Design, synthesis and identification of silicon-containing HCV NS5A inhibitors with pan-genotype activity

Article abstract of DOI:10.1016/j.ejmech.2018.02.025

Modification of a HCV NS5A inhibitor, ombitasvir, led to the identification of 10d with improved pan-genotype NS5A inhibition and better pharmacokinetic properties. The key structural changes to ombitasvir include bioisosteric replacement of carbon with silicon atom. Compared with ombitasvir, the activity of anti-HCV genotypes (GT 1 to 6) of 10d is increased to some extent, especially the inhibitory activity against genotype 3a and 6a is increased by more than seven times, and the dog's in vivo pharmacokinetics properties were also superior to ombitasvir. Further drug evaluation showed that 10d was similar to ombitasvir on plasma protein binding and liver distribution profiles, with no cytotoxicity and no inhibitory effect on both CYP 450 and hERG ligand binding. However, permeability assay results indicated that 10d was not the substrate of P-gp or BCRP transporter, which is different from that of ombitasvir. The results of a 14-day repeat-dose toxicity study identified no toxicity with 10d. Our findings in preclinical tests suggest that the silicon-containing compound 10d could be worthy of continued study as a potential drug candidate.

Full text of DOI:10.1016/j.ejmech.2018.02.025

Accepted Manuscript
Design, synthesis and identification of silicon-containing HCV NS5A inhibitors with
pan-genotype activity
Baomin Liu, Kuo Gai, Hui Qin, Xushi Liu, Yuan Cao, Qin Lu, Dandan Lu, Deyang
Chen, Hengqiao Shen, Wei Song, Yang Zhang, Xiaojin Wang, Hongjiang Xu,
Yinsheng Zhang
PII:
S0223-5234(18)30153-3
10.1016/j.ejmech.2018.02.025
EJMECH 10202
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 28 December 2017
Revised Date: 30 January 2018
Accepted Date: 9 February 2018
Please cite this article as: B. Liu, K. Gai, H. Qin, X. Liu, Y. Cao, Q. Lu, D. Lu, D. Chen, H. Shen, W.
Song, Y. Zhang, X. Wang, H. Xu, Y. Zhang, Design, synthesis and identification of silicon-containing
HCV NS5A inhibitors with pan-genotype activity, European Journal of Medicinal Chemistry (2018), doi:
10.1016/j.ejmech.2018.02.025.
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
Design, synthesis and identification of silicon-containing HCV NS5A
inhibitors with pan-genotype activity
Baomin Liu, Kuo Gai, Hui Qin, Xushi Liu, Yuan Cao, Qin Lu, Dandan Lu, Deyang
Chen, Hengqiao Shen, Wei Song, Yang Zhang, Xiaojin Wang, Hongjiang Xu,
Yinsheng Zhang*
Chia Tai Tianqing Pharmaceutical Group Co., LTD, Jiangsu Key Laboratory of Antiviral Drug
Research, 699-8 Xuanwu Ave. Building #9, Nanjing 210023, Jiangsu Province, China
*Correspondence to : email: zys@cttq.com; tel: 01186-025-68551568;
address: Chia Tai Tianqing Pharmaceutical Group Co., LTD, Jiangsu Key Laboratory of
Antiviral Drug Research, 699-8 Xuanwu Ave. Building #9, Nanjing 210023, Jiangsu Province,
China
Abstract
Modification of a HCV NS5A inhibitor, ombitasvir, led to the identification of 10d
with improved pan-genotype NS5A inhibition and better pharmacokinetic properties.
The key structural changes to ombitasvir include bioisosteric replacement of carbon
with silicon atom. Compared with ombitasvir, the activity of anti-HCV genotypes (GT
1 to 6) of 10d is increased to some extent, especially the inhibitory activity against
genotype 3a and 6a is increased by more than seven times, and the dog’s in vivo
pharmacokinetics properties were also superior to ombitasvir. Further drug evaluation
showed that 10d was similar to ombitasvir on plasma protein binding and liver
distribution profiles, with no cytotoxicity and no inhibitory effect on both CYP 450
and hERG ligand binding. However, permeability assay results indicated that 10d was
not the substrate of P-gp or BCRP transporter, which is different from that of
ombitasvir. The results of a 14-day repeat-dose toxicity study identified no toxicity
with 10d. Our findings in preclinical tests suggest that the silicon-containing
compound 10d could be worthy of continued study as a potential drug candidate.
Keywords
Hepatitis C virus ; NS5A inhibitor; Ombitasvir; Silicon atom; Anti-HCV

ACCEPTED MANUSCRIPT
1. Introduction
Chronic infection caused by hepatitis C virus (HCV) is a liver disease, which can lead
to cirrhosis of the liver and hepatocellular carcinoma. It is estimated that more than
177 million adults have been infected with HCV worldwide [1].
HCV is a single-stranded, positive-sense RNA virus. It is responsible for encoding
a total of three thousand amino acids [2]. At least 10 structural and nonstructural (NS)
proteins (NS2, NS3, NS4A, NS4B, NS5A, NS5B, and protein p7, derived from E2
cleavage) are translated from HCV RNA [3]. Given their involvement in the viral life
cycle, several nonstructural (NS) proteins have been treated as targets for HCV
therapy [4]. In particular, the nonstructural viral protein NS5A is an attractive target
for small-molecule inhibition for its relevant role in viral replication, reorganized
membrane assembly, and complex interactions with cellular functions [5-7].
Traditional treatment options for HCV such as interferon and ribavirin are poorly
tolerated and produces low sustained virological response (SVR) rates (42-46%)[8].
Fortunately, direct-acting antivirals (DAAs) approved by the Food and Drug
Administration (FDA), such as the NS3/4A protease inhibitors simeprevir [9] and
paritaprevir [10], the NS5B inhibitors sofosbuvir [11] and dasabuvir [12], the NS5A
inhibitors daclatasvir [13], ledipasvir [14], elbasvir [15], ombitasvir [16], velpatasvir
[17] and pibrentasvir [18 ] have led to significant improvement in treating chronic
hepatitis C (CHC). However, most of the existing approved drugs showed different
potency against various HCV genotypes (GT 1 to 6), demonstrating a clear unmet
medical need for high potency, pan-genotype activity drugs.
Small-molecule NS5A inhibitors are important components of several combination
therapies such as Harvoni®, Zepatier®, Technivie® & Eplcusa®, and have been
considered as the most promising direct acting antiviral agents [19]. Ombitasvir
(ABT-267) is a NS5A inhibitor recently approved by the FDA for use in the
complicated combination with paritaprevir, ritonavir and dasabuvir in the product
Viekira Pak for the treatment of HCV genotype 1 [20], and with paritaprevir and
ritonavir in the product Technivie ® for the treatment of HCV genotype 4 [21]. In an
effort to develop better NS5A inhibitors, we choose ombitasvir (OMBITASVIR) as a
lead to identify inhibitors with more potent activity against all genotypes. Herein, we
describe our efforts to investigate the impact of introducing a silicon atom into the
ombitasvir molecule.

ACCEPTED MANUSCRIPT
Incorporation of a silicon atom into drug molecules has attracted attention as a drug
design principle that can have the potential to affect and, ideally, improve both the
bioactivity and ADMET (absorption, distribution, metabolism, excretion and toxicity)
profiles of compounds [22]. The changes in biological and pharmaceutical properties
due to sila-substitution may be attributed to the different properties of silicon from
carbon, such as atomic size, electronegativity and hydrophobicity[22]. We adopted a
carbon-silicon (C-Si) switch strategy to design a series of novel silicon-containing
compounds with the goal of identifying novel inhibitors that possess both
pan-genotype activity and improved pharmacokinetic (PK) properties over the known
drug, ombitasvir. The strategy focused on replacing the tert-butyl group of ombitasvir
(ABT-267) with several silyl groups to give its novel silicon analogs (Figure 1). All
the silicon analogues were assessed against various of HCV genotypes. The most
potent compound 10d was identified as a promising candidate after an in depth
investigation of its ADMET profiles.
Figure 1. Design of silicon incorporated ombitasvir analogs.
2. Results and Discussion
2.1. Chemistry
The synthesis of the silicon derivatives 10a-10i was carried out as shown in Scheme 1.
The silyl-aniline 3I was synthesized in a two-step process with good yields by
following Kimes’ procedures [23]. Availability of triethylsaline and
t-butyldimethylsaline allowed us to prepare the silyl-amines (3II & 3III) in one C-Si
cross-coupling step according to the reported method [24] which used Rh(cod)2BF4 as
a catalyst and K3PO4 as a base.
Scheme 1. Synthesis of silicon-containing HCV NS5A inhibitorsa

ACCEPTED MANUSCRIPT
aReagents and conditions: (a) Pd(PPh3)4, hexamethyldisilane, xylene 170°C; (b) 10% Pd/C, H2,
EtOH, rt; (c) Rh(cod)2BF4, hydrosilanes, N-methylpyrolidinone, K3PO4, rt; (d) Compounds
3I-3III, Et3N, DMF, 60°C; (e) PtO2, H2, rt; (f) (phenoxycarbonyl)-L-proline, EDCI, HOBT,
N-methylmorpholine, DMF; (g) H2, 10% Pd/C, EtOH/MeOH, 10 atm; (h)
(methoxycarbonyl)-L-valine, EDCI, HOBT, N-methylmorpholine, DMF.
The di-mesylates 5 (three stereoisomers, 1,4-SS, RR, SR) were then treated with the
silyl-aniline (3I-3III) of choice in the presence of triethylamine to provide pyrrolidine
derivatives 6a-6i as a crude. Without further purification, di-nitro compounds 6a-6i
were reduced to di-aniline compounds 7a-7i under PtO2 catalyzed hydrogenation
condition. Compounds 7a-7i as a three-stereoisomer mixture can be readily separated
into racemic trans-pyrrolidine (7a/7b, 7d/7e, 7g/7h) and cis-pyrrolidine (7c,7f,7i)
isomers by silica gel chromatography. The proline amide side chains were attached to
pyrrolidine derivatives (7a-7i) by peptide coupling with cbz-proline to give 8a-8i,
followed
by
deprotection
and
further
peptide
coupling
with
(methoxycarbonyl)-L-valine to afford the final products 10a-10i. Final compounds
prepared using racemic trans-pyrrolidine isomers were further separated into
enantiomerically pure trans-pyrrolidine analogues on a CHIRALART Amylose-SA

ACCEPTED MANUSCRIPT
column. The cis-pyrrolidine intermediates gave the cis-pyrrolidine analogues
(10c,10f,10i) as final compounds directly. A single stereoisomer (1R,4R) of 5 was also
prepared according to the literature method [25] and applied to prepare 10a, 10d and
10g as a single enantiomer.
2.2. In vitro anti-HCV activity
To determine whether the compounds are effective HCV inhibitors, we investigated
the inhibitory activities of the synthesized compounds using the GT1a and GT1b
subgenomic replicon assays. As indicated in Table 1, most of the compounds showed
similar potency with ombitasvir against GT1b and three compounds (10a, 10d, 10g)
demonstrated higher potency than ombitasvir against GT1a. It’s noted that the 2S,5S
isomers (10a, 10d & 10g) exhibited increased potency against GT1a compared to
their 2R,5R isomers (10b, 10e & 10h), while the cis-isomers showed no obvious
potency against GT1a. In addition, the 50% cytotoxic concentration (CC50) values of
all the compounds tested in an HCV genotype 1a replicon cell line was more than 1
nM.
Table 1. In
vitro activities
of compounds
in
the
replicon
assay.
Compounds Configuration
R1
R2
R3 Replicon
(nM)a
EC50
GT1b
Cytotoxicity
CC50 (nM)a
GT1a
2S,5S
2R,5R
cis
Me Me Me
Me Me Me
Me Me Me
0.014
0.118
>0.5
0.007
0.041
>0.5
0.011
ndb
0.004
0.007
0.008
0.003
0.006
0.007
0.003
ndb
>1
>1
>1
>1
>1
>1
>1
ndb
ndb
>1
10a
10b
10c
10d
2S,5S
2R,5R
cis
Et
Et
Et
Et
Et
Et
Et
Et
Et
10e
10f
2S,5S
2R,5R
cis
Me Me t-Bu
Me Me t-Bu
Me Me t-Bu
10g
10h
ndb
ndb
10i
Ombitasvir
-
-
-
-
0.03
0.005

ACCEPTED MANUSCRIPT
aMean of triplicate well values. All experiments were performed at least twice. EC50 stands
for 50% effective concentration; CC50 stands for 50% cytotoxic concentration in an HCV
genotype 1a replicon cell line.b Not determined.
As a result of their picomolar (pM) potency, compound 10a, 10d and 10g were
selected for further in vitro virologic evaluation. As shown in Table 2, the selected
compounds demonstrated picomolar potency not only against GT1a and GT1b, but
also against GT2 through GT6. It is interesting to note that the three selected
compounds showed more potent inhibitory activity against GT3a and GT6a in
comparison to ombitasvir, with 2.3-, 8.6- and 7.2-fold increased activity against GT3a
and 2.4-, 7.6- and 3.7-fold increased activity against GT6a, respectively.
Table 2. Antiviral activity of 10a, 10d & 10g in HCV replicon cell lines
containing NS5A from GT1-6.
Replicon EC50 (pM)a
Compound
10a
GT1a
14
GT1b
GT2a
2.5
GT3a
38
GT4a
0.1
GT5a
GT6a*
1411
445
4
3
3
5
1
1
1
4
10d
7
0.8
10
0.1
11
1.1
12
0.1
925
10g
Ombitasvir
30
2.6
86
0.3
3392
aMean of triplicate well values. All experiments were performed at least twice. EC50 stands
for 50% effective concentration.
* EC50 data for ombitasvir was reported in 2015[26]. The activity against GT6a from the
published data (55 pM) for ombitasvir is quite different from our own data (3392 pM).
Differences observed between published data and our data might result from differences in
laboratory and cell source.
2.3. Pharmacokinetic studies
Based on their broad genotype activity, pharmacokinetic studies were performed for
the selected compounds (10a,10d,10g). In vitro metabolic stability in mouse, rat and
human microsomes (MLM, RLM and HLM, respectively) for the set of compounds
appear in Table 3. Mouse, rat and human microsomal stability was generally similar
for the compounds. All compounds were stable when it was incubated for 60 min in
the presence of 0.2 mg/ml concentration of mouse, rat or human liver microsomal
fractions.
Table 3. In vitro metabolic stability data.
Compound
MLM
（% remaininga）
93.3
RLM
（% remaininga）
100.9
HLM
(% remaininga)
99.2
10a

ACCEPTED MANUSCRIPT
92.7
92.1
88.7
98.0
87.0
85.6
84.3
96.2
10d
10g
Ombitasvir
100.8
a Percentage of parent compound remaining after incubation for 60 min in the presence of 0.2
mg/ml mouse, rat or human liver microsomal fractions.
Oral pharmacokinetic data in mouse were obtained using solutions in organic
solvents in order to aid in dissolution and absorption of the compounds. The selected
compounds showed longer half-life but lower plasma concentrations (AUC)
compared to ombitasvir (Table 4). Especially for compound 10d and 10g, plasma
half-life were longer 2.9- and 4.5-fold than ombitasvir, respectively.
Table 4. In vivo pharmacokinetic parameters of selected compounds in mouse.
Compound
Tmax (h)
Ombitasvir
3.0
10a
3
10d
3
10g
3
Cmax (ng/mL)
t1/2(h)
262.1
8.8
198.2
22.5
3041
211.4
34.7
3516
583.3
7.7
AUC(0-t) (ng*h/mL)
2914
6480
Considering its better broad genotype activity and pharmacokinetic properties,
compound 10d was selected for further pharmacokinetic properties studies in dog. In
comparison to ombitasvir, it is very interesting that compound 10d showed an
improved plasma exposure (604.7 VS 187.5) with similar half-life (12.7h VS 11.1h) in
dog following oral dosing (Table 5). The pharmacokinetic data in dog are very
different from that in mouse. The results support this canine setting for further
evaluation of compound 10d.
Table 5. In vivo pharmacokinetic parameters of 10d in dog.
Compound
Ombitasvir
10d
Tmax(h)
10.5
29.18
12.7
8.3
Cmax (ng/mL)
t1/2(h)
15.68
11.1
AUC(0-t) (ng*h/mL)
604.7
187.5
2.4. Plasma protein binding
Studies were performed to evaluate plasma protein binding to compound 10d and
ombitasvir and species differences (Table 6) as well. Equilibrium dialysis studies
demonstrated that compound 10d is highly bound (>99.9%) to human, dog, rat and

ACCEPTED MANUSCRIPT
mouse albumin, similar to ombitasvir. High plasma protein binding limits the
partitioning of the testing compounds from the blood into the tissues where they could
be metabolized. The results are consistent with the highly lipophilic property of both
compounds.
Table 6. Plasma protein binding profiles of 10d and ombitasvir.
10d
Ombitasvir
99.998%
99.999%
99.996%
99.999%
99.998%
99.999%
99.997%
99.996%
Rat
Mice
Dog
1 µM
10 µM
1 µM
99.991%
99.983%
99.992%
99.989%
99.996%
99.994%
99.992%
99.992%
10 µM
1 µM
10 µM
1 µM
Human
10 µM
2.5. Apparent permeability in human Caco-2 cells
To obtain information about their apparent permeability (Papp), 10d and ombitasvir
were studied in a human Caco-2 cell model (Table 7). For the apical-to-basolateral
(A→B) transport, the apparent permeability was low for both 10d (Papp= 0.7 nm/s)
and ombitasvir (Papp= 0.8 nm/s). The efflux ratio was low for 10d (0.8) and high for
ombitasvir (6.7). These data suggest 10d is not the substrate of P-gp or BCRP
transporter, while P-gp or BCRP or both may played roles in the absorption of
ombitasvir.
Table 7. Apparent permeability (Papp) and efflux ratio (ER) of 10d and ombitasvir.
Papp (nm/s) (N=2)
Efflux ratio
compound
A→B
0.7
B→A
0.5
10d
0.8
6.7
Ombitasvir
0.8
5.4
2.6. Liver distribution
The liver distribution of 10d and ombitasvir were evaluated in adult male
Sprague-Dawley(SD) rats. The mean concentrations of 10d and ombitasvir in rat
plasma and liver after oral administration are shown in Table 8. The concentrations of
10d in plasma and liver were lower than ombitasvir till the 8 hours after oral dosing,
but similar at 24 hour. The plasma and liver concentrations of both compounds
displayed a similar changing tendency, which the concentration of the liver was much
higher than the plasma at same time, and the maximum concentration occurred at

ACCEPTED MANUSCRIPT
about 2 hours in plasma and liver, implicating that the 10d and ombitasvir might be
distributed in rats with a higher speed. The maximum LPR of 10d at 8 h and
ombitasvir at 0.5 h were 52.9 and 43.4, respectively, suggesting that the two
compounds can distribute into liver easily.
Table 8. Liver distribution of 10d and ombitasvir in rat (mean ± SD, n=3).
Ombitasvir
Liver
10d
Time(h)
Plasma
LPR a
Plasma
Liver
LPR a
0.5
2
50.5 ± 19
2198 ± 864
43.4 ± 1.95
24.6 ± 6.34
33.5 ± 6.36
22.3 ± 8.01
22.3 ± 7.84 160 ± 72.8
7.22 ± 1.67
28.1 ± 9.59
52.9 ± 16.4
31.9 ± 12.2
129 ± 9.17 3134 ± 573
50.8 ± 6.48 1704 ± 414
35.4 ± 2.06
11.2 ± 1.22
982 ± 288
592 ± 191
8
24
10 ± 4.58
199.8 ± 22
5.86 ± 3.37 160 ± 37.7
a Liver concentration/Plasma concentration ratio (LPR).
2.7. Cytotoxicity
Several experiments were conducted to assess the safety of compound 10d. First, we
investigated the effect of the compound 10d and ombitasvir in human
umbilical-vein endothelial cells (HUVEC) from healthy donors. Both compounds at
50 µM level showed no cytotoxicity (16.6% and 11.8% inhibition) to HUVEC (Table
9), compared to HCV replicon cells (EC50 value in pM level).
Table 9. Inhibition of compound 10d toward HUVEC a (mean± 3, n = 3).
Compound
10d
Concentration (µM)
Inhibition (%)
16.6 ± 2.3
50
50
Ombitasvir
aHuman umbilical vein endothelial cells.
11.8 ± 2.1
2.8. CYP inhibition
Then, the affinity of compound 10d and ombitasvir toward several human liver
CYP450 enzymes were tested. As shown in Figure 2, both compounds did not inhibit
any of the studied human liver CYP450 enzymes at concentrations of 1 µM and 10
µM. The results suggest the absence of clinically significant interactions between 10d
and other drugs metabolized by the above mentioned human cytochromes.

ACCEPTED MANUSCRIPT
Figure 2. The inhibition of compound 10d and ombitasvir toward human liver CYP450
panel.
2.9. hERG activity
To assess the potential heart related safety issues of compound 10d, the inhibitory
effect of 10d at the hERG potassium ion channel was tested at 3 and 10 µM
concentration respectively (Table 10). At both test concentrations, the inhibitory
effect of 10d did not exceed 10%, suggesting a relatively low risk of QT interval
prolongation.
Table 10. Results of hERG ligand binding assay (mean ± SD, n = 3).
Compound
Concentration (µM)
Inhibition (%)
6.9 ± 1.6
3
10
3
10d
8.7 ± 1.9
Ombitasvir
1.1 ± 1.2
10
1.8 ± 3.1
2.10.
Repeat-dose toxicity study
A 14-day repeated oral dose toxicity study of 10d was performed. ICR mice, each
group consisting of 5 males and 5 females, were administered 10d once daily by
gavage at dose of 150 mg/kg body weight. Mice were killed after the 14th
administration. Animals in all groups were observed daily and detailed clinical signs
evident. Their body weights and food intake were monitored on day 1 of
administration and thereafter twice per week. As demonstrated in figure 3, all animals

ACCEPTED MANUSCRIPT
continued to gain weight, no significant difference with the control group. Food
consumption were no difference between the two groups (data not shown).
Figure 3. Body weights for mice orally treated with 10d for14 days.
At necropsy, animals were weighed and sacrificed by exsanguination from the
abdominal aorta under ether anesthesia. Before killing, blood samples were collected
from the abdominal aorta to use for hematology, blood chemistry and measurement of
serum hormone levels. Hematologically, decrease in the RET and reticulocyte ratio
was observed from 10d, statistically significant (p < 0.05) (Table 11). Blood
chemistry data indicated a statistically significant increase of direct bilirubin in 10d (P
< 0.01) (Table 12).
Table 11. Hematology data for mice orally treated with 10d for 14 days
(Mean±SD, n = 10).
Control
7.03 ± 1.56a
10d
WBC a (10^9/L)
RBC b (10^12/L)
HGB c (g/L)
PLT d (10^9/L)
NEUT# e (10/µL)
LYMPH# f (10/µL)
MONO# g (10/µL)
EO# h (10/µL)
6.20 ± 1.75
9.04 ± 0.80
9.00 ± 0.76
157.40 ± 15.50
1260.30 ± 137.87
184.30 ± 102.75
412.20 ± 162.4
93.90 ± 47.09
11.90 ± 5.11
154.60 ± 11.61
1263.40 ± 382.86
212.60 ± 150.72
318.80 ± 121.87
76.60 ± 26.90
12.00 ± 7.80
0.10 ± 0.32
BASO# i (10/µL)
NEUT% j (%)
LYMPH% k (%)
0.20 ± 0.42
27.74 ± 16.27
57.65 ± 13.53
33.12 ± 16.45
52.54 ± 15.94

ACCEPTED MANUSCRIPT
MONO% l (%)
EO% m (%)
12.93 ± 3.61
1.66 ± 0.54
12.44 ± 3.27
1.88 ± 0.92
BASO% n (%)
RET# o (10^9/L)
RET% p (%)
0.02 ± 0.04
0.02 ± 0.06
427.84 ± 64.70
4.73 ± 0.56
348.97 ± 72.01*
3.88 ± 0.76*
*Significantly different from the control value at the levels of P< 0.05.
a White blood cell count ; b Red blood cell count; c Hemoglobin; d Platelet count; e Neutrophil count; f
Lymphocyte count; g Monocyte count; h Eosinophil count; n Basophil count; j Neutrophil percentage; k
Lymphocyte percentage; l Monocyte percentage; m Eosinophil percentage; n Basophil percentage; RET#
o Reticulocyte count; p Reticulocyte percentage.
Table 12. Blood chemistry data for mice orally treated with 10d for 14 days
(Mean ± SD, n = 10).
Control
28.33 ± 9.89a
111.44 ± 32.76
25.87 ± 1.67
53.97 ± 1.56
0.00 ± 0.01
10d
ALT b (U/L)
AST c (U/L)
ALB d (g/l)
25.30 ± 3.68
110.60 ± 37.17
25.38 ± 1.45
52.31 ± 2.29
0.03 ± 0.09
TP e (g/l)
GGT f (U/L)
ALP g (U/L)
BUN h (mmol/l)
CR i (µmol/l)
GLU j (mmol/l)
TB k (µmol/L)
DB l (µmol/L)
TBA m (µmol/L)
TG n (mmol/l)
CHO o (mmol/l)
GLDH p (U/L)
CK q (U/L)
114.44 ± 21.78
10.44 ± 1.77
25.22 ± 3.42
3.64 ± 0.79
105.70 ± 23.42
10.16 ± 2.02
23.20 ± 2.25
4.18 ± 1.18
9.95 ± 2.18
11.24 ± 2.70
8.57 ± 0.72**
10.50 ± 11.52
1.53 ± 0.39
7.60 ± 0.59
5.67 ± 2.45
1.67 ± 0.32
0.93 ± 0.31
0.85 ± 0.21
13.78 ± 2.86
705.78 ± 330.84
15.10 ± 5.84
953.30 ± 1014.67
a Mean ± SD.
*,**Significantly different from the control value at the levels of P< 0.05, P< 0.01,
respectively.
b Alanine aminotransferase; c Aspartate aminotransferase; d Albumin; e Total protein;
f
g
h
i
j
Glutamyl transpeptidase; Alkaline phosphatase; Blood urea nitrogen; Creatinine;
k
l
m
n
o
p
Glucose; Total bilirubin; direct bilirubin; Total bile acid; Triglyceride; Cholesterol;
Glutamate dehydrogenase; q Creatine kinase.
Absolute and relative weights were non-significant statistically between control and

ACCEPTED MANUSCRIPT
10d (Table 13). Moreover, no abnormality was observed on the organs, when the
animal sacrificed. Pathological examination showed that the lesions were spontaneous
and non drug-induced. Although some of the indicators were abnormal in the
repeat-dose toxicity study, the pathological examination and other related indicators
were normal. In summary, animal studies identified no toxicity with 10d.
Table 13. Body and organ weights (g, Mean ± SD, n = 10).
group
BW
25.8
±3.5a
24.0
±2.5
heart
liver
spleen kidneys Thymus
0.355 1.510 0.289
brain
1.686
testis
Ovary
0.079
control
0.539
4.365
0.881
±0.094 ±0.222 ±0.100 ±0.211 ±0.081 ±0.241 ±0.037 ±0.007
0.567 4.346 0.345 1.579 0.240 1.807 0.789 0.097
±0.100 ±0.305 ±0.065 ±0.167 ±0.088 ±0.185 ±0.174 ±0.017
10d
a Mean ± SD.
3. Conclusion
In summary, we have designed and synthesized several silicon-containing derivatives
of ombitasvir and identified a few of HCV NS5A inhibitors with pan-genotype
activity. Among them, compound 10d demonstrated the most promising
pan-genotypic HCV inhibitory activity, with an EC50 range of 0.1-10 pM against
genotypes 1a, 1b, 2a, 3a, 4a, and 5a, and 445 pM against genotype 6a. As compared to
the marketed anti-HCV drug, ombitasvir, the activity of anti-HCV genotypes (GT 1 to
6) of 10d is increased to some extent, especially the inhibitory activity against
genotype 3a and 6a is increased by more than seven times. Similarly to ombitasvir,
compound 10d showed high stability in mouse, rat and human liver microsomes and
did not inhibit most of human liver CYP450 enzymes. In addition, compound 10d
exhibited excellent pharmacokinetic properties in dog with good liver specificity in
rat. Permeability assay results indicated that 10d was not the substrate of P-gp or
BCRP transporter. Several experiments were conducted to assess the safety of
compound 10d. Our data suggest that compound 10d has no cytotoxicity in HUVEC
and no inhibition activity detected in hERG ligand binding inhibition assay. Moreover,
no toxicity with 10d was observed in 14-day repeat-dose toxicity study. Based on
these encouraging results, further studies are being pursued.
4. Experimental section
4.1 Chemistry. General Procedures. Unless otherwise noted, all materials were
1
obtained from commercial suppliers and used without further purification. H NMR

ACCEPTED MANUSCRIPT
spectra (300 MHz) were collected on a Bruker-300 FT NMR spectrometer with
tetramethylsilane (TMS) as internal standard. High resolution mass spectra (HRMS)
were recorded on an AB SCIEX Triplet TOF 4600 Mass Spectrometer. All final
compounds were purified to > 96% purity as determined by HPLC analyse. Reverse
phase HPLC purity determinations were performed on an Agilent 1260 HPLC system.
Compounds 3I, 3II and 3III were prepared according to the reported methods [27].
Compound 5 was purchased from Nanjing Ally Chemical S& T Co., Ltd. Ombitasvir
was synthesized according to the reported methods with 99% purity [25].
4,4'-(trans-1-(4-(trimethylsilyl)phenyl)pyrrolidine-2,5-diyl)dianiline (7a /7b) and
4,4'-(cis-1-(4-(trimethylsilyl)phenyl)pyrrolidine-2,5-diyl)dianiline (7c). To
a
solution of 5 (4.5 g, 9.2 mmol) in anhydrous N,N-dimethylformamide (24 mL) was
added 3I (10.66 g, 64.47 mmol), followed by dropwise addition of triethylamine (9.32
g, 92.1 mmol). The resulting mixture was stirred at 60 °C for 12 h. The cooled
mixture was poured into water (50 mL), extracted with ethyl acetate (50 mL× 3), and
the combined organic layers were dried over Na2SO4. The drying agent was filtered
off,
and
the
solvent
was
evaporated
to
give
2,5-bis(4-nitrophenyl)-1-(4-(trimethylsilyl)phenyl)pyrrolidine (6a/6b, 6c, 12 g) as a
crude product which was used without further purification.
A mixture of 6a/6b & 6c (12 g, crude product) and platinum (IV) oxide (4.0g,
17.61 mmol) in tetrahydrofuran (80 mL) was stirred under hydrogen gas at 10 atm for
48 h. The resulting mixture was filtered and concentrated in vacuo to give a crude
product which was purified by column chromatography on silica gel using a solvent
gradient of 10-50% ethyl acetate in petrol. The first eluting component was the
1
trans-pyrrolidine isomers 7a/7b (560 mg, 15.2% yield, two steps). H NMR (300
MHz, DMSO-d6) δ 7.03-7.00 (d, J = 8.2 Hz, 2H), 6.83-6.80 (d, J = 8.1 Hz, 4H),
6.48-6.46 (d, J = 8.1 Hz, 4H), 6.28-6.26 (d, J = 8.3 Hz, 2H), 5.00-4.98 (d, J = 6.3 Hz,
2H), 4.86 (s, 4H), 2.38-2.36 (m, 2H), 1.57-1.55 (d, J = 5.7 Hz, 2H), 0.08 (s, 9H).
HRMS (ESI) m/z: Anal.calcd.for [M+H]+ C25H31N3Si: 402.2365; found 402.2363.
The second eluting component was the cis-pyrrolidine isomer 7c (860 mg, 23.3%, two
1
steps). H NMR (300 MHz, DMSO-d6) δ 7.12-7.08 (m, 6H), 6.56-6.54 (d, 4H),
6.44-6.41 (d, J = 8.0 Hz, 2H), 4.92 (s, 4H), 4.53-4.50 (m, 2H), 2.40-2.20 (m, 2H),
1.81-1.74 (m, 2H), 0.10 (s, 9H). HRMS (ESI) m/z: Anal.calcd.for [M+H]+ C25H31N3Si:
402.2365; found 402.2362.

ACCEPTED MANUSCRIPT
Dibenzyl
2,2'-((((trans-1-(4-(trimethylsilyl)phenyl)pyrrolidine-2,5-diyl)bis(4,1-phenylene))
bis(azanediyl))bis(carbonyl))(2S,2'S)-bis(pyrrolidine-1-carboxylate) (8a/8b). A
mixture of 7a/7b (450 mg, 1.12 mmol), ((benzyloxy)carbonyl)-L-proline (1.34g, 5.6
mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (869.34 mg,
5.6 mmol), 1-hydroxybenzotriazole (756.67 mg, 5.6 mmol) and N-methylmorpholine
(566.44 mg, 5.6 mmol) in N,N-dimethylformamide (15 mL) was stirred at room
temperature for 12 h. The mixture was poured into water (50 mL), extracted with
dichlormethane (50 mL× 3 ), and the combined organic layers were dried with
Na2SO4. The drying agent was filtered off, and the solvent was concentrated in vacuo.
The residue was purified by column chromatography on silica gel, eluting with a
solvent gradient of 60% ethyl acetate in petrol to give compound 8a/8b (790 mg,
81.4%). 1H NMR (300 MHz, DMSO-d6) δ 9.99 (s, 2H), 7.53-7.50 (d, J = 7.2 Hz, 4H),
7.40-7.25 (m, 4H), 7.20-7.01 (m, 12H), 6.34-6.25 (m, 2H), 5.22 (s, 2H), 5.10-5.06 (m,
3H), 4.94-4.90 (d, J = 13.0 Hz, 1H), 4.45-4.20 (m, 2H), 3.48-3.44 (m, 4H), 2.88 (s,
1H), 2.73 (s, 1H), 2.35-2.10 (m, 2H), 1.90-1.88 (m, 6H), 1.67 (s, 2H), 0.08 (s, 9H).
HRMS (ESI) m/z: Anal.calcd.for [M+H]+ C51H57N5O6Si : 864.4156 ; found 864.4096.
(2S,2'S)-N,N'-((trans-1-(4-(trimethylsilyl)phenyl)pyrrolidine-2,5-diyl)bis(4,1-phen
ylene))bis(pyrrolidine-2-carboxamide) (9a/9b). A mixture of 8a/8b (790 mg, 0.914
mmol) and 10% Pd/C (97.2 mg) in a solution of ethanol (30 mL) and methanol (5mL)
was stirred under hydrogen gas at 10 atm for 30 h. The resulting mixture was filtered
1
and concentrated in vacuo to give compound 9a/9b (410 mg, 75.9%). H NMR (300
MHz, DMSO-d6) δ 9.80 (s, 2H), 7.50-7.47 (d, J = 8.1 Hz, 4H), 7.06-7.04 (d, J = 8.1
Hz, 4H), 6.97-6.94 (d, J = 7.9 Hz, 2H), 6.20-6.17 (d, J = 8.0 Hz, 2H), 5.13-5.12 (d, J
= 5.1 Hz, 2H), 3.60-3.55 (m, 2H), 2.81-2.77 (m, 5H), 2.65 (s, 1H), 2.43-2.37 (m, 2H),
1.97-1.88 (m, 2H), 1.70-1.52 (m, 8H), 0.0009 (s, 9H). HRMS (ESI) m/z:
Anal.calcd.for [M+H]+ C35H45N5O2Si 596.3421; found 596.3409.
Dimethyl
((2S,2'S)-((2S,2'S)-(((((2S,5S)-1-(4-(trimethylsilyl)phenyl)pyrrolidine-2,5-diyl)bis(
4,1-phenylene))bis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-diyl))bis(3-methy
l-1-oxobutane-1,2-diyl))dicarbamate
(10a)
dimethyl
((2S,2'S)-((2S,2'S)-(((((2R,5R)-1-(4-(trimethylsilyl)phenyl)pyrrolidine-2,5-diyl)bis
(4,1-phenylene))bis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-diyl))bis(3-meth

ACCEPTED MANUSCRIPT
yl-1-oxobutane-1,2-diyl))dicarbamate (10b). Compound 9a/9b (400 mg, 0.67 mmol)
was dissolved in N,N-dimethylformamide (15 mL) and cooled to 0°C, followed by
addition
of
(methoxycarbonyl)-L-valine
(586.85
mg,
3.35
mmol),
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (520 mg, 3.35 mmol),
1-hydroxybenzotriazole (452.65 mg, 3.35 mmol) and N-methylmorpholine (338.85
mg, 3.35 mmol). The resulting mixture was stirred at room temperature for 12 h. The
mixture was poured into water (50 mL), filtered and the solvent was concentrated in
vacuo. The residue was purified by column chromatography on silica gel, eluting with
a solvent gradient of 60% ethyl acetate in petrol to give a 1:1 mixture of
trans-pyrrolidine isomers (10a/10b, 358 mg, 58.7%). The mixture was separated on a
CHIRALART Amylose-SA column (250*30mm, 5µm), eluting with a mixture of (35%
water) and (65% acetonitrile). Compound 10a was the first of two stereoisomers to
1
elute (116 mg, 99% ee by chiral HPLC). H NMR (300 MHz, DMSO-d6) δ 9.96 (s,
2H), 7.51-7.48 (d, J = 8.2 Hz, 4H), 7.29-7.26 (d, J = 8.0 Hz, 2H), 7.14-7.11 (d, J = 8.2
Hz, 4H), 7.06-7.03 (d, J = 8.0 Hz, 2H), 6.27-6.24 (d, J = 8.0 Hz, 2H), 5.19 (s, 2H),
4.45-4.40 (m, 2H), 4.06-4.00 (m, 2H), 3.90-3.70 (m, 2H), 3.65-3.55 (m, 2H), 3.52 (s,
6H), 2.49-2.45 (m, 2H), 2.13-1.88 (m, 10H), 1.70-1.60 (m, 2H), 0.94-0.87 (m, 12H),
0.07 (s, 9H). HRMS (ESI) m/z: Anal. calcd. for [M+H]+ C49H67N7O8Si: 910.4899;
found 910.4865.
Compound 10b was the second of two stereoisomers to elute (103 mg, 99% ee by
1
chiral HPLC). H NMR (300 MHz, DMSO-d6) δ 9.96 (s, 2H), 7.51-7.48 (d, J = 8.6
Hz, 4H), 7.28-7.25 (d, J = 7.9 Hz, 2H), 7.13-7.11 (d, J = 8.2 Hz, 4H), 7.06-7.03 (d, J
= 8.3 Hz, 2H), 6.27-6.24 (d, J = 8.3 Hz, 2H), 5.19 (s, 2H), 4.45-4.40 (m, 2H),
4.05-4.00 (m, 2H), 3.78-3.76 (m, 2H), 3.63-3.58 (m, 2H), 3.52 (s, 6H), 2.49-2.45 (m,
2H), 2.12-1.88 (m, 10H), 1.65-1.64 (m, 2H), 0.92-0.85 (m, 12H), 0.07 (s, 9H). Anal.
calcd. for [M+H]+ C49H67N7O8Si: 910.4899; found 910.4896.
Dibenzyl
2,2'-(((((2S,5R)-1-(4-(trimethylsilyl)phenyl)pyrrolidine-2,5-diyl)bis(4,1-phenylene
))bis(azanediyl))bis(carbonyl))(2S,2'S)-bis(pyrrolidine-1-carboxylate)
(8c).
Compound 8c was synthesized according to the method to prepare compound 10a.
Compound 7c (860mg, 2.14 mmol) was treated with ((benzyloxy)carbonyl)-L-proline
1
under coupling condition to give compound 8c (1.42 g ,76.5%). H NMR (300 MHz,
DMSO-d6) δ 10.05 (s, 2H), 7.60-7.58 (d, J = 6.5 Hz, 4H), 7.45-7.42 (d, J = 7.9 Hz,
4H), 7.36-7.10 (m, 12H), 6.42-6.40 (m, 2H), 5.12-4.92 (m, 4H), 4.73 (s, 2H),

ACCEPTED MANUSCRIPT
4.38-4.33 (m, 2H), 3.49-3.45 (m, 4H), 2.45-2.35 (m, 2H), 2.26-2.23 (m, 2H),
1.89-1.85 (m, 8H), 0.10 (s, 9H). HRMS (ESI) m/z: Anal. calcd. for [M+H]+
C51H57N5O6Si : 864.4156 ; found 864.4088.
(2S,2'S)-N,N'-(((2S,5R)-1-(4-(trimethylsilyl)phenyl)pyrrolidine-2,5-diyl)bis(4,1-p
henylene))bis(pyrrolidine-2-carboxamide) (9c). Compound 9c was synthesized
according to the method to prepare compound 9a/9b. Compound 8c (1.42 g, 1.64
mmol) was treated using hydrogen gas with catalyst to give compound 9c (758 mg,
77.4%).
1H NMR (300 MHz, DMSO-d6) δ 10.06 (s, 2H), 7.65-7.63 (d, J = 8.3 Hz, 4H),
7.43-7.40 (d, J = 8.4 Hz, 4H), 7.14-7.11 (d, J = 8.3 Hz, 2H), 6.40-6.37 (d, J = 8.3 Hz,
2H), 4.72 (s, 2H), 4.18 (br, 2H), 3.80-3.75 (m, 2H), 3.30-2.91 (m, 4H), 2.45-2.35 (m,
2H), 2.14-2.02 (m, 2H), 1.82-1.65(m, 8H), 0.10(m, 9H). HRMS (ESI) m/z:
Anal.calcd.for [M+H]+ C35H45N5O2Si 596.3421; found 596.3399.
Dimethyl
((2S,2'S)-((2S,2'S)-(((((2S,5R)-1-(4-(trimethylsilyl)phenyl)pyrrolidine-2,5-diyl)bis(
4,1-phenylene))bis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-diyl))bis(3-methy
l-1-oxobutane-1,2-diyl))dicarbamate (10c). Compound 10c was prepared according
to the method to synthesize the mixture of compound 10a and 10b. Compound 9c
(758 mg, 1.27 mmol) reacted with (methoxycarbonyl)-L-valine (586.85 mg, 3.35
1
mmol) under peptide coupling condition to give compound 10c (622 mg ,53.7%). H
NMR (300 MHz, DMSO-d6) δ 10.03 (s, 2H), 7.60-7.57 (d, J = 8.3 Hz, 4H), 7.43-7.40
(d, J = 8.4 Hz, 4H), 7.32-7.30 (d, J = 7.3 Hz, 2H), 7.14-7.11 (d, J = 8.1 Hz, 2H),
6.39-6.36 (d, J = 8.3 Hz, 2H), 4.70 (s, 2H), 4.52-4.35 (m, 2H), 4.06-4.00 (m, 2H),
3.83-3.70 (m, 2H), 3.69-3.62 (m, 2H), 3.53 (s, 6H), 2.45-1.90 (m, 14H), 0.94-0.88 (m,
12H), 0.10 (m, 9H). HRMS (ESI) m/z: Anal. calcd. for [M+H]+ C49H67N7O8Si:
910.4899; found 910.4851.
4,4'-(trans-1-(4-(triethylsilyl)phenyl)pyrrolidine-2,5-diyl)dianiline (7d/7e) and
4,4'-(cis-1-(4-(triethylsilyl)phenyl)pyrrolidine-2,5-diyl)dianiline (7f).
2,5-bis(4-nitrophenyl)-1-(4-(triethylsilyl)phenyl)pyrrolidine (6d/6e, 6f) was
synthesized according to the method to prepare compounds 6a/6b & 6c. The crude
product was used without further purification.
Compound 7d/7e and 7f were synthesized according to the method to prepare
1
compounds 7a/7b and 7c. 7d/7e (568mg): H NMR (300 MHz, DMSO-d6) δ
7.00-6.98 (d, J = 8.2 Hz, 2H), 6.84-6.81 (d, J = 8.1 Hz, 4H), 6.49-6.46 (d, J = 8.1 Hz,

ACCEPTED MANUSCRIPT
4H), 6.30-6.27 (d, J = 8.2 Hz, 2H), 5.00-4.98 (d, J = 6.0 Hz, 2H), 4.86 (s, 4H),
2.42-2.31 (m, 2H), 1.56-1.54 (m, 2H), 0.93-0.63 (m, 15H). HRMS (ESI) m/z: Anal.
1
calcd. for [M+H]+ C28H37N3Si: 444.2835; found 444.2829. 7f (830 mg): H NMR
(300 MHz, DMSO-d6) δ 7.14-7.11 (d, J = 8.2 Hz, 4H), 7.08-7.06 (d, J = 8.2 Hz, 2H),
6.58-6.55 (d, J = 8.2 Hz, 4H), 6.46-6.44 (d, J = 8.2 Hz, 2H), 4.92 (s, 4H), 4.54 (s, 2H),
2.38-2.26 (m, 2H), 1.82-1.74 (m, 2H), 0.91-0.77 (m, 9H), 0.70-0.66 (m, 6H). HRMS
(ESI) m/z: Anal. calcd. for [M+H]+ C28H37N3Si: 444.2835; found 444.2831.
Dibenzyl
2,2'-((((trans-1-(4-(triethylsilyl)phenyl)pyrrolidine-2,5-diyl)bis(4,1-phenylene))bis
(azanediyl))bis(carbonyl))(2S,2'S)-bis(pyrrolidine-1-carboxylate)
(8d/8e).
Compound 8d/8e (979 mg, 84.3%) was synthesized according to the method to
prepare compound 8a/8b. 1H NMR (300 MHz, DMSO-d6) δ 9.99 (s, 2H), 7.54-7.51 (d,
J = 6.4 Hz, 4H), 7.42-7.18 (m, 12H), 7.05-7.03 (m, 4H), 6.33-6.30 (m, 2H), 5.22 (s,
2H), 5.11-5.07 (m, 3H), 4.95-4.90 (d, J = 13 Hz, 1H), 4.35 (s, 2H), 3.50-3.44 (m, 4H),
2.88 (s, 2H), 2.73 (s, 2H), 2.35-2.10 (m, 2H), 1.98-1.84 (m, 6H), 1.66 (s, 2H),
0.93-0.79 (m, 9H), 0.63-0.61 (m, 4H), 0.58-0.49 (m, 2H). HRMS (ESI) m/z: Anal.
calcd. for [M+H]+ C54H63N5O6Si : 906.4626; found 906.4670.
(2S,2'S)-N,N'-((trans-1-(4-(triethylsilyl)phenyl)pyrrolidine-2,5-diyl)bis(4,1-phenyl
ene))bis(pyrrolidine-2-carboxamide) (9d/9e). Compound 9d/9e (535 mg, 77.1%)
1
was synthesized according to the method to prepare compound 9a/9b. H NMR (300
MHz, DMSO-d6) δ 9.93 (s, 2H), 7.57-7.54 (d, J = 8.3 Hz, 4H), 7.16-7.13 (d, J = 8.2
Hz, 4H), 7.02-6.99 (d, J = 8.2 Hz, 2H), 6.29-6.26 (d, J = 8.3 Hz, 2H), 5.21-5.20 (d, J
= 5.6 Hz, 2H), 3.73-3.68 (m, 2H), 2.92-2.89 (m, 4H), 2.50-2.45 (m, 2H), 2.07-1.98 (m,
2H), 1.82-1.63 (m, 8H), 1.05-1.01 (m, 2H) , 0.84-0.79 (m, 9H), 0.63-0.55 (m, 6H).
HRMS (ESI) m/z: Anal. calcd. for [M+H]+ C38H51N5O2Si: 638.3890; found 638.3875.
Dimethyl
((2S,2'S)-((2S,2'S)-(((((2S,5S)-1-(4-(triethylsilyl)phenyl)pyrrolidine-2,5-diyl)bis(4,
1-phenylene))bis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-diyl))bis(3-methyl-
1-oxobutane-1,2-diyl))dicarbamate (10d) and
dimethyl((2S,2'S)-((2S,2'S)-(((((2R,5R)-1-(4-(triethylsilyl)phenyl)pyrrolidine-2,5-
diyl)bis(4,1-phenylene))bis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-diyl))bis(
3-methyl-1-oxobutane-1,2-diyl))dicarbamate (10e). Compound 10d and 10e was
synthesized according to the methods to prepare compound 10a and 10b. Compound
9e/9d was treated with (methoxycarbonyl)-L-valine under peptide coupling condition

ACCEPTED MANUSCRIPT
to give a 1:1 mixture of compound 10d and 10e as trans-pyrrolidine isomers (490 mg,
61.4%). The mixture was separated on a CHIRALART Amylose-SA column to give
compound 10d (176 mg, 99% ee by chiral HPLC) and 10e (98mg, 99% ee by chiral
HPLC). 10d: 1H NMR (300 MHz, DMSO-d6) δ 9.98 (s, 2H), 7.52-7.49 (d, J = 8.19
Hz, 4H), 7.31-7.29 (d, J = 8.2 Hz, 2H), 7.15-7.12 (d, J = 8.2 Hz, 4H), 7.03-7.00 (d, J
= 8.1 Hz, 2H), 6.28-6.25 (d, J = 8.1 Hz, 2H), 5.19-5.17 (d, J = 5.1 Hz, 2H), 4.44-4.41
(m, 2H), 4.05-4.00 (m, 2H), 3.84-3.77 (m, 2H), 3.65-3.58 (m, 2H), 3.52 (s, 6H),
2.48-2.45 (m, 2H), 2.20-1.87 (m, 10H), 1.63-1.61 (m, 2H), 0.94-0.89 (m, 12H),
0.87-0.79 (m, 9H), 0.62-0.54 (m, 6H). HRMS (ESI) m/z: Anal. calcd. for [M+H]+
C52H73N7O8Si: 952.5368; found 952.5343.
1
10e: H NMR (300 MHz, DMSO-d6) δ 9.98 (s, 2H), 7.51-7.49 (d, J = 8.2 Hz, 4H),
7.31-7.28 (d, J = 8.3 Hz, 2H), 7.14-7.12 (d, J = 8.2 Hz, 4H), 7.03-7.00 (d, J = 8.1 Hz,
2H), 6.27-6.25 (d, J = 8.1 Hz, 2H), 5.19-5.18 (d, J = 3.6 Hz, 2H), 4.44-4.40 (m, 2H),
4.05-3.99 (m, 2H), 3.84-3.78 (m, 2H), 3.65-3.58 (m, 2H), 3.52 (s, 6H), 2.48-2.45 (m,
2H), 2.17-2.12 (m, 2H), 2.04-1.86 (m, 8H), 1.63-1.62 (m, 2H), 0.92-0.79 (m, 21H),
0.62-0.55 (m, 6H). HRMS (ESI) m/z: Anal. calcd. for [M+H]+ C52H73N7O8Si:
952.5368; found 952.5343.
Dibenzyl
2,2'-(((((2S,5R)-1-(4-(triethylsilyl)phenyl)pyrrolidine-2,5-diyl)bis(4,1-phenylene))
bis(azanediyl))bis(carbonyl))(2S,2'S)-bis(pyrrolidine-1-carboxylate)
(8f).
Compound 8f (1.34 g, 79.2%) was synthesized according to the method to prepare
1
compound 8c. H-NMR (300 MHz, DMSO-d6) δ 10.03 (s, 2H), 7.60-7.58 (d, J = 7.4
Hz, 4H), 7.46-7.43 (d, J = 8.2 Hz, 4H), 7.31-7.10 (m, 12H), 6.45-6.43 (m, 2H),
5.12-5.08 (m, 4H), 4.74 (s, 2H), 4.39-4.34 (m, 2H), 3.50-3.45 (m, 4H), 2.50-2.33 (m,
2H), 2.31-2.10 (m, 2H), 1.98-1.85 (m, 8H), 0.91-0.81 (m, 9H), 0.65-0.58 (m, 6H).
HRMS (ESI) m/z: Anal. calcd. for [M+H]+ C54H63N5O6Si: 906.4626; found 906.4670.
(2S,2'S)-N,N'-(((2S,5R)-1-(4-(triethylsilyl)phenyl)pyrrolidine-2,5-diyl)bis(4,1-phe
nylene))bis(pyrrolidine-2-carboxamide) (9f). Compound 9f (738 mg, 78.2%) was
1
synthesized according to the method to prepare compound 9c. H NMR (300 MHz,
DMSO-d6) δ 9.97 (s, 2H), 7.65-7.63 (d, J = 8.5 Hz, 4H), 7.44-7.41 (d, J = 8.3 Hz, 4H),
7.11-7.08 (d, J = 8.3 Hz, 2H), 6.42-6.39 (d, J = 8.3 Hz, 2H), 4.73 (s, 2H), 3.74-3.71
(m, 2H), 2.93-2.89 (m, 4H), 2.45-2.35 (m, 2H), 2.11-2.00 (m, 2H), 1.90-1.61(m, 8H),
0.86-0.81(m, 9H), 0.65-0.58 (m, 6H). HRMS (ESI) m/z: Anal. calcd. for [M+H]+
C38H51N5O2Si: 638.3890; found 638.3876.

ACCEPTED MANUSCRIPT
Dimethyl
((2S,2'S)-((2S,2'S)-(((((2S,5R)-1-(4-(triethylsilyl)phenyl)pyrrolidine-2,5-diyl)bis(4,
1-phenylene))bis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-diyl))bis(3-methyl-
1-oxobutane-1,2-diyl))dicarbamate (10f). Compound 10f (665 mg, 60.4%) was
synthesized according to the method to prepare compound 10c.
1H NMR (300 MHz, DMSO-d6) δ 10.03 (s, 2H), 7.60-7.57 (d, J = 8.4 Hz, 4H),
7.44-7.41 (d, J = 8.3 Hz, 4H), 7.32-7.30 (d, J = 7.9 Hz, 2H), 7.11-7.08 (d, J = 8.2 Hz,
2H), 6.41-6.38 (d, J = 8.0 Hz, 2H), 4.71 (s, 2H), 4.48-4.44 (m, 2H), 4.06-4.01(m, 2H),
3.83-3.78 (m, 2H), 3.69-3.62 (m, 2H), 3.59 (s, 6H), 2.48-2.45 (m, 2H), 2.20-2.10 (m,
2H), 2.04-1.78 (m, 10H), 0.95-0.80 (m, 21H), 0.65-0.57 (m, 6H). HRMS (ESI) m/z:
Anal. calcd. for [M+H]+ C52H73N7O8Si: 952.5368; found 952.5335.
4,4'-(trans-1-(4-(tert-butyldimethylsilyl)phenyl)pyrrolidine-2,5-diyl)dianiline
(7g/7h)
and
4,4'-(cis-1-(4-( tert-butyldimethylsilyl)phenyl)pyrrolidine-2,5-diyl)dianiline (7i) .
1-(4-(tert-butyldimethylsilyl)phenyl)-2,5-bis(4-nitrophenyl)pyrrolidine (6g/6h, 6i)
was synthesized according to the method to prepare compound 6a/6b & 6c. The crude
product was used without further purification.
Compound 7g/7h and 7i was synthesized according to the method to prepare
1
compound 7a/7b and 7c. 7g/7h (310 mg): H-NMR (300 MHz, DMSO-d6) δ
7.02-6.99 (d, J = 8.4 Hz, 2H), 6.84-6.81 (d, J = 8.3 Hz, 4H), 6.49-6.46 (d, J = 8.3 Hz,
4H), 6.29-6.27 (d, J = 8.5 Hz, 2H), 5.00-4.98 (d, J = 6.4 Hz, 2H), 4.87 (s, 4H),
1.56-1.54 (d, J = 5.8 Hz, 2H), 1.25-1.17 (m, 2H), 0.75 (s, 9H), 0.08-0.07 (m, 6H).
HRMS (ESI) m/z: Anal. calcd. for [M+H]+ C28H37N3Si: 444.2835; found 444.2820.
1
7i (660 mg): H NMR (300 MHz, DMSO-d6) δ 7.13-7.04 (m, 6H), 6.57-6.54 (d, J =
8.3 Hz, 4H), 6.48-6.43 (m, 2H), 4.99-4.97 (m, 4H), 4.65-4.45 (m, 2H), 2.35-2.18 (m,
2H), 1.82-1.74 (m, 2H), 0.77 (s, 9H), 0.10 (s, 6H). HRMS (ESI) m/z: Anal. calcd. for
[M+H]+ C28H37N3Si: 444.2835; found 444.2825.
Dibenzyl
2,2'-((((trans-1-(4-(tert-butyldimethylsilyl)phenyl)pyrrolidine-2,5-diyl)bis(4,1-phe
nylene))bis(azanediyl))bis(carbonyl))(2S,2'S)-bis(pyrrolidine-1-carboxylate)
(8g/8h). Compound 8g/8h (516 mg, 81.6%) was synthesized according to the method
to prepare compound 8a/8b. 1H NMR (300 MHz, DMSO-d6) δ 9.98 (s, 2H), 7.52-7.50
(d, J = 7.5 Hz, 4H), 7.39-7.36 (m, 6H), 7.17-7.12 (m, 6H), 7.05-7.01 (m, 4H),
6.31-6.29 (d, J = 7.9 Hz, 2H), 5.23 (s, 2H), 5.10-4.99 (m, 4H), 4.35-4.32 (m, 2H),

ACCEPTED MANUSCRIPT
3.59-3.56 (m, 4H), 2..45-2.35 (m, 2H), 2.33-1.55 (m, 10H), 0.74 (s, 9H), 0.08-0.06 (d,
J = 7.9 Hz, 6H). HRMS (ESI) m/z: Anal. calcd. for [M+H]+ C54H63N5O6Si: 906.4626;
found 906.4684.
(2S,2'S)-N,N'-((trans-1-(4-(tert-butyldimethylsilyl)phenyl)pyrrolidine-2,5-diyl)bis
(4,1-phenylene))bis(pyrrolidine-2-carboxamide) (9g/9h). Compound 9g/9h (304
mg, 83.7%) was synthesized according to the methods to prepare compound 9a/9b.
1H NMR (300 MHz, DMSO-d6) δ 9.93 (s, 2H), 7.57-7.55 (d, J = 8.4 Hz, 4H),
7.13-7.10 (d, J = 7.6 Hz, 4H), 7.04-7.01 (d, J = 8.3 Hz, 2H), 6.29-6.26 (d, J = 8.4 Hz,
2H), 5.21-5.19 (d, J = 5.9 Hz, 2H), 3.72-3.67 (m, 2H), 2.91-2.87 (m, 4H), 2.45-2.35
(m, 2H), 2.14-1.97 (m, 2H), 1.90-1.62 (m, 8H), 0.74 (s, 9H), 0.08-0.06 (d, J = 5.4 Hz,
6H). HRMS (ESI) m/z: Anal. calcd. for [M+H]+ C38H51N5O2Si: 638.3890; found
638.3901.
Dimethyl
((2S,2'S)-((2S,2'S)-(((((2S,5S)-1-(4-(tert-butyldimethylsilyl)
phenyl)pyrrolidine-2,5-diyl)bis(4,1-phenylene))bis(azanediyl))bis(carbonyl))
bis(pyrrolidine-2,1-diyl))bis(3-methyl-1-oxobutane-1,2-diyl))dicarbamate (10g)
and
dimethyl
((2S,2'S)-((2S,2'S)-(((((2R,5R)-1-(4-(tert-butyldimethylsilyl)phenyl)pyrrolidine-2,
5-diyl)bis(4,1-phenylene))bis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-diyl))bi
s(3-methyl-1-oxobutane-1,2-diyl))dicarbamate (10h). Compound 10g and 10h were
synthesized according to the method to prepare compound 10a and 10b. A 1:1 mixture
1
of trans-pyrrolidine isomers was obtained (350 mg, 77.1%). 10g (108 mg): H NMR
(300 MHz, DMSO-d6) δ 9.96 (s, 2H), 7.51-7.49 (d, J = 8.2 Hz, 4H), 7.29-7.27 (d, J =
8.2 Hz, 2H), 7.15-7.12 (d, J = 8.2 Hz, 4H), 7.05-7.02 (d, J = 8.1Hz, 2H), 6.27-6.25 (d,
J = 8.0 Hz, 2H), 5.19 (s, 2H), 4.44-4.43 (m, 2H), 4.05-4.00 (m, 2H), 3.85-3.75 (m,
2H), 3.62-3.60 (m, 2H), 3.52 (s, 6H), 2.45-2.35 (m, 2H), 2.12-2.08 (m, 2H), 1.99-1.89
(m, 8H), 1.63-1.61 (m, 2H), 0.94-0.82 (m, 12H), 0.73 (s, 9H), 0.08-0.06 (d, J = 5.4 Hz,
6H). HRMS (ESI) m/z: Anal. calcd. for [M+H]+ C52H73N7O8Si: 952.5368; found
952.5405. 10h (110 mg): 1H NMR (300 MHz, DMSO-d6) δ 9.96 (s, 2H), 7.51-7.48 (d,
J = 8.3 Hz, 4H), 7.29-7.26 (d, J = 7.9 Hz, 2H), 7.14-7.11 (d, J = 8.3 Hz, 4H),
7.05-7.02 (d, J = 8.2 Hz, 2H), 6.27-6.24 (d, J = 8.3 Hz, 2H), 5.19 (s, 2H), 4.44-4.33
(m, 2H), 4.05-3.99 (m, 2H), 3.85-3.75 (m, 2H), 3.63-3.60 (m, 2H), 3.52 (s, 6H),
2.45-2.35 (m, 2H), 2.12-2.08 (m, 2H), 2.00-1.75 (m, 8H), 1.64-1.62 (m, 2H),
0.92-0.85 (m, 12H), 0.73 (s, 9H), 0.08-0.06 (d, J = 5.4 Hz, 6H). HRMS (ESI) m/z:
Anal. calcd. for [M+H]+ C52H73N7O8Si: 952.5368; found 952.5404.

ACCEPTED MANUSCRIPT
Dibenzyl
2,2'-(((((2S,5R)-1-(4-(tert-butyldimethylsilyl)phenyl)pyrrolidine-2,5-diyl)bis(4,1-p
henylene))bis(azanediyl))bis(carbonyl))(2S,2'S)-bis(pyrrolidine-1-carboxylate)
(8i). Compound 8i (1.07 g, 79.4%) was synthesized according to the method to
1
prepare compound 8c. HNMR (300 MHz, DMSO-d6) δ 10.04 (s, 2H), 7.63-7.58 (m,
4H), 7.45-7.43 (m, 4H), 7.36-7.09 (m, 12H), 6.44-6.42 (d, J = 6.5 Hz, 2H), 5.13-5.08
(m, 4H), 4.74(s, 2H), 4.36-4.34 (m, 4H), 3.59-3.56 (m, 4H), 2.45-2.35 (m, 2H),
2.26-1.88 (m, 8H), 0.76 (s, 9H), 0.10 (s, 6H). HRMS (ESI) m/z: Anal. calcd. for
[M+H]+ C54H63N5O6Si: 906.4626; found 906.4671.
(2S,2'S)-N,N'-(((2S,5R)-1-(4-(tert-butyldimethylsilyl)phenyl)pyrrolidine-2,5-diyl)
bis(4,1-phenylene))bis(pyrrolidine-2-carboxamide) (9i). Compound 9i (559 mg,
1
74.2%) was synthesized according to the method to prepare compound 9c. H NMR
(300 MHz, DMSO-d6) δ 9.93 (s, 2H), 7.65-7.63 (d, J = 8.5 Hz, 4H), 7.43-7.40 (d, J =
8.4 Hz, 4H), 7.12-7.10 (m, 2H), 6.42-6.39 (d, J = 8.3 Hz, 2H), 4.73 (s, 2H), 3.70-3.63
(m, 2H), 2.90-2.86 (m, 4H), 2.45-2.35 (m, 2H), 2.07-1.98 (m, 2H), 1.84-1.62 (m, 8H),
0.76 (s, 9H), 0.09 (s, 6H). HRMS (ESI) m/z: Anal. calcd. for [M+H]+ C38H51N5O2Si:
638.3890; found 638.3888.
Dimethyl
((2S,2'S)-((2S,2'S)-(((((2S,5R)-1-(4-(tert-butyldimethylsilyl)phenyl)pyrrolidine-2,
5-diyl)bis(4,1-phenylene))bis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-diyl))bi
s(3-methyl-1-oxobutane-1,2-diyl))dicarbamate (10i). Compound 10i (469 mg,
56.2%) was synthesized according to the method to prepare compound 10c. 1H NMR
(300 MHz, DMSO-d6) δ 10.01 (s, 2H), 7.59-7.57 (d, J = 8.1 Hz, 4H), 7.45-7.35 (m,
4H), 7.33-7.21 (m, 2H), 7.14-7.10 (m, 2H), 6.40-6.37 (d, J = 7.5 Hz, 2H), 4.80-4.60
(m, 2H), 4.50-4.40 (m, 2H), 4.05-3.95 (m, 2H), 3.85-3.73 (m, 2H), 3.63-3.55 (m, 2H),
3.52 (s, 6H), 2.45-2.35 (m, 2H), 2.12-2.08 (m, 2H), 2.00-1.70 (m, 10H), 0.93-0.87 (m,
12H), 0.75 (s, 9H), 0.08-0.06 (d, J = 4.0 Hz, 6H). HRMS (ESI) m/z: Anal. calcd. for
[M+H]+ C52H73N7O8Si: 952.5368; found 952.5400.
4.2 Biological studies
4.2.1. Antiviral activity assay test with HCVcc (GT2a)
4.2.1.1. Cell line and cell culture
The Huh 7.5.1 cell line and the HCVcc reporter virus were kindly provided by Wuhan
Institute of Virology, Chinese Academy of Sciences. Cells were cultured in

ACCEPTED MANUSCRIPT
Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum,
100 µg/mL streptomycin, 100 U/mL penicillin at 37°C with 5% CO2. HCVcc reporter
virus (GT2a, JFH-1) is an infectious HCV virus derived from the JFH1 virus
(genotype 2a). The establishment of the infectious HCVcc (GT2a, JFH-1) virus has
been described previously. Virus was propagated in Huh7.5.1 cells. Briefly, Huh7.5.1
cells were infected with virus at MOI = 0.1 and cultured for 4d. Culture supernatant
was collected and fltered through a 0.45 µm membrane and stored at -80ºC as virus
stock. The infectious titers of cell-culture supernatants were evaluated by classical
titration assay[28].
4.2.1.2 Antiviral activity test.
Huh-7.5.1 cells were infected with the HCVcc reporter virus (MOI = 0.1) and treated
with the compounds at 8 concentrations (3-fold dilution, in duplicate) for 3 days.
Antiviral activity was detected by measuring RLuc activity using Renilla luciferase
assay buffer and substrate by following the manufacture’s instructions. Cytotoxicity
of the compounds was tested in uninfected Huh-7.5.1 cells in parallel. EC50 and CC50
values were calculated with the GraphPad Prism software.
4.2.2. Measurement of the anti-HCV activities of compounds using HCV
replicon
4.2.2.1. Cell line and cell culture
The Huh7 cell line were kindly provided by AppTec company(America). HCV GT1b
and GT1a wild type replicon, GT1b/3a, GT1b/4a, GT1b/5a and GT1b/6a NS5A
chimeric replicons were provided by WuXi AppTec. Huh7 cells were grown in
Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum.
The replicon RNAs were in vitro transcribed using the replicon plasmid DNAs.
4.2.2.2. Antiviral activity assay test with HCV replicon
Compounds were added to 96-well plates by Echo at 9 concentrations with a serial
3-fold dilution, in triplicate. The final concentration of DMSO in the cell culture
medium was 0.5%. The Huh7 cells were transiently transfected with the replicon
RNAs by electroporation and seeded at a density of 10,000 cells/well in 96-well plates.
And then the cells were cultured and treated with the compounds at 37°C and 5% CO2
for 3 days. The cell viability in GT1b replicon assay was tested using CellTiter-Fluor

ACCEPTED MANUSCRIPT
following the manufacture’s instruction. For antiviral activity detection, the
supernatants were removed from the wells, then Bright-Glo will be added to detect
luciferase luminescent signal. The raw data (RLU) will be used for calculating the
antiviral activity (% inhibition) of the compounds. EC50 and CC50values were
calculated using the GraphPad Prism software.
4.2.3. In vitro PK assessment
Pooled mouse, rat and human liver microsomes were obtained from BD Gentest. A
typical standard reaction mixture 300µL consisted of the pooled liver microsomes 0.2
mg/mL, 1mM NADPH, 5mM MgCl2, 100 mM potassium phosphate buffer (pH 7.4)
and 0.2 µM of test compouds. After a 5-min pre-incubation at 37 °C, the reactions
were initiated by addition of NADPH and incubation proceeded for 5, 15, 30, 60 min
at 37 °C in a shaking metal bath. The reaction was stopped by transferring 60 µL
aliquots to the tubes on ice and adding 120 µL amounts of ice-cold acetonitrile
containing internal standards. Concentration of the test compounds was measured by
UPLC-MS/MS.
4.2.4. In vivo PK assessment.
Adult male Mice, provided by sino-British SIPPR/BK Lab. Animal Ltd, were housed
at the centralized animal facilities with a 12 h light-dark cycle. The housing
temperature and relative humidity were controlled at 22 °C and 55%, respectively.
The animals fasted 12 h before drug administration had free access to water. A single
dose of 10 mg/kg was administered through intragastric (i.g.) route of administration.
The concentration of compounds in the plasma and liver was measured by the
ultra-performance
liquid
chromatography-mass
spectrometry
analysis
(UPLC-MS/MS). PK parameters were calculated using DAS 3.2.5.8 beagle dogs.
They were administrated via extravascular routes with a single dose of 2 mg/kg, the
methods of plasma preparation and detection were same as mice plasma samples.
4.2.5. Interaction with CYP450 enzymes
The inhibitory potencies of compound 10d on CYP3A4, CYP2D6, CYP2C9,
CYP2C19, CYP2B6, CYP1A2, CYP2A6 were evaluated in pooled human liver
microsomes using eight CYP substrates and ultra-performance liquid
chromatography-tandem mass spectrometry (UPLC-MS/MS).
The incubation mixtures were prepared in total volumes of 100 µL as follows: pooled
human liver microsomes (0.2 mg/mL), 1 mM NADPH, 5 mM MgCl2, 100 mM

ACCEPTED MANUSCRIPT
potassium phosphate buffer (pH 7.4), 1 µM and 10 µM of compound 12 in dimethyl
sulfoxide (DMSO, DMSO <1% v/v), and seven CYP probe substrates. The CYP
substrates were used at concentrations approximating their respective Km values: 5
µM midazolam, 40 µM Nifedipine, 5 µM dextromethorphan, 100 µM
(S)-mephenytoin, 100 µM tolbutamide, 75 µM bupropion hydrochloride, 20 µM
phenacetin and 2 µM coumarin. After a 5-min pre-incubation at 37 °C, the reactions
were initiated by addition of NADPH and incubation proceeded for 30 min at 37 °C in
a shaking metal bath. The reaction was stopped by placing the tubes on ice and adding
300-µL amounts of ice-cold acetonitrile containing internal standards. Concentration
of the CYP substrates and their metabolites was measured by UPLC-MS/MS.
4.2.6. Cardiotoxicity in vitro
Micropipette was pulled from borosilicate glass with the pipette tip resistance
between 3 ~ 5 MΩ. For each experiment, a single dish of cells is removed from the
incubator, washed twice with room temperature ECS and then placed on the
microscope stage. A commercial patch clamp amplifier was used for the whole cell
recordings.
The tail currents were evoked in room temperature once every 30 s by a 3 s -50 mV
repolarizing pulse following a 2 s +50 mV depolarizing pulse with a hold voltage of
-80 m V. A 50 ms depolarized pulse to -50 mV at the beginning of the voltage
protocol served as a baseline for calculating the amplitude of the peak tail current.
Only stable cells with recording parameters above threshold were allowed to enter the
drug application procedure. The hERG currents were allowed to stabilize over a 3
minutes period in the presence of vehicle alone prior to test article application. The
cells were kept in the test solution until the peak tail current was stable (< 5% change)
for ~5 sweeps or for a maximum of 6 min, whichever came first.
4.2.7. Permeability and efflux in human Caco-2 cells
For the permeability studies, Caco-2 cells were seeded on 24-well Transwell inserts
(Millicell Hanging Cell Culture Insert, PET 1 µm) at a cell density of 104 cells per
well. The cells were cultured at 37°C, 5% CO2 atmosphere and maintained in
Dulbecco’s modified Eagle’s medium (DMEM) with sodium pyruvate containing 10%
fetal bovine serum (FBS), 1% nonessential amino acids, and 100 U/ml and 100 g/ml
penicillin and streptomycin, with replacement every 2-3 day, during 21-23 days, until
the monolayer achieved minimum of 200 Ω cm2 of transepithelial electrical resistance
(TEER) values. The tightness of the Caco-2 monolayer was assessed as the TEER

ACCEPTED MANUSCRIPT
values using a Millicell ERS-2 Volt-Ohm Meter and accessories (Millipore
Corporation, Bedford, MA), equipped with a pair of chopstick electrodes inserted into
the apical medium. Before the transport experiments, the cell monolayers were rinsed
twice using warm (37 °C) HBSS (Gibco 14025-092), and then the cells were
incubated using 2% BSA (with or without inhibitor) at 37°C for 30min. After
preincubation, the cell monolayers were incubated with samples in 2% BSA from
either the apical (AP) or basolateral (BL) side for 90 min at 37 °C. The volume of
incubation media on the AP and BL sides was 0.4ꢀmL and 1.4ꢀmL, respectively, and a
100µL aliquot of the incubation solution was withdrawn from the receiver
compartment for analysis. Each experiment was performed in duplicate. The amount
of the samples was analyzed by UPLC/MS/MS. The apparent permeability (Papp)
was calculated as described elsewhere[29].
4.2.8. Plasma protein binding
In vitro plasma protein binding assay was carried out by equilibrium dialysis method
using HTD96b (interchim inc.USA). Plasma (pH 7.4) was preincubated for 15 min at
37 °C prior to addition of stock DMSO solutions (0.5% final concentration)
containing 10d and ombitasvir(1 and 10 µM ). Drug-spiked plasma was then
aliquoted into the donor chambers of the dialysis plate (100 µL per half-well). An
equal volume of phosphate buffer (100 mM, pH 7.4) was placed in each
corresponding receiver well. The dialysis plate was sealed with the kit adhesive
(HT-Dialysis) and dialysis was conducted in an orbital shaker (100 rpm) maintained
at 37 °C for 6 h. Following incubation, aliquots from the donor and receiver chambers,
as well as the plasma stock solution incubated for 6 h at 37 °C were removed and
added to vials containing internal standard in acetonitrile and were mixed to prevent
nonspecific binding to the vial and pipette tip. All samples were analyzed by UPLC–
MS/MS.
4.2.9. Liver distribution
The liver distribution of ombitasvir and 10d were evaluated in adult male Institute of
Cancer Research (ICR) mice (18.0~23.0g,4~6 weeks). After a 12h overnight fast
(with free access to water), the mice were randomly divided into two groups (15
animals for each), and administrated with a single dose of 10 mg/kg of ombitasvir and
10d respectively by intragastric (i.g.) administration. Plasma and liver samples were
collected at 0.5h, 3h, 8h, 16h and 24h after oral dosing (n=3). The concentration of the
two compounds was measured by the ultra-performance liquid chromatography-mass

ACCEPTED MANUSCRIPT
spectrometry analysis (UPLC-MS/MS) and the concentration ratio of liver and plasma
(LPR) was calculated.
4.2.10. Repeated-dose toxicity study
After acclimation, animals were randomly allocated to two groups, each containing
five males and five females, and orally administered 20ml/kg body weight of 10d
(150 mg/kg body weight) or the vehicle alone once daily by gavage. Mice were killed
after the 14th administration. Animals in all groups were observed daily and detailed
clinical signs evident. Their body weights and food intake were monitored on day 1 of
administration and thereafter twice per week. Body weights were used for calculation
of the volume of the test chemical to be administered.
4.2.11. Hematology, blood biochemistry, and serum hormone measurements
At necropsy, animals were weighed and sacrificed by exsanguination from the
abdominal aorta under ether anesthesia. Before killing, blood samples were collected
from the abdominal aorta to use for hematology, blood chemistry and measurement of
serum hormone levels. The hematological parameters included the white blood cell
(WBC) count, red blood cell (RBC) count, hemoglobin (Hb) concentration, platelet
(Plt) count, reticulocyte ratio and differential leukocyte counts. Blood biochemistry
parameters were total protein (TP), albumin (ALB), total bilirubin (TB), indirect
bilirubin (DB), total billliary acid(TBA), aspartate aminotransferase (AST), alanine
aminotransferase (ALT), γ-glutamyltransferase (γ-GGT), glutamate dehydrogenase
(GLDH) and alkaline phosphatase (ALP), total cholesterol (CHO), triglyceride (TG),
glucose (GLU), blood urea nitrogen (BUN), creatinine (Cre), Creatine Kinase (CK).
4.2.12. Histopathology
At autopsy, gross findings were recorded and abnormal portions were removed along
with all organs/tissues. They were fixed with 10% formalin. Hearts, liver, spleen,
kidneys, thymus, brain, testis and ovary were weighed before fixation. Reference to
the results of blood biochemistry and hematology, the heart, liver, spleen were sent for
pathological examination.
Acknowledgment
This study was partially supported by the Natural Science Foundation of Jiangsu
Province (Grants No BK20170454).
Reference
[1] A. Petruzziello, S. Marigliano, G. Loquercio, A. Cozzolino, C. Cacciapuoti,

ACCEPTED MANUSCRIPT
Global epidemiology of hepatitis C virus infection: An up-date of the distribution and
circulation of hepatitis C virus genotypes, World journal of gastroenterology, 22
(2016) 7824.
[2] J.-M. Pawlotsky, NS5A inhibitors in the treatment of hepatitis C, Journal of
hepatology, 59 (2013) 375-382.
[3] B.D. Lindenbach, C.M. Rice, Unravelling hepatitis C virus replication from
genome to function, Nature, 436 (2005) 933.
[4] J.A. Holmes, A.J. Thompson, Interferon-free combination therapies for the
treatment of hepatitis C: current insights, Hepatic medicine: evidence and research, 7
(2015) 51.
[5] R. Schinazi, L. Bassit, C. Gavegnano, HCV drug discovery aimed at viral
eradication, Journal of viral hepatitis, 17 (2010) 77-90.
[6] M.G. Katze, B. Kwieciszewski, D.R. Goodlett, C.M. Blakely, P. Neddermann,
S.-L. Tan, R. Aebersold, Ser2194 is a highly conserved major phosphorylation site of
the hepatitis C virus nonstructural protein NS5A, Virology, 278 (2000) 501-513.
[7] 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 II, Chemical genetics strategy
identifies an HCV NS5A inhibitor with a potent clinical effect, Nature, 465 (2010) 96.
[8] I.-J. Kang, S.-J. Hsu, H.-Y. Yang, T.-K. Yeh, C.-C. Lee, Y.-C. Lee, Y.-W. Tian,
J.-S. Song, T.-A. Hsu, Y.-S. Chao, A Potent, Selective, and Orally Bioavailable HCV
NS5A
Inhibitor
for
Treatment
of
Hepatitis
C
Virus:(S)-1-((R)-2-(Cyclopropanecarboxamido)-2-phenylacetyl)-N-(4-phenylthiazol-
2-yl) pyrrolidine-2-carboxamide, Journal of medicinal chemistry, 60 (2016) 228-247.
[9] Å. Rosenquist, B. Samuelsson, P.-O. Johansson, M.D. Cummings, O. Lenz, P.
Raboisson, K. Simmen, S. Vendeville, H. de Kock, M. Nilsson, Discovery and
development of simeprevir (TMC435), a HCV NS3/4A protease inhibitor, Journal of
medicinal chemistry, 57 (2014) 1673-1693.
[10] C. Hézode, T. Asselah, K.R. Reddy, T. Hassanein, M. Berenguer, K.
Fleischer-Stepniewska, P. Marcellin, C. Hall, G. Schnell, T. Pilot-Matias, Ombitasvir
plus paritaprevir plus ritonavir with or without ribavirin in treatment-naive and
treatment-experienced patients with genotype 4 chronic hepatitis C virus infection
(PEARL-I): a randomised, open-label trial, The Lancet, 385 (2015) 2502-2509.
[11] I.M. Jacobson, S.C. Gordon, K.V. Kowdley, E.M. Yoshida, M. Rodriguez-Torres,
M.S. Sulkowski, M.L. Shiffman, E. Lawitz, G. Everson, M. Bennett, Sofosbuvir for