± 2.9
0.95
2.6
6.7
3.5
1.2
1.2
11
4.8
± 0.5
± 1.1
± 4.0
± 1.7
± 0.8
± 0.6
± 18
± 11
0.86
2.1
6.0
2.7
1.1
1.2
8.4
6.5
± 0.5
± 1.2
± 4.3
± 1.7
± 0.7
± 0.8
± 6.6
± 5.7
In early safety off-target screening (Table 3), all compounds exhibited a promising profile in terms of in vitro CYP inhibition and
PXR transactivation with no significant inhibition of the hERG channel, the cardiac sodium Nav 1.5 and L-type calcium Cav 1.2
channels. Unfortunately, compounds 1, 2 and 3 still suffered from low crystalline aqueous solubility with moderate permeability similar
to MK-8876. However, compound 4 showed modest improvement on permeability and solubility.
Table 3. In Vitro Profiling Data for Compounds 1, 2, 3 and 4
[a] ND = not determined.
FaSSIF
CYP3A4 hERG
Cmpd IC50 patchXpress EC50 IC50
PXR CAV1.2 Nav1.5
MDCK
(Crystalline)
Solubility
(µg/mL)
1.02
IC50
Papp
Pharmacokinetic studies are illustrated in Table 4. Compounds 1,
3 and 4 showed low plasma clearance, good exposure in plasma, and
a long half-life with modest (in rat and monkey) to excellent (in dog)
bioavailability across all three preclinical species and also exhibited
C24h drug concentrations in plasma well above the serum adjusted
EC50 values, indicating a favorable exposure level to target could be
achieved with these unique tetracyclic structures. Compound 2
showed reasonable PK profiles in dog, but exhibited inferior overall
(µM)
>50
>50
>50
>50
IC50 (µM)
>30
(µM) (µM)
>30 22
>30 >30
>30 18
>30 18
(µM)
(10-6cm/s)
1
2
3
4
>30
>30
>30
>30
10.2
8.9
>30
ND[a]
2.9
>30
<1.9
14.2
>30
4.7
PK profiles especially in rat and monkey as compared to others. Subsequently we discovered compound 2 was not stable in the solvent
and can slowly oxidized back to compound 1 during air exposure. Compound 2 was halted for further evaluation.
Table 4. Pharmacokinetic Profile for Compounds 1, 2, 3 and 4
compd
Species
1
2
3
4
Rat
Dog
Monkey Rat
Dog
Monkey Rat
Dog
Monkey Rat
Dog
Monkey
Clearance
8.5
3.2
4.7
16
7.9
7.7
4.2
2.6
5.6
2.3
2.9
11
(mL min-1 kg-1)
± 0.9
± 0.3
± 0.6
± 1.4
± 2.1
± 0.06
± 0.05
± 0.5
± 0.4
± 0.06
± 0.6
± 1.4
IV
8.4
17
6.1
3.8
4.0
3.9
16
22
6.6
12
13
5.9
T1/2 (h)
± 0.4
± 1.5
± 0.2
± 0.5
± 0.2
± 0.5
± 0.2
± 5.6
± 0.4
± 1.0
± 0.2
± 1.5
0.58
0.84
0.66
0.35
0.41
0.13
0.45
1.0
0.37
0.95
1.3
0.32
Cmax (µM)
± 0.3
± 0.1
± 0.05
± 0.1
± 0.002
± 0.02
± 0.1
± 0.02
± 0.06
± 0.4
± 0.1
± 0.1
AUC
(µM.h)
5.2
9.4
3.4
1.4
2.3
1.0
7.9
6.3
3.5
8.8
9.2
1.7
± 1.4
± 1.3
± 0.7
± 0.5
± 0.07
± 0.1
± 0.9
± 0.6
± 0.1
± 1.5
± 1.1
± 0.5
PO
36
64
20
16
30
9.3
26
43
24
18
54
22
F%
± 9.7
± 13
± 0.3
± 5.1
± 1.4
± 2.0
± 2.1
± 4.4
± 0.1
± 3.3
± 7.0
± 7.3
C24hplasma
(µM)
0.11
0.13
0.01
0.0022
0.011
0.004
0.114
0.14
0.031
0.069
0.076
0.0063
± 0.01
± 0.02
± 0.001
± 0.0001
± 0.003
± 0.001
± 0.01
± 0.01
± 0.005
± 0.01
± 0.05
± 0.001
Rat IV was dosed at 1 mg/kg as 3.33 mg/mL solution in DMSO, dog IV was dosed at 0.5 mg/kg as 1.0 mg/ml solution in DMSO, monkey IV was dosed at 1 mg/kg
as 1.0 mg/ml solution in DMSO/PEG400/H2O (20/60/20); Rat p.o. was dosed at 5 mg/kg as 2.5 mg/ml solution in PEG400/TWEEN (90/10), dog p.o. was dosed at
2 mg/kg as 1.0 mg/ml solution in PEG400/TWEEN (90/10), monkey p.o. was dosed at 3 mg/kg as 1.5 mg/ml solution in PEG400/TWEEN (90/10).
All data are the mean of n ≥ 2 determinations; ND = not determined.
Given the excellent potency and initial good plasma exposure following low oral dosing, compound 1 was first chosen to give at
high dose (100mpk) in a precipitation-resistant solution formulation (conventional formulation: 50mg/ml in 90%PEG400 +
10%TWEEN) to rats. As shown in Table 5, plasma exposure dramatically declined at higher dose, most likely due to its low crystalline
solubility and poor permeability. This dissolution-limited absorption was partially circumvented by the use of unconventional
formulation with amorphous solid dispersion based spray drying technology, employing hydroxypropyl methylcellulose phthalate
(HPMCP) matrix (40% compound 1 / 60% HPMCP in a dosing vehicle with 0.5%MC / 0.25%SLS / 5mM HCl). Finally improved oral
absorption was achieved by spray dried formulation (Table 5). Based on this experience, compounds 1 and 4 were selected to run
higher doses rat PK using spray dried formulation to see the level of plasma exposure in order to support one of our safety studies. From
high dose PK (Table 6), both compounds 1 and 4 demonstrated promising and comparative overall profiles to MK-8876, even though

less than dose proportional response was observed in rats with this spray dried formulation. Compound 4 demonstrated 4-fold higher
crystalline solubility and 2-fold higher Papp permeability, but it did not provide anticipated higher exposure by using spray dried
formulation and only provided a 6.5-fold AUC exposure margin at the maximum feasible dose (MFD) of 650 mg/kg in the rats which
was similar to MK-8876’s projected exposure multiples. However, the projected efficacious human dose, which would maintain a
Trough Plasma Concentration of 100-fold over the least sensitive mutant/genotype EC50, was 150mg / QD for compound 4 compared to
300mg / QD for MK-8876 and compound 4 also gave lower predicted dose number (Do = 257) compared to MK-8876 (Do = 1900).
Table 5. Compound 1 rat dose at 100mpk
Table 6. Compound 1 and 4 high dose rat PK with spray dry formulation
formulation
AUC (µM.h)
conventional
0.67
spray dry
136
All
are
data
the
MK-8876
AUC
Compound 1
Compound 4
Dose
(mpk)
Cmax
AUC
Cmax
AUC
Cmax
mean of n ≥ 2 determinations; ND = not determined.
(µM.h)
(µM.h)
(µM.h)
(µM.h)
(µM.h)
(µM.h)
10
16
1.5
6.7
ND
12
32
2.7
37
4.3
To further investigate the role of tetracyclic structures,
analogs with the different ring sizes were prepared as
Figure 4.
several
shown in
100
300
650
83
136
181
ND
8.5
5.6
ND
100
ND
181
8.2
ND
11
ND
226
O
O
NH
NH
N
N
N
N
F
F
O
O
N
S
O
N
S
O
O
O
F
F
13
14
O
H
O
N
NH
N
O
N
N
N
F
F
O
O
N
N
S
O
S
O
O
F
F
O
16
15
Figure 4. Tetracycles with different ring size and pyridinone core
Compound 13 was prepared via a very straightforward route as shown in Scheme 3. A simple Suzuki cross coupling of 8 gave the
intermediate 17, followed by NaBH4 reduction to yield 18. Subsequent second Suzuki cross coupling afforded 19 and final Mitsunobu
reaction generated cyclized compound 13. Compounds 14 and 15 were prepared by using the different dichloropyridine starting
materials but the same synthetic procedure.
O
O
O
O
O
B
NH
NH
Cl
N
Cl
(ii)
O
Cl
N
F
F
O
O
N
S
O
O
(i)
N
S
O
O
O
17
8
H
OH
OH
N
O
O
O
B
NH
NH
H
N
N
Cl
N
F
F
F
O
O
N
S
N
(iii)
O
O
O
S
O
F
19
18
O
NH
N
(iv)
N
F
O
N
S
O
O
13
F
Scheme 3. Reagents and conditions: (i) Pd(dppf)Cl2, K2CO3, 1,4-Dioxane/water, 80 °C, 16 h (ii) NaBH4, MeOH/THF, RT, 100% (iii) PdCl2(dtbpf)), Cs2CO3, 1,4-
Dioxane/water, 80 °C, 16 h, 78% (iv) DIAD, Ph3P, THF, RT, 16 h, 85%
Compound 16 was prepared as described in Scheme 4. Starting with Boc-protected 4-fluoroindole-2-boronic acid pinacol ester 20, a
selective Suzuki cross coupling afforded 21. TFA treatment removed the Boc at RT and NaNO2 converted aminopyridine to pyridinone
22 in a 5% H2SO4 aqueous solution. Heating with dibromoethane finished the cyclization and yield 23 which coupled with intermediate
8 to give 16.

NH2
O
Cl
N
NH2
N
Boc
HN
Boc
N
N
H
O
O
Cl
N
Cl
Cl
B
(ii)
(i)
22
21
F
20
F
F
O
O
O
B
NH
O
F
N
O
N
S
Br
O
N
Br
8
Cl
O
(iii)
(iv)
23
F
O
O
N
NH
N
F
O
N
S
O
O
F
16
Scheme 4. Reagents and conditions: (i) Pd(dppf)Cl2, K2CO3, 1,4-Dioxane/water, 70 °C, 3 h, 70% (ii) 1. TFA / DCM, RT, 83%; 2. NaNO2 / H2SO4, 0 °C, 12 h,
41% (iii) BrCH2CH2Br, Cs2CO3 / CH3CN, 90 °C, 8 h, 45% (iv) Xphos / Pd2(dba)3, K3PO4, 1,4-Dioxane / water, 90 °C, 6 h, 33%
All compounds underwent a detailed in vitro potency evaluation as illustrated in Table 7 with compound 4 listed as the benchmark.
Generally, potency for all these ring size changes were maintained across all genotype except inhibitory activities against GT1a
background mutants S365 and 316Y which were more sensitive to subtle changes: a smaller 5-member ring (13) decreased potency
against S365A by 5-fold; the larger 7-number ring (14) lost potency against C316Y by 3-fold and against S365A only by 2-fold; 8-
member ring (15) led to more than 6-fold potency loss against S365A. Compound 16, with the same ring size as compound 4, exhibited
a more tolerant potency profile, including two GT1a mutants.
Table 7. Anti-HCV TaqMan based assay of MK-8876 analog with different ring size
EC50 (nM) [a]
[a] Data are the mean of n ≥ 2 determinations.
Compd
1A
1A
1A
1B
2A
2B
3A
4A
Compounds 13 and 16 were selected for in vivo pharmacokinetic
studies in rat (Table 8). Compound 13 retained reasonable plasma
clearance and a half-life of 9.6 h. A 5 mg / kg oral dose of compound
13 gave the Cmax of 0.12 µM and tmax of 3 h with only 4 %
bioavailability as compared to 18% bioavailability of compound 4.
Compound 16 demonstrated more than 7-fold higher plasma clearance
than compound 4 which resulted in poor oral exposure leading to very
low oral bioavailability.
C316Y S365A
4
0.86
2.1
6.0
2.7
1.1
1.2
8.4
9.5
26
6.5
34
16
42
6.4
13
14
15
16
2.2
5.1
1.9
5.2
1.3
8.5
6.8
26
3.9
6.5
3.8
2.2
3.0
2.7
1.5
3.5
1.5
1.1
1.2
2.0
1.9
13
0.61
11
0.62
9.8
Table 8. Pharmacokinetic Properties of Compounds 13 and 16 vs Compound 4 in
Rat (1 mg/kg IV and 5 mg/kg PO)
All data are the mean of n ≥ 2 determinations; ND = not determined.
Compd
13
16
4
Cl (mL min-1 kg-1)
5.2
17
2.3
In conclusion, several novel and potent HCV NS5B non-nucleoside
inhibitors with N-linked and C-linked tetracyclic bezonfuran-based
structures were prepared and evaluated. Similar to MK-8876, all
tetracycles with the same ring size as MK-8876 maintained broad spectrum
antiviral potency profiles and demonstrated reasonable pharmacokinetic
parameters in all three preclinical species. Compound 4 with an improved
solubility and permeability gave a better dose number and lower projected
human dose, but projected exposure multiples maintain the same level as
MK-8876. The tetracycles with the different ring sizes generally retained
pan-genotypic potency profiles, but showed inferior potency against GT1a
mutants and overall inferior PK profiles.
IV
T1/2 (h)
9.6
0.12
3.0
1.1
4
4.3
12
Cmax (µM)
Tmax (h)
0.19
0.95
4
1.0
PO
AUC (µM.h)
F%
0.078
1.0
8.8
18
C24h plasma (µM) 0.01
ND
0.069
Graphical Abstract

To create your abstract, type over the instructions in the template box below.
Design and evaluation of novel tetracyclic benzofurans as palm
site allosteric inhibitors of HCV NS5B polymerase
Hong Liu, Xing Dai, Shuwen He, Linda Brockunier, Karen Marcantonio, Steven W. Ludmerer, Fangbiao Li, Kung-I Feng, Ravi P. Nargund and Anandan
Palani
Leave this area blank for abstract info.
The Hepatitis C virus (HCV) NS5B polymerase is a prime target for the development of direct-acting
H
N
O
N
O
NH
NH
antiviral drugs for the treatment of chronic HCV infection. Several novel and potent HCV NS5B non-
nucleoside inhibitors with unique tetracyclic bezonfuran-based structures were prepared and evaluated.
Similar to clinical developmental compound MK-8876, N-linked (compounds 1 and 2) and C-linked
(compounds 3 and 4) tetracyclic structures maintained broad spectrum anti-repilcon potency profiles
and demonstrated moderate to excellent oral bioavailability and pharmacokinetic parameters across the
three preclinical animal species. To better understand the importance of tetracyclic structures related to
pan genotypic potency profiles especially against clinically relevant GT1a variants, the teracycles with
different ring size were prepared and in vitro evaluations suggested compounds with six-number ring
have better overall potency profiles.
N
N
N
N
F
F
O
O
N
S
N
S
O
O
F
F
O
2
1
O
O
O
NH
NH
N
N
N
N
F
F
O
O
N
S
N
S
O
O
F
F
O
O
4
3
Fonts or abstract dimensions should not be changed or altered.

N-linked and C-linked tetracyclic benzofuran-based compounds were made and evaluated as HCV 5B non-
nucleoside inhibitors



Tetracyclic benzofuran-based structures maintained broad spectrum anti-replicon potency profiles
Compounds demonstrated moderate to excellent oral bioavailability and pharmacokinetic parameters
C-linked tetracycle with 6-number ring displayed improved solubility and permeability compared to a
clinical compound
References and notes
1 Rustogi, V. K. J. The epidemiology of hepatitis C infection in the United States Journal of gastroenterology. 2007, 42, 513.
2 Lindenbach B.D.; Rice C.M. Unravelling hepatitis C virus replication from genome to function. Nature. 200, 436, 933–938.
3 Scheel, T. K. H.; Rice, C. M. Understanding the hepatitis C virus life cycles paves the way for highly effetive therapies. Nat.
Med. 2013, 19, 837-849.
4 Behrens, S. E.; Tomei, L.; Francesco, R. De. Identification and properties of the RNA-dependent RNA polymerase of hepatitis
C virus. EMBO J. 1996; 15: 12-22.
5 Bressanelli, S.; Tomei, L.; Roussel, A.; Incitti, I.; Vitale, R.L.; Mathieu, M. De Francesco R., Rey F.A. Crystal structure of the
RNA-dependent RNA polymerase of hepatitis C virus. Proc. Nat. Acad. Sci. 1999, 96,13034–13039.
6 Lesburg, C.A.; Cable, M.B.; Ferrari, E.; Hong, Z.; Mannarino, A.F.; Weber, P.C. Crystal structure of the RNA-dependent RNA
polymerase from hepatitis C virus reveals a fully encircled active site. Nat. Struct. Biol. 1999, 6, 937–943.
7 Gerber, L.; Welzel, T. M.; Zeuzem, S. Liver Int. 2013, 33, 85-92.
8
Shih, I.H.; Vliegen, I.; Peng, B.; Yang, H.; Hebner, C.; Paeshuyse, J.; Purstinger, G.; Fenaux, M.; Tian, Y.; Mabery, E. et al.
Mechanistic characterization of GS-9190 (Tegobuvir), a novel nonnucleoside inhibitor of hepatitis C virus NS5B polymerase.
Antimicrob. Agents Chemother. 2011, 55, 4196–4203.
9 Ferenci, P.; Bernstein, D.; Lalezari, J.; Cohen, D.; Luo, Y.; Cooper, C.; Tam, E.; Marinho, R. T.; Naoky, T.; Nyberg, A.; Box,
T. D.; Younes, Z.; Enayati, P.; Green, S.; Baruch, Y.; Bhandari, B. R.; Caruntu, F. A.; Sepe, T.; Chulanov, V.; Janczewska, E.;
Rizzardini, G.; Gervain, J.; Planas, R.; Moreno, C.; Hassanein, T.; Xie, W.; King, M.; Podsadecki, T.; Reddy, K. R. N. Engl. J.
Med. 2014, 370, 1983 –1992.
10 Everson, G. T.; Sims, K. D.; Thuluvath, P. J.; Lawitiz, E.; Hassanein, T.; Rodriguez-Torres, M.; Desta, T.; Hawkins, T.; Levin,
J. M.; Hinestrosa, F.; Rustgi, V.; Schwartz, H.; Younossi, Z.; Webster, L.; Gitlin, N.; Eley, T.; Huang, S. P.; McPhee, F.;
Grasela, D. M.; Gardiner, P. F. Liver. Int. 2016, 36, 189– 197.

11 Kneteman, N. M.; Howe, A. Y. M.; Gao, T.; Lewis, J.; Pevear, D.; Lund, G.; Douglas, D.; Mercer, D. F.; Tyrrell, D. L. J.;
Immermann, F.; Chaudhary, I.; Speth, J.; Villano, S. A.; O’Connell, J.; Collett, M. HCV796: A selective nonstructural protein 5B
polymerase inhibitor with potent anti-hepatitis C virus activity in vitro, in mice with chimeric human livers, and in humans
infected with hepatitis C virus. Hepatology 2009, 49, 745−752
12
McComas, C. C.; Palani, A.; Chang, W.; Holloway, M. K.; Lesburg, C. A.; Li, P.; Liverton, N.; Meinke, P. T.; Olsen, D. B.;
Peng, X.; Soll, R. M.; Ummat, A.; Wu, J.; Zorn, N.; Ludmerer, S. W. Development of a novel structural class of broadly acting
HCV non-nucleoside inhibitors leading to the discovery of MK-8876. ChemMedChem 2017, 12, 1436 – 1448
13 (a) Amidon, G. L.; Lennernas H.; Shah V. P.; Crison J. R. A theoretical basis for a biopharmaceutic drug classification: the
correlation of in vitro drug product dissolution and in vivo bioavailability. Pharm Res. 1995, 12:413; (b) Wuelfing, W. P.;
Daublain, P.; Kesisoglou, F.; Templeton, A.; McGregor, C. Preclinical dose number and its application in understanding drug
absorption risk and formulation design for preclinical species. Mol. Pharmaceutics 2015, 12, 1031−1039
14 Vrolijk, J. M.; Kaul, A.; Hansen, B. E.; Lohmann, V.; Haagmans, B. L.; Schalm, S. W.; Bartenschlager, R. J. A replicon-based
bioassay for the measurement of interferons in patients with chronic hepatitis C. Virol. Methods 2003, 110, 201 –209.
15
Liverton, N. J.; Carroll, S. S.; DiMuzio, J.; Fandozzi, C.; Hraham, D. J.; Hazuda, D.; Holloway, M. K.; Ludmerer, S. W.;
McCauley, J. A.; McIntyre, C. J.; Olsen, D. B.; Rudd, M. T.; Stahlhut, M.; Vacca, J. P. MK-7009, a potent and selective inhibitor
of hepatitis C virus NS3/4A protease. Antimicrob. Agents Chemother. 2010, 54, 305–311.