Differentiation- and apoptosis-inducing activities by pentacyclic triterpenes on a mouse melanoma cell line

Article abstract of DOI:10.1021/np0104673

In a study to investigate the relationship between the chemical structure and the differentiation-inducing activity of pentacyclic triterpenes, several lupane, oleanane, and ursane triterpenes were prepared and their effects on B16 2F2 melanoma cell differentiation and growth were examined. Eleven lupane triterpenes used in this study acted on the melanoma cells as a melanogen, but no induction of melanogenesis of B16 2F2 cells by oleanane and ursane was detected. The differences at C-17 of the lupane series and acetylation of the OH group at C-3 did not markedly influence their activities. However, the ED₅₀ value for up-regulation of melanin biosynthesis was markedly decreased by the oxidation of the OH group at C-3 of lupeol (1). Betulinic acid (11), its methyl ester (12), lup-28-al-20(29)-ene-3β-ol (9), and lup-28-al-20(29)-en-3-one (10) inhibited B16 2F2 cell proliferation by induction of apoptosis. These findings suggested that the carbonyl group at C-17 might be essential for the apoptotic effects of these compounds on B16 2F2 cells.

Full text of DOI:10.1021/np0104673

J . Nat. Prod. 2002, 65, 645-648
645
Differ en tia tion - a n d Ap op tosis-In d u cin g Activities by P en ta cyclic Tr iter p en es
on a Mou se Mela n om a Cell Lin e
Keishi Hata, Kazuyuki Hori,* and Saori Takahashi
Akita Research Institute of Food & Brewing (ARIF), 4-26 Sanuki, Araya-machi, Akita 010-1623, J apan
Received September 28, 2001
In a study to investigate the relationship between the chemical structure and the differentiation-inducing
activity of pentacyclic triterpenes, several lupane, oleanane, and ursane triterpenes were prepared and
their effects on B16 2F2 melanoma cell differentiation and growth were examined. Eleven lupane
triterpenes used in this study acted on the melanoma cells as a melanogen, but no induction of
melanogenesis of B16 2F2 cells by oleanane and ursane was detected. The differences at C-17 of the
lupane series and acetylation of the OH group at C-3 did not markedly influence their activities. However,
the ED50 value for up-regulation of melanin biosynthesis was markedly decreased by the oxidation of the
OH group at C-3 of lupeol (1). Betulinic acid (11), its methyl ester (12), lup-28-al-20(29)-ene-3â-ol (9),
and lup-28-al-20(29)-en-3-one (10) inhibited B16 2F2 cell proliferation by induction of apoptosis. These
findings suggested that the carbonyl group at C-17 might be essential for the apoptotic effects of these
compounds on B16 2F2 cells.
Lupane series triterpenes are widely distributed in
Compositae plants, and their biological activities have
attracted a great deal of attention. A number of studies on
their antiinflammatory activities have been reported.1,2
More recently, the anticarcinogenic activity of Taraxacum
plants was investigated, and the triterpenes such as lupeol
(1) were shown to exhibit inhibitory effects on the induction
of Epstein-Barr virus early antigen by tumor promoters.3
Furthermore, betulinic acid (11), a lupane triterpene, has
been shown to possess anti-AIDS activity, and the C-3
hydroxy group and C-17 carboxylic group of 11 contributed
to enhancement of this activity.4 It was reported that 11
inhibited cell proliferation by induction of apoptosis in
human melanoma and other cancer cell lines.5,6 The
F igu r e 1. Effects of 1 and 2 on B16 2F2 cell growth and melano-
genesis following treatment with test compounds at 1.0 or 10 µM for
72 h. The cell growth (0) and melanin contents (9) were measured.
carboxylic group at C-17 was essential for the apoptotic
effects of both 11 and its analogue, ceanothic acid, on cancer
cells.7
The mouse melanoma cell line B16 can be induced to
differentiate into mature melanocyte-like cells by treat-
ments with R-melanocyte-stimulating hormone (R-MSH) or
signal transduction pathway inhibitors. In the differentia-
tion of B16 cells, up-regulation of melanin biosynthesis was
observed.8-10 In our previous study, we isolated a B16-
derived subclone with high differentiation capability (B16
2F2) and screened differentiation-inducing activities of
naturally occurring substances in food materials on mela-
noma cells using this clone.11 The alcoholic extracts of
Taraxacum plants were found to markedly up-regulate
melanogenesis of B16 2F2 cells, and we isolated lupeol as
the active compound by bioassay-guided fractionation of
the ethanol extract of Chinese dandelion root.11 Here we
evaluated the effects of 11 lupane, 5 oleanane, and 5 ursane
triterpenes on B16 2F2 cell differentiation and growth.
with 1.0 µM 1. However 1.0 µM 2 induced cell melanogen-
esis to the same extent as stimulation with 10 µM. These
results indicated that the structural differences between
these molecules at C-3 influenced their activities. To
investigate the relationships between the structures of
these triterpenes and their activities, B16 2F2 cells were
treated with 11 lupane, 5 oleanane, and 5 ursane series
triterpenes for 72 h, and the intracellular melanin content
and the growth of B16 2F2 cells were measured. The
results are summarized in Table 1. Eleven lupane series
triterpenes used in this study induced the up-regulation
of B16 2F2 cell melanin biosynthesis, an indicator of
melanoma cell differentiation.8 ED50 values of 1, 3, 4, 8, 9,
11, and 12 for the induction of melanogenesis ranged from
2.5 to 9.9 µM. These observations indicated that the
differences at C-17 had little influence on differentiation-
inducing activity. Acetylation at C-3 of 1 or 4 showed no
effect on the activity. However, oxidation of the OH group
at C-3 markedly influenced their ED50 values. From these
results, we speculated that ketone formation at C-3 plays
an important role in the induction of B16 2F2 cell melanin
synthesis by lupane series triterpenes. On the other hand,
the differentiation of B16 2F2 cells was not induced by
other pentacyclic triterpenes such as oleanane (14-18) and
ursane (19-23).
Resu lts a n d Discu ssion
Briefly, B16 2F2 cells were incubated with 1.0 or 10 µM
1 (lupeol) or 2 (lupenone) for 3 days. Following addition of
10 µM 1 or 2, the melanin biosynthesis of B16 2F2
melanoma cells was up-regulated as compared with un-
treated controls (Figure 1). No up-regulation was observed
* Corresponding author. Tel: +81-18(888)2001. Fax: +81-18(888)2008.
E-mail: hori@arif.pref.akita.jp.
10.1021/np0104673 CCC: $22.00
© 2002 American Chemical Society and American Society of Pharmacognosy
Published on Web 04/03/2002

646 J ournal of Natural Products, 2002, Vol. 65, No. 5
Hata et al.
Ta ble 1. Effects of Some Triterpenes on B16 2F2 Cell
Differentiation and Growth
compound
skeleton
lupane
ED50 (µM)a
IC50 (µM)b
1
2
3
4
7
9.9
0.35
7.5
4.8
0.19
3.4
38.0
25.4
22.7
27.4
29.3
36.8
6.4
8
9
3.3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
0.35
4.1
2.5
4.1
7.9
4.9
5.6
48.8
38.7
33.4
6.4
0.13
>50
>50
>50
oleanane
ursane
c
-
-
>50
>50
>50
-
4.8
50.0
40.3
46.8
5.5
-
3.1
a
ED50 values were determined from the results of dose-response
experiments in which the up-regulation of melanogenesis by the
b
compound was monitored. IC50 value represents the concentra-
tion that inhibited cell growth by 50%. c The value for the
compound could not be determined because of its cytotoxicity at
10 µM, and the up-regulation of melanogenesis was not induced
by concentrations under 10 µM.
strong cytotoxic effects against B16 2F2 cells, but 9 and
10 inhibited B16 2F2 cell growth to the same extent as
11, 12, and 13. These results suggested that the carbonyl
group and not the carboxyl group at C-17 was essential
for their apoptotic effects, and similar effects were observed
following treatment with other pentacyclic triterpenes with
a carbonyl group at C-17. In addition, the ketone function
at C-3 of lupane series triterpenes enhanced their B16 2F2
cell differentiation-inducing activities. We concluded that
the different moieties of the lupane skeleton separately
regulated the induction of differentiation and apoptosis of
B16 2F2 melanoma cells.
Exp er im en ta l Section
Ma ter ia ls. Lupeol (1), lupenone (2), lupenyl acetate (3),
betulin (4), betulin diacetate (8), betulinic acid methylester
(12), â-amyrin (14), erythrodiol (16), oleanolic acid (18),
R-amyrin (19), uvaol (21), and ursolic acid (23) were purchased
from Extrasynthese S.A. Hoechst 33258 and betulinic acid (11)
were obtained from Sigma Co. Ltd., (dimethylamino)pyridine
and triphenylmethyl chloride (trytyl chloride) were from
Nakalai Tesque Co. Ltd., and pyridium chlorochromate (PCC)
was from Tokyo Kasei Kogyo Co., Ltd.
Gen er a l Exp er im en ta l P r oced u r es. Melting points were
determined on a Yanagimoto micro-melting point apparatus
and recorded as observed. Optical rotations were measured
in a 0.5 cm length cell tube with a J ASCO DIP-1000 polari-
mater. All the mass spectra (EIMS, negative-FABMS, HRE-
IMS, and negative-HRFABMS) were obtained on a J EOL J MS
BU-20 gas chromatograph mass spectrometer. Infrared (IR)
spectra were obtained on a J ASCO FT-IR 300 spectrometer.
Ultraviolet (UV) spectra were obtained on a J ASCO 270-30
UV-vis spectrometer. 1H and 13C NMR spectra were recorded
on a Varian Unity plus 400 spectrometer (1H, 400 MHz; 13C,
100 MHz), with tetramethylsilane (TMS) as an internal
standard. Chemical shifts are given on a δ scale (ppm). The
following abbreviations are used: s ) singlet, d ) doublet, t
) triplet, q ) quartet, m ) multiplet, and br ) broad. Coupling
constants (J value) are given in hertz (Hz).
Previously, 11 was reported to show selective anticancer
effects against human melanoma cells.5 We examined the
cytotoxic effects of these triterpenes on B16 2F2 cells by
comparing their IC50 values for B16 2F2 cell growth.
Compounds 9, 10, 11, 12, 13, 17, 18, 22, and 23, all of
which have a carbonyl group at C-17, inhibited B16 2F2
cell proliferation at lower concentrations than other com-
pounds. Following incubation of B16 2F2 cells with 10 µM
9 or 17 for 48 h, the cells were stained with Hoechst 33258
dye. The condensation and fragmentation of nuclei were
observed in B16 2F2 cells treated with 9 (Figure 2B) or 17
(Figure 2C). These compounds showed apoptotic effects on
B16 2F2 cells, and 70.4% and 62.5% of the cells underwent
apoptosis by 10 µM 9 and 17 for 48 h, respectively. The
carboxyl group at the C-17 position of 11 and ceanothic acid
was reported to be essential for the apoptotic activities
against some cancer cell lines.7 In this study, 11, 12, and
13, all of which possess a carboxyl group at C-17, showed
Tr ityla tion of Betu lin (4). A solution of betulin (4, 100
mg) in dry dichloromethane (3 mL) was treated with trityl
chloride (200 mg) and N,N-(dimethylamino)pyridine (50 mg),

Pentacyclic Triterpenes
J ournal of Natural Products, 2002, Vol. 65, No. 5 647
F igu r e 2. Induction of apoptosis of B16 2F2 cells by 9 and 17 B16 2F2 cells stained with Hoechst 33258 dye after incubation without (A) or with
10 µM 9 (B) or 17 (C) for 48 h.
and the mixture was stirred at 60 °C for 2 h. The reaction
mixture was poured into ice-water and extracted with EtOAc.
The EtOAc extract was washed with saturated saline, then
dried over MgSO4 and filtered. Evaporation of the solvent from
the filtrate under reduced pressure afforded the product, which
was purified by silica gel column chromatography [SiO2 50 g,
n-hexane-EtOAc (10:1)] to furnish 5 [28-O-trityl-lup-20(29)-
en-3â,28-diol (28-O-trityl betulin), 263 mg].
2.50-2.20 (3H, m, H2-2 and H-19), 1.62 (3H, s, H3-30), 1.05
(3H, s), 1.02 (3H, s), 0.91 (3H, s), 0.89 (3H, s), 0.58 (3H, s); 13
C
NMR (CDCl3, 100 MHz) δ 218.2 (s, C-3), 150.7 (s, C-20), 144.4
(s, trityl C-1′), 128.7 (s, trityl C-3′,5′), 127.7 (s, trityl C-2′,6′),
126.8 (s, trityl C-4′), 109.4 (t, C-29), 85.8 (s, C-17), 59.4 (t,
C-28), 54.8 (d, C-5), 49.6 (d, C-9), 48.8 (d, C-19), 47.7 (s, trityl
C-R), 47.5 (d, C-18), 47.3 (s, C-4), 42.5 (s, C-14), 40.5 (s, C-8),
39.5 (t, C-1), 37.3 (d, C-13), 36.8 (s, C-10), 35.2 (t, C-22), 34.1
(t, C-2), 33.4 (t, C-7), 30.0 (t, C-16), 29.9 (t, C-15), 26.8 (t, C-21),
26.7 (q, C-23), 25.1 (t, C-12), 21.2 (t, C-11), 21.0 (q, C-24), 19.6
(t, C-6), 19.1 (q, C-30), 15.9 (q, C-25), 15.6 (q, C-26), 14.6 (q,
C-27); negative-FABMS (diethanolamine as a matrix) m/z 682
(0.6, [M(13C ×1) - H]-), 681 (2.2, [M - H]-), 502 (5.5), 259
(100); negative-HRFABMS (diethanolamine as a matrix) m/z
681.4675 (calcd for C49H61O2, 681.4671).
5: colorless needles (MeOH); mp 155-156 °C; [R]25 -5.4°
D
(c 0.3,CHCl3); UV (MeOH) λmax (log ꢀ) 208 (sh) (4.50) nm; IR
(KBr) νmax 3500-3200, 2942, 1640, 1597, 1490, 1449, 1388,
1374, 1216, 1064, 983, 884, 760, 706, 632 cm-1 1H NMR
;
(CDCl3, 400 MHz) δ 7.51 (6H, d, J ) 7.6 Hz, trityl 2′,6′-
protons), 7.36 (6H, dd, J ) 7.6, 7.6 Hz, trityl 3′,5′-protons),
7.24 (3H, dd, J ) 7.6, 7.6 Hz, trityl 4′-protons), 4.61, 4.56 (each
1H, br s, H2-29), 3.20 (1H, dd, J ) 10.0, 4.2 Hz, 3R-H), 3.17,
2.96 (each 1H, d, J ) 8.6 Hz, H2-28), 2.30-2.17 (3H, m, H2-2
and H-19), 1.66 (3H, s, H3-30), 1.00 (3H, s), 0.95 (3H, s), 0.79
(3H, s), 0.78 (3H, s), 0.57 (3H, s); 13C NMR (CDCl3, 100 MHz)
δ 150.8 (s, C-20), 149.5 (s, trityl C-1′), 128.7 (s, trityl C-3′,5′),
127.7 (s, trityl C-2′,6′), 126.8 (s, trityl C-4′), 109.3 (t, C-29),
85.8 (s, C-17), 78.9 (d, C-3), 59.4 (t, C-28), 55.2 (d, C-5), 50.2
(d, C-9), 48.8 (d, C-19), 47.7 (d, C-18), 47.5 (s, trityl C-R), 42.4
(s, C-14), 40.5 (s, C-8), 38.8 (s, C-4), 38.6 (t, C-1), 37.2 (d, C-13),
37.1 (s, C-10), 35.2 (t, C-22), 34.1 (t, C-7), 30.1 (t, C-16), 29.9
(t, C-15), 28.0 (q, C-23), 27.3 (t, C-2), 26.4 (t, C-21), 25.1 (t,
C-12), 20.7 (t, C-11), 19.1 (q, C-30), 18.3 (t, C-6), 16.0 (q, C-25),
15.8 (q, C-26), 15.3 (q, C-24), 14.7 (q, C-27); negative-FABMS
(diethanolamine as a matrix) m/z 683 (1.5, [M - H]-), 502 (7),
259 (100); negative-HRFABMS (diethanolamine as a matrix)
m/z 683.4842 (calcd for C49H63O2, 683.4828).
Acid ic Tr ea tm en t of 6. A solution of 6 (35 mg) in
tetrahydrofuran (1 mL) was treated with p-toluenesulfonic acid
(100 mg), and the mixture was stirred at 60 °C for 5 h. The
reaction mixture was poured into ice-water and extracted
with EtOAc. The EtOAc extract, obtained after workup as
described above, was purified by column chromatography [SiO2
20 g, n-hexane-AcOEt (5:1)] to give compound 7 [lup-20(29)-
en-28-ol-3-one (betulone), 13.2 mg].12
7: colorless needles (n-hexane); mp 94-96 °C; [R]25 52.7°
D
(c 0.3,CHCl3); IR (KBr) νmax 3600-3250, 2944, 2868, 1704,
1
1641, 1457, 1375, 1242, 1134, 1111, 1026, 881, 755 cm-1; H
NMR (CDCl3, 400 MHz) δ 4.78, 4.62 (each 1H, br s, H2-29),
3.17, 2.94 (each 1H, d, J ) 8.2 Hz, H2-28), 2.50-2.20 (3H, m,
H2-2 and H-19), 1.62 (3H, s, H3-30), 1.05 (3H, s), 1.02 (3H, s),
0.91 (3H, s), 0.89 (3H, s), 0.58 (3H, s); 13C NMR (CDCl3, 100
MHz) δ 218.2 (s, C-3), 150.4 (s, C-20), 109.7 (t, C-29), 60.5 (t,
C-28), 54.9 (d, C-5), 49.7 (d, C-9), 48.7 (d, C-19), 47.7 (s and d,
C-17 and C-18), 47.3 (s, C-4), 42.7 (s, C-14), 40.8 (s, C-8), 39.6
(t, C-1), 37.4 (d, C-13), 36.8 (s, C-10), 34.1 (t, C-2), 33.9 (t, C-22),
33.5 (t, C-7), 29.7 (t, C-19), 29.0 (t x 2, C-16 and C-21), 27.0 (t,
C-15), 26.6 (q, C-23), 25.2 (t, C-12), 21.3 (t, C-11), 21.0 (q, C-24),
19.6 (t, C-6), 19.1 (q, C-30), 15.9 (q, C-25), 15.8 (q, C-26), 14.6
(q, C-27); EIMS m/z 440 (19.2 [M+]), 422 (15), 409 (92), 245
(40), 203 (92), 189 (90), 121 (100), 107 (97), 95 (93); HREIMS
m/z 440.3659 (calcd for C30H48O2, 440.3654).
Oxid a tion of 4 w ith P CC. PCC (100 mg) was added to a
solution of 4 (100 mg) in dry dichloromethane (2 mL), and the
mixture was stirred vigorously at room temperature for 2 h.
The residue obtained after workup as described above was
purified by column chromatography [SiO2 20 g, n-hexane-
EtOAc (5:1)] to provide compound 9 [lup-28-al-20(29)-en-3â-
ol, 30 mg] and compound 10 [lup-28-al-20(29)-en-3-one, 20.8
mg].
Oxid a t ion of 5 w it h P yr id in iu m Ch lor och r om a t e
(P CC). PCC (50 mg) was added to a solution of 5 (100 mg) in
dry dichloromethane (2 mL), and the mixture was stirred
vigorously at room temperature for 2 h. The reaction mixture
was dissolved with diethyl ether (5 mL) and suspended in 10
volumes of diethyl ether, then passed through a short Florisil
(Nakalai Tesque, 60-100 mesh) column. The solvent was
removed from the eluate to furnish 6 [28-O-trityl lup-20(29)-
en-28-ol-3-one, 78.4 mg].
6: colorless needles (MeOH); mp 142-143 °C; [R]25 16.2°
D
(c 0.3,CHCl3; UV (MeOH) λmax (log ꢀ) 210 (sh) (4.32) nm; IR
(KBr) νmax 2944, 2868, 1705, 1640, 1597, 1490, 1449, 1384,
1375, 1216, 1065, 1031, 986, 897, 883, 759, 707, 632 cm-1; 1H
NMR (CDCl3, 400 MHz) δ 7.49 (6H, d, J ) 7.5 Hz, trityl 2′,6′-
protons), 7.35 (6H, dd, J ) 7.5, 7.5 Hz, trityl 3′,5′-protons),
7.22 (3H, dd, J ) 7.5, 7.5 Hz, trityl 4′-protons), 4.60, 4.57 (each
1H, br s, H2-29), 3.17, 2.94 (each 1H, d, J ) 8.2 Hz, H2-28),

648 J ournal of Natural Products, 2002, Vol. 65, No. 5
Hata et al.
9: colorless needles (MeOH); mp 167-169 °C; [R]25 15.9°
solutions of 14, 16, 19, and 21 in dry dichloromethane, and
mixtures were stirred vigorously at room temperature. Resi-
dues obtained after workup in the same manner as described
above were purified by SiO2 column chromatography to give
15 [olean-12-en-3-one (â-amyrenone)],13 17 [olean-28-al-12-en-
3â-ol (oleanoaldehyde)],14 20 [ursan-12-en-3-one (R-amyren-
one)],13 and 22 [ursan-28-al-12-en-3â-ol (ursolaldehyde)],15
respectively. These structures were identified by IR, 1H NMR,
and 13C NMR spectrometry.
Cell Cu ltu r e. The B16-derived subclone B16 2F211 was
maintained in Dulbecco’s modified Eagle’s medium supple-
mented with 10% fetal calf serum, 100 µg/mL of streptomycin,
and 100 U of penicillin.
D
(c 0.3, CHCl3); IR (KBr) νmax 3650-3200, 3402, 2942, 1726,
1642, 1452, 1376, 1215, 1106, 1043, 984, 886,757, 668 cm-1
;
1H NMR (CDCl3, 400 MHz) δ 9.68 (1H, s, -CHO), 4.70, 4.57
(each 1H, br s, H2-29), 3.20 (1H, dd, J ) 10.4, 4.0 Hz, 3R-H),
2.82 (1H, dt, J ) 6.0, 11.2 Hz, H-19), 1.68 (3H, s, H3-30), 0.98
(3H, s), 0.97 (3H, s), 0.91 (3H, s), 0.82 (3H, s), 0.77 (3H, s); 13
C
NMR (CDCl3, 100 MHz) δ 206.7 (s, C-28), 149.7 (s, C-20), 110.2
(t, C-29), 79.0 (d, C-3), 59.3 (s, C-17), 55.3 (d, C-5), 50.4 (d,
C-9), 48.0 (d, C-19), 47.5 (d, C-18), 42.5 (s, C-14), 40.8 (s, C-8),
38.8 (s, C-4), 38.70 (t, C-1), 38.67 (d, C-13), 37.1 (s, C-10), 34.3
(t, C-7), 33.2 (t, C-22), 29.8 (t, C-21), 29.2 (t, C-16), 28.8 (t,
C-15), 27.9 (q, C-23), 27.4 (t, C-2), 25.5 (t, C-12), 20.7 (t, C-11),
19.0 (q, C-30), 18.2 (t, C-6), 16.1 (q, C-25), 15.8 (q, C-26), 15.3
(q, C-24), 14.2 (q, C-27); EIMS m/z 440 (31 [M+]), 422 (12),
412 (28), 220 (25), 207 (68), 189 (100), 175 (41), 135 (49);
HREIMS m/z 440.3647 (calcd for C30H48O2, 440.3654).
Effect of Tr iter p en es on Mela n ogen esis a n d Cell
Gr ow th of B16 2F 2Cells. Aliquots of 1 mL of B16 2F2 cells
(1 × 105 cells) were incubated with various concentrations of
triterpenes for 3 days, and the melanin contents and viable
cell number were measured as described previously.9 ED50
values for melanin biosynthesis were calculated from the
results of dose-response studies for up-regulation of melano-
genesis. IC50 values, representing the concentration that
inhibited melanoma cell growth by 50%, were measured.
10: colorless oil; [R]25 35.2° (c 0.3, CHCl3); IR (KBr) νmax
D
2943, 2867, 1705, 1642, 1453, 1376, 1248, 1139, 1043, 883, 755
cm-1; 1H NMR (CDCl3, 400 MHz) δ 9.68 (1H, s, -CHO), 4.77,
4.64 (each 1H, br s, H2-29), 2.87 (1H, dt, J ) 6.2, 11.0 Hz,
H-19), 2.38-2.58 (3H, m, H2-2 and H-16eq), 1.70 (3H, s, H3-
30), 1.07 (3H, s), 1.03 (3H, s), 0.99 (3H, s), 0.95 (3H, s), 0.93
(3H, s); 13C NMR (CDCl3, 100 MHz) δ 218.1 (s, C-3), 206.5 (s,
C-28), 149.6 (s, C-20), 110.2 (t, C-29), 59.3 (s, C-17), 54.9 (d,
C-5), 49.8 (d, C-9), 48.0 (d, C-19), 47.5 (d, C-18), 47.3 (s, C-4),
42.6 (s, C-14), 40.8 (s, C-8), 39.6 (t, C-1), 38.8 (d, C-13), 36.9
(s, C-10), 34.1 (t, C-2), 33.6 (t, C-7), 33.2 (t, C-22), 29.8 (t, C-21),
29.1 (t, C-16), 28.8 (t, C-15), 26.6 (q, C-23), 25.4 (t, C-12), 21.3
(t, C-11), 21.0 (q, C-26), 19.6 (t, C-6), 19.0 (q, C-30), 16.0 (q,
C-25), 15.7 (q, C-24), 14.2 (q, C-27); EIMS m/z 438 (31 [M+]),
410 (73), 232 (32), 219 (47), 205 (97), 189 (100), 175 (70);
HREIMS m/z 438.3507 (calcd for C30H46O2, 438.3498).
Oxid a tion of 12 w ith P CC. PCC (50 mg) was added to a
solution of 12 (100 mg) in dry dichloromethane (3 mL), and
the mixture was stirred vigorously at room temperature for 2
h. The residue obtained after workup as described above was
purified by column chromatography [SiO2 20 g, solvent system;
n-hexane-CHCl3 (1:3)] to furnish compound 13 [methyl lup-
20(29)-en-3-one-28-oic acid, 88 mg].
Detection of Cell Ap op tosis. B16 2F2 cells were incubated
with or without various triterpenes at 10 µM for 48 h.
Following collection and washing with PBS, the cells were
fixed in 1% glutaraldehyde and stained with 50 µM Hoechst
33258 dye for 30 min. The apoptotic appearance of B16 2F2
melanoma cells was confirmed by fluorescence microscopy, and
percentages of apoptosis-induced cells were counted manually
on a minimum of 200 cells.
Refer en ces a n d Notes
(1) Hasmeda, M.; Okai, G. K.; Macrides, T.; Polya, G. M. Planta Med.
1995, 65, 14-18.
(2) Recio, C. M.; Giner, R. M.; Manez, S.; Rios, J . L. Planta Med. 1995,
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(3) Takasaki, M.; Konoshima, T.; Tokuda, H.; Masuda, K.; Arai, Y.;
Shiojima, K.; Ageta, H. Biol. Pharm. Bull. 1999, 22, 606-610.
(4) Fujioka, Y.; Kashiwada, Y. J . Nat. Prod. 1994, 57, 243-247.
(5) Pisha, E.; Chai, H.; Lee, I. S.; Chagwedera, T. E.; Fransworth, N.;
Cordell, G. A., Beecher, C. W. W.; Fong, H. H. S.; Kinghorn, A. D.,
Brown, D. M.; Wani, M. C.; Wall, M. E.; Hieken, T. J .; Dupta, T. K.
D.; Pezzuto, J . M. Nature Med. 1995, 1, 1046-1051.
(6) Fulda, S.; Scaffidi, C.; Susin, S. A.; Krammer, P. H.; Kroemer, G.;
Peter, M. E.; Debatin, K. M. J . Biol. Chem. 1998, 273, 33942-33948.
(7) Lee, S. S.; Chen, W. C.; Huang, C. F.; Su, Y. J . Nat. Prod. 1998, 61,
1343-1347.
(8) Englaro, W.; Rezzonico, R.; Clement, M. D.; Lallemand, D.; Ortonne,
J . P.; Ballotti, R. J . Biol. Chem. 1995, 270, 24315-24320.
(9) Busca, R.; Bertolotto, C.; Ortonne, J . P.; Bellotti, R. J . Biol. Chem.
1996, 271, 31824-31830.
(10) Engalo, W.; Bertolotto, C.; Busca, R.; Brunet, A.; Pages, G.; Ortonne,
J . P.; Ballotti, R. J . Biol. Chem. 1998, 273, 9966-9970.
(11) Hata, K.; Ishikawa, K.; Hori, K.; Konishi, T. Biol. Pharm. Bull. 2000,
23, 962-967.
(12) Cole, B. J . W.; Bentley, M. D.; Hua, Y. Holzforschung 1991, 45, 265-
268.
13: colorless needles (MeOH); mp 158-159 °C; [R]25 39.7°
D
(c 0.3, CHCl3); IR (KBr) νmax 2947, 2868, 1726, 1705, 1642,
1458, 1376, 1319, 1187, 1156, 984, 883, 755 cm-1 1H NMR
;
(CDCl3, 400 MHz) δ 4.75, 4.60 (each 1H, br s, H2-29), 3.67 (3H,
s, COOCH3), 3.05 (1H, dt, J ) 5.6, 11.0 Hz, H-19), 2.20-2.55
(3H, m, H2-2 and H-16eq), 1.70 (3H, s, H3-30), 1.08 (3H, s),
1.04 (3H, s), 0.97 (3H, s), 0.96 (3H, s), 0.91 (3H, s); 13C NMR
(CDCl3, 100 MHz) δ 218.2 (S, C-3), 176.6 (s, C-28), 150.5 (s,
C-20), 109.6 (t, C-29), 56.5 (s, C-17), 55.0 (d, C-5), 51.3 (q,
COOCH3), 49.8 (d, C-9), 49.4 (d, C-19), 47.3 (d, C-18), 46.9 (s,
C-4), 42.4 (s, C-14), 40.6 (s, C-8), 39.0 (t, C-1), 38.3 (d, C-13),
36.92 (t, C-22), 36.88 (s, C-10), 34.1 (t, C-2), 33.6 (t, C-7), 32.1
(t, C-21), 30.5 (t, C-16), 29.6 (t, C-15), 26.6 (q, C-23), 25.5 (t,
C-12), 21.3 (t, C-11), 21.0 (q, C-24), 19.6 (t, C-6), 19.3 (q, C-30),
15.9 (q, C-25), 15.7 (q, C-26), 14.6 (q, C-27); EIMS m/z 468
(61, [M+]), 453 (10), 409 (44), 262 (59), 249 (45), 205 (46), 203
(47), 189 (100), 175 (35); HREIMS m/z 468.3632 (calcd for
(13) Wahlberg, I.; Karlsson, K.; Enzell, C. R. Acta Chem. Scand. 1972,
26, 1383-1388.
(14) Kamiya, K.; Yoshikawa, K.; Sakai, Y.; Ikuta, A.; Satake, T. Phy-
tochemistry 1997, 44, 141-144.
(15) Dagna, L.; Gasparini, G.; Ichardi, M. L.; Sesia, E. Am. J . Enol. Vitic.
1982, 33, 201-206.
C
31H48O3, 468.3603).
Oxid a tion of â-Am yr in (14), Er yth r od iol (16), r-Am yr in
(19), a n d Uva ol (21) w ith P CC. PCC was added to the
NP0104673