Design, Synthesis and Biological Evaluation of Pentacyclic Triterpene Dimers as HCV Entry Inhibitors

Article abstract of DOI:10.1002/cjoc.201700272

A series of triterpene dimers bearing different scaffold were designed and synthesized via CuAAC reaction. Their anti-HCV entry activities were evaluated by HCVpp and VSVpp entry assays. It was found that echinocystic acid (EA) and its dimer were still necessary for maintaining anti-HCV entry activity, and replacement of EA by other triterpenes might significantly decrease its anti-viral activities. Using a linker bearing a piperazine group, compound 14 dramatically increased its potency with IC₅₀ at 2.87 nmol/L. In addition, the undesired hemolytic effect of all these compounds was removed.

Full text of DOI:10.1002/cjoc.201700272

FULL PAPER
DOI: 10.1002/cjoc.201700272
Design, Synthesis and Biological Evaluation of Pentacyclic
Triterpene Dimers as HCV Entry Inhibitors
Lingkuan Meng,a Qi Wang,b Tao Tang,a Sulong Xiao,b Lihe Zhang,b
Demin Zhou,b and Fei Yu*,a
a Medical Faculty of Kunming University of Science and Technology, Kunming,
Yunnan 650500, China
b State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University,
Beijing 100191, China
A series of triterpene dimers bearing different scaffold were designed and synthesized via CuAAC reaction.
Their anti-HCV entry activities were evaluated by HCVpp and VSVpp entry assays. It was found that echinocystic
acid (EA) and its dimer were still necessary for maintaining anti-HCV entry activity, and replacement of EA by oth-
er triterpenes might significantly decrease its anti-viral activities. Using a linker bearing a piperazine group, com-
pound 14 dramatically increased its potency with IC50 at 2.87 nmol/L. In addition, the undesired hemolytic effect of
all these compounds was removed.
Keywords pentacyclic triterpene, echinocystic acid, dimer, HCV entry inhibitor
Introduction
1934,[13,14] displays moderate anti-HCV activity (IC50＝
1.4 μmol/L) by interruption of the interaction between
one HCV envelope protein, E2, and its host receptor
CD81, thus blocking entry of the virus into the host cell.
The formation of EA dimer via a linker bearing triazole
(Figure 1), dramatically increased its potency with IC50
at 3.04 nmol/L.[15-19]
However, it is still unknown whether EA moieties of
compound 1 for maintaining its high activity was nec-
essary or not, and further structure-activity relationship
(SAR) of these tritepene dimers as HCV entry inhibitors
was needed to explore. In this paper, as a continuation
of our ongoing research in the development of novel
anti-HCV entry compounds, we report the design and
synthesis of a series of dimers bearing different triter-
penes as pharmacophore, and their anti-HCV entry ac-
tivities were evaluated. This study improved the im-
portance of triterpene dimers as new leads for develop-
ing novel HCV entry inhibitors.
Hepatitis C virus (HCV) infection is the leading
cause of liver fibrosis and cirrhosis that eventually lead
to liver carcinomas.[1] There are more than 170 million
people infected with HCV worldwide.[2,3] Several direct
anti-viral agents targeting the NS3-4A protease (bo-
ceprevir, telaprevir and simeprevir) and the NS5B pol-
ymerase (sofosbuvir) were approved by FDA for treat-
ing patients infected with HCV.[4-6] However, as a kind
of RNA viruses, the genome of HCV bares high muta-
tion rate, which can cause antiviral drugs targeting viral
replication to lose activities rapidly.[7,8]
The upstream of virus replication, involving the in-
teraction between virus and host cells, provides a new
opportunity for the research and development of antivi-
ral drugs.[9-12] Our previous studies suggested that echi-
nocystic acid (EA), a natural product of oleanane-type
triterpenes first isolated from Echinocystis fabacea in
Figure 1 Structures of EA, compound 1 and their anti-HCV activities.
*
E-mail: feiyuz8@kmust.edu.cn
Received April 20, 2017; accepted June 8, 2017; published online XXXX, 2017.
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cjoc.201700272 or from the author.
Chin. J. Chem. 2017, XX, 1—7
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Meng et al.
FULL PAPER
Experimental
J＝4.7, 10.8 Hz, 2H), 4.35 (brs, 4H), 5.33 (brs, 2H),
5.55 (dd, J＝15.3, 17.0 Hz, 4H) 7.33 (s, 4H), 7.78 (s,
2H); 13C NMR (100 MHz, CD3OD) δ: 16.09 (2C), 16.52
(2C), 17.60 (2C), 19.53 (2C), 24.12 (4C), 24.53 (2C),
26.57 (2C), 27.88 (2C), 28.43 (2C), 28.89 (2C), 31.62
(2C), 33.66 (2C), 33.78 (2C), 34.10 (2C), 35.13 (2C),
35.85 (2C), 38.10 (2C), 39.85 (2C), 40.57 (2C), 42.54
(2C), 42.86 (2C), 47.44 (2C), 47.65 (2C), 48.93 (2C),
49.00 (2C), 54.37 (2C), 56.66 (2C), 79.58 (2C), 124.15
(2C), 124.57 (2C), 129.81 (2C), 137.07 (2C), 145.00
(2C), 146.55 (2C), 180.14 (2C). ESI-HRMS calcd for
C74H111N8O4 [M＋H]＋ 1175.8707, found 1175.8723.
Compound 5 Obtained from Scheme 1 as a by-
product. m.p. 128.9－131.4 ℃. [α]2D5 37.60 (c＝0.50,
MeOH). 1H NMR (400 MHz, CDCl3) δ: 0.49, 0.78, 0.86,
0.87, 0.89, 0.98, 1.13 (7×CH3), 0.69－2.16 (m, other
aliphatic ring protons), 2.54 (dd, J＝3.1, 13.3 Hz, 1H),
3.20 (dd, J＝4.4, 10.1 Hz, 1H), 4.32－4.37 (m, 3H),
4.45－4.50 (m, 1H), 5.38 (brs, 1H), 5.44－5.54 (m, 2H),
6.66 (t, J＝5.2 Hz, 1H), 7.27－7.33 (m, 4H), 7.52 (s,
1H); 13C NMR (100 MHz, CDCl3) δ: 15.3, 15.5, 16.4,
18.2, 23.3, 23.5, 23.7, 25.6, 27.0, 27.1, 28.0, 30.6, 32.2,
32.3, 32.9, 34.0, 34.9, 36.8, 38.3, 38.6, 39.2, 41.8, 41.9,
46.1, 46.5, 47.4, 53.6, 54.1, 55.0, 78.7, 122.4, 123.1,
128.4 (2C), 128.7 (2C), 134.6, 136.0, 144.1, 145.1,
178.3.
1
Routine H NMR and 13C NMR spectra were rec-
orded. Samples were dissolved in CDCl3 or CD3OD and
tetramethylsilane (TMS) was used as reference. 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 at 400
and 100 MHz for H and 13C nuclei recorded on a
1
Bruker DRX 400 spectrometer (Rheinstetten, Germany)
at ambient temperature. Chemical shifts are reported in
parts per million relative to the tetramethylsilane peak
recorded as δ 0.00 in CDCl3/TMS solvent, or the resid-
ual chloroform (δ 7.26) or methanol (δ 3.31) peaks. The
13C NMR values were referenced to the residual chlo-
roform (δ 77.0), or methanol (δ 49.0) peaks. Melting
points were determined with an X-4 apparatus and un-
corrected. Optical rotations were measured with a Ru-
dolph Autopol VI polarimeter. Analytical TLC was per-
formed on Merck silica gel 60 F254. Compounds were
visualized 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. Chemi-
cals for the synthesis were analytical grade. Echinocys-
tic acid was isolated from Gleditsia sinensis Lam, which
was characterized by comparing their spectral data with
those reported in the literature.
General procedure A for the CuAAC “click” re-
action CuSO4 (1 equiv.) and Na-L-ascorbate (1.5
equiv.) were added to a solution of compound 3 and the
corresponding azide in CH2Cl2 (5 mL) and H2O (5 mL).
The resulting solution was stirred vigorously for 12 h at
r.t. The reaction mixture was diluted with H2O (10 mL),
then extracted with CH2Cl2 (10 mL×3). The combined
organic layer was dried over Na2SO4, filtered, and con-
centrated. The residue was purified by column chroma-
tography.
Compound 3 Synthesized using methods pub-
lished previously.[18] m.p. 201.3－23.7 ℃. [α]2D5 60.80
(c＝0.50, MeOH). 1H NMR (400 MHz, CDCl3) δ: 0.78,
0.79, 0.91, 0.99, 1.17 (7×CH3), 0.72－2.04 (m, other
aliphatic ring protons), 2.22 (t, J＝2.5 Hz, 1H), 2.54 (dd,
J＝3.2, 13.0 Hz, 1H), 3.21 (dd, J＝4.4, 10.7 Hz, 1H),
3.87－3.93 (m, 1H), 4.02－4.08 (m, 1H), 5.42 (brs, 1H),
6.13 (t, J＝4.7 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ:
15.3, 15.5, 16.9, 18.2, 23.4, 23.5, 23.8, 25.7, 27.1, 27.2,
28.0, 29.2, 30.6, 32.1, 32.2, 32.9, 34.0, 36.8, 38.4, 38.7,
39.3, 41.9, 42.0, 46.2, 46.6, 47.4, 55.0, 71.5, 78.8, 79.6,
123.1, 144.6, 178.0.
Compound 6c Obtained according to general pro-
cedure A. The product was purified by column chroma-
tography [V(CH2Cl2)/V(MeOH)＝25/1] to give 6c as a
white solid (93 mg, 79%). m.p. 193.1－195.7 ℃.
1
[α]2D5 33.60 (c＝0.50, MeOH). H NMR (400 MHz,
CD3OD) δ: 0.53, 0.78, 0.85, 0.87, 0.93, 0.99, 1.07 (14×
CH3), 0.69－2.02 (m, other aliphatic ring protons), 3.21
(dd, J＝3.6, 10.2 Hz, 2H), 4.32 (dd, J＝5.2, 15.0 Hz,
2H), 4.45 (dd, J＝5.3, 15.0 Hz, 2H), 5.33 (brs, 2H), 5.47
(brs, 4H), 6.67 (t, J＝5.1 Hz, 2H), 7.25 (s, 4H), 7.48 (s,
2H); 13C NMR (100 MHz, CD3OD) δ: 15.4 (2C), 15.6
(2C), 16.4 (2C), 17.1 (2C), 18.1 (2C), 21.1 (2C), 23.1
(2C), 23.2 (2C), 24.7 (2C), 27.1 (2C), 27.6 (2C), 28.0
(2C), 30.7 (2C), 32.6 (2C), 34.8 (2C), 36.8 (2C), 36.9
(2C), 38.5 (2C), 38.6 (2C), 38.8 (2C), 39.3 (2C), 39.6
(2C), 42.2 (2C), 47.4 (2C), 47.5 (2C), 53.4 (2C), 55.0
(2C), 78.7 (2C), 122.3 (2C), 126.0 (2C), 128.6 (4C),
135.1 (2C), 139.0 (2C), 145.1 (2C), 178.1 (2C).
ESI-HRMS calcd for C74H111N8O4 [M＋H]＋ 1175.8691,
found 1175.8723.
Compound 7c Obtained according to general pro-
cedure A. The product was purified by column chroma-
tography [V(CH2Cl2)/V(MeOH)＝25/1] to give 7c as a
white solid (83 mg, 71%). m.p. 177.6－179.3 ℃. [α]D25
Compound 4 Prepared from 3 (108 mg, 0.22
mmol) and p-xylylene diazide (19 mg, 0.1 mmol) ac-
cording to general procedure A. The product was puri-
fied by column chromatography [V(CH2Cl2)/V(MeOH)
＝25/1] to give 4 as a white solid (99 mg, 84%). m.p.
1
2.40 (c＝0.50, MeOH). H NMR (400 MHz, CD3OD)
δ: 0.75, 0.76, 0.80, 0.93, 0.96, 1.66 (12×CH3), 0.65－
1.99 (m, other aliphatic ring protons), 2.33－2.38 (m,
2H), 3.04－3.10 (m, 2H), 3.18 (dd, J＝5.2, 10.8 Hz,
2H), 4.35－4.47 (m, 4H), 4.58 (s, 2H), 4.71 (s, 2H),
5.47 (dd, J＝15.0, 47.3 Hz, 4H), 6.71 (t, J＝5.6 Hz, 2H),
7.25 (s, 4H), 7.56 (s, 2H); 13C NMR (100 MHz, CD3OD)
δ: 14.5 (2C), 15.4 (2C), 15.7 (2C), 16.1 (2C),
1
206.3－208.7 ℃. [α]D25 43.20 (c＝0.50, MeOH). H
NMR (400 MHz, CD3OD) δ: 0.46, 0.79, 0.88, 0.91, 0.92,
0.98, 1.14 (14×CH3), 0.70－2.06 (m, other aliphatic
ring protons), 2.76 (dd, J＝3.2, 13.3 Hz, 2H), 3.14 (dd,
2
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Chin. J. Chem. 2017, XX, 1—7

Pentacyclic Triterpene Dimers as HCV Entry Inhibitors
Scheme 1
Reagents and conditions: (a) TBTU, DIEA, THF, r.t., 4 h; (b) 2-propynylamine, Na2CO3, DMF, r.t., 20 min; (c) CuSO4, Na-L-ascorbate,
1,4-bis(azidomethyl)benzene, CH2Cl2/H2O, r.t., overnight.
18.2 (2C), 19.3 (2C), 20.8 (2C), 25.5 (2C), 27.3 (2C),
27.9 (2C), 29.2 (2C), 30.7 (2C), 33.3 (2C), 34.2 (2C),
34.5 (2C), 37.1 (2C), 37.6 (2C), 38.1 (2C), 38.6 (2C),
38.7 (2C), 40.6 (2C), 42.3 (2C), 46.6 (2C), 50.0 (2C),
50.5 (2C), 53.5 (2C), 55.2 (2C), 55.5 (2C), 78.7 (2C),
109.3 (2C), 122.5 (2C), 128.6 (4C), 135.1 (2C), 145.8
(2C), 150.8 (2C), 176.5 (2C). ESI-HRMS calcd for
C74H111N8O4 [M＋H]＋ 1175.8691, found 1175.8703.
Compound 8b Starting from compound 8a (251
mg, 0.5 mmol), compound 8b was synthesized using
methods published previously.[18] The product was puri-
fied by column chromatography [V(PE)/V(EtOAc)＝3/1]
to give 8b as a white solid (239 mg, 88%). m.p. 195.1－
25.8 (2C), 30.8 (2C), 30.9 (2C), 31.7 (2C), 32.1 (2C),
32.1 (2C), 33.5 (2C), 35.6 (2C), 36.5 (2C), 36.6 (2C),
37.8 (2C), 39.0 (2C), 39.4 (2C), 41.3 (2C), 41.9 (2C),
48.8 (2C), 49.1 (2C), 51.9 (2C), 53.1 (2C), 53.5 (2C),
74.0 (2C), 85.1 (2C), 123.5 (2C), 128.5 (4C), 136.2 (2C),
146.7 (2C), 173.9 (2C), 177.6 (2C), 210.8 (2C).
＋
ESI-HRMS calcd for C74H107N8O10 [M ＋ H]
1267.8095, found 1267.8105.
Compound 9b Starting from compound 9a (200
mg, 0.4 mmol), compound 9b was synthesized using
methods published previously.[18] The product was puri-
fied by column chromatography [V(PE)/V(EtOAc)＝3/1]
to give 9b as a white solid (179 mg, 83%). m.p. 143.2－
1
1
196.8 ℃. [α]2D5 68.40 (c＝0.50, MeOH). H NMR (400
145.3 ℃. [α]2D5 55.10 (c＝0.50, MeOH). H NMR (400
MHz, CDCl3) δ: 0.92, 1.03, 1.08, 1.27, 1.44, 1.49 (s, 7
×CH3), 1.19－2.33 (m, other aliphatic ring protons),
2.54－2.67 (m, 2H), 2.74－2.77 (m, 1H), 2.87 (d, J＝
4.2 Hz, 1H), 3.93－4.05 (m, 2H), 4.56 (br, 1H), 6.30 (t,
J＝3.0, 5.3 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ:
15.6, 16.5, 20.4, 22.9, 24.1, 25.9, 29.3, 30.3, 30.9, 31.8,
31.9, 32.0 (2C), 32.9, 35.2, 36.0 (2C), 37.7, 38.7, 39.5,
41.2, 41.5, 48.7 (2C), 51.5, 53.9, 71.0, 74.3, 79.9, 85.9,
174.7, 176.7, 211.1.
MHz, CDCl3) δ: 0.72, 0.76, 0.83, 0.94, 0.94, 0.98, 1.11,
1.14 (s, 7×CH3), 0.69－1.91 (m, other aliphatic ring
protons), 2.24 (t, J＝2.4 Hz, 1H), 2.29 (d, J＝13.8 Hz,
1H), 2.56－2.72 (m, 2H), 2.97－3.11 (m, 1H), 3.21 (dd,
J＝4.5, 11.5 Hz, 1H), 3.96－4.09 (m, 2H), 4.12－4.20
(m, 1H), 4.82 (d, J＝5.5 Hz, 1H), 6.77 (t, J＝5.2 Hz,
1H); 13C NMR (100 MHz, CDCl3) δ: 15.3, 16.4, 17.3,
17.9, 19.8, 25.3, 26.9, 27.5, 27.8, 29.3, 29.5, 30.8, 32.2,
33.3, 33.8, 36.2, 36.8, 37.7, 38.7, 38.8 (2C), 43.6, 44.5,
49.1, 49.5, 54.9, 71.3, 71.5, 75.6, 78.3, 79.8, 176.0,
177.2.
Compound 9c Obtained according to general pro-
cedure A. The product was purified by column chroma-
tography [V(CH2Cl2)/V(MeOH)＝25/1] to give 9c as a
white solid (69 mg, 54%). m.p.＞250 ℃. [α]2D5 41.50
(c＝0.50, MeOH). 1H NMR (400 MHz, CD3OD) δ: 0.76,
0.85, 0.87, 0.89, 0.93, 0.98, 1.14 (s, 14×CH3), 0.71－
1.90 (m, other aliphatic ring protons) 2.24 (d, J＝14.0
Hz, 2H), 2.67 (dd, J＝13.6, 9.8 Hz, 2H), 2.83－2.88 (m,
2H), 3.15 (dd, J＝9.4, 6.5 Hz, 2H), 4.18 (dd, J＝7.4, 3.3
Hz, 2H), 4.37 (d, J＝15.2 Hz, 2H), 4.43 (d, J＝15.1 Hz,
Compound 8c Obtained according to general pro-
cedure A. The product was purified by column chroma-
tography [V(CH2Cl2)/V(MeOH)＝25/1] to give 8c as a
white solid (83 mg, 41.4%). m.p. 213.6－216.1 ℃.
1
[α]2D5 36.80 (c＝0.50, MeOH). H NMR (400 MHz,
Pyr-d5) δ: 0.98, 0.99, 1.06, 1.20, 1.33, 1.40, 1.64 (s, 14
×CH3), 1.23－2.71 (m, other aliphatic ring protons),
3.49 (d, J＝4.0 Hz, 2H), 3.56－3.65 (m, 2H), 4.75－
4.92 (m, 4H), 5.25 (br, 2H), 5.68 (d, J＝15.2 Hz, 2H),
5.73 (d, J＝15.2 Hz, 2H), 7.32 (s, 4H), 8.21 (s, 2H),
9.29 (t, J＝5.1 Hz, 2H); 13C NMR (100 MHz, Pyr-d5) δ:
15.2 (2C), 16.4 (2C), 20.3 (2C), 22.9 (2C), 24.4 (2C),
Chin. J. Chem. 2017, XX, 1—7
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3

Meng et al.
FULL PAPER
2H), 4.98 (d, J＝6.2 Hz, 2H), 5.57 (s, 4H), 7.33 (s, 4H),
7.79 (s, 2H); 13C NMR (100 MHz, CD3OD) δ: 16.2 (2C),
16.8 (2C), 17.7 (2C), 19.1 (2C), 19.5 (2C), 26.6 (2C),
27.8 (2C), 28.5 (2C), 29.3 (2C), 30.7 (2C), 31.9 (2C),
33.1 (2C), 35.2 (2C), 35.6 (2C), 36.0 (2C), 37.2 (2C),
38.0 (2C), 38.2 (2C), 40.0 (2C), 40.0 (2C), 40.1 (2C),
44.3 (2C), 45.8 (2C), 50.2 (2C), 50.8 (2C), 54.4 (2C),
56.2 (2C), 72.9 (2C), 78.0 (2C), 79.1 (2C), 129.8 (4C),
137.2 (2C), 178.9 (2C). ESI-HRMS calcd for
C74H111N8O10 [M＋H]＋ 1271.8398, found 1271.8418.
Compound 10b Starting from compound 10a (195
mg, 0.4 mmol), compound 10b was synthesized using
methods published previously.[21] The product was puri-
fied by column chromatography [V(PE)/V(EtOAc)＝3/1]
to give 10b as a white solid (189 mg, 90%). m.p.
2H), 4.12－4.20 (m, 1H), 4.89 (d, J＝5.6 Hz, 1H), 6.76
(t, J＝5.2 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ: 16.2,
19.4, 19.5, 23.0, 25.7, 26.9, 27.2, 29.4, 29.5, 30.5, 31.9,
32.1, 32.2, 33.4, 36.1, 36.7, 37.4, 40.5, 40.8, 43.7, 44.2,
49.5 (2C), 52.0, 71.2, 71.6, 75.6, 79.7, 85.3, 174.7,
175.4, 177.0.
Compound 11c Obtained according to general
procedure A. The product was purified by column
chromatography [V(CH2Cl2)/V(MeOH)＝25/1] to give
11c as a white solid (38 mg, 29%). m.p.＞250 ℃.
[α]2D5 41.70 (c ＝ 50, MeOH). 1H NMR (400 MHz,
CD3OD) δ: 0.86, 0.92, 0.93, 1.09, 1.15, 1.39, 1.50, (s, 7
×CH3), 1.00－1.99 (m, other aliphatic ring protons),
2.24 (d, J＝14.9 Hz, 2H), 2.49－2.35 (m, 2H), 2.71－
2.93 (m, 6H), 4.18 (dd, J＝7.3, 3.3 Hz, 2H), 4.38 (d,
J＝15.2 Hz, 2H), 4.43 (d, J＝15.1 Hz, 2H), 5.03 (d, J＝
6.3 Hz, 2H), 5.57 (s, 4H), 7.32 (s, 4H), 7.81 (s, 2H); 13C
NMR (100 MHz, CD3OD) δ: 16.8 (2C), 19.3 (2C), 20.1
(2C), 24.2 (2C), 26.6 (2C), 27.5 (2C), 29.2 (2C), 30.7
(2C), 30.7 (2C), 32.9 (2C), 33.0 (2C), 33.1 (2C), 33.4
(2C), 35.6 (2C), 35.9 (2C), 37.2 (2C), 37.9 (2C), 38.2
(2C), 41.7 (2C), 42.1 (2C), 44.6 (2C), 45.5 (2C), 50.5
(2C), 50.8 (2C), 52.6 (2C), 54.3 (2C), 72.9 (2C), 77.9
(2C), 87.4 (2C), 124.3 (2C), 129.7 (4C), 137.2 (2C),
1
158.9－160.3 ℃. [α]D25 51.70 (c＝0.50, MeOH). H
NMR (400 MHz, CD3OD) δ: 0.74, 0.85, 0.89, 0.96, 0.97,
1.04, 1.08 (s, 7×CH3), 0.71－2.06 (m, other aliphatic
ring protons), 2.27 (d, J＝13.8 Hz, 1H), 2.56 (s, 1H),
2.65 (dd, J＝13.1, 11.3 Hz, 1H), 2.69－2.80 (m, 1H),
3.15 (dd, J＝8.4, 7.6 Hz, 1H), 3.82－3.95 (m, 1H),
4.02－4.15 (m, 1H), 5.03 (d, J＝6.7 Hz, 1H), 8.18 (t,
13
J＝5.1 Hz, 1H); C NMR (100 MHz, CD3OD) δ: 16.1,
16.3, 17.2, 18.2, 19.1, 22.5, 23.7, 27.7, 28.5, 29.6, 29.6,
31.3, 32.3, 33.8, 34.4, 35.2, 35.6, 36.6, 38.1, 39.8, 39.9,
39.9, 44.4, 45.7, 47.8, 50.4, 55.8, 71.8, 78.0, 79.1, 81.2,
178.8, 179.2.
146.9 (2C), 177.6 (2C), 178.5 (2C), 179.5 (2C).
＋
ESI-HRMS calcd for C74H107N8O12 [M ＋ H]
1299.7970, found 1299.8003.
Compound 10c Obtained according to general
procedure A. The product was purified by column
chromatography [V(CH2Cl2)/V(MeOH)＝25/1] to give
10c as a white solid (68 mg, 59%). m.p. 199.1－
Compound 13 Starting from 5 (50 mg, 0.07 mmol)
and EA-propargylamine conjugate (45 mg, 0.09 mmol)
according to general procedure A. The product was pu-
rified by column chromatography [V(CH2Cl2)/V(MeOH)
＝25/1] to give 13 as a white solid (60 mg, 69%). m.p.
1
201.5 ℃. [α]2D5 35.90 (c＝0.50, MeOH). H NMR (400
1
MHz, CD3OD) δ: 0.71, 0.76, 0.80, 0.87, 0.95, 0.97, 1.01
(s, 14×CH3), 0.68－2.03 (m, other aliphatic ring pro-
tons), 2.22 (d, J＝13.8 Hz, 2H), 2.55 (dd, J＝13.3, 11.0
Hz, 2H), 2.66－2.75 (m, 2H), 3.14 (dd, J＝9.3, 6.9 Hz,
2H), 4.38 (d, J＝15.0 Hz, 2H), 4.44 (d, J＝15.0 Hz, 2H),
4.90 (d, J＝6.8 Hz, 2H), 5.58 (s, 4H), 7.35 (s, 4H), 7.85
(s, 2H); 13C NMR (100 MHz, CD3OD) δ: 16.3 (2C),
16.4 (2C), 17.2 (2C), 17.6 (2C), 19.1 (2C), 22.5 (2C),
23.8 (2C), 27.7 (2C), 28.6 (2C), 29.6 (2C), 31.3 (2C),
32.3 (2C), 33.8 (2C), 34.6 (2C), 35.2 (2C), 35.5 (2C),
35.7 (2C), 36.7 (2C), 38.2 (2C), 39.8 (2C), 39.9 (2C),
44.4 (2C), 45.6 (2C), 47.8 (2C), 50.4 (2C), 54.3 (2C),
55.8 (2C), 77.9 (2C), 79.1 (2C), 124.7 (2C), 129.8 (4C)
(2C), 137.2 (2C), 146.8 (2C), 178.7 (2C), 179.3 (2C).
ESI-HRMS calcd for C74H111N8O8 [M＋H]＋ 1239.8501,
found 1239.8519.
182.1－183.7 ℃. [α]D25 30.40 (c＝0.50, MeOH). H
NMR (400 MHz, CDCl3) δ: 0.50, 0.78, 0.86, 0.87, 0.87,
0.88, 0.89, 0.90, 0.99, 0.99, 1.14 (14×CH3), 0.69－2.05
(m, other aliphatic ring protons), 2.24 (t, J＝13.3 Hz,
1H), 2.54 (dd, J＝3.7, 13.6 Hz, 1H), 2.69 (dd, J＝2.5,
13.2 Hz, 1H), 3.20－3.23 (m, 2H), 4.32－4.50 (m, 5H),
5.38－5.55 (m, 6H), 6.68 (t, J＝5.2 Hz, 1H), 6.90 (t,
J＝5.1 Hz, 1H), 7.25 (s, 4H), 7.43 (s, 1H), 7.49 (s, 1H).
13C NMR (100 MHz, CDCl3) δ: 15.3, 15.6 (3C), 16.4,
16.6, 18.2 (2C), 23.3, 23.4, 23.5, 23.7, 24.9, 25.7, 26.7,
27.1 (3C), 28.0, 29.7, 30.2, 30.6, 32.2, 32.4, 32.5 (2C),
32.9, 34.0, 34.9 (2C), 35.1, 35.2, 36.8, 38.4, 38.5, 38.7,
39.2, 39.4, 41.5, 41.7, 41.8, 41.9, 46.1, 46.5, 46.6, 46.9,
47.4, 48.8, 53.5 (2C), 55.0, 55.1, 75.0, 78.7, 78.8, 122.3,
122.6, 123.1, 123.4, 128.7 (2C), 128.7 (2C), 135.2 (2C),
143.6, 144.1, 145.2, 145.3, 178.0, 178.5. ESI-HRMS
calcd for C74H111N8O5 [M＋H]＋ 1191.8645, found
1191.8672.
Compound 11b Starting from compound 11a (207
mg, 0.4 mmol), compuond 11b was synthesized using
methods published previously.[18] The product was puri-
fied by column chromatography [V(PE)/V(EtOAc)＝3/1]
to give 11b as a white solid (168 mg, 76%). m.p.
Compound 14 Obtained according to general
procedure A. The product was purified by column
chromatography [V(CH2Cl2)/V(MeOH)＝25/1] to give
14 as a white solid (66 mg, 53%). m.p. 185.4 －
1
171.4－173.5 ℃. [α]D25 63.90 (c＝0.50, MeOH). H
1
NMR (400 MHz, CDCl3) δ: 0.94, 0.95, 1.11, 1.15, 1.40,
1.50, (s, 7×CH3), 1.01－1.96 (m, other aliphatic ring
protons), 2.21－2.31 (m, 2H), 2.45－2.55 (m, 1H), 2.61
－2.79 (m, 3H), 3.00－3.10 (m, 1H), 3.96－4.10 (m,
187.7 ℃. [α]2D5 20.80 (c＝0.50, MeOH). H NMR (400
MHz, CDCl3) δ: 0.58, 0.78, 0.89, 0.90, 0.92, 0.99, 1.36
(14×CH3), 0.71－2.11 (m, other aliphatic ring protons),
2.25 (t, J＝13.3 Hz, 2H), 2.50 (brs, 8H), 2.74 (dd, J＝
4
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Chin. J. Chem. 2017, XX, 1—7

Pentacyclic Triterpene Dimers as HCV Entry Inhibitors
3.4, 13.6 Hz, 2H), 2.81 (t, J＝6.2 Hz, 4H), 3.21 (dd, J＝
2.8, 10.1 Hz, 2H), 2.32－4.49 (m, 10H), 5.53 (t, J＝3.0
Hz, 2H), 6.92 (t, J＝5.3 Hz, 2H), 7.61 (s, 2H); 13C NMR
(100 MHz, CDCl3) δ: 15.6 (2C), 15.6 (2C), 16.7 (2C),
18.2 (2C), 23.3 (2C), 25.0 (2C), 26.8 (2C), 27.1 (2C),
28.0 (2C), 29.8 (2C), 30.2 (2C), 32.5 (2C), 32.6 (2C),
35.1 (2C), 35.3 (2C), 36.9 (2C), 38.5 (2C), 38.7 (2C),
39.5 (2C), 41.5 (2C), 41.7 (2C), 46.6 (2C), 46.8 (2C),
47.6 (2C), 48.9 (2C), 52.9 (4C), 55.1 (2C), 57.3 (2C),
75.2 (2C), 78.8 (2C), 122.7 (2C), 123.5 (2C), 143.5 (2C),
144.4 (2C), 177.9 (2C). ESI-HRMS calcd for
C74H119N10O6 [M＋H]＋ 1243.9278, found 1243.9285.
acid (OA) was firstly activated by TBTU (2) then re-
acted with 2-propynylamine to give compound 3 with
high yield.[20] OA dimer (4) was prepared from com-
pound 3 according to our reported method, which un-
derwent a ‘click reaction’ with 1,4-bis(azidomethyl)-
benzene in CH2Cl2/H2O (V/V＝1/1) in the presence of a
catalytic amount of copper sulfate and sodium ascor-
bate.[15,16] By the same strategy, a series of dimers bear-
ing different triterpene scaffolds, such as ursolic acid
(UA), betulinic acid (BA) etc. (compounds 6c－11c),
were designed and synthesized (Table 1).
To further investigate whether the EA moiety of
compound 1 can be replaced by OA or not, compound
13, with half OA and half EA, was synthesized and its
anti HCV entry activity was also evaluated (Scheme 2).
As a preliminary screen for anti-HCV entry activity,
all compounds were tested for their capacities to inhibit
HCV at two concentrations (1 μmol/L and 5 μmol/L)
using an HCV pseudo-particle (HCVpp) entry assays.
vesicular stomatitis virus G protein pseudo particle
(VSVpp) was also tested in parallel to determine the
specificity and toxicity as reported.[10,22,23] 1% DMSO
was used as negative control, and CD81 antibody was
utilized as a positive control because it blocks HCV vi-
rus entry via binding to CD81 receptors (Figure 2). In
this assay (Figure 2A), we found both compound 4 and
6c exhibited certain activities of inhibiting the entry of
HCV into host cells, but were less potent than that of
EA. Although compound 7c, with replacement of EA
with BA, displayed significant anti-HCV activity at 1
μmol/L, it also decreased the report reading of VSVpp
infected cells (Figure 2B), suggesting compound 7c was
a nonselective antiviral agent against HCV or toxicity
rather than potency enhancement. Furthermore, the an-
ti-HCV activity of the dimers 8c－11c completely abol-
ished the activity of compound 1. Interestingly, biologi-
cal evaluation showed that compound 13, a heterodimer
containing both EA and OA moiety, displayed a further
potency enhancement than that of EA but was much less
potent than compound 1. All these results provided the
evidence that EA moiety played an irreplaceable role in
maintaining high anti-HCV entry activity, and any re-
placement of EA by other triterpenes might significantly
decrease its potency.
HCV and VSV pseudovirus entry assay
All compounds were tested by using the HCVpp and
VSVpp entry assay as described. Briefly, pseudotyped
viruses were produced by cotransfecting 293T cells with
plasmid encoding HCV E1, E2 or vesicular stomatitis 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 was harvested from the supernatant of the
transfected cells. For compound library screening, in-
fections 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 ℃. Luciferase activity,
reflecting the degree of the pseudoparticles into host
cells, was measured 3 days after infection using the
Bright-Glo Reagent (Promega). Test compounds were
serially diluted to give a final concentration of 1 μmol/L
and 5 μmol/L in 1% dimethyl sulfoxide (DMSO).
Maximum activity (100% of control) and background
were derived from control wells containing DMSO
alone or from uninfected wells, respectively. The indi-
vidual 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 determine 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 inhibition of VSVpp infection in parallel.
Each sample was done in duplicate, and experiments
were repeated at least three times.
In our previous studies, we found the rigidity, length,
and hydrophobicity of the spacer all had significant in-
fluence on the potency of EA dimers to HCVpp entry,
and this dramatically affects their overall anti-HCV ac-
tivity.[16] However, phenyl group is hydrophobic, which
may affect the potential pharmacokinetic properties of
compound 1. To increase its solubility and maintain
some rigidity, compound 14, replacing the phenyl group
of the linker with a piperazine, was design and synthe-
sized (Scheme 3).
Hemolytic assay
Hemolytic activity was measured as follows: 2%
rabbit red blood cells in erythrocyte buffer (130 mmol/L
NaCl, 20 mmol/L Tris-HCl, pH 7.4) were incubated
with serially diluted compounds. After incubating for 60
min at 37 ℃, hemolysis was monitored by measuring
absorption at 540 nm with a microplate reader Infinite
M200 Pro. Percentage of hemolysis was then calculated
as the routine method.
As shown in Figure 2, compound 14 exhibited sig-
nificantly higher potency with IC50 at 2.87 nmol/L, two
orders of magnitudes more potent than EA (1.4 μmol/L),
and no significant cytotoxicity was observed. To our
Results and Discussion
As shown in Scheme 1, the 28-COOH of oleanolic
Chin. J. Chem. 2017, XX, 1—7
© 2017 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.cjc.wiley-vch.de
5

Meng et al.
FULL PAPER
Table 1 The structures of compounds 6c－11c
Compd
R
Compd
R
6
9
HO
7
10
O
O
8
11
O
HO
O
Scheme 2
N3
N
N
H
H
N
N
N
a
+
O
O
OH
HO
HO
N
5
12
N
N
N
N
H
N
H
N
N
O
O
OH
HO
OH
13
OA
EA
Reagents and conditions: (a) CuSO4, Na-L-ascorbate, CH2Cl2/H2O, r.t., overnight.
Scheme 3
Reagents and conditions: (a) NaN3, Acetone/H2O, 60 °C, overnight; (b) TsCl, TEA, CH2Cl2, overnight; (c) piperazine, TEA, DMF, 50 °C,
overnight; (d) Na-L-ascorbate, CH2Cl2/H2O, r.t., overnight.
6
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© 2017 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2017, XX, 1—7

Pentacyclic Triterpene Dimers as HCV Entry Inhibitors
pounds was removed. As a novel HCV entry inhibitor,
compound 14 is worth for further exploration.
Acknowledgement
This work was supported by the National Natural
Science Foundation of China (No. 81560560),
Open-Fund Program of the State Key Laboratory of
Natural and Biomimetic Drugs (No. K20160209) and
Yunnan Basic Research Project (No. 14051789).
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Figure 2 Anti-HCV entry activities of triterpene dimers.
knowledge, this is the most potent compound based on
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that EA gains substantial hemolytic side effect with
CC50 at 15 mmol/L, which may restrict triterpenoids
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Conclusions
In this study, we reported the design, synthesis and
anti-HCV entry activity studies of novel triterpene di-
mers. Compounds 4, 6c－11c and 13 showed no im-
provement but detrimental effect on potency of com-
pound 1. The results confirmed that EA moiety of
triterpene dimer is highly conserved, and any modifica-
tion or replacement might significantly decrease or even
eliminate its anti-HCV entry activity. Based on all these
data, compound 14, bearing a piperazine group in the
linker, was design and synthesized, which is the most
potent compound based on HCVpp entry assay. In addi-
tion, the undesired hemolytic effect of all these com-
(Cheng, F.)
Chin. J. Chem. 2017, XX, 1—7
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7