Synthesis of dammarane-type triterpene derivatives and their ability to inhibit HIV and HCV proteases

Article abstract of DOI:10.1016/j.bmc.2009.03.019

We synthesized dammarane-type triterpene derivatives and evaluated their ability to inhibit HIV-1 and HCV proteases to understand their structure-activity relationships. All of the mono- and di-succinyl derivatives (5a-5f) were powerful inhibitors of HIV-

Full text of DOI:10.1016/j.bmc.2009.03.019

Bioorganic & Medicinal Chemistry 17 (2009) 3003–3010
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of dammarane-type triterpene derivatives and their ability
to inhibit HIV and HCV proteases
Ying Wei a,b, Chao-Mei Ma a, Masao Hattori a,
*
a Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan
b Department of Pharmacy, Guiyang College of Traditional Chinese Medicine, 50 Shidong Road, Guiyang, Guizhou 550002, China
a r t i c l e i n f o
a b s t r a c t
Article history:
We synthesized dammarane-type triterpene derivatives and evaluated their ability to inhibit HIV-1 and
HCV proteases to understand their structure–activity relationships. All of the mono- and di-succinyl
Received 8 February 2009
Revised 8 March 2009
Accepted 10 March 2009
Available online 14 March 2009
derivatives (5a–5f) were powerful inhibitors of HIV-1 protease (IC50 < 10
(5e) and 2,3-seco-2,3-dioic acid (3b) derivatives similarly inhibited HCV protease (IC50 < 10
dammarane-type triterpenes (4a and 4b, IC50 10.0 and 29.9 M, respectively) inhibited HIV-1 protease
lM). However, only di-succinyl
l
M). A-nor
l
moderately or strongly, but were inactive against HCV protease. All compounds that powerfully inhibited
HIV-1 or HCV protease did not appreciably inhibit the general human proteases, renin and trypsin
(IC50 > 1000 lM). These findings indicated that the mono-succinyl dammarane type derivatives (5a–
5d) selectively inhibited HIV-1 protease and that the di-succinyl (5e, 5f) as well as 2,3-seco-2,3-dioic acid
(3b) derivatives preferably inhibited both viral proteases.
Keywords:
Dammarane
Triterpene
2,3-seco-Derivative
Succinyl derivative
HIV-1 protease
HCV protease
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
tious disease due to inadequate access to HIV prevention and treat-
ment.7 As with hepatitis C, clinical options are currently limited,
The retroviral human immunodeficiency virus type-1 (HIV-1), is
a causative agent of acquired immunodeficiency syndrome (AIDS).
One important enzyme necessary for the maturation and infectiv-
ity of this virus is the aspartic protease, HIV-1 protease (HIV-1 PR),
which functions in the dimeric form composed of monomers of 99
amino acids. Thus, HIV-1 PR is an important target of searches for
anti-HIV drugs.1
On the other hand, the single-stranded RNA flavivirus hepatitis
C virus (HCV), causes type C hepatitis, which often leads to liver
cirrhosis, hepatic failure and hepatocellular carcinoma.2 Its gen-
ome of about 9.6 kb encodes the structural proteins C, E1, E2,
and the nonstructural proteins NS2, NS3, NS4A, NS4B, NS5A and
NS5B. Since the NS3-NS4A protease plays a critical role in HCV viral
replication, this dimeric serine protease has been viewed as a
promising target for screening anti-HCV drugs.3,4
Currently approved anti-HIV drugs, including highly active anti-
retroviral therapy (HAART) with a combination of HIV PR inhibitors
and reverse transcriptase inhibitors, have remarkably decreased
the mortality of AIDS patients and improved the quality of life of
those infected with HIV.5,6 However, because HIV easily mutates
and rapidly develops drug resistance, it remains a leading cause
of death worldwide, resulting in more deaths than any other infec-
and the only available drugs are subcutaneous pegylated interferon
(pegIFN- ) alone or in combination with oral ribavirin (Rbv), the
a
therapeutic effectiveness of which is only 50% for HCV infected pa-
tients.8–10 In light of the potential offered by chemoprevention, no-
vel effective and safe agents are urgently required.11 We reported
that some natural products and their derivatives have anti-HIV-1
PR activity.12 Of these, some triterpenes, such as ganoderic acid B
and ganoderiol B,13 N-(3b-hydroxylolean-12-en-28-oyl)-6-amino-
hexanoic acid,14 hemiesters of ursolic acid,15 3-oxotirucalla-7,24-
dien-21-oic acid,16 16b-hydroxy-2,3-seco-lup-20(29)-en-2,3-dioic
acid17 and colossolactone V,18 are potent inhibitors of HIV-1 PR.
We also reported that embelin, 5-O-methylembelin19 as well as
some indole derivatives20 inhibited HCV PR. Several studies over
the past decade have demonstrated that pentacyclic triterpenes
especially oleanolic acid (OA), ursolic acid (UA), betulinic acid
(BA) and glycyrrhetic acid (GA) as well as their derivatives inhib-
ited HIV.21,22 However, the abilities of tetracyclic triterpenes, espe-
cially dammarane-type triterpene derivatives to inhibit HIV and
HCV PRs have not been reported. Ginseng roots contain abundant
ginsenosides mainly consisting of dammarane-type triterpenes as
the aglycones, such as protopanaxadiol and protopanaxatriol.23
However, these aglycones convert to panaxadiol (PD) and panaxat-
riol (PT), respectively under strong acid conditions.24 PD and PT
have the same A ring moiety, but PT possesses one more hydroxyl
* Corresponding author. Tel.: + 81 76 434 7630; fax: + 81 76 434 5060.
E-mail address: saibo421@inm.u-toyama.ac.jp (M. Hattori).
group in B ring, that is, PT is 6a-hydroxy-PD. Panaxadiol has two
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.03.019

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Y. Wei et al. / Bioorg. Med. Chem. 17 (2009) 3003–3010
hydroxyl groups at C-3 and C-12 in the A and C rings, respectively,
whereas PT has three hydroxyl groups at C-3, C-6 and C-12 in the
A, B and C rings, respectively. These functional groups provide foot-
holds to convert A—seco and acylated derivatives. Here, we de-
scribe the preparation of dammarane-type triterpene derivatives
by modifying the hydroxyl groups in the A, B and C rings, and their
abilities to inhibit HIV-1 and HCV PRs as well as the human aspar-
tyl and serine proteases renin and trypsin, respectively.25,26
derivative 3a was treated with the Jones reagent to yield the 12-
oxo-2,3-seco-2.3-dioic acid derivative 3b (67.8%). To determine
the orientation of the 2-carboxyl or 2-hydroxyl group of A-nor
derivatives, compounds 4a and 4b were treated with trimethylsi-
lyldiazomethane in methanol to afford the methyl esters 4c
(72.5%) and 4d (48.2%), respectively. The orientation of a 2-
methoxycarbonyl group in compounds 4c and 4d was confirmed
by significant NOE correlations in a NOESY experiment, which
identified the following correlations. Proton signals of the 2-meth-
oxylcarboxyl group in compound 4c correlated with methyl signals
at d 0.97 (H3-29, b-orientation) and d 0.89 (H3-19, b-orientation),
suggesting that the 2-methoxylcarboxyl is oriented to the b-face
2. Results and discussion
2.1. Preparation of various dammarane type derivatives
and that the 2-hydroxyl is consequently oriented to the
a-face. A
methoxyl signal of d 3.78 (2-OCH3) in compound 4d correlated
with proton signals at d 2.65 (H-1b, b-orientation), d 2.18 (H-9,
b-orientation), d 0.94 (H3-19, b-orientation) and d 0.89 (H3-29, b-
orientation), suggesting that a 2-COOCH3 group is in the b-orienta-
Acid hydrolysis of a ginseng extract yielded (20R)-PD and (20R)-
PT,24 which were used as starting materials to prepare various 2,3-
seco, A-nor and acylated dammarane-type triterpene derivatives by
modification of the A, B and C rings.
tion and thus a 2-hydroxyl is in the a-orientation. Under the same
reaction conditions described for preparation of the A-nor deriva-
tive 4a from (20R)-PD, the corresponding compound 4b was ob-
tained from (20R)-PT simply by two-step oxidation.
2.1.1. Synthesis of 2,3-seco and A-nor dammarane type
triterpene derivatives
Scheme 1 shows the synthetic routes of various 2,3-seco and
other oxidized derivatives of (20R)-PD and (20R)-PT that were
essentially developed by Urban et al.27 and Nagai et al.28 We ini-
tially oxidized (20R)-PD or (20R)-PT to 3-oxo derivatives 1a
(92.0%) or 1b (75.5%) using pyridinium chlorochromate (PCC).
Thereafter, compound 1a or 1b was further oxidized at the A ring
by introducing air into a t-BuOH solution of 1a or 1b in the pres-
ence of t-BuOK to afford the 2-hydroxy-3-oxo-1-en derivative 2a
(95.0%) or A-nor dammarane-type triterpene 4b (6.2%). Compound
2a was then oxidized by the combined action of hydrogen peroxide
and KOH in refluxing MeOH to obtain the major products, which
were the seco-derivatives 3a (38.8%), and the minor product, the
A-nor derivative 4a (2.0%). Finally, the 2,3-seco-2,3-dioic acid
2.1.2. Synthesis of (20R)-20,25-epoxy-dammaran-3,12-dione
(1c) and (20R)-20,25-epoxy-dammaran-3,6,12-trione (1d)
To investigate the effects of hydroxyl groups in (20R)-PD and
(20R)-PT on inhibitory activity against HIV and HCV PRs, (20R)-
PD and (20R)-PT were treated with the Jones reagent for 3 h at
room temperature to afford the corresponding oxidized com-
pounds 1c (25.0%) and 1d (25.3%) (Scheme 1).
2.1.3. Synthesis of 2,2-dimethylsuccinyl derivatives
Scheme 2 shows the synthetic routes of acylated (20R)-PD and
(20R)-PT with 2,2-dimethylsuccinic acid, followed by oxidation of a
O
H
O
H
O
H
OH
OH
OH
a
HO
O
O
HO
R
(20R)-Panaxadiol:
(20R)-Panaxatriol:
b
R
R
R = H, H
R = H, α OH
1a : R = H, H
2a : R = H, H
c
1b :
R = H, α OH
d
O
H
O
H
O
H
O
OH
OH
b
HOOC
HO
HOOC
HOOC
O
R
R
4a :
4b :
1c :
d
3a
R = H, H
R = O
R = H, H
1d : R = O
e
O
H
O
H
OH
O
H3COOC
HOOC
HOOC
HO
R
4c :
4d :
R = H, H
R = O
3b
Scheme 1. Synthesis of 2,3-seco and other oxidized derivatives of PD and PT. Reagents and conditions: (a) PCC, rt, 6 h; (b) O2, t-BuOK, t-BuOH, 40 °C, 40 min; (c) H2O2, KOH,
MeOH, reflux, 100 min; (d) Jones reagent, rt, 3 h; (e) MeOH, trimethylsilyldiazomethane, rt, 30 min.

Y. Wei et al. / Bioorg. Med. Chem. 17 (2009) 3003–3010
3005
O
H
O
H
O
H
OH
O
OH
a
b
R1
R1
HO
R2
R2
R = H, β DMS R = H, H
R2
R = H, H
R2 = H, α OH
R = H, β DMS R = H, H
R = H, β DMS R = H, α OH
5b :
5f :
PD:
PT:
5a :
5c :
5d :
2
1
2
1
2
R = H, β DMS R = H, α DMS
1 2
1
2
R2 = H, α DMS
R1 = H, β OH
5e : R1 = H, β DMS
R2 = H, α DMS
Scheme 2. Synthesis of succinyl derivatives of PD and PT. Reagents and conditions: (a) 2,2-dimethylsuccinic anhydride, DMAP, Pyridine, reflux, 16 h; (b) Jones reagent, rt, 3 h.
DMS =
O
HOOC
O
.
12-hydroxyl group. As described by Hashimoto et al.,29 (20R)-PD
and (20R)-PT were treated with 2,2-dimethylsuccinic anhydride
in the presence of 4-(dimethylamino) pyridine (DMAP) in dry pyr-
idine to afford the acylated derivatives, 3-O-acyl [5a (35.6%) and 5c
(31.4%)], 6-O-acyl [5d (12.4%)] and 3,6-di-O-acyl [5e (24.8%)] deriv-
atives. Compounds 5a and 5e were further oxidized using the Jones
reagent to afford 12-oxo derivatives 5b (12%) and 5f (9.8%), respec-
tively. The structures of these compounds were determined by
spectroscopic methodologies including MS, 1H NMR, 13C NMR
and IR, and their spectral data are described in Section 4.
Table 1
Abilities of dammarane-type triterpene derivatives to inhibit HIV-1 and HCV PRs
O
H
O
H
R3
O
H
R3
O
H
R3
OH
HO
O
R1
HOOC
HOOC
R1
R2
R2
PD, PT
,
1a-1d, 5a-5f
2a
3a-3b
4a-4d
Compound
R1
R2
R3
IC50 (lM) RSD (%)
HIV PR
HCV PR
PD
PT
1a
1b
1c
1d
2a
3a
3b
4a
4b
4c
4d
5a
H, b OH
H, b OH
O
O
O
O
H, H
H,
H,H
O
H, H
O
H, b OH
H, b OH
H, b OH
H, b OH
O
>217.1 1.3
>209.9 3.1
39.4 2.4
42.3 2.5
>218.0 4.3
24.2 4.5
28.5 6.4
11.9 6.8
22.8 2.2
10.0 1.0
29.9 5.2
>198.0 2.8
>193.0 3.4
2.7 4.3
6.5 0.1
3.9 0.1
2.7 0.4
5.4 3.8
10.9 1.5
1.3 4.0
nt
>217.1 1.3
>209.9 3.1
>218.0 4.3
>213.0 6.2
72.3 4.5
>213.0 4.6
>212.0 3.0
>198.0 3.6
9.1 5.4
>204.0 3.8
>213.0 7.2
>198.0 3.2
>193.0 4.7
30.4 3.0
a
OH
O
H, b OH
O
b COOH,
b COOH,
a
a
OH
OH
H, H
O
H, H
O
H, H
H, H
H, b OH
H, b OH
H, b OH
H, b OH
H, b OH
O
H, b OH
H, b OH
H, b OH
O
b COOCH3,
b COOCH3,
H, b DMS
H, b DMS
H, b DMS
H, b OH
a
a
OH
OH
5b
5c
5d
5e
62.2 3.5
H,
H,
H,
H,
a
a
a
a
OH
>166.0 0.4
>166.0 1.8
1.8 2.6
DMS
DMS
DMS
H, b DMS
H, b DMS
5f
18.9
1.6
aPC A
bPCB
nt
0.5 1.5
Data represent mean values RSD(%) for three independent experiments.
a
PC A, Pepstatin A, positive control for HIV PR.
PC B: Hepatitis Virus C NS 3 Protease Inhibitor 2, positive control for HCV PR; nt: not tested. DMS =
O
b
HOOC
O
.

3006
Y. Wei et al. / Bioorg. Med. Chem. 17 (2009) 3003–3010
HOOC
HIV PR
HIV PR
O
COOMe
HIV PR
HOOC
HCV PR
HCV PR
HOOC
HO
HIV PR
A ring
seco
O
H
HCV PR
OH
12
A
h
r
i
n
n
g
e
c
a
g
d
O
3
mono
6
HIV PR
HO
m
O
O
HOOC
O
HCV PR
o
n
o
R
HIV PR
HOOC
-OH,
HCV PR
O
HIV PR
HIV PR
O
HCV PR
HCV PR
di-HOOC
O
panaxadiol derivatives
panaxatriol derivatives
: Activity increased
: Activity decreased
panaxadiol and panaxatriol derivatives
Figure 1. Structure–activity relationship of dammarane triterpene derivatives against HIV and HCV PRs.
2.2. Inhibitory activities of dammarane type derivatives against
HIV-1, HCV and other proteases
and di- (5e and 5f) acyl compounds strongly inhibited HIV-1 PR
(IC50 2.7–10.9 M). The mono-acyl compounds were more inhibi-
l
tory than the di-acyl compounds. On the other hand, the di-acyl
Table 1 shows the 50% inhibitory concentrations (IC50) in assays
of various dammarane-type triterpene derivatives against HIV-1
and HCV PRs using the assay methods described previously.17,30
The two known protease inhibitors, pepstatin A and hepatitis virus
C NS 3 protease inhibitor 2, served as positive controls for HIV-1
compounds inhibited HCV PR, especially compound 5e, with an
IC50 value of 1.8 lM. A comparison of the mono- and di-acyl com-
pounds (5d vs 5e and 5b vs 5f) showed that the former preferably
inhibited HIV PR, whereas the latter inhibited both proteases, indi-
cating higher selectivity of the mono- than the di-acyl compounds.
Moreover, the 12-hydroxyl derivatives (5a and 5e) were more
inhibitory than the 12-oxo derivatives (5b and 5f) against both
proteases.
and HCV PRs assays, their IC50 values being 1.3 and 0.5
tively.31,32 We regarded compounds with IC50 < 10
M, 10–50
and 50–100 M as strongly, moderately and weakly inhibitory,
lM, respec-
l
lM
l
respectively. Compounds with IC50 > 100
inactive.
The starting materials, PD and PT, did not show inhibitory activity
on either HIV protease24 or HCV protease. Some of their derivatives
showedpotentactivitiesandthestructure–activityrelationshipsare
discussed as following and summarized in Figure 1.
l
M were considered
We determined the selectivity of the eight compounds (3a, 3b
and 5a–5f) with relatively high activity against viral proteases,
by their ability to inhibit the human proteases, renin and trypsin.
Renin is an aspartyl protease like HIV-1 PR and trypsin is a serine
protease like HCV PR. None of the tested derivatives exerted any
appreciable inhibitory activity against renin and trypsin PRs at
concentrations up to 1 mg/mL (1365–2046 lM). These findings
2.2.1. 3-Oxo- and 2,3-seco derivatives
The inhibitory abilities of the dammarane-type triterpenes 1a,
2a and 1b (IC50 39.4, 28.5 and 42.3 lM, respectively) among the
suggested that the dammarane-type triterpenes are highly selec-
tive and potent HIV-1 or HCV PR inhibitors that might not interfere
with functional physiological reactions.
3-oxo derivatives were moderate against HIV PR, but did not inhi-
bit HCV PR. On the other hand, although both 2,3-seco derivatives
3. Conclusion
(3a and 3b, IC50, 11.9 and 22.8
ited HIV PR, only compound 3b was strongly inhibitory against
HCV PR (IC50 9.1 M), suggesting that the 12-oxo group signifi-
cantly increased the ability to inhibit HCV PR compared with the
lM, respectively) moderately inhib-
Starting from the artificial aglycones, (20R)-PD and (20R)-PT de-
rived from an acid hydrolysate of a ginseng extract, we prepared a
series of dammarane type derivatives bearing a 2,3-seco-2,3-dioic
acid (3a and 3b) moiety or 2,2-dimethylsuccinyl functional
group(s) at C-3 (5a–5c) or C-6 (5d) or at both positions (5e and
5f). Of seventeen tested compounds, eight were powerful inhibi-
l
corresponding free hydroxyl group (3a, IC50 > 198
seco derivatives.
lM) in the 2,3-
2.2.2. A-nor dammarane type derivatives
The A-nor dammarane type compounds 4a and 4b, with a car-
boxylic acid group attached to the A ring, moderately inhibited
tors of HIV PR with IC50 values of 2.7–11.9
mono-2,2-dimethylsuccinyl compounds 5a and 5d were the most
active with an IC50 of 2.7 M. On the other hand, only compounds
3b and 5e were powerful inhibitors of HCV PR, (IC50 9.1 and
1.8 M, respectively), and compound 5a was moderately inhibi-
lM. Among these, the
l
HIV PR with IC50 values of 10.0 and 29.9 lM, respectively, but they
were inactive against HCV PR. The oxo group at C-6 in 4b decreased
the inhibitory activity against HIV PR. In addition, the methyl es-
ters 4c and 4d were inactive against both PRs, suggesting that
the free carboxylic acid group at C-2 of 4a and 4b is important
for interaction with HIV PR.
l
tory. The structure–activity relationships (SARs) of these com-
pounds remarkably differed between the two viral proteases;
most of the 2,2-dimethylsuccinyl derivatives (5a–5e) potently
inhibited HIV PR, whereas only the di-2,2-dimethylsuccinyl (5e)
and 12-oxo-2,3-seco-2,3-dioic acid (3b) derivatives potently inhib-
ited HCV PR. Methylation of the carboxyl group in A-nor dammara-
ne type compounds significantly decreased the inhibitory activities
against both proteases. Thus, our study demonstrated that struc-
tural modification of dammarane-type triterpenes, especially
2.2.3. 2,2-Dimethylsuccinyl derivatives
Of the mono-2,2-dimethylsuccinyl and di-2,2-dimeylsuccinyl
derivatives of (20R)-PD and (20R)-PT, in which the hydroxyl groups
at C-3 and C-6 are acylated with succinic acid, the mono- (5a–5d)

Y. Wei et al. / Bioorg. Med. Chem. 17 (2009) 3003–3010
3007
(20R)-PD and (20R)-PT, leads to derivatives with powerful inhibi-
tory activity against either HIV or HCV PR. Compounds with high
inhibitory activity on HIV or HCV PR were inactive against renin
and trypsin, suggesting low physiological toxicity. Based on these
findings, we believe that dammarane-type triterpene derivatives
bearing a 2,3-seco-2,3-dioic acid moiety or 2,2-dimethylsuccinyl
group(s) will provide valuable information for the development
of new anti HIV and HCV agents.
was carried out in the following procedure. The mixture was
poured into H2O and extracted with chloroform. The organic layer
was successively washed with H2O, saturated aqueous NaHCO3,
once again with H2O, and then dried over MgSO4 and filtered.
The filtrate was evaporated under reduced pressure, loaded onto
a column (70 ꢀ 2.5 cm) containing silica gel and eluted with hex-
ane–acetone (99:1–95:5) to afford compound 1a as a white amor-
phous powder (920 mg, 92% yield); ½a D26
ꢁ
+24.7 (c 1.24, CHCl3), lit.28
,
a 2D4
ꢁ
+ 35.2 (c 0.11, MeOH). ESI-MS m/z 459.4 [M+H]+; IR
m
cmꢂ1
:
CS2
max
½
3396, 1715. lit.28, IR
m
cmꢂ1: 3340, 2960, 1710, 1460, 1380.
KBr
max
4. Experimental
4.1. Instruments and chemicals
4.3.2. (20R)-20,25-Epoxy-dammaran-3,12-dione (1c)
The Jones reagent (0.29 mL) was reacted with a solution of
(20R)-panaxadiol (59.0 mg, 0.1281 mmol) in acetone (5.88 mL)
for 4 h at room temperature. The mixture was worked up in the
same way as for 1a and eluted through a column (45 ꢀ 2.0 cm)
of silica gel with hexane–acetone (99:1–97:3) to afford compound
Melting points were measured using a Yanagimoto micro hot-
stage melting point apparatus without correction. Optical rotation
was measured using a Jasco DIP-360 automatic polarimeter. Infra-
red spectra (IR) were measured using a Jasco FT/IR-230 spectrom-
eter. 1H and 13C NMR were measured using UNITY 500 (1H,
500 MHz; 13C, 125 MHz) and UNITY 300 (1H, 300 MHz; 13C,
75 MHz) spectrometers, the chemical shifts being represented as
ppm with tetramethylsilane as the internal standard. Electrospray
ionization mass (ESI-MS) spectra were obtained using an Esquire
3000plus spectrometer (Bruker Daltonik GmbH, Bremen, Germany).
High-resolution-EI-MS and EI-MS spectra were obtained on a
JMS-AX 505 HAD GC/MS system and a JMS DX-300 system at ion-
ization voltage at 70 eV. High-resolution-FAB-MS were measured
on a Jeol JMS-700 with a resolution of 5000 using m-nitrobenzyl
alcohol as the matrix. Compounds were separated by TLC on plates
recoated with Silica Gel 60 F254 (Merck). The following were the
chromatographic carriers used for isolations, silica gel BW-
820MH (Fuji Silysia Chemical Ltd) and ODS DM 1020T (Fuji Silysia).
Preparative HPLC was performed on a Tosoh CCPM-CCPM-II sys-
tem (Tosoh Co.) equipped with a UV 8020 detector and a TSK gel
ODS-80Ts column (21.5 ꢀ 300 mm, Tosoh Co.).
1c as a white amorphous powder (5.0 mg, 8.6% yield); ½a D25
ꢁ
+71.8
(CHCl3), lit.33, ½a D24
ꢁ
+72 (c 0.03, MeOH). ESI-MS m/z 457.5 [M+H]+;
IR mCmSax cmꢂ1: 1717. lit.33, IR
m
cmꢂ1: 2980, 2870, 1710, 1650,
2
KBr
max
1460.
4.3.3. (20R)-20,25-Epoxy-2,12-dihydroxydammaran-1-en-3-
one (2a)
Air was introduced to a solution of 1a (860 mg, 1.87 mmol) and
t-BuOK (7.65 g) in t-BuOH (74.4 mL) and the mixture was stirred
for 40 min at 40 °C and worked up as usual. The crude product
was eluted through an ODS column (20 ꢀ 4.5 cm) with MeOH–
H2O (50–100%) to afford compound 2a as white crystals (884 mg,
100% yield); mp. 193–196 °C; ½a D24
ꢁ
+45.9 (c 0.08, MeOH). HR-EI-
MS m/z 472.35780 [M+H]+ (calcd for C30H48O4, 472.35526); IR
cmꢂ1: 3360, 3100, 2960, 2880, 2800, 2500, 2200, 1900,
KBr
m
max
1660; 1H NMR (CDCl3, 500 MHz) d 0.81 (3H, s), 0.97 (3H, s), 1.05
(3H, s), 1.11 (3H, s), 1.12 (3H, s), 1.13 (3H, s), 1.16 (3H, s), 120
(3H, s), 3.50 (1H, m, H-12), 5.98 (1H, br, OH-2), 6.36 (1H, s, H-1),
6.44 (1H, s, OH-12); 13C NMR (CDCl3, 125 MHz) d: 16.0, 16.2,
16.9, 17.1, 18.7, 19.3, 20.2, 21.5, 25.1, 27.1, 30.4, 30.9, 33.0, 34.3,
35.6, 36.4, 38.5, 40.5, 44.0, 45.0, 49.2, 51.2, 54.4, 54.5, 69.5, 73.1,
76.6, 128.7, 143.8, 200.9.
t-BuOK, t-BuOH, 2,2-dimethylsuccinic anhydride, 4-dimethyla-
minorpyridine (DMAP), pyridinium chlorochromate (PCC) and
chromium(VI) oxide were purchased from Sigma–Aldrich
Company.
4.2. Enzyme assay kits
4.3.4. (20R)-20,25-Epoxy-2a,12b-dihydroxy-A-nordammaran-2-
carboxylic acid (4a)
We purchased HIV protease (Lot# 146125) from Bachem AG.,
Switzerland and Wako Pure Chemical Industries Ltd provided the
HIV protease substrate (Lot# 2000666) and Pepstatin A (Cat#
71147). SensoLyteTM 520 HCV Protease Assay Kit Fluorimetric
(Lot# AK 71145-1013), HCV NS3/4A protease (Lot# 046-079), Hepa-
titis Virus C NS3 protease inhibitor 2 (Cat# 25346), SensoLyteTM 520
Renin Assay Kit Fluorimetric (Lot# 72040) and SensoLyteTM Green
Protease Assay Kit Fluorimetric (Lot# AK 71124-1009) were pur-
chased from AnaSpec Inc., San Jose, CA, USA. Soybean trypsin-chy-
motrypsin inhibitor was obtained from Sigma–Aldrich Co. Becton
Asolutionof 1c(841.0 mg, 1.78 mmol)and KOH (2.57 g) inMeOH
(138.4 mL) was heated under reflux, and hydrogen peroxide
(13.8 mL, 30%) was added over a period of 100 min. The solution
was then poured into cold H2O and the products were extracted with
ethyl acetate (2 ꢀ 100 mL), followed by preparative HPLC [MeOH–
0.1% TFA/H2O, 5 mL/min, monitored at 208 nm] to afford 3a
(350 mg, 38.8% yield) as a major product and 4a (12 mg, 1.4% yield)
as a minor product. Compound 4a: mp. 120–123.8 °C; ½a D24
ꢁ
+17.5 (c
0.07, MeOH). HR-FAB-MS m/z 491.37219 [M+H]+ (calcd for
Dickinson FalconTM MicrotestTM 384-well 120
lL black assay plates
cmꢂ1: 3260, 2940, 2880, 1710, 1690;
KBr
C30H51O5, 491.37365); IR
m
max
1H NMR (CDCl3, 500 MHz) d 0.93 (3H, s), 0.97 (6H, s), 1.01 (3H, s),
1.10 (3H, s), 1.18 (3H, s), 1.21 (3H, s), 1.28 (3H, s), 1.81 (1H, d,
J = 13.0 Hz, H-1a), 1.96 (1H, ddd, J = 7.5, 11.0 Hz), 2.13 (1H, d,
J = 13.0 Hz, H-1b), 3.64 (1H, ddd, J = 5.5, 10.5, 15.5 Hz, H-12); 13C
NMR (CDCl3, 125 MHz) d: 15.7, 16.2, 16.7, 17.4, 18.8, 19.3, 20.9,
25.1, 27.0, 27.2, 31.3, 32.4, 32.8, 34.8, 35.7, 36.4, 40.6, 43.6, 48.2,
48.8, 49.8, 51.4, 54.4, 54.5, 62.5, 70.1, 73.6, 76.7, 86.7, 179.2.
were purchased from Nonsterile, No Lid. Lot# 06 04 11 55, Germany.
4.3. Isolation of (20R)-panaxadiol and (20R)-panaxatriol
Panax ginseng roots were extracted with hot methanol–H2O
(7:3) to obtain a crude saponin fraction. This fraction was hydro-
lyzed with 7% HCl and then separated by various types of column
chromatography to yield the major aglycones, (20R)-panaxadiol
(PD), (20R)-panaxatriol (PT) and oleanolic acid.24
4.3.5. (20R)-20,25-Epoxy-2a,12b-dihydroxy-A-nordammaran-2-
carboxylic acid methyl ester (4c)
4.3.1. (20R)-20,25-Epoxy-12b-hydroxydammaran-3-one (1a)
Trimethylsilyldiazomethane (0.5 mL) was added to a solution of
4a (20 mg, 0.041 mmol) in MeOH (3.0 mL) until the mixture be-
came yellow. After stirring for 30 min at room temperature moni-
toring by TLC, the mixture was worked up as usual and then eluted
A
CH2Cl2 (11.1 mL) solution of (20R)-panaxadiol (1.0 g,
2.17 mmol) and pyridinium chlorochromate (2812.1 mg,
13.0 mmol) was stirred for 6 h at room temperature. Work up

3008
Y. Wei et al. / Bioorg. Med. Chem. 17 (2009) 3003–3010
through an ODS column (16 ꢀ 2.4 cm) with methanol–H2O (4:1–
100:0) to afford 4c as a white amorphous powder (15 mg, 72.5%
umn, 21.5 ꢀ 300 mm; MeOH-0.1% TFA/H2O, 5 mL/min, monitored
at 208 nm] afforded 4b as a white amorphous powder (44.8 mg,
yield); ½a 2D4
ꢁ
+ 21.9 (c 0.10, MeOH). ESI-MS m/z 505.5 [M+H]+ (calcd
6.2% yield);
½
a 2D5
ꢁ
ꢂ26.1 (c 0.15, MeOH). HR-FAB-MS m/z
cmꢂ1: 3460, 2960, 2870; 1H NMR
503.33396 [MꢂH]ꢂ (calcd for C30H47O6, 503.33727); IR
m
cmꢂ1
:
max
KBr
max
KBr
for C31H52O5, 504.38147); IR
m
(CDCl3, 500 MHz) d 0.88 (3H, s), 0.92 (6H, s), 0.96 (3H, s), 0.97 (3H,
s), 1.11 (3H, s), 1.19 (3H, s), 1.27 (3H, s), 1.81 (1H, d, J = 13.0 Hz, H-
1a), 1.94 (1H, ddd, J = 3.5, 11.0, 14.0 Hz, H-17), 2.08 (1H, d,
J = 13.0 Hz, H-1b), 3.60 (1H, ddd, J = 5.0, 11.0, 16.0 Hz, H-12), 3.78
(1H, s, OCH3); 13C NMR (CDCl3, 125 MHz) d: 15.8, 16.3, 16.4, 17.3,
18.8, 19.3, 20.8, 25.1, 27.0, 27.1, 31.2, 32.7, 32.9, 34.8, 35.7, 36.4,
40.6, 43.6, 48.7, 49.1, 49.7, 51.2, 52.2, 53.3, 54.7, 62.5, 69.9, 73.2,
76.7, 87.1, 177.3 (C-2, COOCH3).
3400, 3280, 2980, 2860, 1760, 1680; 1H NMR (MeOD, 500 MHz) d
0.95 (3H, s, Me-28), 0.97 (3H, s, Me-19), 0.98 (3H, s, Me-30), 1.12
(3H, s, Me-29), 1.22 (6H, s, Me-21, Me-26), 1.25 (3H, s, Me-18),
1.30 (3H, s, Me-27), 1.95 (1H, d, J = 14.0 Hz, H-1a), 2.08 (1H, d,
J = 14.5 Hz, H-7a), 2.28 (1H, dd, J = 3.0, 13.5 Hz, H-9), 2.47 (1H, d,
J = 14.5 Hz, H-5), 2.73 (1H, d, J = 14.5 Hz, H-1b), 3.64 (1H, dd,
J = 5.0, 10.0 Hz, H-12); 13C NMR (MeOD, 125 MHz) d: 16.9, 17.1,
17.5, 19.9, 22.1, 24.4, 25.8, 27.6, 28.6, 32.5, 33.5, 35.2, 36.7, 37.4,
44.3, 45.0, 48.5, 50.2, 51.7, 52.7, 53.1, 54.7, 56.0, 68.6, 71.0, 74.7,
78.1, 86.5, 176.5, 217.1.
4.3.6. (20R)-20,25-Epoxy-2,3-seco-dammaran-2,3-dioc acid (3a)
This compound was prepared from 2a in the same manner as
described for 4a. Compound 3a: mp 208–210.5 °C; ½a D25
ꢁ
+10.7 (c
4.3.11. (20R)-20,25-Epoxy-2a,12b-dihydroxy-6-oxo-A-
nordammaran-2-carboxylic acid methyl ester (4d)
0.14, MeOH). HR-FAB-MS m/z 507.36360 [M+H]+ (calcd for
cmꢂ1: 3500, 3260, 2980, 2800,
This compound was prepared from 4b (22.4 mg, 0.044 mmol) in
the same manner as described for 4c. Purification by HPLC [col-
umn, 21.5 ꢀ 300 mm; eluted with MeOH-0.1% TFA/H2O, 5.0 mL/
min, monitored at 208 nm] afforded 4d as a colorless powder
KBr
C30H51O6, 507.36857); IR
m
max
1710, 1690, 1650; 1H NMR (CDCl3, 500 MHz) d 0.90 (3H, s), 0.98
(6H, s), 1.18 (3H, s), 1.20 (3H, s), 1.21 (3H, s), 1.24 (3H, s), 1.25
(6H, s), 3.56 (1H, m, H-12), 6.59 (1H, s, 12-OH); 13C NMR (CDCl3,
125 MHz) d: 15.5, 16.3, 17.1, 19.4, 20.2, 22.2, 25.3, 27.1, 28.7,
31.0, 31.2, 33.0, 34.0, 35.7, 36.5, 39.6, 41.0, 41.6, 45.9, 48.6, 49.1,
51.6, 54.5, 69.7, 73.2, 176.7, 185.6.
(11 mg, 48.2% yield); mp. 124–126.2 °C. ½a D25
ꢁ
+ 53.3 (c 0.04, MeOH).
ESI-MS m/z 519.7 [M+H]+ (calcd for C31H50O6, 518.36074); IR
cmꢂ1: 3400, 2980, 2860, 1780, 1710, 1690; 1H NMR (CDCl3,
KBr
m
max
500 MHz) d 0.89 (3H, s), 0.94 (6H, s), 0.97 (3H, s), 1.11 (3H, s),
1.19 (3H, s), 1.23 (3H, s), 1.24 (3H, s), 1.28 (3H, s), 1.98 (1H, d,
J = 14.5 Hz, H-1a), 2.13 (1H, d, J = 14.5 Hz, H-7a), 2.18 (1H, dd,
J = 3.5, 13.5 Hz, H-9), 2.37 (1H, d, J = 14.5 Hz, H-7b), 2.58 (1H, s,
H-5), 2.65 (1H, d, J = 14.0 Hz, H-1b), 3.62 (1H, dd, J = 4.0, 10.0 Hz,
H-12), 3.78 (1H, s, OCH3); 13C NMR (CDCl3, 125 MHz) d: 16.2,
16.4, 17.2, 19.4, 21.3, 23.7, 24.8, 27.0, 27.8, 32.5, 31.6, 32.9, 34.3,
35.7, 36.4, 42.9, 44.1, 47.8, 49.0, 50.5, 51.7, 52.4, 52.7, 52.8, 54.6,
66.6, 69.3, 73.4, 76.5, 86.2, 173.8, 214.4.
4.3.7. (20R)-20,25-Epoxy-12-oxo-2,3-seco-dammaran-2,3-dioc
acid (3b)
The Jones reagent (0.23 mL) was reacted with a solution of 3a
(100 mg, 0.198 mmol) in acetone (9.0 mL) for 4 h at room tempera-
ture. The mixture was worked up as usual and then purified by HPLC
[column chromatography (21.5 ꢀ 300 mm) with MeOH–0.1% TFA/
H2O, 5.0 mL/min, monitored at 208 nm] to afford 3b as a colorless
crystal (10 mg, 10% yield); mp 127–130.9 °C. ½a D25
ꢁ
+149.8 (c 0.04,
MeOH). HR-FAB-MS m/z 503.33579 [MꢂH]ꢂ (calcd for C30H47O6,
cmꢂ1: 3440, 3180, 2960, 2620, 2040, 1710; 1
H
4.3.12. (20R)-20,25-Epoxy-dammaran-3b,12b-diol-3-(30,30-
dimethyl) succinate (5a)
KBr
503.33727); IR
m
max
NMR (CDCl3, 300 MHz) d 0.73 (3H, s), 1.03 (3H, s), 1.09 (3H, s), 1.16
(9H, s), 1.18 (3H, s), 1.24 (3H, s), 2.31 (1H, d, J = 18.5 Hz, H-1a), 2.60
(1H, d, J = 18.5 Hz, H-1b), 3.31 (1H, m); 13C NMR (CDCl3, 300 MHz)
d: 15.6, 16.4, 16.9, 19.8, 20.7, 21.8, 24.3, 25.9, 27.4, 29.1, 32.2, 33.3,
33.6, 37.0, 40.0, 40.2, 41.1, 41.9, 45.7, 45.9, 46.0, 48.6, 55.7, 56.1,
70.7, 74.8, 76.6, 174.7, 186.1, 214.8.
A mixture of (20R)-panaxadiol (50 mg, 0.109 mmol), 2,2-dim-
ethylsuccinic anhydride (85.3 mg, 0.67 mmol) and 4-dimethyl-
amino pyridine (DMAP), (26.5 mg, 0.217 mmol) was heated in
pyridine (2.0 mL) overnight under reflux. Ethyl acetate (50 mL)
was added and then the mixture was washed with 2 N HCl and
H2O. Evaporating the EtOAc yielded a mixture of the starting mate-
rial and products that were separated by elution through an ODS
column with MeOH/H2O (9:1) and by HPLC [MeOH-0.1% TFA/
H2O, 5 mL/min, monitored at 208 nm] to afford 5a as a white crys-
4.3.8. (20R)-20,25-Epoxy-12b-hydroxydammaran-3,6-dione
(1b)
This compound was prepared from PT (1.0 g, 2.10 mmol) in the
same manner as described for 1a. Purification by elution through
silica gel (50 ꢀ 3.0 cm) with hexane–acetone (99:1 [v/v] to pure
acetone) afforded 1b as a white amorphous powder (749 mg,
talline powder (22.0 mg, 34.3% yield); mp. 145–147.7 °C; ½a D25
ꢁ
+14.5 (c 0.08, MeOH). HR-FAB-MS m/z 589.44494 [M+H]+ (calcd
KBr
max
for C36H61O6. 589.44682); IR
m
cmꢂ1: 3440, 2980, 2880, 1730;
75.5% yield). ½a D25
ꢁ
ꢂ25.1 (c 0.11, MeOH). ESI-MS m/z 473.5
1H NMR (CDCl3, 500 MHz) d 0.83 (3H, s), 0.84 (3H, s), 0.87 (3H,
s), 0.89 (3H, s), 0.97 (3H, s), 1.18 (3H, s), 1.21 (3H, s), 1.26 (1H,
s), 1.27 (1H, s), 1.29 (1H, s), 1.92 (1H, m, H-17), 2.56 (1H, d,
J = 15.5 Hz, H-20), 2.66 (1H, d, J = 15.5 Hz, H-200), 3.55 (1H, m,
J = 5.0, 10.5 Hz, H-12), 4.49 (1H, m, J = 6.0, 11.5 Hz, H-3), 6.43
(1H, s, 12-OH); 13C NMR (CDCl3, 125 MHz) d: 15.4, 15.9, 16.0,
16.3, 16.8, 18.0, 19.2, 23.4, 25.0, 25.1, 25.4, 26.9, 27.7, 30.0, 31.0,
32.7, 34.6, 35.5, 36.2, 36.8, 37.6, 38.4, 39.6, 39.6, 44.7, 48.8, 49.6,
51.1, 54.5, 55.8, 69.9, 73.1, 76.6, 81.3, 171.7, 181.7.
[M+H]+. IR
m
cmꢂ1: 3327, 1718, lit.34 IR
m
cmꢂ1: 3380, 2940,
max
CCl4
max
KBr
2880, 1710.
4.3.9. (20R)-20,25-Epoxy-dammaran-3,6,12-trione (1d)
This compound was prepared from PT (206 mg, 0.433 mmol) in
the same manner as described for 3b. Chromatographic separation
by hexane–acetone (99:1–95:5) elution through
a column
(50 ꢀ 3.0 cm) containing silica gel afforded 1d as a colorless amor-
phous powder (50 mg, 25.3% yield); ½a D25
ꢁ
+25.9 (c 0.06, MeOH). ESI-
MS m/z 471.5 [M+H]+; IR
m
cmꢂ1: 2960, 2800, 2300, 1710. The
4.3.13. (20R)-20,25-Epoxy-3b-hydroxydammaran-12-one-3-
(30,30-dimethyl) succinate (5b)
KBr
max
1H and 13C NMR spectral data of 1d were in agreement with those
published in the literature.35
This compound was prepared from 5a (102 mg, 0.17 mmol) in
the same manner as described for compound 1c. Purification by
HPLC [column, 21.5 ꢀ 300 mm; MeOH-0.1% TFA/H2O, 5.0 mL/min,
monitored at 208 nm] afforded compound 5b as a white amor-
4.3.10. (20R)-20,25-Epoxy-2a,12b-dihydroxy-6-oxo-A-
nordammaran-2-carboxylic acid (4b)
This compound was prepared from 1b (679 mg, 1.44 mmol) in
the same manner as described for 2a. Purification by HPLC (col-
phous powder: (12.0 mg, 12% yield). ½a D25
ꢁ
+46.7 (c 0.03, MeOH),
ESI-MS m/z 587.7 [M+H]+ (calcd for C36H58O6, 586.42334); IR

Y. Wei et al. / Bioorg. Med. Chem. 17 (2009) 3003–3010
3009
cmꢂ1: 3460, 2980, 2880, 2680, 1710, 1650; 1H NMR (CDCl3,
0.04, MeOH). ESI-MS m/z: 729.9 [MꢂH]ꢂ (calcd for C42H66O10
,
KBr
m
max
500 MHz) d 0.89 (3H, s), 0.95 (3H, s), 1.04 (3H, s), 1.06 (3H, s),
1.12 (3H, s), 1.16 (3H, s), 1.17 (3H, s), 1.22 (1H, s), 1.32 (1H, s),
1.38 (1H, s), 2.39–2.49 (2H, m, H-20, H-200), 4.64 (1H, dd, J = 4.5,
12.0 Hz, H-3); 13C NMR (CDCl3, 125 MHz) d: 16.4, 17.0, 17.7, 18.4,
18.7, 19.5, 22.8, 23.1, 24.8, 24.9, 27.2, 27.9, 31.3, 32.8, 34.7, 35.4,
36.4, 36.8, 37.8, 38.2, 40.0, 40.3, 42.0, 42.9, 48.5, 50.9, 54.0, 55.7,
56.2, 58.5, 71.2, 75.2, 80.7, 171.0, 183.4, 210.0.
730.46450); IR
m
cmꢂ1: 3460, 2980, 2880, 2700, 1730, 1710;
KBr
max
1H NMR (CDCl3, 500 MHz) d 0.74 (3H, s), 0.91 (3H, s), 1.02 (6H,
s), 1.07 (6H, s), 1.16 (3H, s), 1.18 (3H, s), 1.26 (6H, s), 1.29 (3H,
s), 1.30 (3H, s), 1.32 (3H, s), (12 ꢀ CH3), 2.46 (1H, d, J = 15.5 Hz,
H-2a0), 2.57 (1H, d, J = 15.5 Hz, H-2a00), 2.66 (1H, d, J = 8.0 Hz, H-
2b0), 2.69 (1H, d, J = 8.0 Hz, H-2b00), 3.02 (1H, d, J = 8.5 Hz, H-11),
4.50 (1H, dd, J = 4.5, 11.5 Hz, H-3), 5.47 (1H, m, J = 5.5, 10.0 Hz,
H-6); 13C NMR (CDCl3, 125 MHz) d: 16.3, 16.5, 16.7, 17.0, 17.1,
22.9, 24.1, 24.8, 24.9, 25.7, 25.7, 25.8, 27.3, 30.0, 32.1, 33.5, 37.0,
37.5, 37.7, 39.3, 39.6, 40.6, 40.8, 41.1, 41.7, 45.1, 45.4, 45.8, 53.2,
55.2, 55.8, 58.3, 70.2, 70.7, 74.7, 80.7, 170.3, 170.5, 183.5, 183.5,
212.0.
4.3.14. (20R)-20,25-Epoxy-dammaran-3b,6a
,12bb-triol-3-(30,30-
dimethyl) succinate (5c)
This compound was prepared from (20R)-panaxatriol (50 mg,
0.105 mmol) in the same manner as described for 5a. Purification
by HPLC [MeOH–0.1% TFA/H2O, 5 mL/min, monitored at 208 nm]
afforded 5c (20.0 mg, 31.4% yield), 5d (8.0 mg, 12.4% in yield)
and 5e (19.0 mg, 24.8% in yield) as white amorphous powders.
4.4. HIV protease assay
½
a 2D5
ꢁ
+7.89 (c 0.08, MeOH), HR-FAB-MS m/z 605.44166 [M+H]+
Inhibitory activity against HIV PR was tested using HPLC meth-
od as described.17
KBr
(calcd for C36H61O7, 605.44173); IR
m
cmꢂ1: 3440, 3340, 2960,
max
2860, 2600, 1730, 1710; 1H NMR (CDCl3, 500 MHz) d 0.90 (3H, s),
0.95 (3H, s), 1.04 (3H, s), 1.06 (3H, s), 1.16 (3H, s), 1.18 (3H, s),
1.21 (3H, s), 1.26 (3H, s), 1.28 (3H, s), 1.29 (3H, s) (10 ꢀ CH3),
1.91 (1H, m, H-17), 2.56 (1H, d, J = 16.0 Hz, H-20), 2.69 (1H,
J = 16.0 Hz, H-200), 3.54 (1H, m, J = 5.5, 10.5, 15.5 Hz, H-12), 4.10
(1H, m, J = 4.0, 9.5 Hz, H-6), 4.47 (1H, dd, J = 5.5, 11.0 Hz, H-3),
6.45 (1H, s, OH-12); 13C NMR (CDCl3, 125 MHz) d: 16.2, 16.6,
17.0, 17.1, 17.2, 17.9, 19.3, 20.9, 23.3, 25.1, 25.8, 27.1, 28.4, 30.2,
30.6, 31.1, 33.0, 35.7, 36.4, 38.1, 38.3, 39.0, 40.9, 44.7, 46.8, 48.6,
49.2, 51.0, 54.6, 55.2, 61.2, 68.4, 69.8, 73.2, 76.6, 81.3, 170.5, 181.7.
4.5. HCV protease assay
Inhibitory activity against HCV PR was tested using a fluoromet-
ric method as described.30
4.6. Renin protease assay
A compound dissolved in DMSO (2.5
10%) was placed in the wells of 384-well microplates, then
14.0 L of rec-renine PR (0.5 g/mL) was added and the plate
was agitated. Thereafter, 8.5 L of fresh dilutions of the renin sub-
ll; final concentration,
l
l
4.3.15. (20R)-20,25-Epoxy-dammaran-3b,6
a
,12b-triol-6-(30,30-
l
dimethyl) succinate (5d)
strate [Arg-Glu(EDANS)-Ile-His-Pro-Phe-His-Leu-Val-Il2-His-Thr-
Lys (Dabcyl)-Arg] was added and the mixture was rotated for
30 min at 37 °C. A renin inhibitor [Ac-HPFV-(Sta)-LF-NH2] served
as the positive control.36 The methods of fluorometric analyses
and inhibitory calculation were as described for the HCV protease
assay.
½
a 2D5 +108.8 (c 0.99, MeOH), HR-FAB-MS m/z 605.43693 [M+H]+
ꢁ
cmꢂ1: 3480, 2980, 2880,
KBr
(calcd for C36H61O7, 605.44173); IR
m
max
2600, 1730, 1710; 1H NMR (CDCl3, 500 MHz) d 0.82 (3H, s), 0.89
(3H, s), 0.97 (3H, s), 1.10 (3H, s), 1.17 (3H, s), 1.18 (3H, s), 1.21
(3H, s), 1.26 (3H, s), 1.30 (3H, s), 1.33 (3H, s) (10 ꢀ CH3), 2.54
(1H, d, J = 16.5 Hz, H-20), 2.61 (1H, d, J = 16.5 Hz, H-200), 3.19 (1H,
dd, J = 4.5, 11.5 Hz, H-3), 3.53 (1H, m, J = 5.0, 10.0 Hz, H-12), 5.36
(1H, ddd, J = 4.5, 10.5, 15.0 Hz, H-6), 6.40 (1H, s, OH-12); 13C
NMR (CDCl3, 125 MHz) d: 15.5, 16.2, 16.8, 17.0, 17.0, 19.4, 25.1,
25.3, 25.5, 26.9, 27.1, 30.2, 30.5, 31.1, 33.0, 35.7, 36.4, 38.5, 38.7,
39.4, 40.5, 40.8, 42.4, 44.8, 48.6, 49.4, 51.0, 54.6, 58.7, 69.8, 71.1,
73.2, 76.6, 78.3, 170.6, 181.0.
4.7. Green protease assay
A compound dissolved in DMSO (2.5
10%) was placed in the wells of 384-well microplates, then
17.5 L of assay buffer and 2.5 L of trypsin (0.1 U/ L) were added
to the wells and the plate was agitated. Finally, fresh 2.5 L dilu-
lL; final concentration:
l
l
l
l
tions of the protease substrate, HiLyte FluorTM 488-labeled casein,
were added under sequential rotary shaking incubated at 37 °C for
30 min. The positive control was soybean trypsin–chymotrypsin
inhibitor.37 The methods for the fluorometric analysis and inhibi-
tory calculations were the same as described for the HCV protease
assay.
4.3.16. (20R)-20,25-Epoxy-dammaran-3b,6a,12b-triol-3,6-di-
(30,30-dimethyl) succinate (5e)
½
a 2D5 +40.0 (c 0.03, MeOH), HR-FAB-MS m/z 733.48796 [M+H]+
(calcd for C42H69O10, 733.48903); IR
ꢁ
KBr
max
m
cmꢂ1: 3460, 3380, 2960,
2880, 2600, 1730, 1710; 1H NMR (CDCl3, 500 MHz) d 0.89 (3H, s),
0.90 (3H, s), 1.01 (6H, s), 1.07 (3H, s), 1.21 (6H, s), 1.26 (12H, s),
1.29 (3H, s) (12 ꢀ CH3), 2.49 (1H, d, J = 15.5 Hz, H-2a0), 2.56 (1H,
d, J = 15.5 Hz, H-2a00), 2.62 (1H, d, J = 16.0 Hz, H-2b0), 2.72 (1H, d,
J = 16.0 Hz, H-2b00), 3.53 (1H, m, J = 5.0, 10.0 Hz, H-12), 4.51 (1H,
m, H-3), 5.39 (1H, m, J = 3.0, 10.5, 14.5 Hz, H-6), 6.46 (1H, s, OH-
12); 13C NMR (CDCl3, 125 MHz) d: 16.2, 16.5, 16.9, 17.2, 19.5,
23.2, 24.9, 25.1, 25.2, 25.6, 25.8, 27.1, 30.1, 30.1, 31.1, 33.0, 35.7,
36.4, 37.6, 38.2, 39.2, 40.6, 40.7, 40.7, 42.5, 45.1, 45.4, 48.6, 49.2,
51.0, 54.6, 58.7, 69.8, 70.4, 73.2, 76.7, 81.2, 170.0, 170.8, 183.1,
183.1.
Acknowledgment
This study received financial support from the Japan China
Medical Association (NO. 103, Japan).
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