Protein tyrosine phosphatase 1B inhibitory activity of triterpenes isolated from Astilbe koreana

Article abstract of DOI:10.1016/j.bmcl.2006.03.036

Bioassay-guided fractionation of a MeOH extract of the rhizomes of Astilbe koreana (Saxifragaceae), using an in vitro protein tyrosine phosphatase 1B (PTP1B) inhibitory assay, resulted in the isolation of a new triterpene, 3α,24-dihydroxyolean-12-en-27-oic acid (4), along with four triterpenes, 3-oxoolean-12-en-27-oic acid (1), 3β-hydroxyolean-12-en-27-oic acid (β-peltoboykinolic acid; 2), 3β-hydroxyurs-12-en-27-oic acid (3), and 3β,6β-dihydroxyolean-12-en-27-oic acid (astilbic acid; 5). Compounds 1-5 inhibited PTP1B with IC₅₀ values of 6.8 ± 0.5, 5.2 ± 0.5, 4.9 ± 0.4, 11.7 ± 0.9, and 12.8 ± 1.1 μM, respectively. Our results indicate that 3-hydroxyl group and a carboxyl group in this type of triterpenes may be required for the activity, while addition of one more hydroxyl group at C-6 or C-24 may be responsible for a loss of activity. Thus, compounds 2 and 3 which possess only one hydroxyl group at C-3 and a carboxyl group at C-27 could be potential PTP1B inhibitors.

Full text of DOI:10.1016/j.bmcl.2006.03.036

Bioorganic & Medicinal Chemistry Letters 16 (2006) 3273–3276
Protein tyrosine phosphatase 1B inhibitory activity
of triterpenes isolated from Astilbe koreana
MinKyun Na,a Long Cui,a Byung Sun Min,b KiHwan Bae,c Jae Kuk Yoo,c
Bo Yeon Kim,a Won Keun Oha,* and Jong Seog Ahna
aKorea Research Institute of Bioscience and Biotechnology (KRIBB), 52 Eoun-dong, Yuseong-gu, Daejeon 305-333, Republic of Korea
bCollege of Pharmacy, Catholic University of Daegu, Gyeongsan 712-702, Republic of Korea
cCollege of Pharmacy, Chungnam National University, Daejeon 305-764, Republic of Korea
Received 13 December 2005; revised 23 February 2006; accepted 13 March 2006
Available online 31 March 2006
Abstract—Bioassay-guided fractionation of a MeOH extract of the rhizomes of Astilbe koreana (Saxifragaceae), using an in vitro
protein tyrosine phosphatase 1B (PTP1B) inhibitory assay, resulted in the isolation of a new triterpene, 3a,24-dihydroxyolean-
12-en-27-oic acid (4), along with four triterpenes, 3-oxoolean-12-en-27-oic acid (1), 3b-hydroxyolean-12-en-27-oic acid (b-peltoboy-
kinolic acid; 2), 3b-hydroxyurs-12-en-27-oic acid (3), and 3b,6b-dihydroxyolean-12-en-27-oic acid (astilbic acid; 5). Compounds 1–5
inhibited PTP1B with IC50 values of 6.8 0.5, 5.2 0.5, 4.9 0.4, 11.7 0.9, and 12.8 1.1 lM, respectively. Our results indicate
that 3-hydroxyl group and a carboxyl group in this type of triterpenes may be required for the activity, while addition of one more
hydroxyl group at C-6 or C-24 may be responsible for a loss of activity. Thus, compounds 2 and 3 which possess only one hydroxyl
group at C-3 and a carboxyl group at C-27 could be potential PTP1B inhibitors.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Protein tyrosine phosphatases (PTPs), which dephos-
phorylate the tyrosine residues of proteins, have an
important role in intracellular signaling and metabo-
lism. Although several PTPs, such as PTP-a, leukocyte
antigen-related tyrosine phosphatase (LAR), and SH2-
has been suggested that compounds that reduce PTP1B
activity or expression levels could not only be used for
treating type 2 diabetes but also obesity. Although there
have been a number of reports on the designing and
development of synthetic PTP1B inhibitors,1,5 only a
few studies have been reported as PTP1B inhibitors
derived from plants.6
domain-containing
phosphotyrosine
phosphatase
(SHP2) have been implicated in the regulation of insulin
signaling, there are substantial evidences supporting
PTP1B as the critical PTP controlling insulin signaling
pathway.1,2 PTP1B can interact with and dephosphory-
late the activated insulin receptor (IR) as well as insulin
receptor substrate (IRS) proteins.1,2 Its overexpression
has been shown to inhibit the IR signaling cascade
and increased expression of PTP1B occurs in insulin-
resistant states.3 Furthermore, recent genetic evidence
has shown that PTP1B gene variants are associated with
changes in insulin sensitivity.4 As with the insulin signal-
ing pathway, the leptin signaling pathway can be atten-
uated by PTPs and there is compelling evidence that
PTP1B is also involved in this process.1,2 Therefore, it
In our screening program to search for PTP1B inhibi-
tors from plants, a MeOH extract of the rhizomes of
Astilbe koreana exhibited PTP1B inhibitory activity
(>70% inhibition at 30 lg/mL), which led us to investi-
gate the PTP1B inhibitory compounds from this plant.
A. koreana belongs to Saxifragaceae and is a perennial
herb growing in the moist fields and mountains, usually
to a height of 60 cm. The plants of this genus have been
used to treat pain, headache, arthralgia, chronic bron-
chitis, and inflammation.7 Bergenin and its galloyl ester,
flavonoids, triterpenes, and phytosterols have been
reported as constituents of the genus Astilbe, and have
been found to possess analgesic and cytotoxic activi-
ties.8,9 Our previous study has demonstrated that the
EtOH extract of A. koreana inhibited UVB-induced gen-
eration of proinflammatory cytokines.10 Despite a num-
ber of studies on the genus Astilbe, there have been no
investigations regarding the chemical constituents and
Keywords: Protein tyrosine phosphatase 1B (PTP1B); Astilbe koreana;
Saxifragaceae; Triterpenes; 3a,24-Dihydroxyolean-12-en-27-oic acid.
*
Corresponding author. Tel.: +82 42 860 4295; fax: +82 42 860
4595; e-mail: wkoh@kribb.re.kr
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.03.036

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M. K. Na et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3273–3276
the PTP1B inhibitory activity of A. koreana. Therefore,
we have investigated the PTP1B inhibitory compounds
of this plant. Bioassay-guided fractionation of a MeOH
extract of the rhizomes of this plant led to the isolation
of a new triterpene, 3a,24-dihydroxyolean-12-en-27-oic
acid (4), along with four triterpenes, 3-oxoolean-12-en-
27-oic acid (1), 3b-hydroxyolean-12-en-27-oic acid (b-
peltoboykinolic acid; 2), 3b-hydroxyurs-12-en-27-oic
acid (3), and 3b,6b-dihydroxyolean-12-en-27-oic acid
(astilbic acid; 5), as the active principles. In this report,
we describe the isolation and structure determination
of these compounds, and the evaluation of their PTP1B
inhibitory activity.
479 [M+Na]+}, and 3b,6b-dihydroxyolean-12-en-27-oic
25
acid {compound 5; mp: 220–223 ꢂC; ½aꢀ +100ꢂ (c 1.0,
D
CHCl3); ESI-MS m/z: 471 [MꢁH]+, 495 [M+Na]+} by
analyses of MS and NMR data, and comparison with
those in the literature (Fig. 1).9,11
Compound 4 was obtained as a white amorphous pow-
25
D
der, mp 245–247 ꢂC, ½aꢀ +110ꢂ (c 1.1, CHCl3). A
molecular formula of C30H48O4 was determined for this
compound from the molecular ion peak at m/z 472.3553
[M]+ (calcd for C30H48O4: 472.3554) obtained by HRE-
IMS. The 1H NMR spectrum of 4 displayed the charac-
teristic signals of an olefinic proton (dH 5.61), an
oxygenated methine (dH 3.74), a hydroxymethylene
group (dH 3.63 and 3.41), and six tertiary methyl groups
(dH 1.10, 1.05, 0.92· 2, 0.91, 0.73). Thirty carbon signals
appeared in 13C and DEPT NMR spectra including a
carbonyl carbon signal at dC 179.6 (C-27), olefinic car-
bon signals at dC 137.8 (C-13) and 126.0 (C-12), an oxy-
genated methine at dC 70.9 (C-3), an oxygenated
methylene at dC 66.5, and six methyls at dC 33.1, 28.5,
25.1, 23.9, 18.4, and 16.7 indicated that compound 4
had a skeleton similar to those of b-peltoboykinolic acid
(2) and astilbic acid (5).9,11 When compared to com-
pound 2 or 5, C-3 of 4 showed a upfield chemical shift
(6–8 ppm), and the signal of H-3 in 4 was displayed as
a broad singlet. This suggested that compound 4 should
have a 3a-OH rather than 3b-OH.12,13 The proton sig-
nals of hydroxymethylene showed HMBC correlation
to a hydroxymethine (dC 70.9, C-3), a quaternary carbon
(dC 44.2, C-4), and an aliphatic methine (dC 49.7, C-5),
indicating that the hydroxymethylene is located at
The rhizomes of A. koreana (3 kg), collected at Mt. Sulak,
Korea, in July 2001 (voucher No. CNU 0880), were
extracted with MeOH at room temperature for two
months. The MeOH extract (IC50 = 22.7 lg/mL, 460 g)
was suspended in H2O (3 L) and sequentially partitioned
with hexane (3· 3 L), EtOAc (3· 3 L), and BuOH (3·
3 L), to obtain hexane-soluble fraction (IC50 = 18.5 lg/
mL), EtOAc-soluble fraction (IC50 = 9.5 lg/mL),
BuOH-soluble fraction (IC50 = 47.3 lg/mL), and H2O-
soluble fraction (IC50 > 100.0 lg/mL), respectively. Since
the EtOAc-soluble fraction showed the strongest PTP1B
inhibitory activity, this fraction (102 g) was further sepa-
rated by silica gel column chromatography (10 · 30 cm;
63–200 lm particle size) using a stepwise gradient of
CH2Cl2/MeOH (from 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 3:1, 1:1
to 0:1; 5 L for each step), to yield seven fractions (Fr.
1–Fr. 7). Of these, Fr. 1, Fr. 2 and Fr. 3 showed the most
potent PTP1B inhibitory activity (80%, 76%, and 74%
inhibition at 10 lg/mL). Fr. 1 [eluted with CH2Cl2/MeOH
(10:1), 10.4 g] was chromatographed over silica gel
(6.5 · 35 cm; 63–200 lm particle size) using a mixture of
CHCl3/MeOH (20:1), to yield four subfractions (Fr.
1-1–Fr. 1-4). Further purification of Fr. 1-1 (2.3 g) by pre-
parative MPLC [LiChroprepꢁ Si 60 column
(25 · 310 mm; 40–63 lm particle size); mobile phase
CHCl3/MeOH (20:1); flow rate 5 mL/min] resulted in
the isolation of compounds 1 (6.2 mg), 2 (7.5 mg), and 3
(3.9 mg). Another active fraction, Fr. 3 [eluted with
CH2Cl2/MeOH (8:1), 9.8 g], was subjected to silica gel col-
umn chromatography using a stepwise gradient of
CHCl3/MeOH (from 20:1, 15:1, 10:1, 9:1, 8:2, 7:3, 6:4 to
1:1; 2 L for each step) to yield six subfractions (Fr.
3-1–Fr. 3-6). Fr. 3-2 [eluted with CHCl3/MeOH (from
15:1 to 10:1), 1.5 g] was purified by preparative MPLC
[LiChroprepꢁ Si 60 column (25 · 310 mm; 40–63 lm par-
ticle size); mobile phase CHCl3/MeOH (15:1); flow rate
5 mL/min], to afford compound 4 (12 mg). Additional sil-
ica gel column chromatography (10 · 30 cm; 40–63 lm
particle size) of Fr. 3-3 [eluted with CHCl3/MeOH (from
10:1 to 9:1), 2.0 g] with hexane/acetone (2:1) resulted in
the isolation of compound 5 (37 mg). The structures of
the isolated compounds 1, 2, 3, and 5 were identified as
COOH
COOR2
O
R1O
1
2
R1 = R2 = H
2a R1 = Ac, R2 = H
2b R1 = H, R2 = Me
30
29
COOH
19
20
21
HO
12
13
17
22
28
11
18
15
25
26
3
1
4
14
16
2
3
9
10
8
COOH
27
R1O
7
5
6
24
23
OR2
3-oxoolean-12-en-27-oic acid {compound 1; mp: 210 ꢂC;
25
½aꢀ +140ꢂ (c 1.3, CHCl3); ESI-MS m/z: 453 [MꢁH]+,
25
D
COOH
477 [M+Na]+}, 3b-hydroxyolean-12-en-27-oic acid
4
R1 = R2 = H
HO
4a R1 = R2 = Ac
{compound 2; mp: 227 ꢂC; ½aꢀ +105ꢂ (c 1.3, CHCl3);
D
OH
ESI-MS m/z: 455 [MꢁH]+, 479 [M+Na]+}, 3b-hydrox-
5
yurs-12-en-27-oic acid {compound 3; mp: 240–241 ꢂC;
25
½aꢀ +102ꢂ (c 0.9, CHCl3); ESI-MS m/z: 455 [MꢁH]+,
Figure 1. Structures of compounds 1–5 isolated from A. koreana.
D

M. K. Na et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3273–3276
3275
Table 1. The inhibitory activity of the isolated compounds 1–5 and
related compounds against PTP1B
C-23 or 24. The upfield shift of the oxygenated methy-
lene (dC 66.5) in 4 suggested that this group could be as-
signed to the axial C-24.12–17 In order to confirm the
position of hydroxymethylene, 4 was acetylated using
pyridine and acetic anhydride at room temperature for
12 h, to yield the diacetate (4a).18 Comparison of the
average chemical shift value for the acetoxymethylene
protons of 4a with those of reported values in analogous
triterpenoids indicated an axial orientation for the
hydroxymethylene group, which is b (C-24) in configu-
ration.12–17 This was further supported by the NOE cor-
relations from Me-25 to both the H-24a and H-24b.
Thus, the structure of this new compound (4) was deter-
mined as 3a,24-dihydroxyolean-12-en-27-oic acid
(Fig. 1).19
Compound
PTP1B
inhibitory
activity
a
IC50 (lM)
3-Oxoolean-12-en-27-oic acid (1)
6.8 0.5
5.2 0.5
8.5 0.7
16.0 1.3
4.9 0.4
11.7 0.9
12.8 1.1
3b-Hydroxyolean-12-en-27-oic acid (2)
3b-Acetoxyolean-12-en-27-oic acid (2a)
Methyl 3b-hydroxyolean-12-en-27-oate (2b)
3b-Hydroxyurs-12-en-27-oic acid (3)
3a,24-Dihydroxyolean-12-en-27-oic acid (4)
3b,6b-Dihydroxyolean-12-en-27-oic acid (5)
Oleanolic acid (3b-hydroxyolean-12-en-28-oic acid)
Methyl 3b-hydroxyolean-12-en-28-oate
Glycyrrhizin
3.9 0.5
14.2 1.1
>100
18b-Glycyrrhetic acid
18a-Glycyrrhetic acid
13.8 1.0
15.6 1.1
PTP1B (human, recombinant) was purchased from
BIOMOLꢁ International LP (USA) and the enzyme
activity was measured using p-nitrophenyl phosphate
(p-NPP) as a substrate.20 To each 96-well (final volume:
200 lL) were added 2 mM p-NPP and PTP1B (0.05–
0.1 lg) in a buffer containing 50 mM citrate (pH 6.0),
0.1 M NaCl, 1 mM EDTA, and 1 mM dithiothreitol
(DTT) with or without test compounds. Following incu-
bation at 37 ꢂC for 30 min, the reaction was terminated
with 10 M NaOH. The amount of produced p-nitro phe-
nol was estimated by measuring the absorbance at
405 nm. The nonenzymatic hydrolysis of 2 mM p-NPP
was corrected by measuring the increase in absorbance
at 405 nm obtained in the absence of PTP1B enzyme.
All the isolates were assayed for their inhibitory activity
against PTP1B, and the results are presented in Table 1.
Of the compounds tested, 3b-hydroxyurs-12-en-27-oic
acid (3) and 3b-hydroxyolean-12-en-27-oic acid (2)
exhibited the strongest inhibitory activity with IC50
values of 4.9 0.4 and 5.2 0.5 lM, respectively, which
were comparable to that of RK-682 (IC50 = 4.5 0.5
lM) used as a positive control.21 This result indicates
that only a positional change of methyl groups at C-29
and C-30 may not affect the PTP1B inhibitory
activity. Compound 1 (IC50 = 6.8 0.5 lM) converted
a hydroxyl group at C-3 to ketone was somewhat less
active than 2. Moreover, addition of an acetyl group
to C-3 (2a,22 IC50 = 8.5 0.7 lM) resulted in loss of
in vitro inhibitory activity. These results suggest that
3-hydroxyl group in this type of triterpenes may be re-
RK-682b
4.5 0.5
a IC50 values were determined by regression analyses and expressed as
means SD of three replicates.
b Positive control.21
inhibitory activity (Table 1). A similar loss of activity
was observed in the 27-methyl ester, 2b,22 as compared
to that in compound 2 (Table 1). These results indicate
that a carboxyl group in oleanane triterpenes is impor-
tant for the PTP1B inhibitory activity, however, the po-
sition of carboxyl group, whether it is at C-27 or C-28,
seems not to be critical for the activity. Additionally,
we tested the ability of a group of related natural triter-
penes to inhibit in vitro PTP1B activity. As a result, 18b-
glycyrrhetic acid (3b-hydroxy-11-oxo-18b,20b-olean-12-
en-30-oic acid) and the 18a-isomer displayed IC50 values
of 13.8 1.0 and 15.6 1.1 lM, respectively, whereas
glycyrrhizin did not show any activity in our assay (Ta-
ble 1). PTP1B is known to have several binding sites
such as electrostatic, hydrophobic, and hydrogen-bond-
ing sites, and to have several N-terminal favorable for
binding to acidic site.1,23 Although the inhibition mode
of action with regard to this type of triterpenes has
not been elucidated yet, considering the molecular fea-
tures of active triterpenes, pentacyclic ring might facili-
tate the hydrophobic interaction, and the hydroxyl
group at C-3 may be presumed to form hydrogen bond.
Besides, a free carboxyl group at C-27 or C-28 might
play a role in binding to N-terminal residues. Of the iso-
lates, thus, compounds 2 and 3 which possess only one
hydroxyl group at C-3 and a carboxyl group at C-27
can be potential PTP1B inhibitors.
quired
for
the
activity.
Compound
5
(IC50 = 12.8 1.1 lM) substituted a hydroxyl group at
C-6 of 2 exhibited significantly lower activity than 2.
Compound 4, which possesses one more hydroxyl group
at C-24, was also found to be less effective than 2.
Although the structure–activity relationships of these
triterpenes bearing a carboxyl group at C-27 were not
thoroughly investigated, our results indicate that intro-
duction of one more hydroxyl group at C-6 or C-24
may be responsible for a loss of activity. To evaluate
the role of a carboxyl group at C-27, we assayed a group
of related triterpenes that are commercially available or
can be easily prepared by standard method.22 Oleanolic
acid with the same substitution pattern as that of com-
pound 2 except for a position of carboxyl group at C-
28 retained the activity, whereas addition of methyl ester
to C-28 carboxylic acid resulted in a loss in its PTP1B
Pentacyclic triterpenes with a carboxyl group at C-27
have been isolated rarely from the genera Astilbe, Pelt-
oboykinia, Boykinia, Chrysosplenium, and Aceriphyllum
of the family Saxifragaceae,9,24–26 the genus Cordia of
Boraginaceae,11 and the genus Cornulaca of Chenopodi-
aceae.27 This type of triterpenes has been reported to
possess ant-repellent,11 cytotoxic, and antitumor activi-
ties,9,25 and to inhibit acyl-CoA cholesterol acyltransfer-
ase (ACAT) related to atherosclerosis.26 However, to
our knowledge, PTP1B inhibitory activity of these triter-
penes is now being reported for the first time in this
study. Our results indicate that the PTP1B inhibitory

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M. K. Na et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3273–3276
12 h. To the mixture 5 mL of H2O was added, which was
then extracted with CH2Cl2 (3· 5 mL). The combined
organic extract was purified over a silica gel column
(1.6 · 15 cm) using a mixture of hexane/CHCl3/MeOH
(5:1:1), to afford the diacetate (4a): white amorphous
powder; Rf: 0.5 [hexane/CHCl3/MeOH (4:1:1)]; FABMS
activity for this unique type of triterpenes warrants
further investigation and optimization.
Acknowledgments
1
m/z: 579 [M+Na]+; H NMR (400 MHz, CDCl3): d 5.71
This research was supported in part by the grants
from the Plant Diversity Research Center of 21st
Frontier Research Program (PF0320903-00), the
Molecular and Cellular BioDiscovery Research Pro-
gram (M1-0311-00-0023) of the Ministry of Science
and Technology of Korea, and from KRIBB Research
Initiative Program.
(1 H, br s, H-12), 4.82 (1H, br s, H-3), 4.11 (1H, d,
J = 10.0 Hz, H-24a), 3.86 (1H, d, J = 10.0 Hz, H-24b),
2.29 (1H, dd, J = 5.2, 11.6 Hz, H-9), 2.10 (1H, m, H-18),
2.00 (3H, s, OAc), 1.99 (3H, s, OAc), 1.07 (3H, s, Me), 1.03
(3H, s, Me), 1.01 (3H, s, Me), 0.86 (3H, s, Me), 0.84 (3H, s,
Me), 0.80 (3H, s, Me).
19. 3a,24-Dihydroxyolean-12-en-27-oic acid (4): white amor-
25
phous powder, mp 245–247 ꢂC; ½aꢀD +110ꢂ (c 1.1, CHCl3);
ESI-MS m/z: 471 [MꢁH]+, 495 [M+Na]+; HREIMS m/z:
1
References and notes
472.3553 [M]+ (calcd for C30H48O4: 472.3554); H NMR
(400 MHz, CDCl3): d 5.61 (1H, br s, H-12), 3.74 (1H, br s,
H-3), 3.63 (1H, d, J = 11.6 Hz, H-24a), 3.41 (1H, d,
J = 11.6 Hz, H-24b), 2.34 (1H, dd, J = 5.2, 12.0 Hz, H-9),
2.13 (1H, m, H-18), 1.10 (3H, s, H-26), 1.05 (3H, s, H-25),
0.92 (6H, s, H-28 and H-29), 0.91 (3H, s, H-30), 0.73 (3H,
s, H-23); 13C NMR (100 MHz, CDCl3): d 36.8 (C-1), 27.9
(C-2), 70.9 (C-3), 44.2 (C-4), 49.7 (C-5),18.0 (C-6), 32.9 (C-
7), 35.9 (C-8), 47.0 (C-9), 37.0 (C-10), 22.9 (C-11), 126.0
(C-12), 137.8 (C-13), 56.1 (C-14), 22.3 (C-15), 27.9 (C-16),
31.3 (C-17), 49.4 (C-18), 42.2 (C-19), 33.5 (C-20), 34.6 (C-
21), 37.0 (C-22), 25.1 (C-23), 66.5 (C-24), 16.7 (C-25), 18.4
(C-26), 179.6 (C-27), 28.5 (C-28), 33.1 (C-29), 23.9 (C-30).
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Prod. 1986, 49, 225.
25
pyridine (DMAP): colorless needles; mp 228–230 ꢂC; ½aꢀD
+101ꢂ (c 0.6, CHCl3), ESI-MS m/z: 497 [MꢁH]+, 521
[M+Na]+; (b) Reaction of compound 2 with diazomethane
in ether yielded the methyl ester, 2b: colorless needles; mp
25
210–213 ꢂC; ½aꢀD +115ꢂ (c 0.5, CHCl3), ESI-MS m/z: 469
[MꢁH]+, 493 [M+Na]+; (3) reaction of oleanolic acid with
diazomethane in ether afforded the methyl ester: colorless
needles; mp 202–205 ꢂC; ½aꢀ2D5 +70ꢂ (c 1.0, CHCl3), ESI-MS
m/z: 469 [MꢁH]+, 493 [M+Na]+.
23. Zhang, Z. Y. Annu. Rev. Pharmacol. Toxicol. 2002, 42,
209.
24. Izawa, K.; Nagai, M.; Inoue, T. Phytochemistry 1973, 12,
1508.
25. Arisawa, M.; Bai, H.; Shimizu, S.; Koshimura, S.; Tanaka,
M.; Sasaki, T.; Morita, N. Chem. Pharm. Bull. 1992, 40,
3274.
26. Han, J. T.; Kim, H. Y.; Park, Y. D.; Lee, Y. H.; Lee,
K. R.; Kwon, B. M.; Baek, N. I. Planta Med. 2002, 68,
558.
16. Kojima, H.; Ogura, H. Phytochemistry 1989, 28, 1703.
17. Singh, S. K.; Tripathi, V. J.; Singh, R. H. Phytochemistry
1990, 29, 3360.
27. Dawidar, A. A.; Reisch, J.; Amer, M. Chem. Pharm. Bull.
1979, 27, 2938.
18. Compound 4 (8 mg) was treated with pyridine (0.5 mL)
and acetic anhydride (0.5 mL) at room temperature for