Elucidation of the pharmacophore of echinocystic acid, a new lead for blocking HCV entry

Article abstract of DOI:10.1016/j.ejmech.2013.03.041

To elucidate the pharmacophore of echinocystic acid (EA), an oleanane-type triterpene displaying substantial inhibitory activity on HCV entry, two microbial strains, Rhizopus chinensis CICC 3043 and Alternaria alternata AS 3.4578, were utilized to modify the chemical structure of EA. Eight new metabolites with regio- and stereo-selective introduction of hydroxyl and lactone groups at various inert carbon positions were obtained. The anti-HCV entry activity of the metabolites 2-13, along with their parental compound EA and other analogs 14-15, were evaluated. Most of the metabolites showed no improvement but detrimental effect on potency except compound 5 and 6, which showed similar and even a litter higher anti-HCV entry activity than that of EA. The results demonstrated that ring A, B, C and the left side of ring E of EA are highly conserved, while ring D and the right side of ring E of EA are flexible. Introduction of a hydroxyl group at C-16 enhanced the triterpene potency. Further analysis indicated that the hemolytic effect of EA disappeared upon such modifications.

Full text of DOI:10.1016/j.ejmech.2013.03.041

European Journal of Medicinal Chemistry 64 (2013) 160e168
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Elucidation of the pharmacophore of echinocystic acid, a new lead for
blocking HCV entry
,
,
,
,
a
Han Wang a 1, Qi Wang a 1, Su-Long Xiao a 1, Fei Yu a, Min Ye a , Yong-Xiang Zheng ,
**
Chuan-Ke Zhao a, Di-An Sun b, Li-He Zhang a, De-Min Zhou a
,
*
a State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191, China
b Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100094, China
a r t i c l e i n f o
a b s t r a c t
Article history:
To elucidate the pharmacophore of echinocystic acid (EA), an oleanane-type triterpene displaying sub-
stantial inhibitory activity on HCV entry, two microbial strains, Rhizopus chinensis CICC 3043 and Alter-
naria alternata AS 3.4578, were utilized to modify the chemical structure of EA. Eight new metabolites
with regio- and stereo-selective introduction of hydroxyl and lactone groups at various inert carbon
positions were obtained. The anti-HCV entry activity of the metabolites 2e13, along with their parental
compound EA and other analogs 14e15, were evaluated. Most of the metabolites showed no improve-
ment but detrimental effect on potency except compound 5 and 6, which showed similar and even a
litter higher anti-HCV entry activity than that of EA. The results demonstrated that ring A, B, C and the
left side of ring E of EA are highly conserved, while ring D and the right side of ring E of EA are flexible.
Introduction of a hydroxyl group at C-16 enhanced the triterpene potency. Further analysis indicated that
the hemolytic effect of EA disappeared upon such modifications.
Received 1 December 2012
Received in revised form
18 March 2013
Accepted 22 March 2013
Available online 3 April 2013
Keywords:
Echinocystic acid
Microbial transformation
HCV entry inhibitor
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
Hepatitis C virus (HCV) infection is the leading cause of liver
fibrosis and cirrhosis which eventually leads to liver cancer.
Pentacyclic triterpenes, mainly including lupane, oleanane, and
ursane types [1,2], are secondary plant metabolites found in
different plants, with a few species containing up to 30% of their dry
weight [3,4]. It has been proposed that triterpenes possess defense
activities and thus protect hosts from various pathogen and her-
bivore infections [5,6]. Betulinic acid, a lupane-type triterpene, has
been shown by many studies to display significant inhibiting ac-
tivity against HIV entry and maturation. One of its derivatives,
bevirimat, is currently in clinical trials [7e9]. Moronic acid and
hawthorn acid, two oleanane-type triterpenes, have also shown
anti-HIV activity with even better antiviral profiles in vitro than
bevirimat [10]. The diverse and promising biological activities of
triterpenes, including anti-inflammatory [11,12], hepatoprotective
[13], analgesic, antimicrobial, antimycotic, virostatic, immuno-
modulatory and tonic effects [14,15], warrant further investigation
of their potential pharmaceutical uses.
Treatment of HCV infection has relied on ribavirin and interferon
for almost 30 years with recently approved telaprevir and boce-
previr representing the beginning of a new era targeting HCV
replication [16]. However, resistance to antiviral drugs is likely to
develop and combination of drugs targeting different stages of HCV
viral life cycle is eventually required. Inhibition of viral entry into
HCV-permissive cells represents an emerging field for reduction or
elimination of productive infection. HCV entry inhibitors could
satisfy the tandem mechanism for use with other inhibitors of viral
replication, ultimately leading to a multifaceted approach to the
eradication of HCV infection.
Recently, we found echinocystic acid (EA, compound 1 in Fig. 1),
an oleanane-type triterpene, displayed substantial inhibitory ac-
tivity on HCV entry with one derivative showing IC50 at sub-
mM
level. Although the mechanism underlying the blocking of HCV
entry by EA is only narrowly understood, our recent study estab-
lished the importance of triterpenes serving as parent compounds
from which further potential entry inhibitors could be developed
(manuscript in preparation). Aimed at rational design of specific
inhibitors for viral entry, efforts are made to further elucidate the
relationship between their structures and activities, and to define
specific factors necessary for regulation of virus/host cell recogni-
tion. However, expansion of triterpene structural diversity via
* Corresponding author. Tel./fax: þ86 10 8280 5519.
** Corresponding author. Tel.: þ86 10 82802024.
E-mail addresses: yemin@bjmu.edu.cn (M. Ye), deminzhou@bjmu.edu.cn
(D.-M. Zhou).
1
Those authors have contributed equally to this work.
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.03.041

H. Wang et al. / European Journal of Medicinal Chemistry 64 (2013) 160e168
161
Fig. 1. The proposed route for R. chinensisemediated biotransformation of EA.
chemistry approaches is limited because of the few chemical active
sites within their skeletons. Here, we report a microbial trans-
formation strategy to allow the regio- and stereo-selective intro-
duction of hydroxyl, ketone and other groups at proper positions of
EA, which are usually difficult to achieve by chemical means.
Through such chemical expansion, ring A and B are confirmed as
highly conserved pharmacophore for blocking HCV entry and ring
D is flexible with introducing a hydroxyl group at C-16 significantly
enhancing the potency of triterpene. More importantly, ring E was
excluded as a potential modifiable site for improving potency.
same yields (Fig. 1). Alternative metabolism pathway by R. chinensis
CICC 3043 directed to C-7, yielding metabolite 6, which was then
dehydrated to form metabolite 7 (Fig. 1).
The major product by A. alternata AS 3.4578-mediated
biotransformation was identified as metabolite 12 (20.0%) and 11
(10.0%), along with minor metabolites: compound 5 (9.1%), 7 (0.8%),
8 (7.3%), 9 (2.6%), and 10 (3.1%). Based on the structures of those
metabolites, we proposed that the main modification by
A. alternata AS 3.4578 on EA was initially directed to C-1, yielding
metabolite 8, which was then transformed into 5 and 11 simulta-
neously in almost the same yields. Alternative metabolite pathway
by A. alternata AS 3.4578 directed to C-29, yielding metabolite 9,
which was then oxidized to metabolite 12. Incubation of EA with
A. alternata AS 3.4578 may also provide metabolite 7 and 10 (Fig. 2).
2. Results and discussion
2.1. Screening of microorganisms
We carried out a preliminary screening to identify which mi-
croorganisms among 13 microbial strains that were widely used in
our lab for biotransformation of steroids [17,18] and tetracyclic
triterpenoids [19] were able to transform EA. We found strain
Rhizopus chinensis CICC 3043 and Alternaria alternata AS 3.4578,
rarely used for fermentations of triterpenes, consumed substrate
EA within one week. After scale-up, 6 and 7 metabolites were
generated respectively, with a total of 8 new compounds.
The major product from R. chinensis CICC 3043-mediated
biotransformation was identified as metabolite 2 (45.2%), along
with minor metabolites: compound 3 (4.5%), 4 (4.2%), 5 (8.1%), 6
(6.5%), and 7 (6.7%). Based on the structures of those metabolites,
we proposed that the main modification by R. chinensis CICC 3043
on EA was initially directed to C-21, yielding metabolite 2, which
was then transformed into 3, 4 and 5 simultaneously in almost the
2.2. Structure elucidation of the metabolites
All the 1H and 13C NMR data of the new compounds 4e9, 11 and
12 are given in Table 1 and Table 2, respectively.
Metabolite 2 was obtained as white powder, and was identified
as the known compound acacic acid lactone, based on the strong
absorption signal at 1763 cmꢀ1 (the diagnostic IR feature for
lactone) and the identical 1H and 13C NMR spectra as previously
reported [20,21]. The change of the olefin chemical shift at dH 5.42
(H-12 of 2) to dH 6.38 (dd, J ¼ 10.5, 2.6 Hz) and dH 5.72 (d,
J ¼ 10.0 Hz), together with an identical
g-lactone IR absorption. The
structure of metabolite 3 was further confirmed by comparison of
1H and 13C NMR spectra with those reported in the literature [22].
Metabolite 4 was isolated as colorless powder. The HR-ESI-MS
showed an [M þ K]þ ion at m/z 525.2981 (calcd for C30H46O5K,

162
H. Wang et al. / European Journal of Medicinal Chemistry 64 (2013) 160e168
Fig. 2. The proposed route for A. alternataemediated biotransformation of EA.
525.2977), together with a
g
-lactone IR absorption at 1755 cmꢀ1
,
molecular formula of C30H48O5, indicating a 16 amu mass increase
comparing to that of 1. The 13C NMR and DEPT spectra exhibited a
new signal at dC 74.42 ppm, which was correlated with a new
proton signal at dH 3.85 ppm (1H, dd, J ¼ 11.1, 4.8 Hz) in the HSQC
spectrum. The HMBC spectrum disclosed long-range 1He13C cor-
relations of dH 3.85 with C-26 (dC 10.54) and C-14 (dC 44.13) and a
correlation of dC 74.42 with C-26 methyl protons (dH 0.81) (Fig. 3).
All these spectra supported hydroxylation occurred at C-7 position.
indicating it was a hydroxylated derivative of metabolite 2. The 13C
NMR spectrum exhibited a new signal at dC 75.15 ppm rather than
the original dC 19.66 ppm, a methylene attributed to C-6 in 2,
suggesting hydroxylation occurred at this position. Such deduction
was consistent with the threeebond correlation between dH
0.99 ppm (collective signals for 23-, 30- and 24-CH3) and dC
75.15 ppm observed in the HMBC spectrum (Fig. 3). The relative
stereochemistry was established as 6
trum which showed NOE enhancements between dH 1.32 (27-CH3),
dH 0.86 (H-5) and dH 3.86 (H-6).
b
-OH based on NOESY spec-
The 7-OH was deduced to be
enhancement between dH 3.85 and 27-CH3
Metabolite 7 was obtained as white solid and was determined to
have the molecular formula C30H46O5 by HR-ESI-MS. There was a
similar quarternary methyl group attached to an oxygenated car-
bon atom in the 1D NMR spectra of both 6 and 7, suggesting
b
-oriented due to the observed NOE
dH 1.43).
(
Compound 5 was obtained as white powder with its molecular
formula identified as C30H46O5 based on its HR-ESI-MS. It also
showed a lactone IR signal at 1766 cmꢀ1, suggesting it was a hy-
droxylated derivative of lactone (2). The new oxygen-bearing
methine carbon at dC 80.21 correlated with a new proton at dH
3.33 (m) in the HSQC spectrum. However, the W-type long-range
coupling of this new proton with H-3 (dH 3.17, m) was observed
in the 1He1H COSY spectrum (Fig. 3), together with the downfield
shift of C-2 (Dd 10.45 ppm) and the shift of dC 12.07 (C-25) sup-
ported a hydroxyl group introduced at C-1 position. The NOE cor-
relations of H-1 with H-5 and H-3 indicated that the introduced
b
-hydroxylation of C-7. The NOE correlation between H-7 (dH 3.73)
and 27-CH3 dH 1.30) confirmed the assignment that 7-OH was in
-orientation (Fig. 3).
Based on the structures of the metabolites in A. alternata AS
(
b
3.4578-mediated biotransformation, the initial metabolic products
were compound 8 (7.3%), 9 (2.6%) and 10 (3.1%), supposedly pro-
duced simultaneously. The secondary metabolic products from 8
were compound 5 (9.1%) and 11 (10.0%) in almost the same yield.
The secondary metabolic product from compound 9 was suppos-
edly compound 12 (20.0%) (Fig. 2). It seemed that compound 10 was
a final transformation product from the starting material. However,
hydroxyl group was b-oriented.
Metabolite 6 was isolated as white powder. The HR-ESI-MS
showed an [M þ H]þ ion at m/z 489.3576, corresponding to the

H. Wang et al. / European Journal of Medicinal Chemistry 64 (2013) 160e168
163
Table 1
1H NMR data (400 MHz in CD3OD) of compounds 4e9, 11 and 12.
Position
4
5
6
7
8
9
11
12
1
0.86(m)
3.33(m)
1.72(m)
1.89(m)
1.00(m)
1.66(m)
3.17(dd; 10.8,5.2)
0.84(m)
1.63(m)
3.73(dd; 10.6,4.4)
1.90(m)
3.34(m)
1.67(m)
1.07(m)
1.59(m)
3.17(dd; 11.7,5.0)
0.76(m)
1.58(m)
1.38(m)
1.67(m)
1.93(m)
3.45(m)
1.67(m)
1.07(m)
1.43(m)
3.21 (m)
0.84(m)
1.58(m)
1.41(m)
1.82(m)
1.93(m)
0.99(m)
5.37(m)
1.95(m)
2
3
5
6
7
9
11
1.63(m)
3.15(m)
0.86(m)
3.86(m)
1.32(m)
1.32(m)
1.98(m)
1.71(m)
3.17(m)
0.62(m)
1.65(m)
1.52(m)
3.30(m)
2.50(m)
2.13(m)
5.41(s)
2.03(dd; 14.2,5.0)
1.09(m)
3.90(dd; 11.7,4.9)
2.48(m)
1.58(m)
3.15(dd; 11.0,9.1)
0.84(m)
1.6(m); 1.50(m)
3.85(dd; 11.1,4.8)
1.65(m)
1.92(m)
1.78(m)
3.19(m)
0.64(d; 11.0)
1.58(m)
1.73(m), 1.90(m)
2.20(m)
3.17(dd; 9.0,7.7)
0.60(d; 8.4)
1.58(m)
1.51(m)
1.72(m)
2.44(m)
2.06(m)
5.28(m)
1.84(m)
1.90(m)
6.49(d; 5.0)
6.37(d; 10.2)
0.99(m)
5.34(m)
1.88(m)
12
15
5.47(s)
1.64(m)
5.34(s)
2.07(m)
1.82(d; 14.8)
4.43(s)
3.00(dd; 14.3,3.5)
5.55(d; 10.6)
2.17(m)
1.77(m)
6.69(d; 11.0)
1.38(m)
1.33(m)
4.45(s)
2.98(dd; 14.3,4.2)
16
18
3.90(m)
2.47(dd; 12.0,6.6)
4.11(s)
4.47(s)
3.05(dd; 14.0,3.5)
4.10(s)
4.47(m)
3.07(dd;
14.3,4.1)
2.27(m)
1.09(m)
1.94(m)
1.82(m)
1.78(m)
1.10(s)
0.89(s)
1.09(s)
1.07(s)
1.40(s)
3.22(s)
1.02(s)
19
1.35(m)
1.82(m)
2.30(m)
1.87(m)
2.56(d; 14.7)
1.38(m)
2.28(m)
1.00(m)
1.72(m)
1.89(m)
1.78(m)
0.94(s)
0.75(s)
0.96(s)
0.79(s)
1.37(s)
0.87(s)
0.96(s)
2.27(m)
1.09(m)
1.94(m)
1.78(m)
1.82(m)
0.99(s)
0.80(s)
0.98(s)
0.83(s)
1.40(s)
3.21(s)
1.02(s)
2.55(d; 14.6)
1.81(m)
1.38(m)
2.08(m)
1.92(m)
0.93(s)
0.74(s)
0.97(s)
0.77(s)
0.83(s)
1.37(dd; 6.7,4.3)
4.25(d; 5.5)
2.32(dd; 12.0,5.6)
2.18(d; 11.9)
0.95(s)
0.77(s)
0.97(s)
0.93(s)
1.25(s)
21
22
4.24(d,5.4)
1.23(m)
1.90(m); 1.15(m)
1.90(m)
1.72(m)
0.83(s)
0.77(s)
0.94(s)
0.81(s)
1.43(s)
0.88(s)
0.95(s)
1.91(m)
23
24
25
26
27
29
30
0.99(s)
0.99(s)
0.95(s)
0.79(s)
1.32(s)
1.01(s)
0.99(s)
0.97(s)
0.76(s)
0.90(s)
0.78(s)
1.30(s)
0.92(s)
0.83(s)
1.00(s)
0.99(s)
0.92(s)
1.25(s)
it is not clear what pathway generated compound 7. It is potentially
from intermediate 6 which was detected at day 3 but not at day 7,
during the time course of monitoring EA biotransformation (Fig. 4).
The molecular formula of metabolite 8, a white powder, was
assigned as C30H48O5 based on its HR-ESI-MS at m/z 489.3577, a 16
NMR spectral data of ring A as that of 5, and the COSY correlations
of H-1 and H-3 supported a hydroxyl group was introduced at C-1
position. The NOE enhancements between H-1 (dH 3.34), H-3
3.17) and H-5 dH 0.60) confirmed the hydroxyl group adopting
-orientation (Fig. 3).
The molecular formula of compound 9, isolated as a white po-
a (dH
a
(
b
mass unit increase over the starting material. The similar 1H and 13
C
wer, was C30H48O5 based on HR-ESI-MS [M þ H]þ at m/z 489.3578.
It was also a monohydroxylated metabolite of the starting material
based on 16 amu mass increase. The disappearance of one methyl
signal instead of an oxygenated methylene signals, dC 74.83 ppm
and dH 3.21 ppm, plus the change of C-19, C-20 and C-21 chemical
shifts in 1H and 13C NMR suggested the hydroxylation site was
either at C-29 or C-30. The NOE correlation of methyl proton signal
dH 1.02 (CH3-30) with dH 3.05 (H-18), indicated the introduced
hydroxyl group was located at C-29 (Fig. 3).
The less polar metabolite from A. alternata AS 3.4578-mediated
biotransformation was 10, which displayed a molecular formula of
C30H47O4 based on the HR-ESI-MS data. Comparison of its NMR
data with that of 1 revealed that metabolite 10 is the previously
reported 3-oxo derivative of echinocystic acid [23,24].
Metabolite 11 was also a white powder with molecular formula
of C30H46O5 based on [M þ H]þ at m/z 487.3412, the same as
metabolite 7 but 2 amu mass less than 8. The almost identical 13C
NMR spectra between metabolite 11 and 7 but an oxygenated
quaternary carbon, dC 80.67 ppm versus dC 74.04 ppm, indicated
their structure similarity. The HSQC correlation of this new
oxygenated carbon with dH 3.45 ppm, which displayed a correlation
Table 2
13C NMR data (100 MHz in CD3OD) of compounds 4e9, 11 and 12.
Position
4
5
6
7
8
9
11
80.67
38.47
76.83 220.56
12
40.62
35.07
1
2
3
4
5
6
7
8
30.29
27.91
79.35
39.60
54.21
75.15
39.97
45.24
48.49
38.58
24.58
80.21
38.34
76.72
44.20
54.68
19.09
33.40
41.78
49.17
39.86
28.08
39.80
27.94
79.46
39.60
54.06
30.53
74.42
46.41
48.32
38.26
39.10
27.77
79.46
39.12
53.98
30.52
74.04
39.67
54.18
37.70
80.60
38.34
76.87
41.15
54.69
19.16
34.38
42.48
48.95
39.85
28.07
39.91
27.89
79.70
39.84
56.89
19.50
34.26
40.67
48.20
38.16
42.51
53.78
18.92
33.43
43.02
55.83
39.97
47.33
56.55
20.24
33.69
40.32
48.47
38.23
24.54
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
24.53 127.00
24.46 125.67
126.31 126.98 123.69 126.37 124.55 123.65 131.89 123.46
140.45 139.56 144.74 137.00 143.98 144.87 137.44 144.95
46.35
39.62
67.86
50.64
43.31
43.60
34.95
85.25
27.61
28.64
16.42
16.13
10.82
29.16
44.29
38.02
67.94
50.72
42.61
44.01
34.86
85.19
27.49
28.56
16.00
12.07
16.93
29.19
44.13
39.53
75.44
49.64
43.68
36.54
71.43
48.20
44.29
36.28
75.33
49.55
41.94
47.69
31.40
36.60
32.28
28.61
16.01
11.97
18.06
27.18
42.63
36.21
75.04
50.08
43.70
37.61
71.41
54.39
42.45
36.61
74.96
50.10
41.38
42.78
36.19
37.90
31.29
27.10
21.87
15.68
17.63
27.25
42.84 131.24
41.30 129.37
47.76
31.40
36.57
32.36
28.66
16.38
16.01
10.54
27.24
41.68
32.81
37.54
29.57
28.33
15.71
18.80
12.41
22.06
42.47
36.60
31.35
31.30
28.74
16.33
16.03
17.77
27.32
41.40
32.79
37.61
29.45
28.40
15.55
14.88
17.54
24.96
with 25-CH3 (dH 0.97 ppm) in HMBC and a W-type long-range
coupling with H-3 (dH 3.19 ppm, m) in the 1He1H COSY spectrum,
demonstrated that C-1 was hydroxylated. The NOE correlations H-1
with H-3 and H-5 indicated its b-orientation (Fig. 3).
Compound 12 had a molecular formula of C30H46O5 as deter-
mined by HR-ESI-MS ([M þ H]þ at m/z 487.3409), 2 amu mass less
than that of metabolite 9. The almost identical 1H and 13C NMR
between compound 12 and 9, together with the appearance of a
carbonyl signal at dC 220.56 ppm but disappearance of a methine
183.22 183.11 181.16 179.94 181.16 181.05 180.00 180.98
28.93
24.35
28.96
24.26
33.41
24.91
32.90
24.99
33.43
24.88
74.83
20.25
32.92
21.47
74.80
20.75

164
H. Wang et al. / European Journal of Medicinal Chemistry 64 (2013) 160e168
7
8
9
11
Fig. 3. Schematic representation of key HMBC, COSY and NOE correlations of EA metabolites 4e9 and 11.
proton signal corresponding to C-3 (dH 3.17) suggested C-3 was a
ketone, which was further supported by the downfield shifts of C-2
and C-4 by 7.01 and 7.26 ppm, respectively.
remained to be elucidated whether the same enzyme is responsible
for formation of such lactones.
In order to test whether the isolated compounds are single iso-
mers, enantiomeric mixtures or diastereomeric mixtures, we per-
formed asymmetric HPLC analyses using a chiral column. As shown
in Fig. R1 (Supporting information), all products were clearly sepa-
rated with only one single peak detected for each of all products,
suggesting all isolated products were optically pure with >95% pu-
rity. Combinations of the clear 1H, 13C NMR data and the asymmetric
HPLC results excluded the possibility that the isolated products were
enantiomeric or diastereomeric mixtures but single isomers.
2.4. The effect of 16-hydorxyl group on biotransformation of
triterpenes
Another remarkable observation in this study was the signifi-
cant contribution of 16-hydroxyl that boosted the efficiency on
biotransformation of triterpene. A 7-day fermentation of EA with
R. chinensis CICC 3043 and A. alternata AS 3.4578 converted around
80% starting material into various metabolites. In contrast, a par-
allel fermentation of oleanolic acid (OA) by both fungi harvested
nothing but the starting material. The molecular structure of EA is
only slightly different from that of OA, eCHOHe versus eCH2e at C-
16. The poor biotransformation of OA is likely due to the lack of the
hydroxyl group at C-16, which presumably activates the backbone
of EA for introducing multiple hydroxyl groups at various positions.
A reciprocal subjection of ursolic acid and betulinic acid, two
lupane- and ursane-type triterpenes both lack of 16-OH, to the
transformation by both fungi afforded nothing but the starting
material, which also supported such conclusion.
2.3. Lactonization of EA by R. chinensis CICC 3043
One major feature observed in R. chinensis CICC 3043emediated
biotransformation was the formation of a lactone group in most of
the metabolites. Such regio- and stereo-selective condensation of
21-CH2 with 28-COOH is almost impossible by routine chemical
means due to the inert characteristics of 21-CH2. The lactone for-
mation suggests the presence of a potential enzyme in R. chinensis
CICC 3043 that displays strong capability for intramolecular ester-
ification. Such enzyme, once identified, should have broad appli-
cations for diversity expansion of a variety of triterpenes. Lipase
named RCL-lip2, isolated from R. chinensis CCTCC M201021, was
found to catalyze the esterification of short-chain fatty acids [24]. It
2.5. The pharmacophore of EA for blocking HCV entry
We have demonstrated that triterpenoid natural products might
serve as a distinct chemical class for the development of potent

H. Wang et al. / European Journal of Medicinal Chemistry 64 (2013) 160e168
165
Fig. 4. LC-MS detected by m/z 486 profile of compound 4 (a) and compound 1’s metabolites obtained from the extraction of the A. alternata culture medium at t ¼ 72 h (b) and
t ¼ 168 h (c); mass spectrum of compound 4 giving m/z 487.53 [M þ H]þ (d); mass spectrum of peak B giving m/z 487.56 [M þ H]þ (e).
HCV entry inhibitors. Evaluation of EA analogs obtained in this
study may help to clarify the triterpene SAR, in particularly, the
pharmacophore of EA for blocking HCV entry. Initial SAR explora-
tion at ring A and B, including hydroxylation at C-1 (8) or C-7 (6)
and oxidation of C-3 (10), indicated all modifications significantly
decreased the potency of EA (Table 3), confirming the previous
observation that the left side of EA is conserved (Fig. 5). Modifica-
tions at the middle side of EA, such as dehydrations at ring C
together with a hydroxyl group at either C-1 (11) or C-7 (7) or with
a lactone between C-28 and C-21 (3), also significantly decreased
the potency of EA (Table 3). This is also consistent with previous
observations that chemical introduction of a hydroxyl (14) or a
ketone (15) group at this region abolished the function of EA [25].
Continuous exploration at ring D including hydroxylation at
C-16 (EA versus OA) resulted in significant increase in potency
(Table 3), suggesting ring D of oleanane-type triterpene is tolerant
to modification and introduction of proper groups other than hy-
droxyl may further enhance its potency (Fig. 5). However, modifi-
cations at ring E, including hydroxylation at C-29 (9) and
lactonization of C-28 with C-21 (2), reduced EA potency by over
50%. Further modifications of metabolite 2, the hydroxylation at C-1
(5) and C-6 (4), showed the similar or even a little higher anti-HCV
entry activities. However, dehydration at C-11 and C-12 and hy-
drolysis of the lactone (13 and acacic acid lactone [26e28]) showed
no improvement but detrimental effect on potency (Table 3). All
data excluded ring E modification, but ring D was a potential
modifiable site for further chemical modification (Fig. 5). This result
led us to focus efforts on chemical optimization of ring D for
improving the potency of EA. One derivative showed IC50 at 10 nM
for blocking HCV entry [25].
Table 3
Effect of metabolites 2e13 and compounds14e15 on blocking HCV and VSV entry.
Compound
Anti-HCV (%)
Anti-VSV (%)
1
mM
5
m
M
1
m
M
5 mM
2
3
4
5
6
7
8
9
13.9 ꢁ 2.1
11.6 ꢁ 1.7
ꢀ1.0 ꢁ 0.4
28.9 ꢁ 3.2
30.4 ꢁ 2.9
22.2 ꢁ 1.3
14.3 ꢁ 0.7
16.5 ꢁ 2.3
7.5 ꢁ 1.3
31.9 ꢁ 2.3
31.7 ꢁ 2.0
4.7 ꢁ 0.9
ꢀ17.3 ꢁ 5.6
ꢀ16.2 ꢁ 3.2
ꢀ8.3 ꢁ 2.6
1.8 ꢁ 1.2
ꢀ12.1 ꢁ 4.9
ꢀ10.3 ꢁ 2.8
ꢀ24.0 ꢁ 3.0
19.2 ꢁ 1.7
5.6 ꢁ 1.9
72.6 ꢁ 2.8
53.5 ꢁ 3.1
31.9 ꢁ 1.9
35.2 ꢁ 1.2
36.5 ꢁ 1.7
41.6 ꢁ 1.8
26.3 ꢁ 0.8
ꢀ3.3 ꢁ 0.7
18.4 ꢁ 1.8
ꢀ8.7 ꢁ 0.7
51.0 ꢁ 0.2
75.5 ꢁ 1.7
1.6 ꢁ 1.1
ꢀ13.7 ꢁ 2.0
7.9 ꢁ 2.6
ꢀ4.4 ꢁ 1.9
11.8 ꢁ 2.1
3.6 ꢁ 1.1
ꢀ2.5 ꢁ 0.9
ꢀ2.5 ꢁ 1.0
ꢀ15.8 ꢁ 2.3
ꢀ20.0 ꢁ 3.7
20.7 ꢁ 0.8
42.7 ꢁ 3.5
35.8 ꢁ 1.5
ꢀ0.1 ꢁ 1.3
10
11
12
13
14a
15a
EA(1)
13.6 ꢁ 1.5
5.9 ꢁ 1.2
2.3 ꢁ 0.6
3.1 ꢁ 0.0
10.7 ꢁ 4.3
33.2 ꢁ 0.6
46.1 ꢁ 4.1
43.5 ꢁ 0.1
ꢀ0.1 ꢁ 0.7
ꢀ5.7 ꢁ 2.3
4.9 ꢁ 0.3
22.6 ꢁ 1.9
21.3 ꢁ 2.1
Fig. 5. Schematic representation of the conserved and modifiable sites within
oleanane-type triterpenes for blocking HCV entry.
a
Compounds 14 and 15 were semi-synthesized from EA [25].

166
H. Wang et al. / European Journal of Medicinal Chemistry 64 (2013) 160e168
2.6. Depletion of the hemolytic effect of EA
enantiomeric mixtures or diastereomeric mixtures, we further
performed HPLC using Agilent 1100 HPLC system and Shiseido
A series of studies have demonstrated that the aglycons of tri-
terpenoids play an essential role in the hemolytic properties [29e
31], one of the well known characteristics of saponins. Compared
with OA which possesses mild hemolytic activity, EA which gains a
hydroxyl group at C-16, showed substantial hemolytic effect with
Capcell Pak Chiral CD-Ph C18 chromatographic column (5 mm,
4.6 mm ꢂ 250 mm) (Shiseido Co., Ltd., Tokyo, Japan), which
frequently utilized in the enantiomeric impurity determination
[32,33], under UV 215 nm. Mobile phase was acetonitrile (A) and
water (B) with gradient as: 0e25 min, 20e60% A; 25e40 min, 60e
90% A; 40e45 min, 90% A, and flow rate at 1.0 mL/min.
CC50 at 15 mM (Fig. 6). Such hemolytic property, once enhanced in
those afforded triterpene metabolites, may restrict their pharma-
ceutical use as potential anti-HCV entry inhibitors. Here, we found
that hydroxylation of EA at C-1 (8), C-7 (6), C-29 (9), oxidation of C-
3 (10) and lactonization of C-28 (2) depleted the hemolytic prop-
erty (Fig. 6). Further modifications of those compounds with for-
mation of lactones (4 and 5) or dehydration (3) did not increase
their hemolytic effect (Fig. 6). It is possible that the biotransfor-
mation depletes the intrinsic hemolytic property of EA, which de-
serves further exploration.
Column chromatography was performed on silica gel (200e300
mesh, Qingdao Haiyang Chemical Co. Ltd, China), and Sephadex LH-
20 (Pharmacia Biotech AB, Sweden). Fractions were monitored by
TLC, and spots were visualized by spraying the plate with 10%
H2SO4 solution in EtOH followed by heating at 110 ꢃC. All reagents
and solvents were of analytical grade.
4.2. Microorganisms and culture media
The substrate EA was isolated from Gleditsia japonica and
Gymnocladus chinensis [34]. A total of 13 different fungal strains,
including Alternaria alternata AS 3.4578, Doratomyces stemonitis AS
3.1411, Fusarium avenaceum AS 3.4594, Mucor spinosus AS 3.3450,
R. chinensis CICC 3043, Syncephalastrum racemosum AS 3.264,
Cunninghamella elegans AS 3.1207, Chaetomium globosum IFFI 2445,
Crebrothecium ashbyii ACCC 2114, Absidia coerulea AS 3.3389,
Aspergillus niger AS 3.795, Gibberella fujikuroi var. fujikuroi AS
3.4748, Saccharomyces cerevisiae ACCC 2168, were screened in the
preliminary test for their capabilities to metabolize EA. All fungal
strains were purchased from China General Microbiological Culture
Collection Center (CGMCCC) or China Center of Industrial Culture
Collection (CCICC). All of the microorganisms were freshly sub-
cultured prior to the biotransformation experiments. All pre-
liminary screening experiments were performed by a two-stage
fermentation procedure in potato media.
3. Conclusion
In summary, we have expanded the structural diversity of EA via
biotransformation and elucidated its pharmacophore that is
needed for blocking HCV entry. We found most of the metabolites
showed no improvement but detrimental effect on the potency
except compound 5 and 6, which showed similar and even a litter
higher anti-HCV entry activity than that of EA. We concluded that
ring A, B, C and the left side of ring E of EA are highly conserved,
while ring D and the right side of ring E of EA, including C-28, are
flexible. Introduction of proper groups at proper positions may
enhance the potency of triterpenes. In addition, the substantial
hemolytic property of EA disappeared upon such bio-modifications.
4. Experimental
Fermentations were carried out in potato media consisting of
20 g of potato extract (prepared from 200 g of potato slices
extracted in boiling water for 30 min), 20 g of glucose, and 1000 mL
of water. The media were sterilized at 121 ꢃC and 1.06 kg/cm2 for
20 min. The strains were maintained on potato dextrose-agar (PDA)
medium slants and stored at 4 ꢃC.
4.1. Chemistry-general
Melting points were determined with an X-4 apparatus and
uncorrected. Optical rotations were measured with a Rudolph
Autopol III polarimeter. UV spectra were detected with a TU-1901
UVeVis spectrophotometer. IR spectra were recorded in KBr with
a NEXUS-470 FT-IR spectrophotometer. 1H and 13C NMR spectra
were recorded in CD3OD on Bruker Avance III spectrometer oper-
ating at 400 and 100 MHz. High Resolution Mass Spectra (HRMS)
were obtained on an APEX IV FT_MS (7.0 T) spectrometer (Bruker)
in positive ESI mode. Reversed-phase preparative HPLC was per-
formed on an Alltech 426 HPLC system, using an Agilent Zorbax SB
4.3. Biotransformation of EA by R. chinensis CICC 3043
Mycelia of R. chinensis CICC 3043 from agar slants were asepti-
cally transferred to 1000 mL Erlenmeyer flasks containing 400 mL
of liquid potato medium. The fungus was incubated at 25 ꢃC on a
rotary shaker (180 rpm). After 48 h incubation, a total amount of
900 mg of EA dissolved in 50 mL of EtOH was distributed equally
among 50 flasks. The incubation was allowed to continue for seven
additional days on the shaker. The cultures were then pooled and
filtered through a filter paper. The filtrate was extracted with equal
volume of EtOAc for three times. The organic layer was dried over
anhydrous Na2SO4, filtered, and evaporated in vacuo to afford a
brown syrup. The syrup was subjected to column chromatography
on silica gel using petroleum ether/EtOAc (98:2 to 0:100, v/v) as
eluting system to afford fifteen fractions, followed by a purification
step on a Sephadex LH-20 column. Fr. 3e5 was subjected to pre-
parative HPLC and eluted with CH3CNeH2O (50:50, v/v) to give 2
(76.5 mg). Fr. 8e10 was purified by preparative HPLC and eluted
with CH3CNeH2O (50:50, v/v) to give 6 (39.4 mg). Fr. 11 was sub-
jected to preparative HPLC and eluted with CH3CNeH2O (50:50,
v/v) to give 5 (50.4 mg). Fr. 12e14 was subjected to preparative
HPLC and eluted with CH3CNeH2O (45:55, v/v) to give 4 (21.6 mg).
Fr. 15 was subjected to preparative HPLC and eluted with CH3CNe
H2O (50:50, v/v) to give 3 (27.7 mg) and 7 (41.5 mg), together with
280 mg untransformed 1.
C18 chromatographic column (5
detection at 215 nm and RI 2000 detector. In order to confirm
whether the synthesized compounds are single isomers,
mm, 9.4 mm ꢂ 250 mm) with UV
Fig. 6. Hemolytic properties of EA metabolites.

H. Wang et al. / European Journal of Medicinal Chemistry 64 (2013) 160e168
167
4.3.1. Acacic acid lactone (2)
744 cmꢀ1; the asymmetric HPLC showed the purity was 95.7%; 1
H
Colorless needles; mp 240e242 ꢃC; ½a D24
ꢄ
54.3 (c 0.08, MeOH);
and 13C NMR data see Tables 1 and 2; HR-ESI-MS m/z 489.3577
[M þ H]þ (calcd for C30H49O5, 489.3575).
the asymmetric HPLC showed the purity was 96.4%; 1H and 13C
NMR data were consistent with reported data.20
4.4.2. 3b,16a,29-Trihydroxy-olean-12-en-28b-oic acid (9)
4.3.2. 3
lactone (3)
b
,16
a
-Dihydroxy-olean-11,13(18)-dien-28
b
-oic acid-21-
White amorphous solid; mp 208e210 ꢃC; ½a D25
ꢄ
156.0 (c 0.04,
MeOH); IR (KBr) nmax 3435, 2930, 2519, 1678, 1639, 1465, 1044, 999,
469 cmꢀ1; the asymmetric HPLC showed the purity was 95.5%; 1H
and 13C NMR data see Tables 1 and 2, HR-ESI-MS m/z 489.3578
[M þ H]þ (calcd for C30H49O5, 489.3575).
White amorphous powder; mp 184e186 ꢃC; ½a D25
ꢀ39.8 (c 0.13,
ꢄ
MeOH); IR (KBr) nmax 3444, 2937, 1763, 1464, 1035, 993, 578 cmꢀ1
;
the asymmetric HPLC showed the purity was 95.4%; 1H and 13C
NMR data were consistent with reported data [21].
4.4.3. 3-Oxo-16a-Hydroxy-olean-12-en-28b-oic acid (10)
4.3.3. 3
b
,6
b
,16
a
-Trihydroxy-olean-12-en-28
b
-oic acid-21-lactone
White amorphous solid; mp 230e233 ꢃC; ½a D24
ꢄ
151.6 (c 0.14,
(4)
MeOH); IR (KBr) nmax 3524, 2949, 1719, 1679, 1449, 1224, 1004,
White amorphous powder; mp 182e184 ꢃC; ½a D25
ꢄ
ꢀ40.9 (c 0.16,
707 cmꢀ1; the asymmetric HPLC showed the purity was 98.1%; 1
H
MeOH); IR (KBr) nmax 3440, 2944, 2527,1755,1640,1466,1392,1175,
1039, 621 cmꢀ1; the asymmetric HPLC showed the purity was
96.6%; 1H and 13C NMR data see Tables 1 and 2; HR-ESI-MS m/z
525.2981 (calcd for C30H46O5K, 525.2977).
NMR, 13C NMR and HRMS data were consistent with reported
data.22,23
4.4.4. 1
b
,3
b
,16
a
-Trihydroxy-olean-11,13(18)-dien-28
b
-oic acid (11)
White amorphous solid; mp 188e190 ꢃC; ½a D25
ꢄ
ꢀ48.3 (c 0.06,
4.3.4. 1
b
,3
b
,16
a
-Trihydroxy-olean-12-en-28
b
-oic acid-21-lactone
MeOH); IR (KBr) nmax 3433, 2923, 1636, 1457, 1060, 585, 457 cmꢀ1
;
(5)
the asymmetric HPLC showed the purity was 95.1%; 1H and 13C
NMR data see Tables 1 and 2; HR-ESI-MS m/z 487.3412 [M þ H]þ
(calcd for C30H47O5, 487.3418).
White amorphous powder; mp 198e199 ꢃC; ½a D25
ꢀ98.8 (c 0.23,
ꢄ
MeOH); IR (KBr) nmax 3462, 2951, 1766, 1467, 1042, 995, 619 cmꢀ1
;
the asymmetric HPLC showed the purity was 95.1%; 1H and 13C
NMR data see Tables 1 and 2; HR-ESI-MS m/z 504.3691 [M þ NH4]þ
(calcd for C30H50NO5, 504.3684).
4.4.5. 3-Oxo-16a,29-Dihydroxy-olean-12-en-28b-oic acid (12)
White amorphous solid; mp 200e202 ꢃC; ½a D24
ꢄ
279.0 (c 0.65,
MeOH); IR (KBr) nmax 3455, 2932, 2866, 1699, 1461, 1386, 1044, 974,
534 cmꢀ1; the asymmetric HPLC showed the purity was 96.6%; 1H
and 13C NMR data see Tables 1 and 2, HR-ESI-MS m/z 487.3409
[M þ H]þ (calcd for C30H47O5, 487.3418).
4.3.5. 3b,7b,16a-Trihydroxy-olean-12-en-28b-oic acid (6)
White amorphous powder; mp 233e235 ꢃC; ½a D24
ꢄ
118.2 (c 0.17,
MeOH); IR (KBr) nmax 3510, 3433, 2949, 1690, 1632, 1467, 1042, 984,
718 cmꢀ1; the asymmetric HPLC showed the purity was 97.8%; 1H
and 13C NMR data see Tables 1 and 2; HR-ESI-MS m/z 489.3576
[M þ H]þ (calcd for C30H49O5, 489.3575).
4.5. Alkaline hydrolysis of compound 2 (13)
A sample of compound 2 (14.9 mg, 0.032 mmol) was saponified
in 1% KOH/EtOH (5 mL) for 2.5 h at 60 ꢃC. After neutralization with
0.5 N HCl/EtOH, the mixture was separated by silica gel chroma-
tography to give compound 13 (12.9 mg, 84%) as white amorphous
powder. mp 213e215 ꢃC; the asymmetric HPLC showed the purity
was 98.2%; 1H NMR, 13C NMR and HRMS data were consistent with
reported data [35].
4.3.6. 3
b
,7
b
,16
a
-Trihydroxy-olean-11,13(18)-dien-28
b
-oic acid (7)
ꢀ141.6 (c 0.28,
White amorphous powder; mp 185e187 ꢃC; ½a D24
ꢄ
MeOH); IR (KBr) nmax 3440, 2942, 1701, 1463, 1042, 979, 721 cmꢀ1
;
the asymmetric HPLC showed the purity was 95.1%; 1H and 13C
NMR data see Tables 1 and 2; HR-ESI-MS m/z 487.3419 [M þ H]þ
(calcd for C30H47O5, 487.3418).
4.4. Biotransformation of EA by A. alternata AS 3.4578
4.6. HCV and VSV pseudovirus entry assay
Different from the process by R. chinensis CICC 3043, mycelia of
A. alternata AS 3.4578 from agar slants were aseptically transferred
to 250 mL Erlenmeyer flasks containing 100 mL of liquid potato
medium. The fungus was incubated at 25 ꢃC on a rotary shaker
(180 rpm) in the dark for 48 h to make a stock inoculum. Then 5 mL
of the inoculum was added to each of the 1000 mL flasks containing
400 mL of potato medium. After 48 h incubation, a total amount of
900 mg of EA dissolved in 50 mL of EtOH was distributed equally
among 50 flasks. The incubation was allowed to continue for seven
additional days on the shaker. The cultures were then pooled and
filtered through a filter paper. The filtrate was extracted with equal
volume of EtOAc for three times. The EtOAc extract as a brown syrup
was subjected to column chromatography, PR-18 and Sephadex LH-
20, which was similar to that described above for the biotransfor-
mation of EA by R. chinensis CICC 3043, to give 5 (28.3 mg), 7
(5.6 mg), 8 (50.8 mg), 9 (18.2 mg), 10 (22.1 mg), 11 (63.1 mg), and 12
(132.6 mg), together with 200 mg untransformed EA.
All compounds were tested using the HCV and VSV pseudo
particle (HCVpp and VSVpp) entry assay as described previously
[36,37].
4.7. Hemolytic assay
Hemolytic activity was measured as following [38]: 2% rabbit
red blood cells (5 ꢂ 108 RBC/mL) in PBS buffer (pH 7.4) were
incubated with serially diluted compounds. After incubating for
60 min at 37 ꢃC, hemolysis was monitored by measuring absorption
at 541 nm with a microplate reader. Percentage of hemolysis was
then calculated by the routine method.
Acknowledgments
This work was supported by the National Basic Research Pro-
gram of China (973 Program; grant no. 2010CB12300), the National
Natural Science Foundation of China (grant nos. 20932001
20852001 and 81101239), Beijing Natural Science Foundation
(grant no. 7102103), and Beijing NOVA Program (grant no.
2009B02).
4.4.1. 1b,3b,16a-Trihydroxy-olean-12-en-28b-oic acid (8)
White amorphous solid; mp 185e187 ꢃC; ½a D25
ꢄ
179.6 (c 0.26,
MeOH); IR (KBr) nmax 3435, 2948, 1695, 1677, 1468, 1275, 998,

168
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