Anti-AIDS agents, 30. Anti-HIV activity of oleanolic acid, pomolic acid, and structurally related triterpenoids

Article abstract of DOI:10.1021/np9800710

Oleanolic acid (1) was identified as an anti-HIV principle from several plants, including Rosa woodsii (leaves), Prosopis glandulosa (leaves and twigs), Phoradendron juniperinum (whole plant), Syzygium clavifiorum (leaves), Hyptis capitata (whole plant), and Ternstromia gymnanthera (aerial part). It inhibited HIV-1 replication in acutely infected H9 cells with an EC₅₀ value of 1.7 μg/mL, and inhibited H9 cell growth with an IC₅₀ value of 21.8 μg/mL [therapeutic index (T. I.) 12.8]. Pomolic acid, isolated from R. woodsii and H. capitata, was also identified as an anti-HIV agent (EC₅₀ 1.4 μg/mL, T. I. 16.6). Although ursolic acid did show anti-HIV activity (EC₅₀ 2.0 μg/mL), it was slightly toxic (IC₅₀ 6.5 μg/mL, T. I. 3.3). A new triterpene (11) was also isolated from the CHCl₃-soluble fraction of R. woodsii, though it showed no anti-HIV activity. The structure of 11 was determined to be 1β-hydroxy-2-oxopomolic acid by spectral examination. Based on these results, we examined the anti-HIV activity of oleanolic acid- or pomolic acid-related triterpenes isolated from several plants. In addition, we previously demonstrated that derivatives of betulinic acid, isolated from the leaves of S. claviflorum as an anti-HIV principle, exhibited extremely potent anti-HIV activity. Accordingly, we prepared derivatives of oleanolic acid and evaluated their anti-HIV activity. Among the oleanolic acid derivatives, 18 demonstrated most potent anti-HIV activity, with an EC₅₀ value of 0.0005 μg/mL and a T. I. value of 22 400.

Full text of DOI:10.1021/np9800710

1
090
J . Nat. Prod. 1998, 61, 1090-1095
An ti-AIDS Agen ts. 30. An ti-HIV Activity of Olea n olic Acid , P om olic Acid , a n d
Str u ctu r a lly Rela ted Tr iter p en oid s1
†
,#
†
†,#
†,⊥
†,|
Yoshiki Kashiwada, Hui-Kang Wang, Tsuneatsu Nagao, Susumu Kitanaka, Ichiro Yasuda,
Toshihiro Fujioka,† Takashi Yamagishi, L. Mark Cosentino, Mutsuo Kozuka, Hikaru Okabe,
∇
,∇
†,O
‡
§
#
O
†
,†
Yasumasa Ikeshiro, Chang-Qi Hu, Eric Yeh, and Kuo-Hsiung Lee*
Natural Products Laboratory, Division of Medicinal Chemistry and Natural Products, School of Pharmacy, University of
North Carolina, Chapel Hill, North Carolina 27599, Biotech Research Laboratories, 1600 East Gude Drive,
Rockville, Maryland 20850, Kyoto Pharmaceutical University, Nakauchi-cho, Misasagi, Yamashina-ku, Kyoto 607, J apan,
College of Pharmacy, Nihon University, Narashinodai, Funabashi, Chiba 274, J apan, Tokyo Metropolitan Govern Research
Laboratory of Public Health, Hyakunin-cho, Shinjuku-ku, Tokyo 169, J apan, Kitami Institute of Technology,
1
65 Koen-cho, Kitami, Hokkaido 090, J apan, Faculty of Pharmaceutical Sciences, Fukuoka University,
Nanakuma, J onan-ku, Fukuoka 814-80, J apan, Niigata College of Pharmacy, Kamishin’ei-cho, Niigata 950-21, J apan, and
Shanghai Medical University, 138 XueYuan Road, Shanghai 200032, People’s Republic of China
Received March 5, 1998
Oleanolic acid (1) was identified as an anti-HIV principle from several plants, including Rosa
woodsii (leaves), Prosopis glandulosa (leaves and twigs), Phoradendron juniperinum (whole
plant), Syzygium claviflorum (leaves), Hyptis capitata (whole plant), and Ternstromia gym-
nanthera (aerial part). It inhibited HIV-1 replication in acutely infected H9 cells with an EC50
value of 1.7 µg/mL, and inhibited H9 cell growth with an IC50 value of 21.8 µg/mL [therapeutic
index (T. I.) 12.8]. Pomolic acid, isolated from R. woodsii and H. capitata, was also identified
as an anti-HIV agent (EC50 1.4 µg/mL, T. I. 16.6). Although ursolic acid did show anti-HIV
activity (EC50 2.0 µg/mL), it was slightly toxic (IC50 6.5 µg/mL, T. I. 3.3). A new triterpene (11)
3
was also isolated from the CHCl -soluble fraction of R. woodsii, though it showed no anti-HIV
activity. The structure of 11 was determined to be 1â-hydroxy-2-oxopomolic acid by spectral
examination. Based on these results, we examined the anti-HIV activity of oleanolic acid- or
pomolic acid-related triterpenes isolated from several plants. In addition, we previously
demonstrated that derivatives of betulinic acid, isolated from the leaves of S. claviflorum as
an anti-HIV principle, exhibited extremely potent anti-HIV activity. Accordingly, we prepared
derivatives of oleanolic acid and evaluated their anti-HIV activity. Among the oleanolic acid
derivatives, 18 demonstrated most potent anti-HIV activity, with an EC50 value of 0.0005 µg/
mL and a T. I. value of 22 400.
We are continuing a search for new anti-HIV agents
from natural products, and also modification of the
active principles to develop more potent anti-HIV
Based on these results, we have examined anti-HIV
activity of oleanolic acid- or pomolic acid-related triter-
penes isolated from several plants. Furthermore, a
series of oleanolic acid derivatives has been prepared
and their anti-HIV activity evaluated.
2
agents. As a part of our continuing screening of plant
extracts as anti-HIV agents, the MeOH extracts of the
leaves of Rosa woodsii Lindl. (Rosaceae), the leaves and
twigs of Prosopis glandulosa Torr. (Leguminosae), the
whole plant of Phoradendron juniperinum Engel. (Lo-
ranthaceae), the leaves of Syzygium claviflorum Wall.
ex A. & J . Cowan (Myrtaceae), the whole plant of Hyptis
capitata J acq. (Labiatae), and the aerial parts of Tern-
stromia gymnanthera Sprague (Theaceae) demonstrated
significant anti-HIV activity (EC50 < 20 µg/mL). Sub-
sequent bioassay-directed fractionation of these extracts
has resulted in the isolation of oleanolic acid (1) as an
anti-HIV principle from all of the foregoing plants.
Pomolic acid (5), isolated from R. woodsii and H.
capitata, was also identified as an anti-HIV agent.
Resu lts a n d Discu ssion
In the course of our continuing screening of plant
extracts as potential anti-HIV agents, the MeOH ex-
tracts of the leaves of R. woodsii (Rosaceae), the leaves
and twigs of P. glandulosa (Leguminosae), the whole
plant of Ph. juniperinum (Loranthaceae), the leaves of
S. claviflorum (Myrtaceae), the whole plant of H.
capitata (Labiatae), and the aerial part of T. gymnan-
thera (Theaceae) demonstrated significant anti-HIV
activity (EC50 < 20 µg/mL) consistent with the presence
of an active substance(s). Subsequent bioassay-directed
solvent extraction of these extracts successively with
n-hexane, CHCl3, EtOAc, and n-BuOH yielded a CHCl3-
soluble anti-HIV active fraction in each case. Further
bioassay-guided chromatography of active fractions
resulted in the isolation of oleanolic acid (1) as an anti-
HIV principle from all of these plant extracts. Pomolic
acid (5), isolated from R. woodsii and H. capitata, was
also identified as an anti-HIV triterpene, while betulinic
acid (15), a previously reported anti-HIV triterpene, was
*
To whom correspondence should be addressed. Tel.: (919) 962-
0
066. Fax: (919) 966-3893. E-mail: khlee@unc.edu.
†
Natural Products Laboratory, University of North Carolina.
Biotech Research Laboratories.
Kyoto Pharmaceutical University.
College of Pharmacy, Nihon University.
Tokyo Metropolitan Govern Research Laboratory of Public Health.
Faculty of Pharmaceutical Sciences, Fukuoka University.
Niigata College of Pharmacy.
Shanghai Medical University.
‡
§
⊥
|
∇
#
O
S0163-3864(98)00071-8 CCC: $15.00
© 1998 American Chemical Society and American Society of Pharmacognosy
Published on Web 07/31/1998

Anti-AIDS Agents. 30
J ournal of Natural Products, 1998, Vol. 61, No. 9 1091
Ta ble 1. Anti-HIV Activities for Oleanolic Acid (1), Pomolic
Acid (5), and Related Triterpenes
of an oxygen-bearing methine carbon signal at δ 85.3
as well as the absence of one methylene carbon signal.
IC50 (µg/mL)a
EC50 (µg/mL)b
T. I.c
12.8
1
compound
The H NMR spectrum also resembled that of 10, but
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
21.8 ( 13.6
1.7 ( 1.7
it revealed an additional one-proton singlet at δ 4.37
d
8
NS
due to a hydroxy-carrying methine signal. The EIMS
36
15
2.4
5.7
16.6
3.3
2
+
of 11 gave an M ion at m/z 502, and the molecular
25
4.4
23.3 ( 16.6
1.4 ( 0.1
2.0 ( 1.0
4
formula was confirmed as C30H45O6 by HRFABMS. The
molecular weight was 16 mass units more than that of
6.5 ( 1.9
8
9
1
0. Treatment of 11 with Ac2O and pyridine yielded a
NS
+
15
40
40
NS
diacetate with an M ion peak at m/z 586. These
observations indicate that 11 possesses an additional
hydroxy group compared to 10. The location of the
additional hydroxy group was determined to be at C-1
by examination of HMBC; the methine proton signal at
δ 4.37 showed long-range correlations with the C-2
carbonyl (δ 212.2), C-9 methine (δ 48.7), and 26-CH3 (δ
0
1
2
3
4
6
8
9
0
1
2
3
4
5
6
7
8
20
2
NS
e
>20
NS
37.5 ( 2.9
NS
f
>100
35
20
>2.9
0.25
22 400
4
11.2
9.7
0.0005
NS
1.5
NS
14.2
22.1
6.5 ( 3.1
14.6
27.5
4.4
4.7
0.8
34.3 ( 12.5
500
9.3
1
7.1) carbon signals, and a long-range correlation
between H-25 (δ 0.96) and the carbon resonance at δ
85.3 was observed. The configurations of the hydroxy
groups at C-1 and C-3 were concluded to be â by
observation of NOEs between H-5 and H-1 as well as
H-3 in an NOE difference experiment. On the basis of
these spectral examinations, the structure of 11 was
concluded to be 1â-hydroxy-2-oxopomolic acid.
6.1 ( 4.0
1.1
7.1
2.1
8.3
3.3
1.1
4.1
1.2
3.9
0.11
0.5 ( 0.26
0.012
7.3
68.6
41 667
41 667
AZT
g
1
871 µM
0.045 ( 0.056
a
We have reported previously that modification of
betulinic acid (15), identified as the anti-HIV principle
of the leaves of S. claviflorum, yielded extremely potent
anti-HIV compounds, including 17, with an EC50 value
The agent concentration that inhibited H9 cell growth by 50%.
The agent concentration that inhibited viral replication in H9
b
c
d
cell by 50%. In vitro therapeutic index (TI) radio: IC50/EC50. NS
no suppression. Due to limited quantities, this agent could not
e
)
be tested at 100 µg/mL concentration; the maximum concentration
2
tested in the presence of DMSO was 20 µg/mL. f Because all agents
of < 0.000 35 µM and a T. I. of > 20 000. Because the
were dissolved in DMSO and their stock concentrations were 10
mg/mL, a minimum of a 1:100 dilution was needed to negate the
effects of DMSO on this assay; thus, the greatest agent concentra-
tion possible was 100 µg/mL. This EC50 value represents the
mean and standard deviation of 65 EC50 values for AZT.
anti-HIV activity of oleanolic acid (1) was comparable
to that of betulinic acid, oleanolic acid was considered
to be a potential anti-HIV lead triterpenoid. Based on
the these findings, derivatives of oleanolic acid were
prepared and evaluated for their inhibitory effect on
HIV replication. The structures of oleanolic acid and
betulinic acid are partially correlated, especially in the
A- through D-ring moiety, but differ mainly in the
E-ring moiety. Therefore, modification of oleanolic acid
focused on introducing an ester group at the C-3
hydroxy group, based on the structures of potent anti-
HIV betulinic acid derivatives.
g
isolated from R. woodsii and T. gymnanthera. Oleanolic
acid (1) and pomolic acid (5) demonstrated anti-HIV
activity, with EC50 values of 1.7 and 1.4 µg/mL, respec-
tively, and inhibited uninfected H9 cell growth with IC50
values of 21.8 and 23.3 µg/mL, respectively. Their
calculated therapeutic index (T. I.) values [defined as
toxicity (IC50) divided by anti-HIV activity (EC50)] were
1
2.8 and 16.6, respectively. Ursolic acid (6), isolated
As seen in previous betulinic acid derivatives, olean-
olic acid 3-O-3′,3′-dimethylsuccinate (18) demonstrated
a most potent anti-HIV activity, with an EC50 value of
from P. glandulosa, S. claviflorum, and H. capitata, was
found to show similar anti-HIV activity, with an EC50
value of 2.0 µg/mL and IC50 values of 6.5 µg/mL (T. I.
value, 3.3).
0
.0005 µg/mL, and showed a greatly improved T. I. value
(22 400), while the lack of any discernible anti-HIV
The HIV inhibitory effects of 11 oleanolic acid- and
pomolic acid-related triterpenes, isolated from various
activity in its isomer (19) was similar to the correspond-
ing betulinic acid derivative. Compounds 20, 25, and
4
,5,6,12
plant sources,
were examined. Among the com-
2
6 displayed relatively potent anti-HIV activity, with
pounds tested, only oleanolic acid and pomolic acid
exhibited anti-HIV activity as shown in Table 1. The
compounds with additional oxygen function(s) to those
in oleanolic acid or pomolic acid showed decreased anti-
HIV activity. Alphitolic acid (16), isolated from R.
woodsii, has the same structure as betulinic acid (15)
except for an additional hydroxy group at C-2; it also
showed decreased anti-HIV activity, which was similar
to observations found for oleanolic acid- and pomolic
acid-related compounds.
EC50 values of 1.5, 1.1, and 1.2 µg/mL, respectively, but
were not as potent as 18. Moreover, the small T. I.
values of 25 and 26 (4.1 and 3.9, respectively) suggested
that they are slightly toxic.
Oxidation of oleanolic acid (1) with pyridinium chlo-
rochromate (PCC) yielded a 3-oxo- derivative (27), which
was more toxic against uninfected H9 cells than its
parent compound, but inhibited HIV-1 replication, with
an improved EC50 value of 0.11 µg/mL. Treatment of 1
with a molar equivalent of potassium hydroxide fur-
nished the potassium salt of oleanolic acid (28). Com-
pound 28 exhibited potent anti-HIV activity, with an
EC50 value of 0.5 µg/mL and a T.I value of 68.6. Such
enhanced anti-HIV activity with the potassium salt
A new triterpenoid (11) was also obtained from the
CHCl3-soluble fraction of R. woodsii by repeated Si gel
and ODS chromatography, although it showed no anti-
HIV activity. The 13C NMR spectrum of 11 was similar
9
to that of 2-oxopomolic acid (10), except for the presence

1
092 J ournal of Natural Products, 1998, Vol. 61, No. 9
Kashiwada et al.
Ta ble 2. Anti-HIV Activities for MeOH Extract and Fractions
from Ph. juniperinum
IC50 (µg/mL)
EC50 (µg/mL)
MeOH extract
100
> 4 but < 20
n-hexane-soluble fraction
CHCl3-soluble fraction
Me2CO-soluble fraction
MeOH-soluble fraction
fraction C-1
100
4
> 20 but < 100
100
< 0.8
11
> 100
4
6
20
10
0.8
> 100
> 100
fraction C-2
> 4 but < 20
> 100
fraction C-3
fraction C-4
> 20 but < 100
20
fraction C-5
fraction C-6
> 20 but < 100
> 20 but < 100
> 1 but < 5
> 3 but < 15
fraction C-7
DIP-1000 polarimeter. MS were determined on a J EOL
1
13
HX-110 spectrometer. H and C NMR spectra were
measured on Bruker AC-300, J EOL A-400, and J EOL
A-500 spectrometers with TMS as internal standard. All
known triterpene acids were identified by comparison
with reported physical and spectral data.
P la n t Ma ter ia ls. R. woodsii was collected at Aspen
Grove, Mt. Timpanogos, Utah, in August 1990 and 1991.
Ph. juniperinum was collected at Blanding, Utah, in
April 1993, and P. glandulosa was collected at Witchita
Mountains Wild Refuge, Oklahoma, in May 1992.
Voucher specimens of R. woodsii and Ph. juniperinum
are deposited in the herbarium of Monte L. Bean Life
Science Museum, Brigham Young University, Provo,
Utah. The voucher specimen of P. glandulosa is depos-
ited in the Natural Products Laboratory, School of
Pharmacy, University of North Carolina at Chapel Hill,
Chapel Hill, North Carolina. Sy. claviflorum was
compared to the free carboxylic acid was also found in
caffeic acid tetramers.3
collected in Lanyu, Taiwan, in February 1990.
A
voucher specimen is deposited in the School of Phar-
macy, Kaohsiung Medical College, Taiwan. T. gymnan-
thera was collected in the People’s Republic of China.
The extraction and isolation of hyptatic acid A (2), 2R-
hydroxy-ursolic acid (7), ursolic acid (6), and tormentic
acid (9) from Hyptis capitata have been reported.4
Bioa ssa y-Gu id ed F r a ction a tion a n d Isola tion .
Fractionation and isolation were guided by the anti-HIV
assay results. A typical example is described here for
the bioassay-guided fractionation and isolation of ole-
anolic acid (1) from Ph. juniperinum. The dried whole
plant of Ph. juniperinum (4.5 kg) was extracted with
MeOH, and the MeOH extract (750 g) was adsorbed on
Celite. The Celite-MeOH mixture was washed succes-
sively with n-hexane, CHCl3, Me2CO, and MeOH. The
CHCl3-soluble fraction (70 g) was chromatographed on
Si gel using CHCl3-MeOH (1:0 f 9:1) to give seven
fractions (fractions C-1-C-7). Fraction C-5 consisted
mainly of oleanolic acid, which was purified by Si gel
chromatography with C6H6-EtOAc (10:1 f 3:1) as
eluent to afford pure compound 1 (12.6 g). The anti-
HIV activities for the MeOH extract and each fraction
from P. juniperinum are shown in Table 2.
,5
Isola tion of Olea n olic Acid (1), Ar ju n olic Acid
(
3), Ur solic Acid (6), Asia n tic Acid (8), a n d Betu -
lin ic Acid (15) fr om Sy. cla viflor u m . The dried
leaves (3.0 kg) of Sy. claviflorum were extracted with
MeOH, and a portion (120g) of this MeOH extract (315
Exp er im en ta l Section
Gen er al Exper im en tal P r ocedu r es. Melting points
were measured on a Fisher-J ohns or a Yanako mi-
cromelting-point apparatus and are uncorrected. Opti-
cal rotations were measured on a J ASCO DIP-360 or
g total) was suspended in H O and partitioned with
hexane, CHCl3, EtOAc, and BuOH, successively. The
CHCl3 extract (25.5 g) was chromatographed on Si gel
using CHCl3-EtOAc (4:1) and CHCl3-MeOH (95:5) as
2

Anti-AIDS Agents. 30
J ournal of Natural Products, 1998, Vol. 61, No. 9 1093
eluents to afford fractions 1-6. Fraction 2 was chro-
matographed on MCI gel CHP-20P (90 f 100% MeOH)
and reversed-phase Si gel (ODS) (90% MeOH in 1% CH3-
COOH) to give oleanolic acid (1) (42 mg), ursolic acid
room temperature overnight. The mixture was concen-
trated to dryness, then was purified by column chro-
matography on Si gel with CHCl3 to furnish the
diacetate (7.5 mg) as colorless needles (from C H -
6
6
5
6
1
(6) (62 mg), and betulinic acid (15) (612 mg). Fraction
hexane): mp 264-265 °C; H NMR (CDCl ) δ 0.73 (3H,
3
5
1
was chromatographed on MCI gel CHP-20P (80 f
s, H-24), 0.80 (3H, s, H-25), 0.95 (3H, d, J ) 6.5 Hz,
00% MeOH) and ODS (60 f 100% MeOH) to yield a
H-30), 0.99 (3H, s, H-26), 1.10 (3H, s, H-23), 1.21 (3H,
s, H-29), 1.29 (3H, s, H-27), 2.16 (6H, s, OAc × 2), 2.52
(1H, s, H-18), 2.55 (1H, m, H-16R), 5.04 (1H, s, H-3),
5.08 (1H, s, H-1), 5.30 (1H, t, J ) 3 Hz, H-12); EIMS
7
mixture (480 mg) of arjunolic acid (3) and asiatic acid
7
(8). The mixture was dissolved and stirred in pyridine
(3 mL) and Ac2O (3 mL) at room temperature for 24 h.
+
The solvent was evaporated, and the residue was
chromatographed on ODS (85% MeOH in 1% CH3-
COOH) to give the acetates of 3 and 8. Both acetates
were treated with 2% NaOMe-MeOH at room temper-
ature for 1 day followed by neutralization with Amber-
lite IR-120B to afford the deacylated products, 3 (262
mg) and 8 (122 mg).
Isola tion of Olea n olic Acid (1), P om olic Acid (5),
Tor m en tic Acid (9), 2-Oxop om olic Acid (10), 1â-
Hyd r oxy-2-oxop om olic Acid (11), 1â,2â-Dih yd r oxy-
p om olic Acid (12), Eu sca p h ic Aid (13), 1â-Hyd r ox-
yeu sca p h ic Acid (14), Betu lin ic Acid (15), a n d
Alp h itolic Acid (16) fr om R. wood sii. The dried
leaves of R. woodsii (2.3 kg) were extracted with MeOH,
and the MeOH extract (390 g) was adsorbed on Celite.
The Celite-MeOH mixture was washed successively
with hexane, CHCl3, Me2CO, and MeOH. The CHCl3-
soluble fraction (22 g) was chromatographed on Si gel
to give fractions 1-7. Fraction 1 was purified by Si gel
column chromatography (C6H6) to give 3-O-acetyl po-
molic acid (14 mg). Fractions 2 and 3 were combined
and chromatographed on Si gel (C6H6-EtOAc, 19:1 f
m/z 586 (M ), 480, 438, 408, 366, 146.
Isola tion of Olea n olic Acid (1) a n d Ur solic Acid
(
6) fr om P . gla n d u losa . The dried leaves of Pr.
glandulosa (1.3 kg) were extracted with MeOH, and the
MeOH extract (225 g) was adsorbed on Celite. The
Celite-MeOH mixture was washed with hexane, CHCl3,
Me2CO, and MeOH, successively. The CHCl3-soluble
fraction (12 g) was repeatedly chromatographed on Si
gel using CHCl3-MeOH (1:0 f 4:1), C6H6-EtOAc (9:1
f 4:1), and hexane-EtOAc (8:1 f 2:1) as eluents to give
oleanolic acid (1) (20 mg) and ursolic acid (6) (186 mg).
Isola tion of Olea n olic Acid (1) fr om P h . ju n i-
per in u m . The dried whole plant of Ph. juniperinum
(4.5 kg) was extracted with MeOH, and the MeOH
extract (750 g) was adsorbed on Celite. The Celite-
MeOH mixture was washed with n-hexane, CHCl3, Me2-
CO, and MeOH, successively. The CHCl3-soluble frac-
tion (70 g) was repeatedly chromatographed on Si gel
using CHCl3-MeOH (1:0 f 9:1) and C6H6-EtOAc (10:1
f 3:1) as eluents to afford oleanolic acid (1) (12.6 g).
Isola tion of Olea n olic Acid (1) a n d Betu lin ic a cid
(
15) fr om T. gym n a n th er a . The 95% EtOH extract
9
:1) to yield oleanolic acid (1) (15 mg) and betulinic acid
6
(45 g) of T. gymnanthera (300 g) was fractioned by
n-hexane, CHCl3, and EtOAc, respectively. The CHCl3-
soluble fraction (6 g) was repeatedly chromatographed
on Si gel using CHCl3-n-hexane (1:1 f 4:0) as eluents
to afford oleanolic acid (1) (106 mg) and betulinic acid
(15) (8 mg). Fraction 4 was chromatographed on Si gel
(
CHCl3-MeOH, 1:0 f 19:1) and ODS (75 f 85% MeOH)
8
to give oleanolic acid (1) (137 mg), pomolic acid (5) (33
mg), and 2-oxopomolic acid (10) (54 mg). Fractions 5
9
and 6 were also combined and repeatedly chromato-
graphed on ODS (65 f 75% MeOH) and finally purified
by Si gel column chromatography (CHCl3-MeOH, 1:0
(15) (87 mg).
Gen er a l P r oced u r e for P r ep a r a tion of 3-O-Acyl
8
f 19:1) to afford tormentic acid (9) (72 mg), euscaphic
Olea n olic Acid Der iva tives (18-26). A mixture of
oleanolic acid, an appropriate acid anhydride (2.5-10
equivalent mol) and (dimethylamino)pyridine (1 equiva-
lent mol) in pyridine (5-10 mL) was refluxed overnight.
The reaction mixture was diluted with ice-water and
extracted with CHCl3. The organic layer was washed
with H2O, dried over MgSO4, and concentrated under
reduced pressure. The residue was purified by Si gel
column chromatography to afford the product. Separa-
tion of a mixture of 18 and 19 was achieved by
semipreparative-scale HPLC.
8
10
acid (13) (124 mg), 1â-hydroxyeuscaphic acid (14) (21
1
1
mg), 1â,2â-dihydroxypomolic acid (12) (20 mg), and a
new triterpene acid (11) (24 mg).
1
â-Hyd r oxy-2-oxop om olic a cid (1â,3â,19r-tr ih y-
d r oxy-2-oxou r s-12-en -28-oic a cid ) (11): a white crys-
talline powder from hexane-Et2O; mp 227-229 °C;
2
3
[
R] D -105.6 °C (c 0.26, MeOH); IR (KBr) νmax 3570,
-
1 1
3
0
7
480, 2930, 1715, 1690 cm ; H NMR (pyridine-d5) δ
.90 (3H, s, H-24), 0.96 (3H, s, H-25), 1.13 (3H, d, J )
Hz, H-30), 1.14 (3H, s, H-26), 1.35 (3H, s, H-23), 1.45
(
(
(
1
2
3H, s, H-29), 1.80 (3H, s, H-27), 3.07 (1H, s, H-18), 3.16
1H, m, H-16a), 4.33 (1H, s, H-3), 4.37 (1H, s, H-1), 5.70
1H, t-like, H-12); 13C NMR δ (pyridine-d5) 12.5 (C-25),
6.8 (C-30), 17.1 (C-24 and 26), 18.7 (C-6), 24.7 (C-27),
6.4 (C-16), 27.0 (C-21), 27.1 (C-29), 27.3 (C-11), 29.4
Olea n olic a cid 3-O-(3′,3′-d im eth yl)su ccin a te (18):
colorless rods (from MeOH-EtOAc); mp 268-270 °C;
27
1
[R]
D
+59.2° [c 0.6, MeOH + CHCl (1:1)]; H NMR
3
(pyridine-d , 400 MHz) δ 0.82 (3H, s, H-25), 0.93 (3H,
5
s, H-24), 0.96 (6H, s, H-23 and H-29), 0.98 (3H, s, H-26),
(C-23), 29.5 (C-15), 33.4 (C-7), 38.5 (C-22), 41.2 (C-8),
1.01 (3H, s, H-30), 1.26 (3H, s, H-27), 1.54 (6H, s,
4
9
2.2 (C-14), 42.4 (C-20), 45.8 (C-4), 48.3 (C-17), 48.7 (C-
), 49.6 (C-10), 51.9 (C-5), 54.6 (C-18), 72.7 (C-19), 81.9
dimethylsuccinyl CH ), 2.88, 2.95 (each 1H, d, J ) 15.5
3
Hz, H -2′), 3.30 (1H, dd, J ) 4.0, 13.5 Hz, H-18), 4.76
2
(C-3), 85.3 (C-1), 129.1 (C-12), 138.9 (C-13), 180.7 (C-
(1H, dd, J ) 4.5, 11.5 Hz, H-3), 5.46 (1H, t, J ) 3 Hz,
+
13
2
3
8), 212.2 (C-2); EIMS m/z 502 (M ), 484, 466, 456, 438,
H-12); C NMR (pyridine-d , 75 MHz) δ 15.4 (C-25),
5
66, 246, 220, 187, 165, 146; negative-ion HRFABMS
17.1 (C-24), 17.4 (C-26), 18.5 (C-6), 23.7 (C-16), 23.7 (C-
-
m/z 501.3212 [M - H] , C30H45O6 requires 501.3216.
1
1), 23.8 (C-2), 23.9 (C-30), 25.9 (CH3-3′), 26.1 (CH3-3′),
Acetyla tion of 11. A mixture of 11 (8 mg) in Ac2O
0.5 mL) and pyridine (0.5 mL) was kept standing at
26.2 (C-27), 28.2 (C-23), 28.3 (C-15), 31.0 (C-20), 33.0
(C-7), 33.2 (C-15), 33.3 (C-22), 34.3 (C-29), 37.2 (C-10),
(

1
094 J ournal of Natural Products, 1998, Vol. 61, No. 9
Kashiwada et al.
3
1
7.9 (C-4), 38.2 (C-1), 39.7 (C-8), 40.9 (C-3′), 42.0 (C-
8), 42.2 (C-14), 45.2 (C-2′), 46.5 (C-19), 46.7 (C-17), 47.9
COOH), 181.2 (C-28); negative HRFABMS m/z 571.3637
([M - H] ), C34H51O7 requires 571.3635.
-
(C-9), 55.6 (C-5), 80.9 (C-3), 122.4 (C-12), 144.8 (C-13),
Olean olic acid 3-O-su ccin ate (22): colorless needles
1
71.5 (dimethylsuccinyl COO), 179.2 (dimethylsuccinyl
23
from C6H6, mp 261-263 °C; [R] D +60.0° (c 0.21,
COOH), 180.1 (C-28); positive FABMS m/z 607.3970 ([M
+
1
MeOH); H NMR (pyridine-d5, 500 MHz) δ 0.83 (3H, s,
+
Na] ), C36H56NaO6 requires 607.3974.
H-25), 0.93 (3H, s, H-24), 0.96 (3H, s, H-29), 0.98 (3H,
s, H-23), 0.99 (3H, s, H-26), 1.02 (3H, s, H-30), 1.27 (3H,
s, H-27), 2.89, 2.93 (each 2H, m, succinyl CH2), 3.29 (1H,
dd, J ) 4, 14 Hz, H-18), 4.78 (1H, dd, J ) 5, 11 Hz,
Olea n olic a cid 3-O-(2′,2′-d im eth yl)su ccin a te (19):
a white crystalline powder (from MeOH-EtOAc); mp
2
8
2
1
0
1
1
75-278 °C; [R] D +59.8° [c 0.7, MeOH + CHCl3 (1:
1
13
)]; H NMR (pyridine-d5, 400 MHz) δ 0.82 (3H, s, H-25),
H-3), 5.47 (1H, t-like, H-12); C NMR (pyridine-d5, 100
.94 (3H, s, H-24), 0.97 (3H, s, H-29), 1.00 (3H, s, H-26),
.02 (3H, s, H-30), 1.04 (3H, s, H-23), 1.27 (3H, s, H-27),
.49 (6H, s, dimethylsuccinyl CH3), 2.91, 2.96 (each 1H,
MHz) δ 15.3 (C-25), 16.9 (C-24), 17.3 (C-26), 18.4 (C-6),
23.6 (C-16), 23.7 (C-11 and C-30), 23.8 (C-2), 26.1 (C-
27), 28.1 (C-23), 28.2 (C-15), 30.9 (C-20), 33.0 (C-22),
33.1 (C-7), 33.2 (C-29), 34.2 (C-21), 37.1 (C-10), 37.9 (C-
4), 38.1 (C-1), 39.7 (C-8), 41.9 (C-18), 42.1 (C-14), 46.4
(C-19), 46.6 (C-17), 47.8 (C-9), 55.5 (C-5), 80.9 (C-3),
122.3 (C-12), 144.8 (C-13), 180.0 (C-28); succinyl moiety,
29.9, 30.3, 172.4, 174.7; negative ion HRFABMS m/z
d, J ) 16.5 Hz, H2-3′), 3.31 (1H, dd, J ) 4.0, 13.5 Hz,
H-18), 4.78 (1H, dd, J ) 4.5, 11.5 Hz, H-3), 5.47 (1H, t,
J ) 3.5 Hz, H-12); 13C NMR (pyridine-d5, 75 MHz) δ
1
1
2
5.3 (C-25), 17.1 (C-24), 17.4 (C-26), 18.4 (C-6), 23.6 (C-
6), 23.7 (C-11), 23.8 (C-2), 23.8 (C-30), 25.9 (C-2′-CH3),
5.9 (C-2′-CH3), 26.2 (C-27), 28.3 (C-23), 28.3 (C-15), 31
-
555.3685 [M - H] , C34H51O6 requires 555.3685.
(
C-20), 33.1 (C-7), 33.2 (C-22), 33.3 (C-29), 34.3 (C-21),
Olean olic acid 3-O-glu tar ate (23): colorless needles
2
4
3
4
1
7.2 (C-10), 38.1 (C-4), 38.1 (C-1), 39.7 (C-8), 41 (C-2′),
2 (C-18), 42.2 (C-14), 44.8 (C-3′), 46.5 (C-19), 46.7 (C-
7), 47.9 (C-9), 55.7 (C-5), 80.6 (C-3), 122.4 (C-12), 144.9
from CHCl , mp 222-223 °C; [R]
+84.8° (c 0.40,
3
D
1
CHCl ); H NMR (CDCl , 300 MHz) δ 0.74 (3H, s, H-25),
3
3
0.85 (3H, s, H-24), 0.88 (3H, s, H-29), 0.91 (3H, s, H-23),
0.94 (3H, s, H-26), 0.95 (3H, s, H-30), 1.13 (3H, s, H-27),
(
C-13), 174 (dimethylsuccinyl COO-), 176.7 (dimethyl-
succinyl COOH), 180.2 (C-28); positive FABMS m/z
2.42 (4H, m, glutaryl CH ), 2.82 (1H, br d, J ) 14 Hz,
2
+
6
07.3973 ([M + Na] ), C36H56NaO6 requires 607.3974.
H-18), 4.52 (1H, dd, J ) 5, 11 Hz, H-3), 5.27 (1H, t-like,
1
3
Olea n olic a cid 3-O-(3′,3′-d im eth yl)glu ta r a te (20):
H-12); C NMR (CDCl3, 75 MHz) δ 15.4 (C-25), 16.8
(C-24), 17.1 (C-26), 18.2 (C-6), 20.16, 22.8 (C-16), 23.4
(C-2), 23.5 (C-11), 23.6 (C-30), 25.9 (C-27), 28.2 (C-23
and C-15), 30.6 (C-20), 32.4 (C-22), 33.1 (C-7), 33.8 (C-
29), 37.0 (C-21), 37.7 (C-10 and C-4), 37.9 (C-1), 39.3
(C-8), 40.9 (C-18), 41.5 (C-14), 45.8 (C-19), 46.5 (C-17),
47.4 (C-9), 55.2 (C-5), 81.0 (C-3), 122.6 (C-12), 143.6 (C-
13), 179.4 (C-28); glutaryl moiety: 27.7, 29.7, 32.4,
172.4, 184.9; anal. C 73.75%, H 9.62%, calcd for
C35H54O6, C 73.65%, H 9.54%.
2
2
colorless needles (from MeOH), mp 244-245 °C; [R]
+
dine-d5, 300 MHz) δ 0.84 (3H, s, H-25), 0.93 (3H, s,
H-24), 0.97, 0.98 (6H, s, H-23 and H-29), 0.99 (3H, s,
H-26), 1.02 (3H, s, H-30), 1.29 (3H, s, H-27), 1.37, 1.38
D
1
54.9° [c 0.51, MeOH + CHCl3 (1:1)]; H NMR (pyri-
(each 3H, s, dimethylglutaryl CH3), 2.78 (4H, m, dim-
ethylglutaryl H2-2′ and 4′), 3.30 (1H, dd, J ) 4, 14 Hz,
H-18), 4.74 (1H, dd, J ) 5, 11.5 Hz, H-3), 5.47 (1H, t-like,
H-12); C NMR (pyridine-d5, 75 MHz) δ 15.5 (C-25),
1
3
1
1
7.3 (C-24), 17.5 (C-26), 18.6 (C-6), 23.8 (C-16), 23.9 (C-
1), 23.9 (C-30), 24.2 (C-2), 26.3 (C-27), 28.1 (dimeth-
Olea n olic a cid 3-O-(3′-m eth yl)glu ta r a te (24): col-
2
4
orless needles from CHCl , mp 205-206 °C; [R]
3
D
1
ylglutaryl CH3), 28.4 (C-23 and 15), 31.1 (dimethylglu-
taryl C-3′), 32.8 (C-20), 33.2 (C-7), 33.3 (C-22), 33.4 (C-
+44.9° (c 0.4, CHCl ); H NMR (CDCl , 300 MHz) δ 0.74
3
3
(3H, s, H-25), 0.85 (3H, s, H-24), 0.87 (3H, s, H-29), 0.91
2
(
9), 34.4 (C-21), 37.3 (C-10), 38 (C-1), 38.4 (C-4), 39.8
C-8), 42.1 (C-18), 42.3 (C-14), 46.1 (dimethylglutaryl
C-2′ and 4′), 46.6 (C-19), 46.8 (C-17), 48 (C-9), 55.7 (C-
), 80.9 (C-3), 122.5 (C-13), 145 (C-14), 172.2 (dimeth-
(3H, s, H-23), 0.92 (3H, s, H-26), 0.94 (3H, s, H-30), 1.06
(3H, d, 3-methyl glutaryl CH ), 1.12 (3H, s, H-27), 2.29,
3
2.45 (each 2H, m, 3-methyl glutaryl CH ), 2.81 (1H, br
2
5
d, J ) 14 Hz, H-18), 4.53 (1H, dd, J ) 5, 11 Hz, H-3),
1
3
ylglutaryl COO), 174.7 (dimethylglutaryl COOH), 180.5
5.27 (1H, t-like, H-12); C NMR (CDCl , 75 MHz) δ 15.3
3
-
(
C-28); negative HRFABMS m/z 597.4157 ([M - H] ),
(C-25), 16.8 (C-24), 17.1 (C-26), 18.2 (C-6), 22.8 (C-16),
C37H57O6 requires 597.4155.
23.4 (C-2), 23.6 (C-11 and C-30), 25.9 (C-27), 28.1 (C-
23), 28.2 (C-15), 30.9 (C-20), 32.5 (C-22), 33.0 (C-7), 33.7
(C-29), 37.0 (C-21), 37.7 (C-10), 37.9 (C-4), 37.9 (C-1),
39.2 (C-8), 40.8 (C-18), 41.5 (C-14), 45.8 (C-19), 46.6 (C-
Olean olic acid 3-O-diglyor ate (21): colorless needles
2
2
(
from MeOH); mp 260-263 °C; [R] D +64.4° [c 0.36,
1
MeOH + CHCl3 (1:1)]; H NMR (CDCl3 + CD3OD, 400
MHz) δ 0.81 (3H, s, H-25), 0.88 (3H, s, H-24), 0.90 (3H,
s, H-29), 0.91 (3H, s, H-23), 0.94 (3H, s, H-26), 0.96 (3H,
s, H-30), 1.17 (3H, s, H-27), 2.85 (1H, dd, J ) 4, 14 Hz,
H-18), 4.19 (2H, s, H-4′), 4.25, 4.29 (each 1H, d, J ) 16.5
Hz, H-2′), 4.63 (1H, dd, J ) 5, 10.5 Hz, H-3), 5.28 (1H,
t, J ) 3.5 Hz, H-12); 13C NMR (CDCl3 + CD3OD, 100
MHz) δ 15.7 (C-25), 16.9 (C-26), 17.2 (C-24), 18.7 (C-6),
1
(
5
7), 47.5 (C-9), 55.2 (C-5), 81.0 (C-3), 122.5 (C-12), 143.5
C-13), 178.8 (C-28); succinyl moiety, 19.9, 27.3, 40.5,
1.53, 171.9, 184.8; anal. C 72.18%, H 9.28%, calcd for
C36H56O6‚3/4H2O, C 72.27%, H 9.68%.
Olea n olic a cid 3-O-(3′-m eth yl-3′-eth yl)glu ta r a te
(25): colorless needles from CHCl , mp 266-267 °C;
3
2
4
1
[R] D +58.7° (c 0.42, CHCl3); H NMR (CDCl3, 300
MHz) δ 0.73 (3H, s, H-25), 0.82 (3H, s, H-24), 0.88 (3H,
s, H-29), 0.91 (3H, s, H-23), 0.94 (3H, s, H-26), 0.97 (3H,
s, H-30), 1.02, 1.09 (1.5 H each, s, (RS)-3′-methyl-3′-
ethyl-glutaryl CH3), 1.13 (3H, s, H-27), 2.43-2.75 (4H,
m, (RS)-3′-methyl-3′-ethyl-glutaryl COCH2), 2.81 (1H,
dd, J ) 2, 14 Hz, H-18), 4.50, 4.53 (0.5 H each, both t,
J ) 11 Hz, H-3), 5.28 (1H, t-like, H-12); 13C NMR
2
2
3
4
3.5 (C-11), 23.8 (C-30), 23.9 (C-16), 23.9 (C-2), 26.3 (C-
7), 28.2 (C-15), 28.6 (C-23), 31.1 (C-20), 33.1 (C-22),
3.2 (C-7), 33.3 (C-29), 34.3 (C-21), 37.4 (C-10), 38.3 (C-
), 38.6 (C-1), 39.8 (C-8), 41.8 (C-14), 42.3 (C-18), 46.5
(
C-19), 46.9 (C-17), 48.2 (C-9), 56 (C-5), 68.2 (diglucoryl
CH2), 70.7 (diglucoryl CH2), 84.7 (C-3), 122.5 (C-12),
44.5 (C-13), 173.2 (diglycoryl COO-), 175.5 (diglycoryl
1

Anti-AIDS Agents. 30
CDCl3, 75 MHz) δ 15.5 (C-25), 17.0 (C-24), 17.5 (C-26),
J ournal of Natural Products, 1998, Vol. 61, No. 9 1095
(
dium. The mock-infected sample was used for toxicity
determinations (IC , see definition below). The stock
1
2
8.3 (C-6), 22.6 (C-16), 23.3 (C-2), 23.6 (C-11 and C-30),
5.9 (C-27), 27.7 (C-23), 28.3 (C-15), 30.6 (C-20), 32.3
5
0
virus used for these studies typically had a TCID50 value
4
(C-22), 33.0 (C-7), 33.7 (C-29), 37.0 (C-21), 37.6, 37.6 (C-
of 10 Infectious Units (IU)/mL. The appropriate amount
1
1
8
0 and C-4), 37.7 (C-1), 39.2 (C-8), 40.8 (C-18), 41.3 (C-
4), 45.8 (C-19), 46.5 (C-17), 47.3 (C-9), 55.1 (C-5), 80.8,
1.0 (0.5 C each, C-3), 122.6 (C-12), 143.7 (C-13), 178.2
of virus for a multiplicity of infection between 0.1 and
0
.01 IU/cell was added to the first aliquot of cells. The
other aliquot of cells received only culture medium and
was then incubated under identical conditions to the
HIV-infected cells. After a 4-h incubation at 37 °C and
(C-28); (RS)-3′-methyl-3′-ethyl-glutaryl moiety: δ 7.9,
8
4
.0, 24.4, 24.6, 32.5, 32.9, 33.4, 35.2, 41.8, 42.3, 42.7,
3.1, 171.9, 172.0 (each 0.5C), 184.8; anal. C 73.84%,
5
% CO2, both cell populations were washed three times
H 9.75%, calcd for C38H60O6‚1/4H2O, C 73.93%, H 9.88%.
Olea n olic a cid 3-O-(3′,3′-t et r a m et h ylen e)glu t -
a r a te (26): colorless needles from CHCl3, mp 259-260
with fresh medium and then added to the appropriate
wells of a 24-well plate containing the various concen-
trations of the test drug or culture medium (positive
infected control/negative-control drug). In addition, AZT
was also assayed during each experiment as a positive-
control drug. The plates were incubated at 37 °C and
2
4
1
°
C; [R] D +44.1° (c 0.20, CHCl3); H NMR (CDCl3 +
CD3OD 1:1, 300 MHz) δ 0.61 (3H, s, H-25), 0.69 (3H, s,
H-24), 0.70 (3H, s, H-29), 0.72 (3H, s, H-23), 0.75 (3H,
s, H-26), 0.76 (3H, s, H-30), 0.97 (3H, s, H-27), 1.44-
5
% CO2 for 4 days. Cell-free supernatants were col-
1
.49 (8H, m, 4 H2 of suberoyl), 2.34, 2.38 (each 2H, m,
lected on day 4 and tested by an in-house p24 antigen
ELISA assay; p24 antigen is a core protein of HIV and,
therefore, is an indirect measure of virus present in the
supernatants. Toxicity was determined by performing
cell counts by a coulter counter on the mock-infected
cells, which had either received culture medium (no
toxicity) or test sample or AZT. If a test sample had
suppressive capability and was not toxic, its effects are
reported in the following terms: IC , the concentration
of test sample that was toxic to 50% of the mock-infected
cells; EC50, the concentration of the test sample that was
able to suppress HIV replication by 50%; and therapeu-
tic index (TI), the ratio of IC50 to EC50.
COCH2 of suberoyl), 2.50 (1H, dd, J ) 4, 14 Hz, H-18),
4
.31 (1H, t, J ) 8.5 Hz, H-3), 5.09 (1H, t-like, H-12);
C NMR (CDCl3, 75 MHz) δ 15.0 (C-25), 16.4 (C-24),
6.5 (C-26), 18.0 (C-6), 22.8 (C-16), 23.3 (C-2), 23.7 (C-
1 and C-30), 25.6 (C-27), 27.4 (C-23), 27.8 (C-15), 30.4
1
3
1
1
(C-20), 32.4 (C-22), 32.7 (C-7), 33.6 (C-29), 37.3 (C-21),
3
1
7.6 (C-10), 37.8 (C-4), 37.9 (C-1), 39.1 (C-8), 41.0 (C-
8), 41.8 (C-14), 45.7 (C-19), 46.2 (C-17), 47.3 (C-9), 55.2
(C-5), 81.1 (C-3), 122.0 (C-12), 143.7 (C-13), 174.6 (C-
5
0
2
1
8); suberoyl moiety: δ 23.2, 23.8, 32.3, 36.7, 41.5, 41.9,
72.7, 180.7; anal. C 72.65%, H 9.45%, calcd for
C39H60O6‚H2O, C 72.86%, H 9.72%.
Oxid a t ion of Olea n olic Acid (27). A mixture of
oleanolic acid (1) (180 mg) and PCC (170 mg) in CH2-
Cl2 (70 mL) was stirred at room temperature for 25 h.
The solid was filtered using Celite and discarded, and
the filtrate was evaporated. The reaction product was
purified by Si gel column chromatography using CHCl3
as eluent to give 3-oxooleanolic acid (27) (102 mg) as
colorless prisms (from n-hexane-EtOAc), mp 201-203
Ack n ow led gm en t . This investigation was sup-
ported by grant AI-33066 from the National Institute
of Allergies and Infectious Diseases awarded to K. H.
Lee, and, in part, by a Grant-in-Aid for International
Scientific Research (J oint Research) from the Ministry
of Education, Science, Sports and Culture, Tokyo,
J apan.
2
5
°
[
C; [R] D +88.1° (c 0.49, MeOH); HREIMS m/z 453.3372
+
M - H] , C30H45O3 requires 453.3368.
P r ep a r a tion of P ota ssiu m Sa lts of Olea n olic
Refer en ces a n d Notes
Acid (28). A solution of oleanolic acid (105 mg) was
treated with 2% KOH in Me2CO-H2O (1:1) at room
temperature for 30 min. After removal of Me2CO by
evaporation, the resulting aqueous solution was sub-
jected to pass over a MCI-gel CHP20P column, and
washed with H2O to remove excess amounts of KOH.
Subsequent elution with MeOH gave potassium olean-
ate (27) (98 mg) as a white powder; mp 285-288 °C
(1) Part 29: Lee, C. T.-L.; Lin, V. C.-K.; Zhang, S. X.; Zhu, X. K.;
VanVliet, D.; Hu, H.; Beers, S. A.; Wang, Z. Q.; Cosentino, L.
M.; Morris-Natschke, S. L.; Lee, K. H. Bioorg. Med. Chem. Lett.
1
997, 7, 2897-2902.
(2) Kashiwada, Y.; Hashimoto, F.; Cosentino, L. M.; Chen, C. H.;
Garrett, P. E.; Lee, K. H. J . Med. Chem. 1996, 39, 1016-1017.
(
3) Kashiwada, Y.; Nishizawa, T.; Yamagishi, T.; Tanaka, T.;
Nonaka, G.; Cosentino, L. M.; Snider, J . V.; and Lee, K. H. J .
Nat. Prod. 1994, 58, 392-400.
+
+
(
decomp); FABMS m/z 495 [M + H] , 533 [M + K] .
(4) Yamagishi, T.; Zhang, D. C.; Chang, J . J .; McPhail, D. R.;
McPhail, A. T.; Lee, K. H. Phytochemistry 1988, 27, 3213-3216.
An ti-HIV Assa y. The T cell line, H9, was main-
tained in continuous culture with complete medium
RPMI 1640 with 10% fetal calf serum [FCS] supple-
(
5) Lee, K. H.; Lin, Y. M.; Wu, T. S.; Zhang, D. C.; Yamagishi, T.;
Hayashi, T.; Hall, I. H.; Chang, J . J .; Wu, R. Y.; Yang, T. H.
Planta Med. 1988, 53, 308-311.
(
(
6) Fujioka, T.; Kashiwada, Y.; Kilkuskie, R. E.; Chen, I. S.; Lee,
K. H. J . Nat. Prod. 1994, 57, 243-247.
mented with L-glutamine) at 5% CO2 and 37 °C. Ali-
quots of this cell line were used in experiments only
when in log-phase of growth.
Test samples were first dissolved in dimethyl sulfox-
ide. The following are the final drug concentrations
routinely used for screening: 100, 20, 4, and 0.8 µg/mL.
For active agents, additional dilutions are prepared for
subsequent testing so that an accurate EC50 values (see
definition below) could be achieved.
(
7) Cheung, H. T.; Feng, M. C. J . Chem. Soc. 1968, 1047-1051.
(8) Takahashi, K.; Kawagushi, S.; Nishura. K.; Kubota, K.; Tanabe,
Y.; Takani, M. Chem. Pharm. Bull. 1974, 22, 650-653.
(
9) J ia, Z. J .; Liu, X. Q.; Liu, Z. M. Phytochemistry 1993, 32, 155-
1
59.
(10) Guang-Yi, L.; Gray, A. I.; Waterman, P. G. J . Nat. Prod. 1989,
2, 162-166.
11) Kouno, I.; Baba, N.; Ohni, Y.; Kawano, N. Phytochemistry 1988,
5
(
(
2
7, 297-299.
12) Kashiwada, Y.; Zhang, D. C.; Chen, Y. P.; Cheng, C. M.; Chen,
H. T.; Chang, H. C.; Chang, J . J .; Lee, K. H. J . Nat. Prod. 1993,
As the test samples were being prepared, an aliquot
of H9 cells was infected with HIV-1 (IIIB isolate), while
another aliquot was mock-infected with complete me-
5
6, 2077-2082.
NP9800710