Discovery of a series of novel compounds with moderate anti-hepatitis C virus NS3 protease activity in vitro

Article abstract of DOI:10.1016/j.bmc.2015.07.032

The hepatitis C virus (HCV) NS3/4A protease that plays an important role in the viral life cycle has been proven to be an excellent target for the discovery of anti-HCV drugs. Enlightened by some P2-triazole and amide compounds, which had been found as HCV NS3 protease inhibitors, we designed and synthesized a series of novel compounds by incorporating different amino acid residues in P1/P1′ and P3/P3′ position to develop novel antiviral agents. The result of enzyme inhibition assay indicated that all the designed compounds showed moderate anti-HCV NS3 protease activity. On the basis of the biological result, a detailed structure-activity relationship (SAR) was derived and discussed.

Full text of DOI:10.1016/j.bmc.2015.07.032

Accepted Manuscript
Discovery of a series of novel compounds with moderate anti-hepatitis C virus
NS3 protease activity in vitro
Fangyuan Shi, Yingjie Zhang, Wenfang Xu
PII:
S0968-0896(15)00616-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.07.032
BMC 12461
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
29 May 2015
16 July 2015
17 July 2015
Please cite this article as: Shi, F., Zhang, Y., Xu, W., Discovery of a series of novel compounds with moderate anti-
hepatitis C virus NS3 protease activity in vitro, Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/
1
0.1016/j.bmc.2015.07.032
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Discovery of a series of novel compounds with moderate anti-hepatitis C virus NS3 protease
activity in vitro
Fangyuan Shi, Yingjie Zhang*and Wenfang Xu*
Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong University, 44,
West Culture Road, Jinan, Shandong 250012, P. R. China
Corresponding author: Tel. /fax: +86 531 88382264. E-mail addresses: wenfxu@163.com
[Abstract] The hepatitis C virus (HCV) NS3/4A protease that plays an important role in the viral life
cycle has been proven to be an excellent target for the discovery of anti-HCV drugs. Enlightened by
some P2-triazole and amide compounds, which had been found as HCV NS3 protease inhibitors, we
designed and synthesized a series of novel compounds by incorporating different amino acid residues
in P1/P1’ and P3/P3’ position to develop novel antiviral agents. The result of enzyme inhibition assay
indicated that all the designed compounds showed moderate anti-HCV NS3 protease activity. On the
basis of the biological result, a detailed structure–activity relationship (SAR) was derived and
discussed.
[Key words] HCV; Protease inhibitors; Peptidomimetics; Triazole; acylsulfonamide.
1.
Introduction
Hepatitis C is a serious liver disease caused by Hepatitis C virus (HCV). HCV can cause inflammation
of the liver, which leads to serious conditions such as acute liver disease, cirrhosis, liver cancer and is
also the leading cause of liver transplants.[1] According to incomplete statistics, an estimated 200
million people worldwide are infected with HCV, which has been a serious global health problem.[2, 3]
HCV is a member of the Flaviviridae family consisting of a positive single strand RNA that encodes a
polyprotein. This polyprotein is cleaved to the structural and nonstructural (NS) proteins by both host
and viral proteases.[4] The NS3 region is a trypsin-like serine protease that plays an essential role in
viral replication and involves in processes to attenuate and evade the host cell’s natural immune
defense. [5] Extensive studies have indicated that blocking the function of NS3 protease would
effectively inhibit the proliferation of HCV. [6-8]
Previously, the standard treatment of chronic HCV infection has been based on the combination of
pegylated interferon-ꢀ (PEG-IFN) and ribavirin (RBV),[9, 10] However, the cure rates of this
procedure is only 40−50% for patients infected with genotype 1, and a little higher for patients infected
with genotypes 2 and 3. [11] Besides, the procedure is associated with severe side effects and long
treatment period. [10, 12]
Since boceprevir and telaprevir were approved (figure 1), HCV protease inhibitors have been the main
options for the treatment of HCV, and also became a hot research topic in the field of anti-HCV
drugs.[13-15] Till now, a number of potent inhibitors have been identified by various research
laboratories around the world.[6, 16-23] However, most of these inhibitors are poorly tolerated and
resistant viral variants are constantly emerging. [24]
Studies have shown that proline played an important role in the skeleton of the compound, so we
maintained it in our compound skeleton. [25, 26]Ayclsulfonamide was a superior recognition group,

whose binding properties exceeded the carboxylic acid and ꢀ-ketone carbonyl, which have been
confirm to related to the side effect. So we selected the ayclsulfonamide as the recognition group. So
many works have proved that substituent in P2 position had a huge impact on the activity, and many
scientists started work around this position including us. Since the P2-triazole and amide compounds
have been proved to have potent inhibitory activity on the NS3 serine protease, [27, 28] based on this,
we designed and synthesized a series of P2-triazole and amide analogs bearing different amino acid
residues in P1/P1’ and P3/P3’ position (figure 1) to develop novel antiviral agents. The result of
enzyme inhibition assay indicated that all the designed compounds showed moderate anti- HCV NS3
protease activity with IC50values in the micromolar range.
O
N
O
N
H
N
N
N
N
H
H
O
N
N
O
NH
H
O
O
O
O
NH
NH2
O
N
H
O
O
VX-950, Telaprevir
SCH 503034, Boceprevir
K = 14 nM, EC = 200 nM
K = 40 nM, EC = 350 nM
i
50
I
50
N
N
N
O
N
N
H
O
N
S
H
NH
O
O
O
O
O
P3'
P3
P2
P1
P1'
Figure1. The chemical structures of boceprevir and telaprevir and P3’−P1’ spanning HCV NS3/4A

protease inhibitor
2
. Result and Discussion
.1 Chemistry
2
The target compounds were synthesized according to Scheme 1. Compound 2 was a key intermediate
which was prepared from N-Boc-trans-4-hydroxy-L-proline methyl ester 1 through two steps,
sulfonation with methanesulfonyl chloride and substitution with sodium azide. Compound 3 was
obtained by the azide alkyne Huisgen cycloaddition using a Copper (Cu) catalyst at room temperature
to afford 1,4-regioisomers of 1,2,3-triazoles as sole products.[29] Intermediates 4a-4c were prepared by
condensation of Boc-protected amino acids with sulfonamide using isobutyl chloroformate and NaH.
Condensation of compound 3 with intermediates 4 afforded intermediates 5. The following
deprotection and introduction of acylated amino acids on the NH group of the pyrrole ring gave the
final products A1-A7 and B1-B7. The azide group of compound 2 was reduced with hydrogen to give
compound 6. Condensation of it with acid fragments on the NH
2
group gave intermediates 7a-7c. The
following steps were the same as described above to obtain compound C1-C3.
Scheme1. Reagents and conditions: (a)$ )MsCl, Et
3
N, DCM, rt; $ )NaN
3
, DMF, 80$ ,yieid 70%; (b)
, yieid 88%; (d) EDCI,
phenylacetylene, CuSO , ascorbic acid, EtOH/H O, rt, yieid 73%; (c) Pd/C, H
4
2
2
NMM, HOBt, DCM, rt, yieid46%-55%; (e) HCl/EtOAc, yieid 92%; (f) NaOH, MeOH/H
2
O, yieid 95%;
(g) 6-Chloropurine, Et N, butyl alcohol, reflux, yieid 40%.
3

2.2 Pharmacology
The newly synthesized compounds were assayed for their ability to inhibit NS3/4A according to the
standard method [30], Table 1-3. In general, all the compounds demonstrated a clear structure–activity
1 2 3 4
relationship (SAR) surrounding the R , R , R and R groups. It could be found that compounds
containing a hydrophobic aromatic group in R
aliphatic hydrocarbon group, such as compounds A1, A2, A3 and compounds B1, B3. Notably,
introduction of 2-methylpropanoyl on R position resulted in decreased activity, such as compounds A5
4
position had better activity relative to the ones with an
4
and B4, with IC50 values ＞100 ꢁM. From compounds A4, A6 and A7, it was noted that their inhibitory
activity was increased with the size of the aromatic group at R position, indole＞benzene＞tert butyl.
The R fragment bearing S-isopentane or R-benzyl and R fragment bearing p-xylene or cyclopropane
3
1
2
had little effect on NS3/4A inhibitory activity, which could be easily identified by compounds of A
series and B series.
In an effort to further improve their inhibit activity, we replaced of the triazole with amide group. From
compounds C1, C2 and C3 (IC50 values ＞100 ꢁM), it was noted that p-methoxyphenyl and naphthyl
had no benefit on the inhibit activity , but mefenamic acid fragment greatly improved the strength of
the activity with IC50 values = 2.1ꢁM. It suggested that the substituent on the benzene ring contribute
to the activity.
In order to gain insight on the binding pattern of these series of inhibitors, compound C3 was docked
into the active site of the NS3 protease (figure 2). In this model, the P1, P2, and P3 moieties of C3 were
docked to the known intermolecular hydrogen bond network with the NS3 protease enzyme.
Importantly, the model indicated that the mefenamic acid substituent on the P2 proline ring had a good
combination with the large, flat lipophilic surface in the protein. However, the S-isopentane substituent
on P3 moiety affected the hydrogen bonding of acylsulfonamide with SER-139, which led to the
decrease of the activity. It seemed that the vinyl cyclopropyl amino acid fragment was necessary for the
activity.
a)
b)
c)

Figure2. a) The docking result of C3 and BMS-650032 (the smaller compound) with the active site of
NS3 (PDB ID: 4A92); b) The bonding mode of BMS-650032 with the active site of NS3; c) The
bonding mode of C3 with the active site of NS3.
Bioisosterism is a useful strategy for the rational design of new drugs and is widely used in the
modification of a lead compound. -O- and -NH- are classic bioisosterism of bivalent groups, so we
replaced the -O- group in proline ring by the-NH- group to get the target compound D1. But it was
depressing that there was a large decrease in the activity with IC50 values ＞100ꢁM, which suggested
that the steric configuration in the C4 position of proline ring was important to the activity.
Table 1
The structures and IC50 values of the target compounds A1-A7 compared with positive control
telaprevir against NS3
compound
A1
R
3
R
4
IC50 (µM)
1
.9
4
A2
A3
3
2.5
A4
A5
A6
Boc
Boc
Boc
5.9
＞100
5.6
A7
＞100

telaprevir
Table 2
0.45
The structures and IC50 values of the target compounds B1-B7 compared with positive control
telaprevir against NS3
compound
B1
R
3
R
4
IC50 (µM)
4.5
Cbz
Boc
72
B2
B3
B4
7
.4
＞
100
Boc
Boc
＞100
B5
B6
7.1
Boc
＞100
B7
telaprevir
0.45
Table 3
The structures and IC50 values of the target compounds C1-C3 compared with positive control
telaprevir against NS3

compound
C1
R
5
IC50 (µM)
＞
100
100
C2
C3
＞
HN
2.1
0.45
telaprevir
3.Conclusion
In summary, we have synthesized a series of novel compounds and evaluated their anti-HCV NS3/4A
activity. The HCV NS3/4A inhibition assay results showed that all the compounds exhibited moderate
NS3/4A inhibitory activity. Among them, compound A1 and C3 were the best with IC50 values of 1.9
and 2.1 ꢁM respectively. NS3 was still an important target for anti-HCV drugs design. Considering the
current anti-HCV situation, it was still necessary to discover novel NS3 inhibitors.
4
. Experiments
.1Chemistry
4
1
Solvent for anhydrous reaction should be processed before use. H-NMR spectra were determined
on a Brucker Avance 300 spectrometer or 600 using TMS as an internal standard. The solvents for
1
NMR were DMSO-d
6
(ꢂ 2.5 for H). HRMS analysis was provided by Agilent 6520 Q-TOF LC/MS
spectrometer (Agilent, Germany). All reactions were monitored by thin-layer chromatography (TLC)
on 25.4×76.2 mm silica gel plates (GF-254). Silica gel used for column chromatography was 200~300
mesh. Melting points were determined on an electrothermal melting point apparatus and were
uncorrected.
(2S)-1-tert-butyl 2-methyl 4-azidopyrrolidine-1,2-dicarboxylate(2)
To a solution of N-Boc-trans-4-hydroxy-L-proline (2.45 g, 10.0 mmol) and Et
3
N (2.02 g, 20.0 mmol)
in dichloromethane (50 mL), methanesulfonyl chloride (1.72 g, 15.0 mmol) was added dropwise , and

the mixture was stirred at room temperature overnight. After evaporation of the solvent, the obtained
residue was extracted with ethyl acetate and the combined extracts were washed with saturated
NaHCO
3
, 1 M aqueous HCl, brine, dried over anhydrous MgSO
4
, and concentrated to yield the crude
product. After dissolving it into 20 mL DMF, NaN
3
(3.3g, 50.0 mmol) was added and the mixture was
heated at 80 °C overnight. The mixture was allowed to cool to room temperature, then diluted with
water (50 mL), and extracted with EtOAc (50 mL × 3). The organic layer was dried over anhydrous
MgSO
4
, filtered, and evaporated to yield the crude product without further purification. (1.92 g, yield:
70.3%).
(
2S,4S)-1-(tert-butoxycarbonyl)-4-(4-phenyl-1H-1,2,3-triazol-1-yl)pyrrolidine-2-carboxylic acid
3)
(
4
Ascorbic acid (0.18 g, 1.0 mmol) and a catalytic amount of CuSO was added to a mixture of
compound 2 (2.72 g, 10.0 mmol), and phenylacetylene (1.53 g, 15.0 mmol) in CH CH
3
2 2
OH + H O (1:1)
(50 mL), and the mixture was stirred at room temperature overnight. Then the mixture was extracted
with ethyl acetate and the combined extracts were washed with saturated NaHCO
3
, 1 M aqueous HCl,
brine, dried over anhydrous MgSO
1:1) (50 mL) and NaOH (0.8 g, 20.0 mmol) was added. After stirring it at room temperature for 0.5
hour, the solvent was evaporated. The obtained residue was extracted with ethyl acetate (50 mL × 3).
The organic layer was dried over anhydrous MgSO , filtered, and evaporated to yield the crude product
4
, and concentrated. The product was dissolved in CH
3
OH + H O
2
(
4
which was purified by column chromatography eluting with PE/EA (2:1 v/v) to give compound 3 as
1
white solid (2.63 g, yield: 73.5%); H-NMR (600 MHz, DMSO): ꢂ 12.79 (brs, 1H), 8.74 (d, 1H, J=7.8),
7.85 (d, 2H, J=7.8), 7.45 (t, 2H, J=7.8), 7.34 (t, 1H, J=7.8), 5.28–5.25 (m, 1H), 4.32–4.29 (m, 1H),
4.09–4.06 (m, 1H), 3.78–3.75 (m, 1H), 2.96–2.93 (m, 1H), 2.57–2.50 (m, 1H), 1.43-1.37 (m, 9H).
(S)-2-amino-4-methyl-N-tosylpentanamide (4a)
To a mixture of N-Boc-L-leucine (0.69 g, 3.0 mmol), 4-Methylmorpholine (0.61 g, 6. 0 mmol) in
anhydrous THF (20 mL) was added isobutyl chloroformate (0.45 g, 3.3 mmol) dropwise, and the
mixture was stirred at -20 °C for 1 hour. Then added it to the p-toluenesulfonamide (0.62 g, 3.6 mmol)
which had been stirred with NaH(0.36 g, 9.0 mmol) in anhydrous THF for 4 hours. The reaction
solution was stirred at room temperature overnight. After evaporation of the solvent, the obtained
residue was extracted with ethyl acetate and the combined extracts were washed with saturated
3 4
NaHCO , 1 M aqueous HCl, brine, dried over anhydrous MgSO , and concentrated to yield the crude
product. The crude product was treated with HCl/EtOAc to give a white solid. (0.69 g, yieid: 82.1%);
1
H-NMR (400 MHz, DMSO): ꢂ 8.34 (s, 3H), 7.83 (d, 2H, J=8), 7.44 (d, 2H, J=8), 7.34 (t, 1H, J=7.8),
3
.83 (s, 1H), 2.40 (s, 3H), 1.60–1.57 (m, 1H), 1.56–1.50 (m, 2H), 0.84-0.82 (m, 6H).
The other compounds (4b-4c) were prepared in the same procedure as described above.
2S,4S)-tert-butyl 2-(((S)-4-methyl-1-(4-methylphenylsulfonamido)-1-oxopentan-2-yl)carbamoyl)-
-(4-phenyl-1H-1,2,3-triazol-1-yl)pyrrolidine-1-carboxylate(5a)
To a solution of compound 3 (0.72 g, 2.0 mmol) and 4-Methylmorpholine (0.61 g, 6. 0 mmol) in
(
4

dichloromethane (50 mL), HOBt (0.32 g, 2.4 mmol) and EDCI (0.49 g, 2.4 mmol) was added , and the
mixture was stirred at room temperature for 0.5 hour. After compound 4a was added, the solution was
stirred overnight. The solvent was evaporated, and the obtained residue was extracted with ethyl
3
acetate. The combined extracts were washed with saturated NaHCO , 1 M aqueous HCl, brine, dried
4
over anhydrous MgSO , and concentrated to yield the crude product which was purified by column
chromatography eluting with PE/EA (1:1 v/v) to give compound 5a as white solid (0.58 g, yield:
46.2%);
1
H-NMR (600 MHz, DMSO): ꢂ 8.69 (s, 1H), 7.86 (d, 2H, J=7.2), 7.575-7.550 (m, 3H), 7.32 (d, 2H,
J=7.2), 7.06-7.05 (m, 2H), 5.21-5.20 (m, 1H), 4.34-4.31 (m, 1H), 4.12-4.09 (m, 1H), 4.00 (s, 1H),
3
.67-3.64 (m, 1H), 2.90-2.89 (m, 1H), 2.50-2.39 (m, 1H), 2.23 (s, 3H), 1.57 (s, 1H), 141–1.25 (m, 11H),
.80-0.78 (m, 6H).
0
The other compounds (5b-5c, 7a-7c, 8a-8c, 10) were prepared in the same procedure as described
above.
(
2S, 4S)-1-tert-butyl 2-methyl 4-aminopyrrolidine-1, 2-dicarboxylate(6)
Compound 2 (2.72 g, 10 mmol) was added in 100 ml dried CH OH and hydrogenated at room
temperature using hydrogen balloon in the presence of 200 mg of 10 % Pd/C. The reaction was
3
monitored by TLC until the starting material was no longer detected. Evaporation of CH
afford light yellow oil (2.1 g, yield: 87.5 %).
3OH could
(2S, 4S)-4-((7H-purin-6-yl) amino)-1-(tert-butoxycarbonyl)pyrrolidine-2-carboxylic acid(9)
6-Chloropurine was added to a solution of compound 6 (2.4 g, 10.0 mmol) and Et N (2.02 g, 20.0
3
mmol) in butyl alcohol (50 mL). After refluxing for 5 hours, the solvent was evaporated to yield the
crude product which was purified by column chromatography eluting with PE/EA (3:1 v/v) to give
compound 9 as white solid (1.1 g, yield: 39.7%).
(2S,4S)-1-((S)-2-cyclohexyl-2-(thiophene-2-carboxamido)acetyl)-N-((S)-4-methyl-1-(4-
methylphenylsulfonamido)-1-oxopentan-2-yl)-4-(4-phenyl-1H-1,2,3-triazol-1-yl)pyrrolidine-2-
carboxamide (A1)
1
White solid, mp=153-154℃ , H-NMR (300 MHz, DMSO): ꢂ 11.90 (s, 1H), 8.843 (d, 1H, J=7.2),
8.761 (s, 1H), 8.01 (dd, 1H, J=3.6, J=0.9), 7.90-7.87 (m,3H), 7.84-7.73 (m,2H), 7.63 (d, 2H, J=8.1),
7.46 (t,2H, J=1.5), 7.36-7.27 (m, 4H), 7.18-7.13 (m,1H), 5.33-5.30 (m, 1H), 4.56-4.50 (m, 2H), 4.45-
43.35 (m, 2H), 3.98-3.93 (m, 1H), 2.89-2.80 (m,1H), 2.45-2.39 (m, 1H), 2.32-2.27 (m, 4H), 1.96-1.92
(m, 2H), 1.72-1.66 (m, 4H), 1.21-1.15 (m,9H), 0.88-0.84 (m,1H), 0.61 (d, 3H, J=6.3), 0.54 (d, 3H,
+
+
48 7 6 2
J=6.3). HRMS calcd for C39H N O S [M+H] 774.3102; found: 774.3121.
(2S,4S)-1-((S)-2-(furan-2-carboxamido)-3,3-dimethylbutanoyl)-N-((S)-4-methyl-1-(4-
methylphenylsulfonamido)-1-oxopentan-2-yl)-4-(4-phenyl-1H-1,2,3-triazol-1-yl)pyrrolidine-2-
carboxamide (A2)
1
White solid,mp= 250-252℃ , H-NMR (300 MHz, DMSO): ꢂ 12.30 (s, 1H), 8.80 (s, 1H), 8.37 (d, 1H,
J=7.5), 7.89-7.85 (m, 3H), 7.77 (d, 2H, J=8.4), 7.62 (d, 1H, J=9), 7.47 (t, 2H, J=7.5), 7.37-7.30 (m,4H),

6.41 (dd, 1H, J=3.3, J=1.8), 5.35-5.24 (m,1H), 4.70-4.60 (m, 2H), 4.49 (t, 1H, J=8.4), 4.41-4.33 (m,
1H), 3.81 (t, 1H, J=9.6), 2.89-2.73 (m, 1H), 2.33-2.20 (m, 4H), 1.73-1.64 (m, 1H), 1.42-1.34 (m, 2H),
1
3
1
.00 (s, 9H), 0.85 (d, 3H, J=6.6), 0.81 (d, 3H, J=6.3). C NMR (75 MHz, DMSO): ꢂ 171.95, 170.87,
69.66, 158.10, 147.47, 146.87, 145.90, 144.64, 136.77, 131.10, 129.91, 129.38, 128.41, 127.88,
25.64, 121.38, 114.73, 112.43, 58.30, 57.31, 52.97, 51.75, 35.79, 34.44, 26.71, 24.34, 23.56, 21.49.
1
1
+
+
46 7 7
HRMS calcd for C37H N O S [M+H] 732.3174; found: 732.3189.
(2S,4S)-1-((S)-2-benzamido-2-cyclohexylacetyl)-N-((S)-4-methyl-1-(4-methylphenylsulfonamido)-
1-oxopentan-2-yl)-4-(4-phenyl-1H-1,2,3-triazol-1-yl)pyrrolidine-2-carboxamide(A3)
1
White solid,mp= 258-259℃ , H-NMR (300 MHz, DMSO): ꢂ 11.88 (s,1H), 8.79-8.75 (m, 2H), 7.88-
7.84 (m, 3H), 7.7 (d, 2H, J=7.2), 7.64 (d, 2H, J=8.4), 7.58-7.53 (m, 1H), 7.48-7.43 (m, 5H), 7.42-
7.26(m, 4H), 5.34-5.30 (m, 1H), 4.57-4.49 (m, 2H), 4.43-4.42 (m, 2H), 4.04-3.94 (m, 1H), 2.88-2.79
(m, 1H), 2.50-2.46 (m, 1H), 2.31 (s, 3H), 1.99-1.93 (m, 2H), 1.72-1.65 (m, 4H), 1.19-1.14 (m, 9H),
+
+
0
49 7 6
.88-0.84 (m, 1H), 0.59 (d, 3H, J=6.3), 0.52 (d, 3H, J=6.3). HRMS calcd for C41H N O S [M+H]
768.3538; found:768.3559.
tert-butyl((S)-1-((2S,4S)-2-(((S)-4-methyl-1-(4-methylphenylsulfonamido)-1-oxopentan-2-
yl)carbamoyl)-4-(4-phenyl-1H-1,2,3-triazol-1-yl)pyrrolidin-1-yl)-1-oxo-3-phenylpropan-2-
yl)carbamate(A4)
1
White solid,mp=142-143℃ , H-NMR (300 MHz, DMSO): ꢂ 12.22 (s, 1H), 8.72 (s, 1H), 8.21-8.19 (m,
1
5
3
H), 7.89-7.63 (m, 2H), 7.76 (d, 2H, J=8.4), 7.50-7.44 (m,2H), 7.37-7.19 (m, 8H), 7.04 (d, 1H, J=8.1),
.37-5.31 (m, 1H), 4.58-4.50 (m,2H), 4.66-4.40 (m,1H), 4.32-4.25 (m, 1H), 3.89 (t, 1H), 2.97-2.69 (m,
1
3
H), 2.34-2.24 (m,4H), 1.62-1.57 (m, 1H), 1.40-1.25 (m, 11H), 0.86-0.75 (m, 6H). C NMR (75 MHz,
DMSO): ꢂ 171.65, 170.57, 170.34, 155.34, 146.45, 137.72, 130.62, 129.30, 128.84, 128.22, 127.95,
127.29, 126.22, 125.17, 120.79, 78.06, 57.89, 57.34, 53.87, 51.65, 51.10, 36.08, 34.02, 28.12, 24.05,
+
+
2
50 7 7
2.88, 21.37, 20.92. HRMS calcd for C40H N O S [M+H] 772.3487; found: 772.3507.
(2S,4S)-1-((S)-2-isobutyramido-3-phenylpropanoyl)-N-((S)-4-methyl-1-(4-
methylphenylsulfonamido)-1-oxopentan-2-yl)-4-(4-phenyl-1H-1,2,3-triazol-1-yl)pyrrolidine-2-
carboxamide(A5)
1
White solid,mp= 122-123℃ , H-NMR (300 MHz, DMSO): ꢂ 12.21 (s,1H), 8.71 (s, 1H), 8.22 (d, 1H,
J=7.2), 7.88-7.80 (m, 2H), 7.76 (d, 2H, J=8.4), 7.49-7.44 (m,2H), 7.37-7.13 (m, 9H), 5.37-5.26 (m, 1H),
4.75-4.68 (m,1H), 4.59-4.46 (m,2H), 4.34-4.27 (m, 1H), 3.89 (t, 1H, J=9.3), 3.14-2.71 (m, 3H), 2.49-
1
.15 (m,4H), 1.64-1.53 (m, 1H), 1.37-1.33 (m, 2H), 0.92-0.80 (m, 12H). C NMR (75 MHz, DMSO):
3
2
ꢂ 175.94, 170.27, 146.43, 137.50, 130.61, 129.35, 128.88, 127.92, 127.33, 126.22, 125.17, 120.80,
48 7 6
57.87, 57.19, 51.73, 33.42, 24.05, 22.88, 21.30 20.94, 19.53, 19.12. HRMS calcd for C39H N O S
+ +
[M+H] 741.3381; found: 742.3888.
tert-butyl((S)-3-(1H-indol-3-yl)-1-((2S,4S)-2-(((S)-4-methyl-1-(4-methylphenylsulfonamido)-1-
oxopentan-2-yl)carbamoyl)-4-(4-phenyl-1H-1,2,3-triazol-1-yl)pyrrolidin-1-yl)-1-oxopropan-2-
yl)carbamate(A6)
1
White solid, mp= 146-147℃ , H-NMR (300 MHz, DMSO): ꢂ 12.22 (s, 1H), 10.85 (s, 1H), 8.68 (s, 1H),

8
.25-8.20 (m, 1H), 7.88-7.85 (m, 2H), 7.77-7.70 (m, 2H), 7.54-7.37 (m,4H), 7.35-7.2 (m, 5H), 7.06-
.95 (m, 2H), 5.32-5.28 (m, 1H), 4.56-4.43 (m,3H), 4.34-4.27 (m,2H), 3.88-3.82 (m, 1H), 3.07-3.02
6
(m,1H), 2.94-2.80 (m, 2H), 2.34-2.20 (m, 4H), 1.63-1.56 (m, 1H), 1.38-1.30 (m, 9H), 1.18-1.15 (m,
1
3
2
1
1
2
H), 0.86-0.75 (m, 6H). C NMR (75 MHz, DMSO): ꢂ 171.01, 170.42, 155.33, 146.46, 135.97,
30.58, 129.33, 128.88, 128.75, 127.91, 127.33, 127.24, 125.17, 124.04, 120.77, 118.30, 117.99,
11.29, 109.83, 78.45, 78.05, 57.95, 57.29, 53.21, 51.61, 51.14, 34.10, 28.13, 26.39, 24.06, 22.89,
+
+
51 8 7
1.34, 20.92. HRMS calcd for C42H N O S [M+H] 811.3596; found: 811.3621.
tert-butyl ((S)-3,3-dimethyl-1-((2S,4S)-2-(((S)-4-methyl-1-(4-methylphenylsulfonamido)-1-oxopentan-2-
yl)carbamoyl)-4-(4-phenyl-1H-1,2,3-triazol-1-yl)pyrrolidin-1-yl)-1-oxobutan-2-yl)carbamate(A7)
1
White solid,mp= 221-222℃, H-NMR (400 MHz, DMSO): ꢂ 8.61 (s, 1H), 7.88-7.81 (m, 2H), 7.66-
7
4
3
.55 (m, 2H), 7.46-7.41 (m, 3H), 7.35-7.31 (t, 1H), 7.21-7.09 (m, 2H), 6.74-6.72 (d, 1H), 5.41 (m, 1H),
.59-4.55 (m,1H), 4.39-4.37 (m, 1H), 4.14-4.02 (m, 3H), 3.07-3.02 (m,1H), 2.50-2.41 (m, 2H), 2.32 (s,
13
C
H), 1.68 (m, 1H), 1.43-1.41 (m, 2H), 1.35-1.24 (m, 9H), 0.97-0.94 (m, 9H), 0.80-0.64 (m, 6H).
NMR (100 MHz, DMSO): ꢂ 170.30, 170.07, 156.39, 146.87, 131.22, 129.35, 129.25, 128.43, 128.32,
1
2
7
27.09, 125.67, 120.16, 78.63, 59.66, 58.58, 54.00, 53.33, 43.06, 35.60, 35.35, 34.95, 28.70, 28.55,
+
+
6.84, 26.74, 24.31, 23.93, 22.31, 21.31. HRMS calcd for C37
38.3657.
52 7 7
H N O S [M+H] 738.3643; found:
benzyl((S)-1-cyclohexyl-2-((2S,4S)-2-(((R)-1-(cyclopropanesulfonamido)-1-oxo-3-phenylpropan-2-
yl)carbamoyl)-4-(4-phenyl-1H-1,2,3-triazol-1-yl)pyrrolidin-1-yl)-2-oxoethyl)carbamate(B1)
1
White solid, mp= 170-171℃, H-NMR (400 MHz, DMSO): ꢂ 8.73 (s, 1H), 7.87 (d, 2H, J=8), 7.48 (t,
3H, J=8), 7.35-7.31 (m, 6H), 7.11-7.07 (m, 4H), 7.01-7.00 (m, 1H), 5.23-5.17 (m, 1H), 5.06-4.97 (m,
2H), 4.59-4.54 (m, 1H), 4.51-4.47 (m, 1H), 4.31 (m, 1H), 4.12-4.08 (m, 1H), 3.07-3.03 (m, 1H), 2.88-
2.83 (m, 2H), 2.72-2.69 (m, 1H), 1.95-1.84 (m, 2H), 1.76-1.73 (m, 1H), 1.62-1.57 (m, 4H), 1.13-1.04
1
3
(
m, 3H), 0.99-0.76 (m, 6H). C NMR (100 MHz, DMSO): ꢂ 170.80, 156.76, 146.96, 137.52, 131.18,
130.06, 129.42, 128.81, 128.23, 128.12, 125.64, 120.99, 65.92, 58.72, 57.70, 35.34, 28.67, 26.32, 4.79.
+ +
[M+H] 782.3330; found: 782.3347.
HRMS calcd for C41
H N
48 7
7
O S
2
tert-butyl
((S)-1-cyclohexyl-2-((2S,4S)-2-(((R)-1-(cyclopropanesulfonamido)-1-oxo-3-
phenylpropan-2-yl)carbamoyl)-4-(4-phenyl-1H-1,2,3-triazol-1-yl)pyrrolidin-1-yl)-2-
oxoethyl)carbamate(B2)
1
White solid,mp= 126-127℃, H-NMR (300 MHz, DMSO): ꢂ 11.83 (s, 1H), 8.63 (s, 1H), 8.47-8.44 (m,
1H), 7.86-7.83 (m, 2H), 7.50-7.45 (m, 2H), 7.38-7.33 (m, 1H), 7.21-7.14 (m, 4H), 7.08-7.02 (m, 1H),
6.77 (d, 1H, J=8.1 ), 5.29-5.18 (m, 1H), 4.75-4.68 (m, 1H), 4.52-4.45 (m, 2H), 4.07-4.00 (m, 1H), 3.82-
3.76 (m, 1H), 3.09-3.08 (m, 1H), 3.05-2.89 (m, 1H), 2.81-2.76 (m, 1H), 2.73-2.62 (m, 1H), 1.83-1.79
1
3
(
m, 2H), 1.76-1.50 (m, 5H), 1.37 (m, 9H), 0.88-0.77 (m, 9H). C NMR (75 MHz, DMSO): ꢂ 170.60,
170.11, 155.60, 146.39, 136.56, 130.63, 129.25, 128.90, 127.94, 126.35, 125.09, 120.54, 77.99, 58.01,
57.14, 56.62, 53.66, 51.68, 51.14, 37.63, 34.43, 30.65, 28.47, 28.17, 25.77, 25.58, 25.46, 5.53, 5.41.
+
+
50 7 7 2
HRMS calcd for C48H N O S [M+H] 748.3487; found: 748.3502.
(2S,4S)-1-((S)-2-benzamido-2-cyclohexylacetyl)-N-((R)-1-(cyclopropanesulfonamido)-1-oxo-3-

phenylpropan-2-yl)-4-(4-phenyl-1H-1,2,3-triazol-1-yl)pyrrolidine-2-carboxamide(B3)
1
White solid,mp= 125-126℃, H-NMR (400 MHz, DMSO): ꢂ 11.90 (s, 1H), 8.67 (s, 1H), 8.51 (d, 2H),
7
.91-7.86 (m, 4H), 7.54-7.44 (m, 5H), 7.38-7.36 (m, 1H), 7.21-7.15 (m, 4H), 7.07-7.06 (m, 1H), 5.31-
.23 (m, 1H), 4.77-4.73 (m, 2H), 4.51-4.47 (m, 2H), 3.91-3.86 (m, 1H), 3.09-3.05 (m, 1H), 2.96-2.93
5
(m, 1H), 2.80-2.77 (m, 1H), 2.70-2.66 (m, 1H), 1.99-1.94 (m, 1H), 1.87-1.80 (m, 3H), 1.66-1.61 (m,
+
+
3
H), 1.16-1.03 (m, 8H), 0.85-0.81 (m, 1H). HRMS calcd for C40
46 7 6 2
H N O S [M+H] 752.3225; found:
752.3247.
(2S,4S)-1-((S)-2-cyclohexyl-2-isobutyramidoacetyl)-N-((R)-1-(cyclopropanesulfonamido)-1-oxo-3-
phenylpropan-2-yl)-4-(4-phenyl-1H-1,2,3-triazol-1-yl)pyrrolidine-2-carboxamide(B4)
1
White solid,mp= 114-115℃, H-NMR (400 MHz, DMSO): ꢂ 11.88 (s, 1H), 8.64 (s, 1H), 8.50 (d, 1H,
J=8 ), 7.93 (d, 1H, J=8 ), 7.85(d, 2H, J=8 ), 7.48 (t, 2H, J=8 ), 7.36 (t, 1H, J=8 ), 7.21-7.15 (m, 4H),
7
.07-7.04 (m, 1H), 5.24-5.21 (m, 1H), 4.74-4.73 (m, 1H), 4.61-4.56 (m, 1H), 4.48-4.43 (m, 1H), 4.29-
.26 (m, 1H), 3.83-3.78 (m, 1H), 3.08-3.04 (m, 1H), 2.96-2.91 (m, 1H), 2.80-2.74 (m, 1H), 2.69-2.62
4
(m, 1H), 1.85-1.73 (m, 3H), 1.63-1.59 (m, 4H), 1.24 (s, 2H), 1.10 (m, 7H), 1.00-0.95 (m, 8H). HRMS
+
+
48 7 6 2
calcd for C37H N O S [M+H] 718.3381; found: 718.3417.
tert-butyl((S)-1-((2S,4S)-2-(((R)-1-(cyclopropanesulfonamido)-1-oxo-3-phenylpropan-2-
yl)carbamoyl)-4-(4-phenyl-1H-1,2,3-triazol-1-yl)pyrrolidin-1-yl)-4-methyl-1-oxopentan-2-
yl)carbamate(B5)
1
White solid,mp= 141-142℃, H-NMR (400 MHz, DMSO): ꢂ 11.93 (s, 1H), 8.60 (s, 1H), 8.44 (d, 1H,
J=8), 7.86(d, 2H, J=8 ), 7.48 (t, 2H, J=8 ), 7.36 (t, 1H, J=8 ), 7.22-7.16 (m, 4H), 7.08-7.04 (m, 1H),
6.95 (d, 1H, J=8 ), 5.29-5.26 (m, 1H), 4.70-4.64 (m, 1H), 4.48-4.44 (m, 2H), 4.24-4.21 (m, 1H), 3.81-
3.78 (m, 1H), 3.08-3.03 (m, 1H), 2.95-2.91 (m, 1H), 2.80-2.74 (m, 1H), 2.70-2.66 (m, 1H), 1.85-1.77
13
C
(m, 1H), 1.66 (m, 1H), 1.40-1.23 (m, 10H), 1.23 (s, 1H), 1.10-1.04 (m, 4H), 0.86-0.80 (m, 6H).
NMR (100 MHz, DMSO): ꢂ 171.68, 171.20, 170.84, 156.01, 146.91, 137.14, 131.10, 129.76, 129.41,
128.47, 126.86, 125.64, 121.15, 78.47, 58.45, 57.74, 54.30, 51.49, 51.00, 38.01, 34.86, 31.16, 28.69,
+
+
2
48 7 7 2
4.63, 23.56, 21.87, 6.06, 5.88. HRMS calcd for C36H N O S [M+H] 722.3330; found: 722.3336.
tert-butyl((S)-1-((2S,4S)-2-(((R)-1-(cyclopropanesulfonamido)-1-oxo-3-phenylpropan-2-
yl)carbamoyl)-4-(4-phenyl-1H-1,2,3-triazol-1-yl)pyrrolidin-1-yl)-1-oxo-3-phenylpropan-2-
yl)carbamate(B6)
1
White solid,mp= 162-163℃, H-NMR (400 MHz, DMSO): ꢂ 8.67 (s, 1H), 7.89-7.85(m, 2H), 7.60-
7.46 (m, 2H), 7.37-7.30 (m, 4H ), 7.26-7.22 (m, 3H), 7.19-7.10 (m, 5H), 7.06-7.03 (m, 1H), 5.32-5.28
(m, 1H), 4.56-4.49 (m, 2H), 4.43-4.38 (m, 2H), 3.90-3.86 (m, 1H), 3.08-3.04 (m, 1H), 2.95-2.93 (m,
1H), 2.89-2.86 (m, 2H), 2.78-2.69 (m, 2H), 2.04-1.99 (m, 1H), 1.31-1.30 (m, 9H), 0.86-0.80 (m, 4H).
+
+
46 7 7 2
HRMS calcd for C39H N O S [M+H] 756.3174; found: 756.3212.
tert-butyl((S)-1-((2S,4S)-2-(((R)-1-(cyclopropanesulfonamido)-1-oxo-3-phenylpropan-2-
yl)carbamoyl)-4-(4-phenyl-1H-1,2,3-triazol-1-yl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-
yl)carbamate(B7)
1
White solid,mp= 154-155℃, H-NMR (400 MHz, DMSO): ꢂ 11.88 (s, 1H), 8.64 (s, 1H), 8.60 (m, 1H),

7
1
3
1
.46(d, 2H, J=8 ), 7.47 (t, 2H, J=8 ), 7.37-7.34 (m, 1H), 7.21-7.16 (m, 4H), 7.06-7.04 (m, 1H), 6.58 (d,
H, J=8 ), 5.26-5.22 (m, 1H), 4.70 (m, 1H), 4.53-4.46 (m, 2H), 4.12 (d, 1H, J=8), 3.81-3.76 (m, 1H),
.08-3.03 (m, 1H), 2.95-2.90 (m, 1H), 2.80-2.75 (m, 1H), 2.69-2.64 (m, 1H), 1.89-1.81 (m, 1H), 1.40-
+
+
48 7 7 2
.30 (s, 9H), 1.08-1.06 (m, 4H), 0.98-0.85 (m, 9H). HRMS calcd for C37H N O S [M+H] 722.3330;
found: 722.3336.
tert-butyl((S)-1-((2S,4S)-4-(2-naphthamido)-2-(((S)-1-(cyclopropanesulfonamido)-4-methyl-
1
oxopentan-2-yl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate(C2)
1
White solid,mp= 155-156℃, H-NMR (400 MHz, DMSO): ꢂ 11.86 (s, 1H), 8.61 (d, 1H, J=8), 8.42 (s,
1H), 8.24 (d, 1H, J=8), 8.04-7.97 (m, 3H), 7.92(d, 1H, J=8 ), 7.64-7.68 (m, 2H), 6.62 (d, 1H, J=8 ),
4.71-4.68 (m, 1H), 4.63-4.61 (m, 1H), 4.40-4.36 (m, 1H), 4.11 (d, 1H, J=8), 3.99-3.96 (m, 1H), 3.74-
3.70 (m, 1H), 2.97-2.91 (m, 1H), 2.29-2.24 (m, 1H), 2.14-2.12 (m, 1H), 1.75-1.73 (m, 1H), 1.52-1.44
1
3
(
m, 2H), 1.30 (s, 9H), 1.08-1.07 (m, 4H), 0.95 (s, 9H), 0.90 (d, 3H, J=8), 0.85 (d, 3H, J=8). C NMR
100 MHz, DMSO): ꢂ 172.79, 171.93, 167.08, 156.13, 134.64, 132.56, 132.09, 129.28, 128.30, 128.10,
(
127.20, 124.66, 78.55, 59.00, 58.16, 51.92, 49.29, 35.05, 34.40, 31.12, 28.59, 26.73, 24.51, 23.58,
+
+
2
52 5 8 2
1.70, 5.93, 5.86. HRMS calcd for C36H N O S [M+H] 714.3531; found: 714.3520
tert-butyl((S)-1-((2S,4S)-2-(((S)-1-(cyclopropanesulfonamido)-4-methyl-1-oxopentan-2-
yl)carbamoyl)-4-(2-((2,3-dimethylphenyl)amino)benzamido)pyrrolidin-1-yl)-3,3-dimethyl-1-
oxobutan-2-yl)carbamate. (C3)
1
White solid,mp= 131-132℃, H-NMR (400 MHz, DMSO): ꢂ 11.85 (s, 1H), 9.44 (s, 1H), 8.43 (d, 1H,
J=8), 8.23 (s, 1H), 7.65 (d, 1H, J=8 ), 7.25 (t, 1H, J=8 ) 7.08-7.04 (m, 2H), 6.93 (d, 1H, J=8 ), 6.84 (d,
1
3
1
H, J=8 ), 6.73-6.70 (m, 1H), 6.65 (d, 1H, J=8 ), 4.64-4.58 (m, 2H), 4.35 (m, 1H), 4.09 (d, 1H, J=8),
.90 (m, 1H), 3.74-3.73 (m, 1H), 2.94-2.89 (m, 1H), 2.27-2.22 (m, 4H), 2.11 (s, 4H), 1.75-1.73 (m, 1H),
.51-1.43 (m, 2H), 1.36 (s, 9H), 1.05-1.04 (m, 4H), 0.95 (s, 9H), 0.90 (d, 3H, J=4), 0.84 (d, 3H, J=8).
C NMR (100 MHz, DMSO): ꢂ 171.93, 156.20, 146.69, 139.67, 138.15, 132.60, 129.89, 129.35,
26.30, 125.61, 120.22, 117.17, 114.40, 78.61, 59.07, 51.92, 49.04, 34.98, 31.09, 28.64, 26.72, 24.50,
13
1
2
7
4
+
+
3.57, 21.69, 20.75, 14.00, 5.89, 5.83. HRMS calcd for C36
83.4125.
52 5 8 2
H N O S [M+H] 783.4110; found:
.2 HCV NS3/4A inhibition assay
NS3-NS4 protease activity was assessed by measuring intramolecular fluorescence resonance energy
transfer for detection of the activity of hepatitis C virus (HCV) serine proteinase using a quenched-
fluorogenic substrate. The assay was carried out using 96- well polypropylene plates. The assay buffer
consisted of 30 mM HEPES, pH 7.5, 40% Glycerol, 10 mM DTT, and 2% DMSO. Compounds
dissolved in DMSO were varied over a ten dose, 4-fold serial dilution. After a 10 minute pre-
incubation, reaction containing ±compound were initiated by the addition of HCV Protease Substrate
(RET S1), (Ac-DED (EDANS) EEAbu Y [COO] ASK (DABCYL). The plate was read in kinetic mode
on the Molecular Devices SpectraMax Gemini XS plate reader. Reader settings: Read time: 30 minutes,
Excitation ꢃ: 335nm, Emission ꢃ: 495nm. The concentration required for 50% inhibition of enzymatic
activity (IC50) was calculated plotting percent inhibition of NS3/4A activity versus the inhibitor

concentration.
5. Acknowledgements
This work was supported by National High-tech R&D Program of China, 863 Program (Grant
No.2014AA020523), National Natural Science Foundation of China (Grant no. 21302111, Grant no.
8
1373282 and Grant no. 21172134), China Postdoctoral Science Foundation funded project (Grant no.
013M540558, Grant no.2014T70654).
2
References
1] D.P. Webster, P. Klenerman, J. Collier, K.J. Jeffery, Development of novel
treatments for hepatitis C, The Lancet. Infectious diseases, 9 (2009) 108-117.
2] H.J. Alter, L.B. Seeff, Recovery, persistence, and sequelae in hepatitis C virus
infection: a perspective on long-term outcome, Seminars in liver disease, 20 (2000)
7-35.
[
[
1
[
3] R.S. Brown, Hepatitis C and liver transplantation, Nature, 436 (2005) 973-978.
[4] B.D. Lindenbach, C.M. Rice, Unravelling hepatitis C virus replication from genome
to function, Nature, 436 (2005) 933-938.
5] A.A. Kolykhalov, K. Mihalik, S.M. Feinstone, C.M. Rice, Hepatitis C virus-encoded
[
enzymatic activities and conserved RNA elements in the 3' nontranslated region are
essential for virus replication in vivo, Journal of virology, 74 (2000) 2046-2051.
[
6] K.X. Chen, F.G. Njoroge, J. Pichardo, A. Prongay, N. Butkiewicz, N. Yao, V.
Madison, V. Girijavallabhan, Design, synthesis, and biological activity of m-tyrosine-
based 16- and 17-membered macrocyclic inhibitors of hepatitis C virus NS3 serine
protease, Journal of medicinal chemistry, 48 (2005) 6229-6235.
[
7] F. Velazquez, S. Venkatraman, M. Blackman, P. Pinto, S. Bogen, M. Sannigrahi, K.
Chen, J. Pichardo, A. Hart, X. Tong, V. Girijavallabhan, F.G. Njoroge, Design,
synthesis, and evaluation of oxygen-containing macrocyclic peptidomimetics as
inhibitors of HCV NS3 protease, Journal of medicinal chemistry, 52 (2009) 700-708.
[
8] S.R. LaPlante, H. Nar, C.T. Lemke, A. Jakalian, N. Aubry, S.H. Kawai, Ligand
bioactive conformation plays a critical role in the design of drugs that target the
hepatitis C virus NS3 protease, Journal of medicinal chemistry, 57 (2014) 1777-1789.
[
9] J.G. McHutchison, S.C. Gordon, E.R. Schiff, M.L. Shiffman, W.M. Lee, V.K. Rustgi,
Z.D. Goodman, M.H. Ling, S. Cort, J.K. Albrecht, Interferon alfa-2b alone or in
combination with ribavirin as initial treatment for chronic hepatitis C. Hepatitis
Interventional Therapy Group, The New England journal of medicine, 339 (1998)
1485-1492.
[
10] M.W. Fried, M.L. Shiffman, K.R. Reddy, C. Smith, G. Marinos, F.L. Goncales, Jr.,
D. Haussinger, M. Diago, G. Carosi, D. Dhumeaux, A. Craxi, A. Lin, J. Hoffman, J. Yu,
Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection, The New
England journal of medicine, 347 (2002) 975-982.
[
11] T. Poynard, P. Marcellin, S.S. Lee, C. Niederau, G.S. Minuk, G. Ideo, V. Bain, J.
Heathcote, S. Zeuzem, C. Trepo, J. Albrecht, Randomised trial of interferon alpha2b
plus ribavirin for 48 weeks or for 24 weeks versus interferon alpha2b plus placebo for
4
8 weeks for treatment of chronic infection with hepatitis C virus. International
Hepatitis Interventional Therapy Group (IHIT), Lancet, 352 (1998) 1426-1432.
12] M.P. Manns, J.G. McHutchison, S.C. Gordon, V.K. Rustgi, M. Shiffman, R.
Reindollar, Z.D. Goodman, K. Koury, M. Ling, J.K. Albrecht, Peginterferon alfa-2b
[

plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of
chronic hepatitis C: a randomised trial, Lancet, 358 (2001) 958-965.
[
13] N. Goudreau, M. Llinas-Brunet, The therapeutic potential of NS3 protease
inhibitors in HCV infection, Expert opinion on investigational drugs, 14 (2005) 1129-
144.
14] M.P. Manns, G.R. Foster, J.K. Rockstroh, S. Zeuzem, F. Zoulim, M. Houghton,
1
[
The way forward in HCV treatment--finding the right path, Nature reviews. Drug
discovery, 6 (2007) 991-1000.
[
15] J. Sheldon, P. Barreiro, V. Soriano, Novel protease and polymerase inhibitors for
the treatment of hepatitis C virus infection, Expert opinion on investigational drugs,
6 (2007) 1171-1181.
16] S.L. Tan, Y. He, Y. Huang, M. Gale, Jr., Strategies for hepatitis C therapeutic
intervention: now and next, Current opinion in pharmacology, 4 (2004) 465-470.
17] M. Llinas-Brunet, M.D. Bailey, E. Ghiro, V. Gorys, T. Halmos, M. Poirier, J.
1
[
[
Rancourt, N. Goudreau, A systematic approach to the optimization of substrate-
based inhibitors of the hepatitis C virus NS3 protease: discovery of potent and
specific tripeptide inhibitors, Journal of medicinal chemistry, 47 (2004) 6584-6594.
[18] W.M. Kazmierski, R. Hamatake, M. Duan, L.L. Wright, G.K. Smith, R.L. Jarvest,
J.J. Ji, J.P. Cooper, M.D. Tallant, R.M. Crosby, K. Creech, A. Wang, X. Li, S. Zhang,
Y.K. Zhang, Y. Liu, C.Z. Ding, Y. Zhou, J.J. Plattner, S.J. Baker, W. Bu, L. Liu,
Discovery of novel urea-based hepatitis C protease inhibitors with high potency
against protease-inhibitor-resistant mutants, Journal of medicinal chemistry, 55
(
2012) 3021-3026.
[19] J. Gising, A.K. Belfrage, H. Alogheli, A. Ehrenberg, E. Akerblom, R. Svensson, P.
Artursson, A. Karlen, U.H. Danielson, M. Larhed, A. Sandstrom, Achiral pyrazinone-
based inhibitors of the hepatitis C virus NS3 protease and drug-resistant variants
with elongated substituents directed toward the S2 pocket, Journal of medicinal
chemistry, 57 (2014) 1790-1801.
[
20] B. Moreau, J.A. O'Meara, J. Bordeleau, M. Garneau, C. Godbout, V. Gorys, M.
Leblanc, E. Villemure, P.W. White, M. Llinas-Brunet, Discovery of hepatitis C virus
NS3-4A protease inhibitors with improved barrier to resistance and favorable liver
distribution, Journal of medicinal chemistry, 57 (2014) 1770-1776.
[21] S. Venkatraman, F.G. Njoroge, V.M. Girijavallabhan, V.S. Madison, N.H. Yao, A.J.
Prongay, N. Butkiewicz, J. Pichardo, Design and synthesis of depeptidized
macrocyclic inhibitors of hepatitis C NS3-4A protease using structure-based drug
design, Journal of medicinal chemistry, 48 (2005) 5088-5091.
[
22] S. Venkatraman, F. Velazquez, W. Wu, M. Blackman, K.X. Chen, S. Bogen, L.
Nair, X. Tong, R. Chase, A. Hart, S. Agrawal, J. Pichardo, A. Prongay, K.C. Cheng, V.
Girijavallabhan, J. Piwinski, N.Y. Shih, F.G. Njoroge, Discovery and structure-activity
relationship of P1-P3 ketoamide derived macrocyclic inhibitors of hepatitis C virus
NS3 protease, Journal of medicinal chemistry, 52 (2009) 336-346.
[23] K.X. Chen, L. Nair, B. Vibulbhan, W. Yang, A. Arasappan, S.L. Bogen, S.
Venkatraman, F. Bennett, W. Pan, M.L. Blackman, A.I. Padilla, A. Prongay, K.C.
Cheng, X. Tong, N.Y. Shih, F.G. Njoroge, Second-generation highly potent and
selective inhibitors of the hepatitis C virus NS3 serine protease, Journal of medicinal
chemistry, 52 (2009) 1370-1379.
[
24] A. Aghemo, R. De Francesco, New horizons in hepatitis C antiviral therapy with
direct-acting antivirals, Hepatology, 58 (2013) 428-438.
25] S. Harper, M. Ferrara, B. Crescenzi, M. Pompei, M.C. Palumbi, J.M. DiMuzio, M.
[
Donghi, F. Fiore, U. Koch, N.J. Liverton, S. Pesci, A. Petrocchi, M. Rowley, V. Summa,
C. Gardelli, Inhibitors of the hepatitis C virus NS3 protease with basic amine

functionality at the P3-amino acid N-terminus: discovery and optimization of a new
series of P2-P4 macrocycles, Journal of medicinal chemistry, 52 (2009) 4820-4837.
[
26] J.A. McCauley, C.J. McIntyre, M.T. Rudd, K.T. Nguyen, J.J. Romano, J.W.
Butcher, K.F. Gilbert, K.J. Bush, M.K. Holloway, J. Swestock, B.L. Wan, S.S. Carroll,
J.M. DiMuzio, D.J. Graham, S.W. Ludmerer, S.S. Mao, M.W. Stahlhut, C.M. Fandozzi,
N. Trainor, D.B. Olsen, J.P. Vacca, N.J. Liverton, Discovery of vaniprevir (MK-7009), a
macrocyclic hepatitis C virus NS3/4a protease inhibitor, Journal of medicinal
chemistry, 53 (2010) 2443-2463.
[
27] J. Naud, C. Lemke, N. Goudreau, E. Beaulieu, P.D. White, M. Llinas-Brunet, P.
Forgione, Potent triazolyl-proline-based inhibitors of HCV NS3 protease, Bioorganic &
medicinal chemistry letters, 18 (2008) 3400-3404.
[
28] Y. Jiang, S.W. Andrews, K.R. Condroski, B. Buckman, V. Serebryany, S.
Wenglowsky, A.L. Kennedy, M.R. Madduru, B. Wang, M. Lyon, G.A. Doherty, B.T.
Woodard, C. Lemieux, M. Geck Do, H. Zhang, J. Ballard, G. Vigers, B.J. Brandhuber,
P. Stengel, J.A. Josey, L. Beigelman, L. Blatt, S.D. Seiwert, Discovery of danoprevir
(
ITMN-191/R7227), a highly selective and potent inhibitor of hepatitis C virus (HCV)
NS3/4A protease, Journal of medicinal chemistry, 57 (2014) 1753-1769.
29] C.W. Tornoe, C. Christensen, M. Meldal, Peptidotriazoles on solid phase: [1,2,3]-
[
triazoles by regiospecific copper(i)-catalyzed 1,3-dipolar cycloadditions of terminal
alkynes to azides, The Journal of organic chemistry, 67 (2002) 3057-3064.
[30] A.K. Lampa, S.M. Bergman, S.S. Gustafsson, H. Alogheli, E.B. Akerblom, G.G.
Lindeberg, R.M. Svensson, P. Artursson, U.H. Danielson, A. Karlen, A. Sandstrom,
Novel Peptidomimetic Hepatitis C Virus NS3/4A Protease Inhibitors Spanning the P2-
P1' Region, ACS medicinal chemistry letters, 5 (2014) 249-254.

Discovery of a series of novel compounds with moderate anti-hepatitis C virus NS3 protease
activity in vitro
Fangyuan Shi, Yingjie Zhang*and Wenfang Xu*
Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong University, 44,
West Culture Road, Jinan, Shandong 250012, P. R. China
Corresponding author: Tel. /fax: +86 531 88382264. E-mail addresses: wenfxu@163.com
N
R5
N
O
N
NH
O
R1 HN S R2
O
O
R1 HN S R2
O
R3
R3
O
N
O
N
NH
O
NH
O
N
H
N
H
R4
O
O
R4
O
O