Synthesis and biological evaluation of novel pentacyclic triterpene α-cyclodextrin conjugates as HCV entry inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.08.020

Hepatitis C virus (HCV) entry is a key target for the treatment of chronic HCV infection. In our continuing efforts to identify novel potential anti-HCV entry inhibitors, a series of water-soluble triazole-bridged α-cyclodextrin-pentacyclic triterpene conjugates were easily synthesized with moderate to good yields. These novel compounds were fully identified and characterized by 1D and 2D NMR spectroscopy and ESI-HRMS. The anti-HCV entry activities were determined based on HCVpp/VSVGpp entry assays. The best results were found for compounds 15 and 18, which displayed the most promising anti-HCV entry activities with average IC₅₀values of 1.18?μM and 0.25?μM, respectively. In addition, the in?vitro cytotoxicity activity of the two compounds against MDCK cells showed no toxicity at 100?μM. Five different binding assays were set up to identify the action mechanism. The results showed that the compounds exert their inhibitory activity at the post-binding step and subsequently prevent virus entry.

Full text of DOI:10.1016/j.ejmech.2016.08.020

European Journal of Medicinal Chemistry 124 (2016) 1e9
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis and biological evaluation of novel pentacyclic triterpene
-cyclodextrin conjugates as HCV entry inhibitors
a
,
,
Sulong Xiao a *, Qi Wang a c, Longlong Si a, Xiaoshu Zhou a, Yongmin Zhang b,
Lihe Zhang a, Demin Zhou a
a State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, 100191, China
,
**
b
ꢀ
ꢀ
Institut Parisien de Chimie Moleculaire, CNRS UMR 8232, Universite Pierre & Marie Curie-Paris 6, 4 Place Jussieu, 75005, Paris, France
c Department of Molecular and Cellular Pharmacology, School of Pharmaceutical Sciences, Peking University, Beijing, 100191, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Hepatitis C virus (HCV) entry is a key target for the treatment of chronic HCV infection. In our continuing
efforts to identify novel potential anti-HCV entry inhibitors, a series of water-soluble triazole-bridged a-
cyclodextrin-pentacyclic triterpene conjugates were easily synthesized with moderate to good yields.
These novel compounds were fully identified and characterized by 1D and 2D NMR spectroscopy and
ESI-HRMS. The anti-HCV entry activities were determined based on HCVpp/VSVGpp entry assays. The
best results were found for compounds 15 and 18, which displayed the most promising anti-HCV entry
Received 1 July 2016
Received in revised form
9 August 2016
Accepted 10 August 2016
Available online 12 August 2016
activities with average IC50 values of 1.18
toxicity activity of the two compounds against MDCK cells showed no toxicity at 100
binding assays were set up to identify the action mechanism. The results showed that the compounds
exert their inhibitory activity at the post-binding step and subsequently prevent virus entry.
© 2016 Elsevier Masson SAS. All rights reserved.
m
M and 0.25
m
M, respectively. In addition, the in vitro cyto-
M. Five different
Keywords:
m
a-Cyclodextrin
HCV entry inhibitor
Pentacyclic triterpene
Click chemistry
1. Introduction
sofosbuvir, has further improved treatment due to its pangenotypic
effect on HCV, better pharmacokinetics, and improved resistance
The Hepatitis C virus (HCV) is a small, enveloped, positive-sense
single-stranded RNA virus of the Flaviviridae family [1]. There are
currently ~185 million individuals infected worldwide, many of
whom suffer from serious sequelae of persistent HCV infection [2],
such as chronic hepatitis, cirrhosis, and liver carcinomas [3]. His-
torically, the standard of care (SOC) for treating chronic HCV
infection has been a combination of pegylated interferon (IFN) and
ribavirin (RBV) with sustained viral response (SVR) rates of only
40e50% among genotype 1-infected patients [4]. Recently, HCV
therapy has undergone a tremendous revolution with the advent of
direct-acting antivirals (DAAs), which directly target proteins
involved in replication of the virus. In 2011, the first generation
DAAs, NS3/4A protease inhibitors boceprevir and telaprevir, have
increased rates of SVR for the most difficult to treat genotype-1 HCV
patients [5]. In 2013, approval of the second generation DAAs, NS3/
4A protease inhibitors simeprevir and NS5B polymerase inhibitor
profiles [6]. However, the substantial cost of these DAAs present a
substantial barrier to their broad distribution, and viral resistance
to these drugs is expected to arise quickly [7e9]. A combination of
drugs targeting different stages of the HCV life cycle would be
required to eradicate HCV infections globally. Recently, polyphenol
dimers [10], diflunisal hydrazide-hydrazones [11], indole [12] and
benzofuran [13] derivatives have shown good anti-HCV activity
with EC50 value at mM or sub-mM level. Viral entry is the first step of
the virus-host cell interactions leading to productive infection, and
thus it represents an emerging opportunity for the prevention of
HCV infection [14,15]. Development of HCV entry inhibitors could
be used in tandem with other inhibitors of viral replication, leading
to a multifaceted approach to controlling HCV infection more
effectively.
We initiated our search for new antivirals by exploring natural
products, which have both great structural diversity and existing
bioactivity. Pentacyclic triterpenes are the most represented group
of phytochemicals, derived biosynthetically from the cyclization of
squalene [16]. It has been estimated that more than 20,000
recognized triterpenoids exist in nature [17]. It is generally believed
that they might function as defensive agents against herbivores or
* Corresponding author.
** Corresponding author.
E-mail addresses: slxiao@bjmu.edu.cn (S. Xiao), deminzhou@bjmu.edu.cn
(D. Zhou).
http://dx.doi.org/10.1016/j.ejmech.2016.08.020
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

2
S. Xiao et al. / European Journal of Medicinal Chemistry 124 (2016) 1e9
pathogens, or as allelopathic agents [18,19]. Many interesting bio-
logical properties have been described for pentacyclic triterpenes,
such as hepatoprotective [20], anti-inflammatory [21], anti-tumor
[17,22], and immunomodulatory activities. Quite recently, penta-
cyclic triterpenes have been found to act as effective antiviral
agents [23]. Bevirimat (PA-457), a lupane-type triterpene, has been
undergoing phase two trials as an anti-HIV entry drug candidate
[24]. Moronic acid (MA) and ursolic acid (UA), two oleanane-type
triterpenes, also show anti-HIV activity in vitro [25,26]. In our
previous studies, we found that oleanolic acid (OA) and echino-
cystic acid (EA), two naturally occurring oleanane-type triterpenes,
display substantial activity in inhibiting HCV entry with an IC50 of
O-6 according to the procedure described by Quan et al. [43], fol-
lowed by nucleophilic substitution with NaN3 in DMF to provide
the intermediate 6A-azide-6A-deoxy-
a-cyclodextrin 3 in quantita-
tive yields and was used without further purification in the next
step. Acetylation of the remaining 17 hydroxyl groups of crude 3
with acetic anhydride in the presence of DMAP and pyridine
afforded 6A-azide-6A-deoxy-per-O-acetylated
a-cyclodextrin 4.
Compounds 11e13, prepared according to our previously reported
method [38], underwent a ‘click chemistry’ reaction with 4 in THF/
H2O (1:1) in the presence of a catalytic amount of copper sulfate
and sodium ascorbate to yield 14e16 in 70e82% yields. The acetyl
groups were removed upon treatment with CH3ONa/CH3OH to
afford 17e19 in quantitative yields. Similarly, the CuAAC reaction in
10 and 1.4 mM, respectively [27,28]. The putative mechanism un-
derlying the inhibition of HCV entry by EA is its tight binding to an
HCV envelope protein, E2 (KD ¼ 2.4 ꢀ 10ꢁ5 M). Recently Richardson
et al. found that naturally occurring terpenoid saikosaponin b2 also
efficiently inhibits HCV entry [29].
Despite their promising bioactivity, these pentacyclic tri-
terpenes are bulky, non-polar, and poorly soluble in water. For
the synthesis of
commercially available betulin (Scheme 2).
The structural determination of all -cyclodextrin-triterpene
a-cyclodextrin-BA 26 was carried out from
a
conjugates was achieved using one and two dimensional NMR
spectroscopy and ESI-HRMS. Fig. 2 represents the HSQC spectrum
of compound 15. The characteristic signals have been assigned
unambiguously with the aid of 1He1H COSY and 1He13C HSQC
spectroscopy. In the aromatic region, the signal at 7.96 ppm inte-
grating to 1H, was assigned to triazole-CH according to the 1He1H
and 1He13C correlation spectra. An additional signal was observed
for the amide proton at 7.28 ppm (overlapping with CDCl3), indi-
cated that it was indeed connected by a triazole moiety. The ESI-
HRMS of compound 15 plus one hydrogen ion clearly shows a m/
z of 2221.8965.
example, the solubility of BA and OA in water is only about 0.02
mg/
mL and 0.05 g/mL, respectively [30,31]. The poor aqueous solu-
m
bility is a major drawback for various applications, in particular, the
development of drugs. Hence, the synthesis of more water-soluble
triterpene derivatives is needed. Some water-soluble derivatives
have been synthesized by introducing polar sugar moieties at C-3
and/or C-28 [32]. Other strategies involve cyclodextrin inclusion
complexes [33e35]. Alternatively, direct covalent linkage with
cyclodextrin has been suggested, which has been widely used in
other water insoluble bioactive molecules, such as 5-FU [36] and
folic acid [37]. In our recently studies, a series of water-soluble
Fig. 3 presents the characteristic portions (80e180 ppm) of the
13C NMR spectra (in CDCl3) of conjugates 14, 15, 16, and 25. The
signals have been fully assigned with the aid of 1He1H COSY and
1He13C HSQC spectra.
triazole-bridged
have been synthesized via click chemistry [38]. Compared with
cyclodextrin, -cyclodextrin has greater aqueous solubility at room
b-cyclodextrin-pentacyclic triterpene conjugates
b
-
a
2.2. Calculated ALogP
temperature (14.5 vs 1.85 g/100 mL) [39]. The biological activity of
such conjugates is expected to increase with increasing solubility.
As a continuation of our ongoing research in the development of
novel anti-HCV entry inhibitors [27,28,38,40e42], we describe
herein the synthesis, anti-HCV entry activity, and mechanistic
Lipophilicity (AlogP) has a major impact on a number of drug-
like features, and previous analyses have found that the
decreased hit rate observed at high AlogP might be due to the
limited solubility of the compounds [44]. Lipophilicity governs the
interaction between the molecule and the intestinal membrane;
the drugs must possess sufficient hydrophobicity for passive
transport across the intestinal epithelium. In our study, the calcu-
lated AlogP values based on Ghose and Crippen's method [45] were
determined using Pipeline Pilot software, Vers. 7.5 (Accelrys Cor-
poration, San Diego, USA [46,47]). All conjugates 14e19 and 25e26
showed increased hydrophilicity to their parent compound
studies of new water-soluble a-cyclodextrin-pentacyclic triterpene
conjugates. The pentacyclic triterpenes used include oleanolic acid
(OA), echinocystic acid (EA), urosolic acid (UA), and betulinic acid
(BA) (Fig. 1).
2. Results and discussion
(Table 1). In addition, as compared with
conjugates [38], increased ALogP values were observed for
cyclodextrin-triterpene conjugates. Due to the 16-hydroxyl group,
18 showed the lowest ALogP values among the a-cyclodextrin-tri-
terpene conjugates.
b-cyclodextrin-triterpene
2.1. Chemistry
a
-
The
from -cyclodextrin 1 in five steps (Scheme 1). Three types of hy-
droxyl groups are present in -cyclodextrins, and the ones at O-6
a-cyclodextrin-triterpene conjugates were synthesized
a
a
are the most basic. Thus, it would undergo substitution using a
weak base and a moderate electrophile. Herein, this strategy was
2.3. In vitro anti-HCV entry activity
used for the preparation of mono-functionalized
a-cyclodextrin
derivatives. First, -cyclodextrin 1 was selective monotosylated at
a
Cyclodextrins and their derivatives have been widely utilized for
Fig. 1. The chemical structures and anti-HCV entry activities of pentacyclic triterpenes [27].

S. Xiao et al. / European Journal of Medicinal Chemistry 124 (2016) 1e9
3
Scheme 1. Reagents and conditions: (a)TsCl, dry pyridine; (b) DMF, NaN3, 80 ꢂC, 18 h; (c) pyridine, DMAP, Ac2O; (d) TBTU, DIPEA, THF; (e) propargylamine, K2CO3, DMF, 1 h; (f) THF-
H2O (1:1, V/V), CuSO4, Na-L-ascorbate; (g) CH3ONa/CH3OH, rt, 8e10 h.
Scheme 2. Reagents and conditions: (a) H2CrO4, acetone, 0 ꢂC to rt, 18 h; (b) NaBH4, THF, rt, 2.5 h; (c) TBTU, DIPEA, THF; (d) Propargylamine, K2CO3, DMF, 1 h; (e) THF-H2O (1:1, V/V),
CuSO4, Na-L-ascorbate, 4; (f) CH3ONa/CH3OH, rt, 8e10 h.
improving drug solubility, stability, and absorption, due to their
remarkable ability to form inclusion complexes. Our previous
studies suggested that EA displayed substantial activity in inhibit-
protein) pseudovirions into Huh7 cells was tested. The VSVG virus
has a broad host range and can infect multiple cell types. We found
that, with the exception of 25 and 26, the other a-cyclodextrin-
ing HCV entry [27], and the introduction of
17 carboxylic acid may further enhance its potency [38]. To further
investigate these molecules, a series of -cyclodextrin-triterpene
b
-cyclodextrin at the C-
triterpene derivatives 14e19 did not block VSVG viral entry
(Fig. 4B), indicating BA derivatives 25 and 26 were nonselective
antiviral agents against HCV.
a
conjugates and their acetylated derivatives were synthesized and
their anti-HCV entry activities were evaluated.
As a preliminary screen for anti-HCV entry activity, all conju-
gates were tested for their capacities to inhibit entry of HCVpp
After the preliminary screen at two concentrations (1 and 5 mM),
the anti-HCV entry activities of 15 and 18 were further character-
ized in a dose-dependent assay. As shown in Fig. 5, both com-
pounds exhibited significant inhibition against HCVpp entry with
(Huh-7 cells) at two concentrations (1 and 5
m
M) (Fig. 4A). DMSO
IC50 values of 1.18
their parent compound EA (IC50 ¼ 1.4
-cyclodextrin or per-O-acetylated
potency by 1.2e5.6-fold.
m
M and 0.25
m
M, respectively. Compared with
M [27]), the introduction of
-cyclodextrin increased the
was used as the negative control. CD81 was the positive control to
indicate maximum blocking of HCVpp entry. In this assay, we found
that compounds 15 and 18 exhibited significant HCVpp entry in-
m
a
a
hibition with more than 80% and 95% of virus entry blocked at 5
respectively. Compounds 25 and 26 showed moderate inhibition
(~60%) of HCVpp entry, whereas compounds 16 and 19 showed no
m
M,
Furthermore, we investigated the toxicity of compounds 15 and
18 against the MDCK cell (a Madin-Daby canine kidney cell) line
using the alamarBlue assay. As shown in Table 2, no significant
inhibition of HCVpp entry at concentrations of 5
the specificity of -cyclodextrin-triterpene conjugates toward HCV,
their ability to block entry of VSVG/HIV (vesicular stomtatis virus G
m
M. To determine
toxicity was observed at concentrations of 100 mM for both com-
pounds. Compared with their parent compounds, both compounds
showed high selectivity indexes (SI > 85 and 400, respectively),
a

4
S. Xiao et al. / European Journal of Medicinal Chemistry 124 (2016) 1e9
Fig. 2. A portion of the 400 MHz HSQC (CDCl3, 25 ꢂC) spectrum of 15, with the 1D 1H and 13C NMR spectra along the side and the top, respectively. The frame corresponds to
1
correlation JCH CD-C1 and CD-H1.
Fig. 3. Characteristic portions of the 400-MHz 13C NMR spectra of 14, 15, 16, and 25 recorded in CDCl3 at 25 ꢂC.
which might have been due to the increased solubility of the tri-
terpene derivatives upon the introduction of -cyclodextrin.
treatment assay, pre-binding assay, post-binding (pre-entry)
assay, and post-entry assay were performed as previously reported
[48,49].
a
2.4. Mechanism investigation of HCV entry assay
In all five conditions (Fig. 6), 0.5% DMSO (final concentration)
was used for normalization. The CD81 antibody was utilized as a
positive control due to its blocking of the HCV virus entry by
binding to CD81 receptor. With the exception of the post-entry
assay, high activity of the CD81 antibody was observed in the
In order to understand at which stage of infection the a-cyclo-
dextrin-triterpene conjugates exert their inhibitory activity, five
different assays, including the standard antiviral assay, pre-

S. Xiao et al. / European Journal of Medicinal Chemistry 124 (2016) 1e9
5
Table 1
Calculated ALogP of pentacyclic triterpene and their a
-cyclodextrin conjugatesa.
Compound
ALog P
Compound
ALog P
Compound
ALog P
OA
EA
UA
BA
6.447
5.345
6.492
6.546
14
15
16
25
2.327
1.226
2.373
2.427
17
18
19
26
ꢁ3.553
ꢁ5.673
ꢁ3.508
ꢁ4.472
a
ALogP (The Ghose and Crippen octanol-water partition coefficient at 25 ꢂC) were
calculated using Pipeline Pilot software, Vers. 7.5 (Accelrys Corporation, San Diego,
USA).
Fig. 5. Inhibition curves of 15 and 18. Concentration of 15 and 18 were 5 nM, 50 nM,
0.5 mM, 5 mM, 50 mM, and 500 mM. Each concentration was tested in triplicate, and the
results are expressed as the mean and standard deviation.
Table 2
In vitro anti-HCV entry activity and cytotoxicity of compounds 15 and 18.
Compound
IC50
(m
M)a
CC50
(
m
M)b
SIc
15
18
EA
1.18 0.13
0.25 0.11
1.40 0.17 [27]
>100
>100
15.41 0.54
>85
>400
11.0
a
Concentration inhibiting viral replication by 50%, The values are means of at
least three independent determinations; the corresponding standard deviations are
noted.
b
50% cytotoxicity concentration.
Selectivity index, defined by CC50/IC50
c
.
3. Conclusion
In this study, we reported the synthesis, anti-HCV entry activity,
cytotoxicity, and mechanistic studies of novel -cyclodextrin-pen-
a
tacyclic triterpene conjugates. All new conjugates were unambig-
uous characterized by 1D and 2D NMR and ESI-HRMS. By
conjugating with water soluble
a-cyclodextrin, these derivatives
showed decreased hydrophobicity (ALog P) and reasonable anti-
HCV entry activities. Compounds 15 and 18 displayed the most
promising anti-HCV entry activities with average IC50 values of
1.18
mM and 0.25
mM, respectively. No significant cytotoxicity of
compounds 15 and 18 were observed at concentrations of 100
mM.
Moreover, entry assays indicated that they exert their inhibitory
activity at the post-binding step and subsequently prevent virus
entry. Our results provide a rationale for further exploring the anti-
HCV entry activity of pentacyclic triterpenes, which is widely found
in plants, as potent HCV inhibitors.
Fig. 4. Anti-HCV entry activity of
a-cyclodextrin-pentacyclic triterpene conjugates
based on HCVpp/VSVGpp entry assays.
4. Experimental
4.1. Chemistry
assays. IM2865 was a non-relevant compound used as the negative
control [14], and no significant inhibitory activity was observed in
all conditions. Compared with the standard antiviral assay, only
very weak activity (~10e30%) of 15 and 18 was observed in the pre-
treatment, pre-binding, and post-entry assays. A strong decrease in
HCV infection (>90%) was noted in the post-binding assay, indi-
cating that 15 and 18 exert their inhibitory activity at the post-
binding (pre-entry) step and interfere with subsequent steps
following virus attachment to target cells, presumably viral
envelop-cell membrane fusion.
4.1.1. General information
The syntheses of 2e4, 11e13, and 21e24 were performed as
previously reported (Supporting information) [27,38,41]. High
resolution mass spectra (ESI-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 were referenced to
the internal standard TMS (dH ¼ 0.00) or the solvent signal (
d
¼ 3.31 for the central line of MeOD). 13C NMR chemical shifts were
H

6
S. Xiao et al. / European Journal of Medicinal Chemistry 124 (2016) 1e9
Fig. 6. Mechanistic studies of compounds 15 and 18-mediated blocking of HCVpp entry by five different assays. CD81 antibody, an entry inhibitor targeting host cell membrane, was
utilized as the positive control and IM2865, a non-relevant compound [14], were used as the negative control. 0.5% DMSO (final concentration) was used for normalization in each
condition.
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). Reactions were
monitored by thin-layer chromatography (TLC) on a pre-coated
silica gel 60 F254 plate (layer thickness 0.2 mm; E. Merck, Darm-
stadt, Germany) and detected by detected by staining with a yellow
solution containing Ce(NH4)2(NO3)6 (0.5 g) and (NH4)6Mo7O·244H2O
(24.0 g) in 6% H2SO4 (500 mL) followed by heating. Flash column
chromatography was performed on silica gel 60 (200e300 mesh,
Qingdao Haiyang Chemical Co. Ltd). The calculated Alog P and
water solubility values were determined using Pipeline Pilot soft-
ware, Vers. 7.5 (Accelrys Corporation, San Diego, CA, USA).
68.91, 68.82, 63.21, 63.08, 62.98, 62.74, 62.68, 62.39, 60.63, 55.10,
47.59, 46.55, 46.29, 42.09, 41.89, 39.40, 38.71, 38.44, 36.89, 35.55,
34.11, 32.95, 32.52, 32.42, 30.65, 28.05, 27.38, 27.17, 25.80, 23.83,
23.47, 21.00, 20.93, 20.84, 20.76, 20.73, 20.62, 20.60, 20.23, 20.18,
19.06, 18.22, 16.31, 15.49, 15.29; ESI-HRMS (m/z) Calcd for
C
103H145N4O48 [MþH]þ: 2205.9023. Found 2205.9021.
4.1.5. Synthesis of [1-(6A-deoxy-per-O-acetylated
a-cyclodextrin-6-
yl)-1H-1,2,3-triazol-4-yl]methyl 3b,16a-dihydroxy-olean-12-en-28-
amide (15)
Prepared from 4 and 12 according to general procedure A, the
residue was purified by flash chromatography (eluent: DCM/
CH3OH ¼ 35:1) to afford 15 as a white solid in 60% yield. Rf ¼ 0.29
4.1.2. General procedure A for the click reaction
(eluent: DCM/CH3OH ¼ 15:1); 1H NMR (400 MHz, CDCl3):
d 7.96 (s,
To a solution of alkyne (0.10 mmol) and azide (0.067 mmol) in
1:1 THF:H2O (6 mL) was added CuSO4 (10.5 mg, 0.067 mmol) and
sodium ascorbate (26.6 mg, 0.13 mmol). The resulting solution was
stirred vigorously for 12 h at room temperature. The reaction
mixture was extracted with CH2Cl2 (10 mL ꢀ 3). The combined
organics was dried over Na2SO4, filtered, and concentrated. The
residue was purified by column chromatography.
1H), 7.28 (br s, 1H, overlap with CDCl3), 5.60 (br s, 1H), 5.40e5.92
(m, 6H), 5.30 (br s, 1H), 5.28 (t, 1H, J ¼ 10.7 Hz), 5.12 (d, 1H,
J ¼ 2.9 Hz), 5.01 (br s, 2H), 4.98 (d, 1H, J ¼ 2.7 Hz), 4.85 (br s, 1H),
4.74e4.82 (m, 4H), 4.71 (dd, 1H, J ¼ 2.8, 10.3 Hz), 4.11e4.61 (m,
24H), 3.72e3.88 (m, 5H), 2.21, 2.20, 2.15, 2.13, 2.12, 2.09, 2.07, 2.05,
2.04, 2.03, 1.95, 1.75 (17 ꢀ COCH3), 1.40, 0.98, 0.91, 0.90, 0.87, 0.76,
0.70 (s, each 3H), 0.75 (m, 1H), 0.99e2.25 (m, other aliphatic ring
protons); 13C NMR (100 MHz, CDCl3):
d 177.39, 170.92, 170.86,
4.1.3. General procedure B for the deacetylation reaction
170.66, 170.57, 170.38, 170.31, 170.28, 169.82, 169.77, 169.59, 169.48,
169.35, 169.27, 143.18, 125.09, 123.43, 97.44, 97.41, 96.96, 96.79,
96.66, 78.87, 77.79, 77.68, 77.53, 76.53, 76.43, 75.55, 71.99, 71.71,
71.18, 71.01, 70.95, 70.75, 70.57, 70.42, 70.37, 70.12, 70.06, 69.89,
69.51, 68.92, 68.78, 63.21, 63.03, 62.62, 62.25, 60.67, 55.24, 48.89,
46.85, 46.80, 41.65, 41.29, 39.60, 38.72, 38.52, 36.91, 35.63, 35.34,
32.64, 30.17, 29.75, 28.02, 27.21, 27.01, 24.92, 23.37, 20.97, 20.88,
20.83, 20.81, 20.77, 20.73, 20.59, 20.20, 18.15, 16.54, 15.50; ESI-
The per-O-acetylated
a-cyclodextrin-triterpene conjugate was
dissolved in dry methanol (~5 mL per 100 mg of compound) and a
solution of sodium methoxide (30% in methanol, 0.1 eq per mol of
acetate) was added. The solution was stirred at room temperature
for 4e6 h. After completion (TLC) the reaction mixture was
neutralized with Amberlite IR-120 (Hþ) ion exchange resin, filtered,
and concentrated. The crude product was purified by RP column
chromatography (eluted by CH3OH).
HRMS Calcd for
2221.8965.
C
103H145N4O49 [MþH]þ: 2221.8972. Found
4.1.4. Synthesis of [1-(6A-deoxy-per-O-acetylated
a-cyclodextrin-6-
yl)-1H-1,2,3-triazol-4-yl]methyl 3
(14)
b-hydroxy-olean-12-en-28-amide
4.1.6. Synthesis of [1-(6A-deoxy-per-O-acetylated
a-cyclodextrin-6-
Prepared from 4 and 11 according to general procedure A, the
residue was purified by flash chromatography (eluent: DCM/
CH3OH ¼ 30:1) to afford 14 as a white solid in 68% yield. Rf ¼ 0.37
yl)-1H-1,2,3-triazol-4-yl]methyl 3b-dihydroxy-urs-12-en-28-amide
(16)
Prepared from 4 and 13 according to general procedure A, the
residue was purified by flash chromatography (eluent: DCM/
CH3OH ¼ 40:1) to afford 16 as a white solid in 66% yield. Rf ¼ 0.30
(DCM:CH3OH ¼ 20:1); 1H NMR (400 MHz, CDCl3):
d 7.96 (s, 1H),
7.09 (br s, 1H), 5.38e5.61 (m, 7H), 5.29 (br s, 1H), 5.19e5.28 (m, 1H),
5.14 (br s, 1H), 5.03 (d, 2H, J ¼ 3.0 Hz), 5.00e5.03 (m, 3H), 4.14e4.86
(m, 29H), 3.72e3.87 (m, 5H), 2.22 (2 ꢀ s), 2.14, 2.13, 2.09, 2.08, 2.07,
2.06, 2.04, 2.01, 1.99, 1.96, 1.78, 1.74 (17 ꢀ COCH3), 1.18, 0.98, 0.94,
0.90, 0.89, 0.87, 0.77, 0.70 (s, each 3H, CH3); 13C NMR (100 MHz,
(DCM:CH3OH ¼ 20:1); 1H NMR (400 MHz, CDCl3):
d 7.94 (s, 1H),
7.30 (br s,1H), 5.36e5.60 (m, 6H), 5.29 (d,1H, J ¼ 3.5 Hz), 5.25 (t,1H,
J ¼ 9.8 Hz), 5.12 (d, 1H, J ¼ 3.5 Hz), 5.03 (d, 2H, J ¼ 3.0 Hz), 5.00 (d,
1H, J ¼ 3.4 Hz), 4.86 (d, 1H, J ¼ 3.1 Hz), 4.77e4.84 (m, 3H), 4.72 (dd,
1H, J ¼ 3.3, 10.3 Hz), 4.11e4.68 (m, 23H), 3.72e3.87 (m, 5H), 2.22,
2.19, 2.17, 2.14 (2 ꢀ s), 2.13, 2.10, 2.09, 2.08, 2.07, 2.06, 2.05 (2 ꢀ s),
2.00, 1.96, 1.78, 1.73 (each s, 3H, 17 ꢀ COCH3), 1.12 (s, 3H, CH3), 0.99
(s, 3H, CH3), 0.97 (d, 3H, J ¼ 6.4 Hz, CH3), 0.88 (s, 3H, CH3), 0.87 (d,
3H, J ¼ 6.5 Hz, CH3), 0.77 (s, 3H, CH3), 0.74 (s, 3H, CH3), 0.73 (d, 1H,
CDCl3):
d 178.18, 170.88, 170.78, 170.65, 170.63, 170.55, 170.43,
170.32, 170.30, 170.18, 169.62, 169.52, 169.50, 169.45, 169.36, 169.21,
143.89, 142.64, 125.28, 123.52, 97.20, 97.12, 96.80, 96.41, 96.33,
78.89, 77.74, 77.52, 77.40, 77.34, 76.53, 76.01, 72.02, 71.62, 71.24,
70.89, 70.76, 70.55, 70.54, 70.26, 70.23, 69.91, 69.85, 69.75, 69.59,

S. Xiao et al. / European Journal of Medicinal Chemistry 124 (2016) 1e9
7
J ¼ 10.3 Hz); 13C NMR (100 MHz, CDCl3):
d
178.01, 170.83, 170.65,
1.13 (s, 3H), 0.97e0.98 (2 ꢀ s, 9H), 0.92 (d, 3H, J ¼ 6.2 Hz), 0.79 (s,
170.58, 170.40, 170.28, 170.27, 170.25, 170.13, 169.57, 169.46, 169.39,
169.30, 169.28, 169.10, 142.89, 138.77, 126.36, 125.17, 97.21, 97.19,
96.79, 96.77, 96.34, 96.29, 78.81, 77.65, 77.40, 77.20, 76.14, 71.91,
71.57, 71.03, 70.86, 70.81, 70.74, 70.54, 70.45, 70.26, 70.20, 69.82,
69.61, 69.59, 68.99, 68.72, 63.18, 63.01, 62.86, 62.69, 62.60, 62.35,
60.64, 55.05, 53.61, 47.63, 47.51, 42.23, 39.57, 39.49, 39.03, 38.65,
38.54, 37.06, 36.81, 35.46, 32.67, 30.75, 28.03, 27.82, 27.13, 24.84,
23.31, 23.28, 21.10, 20.96, 20.79, 20.77, 20.70, 20.67, 20.57, 20.54,
20.30, 20.20, 18.14, 17.12, 16.36, 15.50, 15.35; HRMS Calcd for
6H), 0.75 (d, 1H, J ¼ 11.1 Hz); 13C NMR (100 MHz, CDCl3):
d 180.38,
145.28, 139.93, 127.36, 126.54, 103.73, 103.66, 103.62, 103.57, 103.54,
102.51, 83.31, 83.27, 83.17, 83.12, 82.93, 79.79, 79.70, 75.27, 75.23,
74.96, 74.51, 74.07, 73.99, 73.86, 73.74, 73.66, 73.61, 73.34, 72.54,
67.39, 62.12, 61.92, 61.83, 61.80, 56.69, 54.10, 48.98, 43.24, 40.85,
40.81, 40.15, 39.96, 39.82, 38.27, 38.08, 36.32, 36.20, 34.06, 31.87,
28.97, 28.78, 27.89, 25.31, 24.40, 24.15, 21.65, 19.44, 17.75, 17.70,
16.41, 16.06; ESI-HRMS (m/z) Calcd for C69H111N4O31 [MþH]þ:
1491.7227. Found 1491.7215.
C
103H145N4O48 [MþH]þ: 2205.9023. Found 2205.9077.
4.1.10. Synthesis of N-[1-(6A-deoxy-per-O-acetylated
a-
cyclodextrin-6-yl)-1H-1,2,3-triazol-4-yl]methyl 3b-hydroxy-lup-
20(29)-en-28-amide (25)
4.1.7. Synthesis of [1-(6A-deoxy-
triazol-4-yl]methyl 3 -hydroxy-olean-12-en-28-amide (17)
a-cyclodextrin-6-yl)-1H-1,2,3-
b
Prepared from 14 according to general procedure B, the residue
was purified by RP flash chromatography (eluent: methanol) to
afford 17 as a white solid in 90% yield. 1H NMR (400 MHz, CD3OD):
Prepared from 4 and 24 according to general procedure A, the
residue was purified by flash chromatography (eluent: DCM/
EtOAc ¼ 1:1) to afford 25 as a white solid in 71% yield. Rf ¼ 0.30
(DCM:CH3OH ¼ 20:1); Rf ¼ 0.30 (DCM:CH3OH ¼ 20:1); 1H NMR
d
8.02 (s, 1H), 5.37 (s, 1H), 4.93e4.95 (m, 4H), 4.85 (m, 1H), 4.61 (s,
1H), 4.40 (m, 2H), 4.35 (t, J ¼ 9.1 Hz), 3.75e4.09 (m, 24H), 3.64 (d,
J ¼ 9.2 Hz), 3.50e3.60 (m, 7H), 3.43 (t, 1H, J ¼ 9.4 Hz) 3.12e3.18 (m,
2H), 2.80 (d, J ¼ 11.0 Hz), 2.08 (t, 1H, J ¼ 13.7 Hz), 1.92 (m, 2H), 1.79
(t, 1H, J ¼ 13.4 Hz), 1.17 (s, 3H), 0.97 (2 ꢀ s, 6H), 0.94 (s, 3H), 0.91 (s,
3H), 0.78 (s, 3H), 0.75 (m, 1H), 0.73 (s, 3H); 13C NMR (100 MHz,
(400 MHz, CDCl3):
d
7.97 (s, 1H), 6.54 (t, 1H, J ¼ 4.8 Hz), 5.37e5.59
(m, 6H), 5.30 (d, 1H, J ¼ 3.6 Hz), 5.22e5.27 (m, 1H), 5.12 (d, 1H,
J ¼ 3.5 Hz), 5.02e5.03 (m, 2H), 5.00 (d, 1H, J ¼ 3.5 Hz), 4.87 (d, 1H,
J ¼ 2.9 Hz), 4.77e4.84 (m, 3H), 4.74 (m, 1H), 4.72 (dd, 1H, J ¼ 10.4,
3.3 Hz), 4.14e4.59 (m, 23H), 3.81e3.88 (m, 3H), 3.79 (t, 1H,
J ¼ 4.0 Hz), 3.74 (t, 1H, J ¼ 6.4 Hz), 3.12e3.19 (m, 2H), 2.58 (td, 1H,
J ¼ 12.4, 2.9 Hz), 2.22, 2.19, 2.17, 2.15, 2.14, 2.13, 2.10, 2.09, 2.07, 2.06,
2.05, 2.05, 2.01, 1.96, 1.79, 1.78 (each s, 17 ꢀ COCH3), 1.69, 0.98, 0.95,
0.92, 0.81, 0.75 (each s, 6 ꢀ CH3), 0.89e2.14 (m, other aliphatic ring
protons), 0.67 (d, 1H, J ¼ 11.0 Hz); 13C NMR (100 MHz, CDCl3):
CD3OD):
d 180.71, 145.21, 127.37, 127.35, 124.27, 103.78, 103.66,
103.62, 103.60, 103.55, 102.53, 83.37, 83.21, 83.18, 82.98, 79.74,
75.31, 75.26, 74.98, 74.57, 74.10, 74.01, 73.90, 73.77, 73.70, 73.66,
73.45, 72.53, 67.96, 62.05, 61.95, 61.91, 61.84, 56.73, 49.05, 47.66,
42.89, 42.64, 40.67, 39.84, 38.15, 35.79, 35.07, 33.95, 33.74, 33.56,
31.61, 30.73, 28.74, 28.53, 27.88, 26.50, 24.56, 24.15, 19.47, 17.70,
16.33, 15.93; ESI-HRMS (m/z) Calcd for C69H111N4O31 [MþH]þ:
1491.7227. Found 1491.7223.
d
176.14, 171.02, 170.84, 170.70, 170.63, 170.47, 170.41, 170.37, 169.84,
169.72,169.58, 169.55, 169.33,169.25, 150.97, 143.52, 125.64,109.32,
97.45, 97.33, 96.83, 96.67, 96.61, 96.47, 78.97, 77.73, 77.51, 77.38,
76.53, 76.30, 71.99, 71.62, 71.07, 70.90, 70.82, 70.52, 70.25, 69.97,
69.91, 69.76, 69.68, 69.01, 68.75, 63.25, 63.09, 62.92, 62.65, 62.28,
60.73, 55.64, 55.41, 50.67, 50.19, 46.50, 42.43, 40.69, 38.84, 38.68,
38.29, 37.51, 37.18, 35.04, 34.40, 33.48, 30.82, 29.42, 27.99, 27.41,
25.62, 21.05, 20.92, 20.89, 20.84, 20.80, 20.78, 20.64, 20.49, 20.34,
19.49, 19.14, 18.23, 16.12, 16.09, 15.32, 14.61; ESI-HRMS (m/z) Calcd
for C103H145N4O48 [MþH]þ: 2205.9023. Found 2205.9061.
4.1.8. Synthesis of [1-(6A-deoxy-
triazol-4-yl]methyl 3 ,16 -dihydroxy-olean-12-en-28-amide (18)
a-cyclodextrin-6-yl)-1H-1,2,3-
b
a
Prepared from 15 according to general procedure B, the residue
was purified by RP flash chromatography (eluent: methanol) to
afford 18 as a white solid in 88% yield. 1H NMR (400 MHz, CD3OD):
d
7.97 (s, 1H), 5.49 (br t, 1H, H12), 5.09 (d, 1H, J ¼ 3.1 Hz), 4.95e4.97
(m, 3H), 4.93 (d, 1H, J ¼ 3.3 Hz), 4.81 (t, 1H, J ¼ 10.2 Hz, overlap with
H2O), 4.60 (d, 1H, J ¼ 3.4 Hz), 4.40 (m, 1H), 4.36 (m, 1H), 4.34 (m,
1H), 4.32 (m, 1H), 4.05 (m, 1H), 3.77e4.06 (m, 21H), 3.75 (t, 1H,
J ¼ 9.6 Hz), 3.63 (t, 1H, J ¼ 9.2 Hz), 3.47e3.57 (m, 7H), 3.44 (t, 1H,
J ¼ 9.2 Hz), 3.13e3.18 (m, 2H), 2.92 (dd, 1H, J ¼ 3.1, 13.9 Hz), 2.32 (t,
1H, J ¼ 13.2 Hz), 1.00e2.02 (m, other aliphatic ring protons), 1.36 (s,
3H), 0.97 (s, 6H), 0.95 (s, 3H), 0.89 (s, 3H), 0.78 (s, 3H), 0.75 (m, 1H),
4.1.11. Synthesis of N-[1-(6A-deoxy-
triazol-4-yl]methyl 3 -hydroxy-lup-20(29)-en-28-amide (26)
Prepared from 25 according to general procedure B, the residue
was purified by RP flash chromatography (eluent: methanol) to
afford 26 as a white solid in 87% yield. 1H NMR (400 MHz, CD3OD):
a-cyclodextrin-6-yl)-1H-1,2,3-
b
d
8.17 (s, 1H), 5.11 (d, 1H, J ¼ 3.1 Hz), 4.92e4.97 (m, 4H), 4.71 (br s,
0.74 (s, 3H); 13C NMR (100 MHz, CD3OD):
d
180.40, 144.87, 144.66,
1H), 4.62 (d, 1H, J ¼ 3.4 Hz), 4.54e4.58 (m, 2H), 4.32e4.38 (m, 2H),
3.73e4.11 (m, 23H), 3.62 (t, 1H, J ¼ 7.5 Hz), 3.48e3.56 (m, 6H), 3.42
(t, 1H, J ¼ 9.2 Hz), 3.18 (dd, 1H, J ¼ 10.1, 3.3 Hz), 3.05e3.14 (m, 2H),
2.57 (t, 1H, J ¼ 9.4 Hz), 2.15 (d, 1H, J ¼ 12.9 Hz), 1.82e1.88 (m, 2H),
1.69, 0.99, 0.95, 0.93, 0.86, 0.76 (each s, 6 ꢀ CH3), 0.92e1.73 (m,
other aliphatic ring protons), 0.70 (d, 1H, J ¼ 8.9 Hz); 13C NMR
127.23, 124.29, 103.76, 103.67, 103.66, 103.59, 103.56, 102.52, 83.33,
83.32, 83.19, 83.16, 82.95, 79.71 (2C), 75.52, 75.31, 75.25, 74.97,
74.52, 74.09, 74.01, 73.88, 73.76, 73.69, 73.65, 73.40, 72.54, 72.48,
67.82, 62.06, 61.93, 61.89, 61.82, 56.83, 50.15, 48.25, 47.99, 42.86,
42.32, 40.86, 39.95, 39.85, 38.10, 36.19, 36.12, 36.03, 33.86, 33.29,
31.15, 28.72, 27.91, 27.41, 25.74, 24.51, 19.45, 17.77, 16.35, 16.17; ESI-
HRMS (m/z) Calcd for C69H111N4O32 [MþH]þ: 1507.7176. Found
1507.7202.
(100 MHz, CD3OD):
d 179.44, 152.30, 144.72, 127.91, 110.04, 103.81,
103.67, 103.61, 102.50, 83.52, 83.34, 83.19, 83.17, 83.00, 79.69, 79.64,
75.31, 75.27, 75.21, 74.98, 74.56, 74.07, 74.00, 73.89, 73.81, 73.77,
73.74, 73.70, 73.65, 73.48, 72.51, 72.38, 68.49, 61.92, 61.88, 61.83,
57.01, 56.92, 54.80, 53.15, 52.11, 51.43, 48.08, 43.51, 42.01, 40.10,
39.96, 39.09, 38.96, 38.34, 35.57, 34.91, 33.97, 31.92, 30.61, 28.61,
28.04, 27.00, 22.16, 19.63, 19.43, 16.80, 16.13, 15.04; ESI-HRMS Calcd
4.1.9. Synthesis of [1-(6A-deoxy-
triazol-4-yl]methyl 3 -dihydroxy-urs-12-en-28-amide (19)
a-cyclodextrin-6-yl)-1H-1,2,3-
b
Prepared from 16 according to general procedure B, the residue
was purified by RP flash chromatography (eluent: methanol) to
afford 19 as a white solid in 92% yield. 1H NMR (400 MHz, CDCl3):
for
C
C
69H111N4O31 [MþH]þ: 1653.7755. Found 1653.7821;
75H120N4NaO36 [MþNa]þ: 1675.7574. Found 1675.7494.
d
7.98 (s, 1H), 7.50 (br s, 1H), 5.35 (br t, 1 Hz), 5.09 (d, 1H, J ¼ 2.9 Hz),
4.95e4.97 (m, 3H), 4.93 (d, 1H, J ¼ 3.2 Hz), 4.78 (t, 1H, J ¼ 10.3 Hz),
4.61 (d, 1H, J ¼ 3.2 Hz), 4.30e4.45 (m, 3H), 4.04 (m, 1H), 4.09 (m,
1H), 3.73e3.96 (m, 16H), 3.63 (t, 1H, J ¼ 9.3 Hz), 3.48e3.57 (m, 7H),
3.45 (t, 1H, J ¼ 9.2 Hz), 3.17e3.18 (m, 2H), 2.15 (m, 1H), 1.57 (m, 1H),
4.2. Biological assays
4.2.1. HCV and VSV pseudovirus entry assays
All compounds were tested using the HCV and VSV pseudo

8
S. Xiao et al. / European Journal of Medicinal Chemistry 124 (2016) 1e9
particle (HCVpp and VSVGpp) entry assay as described previously
[27,28,40e42]. Briefly, pseudotyped viruses were produced by co-
transfecting plasmids expressing HCV E1E2 or vesicular stromatis
G protein (VSVG) with pNL4-3 HIV proviral DNA (AIDS Reagent
Program, NIH, Bethesda, MD). The envelope protein and the Vpr
deficient HIV vector carrying a luciferase reporter gene was inser-
ted into the Nef position in 293 T producer cells. For compound
library screening, Huh-7 cells (5 ꢀ 103 cells/well) were seeded into
96-well plates for 24 h, and were then infected with HCVpp or
VSVGpp in the presence or absence of compounds, followed by
incubation at 37 ꢂC. Test compounds were diluted to a final con-
compound, followed by addition of 100
37 ꢂC for 72 h to allow viral internalization and replication. The
post-binding assay: Huh-7 cells were first incubated with 50
HCVpp and 50
L medium at 4 ꢂC for 3 h. After incubation, cells
were washed with culture medium thoroughly to remove unbound
virus and then exposed to 50 L medium and 50 L compound at
37 ꢂC for 72 h. The post-entry assay: Huh-7 cells were first treated
with 50 L HCVpp and 50
L medium at 37 ꢂC for 6 h to allow virus
mL medium, and cultured at
mL
m
m
m
m
m
entry into cells but not reverse transcription and integration of the
viral genome within cells. After the incubation period, cells were
washed to remove unbound virus, and incubated at 37 ꢂC with
centration of 1
mM and 5
mM in 1% dimethyl sulfoxide (DMSO).
50 mL compound and 50 mL medium for further 72 h. In all five
Luciferase activity, reflecting the amount of pseudovirus entering
into host cells, was measured 3 days after infection using the
Bright-Glo Reagent (Promega). Maximum activity (100% of control)
and background were determined from the control wells contain-
ing DMSO alone or from uninfected wells, respectively. CD81 was
used as the positive control as previously reported [14]. The indi-
vidual signals in each of the compound test wells were then divided
by the averaged control values (wells lacking inhibitor), back-
ground subtracted, and multiplied by 100% to determine percent
activity. The corresponding % inhibition values were then calcu-
lated by subtracting this value from 100. The specificity of the
compounds for inhibiting HCV was determined by evaluating in-
hibition of VSVGpp infection in parallel. Each sample was analyzed
in duplicate, and the experiments were repeated at least three
times.
conditions, CD81 antibody was utilized as a positive control due to
its blocking HCV virus entry via binding to CD81 receptor. IM2865
was a non-relevant compound as negative control and 0.5% DMSO
(final concentration) was used for normalization in each condition.
Acknowledgments
This work was supported by the National Natural Science
Foundation of China (grant no. 81373271, 81361168002, 81573269
and 21572015). The authors gratefully acknowledge Dr. Hongwei Jin
(State Key Laboratory of Natural and Biomimetic Drugs, Peking
University) for performing the ALog P determinations.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.08.020.
4.2.2. Cytotoxicity assays
All of the reported conjugates were evaluated for cytotoxicity in
MDCK Madin-Daby canine kidney cells. The cells (1 ꢀ 104 cells per
well) were seeded in 96-well tissue culture plates and incubated for
16 h at 37 ꢂC in an atmosphere of 5% CO2 to allow the cells to adhere
to the surface of the wells. Then, the culture medium was replaced
with fresh medium containing the compounds at the concentra-
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