High-Throughput Screening, Discovery, and Optimization to Develop a Benzofuran Class of Hepatitis C Virus Inhibitors

Article abstract of DOI:10.1021/acscombsci.5b00101

Using a high-throughput, cell-based HCV luciferase reporter assay to screen a diverse small-molecule compound collection (300 000 compounds), we identified a benzofuran compound class of HCV inhibitors. The optimization of the benzofuran scaffold led to t

Full text of DOI:10.1021/acscombsci.5b00101

Subscriber access provided by - Access paid by the | UCSB Libraries
Article
High-throughput Screening, Discovery and Optimization to
Develop a Benzofuran Class of Hepatitis C Virus Inhibitors
Shanshan He, Prashi Jain, Billy Lin, Marc Ferrer, Zongyi Hu , Noel Southall, Xin Hu, Wei Zheng,
Benjamin Neuenswander, Chul-Hee Cho, Yu Chen, Shilpa Arvind Worlikar, Jeffrey Aubé,
Richard C. Larock, Frank J. Schoenen, Juan J Marugan, T. Jake Liang, and Kevin J. Frankowski
ACS Comb. Sci., Just Accepted Manuscript • DOI: 10.1021/acscombsci.5b00101 • Publication Date (Web): 02 Sep 2015
Downloaded from http://pubs.acs.org on September 17, 2015
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
ACS Combinatorial Science is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 27
ACS Combinatorial Science
1
2
3
4
5
6
7
8
9
Highꢀthroughput Screening, Discovery and
Optimization to Develop a Benzofuran
Class of Hepatitis C Virus Inhibitors
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
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
58
59
60
a, †
b, †
a
c
a
c
c
Shanshan He, Prashi Jain, Billy Lin, Marc Ferrer, Zongyi Hu, Noel Southall, Xin Hu,
c
d
d
d
d
Wei Zheng, Benjamin Neuenswander, Chul-Hee Cho, Yu Chen, Shilpa A. Worlikar, Jeffrey
b, d
d
b
c
a *
Aubé, Richard C. Larock, Frank J. Schoenen, Juan J. Marugan, T. Jake Liang, and Kevin
b*
J. Frankowski
a
Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National
b
Institutes of Health, 10 Center Drive, Bethesda, Maryland 20892; University of Kansas Specialized
c
Chemistry Center, University of Kansas, Lawrence, KS 66047; NIH Chemical Genomics Center,
Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National
d
Institutes of Health, 9800 Medical Center Drive, Rockville, MD, 20850; University of Kansas Chemical
Methodologies and Library Development Center, University of Kansas, Lawrence, KS 66047. † These
authors contributed equally to this work.
1
ACS Paragon Plus Environment

ACS Combinatorial Science
Page 2 of 27
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ABSTRACT
HCV-Luc activity
Cytotoxicity
1
00
1
00
80
60
40
20
0
HCV-Luc activity
Cytotoxicity
X = N
8
6
4
0
0
0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
20
0
-3
-2
-1
0
1
2
-3
-2
-1
0
1
2
X = CH
Log [Concentration] (µM)
Log [Concentration] (µM)
Using a highꢀthroughput, cellꢀbased HCV luciferase reporter assay to screen a diverse smallꢀ
molecule compound collection (ca. 300,000 compounds), we identified a benzofuran compound
class of HCV inhibitors. The optimization of the benzofuran scaffold led to the identification of
several exemplars with potent inhibition (EC < 100 nM) of HCV, low cytotoxicity (CC > 25
5
0
50
ꢁ
M) and excellent selectivity (Selective Index = CC /EC , > 371ꢀfold). The structureꢀactivity
50 50
studies culminated in the design and synthesis of a 45 compound library to comprehensively
explore the anti HCV activity. The identification, design, synthesis and biological
characterization for this benzofuran series is discussed.
2
ACS Paragon Plus Environment

Page 3 of 27
ACS Combinatorial Science
1
2
3
4
5
6
7
8
9
INTRODUCTION
Hepatitis C virus (HCV) is the causative agent of hepatitis C and represents the major underlying
cause of hepatocellular carcinoma and chronic liver disease. Infection with HCV leads to chronic
hepatitis in 85% of cases. HCV remains a significant public health burden and is a leading cause
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
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
58
59
60
1
of liver transplantations and is responsible for at least 10,000 deaths annually in the US.
Globally, an estimated 180 million people are chronically infected by HCV, with an additional 3ꢀ
2
4
million people being newly infected annually. There is currently no vaccine to prevent HCV
3
infection. Effective broadꢀspectrum therapy against different HCV genotypes is currently
4
unavailable. The previous standard of care, peginterferon (pegIFN) and ribavirin (RBV) for 24 –
4
8 weeks depending on HCV genotype, showed low sustained virological response (SVR) rates
for patients infected with genotype 1, the most prevalent genotype in the US. This treatment is
associated with severe adverse effects including depression, myalgia and hemolytic anemia.
Recent research efforts have been directed towards the development of interferonꢀsparing
regimens that contain one or more directꢀacting antivirals (DAAs) that target one or more
discrete biological targets for HCV (Figure 1). Prior clinical trials with DAAs as monotherapy
were associated with rapid development of resistance, thereby necessitating their use as
5
combinations. Two combinations of firstꢀgeneration DAA, pegIFN and ribavirin approved in
4
2011 have launched the era of DAA combination therapy for HCV. Current efforts towards
interferonꢀfree regimens involve combination therapy of two or three DAAs. Several such
regimens that are associated with high treatment response (~90% sustained virologic response)
6
have been approved over the last two years. These regimens, while very effective and more
tolerable, are associated with high costs. In spite of therapeutic advances for orally active DAAs,
the high cost, potential for drugꢀresistance emergence and drugꢀdrug interactions suggest that a
significant unmet need for additional therapies that act on novel HCV targets remains.
3
ACS Paragon Plus Environment

ACS Combinatorial Science
Page 4 of 27
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 1. Structures of representative HCV directꢀacting antivirals.
RESULTS AND DISCUSSION
7
Hit compound identification and initial SAR screening. Liang and coworkers have developed
a quantitative highꢀthroughput screening (qHTS) assay platform representing the complete HCV
replication cycle. The phenotypic assay incorporated two components, a recombinant infectious
virus (HCVccꢀCre virus) and a stable host cell line (Huh7.5.1 LoxPꢀGluc). This assay platform
4
ACS Paragon Plus Environment

Page 5 of 27
ACS Combinatorial Science
1
2
3
4
5
6
7
8
9
can identify inhibitors of all stages of the HCV replication cycle as well as compounds that
7
interrupt host functions important for HCV infection and propagation. The NIH Molecular
Libraries Small Molecule Repository (MLSMR) library of 339,561 compounds was screened
with this assay platform, affording 11,624 preliminary hit compounds (PubChem AID 651820).
Elimination of known reactive groups and focusing on hits with a minimum of 50% inhibition
reduced the number to 655 promising hits. Concentrationꢀresponse confirmatory assays
(PubChem AID 720575) and cytotoxicity countersceening (PubChem AID 720576) further
narrowed the field to a set of 206 validated compound hits. The general structure 1 was shared by
a structural cluster of several hit compounds, including two benzofuranꢀbased compounds (1a
and 1b) displaying lowꢀmicromolar inhibitory activities in the HCVꢀLuc assay (recombinant
HCV carrying the Renilla luciferase reporter, Figure 2). The hit compounds were originally
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
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
58
59
60
8
synthesized and submitted to the MLSMR by Larock and coworkers as part of library
development efforts within the KU Chemical Methodologies and Library Development Center
(KU CMLD). Common functional groups between the two compounds include an acetyleneꢀ
linked aliphatic alcohol system at the C3ꢀposition, aryl substitution at the C2ꢀposition, and an
aryl/heteroaryl system at C5ꢀposition of benzofuran scaffold.
Figure 2. Confirmed qHTS hit compounds from the benzofuran series.
We decided to further explore this chemotype because substituted benzofurans, such as nesbuvir
(HCVꢀ796, Figure 1), display potent HCV NS5B polymerase inhibitory activity and have
9
advanced to clinical trials. Thus, the structureꢀactivity relationship (SAR) around the benzofuran
scaffold was explored through a twoꢀpronged approach. The biological activity of existing
benzofuran exemplars from the KU CMLD Center compound collection was surveyed first,
providing a rapid survey of diverse chemical space. Next, a focused compound library was
5
ACS Paragon Plus Environment

ACS Combinatorial Science
Page 6 of 27
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
constructed based on the results from the first step to systematically explore the SAR around the
most promising chemical space. Figure 3 summarizes the analogs explored via this strategy; all
three pendant groups on the benzofuran were investigated as well as nitrogenꢀatom incorporation
into the benzofuran centroid.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3: Summary of explored modifications around 1a (PubChem compound CID 24747883)
In the initial phase, 35 2,3,5ꢀtrisubstituted benzofuran compounds were selected from the KU
CMLD Center compound collection and screened in the HCVꢀLuc assay in parallel with the
1
0
ATPlite cytotoxicity assay. The results are summarized in Tables 1ꢀ3. The activity for our
initial hit molecules 1a and 1b were independently validated (2{1} and 2{6}, respectively) with
material directly obtained from the KU CMLD compound repository, showing comparable
potency and toxicity results to the data from the qHTS (lowꢀthroughput assay data shown
throughout the manuscript for consistency, qHTS data available online PubChem AIDs 651820
and 720575). Five analogs (2{2} to 2{5} and 3{4}) displayed improved potency (EC50 ≤ 980 nM)
compared to 2{1}. Gratifyingly, the five analogs that displayed improved potency also
maintained a favorable selectivity index, or in some cases possessed reduced cytotoxicity (CC )
5
0
against Huh7.5.1 cells (Table 1). All five analogs contain a propargyl alcohol moiety at the R3
position, suggesting that this group may be important for activity.
Table 1. HCV inhibition and cytotoxicity of 2ꢀ(4ꢀmethoxyphenyl)ꢀcontaining benzofuran
analogs
Structure
HCVꢀLuc
EC (ꢁM)
ATPlite
Selective
Index (SI)
Cmpd
CC (ꢁM)
50
50
2
3
R
R
6
ACS Paragon Plus Environment

Page 7 of 27
ACS Combinatorial Science
1
2
3
4
5
6
7
8
9
0
.98
2{1}
70.9
26.1
72
(
n=2)
2
2
{2}
{3}
0.074
373
0.17
> 100
> 100
> 100
> 588
> 192
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
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
58
59
60
(
n=3)
2
2
{4}
{5}
0.52
0.084
> 1190
(
n=3)
1
0.9
2
{6}
> 100
> 9
(
n=3)
2{7}
>31.6
6.99
> 100
> 100
N.A.
> 14
2
2
{8}
{9}
21.50
> 31.6
> 31.6
> 100
> 100
> 100
> 5
2
2
{10}
{11}
N.A.
N.A.
2
{12}
5.94
> 100
> 17
2{13}
> 31.6
5.70
> 100
31.90
N.A.
6
2
{14}
2{15}
> 31.6
> 100
N.A.
2
2
{16}
{17}
3.04
4.39
31.70
39.20
10
9
7
ACS Paragon Plus Environment

ACS Combinatorial Science
Page 8 of 27
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2{18}
> 31.6
> 100
N.A.
Compounds in Table 1 contain a 4ꢀmethoxyphenyl group at the C2ꢀposition of the benzofuran
scaffold, while the substituents vary at its C3ꢀ and C5ꢀpositions. Compound 2{2} which has a
propargyl alcohol at the C3 position and morpholino substitution at C5 showed improved
potency but relatively higher cytotoxicity. In 2{3} – 2{5}, introduction of a cyclohexyl group at
the 2’ꢀposition of the propargyl alcohol (resulting in 1ꢀethynylcyclohexanol) provided improved
potency and greatly improved selectivity. Replacing the 4ꢀmethoxyphenyl group at the C5ꢀ
position with the 2ꢀmethoxyꢀ3ꢀpyridyl ring (2{4}) reduced potency and cytotoxicity, resulting in
a modest selectivity improvement. The best compound in Table 1, 2{5}, showed an EC of 84
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
nM and >1,190ꢀfold selectivity. Replacement of the alcohol with electronꢀrich aryl groups
adjacent to the alkyne linker at the R position (2{6} – 2{8}) led to reduced activity against HCV
3
though preserved the low cytotoxicity (> 100 µM), indicating that the alcohol group is important
for HCV inhibition. Even more dramatically, the lack of C3ꢀalkynyl moiety (2{9} – 2{18}) led to
either a significant or complete loss of HCV inhibitory activity.
Table 2. HCV inhibition and cytotoxicity of 2ꢀthiopheneꢀcontaining benzofuran analogs
Structure
HCVꢀLuc
EC (ꢁM)
ATPlite
Selective
Cmpd
CC (ꢁM)
Index (SI)
50
50
2
3
R
R
3
3
{1}
{2}
NH2
>31.6
>31.6
>100
34.20
N.A.
< 1
3
{3}
2.44
43.60
18
0
.177
3
3
{4}
{5}
>100
>100
> 568
> 9
(
n=3)
11.7
8
ACS Paragon Plus Environment

Page 9 of 27
ACS Combinatorial Science
1
2
3
4
5
6
7
8
9
3{6}
8.56
1.52
>100
26.40
> 12
17
3
{7}
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
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
58
59
60
Compounds in Table 2 contain a 3’ꢀthiophene group at the C2ꢀposition of the benzofuran
scaffold and vary at the C3ꢀ and C5ꢀpositions. Compounds with a C5ꢀmorpholine group (3{1} –
{3} and 3{5} – 3{7}) show reduction in HCV inhibitory activity compared to the analog 2{1}.
3
C5ꢀmorpholineꢀcontaining compounds 3{2}, 3{3} and 3{7} show increased cytotoxicity compared
to 2{1}. The best compound in this series 3{4}, with 3ꢀ(1ꢀethynylcyclohexanol) and 5ꢀ(3’,4’,5’ꢀ
trimethoxyphenyl) substitution on the benzofuran scaffold showed EC of 177 nM and >568ꢀ
5
0
fold selectivity against Huh7.5.1 cells. Direct comparison between 3{3} and 3{4} where the only
2
aryl group at R is replaced with a morpholine moiety suggest that this replacement is not
favorable for antiꢀHCV potency.
Table 3. HCV inhibition and cytotoxicity of 2ꢀarylꢀcontaining benzofuran analogs
Structure
Selective
Index
SI)
HCVꢀLuc
ATPlite
Cmpd
EC (ꢁM)
CC (ꢁM)
50
50
(
1
2
3
R
R
R
4
{1}
>31.6
12.00
3.59
>100
>100
38.10
>100
>100
N.A.
> 8
4{2}
4
4
{3}
{4}
11
2.34
> 43
> 54
4{5}
1.84
9
ACS Paragon Plus Environment

ACS Combinatorial Science
Page 10 of 27
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Analogs in Table 3 explored substituent changes at the C2ꢀ, C3ꢀ and C5ꢀpositions
simultaneously, often providing single forays into more drastic structural changes compared to
the qHTS hit compounds. Compound 4{1} lacks the C5ꢀsubstituent on the benzofuran scaffold
and was found to be inactive in the HCVꢀLuc assay. Compounds 4{2} – 4{5} which contain a C5ꢀ
moropholine moiety and vary at the C2ꢀ and C3ꢀpositions of the benzofuran scaffold were found
to lose activity against HCV. Thus, the screening results for compounds in Tables 1ꢀ3 indicate
that HCV inhibitory activity is sensitive to substitutions at all three positions on the benzofuran
scaffold. An aryl or heteroaryl group at the C5ꢀposition of the benzofuran scaffold is important
for the inhibitory activity in the HCVꢀLuc assay. Substitution at the C2ꢀposition of the
benzofuran is wellꢀtolerated with the introduction of various substituted aryl groups imparting
nanomolar inhibitory potency. However, replacement of the propargyl alcohol moiety at the C3ꢀ
position of the benzofuran with an aryl or heteroaryl group led to a complete loss of potency.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Library design, synthesis and evaluation. Based on these encouraging results, we designed a
library of 2,3,5ꢀtrisubstituted benzofuran compounds to systematically explore the SAR, utilizing
8
the previously established synthetic sequence to readily access novel analogs of this chemotype
(Figure 4). All target analogs were synthesized via the synthetic route outlined in Scheme 1. The
synthesis of the 4ꢀbromoꢀ2ꢀiodoꢀ1ꢀmethoxybenzene intermediate 6 was achieved by
regioselective iodination of commercially available 4ꢀbromoanisole 5 using Selectfluor and
1
1
iodine at room temperature. Sonogashira coupling of 6 with substituted aryl alkynes afforded 7,
which was further diversified by SuzukiꢀMiyaura metalꢀcatalyzed crossꢀcoupling using various
aryl boronic acid components to give 8. Iodocyclization of 8 using iodine monochloride provided
the intermediate 9. The final diversification step was achieved in parallel using Sonogashira
TM
crossꢀcoupling with various alkyne fragments on the Bohdan MiniBlock platform. The target
compounds 10 were purified by reverse phase, preparative, massꢀdirected HPLC fractionation
(MDF) and purity was determined using analogous analytical HPLC conditions.
Scheme 1: General synthetic scheme
1
0
ACS Paragon Plus Environment

Page 11 of 27
ACS Combinatorial Science
1
2
3
4
5
6
7
8
9
OMe
OMe
OMe
OMe
I
R2
OH
a
R1
b
c
B
+
Br
R2
OH
Br
Br
R1
R1
5
6
7
8
O
R1
O
I
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
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
58
59
60
R1
R2
d
e
R3
R2
R3
9
10
a) I , Selectfluor, rt, 4 h (77% yield); b) PdCl (PPh ) (3 mol %), CuI (2 mol%), Et N, 23 °C 18 h (75ꢀ93% isolated
2
2
3 2
3
yield); c) "various SuzukiꢀMiayura cross coupling conditions" (41ꢀ93% isolated yield); d) ICl, DCM, rt, 4h (30ꢀ89%
yield); e) PdCl (PPh ) (3 mol %), CuI (2 mol%), Et NH, DMF, 80 °C 15 h (library synthesis and MDF
2
3 2
2
8
purification)
Figure 4. Library design and building blocks.
1
2ꢀ14
Thiophene groups have been reported to present metabolic challenges.
Hence, we decided to
1
explore more stable replacements for the thiophene group at the C2ꢀposition of analog 3{4} (R
group in Figure 4), such as the 4ꢀmethoxyphenyl building block 11a (Figure 4), which was
1
1
ACS Paragon Plus Environment

ACS Combinatorial Science
Page 12 of 27
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
present in the C2ꢀposition of several promising compounds (2{1} to 2{5}, Table 1) and the more
sterically similar imidazole 11b (Figure 4). We also attempted to improve the aqueous solubility
through replacement of the benzofuran scaffold with a furo[3,2ꢀb]pyridine scaffold using the
building block 11c (Figure 4). During physiochemical property assessment for KU CMLD
Center compounds in Tables 1ꢀ3, it was observed that the potent compound 2{5} showed poor (<
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
mg/mL) aqueous solubility (Table 6). Thus, it was also of interest to improve solubility as a
secondary design element for compounds in the library. Introduction of the polar C2ꢀimidazole
moiety and the furo[3,2ꢀb]pyridine moieties into the scaffold were anticipated to improve
solubility for these analogs.
2
Substituents at the R ꢀposition were incorporated from the most active compounds from Tables
3
1ꢀ3. Substitutions at the R ꢀposition retain the propargyl alcohols (13a and 13b in Figure 4),
which were found to be important in the preliminary SAR studies. We focused instead on
exploring the effects of increased steric bulk (13c in Figure 4) or introduction of additional cyclic
oxygen atoms (13d and 13e in Figure 4) on potency and cellular toxicity.
Results from the biological evaluation of the library compounds 10{1} – 10{45} are shown in
1
2
Table 4. Compounds 10{1} – 10{5} (R and R = 4ꢀ methoxyphenyl) were found to be less potent
compared to the leading compounds from previous SAR exploration (i.e. 2{2}, 2{3} and 2{5}).
The most active compound among 10{1} – 10{5} was the propargylic alcohol 10{2}, possessing
an EC of 210 nM and good selectivity. Steric bulk at the carbon atom adjacent to the alcohol
5
0
moiety (10{3} and 10{1}), or introducing the oxygenꢀcontaining heterocycles oxetane (10{4}) or
1
tetrahydropyran (10{5}) were all detrimental to potency. Among compounds 10{6} – 10{10} (R
2
=
4ꢀ methoxyphenyl; R = dioxolophenyl), the tetrahydropyranꢀcontaining analog (10{10}) was
the most potent (130 nM), with compounds 10{7} and 10{9} only slightly less potent. Other
3
modifications at the R position were less conducive to HCV inhibition. Among compounds
1
2
1
0{11} – 10{15} (R = 4ꢀ methoxyphenyl, and R = 3’,4’,5’ꢀtrimethoxyphenyl), compound 10{13}
3
(
R = 2,2ꢀdimethylpropargyl alcohol) was the most potent compound (EC = 98 nM) and
5
0
possessed excellent selectivity (SI = 879). In this subseries, HCV inhibition was reduced by 6ꢀ
3
fold when the cyclohexyl ring on the propargyl alcohol at R (10{11}) was replaced with an
oxetane ring (10{14}).
Table 4. HCV inhibition and cytotoxicity of benzofuran library analogs
1
2
ACS Paragon Plus Environment

Page 13 of 27
ACS Combinatorial Science
1
2
3
4
5
6
7
8
9
Cmpd
HCVꢀLuc EC (ꢁM)
ATPlite CC (ꢁM)
50
50
3
R
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
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
58
59
60
1
2
R
R
A
OH
Me Me
1
a
0{1}
10{2}
0.21 56.6
10{7}
0.17 26.6
10{12}
10{3}
10{4}
10{5}
C
C
0
0
.34 >100
0.52 >100 0.44 >100 0.95 >100
10{8} 10{9} 10{10}
0.50 65.4 0.14 60.1 0.13 41.1
10{13} 10{14} 10{15}
10{6}
a
.47 >100
1
0{11}
C
N
N
N
C
C
a
a
0
.16 >100
0.72
>100 0.098 86.1 1.08 >100 0.13 >100
1
0{16}
10{17}
0.45
10{18}
10{19}
10{20}
0
.41 >100
10{21}
15.4
0.81 44.6 0.66 32.7 2.23 >100
10{22}
10{23}
10{24}
10{25}
0
0
.18 >100
0.22
4.63
0.27 45.3 0.66 >100 0.85 68.5
1
0{26}
10{27}
10{28}
10{29}
10{30}
.35 83.5
0.83
>100
0.24 31.6 0.53 >100 0.82 >100
1
0{31}
4.39 >100
0{36}
.91 76
10{32}
10{33}
10{34}
10{35}
9.4 >100
10{40}
37.3 10.7 33.7 13.7 48.9
0.78
>100
1.99
46.8 N.A. N.A.
1
10{37}
10{38}
10{39}
2
1.12
41.8
1.83
1
0{41}
10{42}
10{43}
0.32 35.7
10{44}
10{45}
C
4
.12 25.9
5.31
>100
9.0 37.8 5.28 21.7
a
Sample was tested independently (n ≥ 2) and value is an average.
Compounds 10{16} – 10{30} built upon the pyrido[3,2ꢀb]furan moiety generally showed HCV
1
inhibitory potency better than the imidazole analogs 10{31} – 10{45} (R = N1ꢀmethyl
imidazole), though they were slightly less active when compared to the corresponding
benzofuran compounds 10{1} – 10{15}. The pyrido[3,2ꢀb]furan analogs displayed similar
cytotoxicity level comparing with the corresponding benzofuran compounds 10{1} – 10{15}. The
1
3
ACS Paragon Plus Environment

ACS Combinatorial Science
Page 14 of 27
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
2
3
best compound in this series was 10{21} (R = 4ꢀ methoxyphenyl; R = dioxolophenyl; R = 1ꢀ
ethynylcyclohexanol) with an HCV EC of 180 nM and about 555ꢀfold selectivity.
5
0
1
Compounds 10{31} – 10{45} (R = N1ꢀmethyl imidazole) showed reduced potency against HCV
inhibition and reduced selectivity compared to the most active compounds from previous SAR
exploration (i.e. 2{2}, 2{3} and 2{5}), indicating that an imidazole ring is not well tolerated in this
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
position. The most active compound in this series was 10{43} (R = 3’,4’,5’ꢀtrimethoxyphenyl;
3
R = 2,2ꢀdimethylpropargyl alcohol) with an HCV EC of 320 nM and about 100ꢀfold
50
selectivity. Overall, the systematic SAR investigation revealed the activity of individual analogs
to be intricately dependent on the substitution at all positions combined. This is illustrated across
3
the substitution at R for each series, where, in some cases, the unsubstituted propargylic alcohol
moiety (13b) was found to be most potent while in other cases the more substituted moieties (i.e.
1
ꢀethynylcyclohexanol (13a), 2ꢀmethylbutꢀ3ꢀynꢀ2ꢀol (13c) or 4ꢀethynyltetrahydroꢀ2Hꢀpyranꢀ4ꢀol
(13e)) were found to be preferred.
1
4
ACS Paragon Plus Environment

Page 15 of 27
ACS Combinatorial Science
1
2
3
4
5
6
7
8
9
AntiꢀHCV characterization. From the SAR campaign, promising compounds were selected
based on the antiviral activity, selectivity and potential improvement in physicochemical
properties. The activity of the promising compounds was confirmed for wildꢀtype HCV (HCVcc,
genotype 2a) infection in Huh7.5.1 cells. Both extracellular and intracellular viral levels were
dramatically inhibited by the test compounds, among which 10{13} and 10{21} showed the most
promising activity (Figure 5). It is worth noting that more reduction was observed in the
intracellularꢀviral levels with most compounds, suggesting this chemotype might be targeting
HCV earlyꢀstage infection. One advantage of this series of antiviral is their low cytotoxicity
towards human hepatocytes. For further confirmation, the cytotoxicity of advanced compounds
was determined in HepG2 cells and primary human hepatocytes. As shown in Table 5, all test
compounds exhibited low cytotoxicity towards both types of cells (CC > 31.6 ꢁM).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
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
58
59
60
5
0
Extracellular
100
80
60
40
20
0
Intracellular
100
8
6
4
2
0
0
0
0
0
Figure 5. Inhibition of wildꢀtype HCV infection in human hepatocytes by selected analogs.
5
Huh7.5.1 cells were seeded in 12ꢀwell plates (10 cells/well) and cultured overnight. HCVcc was
used to infect the cells with the treatment of compounds at 10 ꢁM. Virusꢀcontaining medium
were removed after 4 h incubation and compound treatment were added back followed by
incubation for additional 48 h. Intracellular and extracellular viral RNA levels were evaluated by
1
5
ACS Paragon Plus Environment

ACS Combinatorial Science
Page 16 of 27
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
quantitative realꢀtime PCR. The results shown are the means of three replicates ± SE.
Cyclosporin A at 10 ꢁM was used as a positive control.
To explore the biological target of these compounds in the HCV replication cycle, HCV
replication cycle assays were performed by infecting Huh7.5.1 cells with different types of
modified viral particles. In the HCV singleꢀcycle assay, singleꢀround infectious HCV (HCVsc)
was used to detect inhibition in the HCV replication steps prior to assembly. The test compounds
all showed inhibitory effect in HCVsc assay (< 35% of DMSO control results), suggesting a
target in earlyꢀstage HCV infection (Table 5). This is consistent with what was observed with
HCVcc infection assay. HCV pseudoparticle (HCVpp) and subgenomic replicon assays were
then employed to detect whether HCVpp entry and HCV replication were targeted by these
compounds, respectively. The HCVpp entry was not significantly inhibited by the test
compounds (≥ 69% of DMSO treatment), except for 10{32} and, to lesser extent 10{21} (Table
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5). Even less inhibition was observed in either HCV replicon cell lines of genotype 1b or 2a as
well as in the transient genotype 1a replicon assay (all ≥ 74% of DMSO treatment, Table 5).
Together, these replicon experiments indicate that replication is highly unlikely to be the target
of these compounds. Overall, this series of compounds inhibited infection at early stage and
targeted neither HCVpp viral entry or HCV RNA replication. One possible mechanism of action
could be through inhibition of viral late entry and/or trafficking in the infected cells. We
previously reported a similar inhibitory pattern in the HCV replication cycle assays in a series of
1
5
promising antiviral chlorcyclizine analogs.
1
6
ACS Paragon Plus Environment

Page 17 of 27
ACS Combinatorial Science
1
2
3
4
5
6
7
8
9
Table 5: Selectivity and HCV life cycle target for selected compounds
ATPlite
HCVpp
DMSO (%)
HCV Replicon
DMSO (%)
HCVꢀLuc
EC50
HCVsc
DMSO
(%)
b
b
VSVpp
DMSO
(%)
CC (ꢁM)
50
Cmpd
Structure
Primary
Human
Hepatocytes
GT
1b
b
GT
1b
b
GT
GT Transient
(
ꢁM)
Huh7.5.1 HepG2
1
a
2a
1a
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
a
a
a
3{4}
0.177
0.171
0.084
>100
>100
>100
>100
>100
>100
>100
> 100
> 100
27.3
33.7
24.9
80.3
86.1
85.5
88.4
76.5
83.8
119
104
106
103
109
109
74.7
76.6
2
2
{3}
{5}
89.0
100
137
108
a
a
1
1
0{11}
0{13}
0.162
0.098
>100
86.1
> 31.6
> 31.6
> 31.6
> 31.6
33.2
26.1
69.3
83.7
77.0
79.1
83.4
116
121
106
112
118
121
86.4
106
194
78.7
10{21}
0.184
0.777
> 100
>100
92.3
N.D.
N.D.
20.0
41.2
23.7
78.1
41.3
82.1
106
1
0{32}
> 100
81.3
99.3
136
9
.30
1
7
ACS Paragon Plus Environment

ACS Combinatorial Science
Page 18 of 27
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
a
b
Sample was tested independently (n ≥ 2) and value is an average. Sample was tested at 10 ꢁM .
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
1
8
ACS Paragon Plus Environment

Page 19 of 27
ACS Combinatorial Science
1
2
3
4
5
6
7
8
9
Physicochemical properties. To assess the ADME (absorption, distribution, metabolism and
excretion) of the test compounds, their physicochemical properties were evaluated (Table 6).
Halfꢀlives (microsomal stability) greater than 30 min were observed with all selected analogs,
suggesting a potentially useful therapeutic halfꢀlife in vivo. The aqueous solubility was relatively
low for all the compounds in Table 6. This solubility may be improved by further structural
modifications, through formulation, or by preparing salt forms of the compounds (only
applicable to compounds 10{21} and 10{32}). The low experimental solubility for all compounds
was observed, despite improvements in the calculated cLogP values (e.g. 10{13} and 10{32})
Compounds 10{6}, 10{13} and 10{21} showed moderate permeability, while compounds 3{4} and
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
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
58
59
60
10{32} exhibited relatively lower permeability (Table 6).
Table 6: Physicochemical properties of selected compounds
Microsomal Aqueous
HCVꢀLuc
ATPlite
Selective
cLogP
Permeability
c
Cmpd
3{4}
Structure
Stability
t1/2 (min)
Solubility
b
e
ꢀ6
EC (ꢁM) CC (ꢁM) Index (SI)
(10 cm/s)
50
50
d
(µg/ml)
a
a
a
a
0
0
0
0
.177
.171
.084
.162
>100
> 565
> 585
6.47
7.36
7.41
>30.0
>30.0
>30.0
<1.0
<1.0
<1.0
<5.6
N.D.
>100
>100
2{3}
> 1190
< 13.9
2
{5}
>100
86.1
> 617
883
6.74
5.43
6.40
>30.0
>30.0
>30.0
<1.0
<1.0
<1.0
N.D.
< 19.8
< 15.4
1
1
0{11}
0{13}
a
0.0975
0
.184
> 100
> 543
10{21}
1
9
ACS Paragon Plus Environment

ACS Combinatorial Science
Page 20 of 27
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
10{32}
0.777
>100
> 129
3.47
>30.0
<1.0
<1.0
a
b
Sample was tested independently (n ≥ 2) and value is an average. cLogP values calculated using CambridgeSoft
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
c
d
e
Chemdraw; details; ꢁSOL Evolution protocol (fully automated HTP platform, pION Inc); double sink method
(fully automated HTP platform, pION Inc)
CONCLUSION
In conclusion, we discovered a new chemical series with antiviral activity against HCV, and
explored the SAR around this chemotype utilizing our compound collection and the synthesis of
a 45ꢀmember library of 2,3,5ꢀtrisubstituted compounds. In general, these compounds showed
good potency (subꢀmicromolar to lowꢀnanomolar) in the HCV infection assay and showed good
selectivity compared to the initial qHTS hit. Representative compounds appear to inhibit an early
stage of the viral lifecycle, possibly viral entry or trafficking. Further characterization of the
precise biological target of these compounds is currently underway.
EXPERIMENTAL PROCEDURES
General biological assay procedures. Human hepatoma cell lines Huh7.5.1 and HepG2 cells
were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Life technologies, Grand
Island, NY, USA) with 10% fetal bovine serum (FBS) (Life technologies, Grand Island, NY,
USA) and antibiotics in 5% CO at 37°C. The primary human hepatocytes (obtained from S.
2
Strom in University of Pittsburgh through the NIH Liver Tissue and Cell Distribution System)
were maintained in Williams E medium containing cell maintenance supplement reagents (Life
technologies, Grand Island, NY, USA). The primary screen and related bioinformatics were
7
carried out as reported. Modified HCV particles for infection assays were produced and stocked
7, 16, 17
as reported before.
In all HCV infection assays, Huh7.5.1 cells were plated in 96ꢀwell
4
plates at 10 cells/well and incubated overnight unless noted otherwise. HCV viral particles were
used to infect the cells in the presence of compounds of interest in a fixed concentration (10 ꢁM)
or in concentrationꢀresponse. After 48 h incubation, viral level was measured by luminescence
unless noted otherwise.
2
0
ACS Paragon Plus Environment

Page 21 of 27
ACS Combinatorial Science
1
2
3
4
5
6
7
8
9
HCVꢀLuc infection and ATPlite assays. HCVꢀLuc infection was carried out as described in the
general procedure. Renilla luciferase signal was measured for viral load using using the Renilla
Luciferase assay system (Promega, Madison, WI, USA). Cytotoxicity was tested with the
ATPlite assay kit (PerkinElmer, Waltham, MA, USA) in parallel. The compound concentration
causing 50% effect (antiviral and cytotoxicity) was calculated as EC and CC values using the
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
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
58
59
60
5
0
50
nonlinear regression equation in GraphPad Prism 5.0 software (GraphPad Software Inc., La
Jolla, CA, USA).
HCV replication cycle assays. In the HCVcc assay, Huh7.5.1 cells were cultured in 12ꢀwell
5
plates at 10 cells/well overnight before infection in the presence of compounds. After infection
overnight, virusꢀcontaining medium was removed and compound was added back followed by
48 h incubation. Quantitative realꢀtime PCR was carried out to measure the viral RNA levels.
The HCV singleꢀcycle assay was performed as described in the general procedure. In the HCVpp
assay, HCVpp was added to the cells in the presence of compound for 4 h and washed away.
Cells were incubated for an additional 48 h followed by a luciferase assay to detect inhibition on
HCVpp entry. In the HCV subgenomic replicon assay, HCV replicon cells were plated as
described in the general procedure and treated with tested compounds for 48 h before luciferase
read out. In the transient replicon assay, Huh7.5.1 cells were transiently transfected with
subgenomic replicon RNA before 48 h incubation with compound treatment. Results were
obtained as decribed in general procedure. Cyclosporin A at 10 ꢁM (in HCVsc and replicon
assays) and bafilomycin A1 at 10 nM (in HCVpp assay) were tested as positive controls.
Physicochemical properties. The cLogP values were calculated using CambridgeSoft
Chemdraw software. For the measurement of in vitro microsomal stability, compounds were
incubated with human, mouse or rat microsomes at 37°C with cofactor, NADPH. At 0, 5,15, 30,
and 45 minute, the concentration of compounds was measured by LCꢀMS/MS. Halfꢀlife (t )
1
/2
1
8
was calculated as described before. Fully automated HTP platform (pION Inc) was used to
measure the solubility and permeability of compounds following the protocol of ꢁSOL Evolution
and the doubleꢀsink PAMPA (parallel artificial memebrane permeability assay), respectively.
General synthetic procedures: All chemicals were used as received from commercial sources.
Commercial anhydrous organic solvents (EtOAc, CHCl , MeOH, EtOH, MeCN, DMF, hexane,
3
toluene, etc.) were used for all reactions. The parallel chemistry reactions were carried out in a
2
1
ACS Paragon Plus Environment

ACS Combinatorial Science
Page 22 of 27
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
®
Mettler Toledo Miniblock or sealed microwave vials. Stirring was achieved with ovenꢀdried,
magnetic stir bars. Analytical thin layer chromatography (TLC) was performed using
commercially prepared polyester backed silica gel plates (200 microns), and visualization was
effected with short wavelength UV light (254 nm). Flash column chromatography was carried
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
13
out using Teledyne Isco CombiFlash R employing normal phase disposable columns. H and C
f
1
NMR spectra were recorded on a Bruker Avance spectrometer (400 MHz/500 MHz H and
1
3
101/126 MHz C). Chemical shifts are reported in parts per million (ppm), and referenced to the
1
13
solvent: CDCl with TMS as internal reference (0.0 ppm for H and 0.0 ppm for C) and
3
DMSOꢀd . Coupling constants (J) are reported in Hertz (Hz). Purification via preparative HPLC
6
was achieved utilizing a Waters XꢀBridge C18 column (19 x 150 mm, 5 µm, with 19 x 10 mm
guard column) at a flow rate of 20 mL/min. Samples were diluted in DMSO and purified using
an elution mixture of water and MeCN, running a concentration gradient which increased by
2
0% MeCN over a 4 minute period. Analytical analysis after preparative chromatography
utilized a Waters Acquity system with UVꢀdetection and massꢀdetection (Waters LCT Premier).
The analytical method conditions included a Waters Aquity BEH C18 column (2.1 x 50 mm, 1.7
µm) and elution with a linear gradient of 5% water to 100% MeCN at 0.6 mL/min flow rate. The
purity of each sample was determined using UV peak area detected at 214 nm wavelength. High
resolution mass spectra for the diversified products were recorded using timeꢀofꢀflight mass
spectrometer.
All synthesized compounds tested were determined to be greater than 95% purity by
HPLC/HRMS determination, with the following exceptions: 10{13} (92.8%), 10{21} (93.9%),
1
0{34} (90.2%), 10{41} (92.9%), 10{42} (94.0%), 10{43} (94.4%) and 10{44} (94.6%). A table of
the purity for all synthesized final analogs is provided in the Supporting Information.
Representative synthetic procedure, synthesis of 10{21} (experimental details for all synthetic
intermediates and the preparation of an additional 19 library compounds is provided in the
Supporting Information):
6
ꢀChloroꢀ2ꢀiodoꢀ3ꢀmethoxypyridine (6b). In an oven dried round bottomed flask, 6ꢀchloroꢀ2ꢀ
iodopyridinꢀ3ꢀol (2.5 g, 9.8 mmol) was dissolved in THF (20 mL) and then a 1M solution of
potassium tertꢀbutoxide in THF (15 mL, 15 mmol) was added slowly to the reaction mixture at
2
2
ACS Paragon Plus Environment

Page 23 of 27
ACS Combinatorial Science
1
2
3
4
5
6
7
8
9
room temperature. The resulting reaction mixture was stirred for 15 mins and then iodomethane
(0.914 mL, 15 mmol) was added slowly to the mixture, upon which the colorless solution turned
into a white suspension. The reaction was quenched after 8 h with 20 mL of water and extracted
with EtOAc (2 x 40 mL). The organic layers were pooled, washed with brine, dried over Na SO
2
4
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
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
58
59
60
and concentrated in vacuo. Silica gel column chromatography with EtOAc/hexane provided
o
desired compound as an off white solid (1.5g, 5.6 mmol, 57% yield); Mp = 64ꢀ71 C
1
(uncorrected); H NMR (500 MHz, Chloroformꢀd) δ 7.16 (d, J = 8.5 Hz, 1H), 6.91 (d, J = 8.5 Hz,
1
3
1H), 3.84 (s, 3H); C NMR (126 MHz, CDCl ) δ 155.04, 141.38, 123.65, 119.26, 108.97, 56.83;
3
+
+
HRMS (ESI) m/z calcd for C H ClINO [M+H] : 269.9177, found: 269.9171.
6
6
6ꢀChloroꢀ3ꢀmethoxyꢀ2ꢀ((4ꢀmethoxyphenyl)ethynyl)pyridine (7c). A solution of 6ꢀchloroꢀ2ꢀ
iodoꢀ3ꢀmethoxypyridine 6b (1.43 g, 5.31 mmol), PdCl (PPh ) (112 mg, 0.16 mmol, 3 mol %)
2
3 2
and CuI (61 mg, 0.31 mmol,6 mol %) in Et N (12 mL) was stirred briefly at 0 °C and then 1ꢀ
3
ethynylꢀ4ꢀmethoxybenzene 11a (1.0 mL , 8 mmol) was added to the reaction mixture. The
reaction mixture was warmed to rt and allowed to proceed under vigorous stirring for ca. 18 h
under an argon atmosphere. The resulting mixture was diluted with EtOAc (2 × 50 mL). The
separated organic layers were washed with water and brine, dried over Na SO , and concentrated
2
4
in vacuo. The crude product was purified by column chromatography on silica gel using
EtOAc/hexane as the eluent to afford the corresponding product 7c as a yellow, amorphous solid
1
(
1.35g, 4.93 mmol, 93% yield). H NMR (400 MHz, Chloroformꢀd) δ 7.64 – 7.54 (m, 2H), 7.27
13
–
1
7.18 (m, 2H), 6.95 – 6.86 (m, 2H), 3.96 (s, 3H), 3.86 (s, 3H). C NMR (126 MHz, CDCl ) δ
3
60.31, 155.90, 141.65, 133.76, 133.43, 123.67, 120.73, 114.23, 114.02, 95.60, 83.33, 56.39,
+
+
55.34; HRMS (ESI) m/z calcd for C H ClNO [M+H] : 274.0629, found: 274.0602.
1
5
13
2
6ꢀ(Benzo[d][1,3]dioxolꢀ5ꢀyl)ꢀ3ꢀmethoxyꢀ2ꢀ((4ꢀmethoxyphenyl)ethynyl)pyridine (8g). To a
solution of chloroalkyne 7c (370 mg, 1.35 mmol), XꢀPHOS (129 mg, 0.27 mmol, 20 mol%) and
Pd(OAc) (30 mg, 0.135 mmol, 10 mol %) in dioxane (10 mL):water (2 mL) mixture, K PO
4
2
3
(860 mg, 4.0 mmol) was added under an argon atmosphere in 20 mL microwave vial. To the
resulting mixture was added boronic acid 12b (450 mg, 2.7 mmol) and vaccum purged thrice.
The reaction mixture was heated at 100 °C for 120 mins under microwave irradiation. Upon
cooling to room temperature, the reaction mixture was extracted with EtOAc (2 × 40 mL). The
combined organic extracts were dried over Na SO , concentrated, and purified by silica gel flash
2
4
2
3
ACS Paragon Plus Environment

ACS Combinatorial Science
Page 24 of 27
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
column chromatography using EtOAc/hexane as the eluent to afford the corresponding product
1
8
g as a yellow solid (335 mg, 0.932 mmol, 69% yield). Mp = 188ꢀ192 °C (uncorrected); H NMR
(400 MHz, Chloroformꢀd) δ 7.55 – 7.50 (m, 2H), 7.47 (d, J = 8.7 Hz, 1H), 7.43 (d, J = 1.7 Hz,
1
5
1
H), 7.38 (dd, J = 8.1, 1.8 Hz, 1H), 7.18 (d, J = 7.5 Hz, 1H), 6.83 (s, 1H), 6.83 – 6.76 (m, 2H),
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
13
.93 (s, 2H), 3.88 (s, 3H), 3.77 (s, 3H); C NMR (126 MHz, CDCl ) δ 160.03, 155.63, 149.59,
3
48.16, 147.92, 133.71, 133.26, 133.20, 120.48, 119.86, 118.50, 114.81, 113.93, 108.35, 107.30,
+
4
+
101.22, 93.93, 84.57, 56.06, 55.32; HRMS (ESI) m/z calcd for C H NO [M+H] : 360.1230,
2
2
18
found: 360.1199.
5ꢀ(Benzo[d][1,3]dioxolꢀ5ꢀyl)ꢀ3ꢀiodoꢀ2ꢀ(4ꢀmethoxyphenyl)furo[3,2ꢀb]pyridine (9i). To a
solution of the alkyne 8g (359 mg, 1.0 mmol) in CH Cl (15 mL) was added gradually iodine
2
2
monochloride (244 mg, 1.5 mmol) dissolved in CH Cl (10 mL). The reaction mixture was
2
2
allowed to stir at rt for 2 h and monitored by TLC to establish completion. The excess iodine
monochloride was removed by washing with saturated aqueous Na S O solution (2 × 30 mL).
2
2
3
The organic layer were dried over anhydrous Na SO and concentrated under vacuum to afford
2
4
the crude product, which was purified by flash chromatography on silica gel using
EtOAc/hexanes as the eluent to provide the benzofuran product 9i as an offꢀwhite solid (350 mg,
1
0.743 mmol, 74% yield). Mp = 178ꢀ182 °C (uncorrected); H NMR (500 MHz, Chloroformꢀd) δ
8.23 – 8.21 (m, 2H), 7.73 – 7.68 (m, 2H), 7.63 – 7.58 (m, 2H), 7.08 – 7.01 (m, 2H), 6.96 – 6.88
13
(
m, 1H), 6.04 (d, J = 2.7 Hz, 2H), 3.90 (s, 3H); C NMR (126 MHz, CDCl ) δ 160.89, 156.78,
3
154.10, 149.28, 148.25, 146.00, 133.90, 129.25, 122.22, 121.18, 118.43, 116.94, 114.06, 110.14,
+
+
108.42, 107.85, 101.26, 62.92, 55.44; HRMS (ESI) m/z calcd for C H INO [M+H] :
2
1
15
4
472.0040, found: 472.0038.
1ꢀ((5ꢀ(Benzo[d][1,3]dioxolꢀ5ꢀyl)ꢀ2ꢀ(4ꢀmethoxyphenyl)furo[3,2ꢀb]pyridinꢀ3ꢀ
yl)ethynyl)cyclohexanol (10{21}). To a 4 dram vial was added successively the 3ꢀ
iodobenzofuran 9i (42 mg, 0.09 mmol), the alkyne 13a (22 mg, 0.18 mmol), PdCl (PPh ) (35
2
3 2
mg, 0.009 mmol, 5 mol %), CuI (2 mg, 0.009 mmol, 5 mol %), DMF (1 mL) and Et NH (0.5
2
o
mL). The solution was stirred and flushed with argon, and then heated to 80 C for 15 h. The
TLC revealed complete conversion of the starting material. The solution was allowed to cool and
diluted with EtOAc (3 mL). The combined organic layers were concentrated in vacuo, and
purified by massꢀdirected, preparative HPLC to afford the trisubstituted product 10{21} (19 mg,
2
4
ACS Paragon Plus Environment

Page 25 of 27
ACS Combinatorial Science
1
2
3
4
5
6
7
8
9
1
0
.04 mmol, 44% yield). H NMR (500 MHz, Chloroformꢀd) δ 8.32 – 8.21 (m, 2H), 7.78 (d, J =
.6 Hz, 1H), 7.58 – 7.49 (m, 3H), 7.05 – 6.98 (m, 2H), 6.92 (d, J = 8.1 Hz, 1H), 6.04 (s, 2H),
8
3.91 (s, 3H), 2.19 (d, J = 12.3 Hz, 2H), 1.88 – 1.73 (m, 6H), 1.71 – 1.61 (m, 2H), 1.45 – 1.28 (m,
1
3
1
H); C NMR (126 MHz, CDCl ) δ 161.20, 160.31, 153.61, 148.58, 148.19, 147.78, 145.98,
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
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
58
59
60
1
28.05, 122.01, 121.89, 119.28, 117.07, 114.19, 108.45, 108.06, 102.73, 101.40, 97.93, 74.40,
+
+
69.19, 55.48, 40.98, 39.94, 25.38, 23.55; HRMS (ESI) m/z calcd for C H NO [M+H] :
29 26 5
468.1806, found: 468.1843.
ASSOCIATED CONTENT
Experimental details and characterization for all new synthetic intermediates and the preparation
of an additional 19 library compounds are provided in the Supporting Information. This material
is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
*
Eꢀmail: jakel@bdg10.niddk.nih.gov (T.J.L.)
*Eꢀmail: kevinf@email.unc.edu (K.J.F)
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
We thank Patrick Porubsky for compound management services. This work was supported by
grants from the Molecular Libraries Initiative 5U54HG005031 (J.A.) and the National Institute
for General Medical Sciences KUꢀCMLD PO50ꢀGM069663 (J.A.). Support for the NMR
instrumentation was provided by NIH Shared Instrumentation Grant # S10RR024664 and NSF
Major Research Instrumentation Grant # 0320648.
2
5
ACS Paragon Plus Environment

ACS Combinatorial Science
Page 26 of 27
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
REFERENCES
1.
2.
Belousova, V.; AbdꢀRabou, A. A.; Mousa, S. A. Recent advances and future directions in
the management of hepatitis C infections. Pharmacol. Ther. 2015, 145, 92ꢀ102.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Mohd Hanafiah, K.; Groeger, J.; Flaxman, A. D.; Wiersma, S. T. Global epidemiology of
hepatitis C virus infection: new estimates of ageꢀspecific antibody to HCV
seroprevalence. Hepatology 2013, 57, 1333ꢀ1342.
3.
Liang, T. J. Current progress in development of hepatitis C virus vaccines. Nat. Med.
2
013, 19, 869ꢀ878.
4.
5.
6.
7.
Liang, T. J.; Ghany, M. G. Current and future therapies for hepatitis C virus infection. N.
Engl. J. Med. 2013, 368, 1907ꢀ1917.
De Francesco, R.; Migliaccio, G. Challenges and successes in developing new therapies
for hepatitis C. Nature 2005, 436, 953ꢀ960.
Liang, T. J.; Ghany, M. G. Therapy of hepatitis Cꢀꢀback to the future. N. Engl. J. Med.
2
014, 370, 2043ꢀ2047.
Hu, Z.; Lan, K.ꢀH.; He, S.; Swaroop, M.; Hu, X.; Southall, N.; Zheng, W.; Liang, T. J.
Novel CellꢀBased Hepatitis C Virus Infection Assay for Quantitative HighꢀThroughput
Screening of AntiꢀHepatitis C Virus Compounds. Antimicrob Agents Chemother. 2014,
58, 995ꢀ1004.
8
.
.
Cho, C.ꢀH.; Neuenswander, B.; Lushington, G. H.; Larock, R. C. Parallel Synthesis of a
MultiꢀSubstituted Benzo[b]furan Library. J. Comb. Chem. 2008, 10, 941ꢀ947.
9
Ismail, M. A. H.; Adel, M.; Ismail, N. S. M.; Abouzid, K. A. M. Molecular Design,
Synthesis and Cell Based HCV Replicon Assay of Novel Benzoxazole Derivatives. Drug
Res. (Stuttg) 2013, 34, 109ꢀ120.
10.
Hu, Z.; Lan, K.ꢀH.; He, S.; Swaroop, M.; Hu, X.; Southall, N.; Zheng, W.; Liang, T. J.
Novel CellꢀBased Hepatitis C Virus Infection Assay for Quantitative HighꢀThroughput
Screening of AntiꢀHepatitis C Virus Compounds. Antimicrob. Agents Chemother. 2014,
5
8, 995ꢀ1004.
11.
12.
Zupan, M.; Iskra, J.; Stavber, S. Room Temperature Regioselective Iodination of
Aromatic Ethers Mediated by SelectfluorTM Reagent FꢀTEDAꢀBF4. Tetrahedron Lett.
1
997, 38, 6305ꢀ6306.
Valadon, P.; Dansette, P. M.; Girault, J.ꢀP.; Amar, C.; Mansuy, D. Thiophene Sulfoxides
as Reactive Metabolites:ꢂ Formation upon Microsomal Oxidation of a 3ꢀAroylthiophene
and Fate in the Presence of Nucleophiles in Vitro and in Vivo. Chem. Res. Toxicol. 1996,
9
, 1403ꢀ1413.
13.
Smith, G. F. Designing Drugs to Avoid Toxicity. In Prog. Med. Chem., Lawton, G.;
Witty, D. R., Eds. Elsevier: 2011; Vol. Volume 50, pp 1ꢀ47.
14.
Treiber, A.; Dansette, P. M.; El Amri, H.; Girault, J.ꢀP.; Ginderow, D.; Mornon, J.ꢀP.;
Mansuy, D. Chemical and Biological Oxidation of Thiophene:ꢂ Preparation and Complete
2
6
ACS Paragon Plus Environment

Page 27 of 27
ACS Combinatorial Science
1
2
3
4
5
6
7
8
9
Characterization of Thiophene SꢀOxide Dimers and Evidence for Thiophene SꢀOxide as
an Intermediate in Thiophene Metabolism in Vivo and in Vitro. J. Am. Chem. Soc. 1997,
1
19, 1565ꢀ1571.
15.
He, S. L., B.; Chu, V.; Hu, Z.; Hu, X.; Xiao, J.; Wang, A.Q.; Schweitzer, C.J.; Li, Q.;
Imamura, M.; Hiraga, N.; Southall, N.; Ferrer, M; Zheng, W.; Chayama, K.; Marugan,
J.J.; Liang, T.J. Repurposing of the Antihistamine Chlorcyclizine and Related
Compounds for Treatment of Hepatitis C Virus Infection. Sci. Transl. Med. 2015, 7,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
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
58
59
60
2
82ra49.
16.
17.
18.
Jones, C. T.; Murray, C. L.; Eastman, D. K.; Tassello, J.; Rice, C. M. Hepatitis C virus p7
and NS2 proteins are essential for production of infectious virus. J. virol. 2007, 81, 8374ꢀ
8
383.
He, J.; Choe, S.; Walker, R.; Di Marzio, P.; Morgan, D. O.; Landau, N. R. Human
immunodeficiency virus type 1 viral protein R (Vpr) arrests cells in the G2 phase of the
cell cycle by inhibiting p34cdc2 activity. J. Virol. 1995, 69, 6705ꢀ6711.
Obach, R. S. Prediction of human clearance of twentyꢀnine drugs from hepatic
microsomal intrinsic clearance data: An examination of in vitro halfꢀlife approach and
nonspecific binding to microsomes. Drug Metab. Dispos. 1999, 27, 1350ꢀ1359.
2
7
ACS Paragon Plus Environment