Development of bivalent oleanane-type triterpenes as potent HCV entry inhibitors

Article abstract of DOI:10.1016/j.ejmech.2014.03.017

The development of entry inhibitors is an emerging approach to the prevention and reduction of HCV infection. Starting from echinocystic acid (EA), a μM HCV entry inhibitor, we have developed a series of bivalent oleanane-type triterpenes which, upon optimization of the length, rigidity and hydrophobicity of the linker, exert dramatically potent enhancement of inhibition with IC₅₀ values extending into the nM level. This study establishes the importance of triterpene natural products as new leads in the development of potential HCV entry inhibitors.

Full text of DOI:10.1016/j.ejmech.2014.03.017

European Journal of Medicinal Chemistry 77 (2014) 258e268
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Development of bivalent oleanane-type triterpenes as potent HCV
entry inhibitors
,
d
,
,
,b,
Fei Yu a 1, Yiyun Peng a 1, Qi Wang a 1, Yongying Shi a, Longlong Si a, Han Wang a,
Yongxiang Zheng a, Emily Lee c, Sulong Xiao a , Maorong Yu , Yingbo Li ,
,
a
a
*
Chuanling Zhang a, Hengli Tang c, Chunguang Wang a, Lihe Zhang a, Demin Zhou a
,
*
a State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191, China
b Department of Molecular and Cellular Pharmacology, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China
c Department of Biological Science, Florida State University, Tallahassee, FL 32306, USA
d Medical Faculty of Kunming University of Science and Technology, Kunming 650500, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The development of entry inhibitors is an emerging approach to the prevention and reduction of HCV
Received 30 November 2013
Received in revised form
24 February 2014
Accepted 6 March 2014
Available online 7 March 2014
infection. Starting from echinocystic acid (EA), a mM HCV entry inhibitor, we have developed a series of
bivalent oleanane-type triterpenes which, upon optimization of the length, rigidity and hydrophobicity
of the linker, exert dramatically potent enhancement of inhibition with IC50 values extending into the nM
level. This study establishes the importance of triterpene natural products as new leads in the devel-
opment of potential HCV entry inhibitors.
Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords:
Triterpene
Echinocystic acid
Dimer
HCV entry inhibitor
1. Introduction
Recently we found echinocystic acid (EA), a natural product of
oleanane-type triterpenes, displayed moderate antiꢀHCV activity
The hepatitis C virus (HCV) is an enveloped RNA virus, a member
of the Flaviviridae family. It is the leading cause of liver fibrosis and
cirrhosis that may eventually lead to liver carcinomas [1]. It has
been estimated that over 170 million individuals worldwide are
chronically infected with HCV [2]. The most common treatment for
HCV infection is pegylated interferon and ribavirin; these have
been used for almost 30 years [3]. Telaprevir and Boceprevir, two
recently approved antiviral drugs targeting HCV replication, mark
the beginning of a new era in the control of HCV infection [3e5].
But resistance to individual antiviral drugs is likely to appear [6,7],
and a combination of drugs targeting different stages of the HCV life
cycle will be required. Inhibition of entry of the virus into HCV-
permissive cells is an emerging opportunity for prevention and
reduction of infection [8e11]. HCV entry inhibitors can be used in
tandem with other inhibitors of viral replication, producing a
multifaceted approach to effective control of HCV infection.
(IC50 w 1.4 mM) by interruption of the interaction between one HCV
envelope protein, E2, and its host receptor CD81, thus blocking
entry of the virus into the HCV envelope [12,13]. However, in-
teractions between E2 and CD81 involve at least two large inter-
acting interfaces [14]. As a result, it is a significant challenge for
small molecules like EA to efficiently and competitively occupy and
control the interface and so disrupt the E2/CD81 interactions. In an
attempt to develop more potent HCV entry inhibitors, we utilized
EA as a pharmacophore and explored whether multiple EA moieties
tethered together might enhance the potency. This study led to a
series of bivalent oleanane-type triterpenes which, upon optimi-
zation of the length, rigidity and hydrophobicity of the constituent
linker, have been shown to exert dramatically potent enhancement,
producing compounds with IC50 values in the sub-nM ranges.
2. Results and discussion
2.1. Tethering multiple EA moieties and anti-HCV activity
* Corresponding authors.
E-mail addresses: slxiao@bjmu.edu.cn (S. Xiao), deminzhou@bjmu.edu.cn
(D. Zhou).
We have previously elucidated the structureeactivity re-
lationships of EA as an HCV entry inhibitor, and found that when
1
Those authors have contributed equally to this work.
http://dx.doi.org/10.1016/j.ejmech.2014.03.017
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

F. Yu et al. / European Journal of Medicinal Chemistry 77 (2014) 258e268
259
its ring D is modified at the 28-COOH, the potency is significantly
2.2. Triazolyl vs. alkyl as a constituent linker
increased [12,13]. Due to the relatively small size of EA moiety and
the large interacting interfaces of E2 and its target CD81, we
inquired if the potency might be enhanced when two or more EA
pharmacophores were linked. As shown in Fig. 1, a series of EA
derivatives, including the dimer (1), the trimers (2 and 3) and the
tetramer (4), were synthesized by a click reaction that formed a
triazole linker through which 2e4 EA moieties were linked. The
28-COOH of EA was first activated by TBTU and then reacted with
propynylamine, giving the sequential EA intermediates 1a and 1b
(Scheme 1). Di-, tri- or tetramerization of EA was then carried out
by coupling compound 1b by a CuAAC reaction with a variety of
azides.
HCV entry assay indicated that the product of dimerization of
EA with a bis-triazole-bridged propane linker (1) strikingly
enhanced the potency: the IC50 of dimer 1 is 10 nM (Fig. 2),
almost 150-fold higher than EA. In contrast, trimers (2 and 3) and
tetramer (4) moieties of EA, showed no improvement but rather
a decrease in the potency as measured in the same biological
assay (Table 1). Since compound 1 is a symmetric dimer, we
questioned whether the constitutive monomer, i.e. an EA moiety
with a substituted triazole tail, could maintain such high activity.
Several such EA monomers with different alkyl tails (5e9) were
synthesized (Scheme 1) and HCV entry assays indicated that
monomers 5e7 with ethyl, n-propyl or benzyl tails were devoid
of activity. In the case of monomers 8 or 9, which contain a
relatively hydrophilic ethanol- or propanol-tail, moderate activ-
ity was detected though it was far lower than that of the parent
dimer 1.
Given the significant potency gain achieved by dimerization of
EA, we were curious as to whether the potency could be further
enhanced by optimization of the linker. First, the optimal length of
the linker was determined by the synthesis of a series of EA dimers,
10e13, from the intermediate 1a, which was allowed to react with
alkyldiamines of different lengths (Scheme 2). Subsequent biolog-
ical assays indicated that most of EA dimers tethered by an alkyl
linker had minimal activity, none displaying activity comparable to
that of dimer 1. The major difference between dimer 1 and these
bivalent EA derivatives is the linker component: the bistriazolyl
propyl in 1 vs. the flank alkyl chain in 10-13. This suggests that
absence of a triazole in the linker may leave these EA dimer de-
rivatives much less potent.
Next, the triazoles within the linker were retained while the
length of the linker was re-explored with several bivalent EA de-
rivatives, 16e19 (Scheme 3). HCV entry assay indicated that dimer
1, which contains an n-propyl chain between the triazole rings,
showed the most potency and was followed by compounds 18 and
19, two dimers containing a e(CH2)5e and e(CH2)6e linker,
respectively. These compounds have potencies that are similar
(IC50 ¼ 0.13 and 0.12
mM, respectively), but about one order of
magnitude less than that of compound 1. For compounds 16 and 17,
EA dimers containing a linker with a e(CH2)e shorter or longer
than that in 1, a striking decrease in potency was observed with
their IC50 values back at the mM level. These data suggest that the
length of the linker affects the potency with a propyl chain between
triazoles being the optimum. We also introduced extra triazole
OH
N
OH
N
N
H
O
N
N
H
N
N
N
N
N
N
H
N
O
N
N
O
OH
OH
COOH
OH
N
HO
HO
HO
HN
N
N
H
N
HO
O
N
O
OH
OH
EA
1
2
OH
OH
HO
HO
OH
N
OH
N
H
O
N
N
N
N
N
H
N
O
HO
O
OH
N
N
HO
N
HO
NH
N
O
NH
N
N
HN
N
N
N
O
OH
N
N
N
N
N
N
HN
O
HN
O
OH
OH
3
OH
4
OH
HO
Fig. 1. Chemical structures of EA and EA oligomers.

260
F. Yu et al. / European Journal of Medicinal Chemistry 77 (2014) 258e268
Scheme 1. Reagents and conditions: (a) TBTU, DIEA, THF, rt, 4 h; (b) 2-propynylamine, Na2CO3, DMF, rt, 20 min; (c) CuSO4, Na-L-ascorbate, azides, CH2Cl2/H2O, rt, 8 h.
groups into the monomeric EA (for example, 29, Scheme 4) [15] but
found that this failed to promote EA potency, confirming that it is
the dimerization of EA plus a tethered triazole group that delivers
the striking enhancement to the potency.
IC50 ¼ 3.0 nM (Fig. 2), 3-fold higher than that of the dimer 3. How-
ever, a change of the orientation of the dual triazoles in the benzene
ring from para- to meta- (23) or ortho- (24) decreased the potency of
EA dimmers remarkably. Increasing the rigidity of the linker by
introduction of dual methyl (25) or methoxyl (26) groups at the
para-positions of the benzene ring failed to improve the potency
further. An increase (27) or decrease (28) by a single methylene
group in the chain length from both sides of the phenyl linker failed
to improve the potency of dimer 22. It was concluded that the ri-
gidity of the linker, as well as its length and hydrophilicity, exerts a
significant impact on the potency of EA dimers.
2.3. Effect of a hydrophilic vs. hydrophobic linker on the potency
After exploration of the optimal length of the linker, we sought
to elucidate the effect of hydrophilicity on the potency. We syn-
thesized three EA dimers 14, 15 and 20, which are the counterparts
of compounds 12, 13 and 18 with one or more carbon atoms in the
linkers being replaced by oxygen atoms. The HCV entry assay
revealed all three dimers with such a relatively hydrophilic linker to
be more potent than their hydrophobic counterparts (Table 1). In
addition, compound 21, an EA dimer with additional hydrophilic
components [e(CH2eCH2eO)-] in the linker, exerted much greater
potency compared with that of its counterparts 16e19, supporting
the observation that an increase in the hydrophilicity of the linker
increases the potency. We also found that compound 15, which
contains more hydrophilic e(CH2eCH2eO)- components in the
linker than compounds 10e14, exerted more potent than the dimer
counterparts (Table 1). Clearly, an increase of hydrophilicity in the
linker has a profound effect on the potency.
2.5. Mechanistic exploration of the potency gain from dimerization
of EA
Our pull-down assays reveal the HCV envelope E2 protein to be
a direct target of EA [12]. A BIAcore assay indicated that EA binds
tightly to the E2 protein immobilized on a SA sensor chip, with a KD
value of w24
22, the most potent bivalent EA, binds to E2 protein more tightly
with KD ¼ 2.4 M, almost 10-fold less than that of EA (Fig. 3). In
addition, the KD value for E2 and CD81 binding increased from
20 nM in the absence of any ligand to 0.1 M and 0.5 M, respec-
tively, when 10 M of EA or dimer 22 was added to the assay so-
mM [12]. In the same affinity assay, we found EA dimer
m
m
m
m
2.4. Effect on the potency of flexible linker vs. rigid linker
lution. We also found that both EA and compound 22 failed to bind
to CD81. Based on such data from the affinity and biological assays,
we propose that it is the remarkable disruption of the interaction of
E2 and CD81, potentially due to the significant binding of E2 with
bivalent EA, and potentially also the increase in the size of EA and/
or the substituent moiety, that leads to the potency gain from
dimerization of EA (Fig. 4). The critical role of the triazole within the
linker that promotes bivalent EA so significantly remains to be
determined.
Though dimer 1 has significant potency, the rotational flexibility
of its propyl chain may be significant. We explored this by replace-
ment of the flexible propane chain with a rigid linker, and designed
and synthesized the EA bivalent compounds 22e28 (Scheme 3).
Biological assays (Table 1) indicated that compound 22, an EA dimer
containing a relatively rigid phenyl linker with dual triazoles located
para- to one another, displayed a further potency enhancement with
Fig. 2. The inhibition curves for compound 1 and 22. The concentration of 1 and 22 were 0.5 nM, 5 nM, 50 nM, 0.5 mM, 5 mM and 50 mM. Each concentration was tested triplicate and
results were expressed as mean and standard deviation from triplicate assays.

F. Yu et al. / European Journal of Medicinal Chemistry 77 (2014) 258e268
261
Table 1
and 0.8 mM, respectively in the HCVcc assay (Table 2). We also found
Structures and anti-HCVpp entry activities of EA oligomers.
the similar potency trend against HCVpp and HCVcc for the tested
bivalent EA derivatives: the more potent compounds in the HCV
entry assay are more potent in the HCVcc infectious assay (Table 2).
An AlamarBlue assay indicated no detectable toxicity for these
Comp.
Inhibition of HCVpp
entry (%)a
Inhibition of VSVpp
entry (%)a
1
m
M
5
m
M
1
m
M
5 mM
potent bivalent compounds even at concentrations of 100
mM
EA
1
2
3
4
5
6
7
8
27.5 ꢂ 2.1
86.3 ꢂ 1.8
19.8 ꢂ 1.4
45.4 ꢂ 2.1
22.8 ꢂ 1.9
0.5 ꢂ 4.1
77.7 ꢂ 3.3
89.0 ꢂ 0.7
32.2 ꢂ 2.4
52.7 ꢂ 1.2
58.4 ꢂ 5.0
12.2 ꢂ 1.0
4.9 ꢂ 1.2
7.2 ꢂ 1.7
9.0 ꢂ 0.8
(Table 2). This result was supported by a VSVpp entry assay
(Table 1), a useful assay reflecting the health of host cells when
treated by chemicals. A series of studies have demonstrated that
the aglycones of triterpenoids have a crucial influence on the he-
molytic properties, one of the well-known characteristics of sapo-
nins [17]. The hemolysis assay showed that almost all the EA dimers
depleted the hemolytic activity (Fig. 6). These data are consistent
with the fact that most EA dimers showed far less cytotoxicity.
16.5 ꢂ 1.3
ꢀ5.2 ꢂ 0.3
3.9 ꢂ 0.2
30.5 ꢂ 2.3
0.1 ꢂ 3.1
3.2 ꢂ 1.1
0.9 ꢂ 0.7
1.5 ꢂ 1.9
0.7 ꢂ 2.1
18.7 ꢂ 0.9
ꢀ1.6 ꢂ 0.1
4.1 ꢂ 2.2
1.8 ꢂ 2.1
0.8 ꢂ 1.3
1.8 ꢂ 1.2
ꢀ6.5 ꢂ 0.4
22.6 ꢂ 2.1
19.1 ꢂ 0.7
17.0 ꢂ 0.9
13.1 ꢂ 0.9
2.1 ꢂ 1.1
7.9 ꢂ 0.4
17.8 ꢂ 0.8
18.8 ꢂ 1.4
0.9 ꢂ 3.1
2.2 ꢂ 1.1
21.1 ꢂ 1.2
28.8 ꢂ 1.9
ꢀ2.7 ꢂ 1.5
11.9 ꢂ 0.2
ꢀ2.5 ꢂ 3.1
30.5 ꢂ 1.3
42.6 ꢂ 1.7
ꢀ1.5 ꢂ 2.1
1.9 ꢂ 1.8
3.8 ꢂ 0.2
2.1 ꢂ 1.7
1.1 ꢂ 0.5
27.6 ꢂ 1.6
2.5 ꢂ 1.4
3.7 ꢂ 1.2
ꢀ1.1 ꢂ 0.8
4.6 ꢂ 0.2
4.7 ꢂ 2.3
4.4 ꢂ 1.3
28.0 ꢂ 1.7
24.9 ꢂ 1.3
19.5 ꢂ 1.2
12.5 ꢂ 1.0
1.9 ꢂ 0.7
11.6 ꢂ 1.0
20.7 ꢂ 1.1
26.9 ꢂ 2.1
3.2 ꢂ 1.0
5.0 ꢂ 0.3
46.0 ꢂ 3.1
45.5 ꢂ 2.6
ꢀ0.6 ꢂ 0.4
2.2 ꢂ 3.2
2.2 ꢂ 0.8
2.7 ꢂ 0.2
34.9 ꢂ 2.4
35.7 ꢂ 3.1
44.6 ꢂ 7.4
46.5 ꢂ 1.5
61.7 ꢂ 1.6
56.2 ꢂ 2.8
73.7 ꢂ 1.8
77.5 ꢂ 4.7
57.1 ꢂ 4.2
47.8 ꢂ 1.4
65.5 ꢂ 3.4
72.7 ꢂ 2.3
70.6 ꢂ 3.6
84.1 ꢂ 0.6
88.9 ꢂ 1.7
59.2 ꢂ 4.6
51.5 ꢂ 4.3
72.2 ꢂ 2.7
68.5 ꢂ 5.9
65.2 ꢂ 5.5
68.9 ꢂ 5.2
1.2 ꢂ 4.1
62.5 ꢂ 5.9
67.1 ꢂ 5.3
58.2 ꢂ 2.8
59.6 ꢂ 3.7
72.2 ꢂ 5.1
65.1 ꢂ 5.3
83.6 ꢂ 5.4
84.1 ꢂ 6.1
68.2 ꢂ 4.5
69.9 ꢂ 3.1
71.9 ꢂ 5.7
76.9 ꢂ 6.3
83.7 ꢂ 6.2
91.9 ꢂ 1.4
90.7 ꢂ 0.8
74.6 ꢂ 4.3
65.7 ꢂ 5.0
81.3 ꢂ 5.3
83.5 ꢂ 4.1
81.1 ꢂ 5.7
78.1 ꢂ 4.8
57.9 ꢂ 4.7
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
3. Conclusion
A series of bivalent EAs with different linkers were designed,
synthesized and evaluated. The results showed the length, hydro-
phobicity and rigidity of the linker all have a significant influence
on the potency of these bivalent EAs to HCVpp entry, and this
dramatically affects their overall anti-HCV activity. Several of these
new bivalent EAs, such as compounds 1 and 22, are most potent
with IC50 values of 10 and 3 nM in the HCVpp assay and EC50 of 0.9
and 0.8
mM in the HCVcc assay and no significant cytotoxicity.
Further evaluation and optimization of these compounds may lead
to the development of a new class of anti-HCV entry drugs.
4. Experimental
4.1. Chemistry-general
a
Inhibition (%) ¼ 100% ꢀ DMSO negative control (%) of each tested compound.
High Resolution Mass Spectra (HRMS) were obtained with an
APEX IV FT_MS (7.0 T) spectrometer (Bruker) in positive ESI mode.
NMR spectra were recorded on a Bruker DRX 400 spectrometer at
ambient temperature. 1H NMR chemical shifts are reported relative
to the internal standard TMS (dH ¼ 0.00) or the solvent signal
2.6. Validation of the potency of bivalent EA derivatives against
infectious clones
While HCVpp enters cells in a manner analogous to authentic
HCV, it is important to validate the potency of the lead compounds
using the fully replicating cell culture-adapted GT2a infectious HCV
(HCVcc JFH-1) [11]. Huh-7.5 cells were infected in the presence of
(
dH ¼ 3.31 for the central line of MeOD and dH ¼ 7.22 for the central
line of pyridineꢀd5). 13C NMR chemical shifts are referenced to the
solvent signal (dC ¼ 77.00 for the central line of CDCl3, dC ¼ 49.00 for
the central line of MeOD, and dC ¼ 123.9 for the central line of
pyridineꢀd5). Reactions were monitored by thinꢀlayer chroma-
tography (TLC) on a precoated silica gel 60 F254 plate (layer thick-
ness 0.2 mm; E. Merck, Darmstadt, Germany) and detected by
staining with a yellow solution containing Ce(NH4)2(NO3)6 (0.5 g)
and (NH4)6Mo7O24.4H2O (24.0 g) in 6% H2SO4 (500 mL) followed by
heating. Flash column chromatography was performed on silica gel
60 (200 ꢀ 300 mesh, Qingdao Haiyang Chemical Co. Ltd). The
method employed in determining the purity of synthesized com-
pounds was HPLC using an Agilent Technologies 1260 Infinity in-
struments, with chromatography performed on an Agilent
the lead compound at concentrations 0e20 mM and the inhibition
of HCV infection was determined two days post infection by
quantitative measurement of the NS3 protein in infected cells [16].
We tested 6 bivalent EA derivatives and found all compounds dose-
dependently inhibited infection (Fig. 5) with 1 and 22, the two most
potent compounds in the HCVpp assay, showing EC50 values of 0.9
100 ꢁ 4.6 mm, 3.5
mm C18 column, and eluted with CH3OH:H2O
(82:18 to 100:0) at a flow rate of 1.0 mL/min.
4.2. Synthesis
Compounds 1, 1a and 1b were synthesized using methods
published previously. [12]
4.2.1. General procedure for the preparation of compounds 2e9,
16e28 (A)
CuSO4 (1 eq.) and sodium ascorbate (1.5 eq.) were added to a
solution of compound 1b and the corresponding azide in CH2Cl2
(5 mL) and H2O (5 mL). The resulting solution was stirred vigor-
ously for 8 h at rt. The reaction mixture was diluted with H2O
Scheme 2. Reagents and conditions: (a) diamines, Na2CO3, DMF, rt, 3 h.

262
F. Yu et al. / European Journal of Medicinal Chemistry 77 (2014) 258e268
Scheme 3. Reagents and conditions: (a) CuSO4, Na-L-ascorbate, azides, CH2Cl2/H2O, rt, 8 h.
(10 mL), then extracted with CH2Cl2 (10 mL ꢁ 3). The combined
organic layer was dried over Na2SO4, filtered, and concentrated. The
residue was purified by column chromatography.
procedure A. The residue was purified by column chromatography
(CH2Cl2/MeOH, 25/1 v/v) to give 2 as a white solid (101 mg, 60%). 1H
NMR (400 MHz, MeOD):
d
0.60 (s, 6H, 2 ꢁ CH3), 0.71 (s, 3H, CH3),
0.78, 0.89 (s, 9H each, 3 ꢁ CH3), 0.92 (s, 6H, 2 ꢁ CH3), 0.94 (s, 3H,
CH3), 0.95, 0.98,1.37 (s, 9H each, 3 ꢁ CH3), 2.32e2.38 (m, 3H), 2.83e
2.86 (m, 3H), 3.16 (dd, 3H, J ¼ 10.3, 4.3 Hz), 4.18e4.39 (m, 9H), 5.09
(brs, 3H), 5.46 (brs, 3H), 5.64e5.70 (m, 1H), 7.53 (s, 2H), 7.55 (s, 1H).
4.2.2. General procedure for the preparation of compounds 10e15
(B)
The appropriate diamines were added to compound 1a and
Na2CO3 (2 eq.) with stirring in DMF (5 mL). The mixture was stirred
overnight at room temperature. After completion, as judged by TLC,
the solvent was evaporated under reduced pressure. The mixture
was resolved in EtOAc and washed twice with water and brine. The
organic layer was dried over Na2SO4, then filtered and concentrated.
The crude product was purified bycolumn chromatography (CH2Cl2/
MeOH, 25/1 v/v) to give the target compound as a white solid.
13C NMR (100 MHz, MeOD):
d 16.3 (2C), 16.4, 16.5 (3C), 17.7 (2C),
18.0, 19.5 (3C), 24.5 (3C), 25.6 (2C), 25.9, 27.4 (2C), 27.5, 27.9 (3C),
28.8 (3C), 31.2, 31.3 (2C), 31.5, 31.8 (2C), 33.4 (3C), 34.0 (3C), 36.0
(3C), 36.1 (3C), 36.3 (3C), 38.1 (3C), 39.9 (3C), 40.0 (3C), 40.8 (2C),
40.9, 42.4 (3C), 42.8 (3C), 48.0 (3C), 48.2(3C), 49.9 (2C), 50.0, 51.9,
52.0, 56.8 (3C), 62.1, 75.4, 75.5 (2C), 79.6 (3C), 124.2 (3C), 124.5,
125.0, 125.1, 144.9 (3C), 146.2, 146.3, 146.5, 179.9, 180.0 (2C).
ESIꢀHRMS (m/z) [M þ H]þ calcd for C102H159N12O9, 1696.2375;
found 1696.2348.
4.2.3. 1,2,3-Tri-[(N-(3b,16a-dihydroxyl-olean-12-en-28-oyl)-
amino)methyl-(1H-1,2,3-triazol-4-yl)] propane (2)
Prepared from 1b (160 mg, 0.31 mmol) and 1,2,3-
triazidopropane (17 mg, 0.1 mmol) according to general
4.2.4. 2,2,2-Tri-[(N-(3b,16a-dihydroxyl-olean-12-en-28-oyl)-
amino)methyl-(1H-1,2,3-triazol-4-yl)-methyl]-ethanol (3)
Prepared from 1b (160 mg, 0.31 mmol) and 2,2,2-
tris(azidomethyl)ethanol (21 mg, 0.1 mmol) according to general
procedure A. The residue was purified by column chromatography
(CH2Cl2/MeOH, 25/1 v/v) to give 3 as a white solid (98 mg, 56%). 1H
NMR (400 MHz, MeOD):
d
0.63, 0.76 (s, 9H each, 3 ꢁ CH3), 0.89 (s,
18H, 6 ꢁ CH3), 0.94, 0.97, 1.37 (s, 9H each, 3 ꢁ CH3), 2.34 (t, 3H,
J ¼ 13.3 Hz), 2.89 (dd, 3H, J ¼ 2.8, 13.4 Hz), 3.15 (dd, 3H, J ¼ 4.8,
10.8 Hz), 3.21 (s, 2H), 4.34e4.46 (m, 15H), 5.48 (brs, 3H), 8.05 (s,
3H). 13C NMR (100 MHz, MeOD):
d 16.3, 16.5, 17.8, 19.5, 24.6, 25.6,
27.4, 27.9, 28.8, 31.2, 31.8, 33.4, 33.9, 36.1, 36.2, 36.3 38.1, 39.9, 40.0,
40.8, 42.4, 42.8 (3C, each), 46.8 (1C), 48.1 (6C), 50.1, 51.2, 56.8(3C,
each), 61.6 (1C), 75.5, 79.6,124.3,126.7,145.0,146.1,180.2 (3C, each).
ESIꢀHRMS (m/z) [M þ H]þ calcd for C104H163N12O10, 1740.2610;
found 1740.2561.
4.2.5. Tetra-[(N-(3b,16a-dihydroxyl-olean-12-en-28-oyl)-amino)
methyl-(1H-1,2,3-triazol-4-yl)-methyl]-methane (4)
Prepared from 1b (214 mg, 0.42 mmol) and pentaerythrityl
tetraazide (24 mg, 0.1 mmol) according to general procedure A. The
Scheme 4. Reagents and conditions: (a) 2-azidoethanamine, Na2CO3, DMF, rt, 0.5 h;
(b) CuSO4, Na-L-ascorbate, CH2Cl2/H2O, rt, 12 h.

F. Yu et al. / European Journal of Medicinal Chemistry 77 (2014) 258e268
263
Fig. 3. Kinetic assays by Biacore for valuation of E2, CD81, and potent compounds interactions. (A) Binding curves for E2 and EA interactions. E2 was loaded onto a CM5 chip, then
EA was injected as analyte at concentrations from 3.125 M to 100 M. (B) Binding curves for CD81 and EA interactions. CD81 was loaded onto CM5 chip then EA was
M. KD ¼ 24
injected as analyte at concentrations from 3.125 M to 100 M. No binding signal was detected. (C) Binding curves for interaction of E2 and 22. E2 was loaded onto CM5 chip and
compound 22 was injected as analyte at concentrations from 0.78 M to 25 M. (D) Binding curves for CD81 and 22 interactions. CD81 was loaded onto CM5 chip and
M. KD ¼ 2.4
compound 22 was injected as analyte at concentrations from 0.78 M to 25 M. No binding signal was detected.
m
m
m
m
m
m
m
m
m
m
residue was purified by column chromatography (CH2Cl2/MeOH,
20/1 v/v) to give 4 as a white solid (114 mg, 55%). 1H NMR (400 MHz,
d 16.3, 16.5, 17.9, 19.5, 24.6, 25.7, 27.4, 27.9, 28.8, 31.2, 31.7, 33.4, 33.9,
36.1, 36.2, 36.3, 38.1, 39.9, 40.0, 40.8, 42.4, 42.8 (4C each), 46.3 (1C),
48.0, 48.2, 50.1, 51.4, 56.8, 75.5, 79.6, 124.2, 127.1, 145.0, 146.2, 180.2
MeOD):
d
0.65, 0.77 (s, 12H each, 4 ꢁ CH3), 0.88 (s, 24H, 8 ꢁ CH3),
0.92, 0.97, 1.37 (s, 12H each, 4 ꢁ CH3), 2.34 (t, 4H, J ¼ 13.2 Hz), 2.89
(dd, 4H, J ¼ 13.6, 2.8 Hz), 3.14 (dd, 4H, J ¼ 10.7, 4.8 Hz), 4.33e4.50
(m, 20H), 5.48 (brs, 4H), 8.09 (s, 4H). 13C NMR (100 MHz, MeOD):
(4C, each). ESIꢀHRMS (m/z) [M þ H]þ calcd for C137H213N16O12
,
2274.6543; found 2274.6494.
4.2.6. N-(3b,16a-dihydroxy-olean-12-en-28-oyl)-(1-ethyl-1H-
1,2,3-triazol-4-yl)-methylamine (5)
Prepared from 1b (51 mg, 0.1 mmol) and azidoethane (3 eq.)
according to general procedure A. The product was purified by
column chromatography (petroleum ether/EtOAc, 2/1 v/v) to give 5
as a white solid (40 mg, 69%). 1H NMR (400 MHz, Pyridineꢀd5):
Fig. 5. Measuring antiviral activity of EA dimer compounds by immune blotting of the
NS3 protein produced by HCVcc. Huh-7.5 cells were infected with the JFH-wt virus,
cultured, and incubated in 24-well plates treated with inhibitors at the concentrations
indicated. Treated at time of infection with HCV (JJ) at 1:1 inoculation. After two days
of incubation at 37 ꢃC, HCV replication was measured by Western blotting of NS3
protein. NS3 protein levels detected by immune blotting were used to estimate the IC50
values which were calculated from a linear fit of the inhibitor response. The level of
NS3 was normalized to the level of GAPDH and the level of NS3 protein relative to
DMSO controls (no inhibitor, 100% level of NS3) was plotted against inhibitor con-
centrations. DEBO, an NS3 inhibitor, was as a positive control.
Fig. 4. The putative mechanism of bivalent EA derivatives on blocking virus entry:
disruption of the interacting binding between HCV envelope E2 and its receptor CD81.

264
F. Yu et al. / European Journal of Medicinal Chemistry 77 (2014) 258e268
Table 2
(100 MHz, CDCl3):
d 15.6 (2C), 16.6, 18.2, 23.3, 25.0, 26.7, 27.1, 28.0,
Activities of selected compounds against HCVpp and HCVcc.a
29.6, 30.2, 32.5 (2C), 35.0 (2C), 35.3, 36.8, 38.5, 38.7, 39.5, 41.4, 41.7,
46.6, 46.8, 48.9, 54.2, 55.1, 75.2, 78.8, 123.5, 128.0 (2C), 128.7 (2C),
129.0 (2C), 134.4, 143.4 (2C), 177.9. ESIꢀHRMS (m/z) [M þ H]þ calcd
for C40H59N4O3, 643.4582; found 643.4582.
Comp.
HCVpp IC50 (nM)
HCVcc EC50
(m
M)
CC50 (mM)
3
10.3
1200.4
130.1
3.0
0.9
1.1
1.0
0.8
2.6
>100
>100
>100
>100
>100
19
20
24
25
4.2.9. N-(3b,16a-dihydroxy-olean-12-en-28-oyl)-[1-(2-
hydroxyethyl)-1H-1,2,3-triazol-4-yl]-methylamine (8)
Prepared from 1b (51 mg, 0.1 mmol) and 2ꢀazidoethanol
(13 mg, 0.15 mmol) according to general procedure A. The product
was purified by column chromatography (petroleum ether/EtOAc,
1/1 v/v) to give 8 as a white solid (36 mg, 60%). 1H NMR (400 MHz,
750.7
a
All data presented are averages of at least three separate experiments. IC50
:
concentration that inhibited HCVpp entry by 50%. EC50: concentration that inhibits
HCVcc replication by 50%. CC50: concentration that inhibits 293T cell growth by 50%.
d
0.56, 0.77, 0.88, 0.90, 0.92, 0.98, 1.36 (s, 3H each, CH3), 1.54 (t, 3H,
J ¼ 7.4 Hz) 2.24 (t,1H, J ¼ 13.2 Hz), 2.70e2.72 (m,1H), 3.20e3.22 (m,
1H), 4.33e4.49 (m, 5H), 5.53 (brs, 1H), 6.83 (m, 1H), 7.48 (s, 1H). 13
16.0, 16.3, 17.0, 17.7, 19.2, 24.3, 25.7,
MeOD): d 0.57, 0.77, 0.88, 0.92, 0.96, 0.97, 1.36 (s, 3H each, CH3), 2.36
(t, 1H, J ¼ 13.1 Hz), 2.87 (dd, 1H, J ¼ 3.8, 13.6 Hz), 3.15 (dd, 1H, J ¼ 5.1,
11.4 Hz), 3.91 (t, 1H, J ¼ 5.3 Hz), 4.35e4.37 (m, 3H), 4.45 (t, 1H,
J ¼ 5.2 Hz), 5.46 (t,1H, J ¼ 3.4 Hz), 7.54 (t,1H, J ¼ 5.5 Hz), 7.81 (s, 1H).
C
NMR (100 MHz, Pyridineꢀd5):
d
13C NMR (100 MHz, MeOD):
d. 16.1, 16.3, 17.6, 19.4, 24.5, 25.3, 27.3,
27.7, 28.6, 29.2, 31.2, 32.4, 33.6, 33.8, 36.3, 36.4, 37.8, 39.6, 39.9, 40.5,
42.2, 42.7, 45.6, 47.7, 47.9, 49.7, 50.1, 56.3, 75.4, 78.5, 123.0, 123.6,
145.2, 146.3, 178.4. ESIꢀHRMS (m/z) [M þ H]þ calcd for C35H57N4O3,
581.4426; found 581.4425.
27.9, 28.7, 31.2, 31.9, 33.3, 34.0, 35.9, 36.0 (2C), 36.4, 38.1, 39.8, 39.9,
40.8, 42.4, 42.8, 48.1, 48.2, 53.8, 56.8, 61.6, 75.7, 79.7, 124.4, 125.1,
145.0 (2C), 180.1. ESIꢀHRMS (m/z) [M þ H]þ calcd for C35H57N4O4,
597.4374; found 597.4379.
4.2.7. N-(3b,16a-dihydroxy-olean-12-en-28-oyl)-(1-propyl-1H-
1,2,3-triazol-4-yl)-methylamine (6)
4.2.10. N-(3b,16a-dihydroxy-olean-12-en-28-oyl)-[1-(3-
hydroxypropyl)-1H-1,2,3-triazol-4-yl]-methylamine (9)
Prepared from 1b (61 mg, 0.12 mmol) and 3ꢀazidopropanol
(18 mg, 0.18 mmol) according to general procedure A. The product
was purified by column chromatography (petroleum ether/EtOAc,
1/1 v/v) to give 9 as a white solid (45 mg, 62%). 1H NMR (400 MHz,
Prepared from 1b (61 mg, 0.12 mmol) and azidopropane ac-
cording to general procedure A. The product was purified by col-
umn chromatography (petroleum ether/EtOAc, 2/1 v/v) to give 6 as
a white solid (58 mg, 81%). 1H NMR (400 MHz, CDCl3):
d 0.58, 0.77,
0.88, 0.90, 0.92, 0.98, 1.35 (s, 3H each, CH3), 0.94 (t, 3H, J ¼ 7.4 Hz),
2.23 (t, 1H, J ¼ 13.4 Hz), 2.75 (dd, 1H, J ¼ 3.5, 13.6 Hz), 3.20e3.22 (m,
1H), 4.26e4.50 (m, 5H), 5.53 (brs,1H), 6.93 (t,1H, J ¼ 5.2 Hz), 7.49 (s,
CDCl3):
d 0.58, 0.77, 0.88, 0.90, 0.92, 0.99, 1.35 (s, 3H each, CH3),
2.73e2.76 (m, 1H), 3.21e3.23 (m, 1H), 3.61 (brs, 2H), 4.38e4.52 (m,
1H). 13C NMR (100 MHz, CDCl3):
d 11.0, 15.5, 16.7, 18.2, 23.3, 23.6,
5H), 5.33 (brs, 1H), 6.96 (brs, 1H), 7.60 (brs, 1H). 13C NMR (100 MHz,
25.0, 26.8, 27.1, 28.0, 29.6, 30.2, 32.5, 35.1, 35.1, 35.3 (2C), 36.9, 38.5,
38.7, 39.5, 41.5, 41.7, 46.7, 46.8, 48.9, 51.9, 55.1, 75.1, 75.3, 78.8, 122.1,
123.5, 143.4, 144.5, 177.9. ESIꢀHRMS (m/z) [M þ H]þ calcd for
CDCl3):
d 15.6 (2C), 16.8, 18.2, 23.3, 25.1, 26.8, 27.1, 28.0, 29.5, 30.2,
32.5 (3C), 35.2, 35.3, 36.9, 38.6, 38.7, 39.6, 41.5, 41.8, 46.7, 46.8, 46.9,
49.0, 55.2, 58.4 (2C), 75.2, 78.8, 123.6 (2C), 143.4 (2C), 178.1.
ESIꢀHRMS (m/z) [M þ H]þ calcd for C36H59N4O4, 611.4531; found
611.4531.
C
70H111N8O6, 595.4582; found 595.4582.
4.2.8. N-(3b,16a-dihydroxy-olean-12-en-28-oyl)-(1-benzyl-1H-
1,2,3-triazol-4-yl)-methylamine (7)
4.2.11. N,N0-bis(3
b,16a-dihydroxy-olean-12-en-28-oyl)-
1,3-diaminopropane (10)
Prepared from 1a (75 mg, 0.13 mmol), 1,3-propanediamine
(5.5 mL, 0.06 mmol) according to general procedure B. The prod-
Prepared from 1b (51 mg, 0.1 mmol) and (azidomethyl)benzene
(20 mg, 0.15 mmol) according to general procedure A. The product
was purified by column chromatography (petroleum ether/EtOAc,
1/1 v/v) to give 7 as a white solid (60 mg, 93%). 1H NMR
uct was purified by column chromatography (CH2Cl2/MeOH, 30/1
(400 MHz,CDCl3): d 0.51, 0.78, 0.86, 0.89, 0.90, 0.99, 1.34 (s, 3H each,
v/v) to give 10 as a white solid (35 mg, 55%). 1H NMR (400 MHz,
CH3), 2.22 (t, 1H, J ¼ 13.3 Hz), 2.72 (dd, 1H, J ¼ 3.1, 13.4 Hz), 3.21 (dd,
1H, J ¼ 4.1,10.6 Hz), 4.37e4.42 (m, 3H), 5.43e5.53 (m, 3H), 6.95 (brs,
1H), 7.24e7.26 (m, 2H), 7.34e7.37 (3H), 7.48 (brs, 1H). 13C NMR
MeOD):
d
0.76 (s, 12H, 4 ꢁ CH3), 0.88, 0.92, 0.95, 0.96, 1.35 (s, 6H
each, 2 ꢁ CH3), 2.27 (t, 2H, J ¼ 13.3 Hz), 2.86e2.89 (m, 2H), 3.03e
3.17 (m, 6H), 4.30 (brs, 2H), 5.51 (brs, 2H), 7.09 (brs, 2H). 13C NMR
(100 MHz, MeOD):
d 16.1 (4C), 17.7, 19.0, 24.1, 25.6, 27.1, 27.3, 28.5
(2C each), 30.0 (1C), 30.8, 31.0, 33.0, 33.4, 35.6, 35.8, 37.2, 37.6, 39.4,
39.4, 40.3, 41.8, 42.4, 47.5, 47.5, 56.1, 75.0, 79.2, 123.6, 144.4, 179.8
(2C each). ESIꢀHRMS (m/z) [M þ H]þ calcd for C63H103N2O6,
983.7811; found 983.7804.
4.2.12. N,N0-bis(3
1,4-diaminobutane (11)
Prepared from 1a (130 mg, 0.22 mmol), and 1,4-butanediamine
(8.8 L, 0.1 mmol) according to general procedure B. The product
b,16a-dihydroxy-olean-12-en-28-oyl)-
m
was purified by column chromatography (CH2Cl2/MeOH, 30/1 v/v)
to give 11 as a white solid (79 mg, 79%). 1H NMR (400 MHz, MeOD):
d
0.79, 0.80, 0.90, 0.97, 0.97, 0.98, 1.39 (s, 6H each, 2 ꢁ CH3), 1.89e
1.99 (m, 8H), 2.37 (t, 2H, J ¼ 13.2 Hz), 2.85 (dd, 2H, J ¼ 3.3, 13.5 Hz),
2.99e3.06 (m, 2H), 3.15e3.23 (m, 4H), 4.37 (brs, 2H), 5.48 (brs, 2H),
7.07 (t, 2H, J ¼ 5.2 Hz). 13C NMR (100 MHz, MeOD):
d 16.3, 16.4, 18.0,
19.6, 24.6, 25.5, 27.4, 27.8, 27.9, 28.8, 31.3, 32.2, 33.4, 34.0, 36.0, 36.4,
38.1 (2C each), 39.9 (4C), 40.4, 40.8, 42.4, 42.9 (2C each), 48.1 (4C),
Fig. 6. Hemolysis assay of EA and EA dimers.

F. Yu et al. / European Journal of Medicinal Chemistry 77 (2014) 258e268
265
49.9, 56.8, 75.6, 79.6, 123.9, 145.4, 180.1 (2C each). ESIꢀHRMS (m/z)
[M þ H]þ calcd for C64H105N2O6, 997.7968; found 997.7967.
70.4, 71.4, 71.6, 75.6, 79.5, 124.2, 145.3, 180.1 (2C each). ESIꢀHRMS
(m/z) [M þ H]þ calcd for C68H113N2O9, 1101.8441; found 1101.8441.
4.2.13. N,N0-bis(3
1,6-diaminohexane (12)
Prepared from 1a (130 mg, 0.22 mmol), and 1,6-hexanediamine
(12 mg, 0.1 mmol) according to general procedure B. The product
was purified by column chromatography (CH2Cl2/MeOH, 30/1 v/v)
to give 12 as a white solid (82 mg, 84%). 1H NMR (400 MHz, MeOD):
b,16
a
-dihydroxy-olean-12-en-28-oyl)-
4.2.17. N,N0-bis(3 -dihydroxy-olean-12-en-28-oyl)-(1,10-
b,16a
(ethane-1,2-diyl)bis(1H-1,2,3-triazole-4,1-diyl))dimethanamine (16)
Prepared from 1b (118 mg, 0.22 mmol) and 1,2-diazidoethane
(12 mg, 0.1 mmol) according to general procedure A. The prod-
uct was purified by column chromatography (CH2Cl2/MeOH, 25/1
v/v) to give 16 as a white solid (97 mg, 87%). 1H NMR (400 MHz,
d
0.79, 0.81, 0.90 (s, 6H each, 2 ꢁ CH3), 0.97 (s, 12H, 4 ꢁ CH3), 0.99,
MeOD): d 0.58, 0.78, 0.89, 0.92, 0.96, 0.97, 1.37 (s, 6H each,
1.39 (s, 6H each, 2 ꢁ CH3), 2.36 (t, 2H, J ¼ 13.2 Hz), 2.84 (dd, 2H,
J ¼ 2.5, 13.0 Hz), 3.00e3.08 (m, 2H), 3.15e3.24 (m, 4H), 4.36 (brs,
2H), 5.48 (brs, 2H), 7.00 (t, 2H, J ¼ 5.2 Hz). 13C NMR (100 MHz,
2 ꢁ CH3), 2.35 (t, 2H, J ¼ 13.2 Hz), 2.86 (dd, 2H, J ¼ 2.9, 13.4 Hz),
3.15 (dd, 2H, J ¼ 4.9, 11.0 Hz), 4.26e4.38 (m, 6H), 4.89 (s, 4H), 5.46
(brs, 2H), 7.56 (t, 2H, J ¼ 5.4 Hz), 7.60 (s, 2H). 13C NMR (100 MHz,
MeOD):
d
16.3, 16.5, 18.1, 19.5, 24.6, 25.5, 27.3 (2C each), 27.9 (4C),
MeOD): d 16.3, 16.4, 17.7, 19.5, 24.5, 25.5, 27.4, 27.9, 28.8, 31.3, 31.9,
28.8, 30.3, 31.3, 32.0, 33.3, 34.0, 36.0, 36.4, 38.1, 39.9, 40.0, 40.7, 40.8,
42.5, 42.9, 48.1, 48.1, 50.0, 56.8, 75.6, 79.6, 123.9, 145.4, 180.0 (2C
each). ESIꢀHRMS (m/z) [M þ H]þ calcd for C66H109N2O6, 1025.8280;
found 1025.8280.
33.4, 33.9, 36.0, 36.3, 38.1, 39.8, 39.9, 40.7, 42.3, 42.7, 48.0, 48.1,
49.8, 49.9, 50.8, 56.7, 75.5, 79.6, 124.2, 124.7, 144.9, 146.2, 180.0 (2C
each). ESIꢀHRMS (m/z) [M
þ
H]þ calcd for C68H107N8O6,
1131.8308; found 1131.8291.
4.2.14. N,N0-bis(3
b,16a-dihydroxy-olean-12-en-28-oyl)-
4.2.18. N,N0-bis(3 -dihydroxy-olean-12-en-28-oyl)-(1,10-
b,16a
1,12-diaminododecane (13)
(propane-1,4-diyl)bis(1H-1,2,3-triazole-4,1-diyl))dimethanamine (17)
Prepared from 1b (118 mg, 0.22 mmol) and 1,4-diazidobutane
(14 mg, 0.1 mmol) according to general procedure A. The prod-
uct was purified by column chromatography (CH2Cl2/MeOH, 30/1
v/v) to give 17 as a white solid (82 mg, 71%). 1H NMR (400 MHz,
Prepared from 1a (130 mg, 0.22 mmol), and 1,12-
dodecanediamine (20 mg, 0.1 mmol) according to general proce-
dure B. The product was purified by column chromatography
(CH2Cl2/MeOH, 25/1 v/v) to give 13 as a white solid (97 mg, 87%). 1H
NMR (400 MHz, MeOD):
d
0.79, 0.82, 0.90, 0.97, 0.99,1.31,1.39 (s, 6H
MeOD): d 0.53, 0.77, 0.89, 0.91, 0.96, 0.97, 1.37 (s, 6H each,
each, 2 ꢁ CH3), 1.31 (brs, 16H), 2.37 (t, 2H, J ¼ 13.2 Hz), 2.85 (dd, 2H,
J ¼ 3.2, 13.6 Hz), 2.99e3.07 (m, 2H), 3.14e3.25 (m, 4H), 4.36 (brs,
2H), 5.48 (brs, 2H), 6.98 (t, 2H, J ¼ 5.3 Hz). 13C NMR (100 MHz,
2 ꢁ CH3), 2.36 (t, 2H, J ¼ 13.2 Hz), 2.87 (dd, 2H, J ¼ 3.1, 13.5 Hz),
3.15 (dd, 2H, J ¼ 4.9, 10.9 Hz), 4.35e4.40 (m, 10H), 5.46 (brs, 2H),
7.55 (t, 2H, J ¼ 5.2 Hz), 7.78 (s, 2H). 13C NMR (100 MHz, MeOD):
MeOD):
d
16.4, 16.5, 18.1, 19.5, 24.6, 25.5, 27.3, 27.9, 28.3, 28.8, 30.3,
d 16.3, 16.4, 17.7, 19.5, 24.5, 25.4, 27.4, 27.9, 28.2, 28.8, 31.2, 31.9,
30.5, 30.8, 30.8, 31.3, 32.0, 33.3, 34.0, 36.0, 36.4, 38.2, 39.9, 40.0,
40.8, 40.8, 42.5, 42.9, 48.1, 48.2, 50.0, 56.8, 75.6, 79.6, 123.9, 145.5,
180.0 (2C each). ESIꢀHRMS (m/z) [M þ H]þ calcd for C72H121N2O6,
1109.9220; found 1109.9219.
33.4, 33.9, 36.0, 36.0, 36.4, 38.1, 39.8, 39.9, 40.7, 42.3, 42.8, 48.0,
48.1, 49.9, 50.4, 56.7, 75.6, 79.6, 124.2, 124.4, 144.9, 146.0, 180.0 (2C
each). ESIꢀHRMS (m/z) [M
þ
H]þ calcd for C70H111N8O6,
1159.8621; found 1159.8605.
4.2.15. N,N0-bis(3
b,16a-dihydroxy-olean-12-en-28-oyl)-
4.2.19. N,N0-bis(3 -dihydroxy-olean-12-en-28-oyl)-(1,10-
b,16a
1,5-diamine-3-oxapentane (14)
(pentane-1,5-diyl)bis(1H-1,2,3-triazole-4,1-diyl))dimethanamine (18)
Prepared from 1b (118 mg, 0.22 mmol) and 1,5-diazidopentane
(15 mg, 0.1 mmol) according to general procedure A. The product
was purified by column chromatography (CH2Cl2/MeOH, 30/1 v/v)
to give 18 as a white solid (75 mg, 65%). 1H NMR (400 MHz, MeOD):
Prepared from 1a (130 mg, 0.22 mmol) and 1,5-diamine-3-
oxapentane (11 mg, 0.1 mmol) according to general procedure B.
The product was purified by column chromatography (CH2Cl2/
MeOH, 25/1 v/v) to give 14 as a white solid (62 mg, 61%). 1H NMR
(400 MHz, MeOD):
d
0.79, 0.82, 0.90 (s, 6H each, 2 ꢁ CH3), 0.97 (s,
d
0.53, 0.76, 0.89, 0.91, 0.96, 0.96, 1.37 (s, 6H each, 2 ꢁ CH3), 2.36 (t,
12H, 4 ꢁ CH3), 0.99, 1.40 (s, 6H each, 2 ꢁ CH3), 2.37 (t, 2H,
J ¼ 13.2 Hz), 2.82 (dd, 2H, J ¼ 2.3, 13.9 Hz), 3.15e3.26 (m, 4H), 3.37e
3.43 (m, 2H), 3.47e3.49 (m, 4H), 4.35 (brs, 2H), 5.49 (brs, 2H), 7.09
2H, J ¼ 13.2 Hz), 2.87 (dd, 2H, J ¼ 13.5, 3.5 Hz), 3.15 (dd, 2H, J ¼ 11.0,
5.0 Hz), 4.35e4.38 (m, 10H), 5.46 (t, 2H, J ¼ 3.1 Hz), 7.56 (t, 2H,
J ¼ 5.4 Hz), 7.77 (s, 2H). 13C NMR (100 MHz, MeOD):
d 16.3,16.4,17.7,
(brs, 2H). 13C NMR (100 MHz, MeOD):
d
16.4, 16.5, 18.0, 19.5, 24.6,
19.5 (2C each), 24.4 (1C), 24.5, 25.4, 27.3, 27.9, 28.8, 30.7, 31.3, 32.0,
33.3, 34.0, 35.9, 36.0, 36.4, 38.1, 39.8, 39.9, 40.7, 42.3, 42.8 (2C each),
48.1 (4C), 49.9, 50.9, 56.7, 75.6, 79.6, 124.2, 124.3, 145.0, 145.9, 180.0
(2C each). ESIꢀHRMS (m/z) [M þ H]þ calcd for C71H113N8O6,
1173.8778; found 1173.8753.
25.5, 27.4, 27.9, 28.8, 31.3, 31.9, 33.3, 34.0, 36.0, 36.4, 38.1, 39.9, 40.0,
40.6, 40.8, 42.6, 42.9, 48.1, 48.1, 50.1, 56.8, 70.2, 75.6, 79.5, 124.1,
145.3, 180.3 (2C each). ESIꢀHRMS (m/z) [M þ H]þ calcd for
C
64H105N2O7, 1013.7905; found 1013.7916.
4.2.16. N,N0-bis(3
b,16a-dihydroxy-olean-12-en-28-oyl)-
4.2.20. N,N0-bis(3 -dihydroxy-olean-12-en-28-oyl)-(1,10-
b,16a
tetraethylene glycol bisamine (15)
(hexane-1,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))dimethanamine (19)
Prepared from 1b (118 mg, 0.22 mmol) and 1,6-diazidohexane
(17 mg, 0.1 mmol) according to general procedure A. The product
was purified by column chromatography (CH2Cl2/MeOH, 30/1 v/v)
to give 19 as a white solid (83 mg, 70%). 1H NMR (400 MHz, MeOD):
Prepared from 1a (130 mg, 0.22 mmol) and tetraethylene glycol
bisamine (19 mg, 0.1 mmol) according to general procedure B. The
product was purified by column chromatography (CH2Cl2/MeOH,
20/1 v/v) to give 15 as a white solid (87 mg, 79%). 1H NMR
(400 MHz, MeOD):
d
0.79, 0.82, 0.90 (s, 6H each, 2 ꢁ CH3), 0.97 (s,
d
0.52, 0.77, 0.88, 0.91, 0.96, 0.97, 1.37 (s, 6H each, 2 ꢁ CH3), 2.36 (t,
2H, J ¼ 13.1 Hz), 2.87 (m, 2H), 3.15 (dd, 2H, J ¼ 10.9, 4.9 Hz), 4.36 (m,
1H), 5.46 (brs, 2H), 7.80 (s, 2H). 13C NMR (100 MHz, MeOD):
16.3,
12H, 4 ꢁ CH3), 0.99, 1.41 (s, 6H each, 2 ꢁ CH3), 2.40 (t, 2H,
J ¼ 13.2 Hz), 2.80 (dd, 2H, J ¼ 3.0, 13.4 Hz), 3.15e3.27 (m, 4H), 3.37e
3.44 (2H), 3.51 (t, 2H, J ¼ 5.3 Hz), 3.62e3.66 (m, 8H), 4.36 (brs, 2H),
5.50 (brs, 2H), 7.11 (t, 2H, J ¼ 5.0 Hz). 13C NMR (100 MHz, MeOD):
d
16.5, 17.7, 19.5, 24.5, 25.4, 26.9, 27.4, 27.9, 28.8, 31.1, 31.3, 32.0, 33.4,
34.0, 36.0, 36.4, 38.1, 39.8, 39.9, 40.7, 42.3, 42.8, 48.1, 51.1, 56.7, 75.6,
79.6, 124.2, 145.0, 179.3 (2C each). ESIꢀHRMS (m/z) [M þ H]þ calcd
for C72H115N8O6, 1187.8934; found 1187.8928.
d
16.4,16.5,17.9,19.5, 24.7, 25.4, 27.3, 27.9, 28.8, 31.3, 32.1, 33.4, 33.9,
36.0, 36.4, 38.1, 39.9, 40.0, 40.6, 40.8, 42.6, 42.9, 48.1, 48.2, 50.0, 56.7,

266
F. Yu et al. / European Journal of Medicinal Chemistry 77 (2014) 258e268
4.2.21. N,N0-bis(3
(oxybis(ethane-2,1-diyl))bis(1H-1,2,3-triazole-4,1-diyl))
dimethanamine (20)
b
,16
a
-dihydroxy-olean-12-en-28-oyl)-(1,10-
129.2 (1C), 129.5 (2C), 130.8 (1C), 137.6, 145.0, 146.5, 180.1 (2C each).
ESIꢀHRMS (m/z) [M þ H]þ calcd for C74H111N8O6, 1207.8621; found
1207.8617.
Prepared from 1b (118 mg, 0.22 mmol) and 1-azido-2-(2-
azidoethoxy)ethane (16 mg, 0.1 mmol) according to general pro-
cedure A. The product was purified by column chromatography
(CH2Cl2/MeOH, 25/1 v/v) to give 20 as a white solid (93 mg, 79%). 1H
4.2.25. N,N0-bis(3 -dihydroxy-olean-12-en-28-oyl)-(1,10-(1,2-
b,16a
phenylenebis(methylene))bis (1H-1,2,3-triazole-4,1-diyl))
dimethanamine (24)
NMR (400 MHz, MeOD):
d
0.55, 0.77, 0.89, 0.92, 0.96, 0.97, 1.37 (s,
Prepared from 1b (118 mg, 0.22 mmol) and o-xylylene diazide
(19 mg, 0.1 mmol) according to general procedure A. The product
was purified by column chromatography (CH2Cl2/MeOH, 25/1 v/v)
to give 24 as a white solid (95 mg, 79%). 1H NMR (400 MHz, MeOD):
6H each, 2 ꢁ CH3), 2.37 (t, 2H, J ¼ 13.1 Hz), 2.87e2.90 (m, 2H), 3.15
(dd, 2H, J ¼ 4.5, 10.8 Hz), 3.80e3.82 (m, 4H), 4.31e4.42 (m, 6H),
4.50e4.53 (m, 4H), 5.47 (brs, 2H), 7.60 (brs, 2H), 7.70 (s, 2H). 13C
NMR (100 MHz, CDCl3):
d
16.3, 16.4, 17.6, 19.5, 24.5, 25.5, 27.4, 27.9,
d
0.44, 0.78, 0.85, 0.87, 0.92, 0.97, 1.35 (s, 6H each, 2 ꢁ CH3), 2.34 (t,
28.8, 31.3, 32.1, 33.4, 33.9, 36.0, 36.1, 36.4, 38.1, 39.8, 39.9, 40.7, 42.3,
42.8, 48.0, 48.1, 49.9, 51.2, 56.7, 70.3, 75.6, 79.6, 124.2, 125.0, 145.0,
146.0, 180.0 (2C each). ESIꢀHRMS (m/z) [M þ H]þ calcd for
2H, J ¼ 13.3 Hz), 2.84 (dd, 2H, J ¼ 13.5, 3.3 Hz), 3.14 (dd, 2H, J ¼ 11.0,
4.8 Hz), 4.35e4.36 (m, 6H), 5.45 (brs, 2H), 5.70 (d, 2H, J ¼ 15.2 Hz),
5.78 (d, 2H, J ¼ 15.2 Hz), 7.27e7.29 (m, 2H), 7.37e7.39 (m, 2H), 7.55
C
70H111N8O7, 1175.8570; found 1175.8550.
(t, 2H, J ¼ 5.4 Hz), 7.68 (s, 2H). 13C NMR (100 MHz, MeOD):
d 16.3,
16.5, 17.6, 19.5, 24.5, 25.4, 27.3, 27.9, 28.8, 31.2, 31.9, 33.3, 33.9 (2C
each), 35.9 (4C), 36.1, 36.4, 38.1, 39.8, 39.9, 40.7, 42.3, 42.8, 48.1,
50.0, 51.9, 56.8, 75.6, 79.6, 124.2, 124.5, 130.6, 131.3, 135.2, 144.9,
146.5, 180.1 (2C each). ESIꢀHRMS (m/z) [M þ H]þ calcd for
4.2.22. N,N0-bis(3 -dihydroxy-olean-12-en-28-oyl)-(1,10-
b,16a
(((oxybis(ethane-2,1-diyl))bis(oxy)) bis(ethane-2,1-diyl))bis(1H-
1,2,3-triazole-4,1-diyl))dimethanamine (21)
Prepared from 1b (118 mg, 0.22 mmol) and tetraethylene glycol
bisazide (24 mg, 0.1 mmol) according to general procedure A. The
product was purified by column chromatography (CH2Cl2/MeOH,
25/1 v/v) to give 21 as a white solid (100 mg, 79%). 1H NMR
C
74H111N8O6, 1207.8621; found 1207.8614.
4.2.26. N,N0-bis(3 -dihydroxy-olean-12-en-28-oyl)-(1,10-((2,5-
dimethyl-1,4-phenylene) bis(methylene))bis(1H-1,2,3-triazole-4,1-
diyl))dimethanamine (25)
b
,16a
(400 MHz, MeOD): d 0.53, 0.77, 0.89, 0.92, 0.96, 0.97,1.37 (s, 6H each,
2 ꢁ CH3), 2.37 (t, 2H, J ¼ 13.2 Hz), 2.88 (dd, 2H, J ¼ 3.3, 13.5 Hz), 3.15
(dd, 2H, J ¼ 4.9, 11.0 Hz), 3.58e3.59 (m, 8H), 3.85e3.88 (m, 4H),
4.35e4.38 (m, 6H), 4.53e4.56 (m, 4H), 5.47 (brs, 2H), 7.54 (t, 2H,
Prepared from 1b (118 mg, 0.22 mmol) and 1,4-bis(azidomethyl)-
2,5-dimethylbenzene (22 mg, 0.1 mmol) according to general pro-
cedure A. The product was purified by column chromatography
(CH2Cl2/MeOH, 30/1 v/v) to give 25 as a white solid (95 mg, 77%). 1H
J ¼ 5.3 Hz), 7.85 (s, 2H). 13C NMR (100 MHz, MeOD):
d 16.3, 16.4, 17.6,
19.5, 24.5, 25.4, 27.4, 27.9, 28.8, 31.3, 32.1, 33.4, 33.9, 35.9, 36.0, 36.4,
38.1, 39.8, 39.9, 40.7, 42.3, 42.7, 48.1, 48.2, 49.9, 51.3, 56.7, 70.4, 71.5,
71.5, 75.6, 79.5, 124.2, 125.1, 144.9, 145.8, 179.9 (2C each). ESIꢀHRMS
(m/z) [M þ H]þ calcd for C74H119N8O7, 1263.9095; found 1263.9075.
NMR (400 MHz, MeOD): d 0.46, 0.78, 0.88, 0.89, 0.93, 0.98,1.35 (s, 6H
each, 2 ꢁ CH3), 2.28 (s, 6H), 2.34 (t, 2H, J ¼ 13.3 Hz), 2.82 (dd, 2H,
J ¼ 13.1, 2.6 Hz), 3.15 (dd, 2H, J ¼ 10.9, 4.7 Hz), 4.25e4.38 (m, 6H), 5.44
(brs, 2H), 5.51 (d, 2H, J ¼ 14.9 Hz), 5.55 (d, 2H, J ¼ 14.9 Hz), 7.11 (s, 2H),
7.52 (t, 2H, J ¼ 5.2 Hz), 7.63 (s, 2H).13C NMR (100 MHz, MeOD):
d 16.2,
4.2.23. N,N0-bis(3
b,16a
-dihydroxy-olean-12-en-28-oyl)-(1,10-(1,4-
16.4, 17.6, 18.8, 19.5, 24.5, 25.4, 27.3, 27.9, 28.8, 31.2, 31.9, 33.3, 33.9,
35.9, 36.1, 36.4, 38.1, 39.9, 39.9, 40.7, 42.3, 42.8 (2C each), 48.1 (4C),
50.0, 52.6, 56.8, 75.6, 79.6, 124.2, 124.3, 133.2, 135.1, 136.3, 144.9,
146.3, 180.1 (2C each). ESIꢀHRMS (m/z) [M þ H]þ calcd for
phenylenebis(methylene)) bis(1H-1,2,3-triazole-4,1-diyl))
dimethanamine (22)
Prepared from 1b (118 mg, 0.22 mmol) and p-xylylene diazide
(19 mg, 0.1 mmol) according to general procedure A. The product
was purified by column chromatography (CH2Cl2/MeOH, 25/1 v/v) to
give 22 as a white solid (96 mg, 80%). 1H NMR (400 MHz, MeOD):
C74H111N8O6, 1235.8934; found 1235.8915.
4.2.27. N,N0-bis(3 -dihydroxy-olean-12-en-28-oyl)-(1,10-((2,5-
b
,16a
d
0.45, 0.79 (s, 6H each, 2 ꢁ CH3), 0.88 (s,12H, 4 ꢁ CH3), 0.93, 0.98,1.35
dimethoxy-1,4-phenylene) bis(methylene))bis(1H-1,2,3-triazole-
4,1-diyl))dimethanamine (26)
(s, 6H each, 2 ꢁ CH3), 2.34 (t, 2H, J ¼ 13.2 Hz), 2.82 (dd, 2H, J ¼ 13.5,
3.4 Hz), 3.19 (dd, 2H, J ¼ 11.0, 4.8 Hz), 4.29e4.37 (m, 6H), 5.44 (brs,
2H), 5.53 (d, 2H, J ¼ 15.0 Hz), 5.57 (d, 2H, J ¼ 15.0 Hz), 7.33 (s, 4H), 7.75
Prepared from 1b (118 mg, 0.22 mmol) and 1,4-
bis(azidomethyl)-2,5-dimethoxybenzene (25 mg, 0.1 mmol) ac-
cording to general procedure A. The product was purified by col-
umn chromatography (CH2Cl2/MeOH, 30/1 v/v) to give 26 as a
(s, 2H). 13C NMR (100 MHz, MeOD):
d 16.3, 16.5, 17.6, 19.5, 24.5, 25.4,
27.3, 27.9, 28.8, 31.3, 31.9, 33.3, 33.9, 35.9, 36.1, 36.4, 38.1 (2C each),
39.9 (4C), 40.7, 42.3, 42.8, 48.1, 49.9, 54.4, 56.8, 75.6, 79.6,124.2,124.4
(2C each), 129.8 (4C), 137.1, 144.9, 146.4, 180.0 (2C each). ESIꢀHRMS
(m/z) [M þ H]þ calcd for C74H110N8O6, 1207.8621; found 1207.8599.
white solid (92 mg, 73%). 1H NMR (400 MHz, MeOD):
d 0.30, 0.78,
0.84, 0.88, 0.93, 0.98, 1.34 (s, 6H each, 2 ꢁ CH3), 2.35 (t, 2H,
J ¼ 13.1 Hz), 2.79 (dd, 2H, J ¼ 3.4, 13.4 Hz), 3.14 (dd, 2H, J ¼ 4.7,
10.7 Hz), 3.84 (s, 6H), 4.25e4.38 (m, 6H), 5.43 (brs, 2H), 5.51 (dd,
4H, J ¼ 14.8, 16.1 Hz), 7.03 (s, 2H), 7.47 (t, 2H, J ¼ 5.4 Hz), 7.77 (s, 2H).
4.2.24. N,N0-bis(3 -dihydroxy-olean-12-en-28-oyl)-(1,10-(1,3-
b,16a
phenylenebis(methylene)) bis(1H-1,2,3-triazole-4,1-diyl))
dimethanamine (23)
Prepared from 1b (118 mg, 0.22 mmol) and m-xylylene diazide
(19 mg, 0.1 mmol) according to general procedure A. The product
was purified by column chromatography (CH2Cl2/MeOH, 25/1 v/v)
to give 23 as a white solid (91 mg, 75%). 1H NMR (400 MHz, MeOD):
13C NMR (100 MHz, MeOD):
d 16.2, 16.4, 17.4, 19.5, 24.4, 25.2, 27.2,
27.9, 28.8, 31.3, 32.1, 33.3, 33.9, 35.8, 35.9, 36.5, 38.0, 39.8, 39.9, 40.6,
42.3, 42.8, 48.0, 48.1, 49.9, 50.1, 54.8, 56.7, 56.8, 75.7, 79.6, 114.8,
124.3, 124.8, 125.9, 144.9, 152.7, 180.0 (2C each). ESIꢀHRMS (m/z)
[M þ H]þ calcd for C76H115N8O8, 1267.8831; found 1267.8832.
d
0.43, 0.78, 0.86, 0.87, 0.93, 0.97, 1.35 (s, 6H each, 2 ꢁ CH3), 2.34 (t,
4.2.28. N,N0-bis(3 -dihydroxy-olean-12-en-28-oyl)-(1,10-(1,4-
b,16a
phenylenebis(ethane-2,1-diyl)) bis(1H-1,2,3-triazole-4,1-diyl))
dimethanamine (27)
Prepared from 1b (118 mg, 0.22 mmol) and 1,4-bis(2-azidoethyl)
benzene (22 mg, 0.1 mmol) according to general procedure A. The
product was purified by column chromatography (CH2Cl2/MeOH,
30/1 v/v) to give 27 as a white solid (98 mg, 79%). 1H NMR
2H, J ¼ 13.2 Hz), 2.84 (dd, 2H, J ¼ 2.7, 13.4 Hz), 3.15 (dd, 2H, J ¼ 4.6,
10.7 Hz), 4.34e4.35 (m, 6H), 5.44 (brs, 2H), 5.54 (dd, 4H, J ¼ 15.1,
17.6 Hz), 7.28e7.38 (m, 4H), 7.54 (t, 2H, J ¼ 5.2 Hz), 7.77 (s, 2H). 13
C
NMR (100 MHz, MeOD): d 16.4, 16.6, 17.7, 19.6, 24.5, 25.5, 27.4, 28.0,
28.9, 31.3, 32.0, 33.4, 34.0, 36.0, 36.2, 36.5, 38.1, 39.9, 40.0, 40.7, 42.4,
42.8, 48.1, 50.0, 54.6, 54.9, 56.8, 75.6, 79.7, 124.3, 124.5 (2C each),

F. Yu et al. / European Journal of Medicinal Chemistry 77 (2014) 258e268
267
(400 MHz, CDCl3):
d
0.57, 0.77, 0.88, 0.90, 0.92, 0.98,1.36 (s, 6H each,
E1, E2 or vesicular stromatis G protein (VSVG) and the envelope and
Vpr deficient HIV vector carrying a luciferase reporter gene inserted
into the Nef position 72 h after transfection; HCVpp or VSVpp were
harvested from the supernatant of the transfected cells. For com-
pound library screening, infections were performed in 96-well
plates by adding diluted HCVpp or VSVpp into 5 ꢁ 103 Huh-7 cells/
well in the presence or absence of test compounds, followed by
incubation for 72 h at 37 ꢃC. Luciferase activity, reflecting the de-
gree of the pseudoparticles into host cells, was measured 3 days
after infection using the Bright-Glo Reagent (Promega). Test com-
2 ꢁ CH3), 2.25 (t, 2H, J ¼ 13.2 Hz), 2.73e2.76 (m, 2H), 3.14e3.23 (m,
6H), 4.30e4.44 (m, 6H), 4.49e4.59 (m, 4H), 5.53 (brs, 2H), 7.00 (s,
4H), 7.34 (s, 2H). 13C NMR (100 MHz, CDCl3):
d 15.5, 15.6, 16.7, 18.2,
23.3, 25.0, 26.7, 27.1, 28.0, 29.6, 30.2 (2C each), 32.5 (4C), 35.0 (4C),
35.2, 36.2, 36.8, 38.5, 38.7, 39.4, 41.4, 41.6, 46.6, 46.7, 48.8, 51.4, 55.1,
74.9, 78.7, 122.6, 123.3 (2C each), 129.0 (4C), 135.7 (2C), 143.4 (4C),
177.9 (2C). ESIꢀHRMS (m/z) [M þ H]þ calcd for C76H115N8O6,
1235.8934; found 1235.8936.
4.2.29. N,N0-bis(3
b
,16
a
-dihydroxy-olean-12-en-28-oyl)-(1,10-(1,4-
pounds were serially diluted to final concentrations of 1
mM and
phenylene)bis(1H-1,2,3-triazole-4,1-diyl))dimethanamine (28)
CuSO4 (32 mg, 0.2 mmol) and sodium ascorbate (60 mg,
0.3 mmol) were added to the solution of 1b (118 mg, 0.22 mmol)
and 1,4-bisazidobenzene (16 mg, 0.1 mmol) in THF (8 mL). The
suspension was refluxed for 1 d then the mixture was filtered and
the solid was collected and washed with H2O (20 mL) and MeOH
(20 mL) to give 28 as a white solid (102 mg, 81%). 1H NMR
5 mM in 1% dimethyl sulfoxide (DMSO). Maximum activity (100% of
control) and background were derived from control wells con-
taining DMSO alone or from uninfected wells, respectively. The
individual signals in each of the compound test wells were then
divided by the averaged control values (wells lacking inhibitor)
after background subtraction, and multiplied by 100% to arrive at
percent activity. The corresponding inhibition values were then
calculated by subtracting this value from 100. The specificity of the
compounds for inhibiting HCV was determined by evaluating in-
hibition of VSVpp infection in parallel. Each sample was analyzed in
duplicate, and experiments were repeated at least three times.
(400 MHz, pyridineꢀd5):
d 0.88, 0.94, 1.01, 1.01, 1.11, 1.21, 1.77 (s, 6H
each, 2 ꢁ CH3), 2.82 (t, 2H, J ¼ 13.2 Hz), 3.44e3.48 (m, 4H), 4.85 (dd,
2H, J ¼ 5.2, 14.9 Hz), 4.97 (dd, 2H, J ¼ 5.4, 14.9 Hz), 5.17 (brs, 2H),
5.68 (brs, 2H), 8.14 (s, 4H), 8.46 (t, 2H, J ¼ 5.3 Hz), 8.74 (s, 2H). 13
C
NMR (100 MHz, Pyridine-d5): 16.1, 16.9, 17.7, 19.1, 24.2, 25.6, 27.6,
28.5, 29.1, 31.1, 32.4, 33.5, 33.6, 36.1, 36.2, 36.3, 37.7, 39.4, 39.7, 40.3,
42.0, 42.6, 47.6, 47.8, 49.6, 56.2, 75.2, 78.4 (2C each), 122.0 (4C),
122.2, 123.4, 137.5, 145.1, 147.5, 178.4 (2C each). ESIꢀHRMS (m/z)
[M þ H]þ calcd for C72H107N8O6, 1179.8309; found 1179.8308.
4.4. SPR
Surface plasmon resonance experiments were performed using
a Biacore T200 system (GE Healthcare, Uppsala, Sweden), at 25 ꢃC.
E2 was immobilized through activation of the sensor chip with
4.2.30. N-(2-azidoethyl)-3
(29a)
Prepared from 1a (589 mg, 1 mmol) and 2-azidoethanamine
(170 mg, 2 mmol) according to general procedure B. The product
was purified by column chromatography (petroleum ether/EtOAc,
3/1 v/v) to give 29a as a white solid (481 mg, 89%). 1H NMR
b
,16
a
-dihydroxy-olean-12-en-28-amide
60
propyl)-carbodiimide at 10
E2 diluted in 10 mM NaOAc buffer pH 4.5. Unreacted activated
groups were blocked by a 60 L injection of 1 M ethanolamine at
10 L/min. Final E2 immobilized levels were typically w15,000RU.
Subsequently, compound was injected as analyte at various con-
centrations (EA from 3.125 M to 100 M, 1 and 22 from 0.78 M to
25 M, respectively), and using PBS-P (10 mM phosphate buffer
m
L of N-hydroxysuccinimide and N-ethyl-N-(dimethylamino-
m
L/min, followed by 30 L injection of
m
m
m
(400 MHz, CDCl3):
d
0.79, 0.80, 0.91, 0.93, 0.95,1.00,1.38 (s, 3H each,
m
m
m
CH3), 2.71e2.74 (m, 1H), 3.17e3.24 (m, 2H), 3.24e3.56 (m, 3H), 4.41
m
(brs, 1H), 5.56 (brs, 1H), 6.55 (brs, 1H). 13C NMR (400 MHz, CDCl3):
with 2.7 mM KCl,137 mM NaCl, and 0.05% Surfactant P20, pH 4.5) as
running buffer. For binding studies, analytes were applied at cor-
d
15.6 (2C), 16.9, 18.2, 23.4, 24.9, 26.8, 27.1, 28.0, 29.6, 30.2, 32.5,
32.5, 35.2, 35.3, 36.9, 38.6, 38.7, 39.0, 39.6, 41.7, 41.8, 46.7, 46.9, 49.2,
50.8, 55.2, 75.4, 78.8, 123.5, 143.8, 178.1.
responding concentrations in running buffer at a flow rate of 30 mL/
min with a contact and dissociation times of 60 s. Chip platforms
were washed with running buffer and additionally washed with
50% DMSO. The Biacore evaluation software (T200 Version 1.0) was
used to analyze the results. The assay of CD81 interaction with
compound is same as the assay of the interaction with E2.
4.2.31. (3b,16a-dihydroxy-olean-12-en-28-oyl)-2-(4-((bis((1-(tert-
butyl)-1H-1,2,3-triazol-4-yl) methyl)amino)methyl)-1H-1,2,3-
triazol-1-yl)ethanamine (29)
Prepared from 29a (54 mg, 0.1 mmol) and N,N-bis((1-(tert-
butyl)-1H-1,2,3-triazol-4-yl)methyl)prop-2-yn-1-amine
[15]
4.5. Hemolytic assay
(50 mg, 0.15 mmol) according to general procedure A. The prod-
uct was purified by column chromatography (petroleum ether/
EtOAc, 1/1 v/v) to give 29 as a white solid (60 mg, 93%). 1H NMR
Hemolytic activity was measured as following: 2% rabbit red
blood cells in erythrocyte buffer (130 mM NaCl, 20 mM TriseHCl, pH
7.4) were incubated with serial diluted compounds. After incu-
bating for 60 min at 37 ꢃC, hemolysis was monitored by measuring
absorption at 540 nm with a microplate reader. Percentage of he-
molysis was then calculated as the routine method [18].
(400 MHz, CDCl3):
d 0.77, 0.84, 0.88, 0.89, 0.96, 0.99, 1.26 (s, 3H
each, CH3), 1.70 (s, 18H, 6 ꢁ CH3), 3.16e3.23 (m, 2H), 3.39e3.47 (m,
1H), 3.67e3.79 (m, 6H), 3.98e4.01 (m, 1H), 4.19 (dd, 1H, J ¼ 2.9 Hz),
4.41e4.56 (m, 3H), 5.53 (t, 1H, J ¼ 3.0 Hz), 7.22 (t, 1H, J ¼ 5.4 Hz),
7.73 (s, 1H), 7.89 (s, 2H). 13C NMR (100 MHz, CDCl3):
d 15.5, 15.7,17.3,
18.2, 23.2, 26.0, 27.1, 27.2, 27.3, 28.0, 29.6, 29.9 (6C), 32.3, 32.5 (2C),
33.6, 35.8, 36.9, 38.6, 38.7, 39.7, 40.1, 40.9, 42.1, 45.8, 46.9 (2C), 47.1,
49.9, 50.2, 55.3, 59.4 (2C), 73.0, 78.7, 121.1 (2C), 122.9, 124.1, 142.2,
143.0 (2C), 143.5, 178.6. ESIꢀHRMS (m/z) [M þ H]þ calcd for
Acknowledgments
This work was supported by the National Basic Research Pro-
gram of China (973 Program; grant no. 2010CB12300) and the
National Natural Science Foundation of China (grant no. 81101239,
20932001, 81373271, 20852001 and 81361168002).
C
49H80N11O3, 870.6440; found 870.6432.
4.3. HCV and VSV pseudovirus entry assay
Appendix A. Supplementary data
All compounds were tested using the HCVpp and VSVpp entry
assay as described previously. Briefly, pseudotyped viruses were
produced by cotransfecting 293T cells with plasmid encoding HCV
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.03.017.

268
F. Yu et al. / European Journal of Medicinal Chemistry 77 (2014) 258e268
[11] C.J. Baldick, M.J. Wichroski, A. Pendri, A.W. Walsh, J. Fang, C.E. Mazzucco,
References
K.A. Pokornowski, R.E. Rose, B.J. Eggers, M. Hsu, A novel small molecule
inhibitor of Hepatitis C virus entry, PLOS Pathogens 6 (2010) e1001086.
[12] F. Yu, Q. Wang, Z. Zhang, Y. Peng, Y. Qiu, Y. Shi, Y. Zheng, S. Xiao, H. Wang,
X. Huang, L. Zhu, K. Chen, C. Zhao, C. Zhang, M. Yu, D. Sun, L. Zhang, D. Zhou,
Development of oleanane-type triterpenes as a new class of HCV entry in-
hibitors, Journal of Medicinal Chemistry 56 (2013) 4300e4319.
[13] H. Wang, Q. Wang, S. Xiao, F. Yu, M. Ye, Y. Zheng, C. Zhao, D. Sun, L. Zhang,
D. Zhou, Elucidation of the pharmacophore of echinocystic acid, a new lead for
blocking HCV entry, European Journal of Medicinal Chemistry 64 (2013) 160e
168.
[14] P. Pileri, Y. Uematsu, S. Campagnoli, G. Galli, F. Falugi, R. Petracca, A.J. Weiner,
M. Houghton, D. Rosa, G. Grandi, Binding of hepatitis C virus to CD81, Science
282 (1998) 938e941.
[15] D. Soriano del Amo, W. Wang, H. Jiang, C. Besanceney, A.C. Yan, M. Levy, Y. Liu,
F.L. Marlow, P. Wu, Biocompatible copper (I) catalysts for in vivo imaging of
glycans, Journal of the American Chemical Society 132 (2010) 16893e16899.
[16] S. Liu, C.A. Nelson, L. Xiao, L. Lu, P.P. Seth, D.R. Davis, C.H. Hagedorn, Measuring
antiviral activity of benzimidazole molecules that alter IRES RNA structure
with an infectious hepatitis C virus chimera expressing Renilla luciferase,
Antiviral Research 89 (2011) 54e63.
[1] A.J. Rodger, S. Roberts, A. Lanigan, S. Bowden, T. Brown, N. Crofts, Assessment
of long-term outcomes of community-acquired hepatitis C infection in a
cohort with sera stored from 1971 to 1975, Hepatology 32 (2000) 582e587.
[2] T. Sy, M.M. Jamal, Epidemiology of hepatitis C virus (HCV) infection, Inter-
national Journal of Medical Sciences 3 (2006) 41e46.
[3] C. Sheridan, New Merck and Vertex drugs raise standard of care in hepatitis C,
Nature Biotechnology 29 (2011) 553e554.
[4] M.W. Fried, M.L. Shiffman, K.R. Reddy, C. Smith, G. Marinos, F.L. Gonçales,
D. Häussinger, M. Diago, G. Carosi, D. Dhumeaux, A. Craxi, A. Lin, J. Hoffman,
J. Yu, Peginterferon Alfa-2a plus ribavirin for chronic hepatitis C virus infec-
tion, The New England Journal of Medicine 347 (2002) 975e982.
[5] A.M. Di Bisceglie, J. McHutchison, C.M. Rice, New therapeutic strategies for
hepatitis C, Hepatology 35 (2002) 224e231.
[6] P. Halfon, S. Locarnini, Hepatitis C virus resistance to protease inhibitors,
Journal of Hepatology 55 (2011) 192e206.
[7] P. Halfon, C. Sarrazin, Future treatment of chronic hepatitis C with direct acting
antivirals: is resistance important? Liver International 32 (2012) 79e87.
[8] J. Yang, D. Zhou, F. Wong-Staal, Screening of small-molecule compounds as
inhibitors of HCV entry, Methods in Molecular Biology 510 (2009) 295e304.
[9] R. Schinazi, L. Bassit, C. Gavegnano, HCV drug discovery aimed at viral erad-
ication, Journal of Viral Hepatitis 17 (2010) 77e90.
[17] G. Francis, Z. Kerem, H.P.S. Makkar, K. Becker, The biological action of saponins
in animal systems: a review, British Journal of Nutrition 88 (2002) 587e605.
[18] G. Luo, R. Colonno, M. Krystal, Characterization of a hemagglutinin-specific
inhibitor of influenza A virus, Virology 226 (1996) 66e76.
[10] S. Xiao, Q. Wang, F. Yu, Y. Peng, M. Yang, M. Sollogoub, P. Sinaÿ, Y. Zhang,
L. Zhang, D. Zhou, Conjugation of cyclodextrin with fullerene as a new class of
HCV entry inhibitors, Bioorganic & Medicinal Chemistry 20 (2012) 5616e
5622.