New potent biaryl sulfate-based hepatitis C virus inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.09.031

The discovery of a new series of potent hepatitis C virus (HCV) NS5A inhibitors containing biaryl sulfone or sulfate cores is reported. Structure-activity relationship (SAR) studies on inhibitors containing various substitution patterns of the sulfate or sulfone core structure established that m-,m′- substituted biaryl sulfate core-based inhibitors containing an amide moiety (compound 20) or an imidazole moiety (compound 24) showed extremely high potency. Compound 20 demonstrated double-digit pM potencies against both genotype 1b (GT-1b) and 2a (GT-2a). Compound 24 also exhibited double-digit pM potencies against GT-1b and sub nM potencies against GT-2a. Furthermore, compounds 20 and 24 exhibited no cardiotoxicity in an hERG ligand binding assay and showed acceptable plasma stability and no mutagenic potential in the Ames test. In addition, these compounds showed distinctive additive effects in combination treatment with the NS5B targeting drug sofosbuvir (Sovaldi). The results of this study showed that the compounds 20 and 24 could be effective HCV inhibitors.

Full text of DOI:10.1016/j.ejmech.2016.09.031

Accepted Manuscript
New potent biaryl sulfate-based hepatitis C virus inhibitors
Youngsu You, Hee Sun Kim, Il Hak Bae, Seung Gi Lee, Min Hyeok Jee, Gyochang
Keum, Sung Key Jang, B. Moon Kim
PII:
S0223-5234(16)30761-9
10.1016/j.ejmech.2016.09.031
EJMECH 8897
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 June 2016
Revised Date: 8 September 2016
Accepted Date: 9 September 2016
Please cite this article as: Y. You, H.S. Kim, I.H. Bae, S.G. Lee, M.H. Jee, G. Keum, S.K. Jang, B.M.
Kim, New potent biaryl sulfate-based hepatitis C virus inhibitors, European Journal of Medicinal
Chemistry (2016), doi: 10.1016/j.ejmech.2016.09.031.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
New potent biaryl sulfate-based hepatitis C virus inhibitors
Youngsu You,†‡ Hee Sun Kim,§‡ Il Hak Bae,† Seung Gi Lee,ꢀ Min Hyeok Jee,ꢀ Gyochang Keum, Sung Key Jang,
*
⊥
ꢀ and B. Moon Kim*†
‡These two individuals contributed equally to this work.
†Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151-747, South Korea
§
Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology, Pohang
790-784, South Korea
ꢀDepartment of Life Sciences, Pohang University of Science and Technology, Pohang 790-784, South Korea
⊥
Center for Neuro-Medicine, Brain Science Institute, Korea Institute of Science and Technology (KIST), Hwarangno
14-gil 5, Seongbuk-gu, Seoul 136-791, South Korea
Keywords: HCV, NS5A inhibitor, biaryl sulfate, structure-activity relationship

ACCEPTED MANUSCRIPT
Abstract
The discovery of a new series of potent hepatitis C virus (HCV) NS5A inhibitors containing biaryl sulfone or
sulfate cores is reported. Structure-activity relationship (SAR) studies on inhibitors containing various substitution
patterns of the sulfate or sulfone core structure established that m-,m’- substituted biaryl sulfate core-based inhibitors
containing an amide moiety (compound 20) or an imidazole moiety (compound 24) showed extremely high potency.
Compound 20 demonstrated double-digit pM potencies against both genotype 1b (GT-1b) and 2a (GT-2a).
Compound 24 also exhibited double-digit pM potencies against GT-1b and sub nM potencies against GT-2a.
Furthermore, compounds 20 and 24 exhibited no cardiotoxicity in an hERG ligand binding assay and showed
acceptable plasma stability and no mutagenic potential in the Ames test. In addition, these compounds showed
distinctive additive effects in combination treatment with the NS5B targeting drug sofosbuvir (Sovaldi®). The
results of this study showed that the compounds 20 and 24 could be effective HCV inhibitors.

ACCEPTED MANUSCRIPT
1. Introduction
Hepatitis C virus (HCV) affects essential liver health leading to infectious hepatitis [1, 2]. Since the first
identification of HCV in 1989, approximately 160-200 million people have been infected worldwide. Nearly 20
percent of those patients developed cirrhosis and hepatocellular carcinoma (HCC) [3, 4]. In 2014, the World Health
Organization (WHO) announced that hepatitis C-related liver diseases kill 350-500 thousand people each year [5].
HCV is a positive sense RNA virus about 50 nm in size. It encodes a total of approximately three thousand amino
acids [6]. Polyproteins translated from HCV RNA include structural proteins and nonstructural (NS) proteins such as
NS2, NS3, NS4A, NS4B, NS5A, and NS5B. These proteins are associated with the production of new virion particle
undergoing replication, transcription, and translation processes [1, 7, 8]. In particular, NS5A proteins are expected to
be involved in replication, reorganized membrane assembly, and secretion of HCVs from infected cells [9].
HCV is classified into 7 genotypes (1a, 1b, 2, 3, 4, 5, and 6) comprising 67 subtypes [10]. According to the WHO
report, HCV patients infected with genotypes 1a (GT-1a) and 1b (GT-1b) are the most predominant and represent
60% of all cases, worldwide [11, 12].
In accordance with international therapy guidelines, the first standard of care (SOC) against HCV comprised a
combination of the pegylated interferon (PEG-IFN) α series (α2a or α2b) and ribavirin (RBV), a nucleoside-type
drug [13, 14]. Nevertheless, this remedy has not only 50 percent cure rate for the common GT-1 patients, but also
other severe adverse effects such as insomnia, depression, neutropenia, flu-like symptoms, fatigue, lymphopenia,
and other unexpected drug interactions [15, 16]. Therefore, Direct Acting Antivirals (DAAs) have been widely
studied to overcome drawbacks of conventional therapy. The first generation of DAAs included NS3/NS4A protease
inhibitors such as telaprevir ans boceprevir. These were approved by the U.S. Food and Drug Administration
(USFDA) in 2011 [17]. However, these drugs still require the use of RBV and PEG-IFN combinations, and have a
54−63% sustained virologic response (SVR) in GT-1 patients [18].
Recently, a new NS5A inhibitor, daclatasvir (BMS790052, Daklinza®), was approved in Europe in 2014 (Figure
1). As a suppressor of the NS5A replication complex, daclatasvir showed impressive results at single-digit picomolar
effective concentrations (EC50) (9 pM and 50 pM against GT-1b and GT-1a, respectively). It effected 2-fold
decreases in levels of viral RNA within 6 h of oral administration in clinical trials [19, 20]. Consequently, the NS5A
phosphoprotein became one of the most desirable drug targets of HCV for many pharmaceutical companies [20].
Daclatasvir has a C-2 symmetric structure that consists of a biphenyl core linked to a 2-(1H-imidazol-2-yl)-
pyrrolidine moiety, and an N-(methoxycarbonyl)-L-valine capping group (Figure 1). In particular, the imidazole and
carbamate groups play an important role in the interaction of daclatasvir with the HCV NS5A complex [21, 22].
Although daclatasvir had no reported serious side-effects or drug-drug interactions (DDI), resistance to the drug
appeared in patients infected with GT-1a and GT1-b HCV [23]. The main amino acid mutations were Y93H and
L31V in GT-1b, which resulted in 19-fold and 23-fold increases in resistance to daclatasvir, respectively [24]. When
there was combination of both Y93H and L31V mutations in GT-1b, resistance to daclatasvir increased 8,336-fold
[25]. To address this issue, many research groups have developed HCV NS5A targeting drug candidates such as
ledipasvir (combination with sofosbuvir and called Harvoni, FDA approval in 2014), ombitasvir (combination with

ACCEPTED MANUSCRIPT
ritonavir and paritaprevir and called Technivie, FDA approval in 2015), elbasvir (MK-8742), samatasvir (IDX-719),
and AV4025 (Figure 1) [26].
Recently, our laboratory reported a series of NS5A inhibitors represented by BMK-20113 (EC50 against GT-1b
and GT-2a strains: 28 pM and 260 pM, respectively), which comprises a benzidine prolinamide skeleton and an N-
(methoxycarbonyl)-D-phenylglycine based capping group (Figure 2) [27]. By considering the structural features of
other known inhibitors, we hypothesized that a longer biaryl core could improve inhibition of the HCV NS5A
replication complex as shown with ombitasvir (EC50 of GT-1b and GT-2a: 5 pM and 12 pM, respectively) and
AV4025 (EC50 of GT-1b and GT-2a: 3.4 pM and 51 pM, respectively) [28]. Herein, we report the discovery of new
NS5A inhibitors embedding biaryl sulfone or sulfate core structures (Ar-SO2-Ar, ArO-SO2-OAr) with imidazole or
amide connections [29, 30].
2. Chemistry
Compounds containing the biaryl sulfone core were prepared following a general amide synthesis procedure as
shown in Scheme 1 [31]. Coupling of 4,4'-diaminodiphenyl sulfone 1 with N-Boc-
ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) provided N-Boc-protected intermediate 2 in moderate yield
(44%). After removal of the Boc group, free amine moieties were coupled with -phenylglycine, -valine, or L-
L-proline in the presence of 1-
D
D
valine, each of which was capped with an N-(methoxycarbonyl) group [27, 32] furnishing the final compounds 3, 4,
and 5 at yields of 50%, 55%, and 53%, respectively.
Synthesis of prolinamide or 2-(pyrrolidin-2-yl)-1H-imidazole containing precursors (8a-d or 11a-b, respectively)
for the sulfate class inhibitors was accomplished according to Scheme 2. Various aminophenol derivatives (7a-d),
which were either commercially available or prepared from reduction (Pd/C, H2) of nitrophenol derivatives [33],
were coupled with N-Boc-L-proline through a standard amide coupling method to yield prolinamide derivatives 8a-d
in moderate to excellent yields (59-99%). Imidazole-containing phenol derivatives 11a and 11b were prepared from
esters 10a and 10b, respectively, which in turn were synthesized through esterification of Boc-proline with α-
bromoacetophenone derivatives 9a and 9b in moderate to high yields (72-91%) [34, 35]. α-Keto ester derivatives
(10a-b) were treated with ammonium acetate (NH4OAc) in toluene to furnish the imidazole-based phenols in
moderate yields (45-52%).
Inhibitors featuring a C2-symmetric biaryl sulfate linkage (13-18 and 20-26) were synthesized in three steps from
prolinamide- or imidazole-containing phenol derivatives (Scheme 3). Coupling of phenol derivatives with 1,1′-
sulfonyldiimidazole (SDI) provided the corresponding biaryl sulfates in low to good yields (30-89%) [36]. Removal
of the Boc group of the diaryl sulfate intermediates (12a-f and 19a-d) provided free amines, which were converted
to final inhibitor compounds (13-18, 20-26) through a standard amide synthesis protocol described above in Scheme
1.

ACCEPTED MANUSCRIPT
3. Results and Discussion
To determine whether the compounds with sulfone or sulfate core structures are effective HCV inhibitors, we
investigated the inhibitory activities of the synthesized compounds using the GT-1b replicon assay and the GT-2a
HCV cell culture system (HCVcc) [37-39]. For the HCV replicon systems (GT-1b), Huh 7.5.1 cells were employed
to investigate the HCV inhibitory activities of the compounds [40]. These Huh 7.5.1 cells contain a bicistronic HCV
replicon NK5.1-Gluc that encodes secretory Gaussia luciferase (Gluc) as a reporter gene. In the case of the GT-2a
HCVcc system, the EC50 values of compounds were measured using the same method employed in our previous
report [27, 41, 42]. The modified JFH1 clone encoding JFH5a-Rluc-ad34 (a variant of JFH1 with 2 cell culture-
adaptive mutations) was added to Huh7.5.1 [38]. After infection for 4 h, Huh7.5.1 cells were added to culture
medium containing compounds of interest at different concentrations. After the cells were incubated for 3 d, EC50
values of compounds were measured using luciferase assays.
The EC50 values of tested compounds against GT-1b and GT-2a are listed in Table 1. Compounds showing good
potencies against GT-1b were selected for further testing against GT-2a. The inhibitory activity of compound 3,
which features a biarylsulfone core with a D-phenylglycine capping group was 36.05 nM against GT-1b (Table 1,
entry 1), whereas both compounds 4 and 5, which contain valine isomers, showed no apparent inhibitory activities
against GT-1b (Table 1, entries 2 and 3). Since the inhibitors containing a biaryl sulfone core did not appear to be
effective as NS5A inhibitors, compounds with a biaryl sulfate core were examined. Compound 13 containing a
biaryl sulfate core with an
L-valine capping group exhibited poor inhibitory activity in the GT-1b assay (Table 1,
entry 4). When the -valine moiety 13 was replaced with an enantiomer (compound 14), a slight increase in
L
inhibition (EC50 574.8 nM) was observed (Table 1, entry 5). The inhibitory activities of compounds 5 and 14 provide
a direct comparison of the influence between the sulfone and sulfate core structures and appear to indicate that the
sulfate core is more effective. However, when the
D-valine residue in compound 14 was replaced by a D-
phenylglycine moiety as in compound 15, excellent sub nanomolar activities were observed in both GT-1b and GT-
2a assays (Table 1, entry 6). The replacement of the amide moiety in compound 15 with an imidazole ring as in
compound 16, a replacement that may improve the water solubility of the inhibitor molecule, decreased the
inhibitory activity 27-fold in the GT-1b assay (Table 1, entry 7). Likewise, similar inhibitory activity was observed
for compound 17 and 14 in the GT-1b assay (Table 1, entries 5 and 8). However, the L-valine diastereomer 18
showed an enhanced activity against GT-1b (Table 1, entry 9).
Next, inhibitors containing meta-,meta’- (m-,m-) substituted biaryl sulfate as a core structure were investigated in
the same HCV replication systems (Table 2). Compound 20, possessing an m-,m’-disubstituted sulfate core and an
N-(methoxycarbonyl)-D-phenylglycine capping group, exhibited extremely high inhibitory activities (EC50 of GT-2a
and GT-1b: 0.0199 nM and 0.01983 nM, respectively; Table 2, entry 1). This is significant since this activity is an
approximate 6-fold improvement relative to that of the p-,p’-disubstituted biaryl sulfate 14 against GT-2a. When the
D-phenylglycine capping of compound 20 was replaced with L-tert-leucine moiety (compound 21), 4- and 47-fold

ACCEPTED MANUSCRIPT
decreased activity, respectively, against GT-1b and GT-2a were surveyed (Table 2, entry 2). In the case of inhibitors
containing an imidazole linker instead of an amide one, compound 22 possessing an N-(methoxycarbonyl)-
phenylglycine capping group, proved to be 18- and 503-fold less potent against GT-1b and GT-2a, respectively, than
inhibitor 20 (Table 2, entry 3). Compound 23, which had an -tert-leucine capping group, showed significantly
decreased activity (EC50 37.1 nM) in the GT-1b assay (Table 2, entry 4). Notably, compound 24, which has an
imidazole linker with -valine capping, showed the highest increase in inhibitory activity (132-fold) in the GT-1b
D-
L
L
assay when compared with compound 18 which has a p-,p’- substituted biaryl sulfate core structure (Table 1, entry 7
and Table 2, entry 5, respectively).
According to our SAR study, the m-,m’-disubstituted biaryl sulfate moiety was confirmed to be the best structure
for the sulfate core part. Earlier observations have suggested that hindered free rotation around the biaryl moiety
significantly influences the inhibitory activity against NS5A [27]. We therefore evaluated whether blocking the free
rotation around the sulfate group may affect the inhibitory potency. We introduced a methyl substitution at the ortho-
position of the biaryl groups. Of the compounds containing the m-,m’-disubstituted aryl sulfate with prolinamide
linker, those possessing an o-methyl group were first investigated. However, compounds 25 and 26, which have
methyl substitution at the 2- and 2’-positions (compound 25) and 6- and 6’-positions of the aryl group (compound
26), respectively, showed a significant loss in inhibitory activity. For compound 25, the EC50 was 22.9 nM against
GT-1b (Table 2, entry 6), and for compound 26, the EC50 was 109 pM against GT-1b and 2.23 nM against GT-2a
(Table 2, entry 7). None of the inhibitors listed in Table 1 and Table 2 were cytotoxic at 20 µM.
Based on these results, we evaluated the top two compounds, 20 and 24, which contain the amide and imidazole
linker, respectively, in a battery of pharmacological studies. First, we assessed the hERG ligand binding of
compounds 20 and 24 at 10 µM (Table 3). The hERG ligand binding assay is used for the evaluation of potential
heart related safety issues [43, 44]. By using “red fluorescent hERG channel ligand tracer”, the inhibition potential
of test compounds could be evaluated [45]. In the case of hERG inhibiting chemicals, the tracers do not bind to the
membrane resulting in low polarization [43, 46]. The % inhibition values of compounds on hERG ligand binding are
listed in Table 3. Compounds having inhibition value more than 50% of the positive control such as astemizole are
considered to possess potential cardiotoxicity [47]. However, compounds 20 and 24 showed no significant inhibition
potential in this study.
Next, we measured the stabilities of compounds 20 and 24 against hydrolytic enzymes, such as aldolase,
cholinesterase, acid phosphatase, alkaline, dehydropeptidase and lipase in rat and human plasma (Table 4). Because
the hydrolysis of candidate molecules in plasma can result in low stability, it is important to assess liability of test
compounds [48, 49]. When compounds 20 and 24 were tested, results showed high stability in rat and human plasma
when compared with positive controls (Table 4).
We then proceeded to evaluate the two compounds for genotoxicity in the Ames test (Table 5). We chose the
commonly used Salmonella typhimurium strains TA98 and TA100 for the detection of frameshift mutations and base

ACCEPTED MANUSCRIPT
substitution mutations, respectively [50]. In this study, the positive controls increased the amount of revertant
colonies significantly in TA98 and TA100 [51], [52]. There was no increase in the quantity of revertant colonies
observed when compounds 20 or 24 (1,000 µg/plate) were applied. In other words, neither test compound had
mutagenic potential against TA98 and TA100. In addition, the evaluation of bis(4-aminophenyl) sulfate, which is a
potential metabolite of compound 20, in the Ames MPF™ 98/100 Mutagenicity Assay showed no mutagenicity at up
to 5 mg/plate.
To evaluate compounds 20 and 24 in combination with a representative NS5B inhibitor, sofosbuvir, we adopted the
Chou method [53]. Compound 20 or 24 was tested at a constant ratio of sofosbuvir to NK5.1 Gluc replicon and,
each inhibition effect was measured through the Gaussia luciferase assay. We calculated the relative inhibition effect
of cells treated with single or combination administrations compared with mock treated replicon cells (Figure 3). To
evaluate the combination index (CI) values, such as effective dose (ED) at each point (ED50, ED75, ED90, and ED95),
we used the Compusyn Freeware Program (ComboSyn, Inc). Compound 20 showed CI values below 1 at 50%, 75%,
90%, and 95% ED concentrations. On the other hand, compound 24 exhibited CI values almost similar to 1 in ED50
and ED75, and above 1 in ED90 and ED95. In conclusion, we suggest that compound 20 has a synergic effect with
sofosbuvir, but that compound 24 shows a dose-dependent additive effect (CI > 1: antagonism effect. CI=1: additive
effect, CI <1: synergistic effect).
4. Conclusion
In summary, we investigated new compounds containing biaryl sulfone or sulfate cores as inhibitors of the
hepatitis C virus NS5A protein. Through SAR study, we discovered that two compounds containing an m-,m’-
disubstituted biaryl sulfate core, 20 and 24, exhibit excellent inhibitory potencies against both GT-1b and GT-2a. In
hERG ligand binding inhibition assays, no inhibition activity was detected for either 20 or 24. These compounds
showed high plasma stability and no mutagenic potential as evaluated by the Ames test. In addition, compounds 20
and 24 had synergistic or nearly additive combination effect with sofosbuvir, respectively. Based on these
encouraging results, further studies are being pursued in our research laboratory.
5. Experimental section
5.1. Chemical studies
5.1.1. General chemical methods
1
The H and 13C NMR-spectra were measured with a Varian/Oxford As-500 (500 MHz), and an Agilent 400-MR
DD2 Magnetic Resonance System (400 MHz) spectrophotometer. Chemical shifts were measured as part per million

ACCEPTED MANUSCRIPT
(δ values) from Tetramethylsilane (TMS) as an internal standard at probe temperature in CD3OD or CDCl3 or
DMSO-D6 for neutral compounds. Coupling constants are provided in Hz, with the following spectral pattern
designations: s, singlet; d, doublet; t, triplet; q, quartet; quint, quintet; m, multiplet; br, broad; app, apparent.
Reactions were conducted, purified, and analyzed according to methods widely practiced in the field, while taking
necessary precautions in the exclusion of moisture and/or oxygen where appropriate. Evaporation of solvents was
performed at reduced pressure using a rotary evaporator. TLC was performed using silica gel 60F254 coated on an
aluminum sheet (E. Merck, Art.5554). High resolution mass spectra (HRMS) were recorded on a ThermoFinnigan
LCQ™ Classic, Quadrupole Ion-Trap Mass Spectrometer. HPLC analyses were carried out on an Agilent HP1100
system (Santa Clara, CA, USA), composed of an auto sampler, quaternary pump, photodiode array detector (DAD),
and HP Chemstation software. The separation was carried out on a poroshell 120 EC-C18 column 50 X 4.6 mm, 2.7
µm with acetonitrile (A), 0.1% TFA in water (B), as a mobile phase at a flow rate of 1 mL/min at 20ꢁ. Method: 5%
A and 95% B (0 min), 50% A and 50% B (15 min), 95% A and 5% B (24min), 95% A and 5% B (25min), 5% A and
95% B (26 min), 5% A and 95% B (27 min). All materials were purchased from a commercial supplier and used
without further purification unless otherwise noted.
5.1.1.1. (Methoxycarbonyl)-
D-valine (Cap-1). Na2CO3 (276 mg, 2.6 mmol) was added to aq NaOH (5 mL of 1
M/H2O, 5 mmol) solution of
D-valine (586 mg, 5.00 mmol) and the resulting solution was cooled with ice-water
bath. Methyl chloroformate (0.420 mL, 5.40 mmol) was added dropwise, the cooling bath was removed and the
reaction mixture was stirred at ambient temperature for 3.25 h. The reaction mixture was washed with ether (3x9
mL), and the aqueous phase was cooled with ice-water bath and acidified with conc HCl to a pH region of 1-2, and
extracted with CH2Cl2 (3x9 mL). The organic phase was dried (MgSO4), filtered, and concentrated in vacuo to
1
afford Cap-1 as a white solid (760 mg, 87%). H NMR (DMSO-d6, δ=2.5 ppm, 300 MHz): 12.54 (s, 1H), 7.32 (d,
1H), 3.84 (t, 1H), 3.54 (s, 3H), 2.03 (m, 1H), 0.87 (d, 6H).
5.1.1.2. (Methoxycarbonyl)-
(br s, 1H), 7.32 (d, 1H), 3.84 (t, 1H), 3.54 (s, 3H), 2.03 (m, 1H), 0.88 (d, J=12, 6H).
L
-valine (Cap-2). Yield 5.0 g (86%) 1H NMR (DMSO-d6, δ=2.5 ppm, 400 MHz): 12.51
1
5.1.1.3. (R)-2-((methoxycarbonyl)amino)-2-phenylacetic acid (Cap-3). Yield 1.4 g (67%). H NMR (DMSO-d6,
δ=2.5 ppm, 500 MHz): 12.79 (br s, 1H), 7.96 (d, J=12, 1H), 7.40-7.29 (m, 5H), 5.13 (d, J=12, 1H), 3.55 (s, 3H).
5.1.1.4. (S)-2-((methoxycarbonyl)amino)-3,3-dimethylbutanoic acid (Cap-4). Yield 670 mg (71%). 1H NMR
(CDCl3, δ=7.26 ppm, 500 MHz): 9.57 (br s, 1H), 5.31 (d, 1H), 4.20 (d, 1H), 3.70 (s, 3H), 1.03 (s, 9H).
5.1.1.5. Di-tert-butyl 2,2'-(((sulfonylbis(4,1-phenylene))bis(azanediyl))bis(carbonyl))(2S,2'S)-bis(pyrrolidine-1-
carboxylate) (2). A mixture of 4,4'-Diaminodiphenyl Sulfone 1 (481 mg, 1.94 mmol), N-Boc-L-proline (1.00 g, 4.65
mmol), and EDCI (945 mg, 5.03 mmol) in CH2Cl2 (10 mL) was stirred at room temperature for 16 h. The mixture
was then poured in 1 N aq. HCl solution and extracted with CH2Cl2. The organic layer dried over magnesium
sulfate, filtered, and concentrated in vacuo. Then without any purification, tert-Butyl (S)-2-((4-((4-((R)-1-(tert-
butoxycarbonyl)pyrrolidine-2-carboxamido)phenyl)sulfonyl)phenyl)carbamoyl)pyrrolidine-1-carboxylate
2
was

ACCEPTED MANUSCRIPT
1
obtained as white solid (544 mg, 44%). H NMR (DMSO-d6, δ=2.5 ppm, 400 MHz): 10.43 (s, 2H), 7.89-7.79 (m,
8H), 4.27-4.18 (m, 2H), 3.44-3.39 (m, 4H), 2.19-2.16 (m, 2H), 1.99-1.78 (m, 6H), 1.38-1.23 (app br s, 18H). 13C
NMR (DMSO-d6, δ=39.52 ppm, 100 MHz): 172.3, 153.0, 143.4, 135.3, 128.5, 119.2, 113.0, 78.6, 62.8, 60.4, 46.5,
45.1, 33.1, 30.9, 30.1, 27.9, 26.7, 26.6, 24.0, 23.4, 13.1. HRMS (ESI) m/z: Anal. calcd. for [M+Na]+
C32H42N4NaO8S: 665.2616; found 665.2613.
5.1.1.6. Dimethyl ((1R,1'R)-((2S,2'S)-(((sulfonylbis(4,1-phenylene))bis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-
1
diyl))bis(2-oxo-1-phenylethane-2,1-diyl))dicarbamate (3). Yield 111 mg (50 %). H NMR (DMSO-d6, δ=2.5 ppm,
400 MHz): 10.37 (s, 2H), 7.87-7.85 (d, 4H), 7.82-7.80 (d, 4H), 7.73-7.57 (q, 2H), 7.40-7.29 (m, 9H), 7.20-6.99 (q,
1H), 5.50-5.48 (d, 2H), 4.38-4.37 (d, 2H), 3.80-3.70 (m, 2H), 3.52 (s, 6H), 3.20-3.14 (q, 2H), 2.02-1.94 (m, 4H),
1.91-1.78 (m, 4H). 13C NMR (DMSO-d6, δ=39.52 ppm, 100 MHz): 171.0, 168.3, 156.0, 143.4, 137.2, 135.3, 128.6,
128.0, 119.2, 60.7, 56.6, 51.6, 46.9, 29.2, 24.3. HRMS (ESI) m/z: Anal. calcd. for [M+Na]+ C42H44N6NaO10S:
847.2732; found 847.2734.
5.1.1.7. Dimethyl ((2R,2'R)-((2S,2'S)-(((sulfonylbis(4,1-phenylene))bis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-
1
diyl))bis(3-methyl-1-oxobutane-1,2-diyl))dicarbamate (4). Yield 53.1 mg (55 %). H NMR (DMSO-d6, δ=2.5 ppm,
500 MHz): 10.72-10.24 (app s, 2H), 7.99-7.84 (m, 4H), 7.82-7.71 (m, 4H), 7.56-7.33 (m, 2H), 4.41-4.40 (d, 2H),
4.10-4.07 (t, 2H), 3.78-3.68 (m, 2H), 3.63-3.59 (m, 1H), 3.54 (s, 6H), 3.46-3.44 (t, 1H), 2.18-2.09 (m, 2H), 2.04-
1.82 (m, 8H), 0.88-0.85 (t, 10H), 0.77-0.76 (d, 1H), 0.68-0.67 (d, 1H). 13C NMR (DMSO-d6, δ=39.52 ppm, 125
MHz): 171.2, 170.2, 156.9, 143.3, 135.3, 128.4, 119.1, 60.3, 57.9, 51.6, 47.7, 29.7, 29.3, 24.3, 19.1, 18.3. HRMS
(ESI) m/z: Anal. calcd. for [M+Na]+ C36H48N6NaO10S: 779.3045; found 779.3045.
5.1.1.8. Dimethyl ((2S,2'S)-((2S,2'S)-(((sulfonylbis(4,1-phenylene))bis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-
1
diyl))bis(3-methyl-1-oxobutane-1,2-diyl))dicarbamate (5). Yield 65.8 mg (53 %). H NMR (DMSO-d6, δ=2.5 ppm,
400 MHz): 10.49 (s, 2H), 7.85-7.76 (m, 8H), 7.33-7.32 (d, 2H), 4.43 (s, 2H), 4.04-4.02 (t, 2H), 3.80 (s, 2H), 3.61 (s,
2H), 3.52 (s, 6H), 2.16 (s, 2H), 1.96-1.87 (m, 8H), 0.93-0.87 (m, 12H). 13C NMR (DMSO-d6, δ=39.52 ppm, 100
MHz): 171.2, 170.4, 156.8, 143.5, 135.2, 128.4, 119.0, 60.3, 57.9, 51.5, 51.4, 47.2, 29.8, 29.3, 24.7, 18.9, 18.6.
HRMS (ESI) m/z: Anal. calcd. for [M+H]+ C36H49N6O10S: 757.3225; found 757.3228.
5.1.1.9. 3-Amino-2-methylphenol (7a). A stirred mixture of 2-Methyl-3-nitrophenol 5 (2.0 g, 13.1 mmol) in MeOH
(50 mL) at room temperature was treated with 10 wt% palladium on charcoal (100 mg). Hydrogen gas was passed
through the mixture for 24 h, and then the palladium on charcoal was removed by filtration through Celite. The
filtrate was concentrated in vacuo, and was obtained as darked solid (1.62 g, 99%). 1H NMR (CD3OD, δ=3.31 ppm,
400 MHz): 6.77-6.73, (t, 1H), 6.29-6.27 (d, 1H), 6.22-6.20 (d, 1H), 2.02 (s, 3H). 13C NMR (CD3OD, δ=49.00 ppm,
100 MHz): 156.7, 147.5, 127.2, 110.9, 108.8, 106.7, 9.2. HRMS (ESI) m/z: Anal. calcd. for [M+H]+ C7H10NO:
124.0757; found 124.0759.
5.1.1.10. tert-Butyl (S)-2-((3-hydroxy-2-methylphenyl)carbamoyl)pyrrolidine-1-carboxylate (8a). A mixture of 3-
amino-2-methylphenol 7a (1.19 g, 5.52 mmol), N-Boc-L-proline (1.43g, 6.63 mmol), and EDCI (928 mg, 7.18

ACCEPTED MANUSCRIPT
mmol) in CH2Cl2 (20 mL) was stirred at room temperature for 5 h. The mixture was then poured in 1 N aq HCl
solution and Extracted with CH2Cl2. The organic layer dried over magnesium sulfate, filtered, and concentrated in
vacuo. Without any purification, the residue 8a was obtained as a white solid (1.76 g, 99%). 1H NMR (DMSO-d6,
δ=2.5 ppm, 400 MHz): 9.36-9.24 (t, 2H), 6.94 (s, 1H), 6.82-6.78 (d, 1H), 6.65 (s, 1H), 4.29 (s, 1H), 3.42-3.32 (m,
2H), 2.23-2.14 (d, 1H), 1.99 (s, 3H), 1.89-1.81 (m, 3H), 1.42-1.37 (app br s, 9H). 13C NMR (DMSO-d6, δ=39.52
ppm, 100 MHz): 171.3, 155.7, 153.3, 137.1, 125.5, 119.3, 116.2, 111.8, 78.5, 60.0, 46.6, 31.4, 28.1, 23.3, 10.7.
HRMS (ESI) m/z: Anal. calcd. for [M+Na]+ C17H24N2NaO4: 343.1628; found 343.1627.
5.1.1.11. tert-Butyl (S)-2-((3-hydroxy-4-methylphenyl)carbamoyl)pyrrolidine-1-carboxylate (8b). Yield 1.92 g
(74%). 1H NMR (DMSO-d6, δ=2.5 ppm, 400 MHz): 9.73 (s, 1H), 9.27 (s, 1H), 7.21 (s, 1H), 6.94-6.92 (d, 1H), 6.86-
6.84 (d, 1H), 4.23-4.13 (m, 1H), 3.39 (s, 2H), 2.20-2.17 (d, 1H), 2.04 (s, 3H), 1.86-1.80 (m, 3H), 1.39-1.23 (app br s,
9H). 13C NMR (DMSO-d6, δ=39.52 ppm, 100 MHz): 171.1, 155.2, 153.2, 137.6, 130.2, 118.6, 109.9, 106.2, 78.4,
60.3, 46.5, 31.0, 28.0, 23.4, 15.5. HRMS (ESI) m/z: Anal. calcd. for [M+Na]+ C17H24N2NaO4: 343.1628; found
343.1626.
1
5.1.1.12. tert-Butyl (S)-2-((3-hydroxyphenyl)carbamoyl)pyrrolidine-1-carboxylate (8c). Yield 5.41 g (96%). H
NMR (DMSO-d6, δ=2.5 ppm, 400 MHz): 9.83 (s, 1H), 9.36 (s, 1H), 7.18 (s, 1H), 7.08-7.04 (t, 1H), 6.96-6.94 (d,
1H), 6.45-6.43 (d, 1H), 4.24-4.15 (m, 1H), 3.37 (m, 1H), 3.33-3.32 (m, 1H), 2.17-2.08 (m, 1H), 1.86-1.75 (m, 3H),
1.39-1.27 (app br s, 9H). 13C NMR (DMSO-d6, δ=39.52 ppm, 100 MHz): 171.4, 157.6, 153.2, 140.2, 129.3, 110.4,
110.1, 106.6, 78.5, 60.4, 46.6, 31.1, 28.0, 23.4. HRMS (ESI) m/z: Anal. calcd. for [M+Na]+ C16H22N2NaO4:
329.1472; found 329.1474.
1
5.1.1.13. tert-Butyl (S)-2-((4-hydroxyphenyl)carbamoyl)pyrrolidine-1-carboxylate (8d). Yield 5.36 g (87%). H
NMR (DMSO-d6, δ=2.5 ppm, 400 MHz): 9.68 (s, 1H), 9.16 (s, 1H), 7.37-7.34 (d, 2H), 6.70-6.67 (d, 2H), 4.22-4.12
(m, 1H), 3.43-3.37 (m, 1H), 3.35-3.29 (m, 1H), 2.19-2.12 (m, 1H), 1.91-1.74 (m, 3H), 1.39-1.28 (app br s, 9H). 13
C
NMR (DMSO-d6, δ=39.52 ppm, 100 MHz): 170.8, 153.3, 130.7, 121.0, 115.0, 78.4, 60.3, 46.6, 31.0, 30.2, 28.0,
24.0, 23.4. HRMS (ESI) m/z: Anal. calcd. for [M+Na]+ C16H22N2NaO4: 329.1472; found 329.1472.
5.1.1.14. 1-(tert-Butyl) 2-(2-(4-hydroxyphenyl)-2-oxoethyl) (S)-pyrrolidine-1,2-dicarboxylate (10a). 2-Bromo-4-
hydroxyacetophenone 9a (1.00 g, 4.65 mmol) and N-Boc-L-proline (1.10 g, 5.12 mmol) in Acetonitrile (50 mL) put
into the flask. Diisopropylethylamine (1.44 mL, 5.58 mmol) was added dropwise to the mixture and was stirred at
room temperature for 5 h. The mixture was then poured in 1 N aq HCl solution and extracted with CH2Cl2. The
organic layer dried over magnesium sulfate, and then was purified by the column chromatography (n-hexane / ethyl
acetate). The residue 10a was obtained as white solid (1.20 g, 74%). 1H NMR (DMSO-d6, δ=2.5 ppm, 400 MHz):
10.49 (s, 1H), 7.86-7.85 (d, 2H), 6.88-6.86 (t, 2H), 5.53-5.32 (m, 2H), 4.32-4.29 (m, 1H), 3.11 (m, 2H), 2.29-2.14
(m, 2H), 1.90-1.85 (m, 2H), 1.36 (app br s, 9H). 13C NMR (DMSO-d6, δ=39.52 ppm, 100 MHz): 190.5, 172.3,
162.7, 153.0, 130.4, 125.4, 115.4, 78.9, 66.1, 58.6, 46.2, 30.4, 27.9, 23.1. HRMS (ESI) m/z: Anal. calcd. for
[M+Na]+ C18H23NNaO6: 372.1418; found 372.1419.

ACCEPTED MANUSCRIPT
5.1.1.15. 1-(tert-Butyl) 2-(2-(3-hydroxyphenyl)-2-oxoethyl) (S)-pyrrolidine-1,2-dicarboxylate (10b). Yield 1.47 g
(91%). 1H NMR (DMSO-d6, δ=2.5 ppm, 400 MHz): 9.88 (s, 1H), 7.45-7.43 (d, 1H), 7.37-7.33 (m, 2H), 7.10-7.08 (d,
1H), 5.58-5.38 (m, 2H), 4.37-4.31 (m, 1H), 3.41-3.31 (m, 2H), 2.34-2.10 (m, 2H), 1.92-1.82 (m, 2H), 1.40-1.37 (app
br s, 9H).13C NMR (DMSO-d6, δ=39.52 ppm, 100 MHz): 192.4, 172.3, 157.8, 153.0, 135.2, 130.0, 121.1, 118.7,
113.9, 79.0, 66.6, 58.6, 46.2, 30.5, 29.6, 28.1, 27.9, 23.9, 23.1. HRMS (ESI) m/z: Anal. calcd. for [M+Na]+
C18H23NNaO6: 372.1418; found 372.1417.
5.1.1.16. tert-Butyl (S)-2-(5-(4-hydroxyphenyl)-1H-imidazol-2-yl)pyrrolidine-1-carboxylate (11a). A mixture of (S)-
1-tert-Butyl 2-(2-(4-hydroxyphenyl)-2-oxoethyl) pyrrolidine-1,2-dicarboxylate 10a (2.00 g, 5.72 mmol) and
Ammonium acetate (6.62 g, 85.9 mmol) were suspended in Toluene (20 mL). The reaction mixture was heated to
90 °C and stirred for 20 h. The mixture was then poured in H2O and extracted with ethyl acetate. The organic layer
dried over magnesium sulfate and evaporated in vacuo. After evaporation, the residue was purified by the column
1
chromatography (n-hexane / ethyl acetate). The residue 11a was obtained as yellow solid (887 mg, 47%). H NMR
(DMSO-d6, δ=2.5 ppm, 400 MHz): 11.76 (br s, 1H), 9.48 (br s, 1H), 7.53 (s, 2H), 7.20 (s, 1H), 6.77-6.75 (d, 2H),
4.83-4.76 (d, 1H), 3.54-3.35 (d, 2H), 2.20-1.81 (m, 4H), 1.39-1.17 (app br s, 9H). 13C NMR (DMSO-d6, δ=39.52
ppm, 100 MHz): 155.9, 153.8, 153.5, 150.1, 125.5, 115.3, 78.2, 55.3, 46.3, 33.4, 31.9, 27.9, 23.9, 23.2. HRMS (ESI)
m/z: Anal. calcd. for [M+Na]+ C18H23N3NaO3: 352.1632; found 352.1634.
5.1.1.17. tert-Butyl (S)-2-(5-(3-hydroxyphenyl)-1H-imidazol-2-yl)pyrrolidine-1-carboxylate (11b). Yield 456 mg
1
(45%). H NMR (DMSO-d6, δ=2.5 ppm, 400 MHz): 11.88 (br s, 1H), 9.41 (br s, 1H), 7.34-7.11 (m, 4H), 6.60 (s,
1H), 4.85-4.77 (d, 1H), 3.55 (s, 1H), 3.35 (s, 1H), 2.22-2.14 (m, 1H), 1.99-1.82 (m, 3H), 1.39-1.16 (app br s, 9H).
13C NMR (DMSO-d6, δ=39.52 ppm, 100 MHz): 157.7, 153.5, 129.4, 115.3, 113.1, 111.3, 78.3, 55.3, 48.7, 46.4,
33.5, 31.9, 28.0, 23.9, 23.3. HRMS (ESI) m/z: Anal. calcd. for [M+Na]+ C18H23N3NaO3: 352.1632; found 352.1634.
5.1.1.18.
Di-tert-butyl
2,2'-((((sulfonylbis(oxy))bis(4,1-phenylene))bis(azanediyl))bis(carbonyl))(2S,2'S)-
bis(pyrrolidine-1-carboxylate) (12a). A mixture of tert-Butyl (S)-2-((4-hydroxyphenyl)carbamoyl)pyrrolidine-1-
carboxylate 7d (464 mg, 1.51 mmol), Cs2CO3 (164 mg, 0.505 mmol) and 1,1′-Sulfonyldiimidazole (100 mg, 0.505
mmol) were suspended in THF (25 mL). The reaction mixture was heated to 66 °C and refluxed for 12 h. The
mixture was then filtered by short silica column, and evaporated in vacuo. After evaporation, the residue was
purified by the column chromatography (n-hexane / ethyl acetate). The product 12a was obtained as yellow solid
1
(102 mg, 30%). H NMR (DMSO-d6, δ=2.5 ppm, 400 MHz): 10.28 (s, 1H), 7.72-7.70 (d, 4H), 7.39-7.37 (d, 4H),
4.23-4.16 (m, 2H), 3.39-3.32 (m, 4H), 2.21-2.16 (t, 2H), 1.86-1.78 (m, 6H), 1.38-1.25 (app br s, 18H). 13C NMR
(DMSO-d6, δ=39.52 ppm, 100 MHz): 172.2, 153.5, 145.2, 138.8, 121.9, 120.9, 79.0, 63.0, 60.7, 46.8, 31.2, 28.2,
23.7. HRMS (ESI) m/z: Anal. calcd. for [M+Na]+ C32H42N4NaO10S: 697.2514; found 697.2515.
5.1.1.19.
Di-tert-butyl
2,2'-(((sulfonylbis(oxy))bis(4,1-phenylene))bis(1H-imidazole-5,2-diyl))(2S,2'S)-
1
bis(pyrrolidine-1-carboxylate) (12b). Yield 254 mg (82%). H NMR (DMSO-d6, δ=2.5 ppm, 400 MHz): 11.99 (s,
2H), 7.88-7.85 (d, 3H), 7.53 (s, 2H), 7.39-7.38 (d, 3H), 7.30-7.27 (d, 1H), 6.91-6.87 (d, 1H), 4.84-4.77 (d, 2H), 4.03-

ACCEPTED MANUSCRIPT
3.98 (t, 1H), 3.53 (s, 2H), 3.29-3.25 (m, 1H), 2.22-2.14 (m, 2H), 2.05-1.93 (m, 2H), 1.85-1.71 (m, 4H), 1.39-1.15
(app br s, 18H). 13C NMR (DMSO-d6, δ=39.52 ppm, 100 MHz): 174.6, 153.4, 147.9, 125.7, 121.1, 78.3, 59.6, 55.2,
46.4, 33.3, 31.0, 30.0, 28.0, 23.9, 23.2. HRMS (ESI) m/z: Anal. calcd. for [M+H]+ C36H45N6O8S: 721.3014; found
721.3014.
5.1.1.24. Bis(4-((S)-1-((methoxycarbonyl)-
D-valyl)pyrrolidine-2-carboxamido)phenyl) sulfate (13). A mixture of Di-
tert-butyl 2,2'-((((sulfonylbis(oxy))bis(4,1-phenylene))bis(azanediyl))bis(carbonyl))(2S,2'S)-bis(pyrrolidine-1-
carboxylate) 12a (48.9 mg, 0.0725 mmol) in CF3CO2H (1 mL) / CH2Cl2 (1 mL) was stirred at room temperature for
30 m. The volatile component was removed in vacuo, 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (36.1 mg,
0.188 mmol), Hydroxybenzotriazole hydrate (25.5 mg, 0.188 mmol), Cap-1 (30.5 mg, 0.174 mmol) were added in
batches over 4 min to a solution of N,N-Diisopropylethylamine (63.1 µL, 0.362 mmol) in CH2Cl2 (5 mL) and the
reaction mixture stirred at room temperature for overnight. The mixture was then poured in 1 N aq HCl solution and
extracted with CH2Cl2. The organic layer dried over magnesium sulfate, and then was purified by the column
1
chromatography (CH2Cl2 / MeOH). The compound 13 was obtained as white solid (58.0 mg, 86 %). H NMR
(DMSO-d6, δ=2.5 ppm, 400 MHz): 10.00 (s, 2H), 7.74-7.72 (d, 4H), 7.42-7.34 (q, 6H), 4.42-4.40 (d, 2H), 4.11-4.07
(t, 2H), 3.79 (s, 2H), 3.63-3.60 (d, 2H), 3.54 (s, 6H), 2.13-1.91 (m, 10H), 0.89-0.86 (t, 10H), 0.80-0.78 (d, 1H), 0.72-
0.70 (d, 1H). 13C NMR (DMSO-d6, δ=39.52 ppm, 100 MHz): 170.69, 170.2, 156.9, 145.0, 138.6, 121.5, 120.5, 60.3,
58.0, 54.9, 51.6, 47.1, 29.7, 29.4, 24.3, 19.1, 18.3. HRMS (ESI) m/z: Anal. calcd. for [M+Na]+ C36H48N6NaO12S:
811.2943; found 811.2941.
5.1.1.25. Bis(4-((S)-1-((R)-2-((methoxycarbonyl)amino)-2-phenylacetyl)pyrrolidine-2-carboxamido)phenyl) sulfate
1
(14). Yield 37.6 mg (59%). H NMR (DMSO-d6, δ=2.5 ppm, 400 MHz): 10.14 (s, 1H), 7.76-7.52 (m, 7H), 7.42-
7.32 (m, 12H), 7.23-7.09 (m, 1H), 5.51-5.49 (d, 2H), 4.40-4.38 (d, 2H), 3.82 (br s, 2H), 3.54 (s, 6H), 3.20-3.18 (d,
2H), 2.04-1.80 (m, 8H). 13C NMR (DMSO-d6, δ=39.52 ppm, 100 MHz): 170.6, 168.2, 156.4, 144.9, 138.7, 136.9,
128.6, 128.5, 128.1, 121.6, 120.5, 62.8, 62.8, 60.8, 56.7, 51.6, 46.9, 29.3, 29.1, 24.7. HRMS (ESI) m/z: Anal. calcd.
for [M+Na]+ C42H44N6NaO12S: 879.2630; found 879.2630.
5.1.1.26. Bis(4-((S)-1-((methoxycarbonyl)-L-valyl)pyrrolidine-2-carboxamido)phenyl) sulfate (15). Yield 85.5 mg
1
(69%). H NMR (DMSO-d6, δ=2.5 ppm, 400 MHz): 10.30 (s, 2H), 7.72-7.71 (d, 4H), 7.39-7.32 (m, 6H), 4.44 (s,
2H), 4.05-4.02 (t, 2H), 3.81 (s, 2H), 3.63 (s, 2H), 3.52 (s, 6H), 2.19-2.08 (m, 2H), 1.99-1.89 (m, 8H), 1.26-1.23 (m,
2H), 0.94-0.88 (m, 12H). 13C NMR (DMSO-d6, δ=39.52 ppm, 125 MHz): 170.7, 170.3, 156.8, 144.8, 138.7, 121.5,
120.3, 60.2, 57.9, 51.4, 47.2, 29.9, 29.4, 24.6, 18.9, 18.6. HRMS (ESI) m/z: Anal. calcd. for [M+H]+ C36H49N6O12S:
789.3124; found 789.3129.
5.1.1.27.
Bis(4-(2-((S)-1-((R)-2-((methoxycarbonyl)amino)-2-phenylacetyl)pyrrolidin-2-yl)-1H-imidazol-5-
1
yl)phenyl) sulfate (16). Yield (56.1 mg, 43%). H NMR (DMSO-d6, δ=2.5 ppm, 400 MHz): 8.14 (s, 1H), 8.07 (s,
1H), 7.98-7.96 (d, 2H), 7.92-7.85 (q, 2H), 7.80-7.78 (d, 1H), 7.71-7.60 (m, 5H), 7.39 (s, 4H), 7.35-7.34 (d, 1H),
7.30-7.29 (d, 4H), 7.04 (s, 1H), 5.54-5.50 (t, 2H), 5.21-5.19 (m, 2H), 3.96-3.77 (m, 2H), 3.51 (s, 6H), 3.39-3.14 (m,

ACCEPTED MANUSCRIPT
2H), 2.38-2.23 (m, 2H), 2.09-1.86 (m, 6H). 13C NMR (DMSO-d6, δ=39.52 ppm, 100 MHz): 169.0, 158.6, 156.3,
149.2, 149.4, 136.8, 131.43, 128.68, 128.32, 128.12, 127.45, 127.25, 122.10, 115.93, 57.00, 53.48, 51.62, 47.00,
30.81, 24.14. HRMS (ESI) m/z: Anal. calcd. for [M+H]+ C46H47N8O10S : 903.3130; found 903.3133.
5.1.1.28. Bis(4-(2-((S)-1-((methoxycarbonyl)-D-valyl)pyrrolidin-2-yl)-1H-imidazol-5-yl)phenyl) sulfate (17). Yield
1
76.9 mg (74%). H NMR (DMSO-d6, δ=2.5 ppm, 400 MHz): 8.09 (s, 2H), 7.96-7.94 (d, 4H), 7.65-7.62 (d, 3H),
7.58-7.52 (m, 1H), 7.25-7.23 (d, 2H), 5.19-5.16 (q, 2H), 4.19-4.15 (t, 2H), 3.91-3.83 (m, 2H), 3.70-3.61 (m, 2H),
3.53 (s, 6H), 2.38-2.36 (t, 2H), 2.08-1.94 (m, 8H), 0.90-0.85 (q, 10H), 0.71-0.70 (d, 1H), 0.35-0.34 (d, 1H).13C NMR
(DMSO-d6, δ=39.52 ppm, 100 MHz): 170.8, 156.8, 149.6, 149.6, 127.3, 122.0, 115.8, 57.8, 53.2, 51.6, 47.0, 30.9,
29.7, 24.4, 19.3, 17.8. HRMS (ESI) m/z: Anal. calcd. for [M+H]+ C40H51N8O10S: 835.3443; found 835.3444.
5.1.1.29. Bis(4-(2-((S)-1-((methoxycarbonyl)-L-valyl)pyrrolidin-2-yl)-1H-imidazol-5-yl)phenyl) sulfate (18). Yield
25 mg, (13%). 1H NMR (DMSO-d6, δ=2.5 ppm, 400 MHz): 11.88 (s, 2H), 7.88-7.81 (m, 4H), 7.52 (s, 2H), 7.39-7.37
(d, 4H), 7.29-7.27 (d, 2H), 5.07 (s, 2H), 4.08-3.99 (m, 2H), 3.80-3.74 (m, 4H), 3.54 (s, 6H), 2.14 (s, 2H), 1.99-1.91
(m, 6H), 1.86-1.76 (m, 2H), 0.89-0.83 (q, 12H). 13C NMR (DMSO-d6, δ=39.52 ppm, 100 MHz): 173.5, 170.4,
170.2, 156.8, 147.9, 125.7, 121.2, 59.2, 58.0, 54.2, 51.5, 46.8, 30.9, 29.9, 24.3, 19.0, 18.5. HRMS (ESI) m/z: Anal.
calcd. for [M+H]+ C40H51N8O10S: 835.3443; found 835.3445.
5.1.1.20. Di-tert-butyl 2,2'-((((sulfonylbis(oxy))bis(2-methyl-3,1-phenylene))bis(azanediyl))bis(carbonyl))(2S,2'S)-
1
bis(pyrrolidine-1-carboxylate) (19a). Yield 321 mg (75%). H NMR (DMSO-d6, δ=2.5 ppm, 400 MHz): 9.66-9.61
(d, 2H), 7.41 (s, 2H), 7.37-7.33 (t, 2H), 7.26-7.24 (d, 2H), 4.31-4.29 (t, 2H), 3.42 (s, 2H), 3.34 (s, 2H), 2.25-2.23 (br
d, 2H), 2.11 (s, 6H), 1.97-1.82 (m, 6H), 1.41-1.35 (app br s, 18H). 13C NMR (DMSO-d6, δ=39.52 ppm, 100 MHz):
171.7, 153.8, 153.2, 148.6, 138.3, 126.8, 125.9, 125.1, 118.1, 78.6, 59.9, 46.6, 31.3, 30.0, 28.1, 24.1, 23.2, 11.4.
HRMS (ESI) m/z: Anal. calcd. for [M+Na]+ C34H46N4NaO10S: 725.2827; found 725.2826.
5.1.1.21. Di-tert-butyl 2,2'-((((sulfonylbis(oxy))bis(4-methyl-3,1-phenylene))bis(azanediyl))bis(carbonyl))(2S,2'S)-
1
bis(pyrrolidine-1-carboxylate) (19b). Yield 229 mg (72%). H NMR (DMSO-d6, δ=2.5 ppm, 400 MHz): 10.22 (s,
2H), 7.95 (s, 2H), 7.54-7.48 (q, 2H), 7.34-7.32 (d, 2H), 4.24-4.13 (m, 2H), 3.42-3.31 (m, 4H), 2.20-2.17 (d, 8H),
1.91-1.77 (m, 6H), 1.38-1.23 (app br s, 18H). 13C NMR (DMSO-d6, δ=39.52 ppm, 100 MHz): 171.8, 153.1, 148.2,
138.4, 131.9, 124.5, 118.5, 111.6, 78.5 60.4, 46.5, 30.9, 27.9, 23.4, 15.1. HRMS (ESI) m/z: Anal. calcd. for [M+Na]+
C34H46N4NaO10S: 725.2827; found 725.2827.
5.1.1.22.
Di-tert-butyl
2,2'-((((sulfonylbis(oxy))bis(3,1-phenylene))bis(azanediyl))bis(carbonyl))(2S,2'S)-
1
bis(pyrrolidine-1-carboxylate) (19c). Yield 378 mg (56%). H NMR (DMSO-d6, δ=2.5 ppm, 400 MHz): 10.32 (s,
1H), 8.21-7.77 (m, 3H), 7.59-7.57 (d, 2H), 7.47 (s, 1H), 7.40-7.22 (m, 1H), 7.12-7.08 (t, 1H), 4.24-4.18 (d, 2H), 3.41
(br s, 4H), 2.20 (s, 2H), 1.88-1.80 (m, 6H), 1.39- 1.24 (app br s, 18H). 13C NMR (DMSO-d6, δ=39.52 ppm, 100
MHz): 172.0, 153.1, 149.9, 140.8, 130.6, 118.4, 115.3, 111.4, 78.6, 60.5, 46.6, 30.9, 27.9, 23.4. HRMS (ESI) m/z:
Anal. calcd. for [M+Na]+ C32H42N4NaO10S: 697.2514; found 697.2514.

ACCEPTED MANUSCRIPT
5.1.1.23.
Di-tert-butyl
2,2'-(((sulfonylbis(oxy))bis(3,1-phenylene))bis(1H-imidazole-5,2-diyl))(2S,2'S)-
bis(pyrrolidine-1-carboxylate) (19d). Yield 388 mg (89%). 1H NMR (DMSO-d6, δ=2.5 ppm, 400 MHz): 12.04-11.97
(br d, 2H), 8.21-7.76 (t, 4H), 7.64-7.61 (d, 1H), 7.52-7.44 (q, 2H), 7.37-7.20 (m, 2H), 6.93-6.74 (q, 1H), 4.83-4.77
(d, 2H), 3.53 (s, 2H), 3.39-3.36 (m, 2H), 2.22-2.15 (m, 2H), 1.98-1.79 (m, 6H), 1.38-1.14 (app br s, 18H). 13C NMR
(DMSO-d6, δ=39.52 ppm, 100 MHz): 153.4, 150.4, 138.1, 131.3, 130.5, 123.5, 119.2, 117.8, 116.4, 116.2, 78.2,
55.2, 46.3, 33.3, 27.9, 23.1. HRMS (ESI) m/z: Anal. calcd. for [M+H]+ C36H45N6O8S: 721.3014; found 721.3014.
5.1.1.30. Bis(3-((S)-1-((R)-2-((methoxycarbonyl)amino)-2-phenylacetyl)pyrrolidine-2-carboxamido)phenyl) sulfate
(20). Yield 1.08 g (70%). 1H NMR (DMSO-d6, δ=2.5 ppm, 400 MHz): 10.25 (s, 1H), 7.88-7.85 (d, 3H), 7.72-7.70
(d, 2H), 7.63-7.61 (d, 2H), 7.50-7.47 (t, 2H), 7.40-7.30 (m, 8H), 7.16-7.10 (q, 2H), 5.50-5.48 (d, 2H), 4.38-4.36 (m,
2H), 3.81-3.73 (d, 2H), 3.52 (s, 6H), 3.20-3.17 (m, 2H), 2.02-1.79 (m, 8H). 13C NMR (DMSO-d6, δ=39.52 ppm, 100
MHz): 170.8, 168.4, 149.9, 140.7, 137.2, 130.7, 128.6, 128.4, 128.0, 118.4, 115.5, 111.3, 60.7, 56.7, 51.6, 47.0, 29.2,
24.3. HRMS (ESI) m/z: Anal. calcd. for [M+Na]+ C42H44N6NaO12S: 879.2630; found 879.2629.
5.1.1.31. Bis(3-((S)-1-((S)-2-((methoxycarbonyl)amino)-3,3-dimethylbutanoyl)pyrrolidine-2-carboxamido)phenyl)
1
sulfate (21). Yield 29.7 mg (22%). H NMR (DMSO-d6, δ=2.5 ppm, 400 MHz): 10.41-10.16 (d, 2H), 7.88-7.82 (d,
2H), 7.62-7.60 (d, 2H), 7.48-7.44 (t, 2H), 7.27-7.10 (q, 4H), 4.45-4.41 (t, 2H), 4.23-4.20 (d, 2H), 3.79-3.76 (m, 2H),
3.65-3.62 (m, 2H), 3.54 (s, 6H), 2.20-2.14 (m, 2H), 2.02-1.84 (m, 6H), 0.96 (s, 18H). 13C NMR (DMSO-d6, δ=39.52
ppm, 125 MHz): 170.9, 169.6, 156.9, 149.9, 140.9, 130.7, 118.2, 115.2, 111.1, 60.3, 59.1, 51.5, 47.9, 34.7, 29.5,
26.4, 26.2, 24.8. HRMS (ESI) m/z: Anal. calcd. for [M+Na]+ C38H52N6NaO12S: 839.3256; found 839.3255.
5.1.1.32.
Bis(3-(2-((S)-1-((R)-2-((methoxycarbonyl)amino)-2-phenylacetyl)pyrrolidin-2-yl)-1H-imidazol-5-
yl)phenyl) sulfate (22). Yield 97.2 mg (67%). 1H NMR (DMSO-d6, δ=2.5 ppm, 400 MHz): 12.05 (br s, 1H), 7.96 (s,
1H), 7.94-7.64 (m, 5H), 7.50 (s, 1H), 7.39-7.25 (m, 13H), 7.00-6.93 (d, 1H), 5.49-5.40 (m, 2H), 5.14-5.05 (d, 2H),
4.18-3.67 (m, 2H), 3.55 (s, 6H), 3.43-3.41 (t, 1H), 3.11 (s, 1H), 1.98-1.72 (m, 8H). 13C NMR (DMSO-d6, δ=39.52
ppm, 100 MHz): 173.4, 172.1, 168.4, 156.0, 150.5, 149.6, 137.2, 130.6, 128.4, 127.7, 124.2, 123.6, 119.0, 116.3,
109.8, 72.3, 60.3, 58.1, 57.0, 55.2, 51.6, 46.7, 31.1, 29.2, 24.0. HRMS (ESI) m/z: Anal. calcd. for [M+H]+
C46H47N8O10S: 903.3130; found 903.3133.
5.1.1.33. Bis(3-(2-((S)-1-((S)-2-((methoxycarbonyl)amino)-3,3-dimethylbutanoyl)pyrrolidin-2-yl)-1H-imidazol-5-
yl)phenyl) sulfate (23). Yield 43.8 mg (73%). 1H NMR (DMSO-d6, δ=2.5 ppm, 400 MHz): 8.07-7.83 (m, 1H), 7.78-
7.71 (m, 4H), 7.66-7.63 (t, 1H), 7.57-7.40 (m, 3H), 7.29-7.21 (m, 2H), 7.08-7.06 (d, 1H), 5.42-5.05 (m, 2H), 4.21-
4.19 (d, 2H), 3.81-3.68 (m, 4H), 3.54 (s, 6H), 2.18-2.08 (m, 3H), 2.02-1.66 (m, 5H), 0.94-0.88 (app br s, 18H). 13
C
NMR (DMSO-d6, δ=39.52 ppm, 125 MHz): 172.7, 169.7, 156.9, 150.4, 130.5, 128.6, 127.4, 125.1, 124.5, 123.5,
119.6, 119.1, 116.4, 109.9, 83.4, 62.8, 59.2, 53.8, 52.5, 51.5, 47.5, 34.6, 30.9, 26.3, 24.4, 16.5. HRMS (ESI) m/z:
Anal. calcd. for [M+H]+ C42H55N8O10S: 863.3756; found 863.3757.
5.1.1.34. Bis(3-(2-((S)-1-((methoxycarbonyl)-
L-valyl)pyrrolidin-2-yl)-1H-imidazol-5-yl)phenyl) sulfate (24). Yield
60.8 mg (44%). 1H NMR (DMSO-d6, δ=2.5 ppm, 400 MHz): 11.98 (br s, 1H), 7.98-7.96 (d, 1H), 7.77-7.75 (d, 3H),

ACCEPTED MANUSCRIPT
7.71-7.69 (d, 1H), 7.59 (s, 1H), 7.53-7.45 (q, 3H), 7.42-7.38 (t, 1H), 7.28-7.26 (d, 1H), 7.21-7.18 (d, 1H), 5.06 (s,
2H), 4.05-4.01 (t, 2H), 3.86-3.76 (m, 4H), 3.53 (s, 6H), 2.12-2.07 (m, 4H), 1.94-1.87 (m, 6H), 0.91-0.84 (m, 8H),
0.81-0.80 (d, 4H). 13C NMR (DMSO-d6, δ=39.52 ppm, 125 MHz): 170.4, 156.8, 150.4, 149.8, 131.1, 129.9, 127.8,
126.6, 123.9, 118.8, 116.8, 110.3, 57.7, 57.2, 54.7, 53.6, 51.3, 29.9. HRMS (ESI) m/z: Anal. calcd. for [M+H]+
C40H51N8O10S: 835.3443; found 835.3442.
5.1.1.35.
Bis(3-((S)-1-((R)-2-((methoxycarbonyl)amino)-2-phenylacetyl)pyrrolidine-2-carboxamido)-2-
1
methylphenyl) sulfate (25). Yield 84.5 mg (85%). H NMR (DMSO-d6, δ=2.5 ppm, 500 MHz): 9.73-9.56 (d, 2H),
7.76-7.58 (m, 2H), 7.48-7.21 (m, 15H), 7.12-6.67 (m, 1H), 5.52-5.43 (m, 2H), 4.65-4.47 (m, 2H), 3.85 (s, 1H), 3.75-
3.62 (m, 1H), 3.55-3.50 (app br s, 6H), 3.17 (s, 2H), 2.16 (s, 6H), 2.00-1.73 (m, 8H). 13C NMR (DMSO-d6, δ=39.52
ppm, 125 MHz): 170.4, 168.8, 156.1, 148.6, 138.2, 137.1, 128.6, 128.1, 126.8, 126.0, 125.3, 118.3, 60.6, 56.8, 51.6,
47.0, 29.1, 24.3, 11.3. HRMS (ESI) m/z: Anal. calcd. for [M+Na]+ C44H48N6NaO12S: 907.2943; found 907.2940.
5.1.1.36.
Bis(5-((S)-1-((R)-2-((methoxycarbonyl)amino)-2-phenylacetyl)pyrrolidine-2-carboxamido)-2-
methylphenyl) sulfate (26). Yield 105 mg (74%). 1H NMR (DMSO-d6, δ=2.5 ppm, 400 MHz): 10.36-10.19 (d, 2H),
7.94-7.89 (d, 2H), 7.75-7.67 (q, 2H), 7.58 (s, 2H), 7.42-7.41 (d, 3H), 7.36-7.07 (m, 9H), 5.52-5.47 (t, 2H), 4.47-4.38
(d, 2H), 3.82-3.66 (d, 2H), 3.53 (s, 6H), 3.18-3.09 (d, 2H), 2.22 (s, 6H), 1.99-1.78 (m, 8H). 13C NMR (DMSO-d6,
δ=39.52 ppm, 100 MHz): 170.6, 168.4, 156.1, 148.3, 138.5, 136.9, 132.0, 128.6, 128.1, 124.6, 118.5, 111.4, 60.7,
56.7, 51.6, 47.0, 29.3, 24.3, 15.3. HRMS (ESI) m/z: Anal. calcd. for [M+Na]+ C44H48N6NaO12S: 907.2943; found
907.2943.
5.2. Biological Studies
5.2.1. Measurement of the anti-HCV activities of compounds using HCVcc
5.2.1.1. Cell line and cell culture
Huh 7.5.1 cells were grown in Dulbecco’s Modified Eagle’s medium (DMEM; Gibco) containing antibiotics (100
U/mL penicillin, 10 g/mL streptomycin) and 10% heat-inactivated fetal bovine serum (ꢀFBS; Hyclone) at 37°C in
µ
a humidified 6.0% CO2 incubator.
5.2.1.2. Virus production
In vitro transcription and RNA electroporation were processed as described in a previous report [54]. In vitro
transcribed RNA of JFH5a-Rluc-ad34, which is a derivative of JFH1 with adaptive mutations in the E2 and p7
regions [38], was transfected into Huh7.5.1 cells by electroporation. JFH5a-Rluc-ad34 virus contains a reporter
Renilla luciferase (Rluc) for convenient virus proliferation assay [38]. HCV-containing cell culture media were
collected 3~5 days after electroporation and filtered through a 0.45 µM pore size filter.
5.2.1.3. Antiviral activity test with infectious HCV particles
Huh 7.5.1 cells were inoculated with a JFH5a-Rluc-ad34 virus stock and cultivated for 3 h. At 3 h after the virus
inoculation, HCV-infected Huh7.5.1 cells were cultured with media containing serially diluted compounds. At 3

ACCEPTED MANUSCRIPT
days after the chemical treatment, the cells were harvested and their luciferase activities were measured using a
Renilla luciferase assay system (Promega) according to the manufacturer’s direction. Finally the luciferase activities
were normalized to those obtained from mock-treated cells.
5.2.2. Measurement of anti-HCV activities of compounds using HCV replicon
5.2.2.1. Cell line and cell culture
Huh 7.5.1 cells containing a bicistronic HCV replicon NK5.1-Gluc cell line (genotype 1b) were employed for the
interrogation of the anti-HCV activities of the compounds (Fig. 4) [55, 56]. The first open reading frame (ORF) of
the replicon contains the Gaussia luciferase gene fused with foot-and-mouth disease virus (FMDV) 2a gene and the
neomycin phosphotransferase gene, and the second ORF contains HCV nonstructural genes NS3-5. The replicon-
containing cells were cultivated under the same conditions as described above in presence of an additional antibiotic
G418 (0.5 mg/ml, Calbiochem).
Please, insert Figure 4 here.
5.2.2.2. Antiviral activity assay test with HCV replicon
Huh 7.5.1 cells containing HCV replicon (NK5.1-Gluc) were planted on a 12-well plate (figure 4). At 16 h after
cultivation, the replicon containing NK/R2AN-containing cells were incubated with media containing serially
diluted compounds for 3 days. After the chemical treatment, the cell culture media were collected and luciferase
activities were measured through the use of the Renilla luciferase assay system (Promega) according to
manufacturer’s direction, and then normalized to those obtained from mock-treated cells.
5.2.3. hERG ligand binding assay [57]
The hERG ligand binding assay was performed by using the hERG Fluorescence Polarization Assay Kit (Cat No.
PV5365), containing predictorTM hERG tracer red, predictorTM hERG membrane, and predictorTM hERG FP assay
buffer. Test compounds (10
volume polystyrene plate, corning #3677). Then, membrane (10
through the use of a multi-pipette (Multi 16 channel pipette type 5ꢂ50
µ
M) and positive control (Astemizole) were added to the 384-well plate (5
L) and tracer (5 L) were added to each plate
L, Thermo). The plate was then covered with
µL each; low-
µ
µ
µ
aluminum foil and was kept for 2–3 h at room temperature. Lastly, the fluorescence polarization of the hERG red
tracer was measured using a microplate reader (molecular devices, SpectraMax M5e, excitation (540 nm) emission
(585 nm) filters).
5.2.4. Plasma stability measurement
Rat plasma (297
µL) was pre-incubated for 5 min at 37°C in a water bath. The plasma and 100 µM test compounds
(3.0 L) were agitated through the use of a vortex, and incubated for 37°C in a water bath. Then the mixture was
µ

ACCEPTED MANUSCRIPT
sampled multiple times (0, 15, 30, 60, and 120 min). A mixture of crude sample (50 µL) and acetonitrile (100 µL)
was then treated by centrifugation at 10,000 rpm for 10 min at 25°C. Finally, the supernatant was isolated and
analyzed using a LC-MS/MS.
5.2.5. Ames test [58]
The Ames test was carried out with the Maron’s method [58] using Ames MPF™ 98/100 Mutagenicity Assay kit.
Histidine-dependent strains (TA98, and TA100) of Salmonella typhimurium were treated with inhibitors compounds
20 and 24 at 1,000
µg/plate and bis(4-aminophenyl) sulfate at 5,000 µg/plate and with positive controls (2-
nitrofluorene, sodium azide, and benzo[a]pyrene). The number of colonies was counted three times for each medium
to provide acceptable data for statistical analysis.
5.2.6. Observation of the effects of co-treatment of two compounds
Huh 7.5.1 cells containing the HCV replicon (NK5.1-Gluc) were plated on a 12-well plate (5 x 104 cells per well).
At 16 h after cultivation, the replicon containing cells were treated with inhibitors alone or with mixtures of
inhibitors and sofosbuvir at serially diluted concentrations. At 3 days after treatment, Gaussia luciferase activities
were measured using the Renilla luciferase assay system (Promega) according to the manufacturer’s direction, and
then normalized to those obtained from mock-treated cells. Combination index was calculated by using Compusyn
Freeware Program (ComboSyn, Inc).
6. Appendix A. Supplementary data.
Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.ejmech.
7. AUTHOR INFORMATION
Corresponding Author
* E-mail: kimbm@snu.ac.kr.
* E-mail: sungkey@postech.ac.kr.
Author Contributions
‡These two individuals contributed equally to this work.
8. ABBREVIATIONS
HCV, hepatitis C virus; Peg-IFNα, pegylated interferon α; SVR, sustained virological response; SOC, standard of
care; GT1a, hepatitis C virus genotype 1a, a subtype of hepatitis C virus genotype 1; GT1b, hepatitis C virus
genotype 1b, a subtype of hepatitis C virus genotype 1; DAA, direct-acting antiviral; NS5A, hepatitis C virus
nonstructural protein 5A; pM, picomolar; RNA, ribonucleic acid; Boc, tert-Butoxycarbonyl; TFA, Trifluoroacetic
acid; EDCI, 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide; DIPEA, N,N-Diisopropylethylamine; DCM,

ACCEPTED MANUSCRIPT
Dichloromethane; DMF, N,N-Dimethylformamide; EC50, concentration at which inhibition is half-maximal;
micromolar; nM, nanomolar; SAR, structure−activity relationship; hERG, the human ether-à-go-go-related gene
µM,
9. Acknowledgment
This study was supported by the Bio R&D Program (No. 2012M3A9A9054974) through the National Research
Foundation funded by the MEST, Republic of Korea.
10. References
[1] A.M. Di Bisceglie, Hepatitis C, The Lancet, 351 (1998) 351-355.
[2] A.U. Neumann, N.P. Lam, H. Dahari, D.R. Gretch, T.E. Wiley, T.J. Layden, A.S. Perelson, Hepatitis C viral
dynamics in vivo and the antiviral efficacy of interferon-alpha therapy, Science, 282 (1998) 103-107.
[3] T.L. Tellinghuisen, J. Marcotrigiano, C.M. Rice, Structure of the zinc-binding domain of an essential component
of the hepatitis C virus replicase, Nature, 435 (2005) 374-379.
[4] G.L. Davis, M.J. Alter, H. El-Serag, T. Poynard, L.W. Jennings, Aging of hepatitis C virus (HCV)-infected
persons in the United States: a multiple cohort model of HCV prevalence and disease progression, Gastroenterology,
138 (2010) 513-521, 521 e511-516.
[5] D. Lavanchy, The global burden of hepatitis C, Liver Int, 29 Suppl 1 (2009) 74-81.
[6] H.R. Rosen, Clinical practice. Chronic hepatitis C infection, N Engl J Med, 364 (2011) 2429-2438.
[7] G.M. Lauer, B.D. Walker, Hepatitis C virus infection, N. Engl. J. Med., 345 (2001) 41-52.
[8] C. Wang, L. Jia, H. Huang, D. Qiu, L. Valera, X. Huang, J.H. Sun, P.T. Nower, D.R. O'Boyle, 2nd, M. Gao, R.A.
Fridell, In vitro activity of BMS-790052 on hepatitis C virus genotype 4 NS5A, Antimicrob. Agents Chemother., 56
(2012) 1588-1590.
[9] S.L. Tan, M.G. Katze, How hepatitis C virus counteracts the interferon response: the jury is still out on NS5A,
Virology, 284 (2001) 1-12.
[10] E.R. Feeney, R.T. Chung, Antiviral treatment of hepatitis C, BMJ, 348 (2014) g3308.
[11] C.A. Coburn, P.T. Meinke, W. Chang, C.M. Fandozzi, D.J. Graham, B. Hu, Q. Huang, S. Kargman, J.
Kozlowski, R. Liu, J.A. McCauley, A.A. Nomeir, R.M. Soll, J.P. Vacca, D. Wang, H. Wu, B. Zhong, D.B. Olsen,
S.W. Ludmerer, Discovery of MK-8742: an HCV NS5A inhibitor with broad genotype activity, ChemMedChem, 8
(2013) 1930-1940.
[12] T. Nakano, G.M. Lau, G.M. Lau, M. Sugiyama, M. Mizokami, An updated analysis of hepatitis C virus
genotypes and subtypes based on the complete coding region, Liver Int, 32 (2012) 339-345.
[13] V.K. Rustgi, Safety of small molecules in combination with interferon-based therapy for hepatitis C virus,
Expert Opin. Drug Saf., 9 (2010) 883-892.
[14] F. Amblard, H. Zhang, L. Zhou, J. Shi, D.R. Bobeck, J.H. Nettles, S. Chavre, T.R. McBrayer, P. Tharnish, T.
Whitaker, S.J. Coats, R.F. Schinazi, Synthesis and evaluation of non-dimeric HCV NS5A inhibitors, Bioorganic &
medicinal chemistry letters, 23 (2013) 2031-2034.

ACCEPTED MANUSCRIPT
[15] R.E. Nettles, M. Gao, M. Bifano, E. Chung, A. Persson, T.C. Marbury, R. Goldwater, M.P. DeMicco, M.
Rodriguez-Torres, A. Vutikullird, E. Fuentes, E. Lawitz, J.C. Lopez-Talavera, D.M. Grasela, Multiple ascending
dose study of BMS-790052, a nonstructural protein 5A replication complex inhibitor, in patients infected with
hepatitis C virus genotype 1, Hepatology, 54 (2011) 1956-1965.
[16] S. Pol, R.H. Ghalib, V.K. Rustgi, C. Martorell, G.T. Everson, H.A. Tatum, C. Hezode, J.K. Lim, J.-P.
Bronowicki, G.A. Abrams, N. Braeu, D.W. Morris, P.J. Thuluvath, R.W. Reindollar, P.D. Yin, U. Diva, R. Hindes, F.
McPhee, D. Hernandez, M. Wind-Rotolo, E.A. Hughes, S. Schnittman, Daclatasvir for previously untreated chronic
hepatitis C genotype-1 infection: a randomised, parallel-group, double-blind, placebo-controlled, dose-finding,
phase 2a trial, Lancet Infect. Dis., 12 (2012) 671-677.
[17] C. Wang, H. Huang, L. Valera, J.-H. Sun, D.R. O'Boyle, II, P.T. Nower, L. Jia, D. Qiu, X. Huang, A. Altaf, M.
Gao, R.A. Fridell, Hepatitis C virus RNA elimination and development of resistance in replicon cells treated with
BMS-790052, Antimicrob. Agents Chemother., 56 (2012) 1350-1358.
[18] J.O. Link, J.G. Taylor, L. Xu, M. Mitchell, H. Guo, H. Liu, D. Kato, T. Kirschberg, J. Sun, N. Squires, J.
Parrish, T. Keller, Z.-Y. Yang, C. Yang, M. Matles, Y. Wang, K. Wang, G. Cheng, Y. Tian, E. Mogalian, E. Mondou,
M. Cornpropst, J. Perry, M.C. Desai, Discovery of Ledipasvir (GS-5885): A Potent, Once-Daily Oral NS5A Inhibitor
for the Treatment of Hepatitis C Virus Infection, Journal of medicinal chemistry, 57 (2014) 2033-2046.
[19] P. McCormack, Daclatasvir: A Review of Its Use in Adult Patients with Chronic Hepatitis C Virus Infection,
Drugs, 75 (2015) 515-524.
[20] D. Ross-Thriepland, M. Harris, Hepatitis C virus NS5A: enigmatic but still promiscuous 10 years on!, J Gen
Virol, 96 (2015) 727-738.
[21] I.H. Bae, J.K. Choi, C. Chough, S.J. Keum, H. Kim, S.K. Jang, B.M. Kim, Potent Hepatitis C Virus NS5A
Inhibitors Containing a Benzidine Core, ACS Med Chem Lett, 5 (2014) 255-258.
[22] M. Zhong, E. Peng, N. Huang, Q. Huang, A. Huq, M. Lau, R. Colonno, L. Li, Discovery of functionalized
bisimidazoles bearing cyclic aliphatic-phenyl motifs as HCV NS5A inhibitors, Bioorganic & medicinal chemistry
letters, 24 (2014) 5731-5737.
[23] R.J. Fontana, E.A. Hughes, M. Bifano, H. Appelman, D. Dimitrova, R. Hindes, W.T. Symonds, Sofosbuvir and
daclatasvir combination therapy in a liver transplant recipient with severe recurrent cholestatic hepatitis C, American
journal of transplantation: official journal of the American Society of Transplantation and the American Society of
Transplant Surgeons, 13 (2013) 1601-1605.
[24] M. Gao, R.E. Nettles, M. Belema, L.B. Snyder, V.N. Nguyen, R.A. Fridell, M.H. Serrano-Wu, D.R. Langley,
J.H. Sun, D.R. O'Boyle, 2nd, J.A. Lemm, C. Wang, J.O. Knipe, C. Chien, R.J. Colonno, D.M. Grasela, N.A.
Meanwell, L.G. Hamann, Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect,
Nature, 465 (2010) 96-100.
[25] M. Issur, M. Götte, Resistance Patterns Associated with HCV NS5A Inhibitors Provide Limited Insight into
Drug Binding, Viruses, 6 (2014) 4227-4241.
[26] R. Schinazi, P. Halfon, P. Marcellin, T. Asselah, HCV direct-acting antiviral agents: the best interferon-free
combinations, Liver international: official journal of the International Association for the Study of the Liver, 34

ACCEPTED MANUSCRIPT
Suppl 1 (2014) 69-78.
[27] I.H. Bae, H.S. Kim, Y. You, C. Chough, W. Choe, M.K. Seon, S.G. Lee, G. Keum, S.K. Jang, B. Moon Kim,
Novel benzidine and diaminofluorene prolinamide derivatives as potent hepatitis C virus NS5A inhibitors, Eur. J.
Med. Chem., 101 (2015) 163-178.
[28] A.V. Ivachtchenko, O.D. Mitkin, P.M. Yamanushkin, I.V. Kuznetsova, E.A. Bulanova, N.A. Shevkun, A.G.
Koryakova, R.N. Karapetian, V.V. Bichko, A.S. Trifelenkov, D.V. Kravchenko, N.V. Vostokova, M.S. Veselov, N.V.
Chufarova, Y.A. Ivanenkov, Discovery of Novel Highly Potent Hepatitis C Virus NS5A Inhibitor (AV4025), Journal
of medicinal chemistry, 57 (2014) 7716-7730.
[29] K.A.R. Mitchell, Use of outer d orbitals in bonding, Chemical reviews, 69 (1969) 157-178.
[30] C.A. Coulson, d Electrons and Molecular Bonding, Nature, 221 (1969) 1106-1110.
[31] D.R. St. Laurent, M. Belema, M. Gao, J. Goodrich, R. Kakarla, J.O. Knipe, J.A. Lemm, M. Liu, O.D. Lopez,
V.N. Nguyen, P.T. Nower, D. O'Boyle, Y. Qiu, J.L. Romine, M.H. Serrano-Wu, J.-H. Sun, L. Valera, F. Yang, X.
Yang, N.A. Meanwell, L.B. Snyder, HCV NS5A replication complex inhibitors. Part 2: Investigation of stilbene
prolinamides, Bioorg. Med. Chem. Lett., 22 (2012) 6063-6066.
[32] A. El-Faham, F. Albericio, Peptide coupling reagents, more than a letter soup, Chemical reviews, 111 (2011)
6557-6602.
[33] D.R. St Laurent, M.H. Serrano-Wu, M. Belema, M. Ding, H. Fang, M. Gao, J.T. Goodrich, R.G. Krause, J.A.
Lemm, M. Liu, O.D. Lopez, V.N. Nguyen, P.T. Nower, D.R. O'Boyle, 2nd, B.C. Pearce, J.L. Romine, L. Valera, J.H.
Sun, Y.K. Wang, F. Yang, X. Yang, N.A. Meanwell, L.B. Snyder, HCV NS5A replication complex inhibitors. Part 4.
Optimization for genotype 1a replicon inhibitory activity, Journal of medicinal chemistry, 57 (2014) 1976-1994.
[34] J. Shi, L. Zhou, F. Amblard, D.R. Bobeck, H. Zhang, P. Liu, L. Bondada, T.R. McBrayer, P.M. Tharnish, T.
Whitaker, S.J. Coats, R.F. Schinazi, Synthesis and biological evaluation of new potent and selective HCV NS5A
inhibitors, Bioorg. Med. Chem. Lett., 22 (2012) 3488-3491.
[35] T.R. Webb, C. Eigenbrot, Conformationally restricted arginine analogs, The Journal of organic chemistry, 56
(1991) 3009-3016.
[36] B.T. Guan, X.Y. Lu, Y. Zheng, D.G. Yu, T. Wu, K.L. Li, B.J. Li, Z.J. Shi, Biaryl construction through Kumada
coupling with diaryl sulfates as one-by-one electrophiles under mild conditions, Org Lett, 12 (2010) 396-399.
[37] S.A. Qureshi, Hepatitis C virus-biology, host evasion strategies, and promising new therapies on the horizon,
Med. Res. Rev., 27 (2007) 353-373.
[38] C.S. Kim, S.J. Keum, S.K. Jang, Generation of a cell culture-adapted hepatitis C virus with longer half life at
physiological temperature, PloS one, 6 (2011) e22808.
[39] R. Bartenschlager, V. Lohmann, F. Penin, The molecular and structural basis of advanced antiviral therapy for
hepatitis C virus infection, Nat. Rev. Microbiol., 11 (2013) 482-496.
[40] C. Liang, E. Rieder, B. Hahm, S.K. Jang, A. Paul, E. Wimmer, Replication of a novel subgenomic HCV
genotype 1a replicon expressing a puromycin resistance gene in Huh-7 cells, Virology, 333 (2005) 41-53.
[41] K.J. Blight, A.A. Kolykhalov, C.M. Rice, Efficient initiation of HCV RNA replication in cell culture, Science,
290 (2000) 1972-1974.

ACCEPTED MANUSCRIPT
[42] B.D. Lindenbach, M.J. Evans, A.J. Syder, B. Woelk, T.L. Tellinghuisen, C.C. Liu, T. Maruyama, R.O. Hynes,
D.R. Burton, J.A. McKeating, C.M. Rice, Complete Replication of Hepatitis C Virus in Cell Culture, Science, 309
(2005) 623-626.
[43] T. Burke, K. Loniello, J. Beebe, K. Ervin, Development and Application of Fluorescence Polarization Assays in
Drug Discovery, Combinatorial chemistry & high throughput screening, 6 (2003) 183-194.
[44] R.J. Vaz, Y. Li, D. Rampe, Human Ether-a-go-go Related Gene (HERG): A Chemist's Perspective, 43 (2005) 1-
18.
[45] D.R. Piper, S.R. Duff, H.C. Eliason, W.J. Frazee, E.A. Frey, M. Fuerstenau-Sharp, C. Jachec, B.D. Marks, B.A.
Pollok, M.S. Shekhani, D.V. Thompson, P. Whitney, K.W. Vogel, S.D. Hess, Development of the predictor HERG
fluorescence polarization assay using a membrane protein enrichment approach, Assay Drug Dev Technol, 6 (2008)
213-223.
[46] F. Weinsberg, C.K. Bauer, J.R. Schwarz, The class III antiarrhythmic agent E-4031 selectively blocks the
inactivating inward-rectifying potassium current in rat anterior pituitary tumor cells (GH 3 /B 6 cells), Pflügers
Archiv European Journal of Physiology, 434 (1997) 1-10.
[47] D.R. Piper, S.R. Duff, H.C. Eliason, W.J. Frazee, E.A. Frey, M. Fuerstenau-Sharp, C. Jachec, B.D. Marks, B.A.
Pollok, M.S. Shekhani, D.V. Thompson, P. Whitney, K.W. Vogel, S.D. Hess, Development of the predictor HERG
fluorescence polarization assay using a membrane protein enrichment approach, Assay and drug development
technologies, 6 (2008) 213-223.
[48] L. Di, E.H. Kerns, Y. Hong, H. Chen, Development and application of high throughput plasma stability assay
for drug discovery, International journal of pharmaceutics, 297 (2005) 110-119.
[49] N.J. Waters, R. Jones, G. Williams, B. Sohal, Validation of a rapid equilibrium dialysis approach for the
measurement of plasma protein binding, J Pharm Sci, 97 (2008) 4586-4595.
[50] D.M. Maron, B.N. Ames, Revised methods for the Salmonella mutagenicity test, Mutation
Research/Environmental Mutagenesis and Related Subjects, 113 (1983) 173-215.
[51] K. Mortelmans, E. Zeiger, The Ames Salmonella/microsome mutagenicity assay, Mutation
Research/Fundamental and Molecular Mechanisms of Mutagenesis, 455 (2000) 29-60.
[52] K. Mortelmans, E.S. Riccio, The bacterial tryptophan reverse mutation assay with Escherichia coli WP2,
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 455 (2000) 61-69.
[53] T.C. Chou, Theoretical basis, experimental design, and computerized simulation of synergism and antagonism
in drug combination studies, Pharmacol Rev, 58 (2006) 621-681.
[54] T. Wakita, T. Pietschmann, T. Kato, T. Date, M. Miyamoto, Z. Zhao, K. Murthy, A. Habermann, H.G.
Krausslich, M. Mizokami, R. Bartenschlager, T.J. Liang, Production of infectious hepatitis C virus in tissue culture
from a cloned viral genome, Nature medicine, 11 (2005) 791-796.
[55] N. Horscroft, V.C.H. Lai, W. Cheney, N. Yao, J.Z. Wu, Z. Hong, W. Zhong, Replicon cell culture system as a
valuable tool in antiviral drug discovery against hepatitis C virus, Antiviral Chem. Chemother., 16 (2005) 1-12.
[56] W. Cun, J. Jiang, G. Luo, The C-terminal α-helix domain of apolipoprotein E is required for interaction with
nonstructural protein 5A and assembly of hepatitis C virus, J. Virol., 84 (2010) 11532-11541.

ACCEPTED MANUSCRIPT
[57] T.J. Burke, K.R. Loniello, J.A. Beebe, K.M. Ervin, Development and application of fluorescence polarization
assays in drug discovery, Combinatorial chemistry & high throughput screening, 6 (2003) 183-194.
[58] D.M. Maron, B.N. Ames, Revised methods for the Salmonella mutagenicity test, Mutat. Res., 113 (1983) 173-
215.

ACCEPTED MANUSCRIPT
Table 1. In vitro activities of inhibitors containing a p-,p’-disubstituted biaryl sulfone or sulfate core
Cytotoxicity
Replicon EC50 HCVCC EC50
Entry Compounds
X
Linker-L-Pro-Cap
(GT-1b) (nM)
(GT-2a) (nM)
(µM)
1
36.1
-
> 30
3
2
3
4
5
> 105
> 105
2,570
575
-
-
-
-
> 30
> 30
> 30
> 30
4
5
13
14
6
7
0.0100
0.270
0.128
0.147
> 30
> 30
15
16
8
9
417
427
-
> 30
> 30
17
18
4.26

ACCEPTED MANUSCRIPT
Table 2. In vitro activities of inhibitors containing an m-,m’-disubstituted biaryl sulfate core
Cytotoxicity
Replicon EC50 HCVCC EC50
Entry Compounds
R
Linker-L-Pro-Cap
(GT-1b) (nM)
(GT-2a) (nM)
(µM)
R1 = H
R2 = H
1
0.0198
0.0199
> 30
20
R1 = H
R2 = H
2
3
0.0772
0.360
0.943
10.0
> 30
> 30
21
22
R1 = H
R2 = H
R1 = H
R2 = H
R1 = H
R2 = H
4
5
37.1
-
> 30
> 20
23
24
0.0323
0.882
R1 = H
6
7
22.9
-
> 30
> 30
25
26
R2 = CH3
R1 = CH3
R2 = H
0.109
2.23

ACCEPTED MANUSCRIPT
Table 3. Result of hERG ligand binding assay (mean ± SD, n=3)
Treatment
% inhibition
2.73 ± 3.16
18.7 ± 4.58
66.0 ± 5.22
Concentration (µM)
10
10
10
20
24
Compound
Positive control
Astemizole

ACCEPTED MANUSCRIPT
Table 4. Plasma stability (% remaining after 4 h incubation at 37°C)
Treatment
Rat (%)
76.7 ± 10.6
87.2 ± 12.6
0.032 ± 0.003
-
Human (%)
86.3 ± 12.3
92.6 ± 9.69
-
20
Compound
24
Enalapril
Procaine
Positive control
0.003 ± 0.002

ACCEPTED MANUSCRIPT
Table 5. Result of bacterial reverse mutation assay with compounds 20 and 24 (Mean ± SD, n=3)
Revertant colonies/plate (Mean ± SD)
Dose
(µg/plate)
0
Tester strain
compound
Without S-9 mix
With S-9 mix
25 ± 3
vehicle control
21 ± 1
26 ± 3
1,000
1,000
29 ± 2
20
24
TA98
28 ± 4
29 ± 4
1 ± 1
1 ± 1
Bis(4-aminophenyl)
sulfate
5,000
(vehicle control: 1 ± 1)
125 ± 9
(vehicle control: 1 ± 1)
129 ± 3
vehicle control
0
1,000
1,000
126 ± 7
130 ± 9
20
24
TA100
125 ± 12
110 ± 2
10 ± 7
8 ± 1
Bis(4-aminophenyl)
sulfate
5,000
(vehicle control: 7 ± 2)
Without S-9 mix
183 ± 24
(vehicle control: 5 ± 2)
With S-9 mix
ND
Positive control
2-nitrofluorene
Benzo(a)pyrene
Sodium azide
Dose
1 µg/plate
TA98
2 µg/plate
ND[a]
198 ± 15
ND
1
2
748 ± 25
TA100
Benzo(a)pyrene
ND
778 ± 14
[a] ND: Non Determined.

ACCEPTED MANUSCRIPT
Figure 1. Structure of NS5A inhibitors in market or clinical trials.