The absolute stereostructures of three rare D:B-friedobaccharane skeleton triterpenes from the leaves of Salacia chinensis

Article abstract of DOI:10.1016/j.tet.2008.05.054

Three new rare D:B-friedobaccharane skeleton triterpenes named foliasalacins D₁ (1), D₂ (2), and D₃ (3) together with the 20 known ones (4-23) were isolated from the leaves of Salacia chinensis Linn., collected in Thailand

Full text of DOI:10.1016/j.tet.2008.05.054

Tetrahedron 64 (2008) 7347–7352
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Tetrahedron
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The absolute stereostructures of three rare D:B-friedobaccharane
skeleton triterpenes from the leaves of Salacia chinensis
*
Yi Zhang, Seikou Nakamura, Tao Wang, Hisashi Matsuda, Masayuki Yoshikawa
Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607-8412, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Three new rare D:B-friedobaccharane skeleton triterpenes named foliasalacins D1 (1), D2 (2), and D3 (3)
together with the 20 known ones (4–23) were isolated from the leaves of Salacia chinensis Linn., collected
in Thailand. The absolute stereostructures of the D:B-friedobaccharane-type compounds (1–3) were
characterized on the basis of chemical and physiochemical evidences.
Received 21 March 2008
Received in revised form 12 May 2008
Accepted 12 May 2008
Available online 15 May 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Salacia chinensis
Hippocrateaceae
D:B-friedobaccharane skeleton
Triterpene
1. Introduction
give three new triterpenes, foliasalacins D1 (1, 0.00042%), D2 (2,
0.00025%), and D3 (3, 0.00024%) (Chart 1), together with the 20
In the course of our characterization studies on bioactive
constituents from Salacia species,1–13 we have reported the
isolation and absolute stereostructural elucidation of 13 mega-
stigmane glycosides, 7 phenolic glycosides, and 8 triterpenes from
the leaves of Salacia chinensis Linn. (Hippocrateaceae) together
with the 20 known constituents.14–17 As a continuing study on this
herbal medicine, three new triterpenes possessing the rare
D:B-friedobaccharane skeleton, named foliasalacins D1 (1), D2 (2),
and D3 (3) were isolated together with the 20 known ones (4–23).
This paper deals with the absolute stereostructural elucidation of
these three new constituents (1–3).
known ones, 3b
-hydroxy-20-oxo-30-norlupane (4, 0.0028%),18 29-
norlupan-3,20-dione (5, 0.00004%),19 lup-20(29)-en-3
b,15a-diol (6,
0.00053%),20 betulin (7, 0.0013%),21 lup-20(29)-en-3-on-28-ol (8,
0.00007%),22 betulinic acid (9, 0.00087%),21 lup-20(29)-ene-
3b
,30-diol (10, 0.00028%),23 30-hydroxy lup-20(29)-en-3-one (11,
0.014%),21
3b
,20-dihydroxylupane (12, 0.00009%),24 friedelin (13,
0.042%),21 4-epifriedelin (14, 0.0011%),25 friedelan-3-one-29-ol (15,
0.00009%),21 octandronol (16, 0.00009%),26 12
b-hydroxy D:A-frie-
dooleanan-3-one (17, 0.00004%),26 oleanoic acid (18, 0.00033%),21
erythrodiol (19, 0.00037%),21 ursolic acid (20, 0.00034%),27 uvaol
(21, 0.00037%),21 19a(H)-taraxastane-3 -diol (22, 0.00007%),28
b,20a
and isoursenol (23, 0.00005%)29 (Chart 2).
23
Foliasalacin D1 (1), [
a]
þ24.6 (CHCl3), was isolated as a white
D
2. Results and discussion
powder. Absorption for hydroxyl (3420 cmꢀ1), double bond
(1640 cmꢀ1), and ether function (1078 cmꢀ1) was revealed in the IR
spectrum. The 1H (CDCl3) and 13C NMR (Table 1) spectra30 of 1
The dried leaves of S. chinensis Linn. collected in Thailand were
finely cut and extracted with methanol (MeOH) under reflux to
provide a methanolic extract (13.0%). The MeOH extract was
partitioned into an EtOAc–H2O (1:1, v/v) mixture to furnish an
EtOAc-soluble fraction (4.1%) and an aqueous phase. The aqueous
phase was further extracted with n-BuOH to give an n-BuOH-
soluble fraction (2.4%) and an H2O-soluble fraction (6.6%). The
EtOAc-soluble fraction was subjected to normal- and reversed-
phase silica gel column chromatographies, and finally to HPLC to
indicated the presence of 8 methyl groups [
1.08, 1.14, 1.32, 1.32 (3H each, all s, H3-28, 25, 26, 23, 27, 24, 29, 30)],
a methine bearing an oxygen function [
3.47 (1H, t-like, J¼2.5 Hz,
H-3)], a disubstituted double bond [
5.58 (1H, d, J¼15.8 Hz, H-21),
5.64 (1H, dd-like, J¼6.9, 15.8 Hz, H-20)], and a tri-substituted
double bond [ 5.62 (1H, m, H-6)], together with 9 methylenes, 2
d 0.87, 0.91, 0.98, 1.04,
d
d
d
methine, and 6 quaternary carbons. As shown in Figure 1, the 1H–1H
COSY experiment on 1 indicated the presence of partial structure
drawn in bold lines, and in the HMBC experiment, long-range
correlations were observed between the following protons and
carbons: H-6 and C-8,10; H2-18 and C-12,14,16,19; H2-19 and C-16,
* Corresponding author. Tel.: þ81 75 595 4633; fax: þ81 75 595 4768.
E-mail address: myoshika@mb.kyoto-phu.ac.jp (M. Yoshikawa).
0040-4020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.05.054

7348
Y. Zhang et al. / Tetrahedron 64 (2008) 7347–7352
OH
OH
H
OH
H
H
H
H
H
HO
HO
HO
foliasalacin D1 (1)
foliasalacin D2 (2)
foliasalacin D3 (3)
Chart 1.
18, 21; H-20 and C-17, 22; H-21 and C-19, 29, 30; H3-23 and C-3–5,
24; H3-24 and C-3–5, 23; H3-25 and C-8–11; H3-26 and C-8, 13–15;
H3-27 and C-12–14, 18; H3-28 and C-16–19; H3-29 and C-21, 22, 30;
and H3-30 and C-21, 22, 29. The spectra of 1 were similar to those of
baruol,31 which was determined as the uncommon natural tri-
terpenoid with D:B-friedobaccharane skeleton. EIMS of 1 exhibited
molecular ion peak at m/z 442 and HREIMS analysis revealed the
molecular formula to be C30H50O2. In addition, the EIMS of 1
showed the characteristic fragments at m/z 152 for C10H16O (i) and
290 for C20H34O (ii) as shown in Figure 2, which were derived from
the retro Diels–Alder cleavage, regarded as suggestive of a D5
-
compound. Consequently, 1 was characterized as D:B-friedo-bac-
char-5,20-dien-3 ,22-diol. Next, the NOE correlations between the
following proton pairs (H-3 and H3-23, H3-24, H-2 , H-2 ; H-8 and
H-10, H3-27; H-10 and H3-23; H-18 and H3-28; H-18 and H2-19;
b
a
b
b
a
and H2-19 and H3-27) were observed in the NOESY experiment on
O
HO
R3
R2
R1
R
R
R1
R2
-OH
H
H
R3
R
R
6:
betulin (7):
10 :
11 :
CH3
-OH
-OH
O
-OH
O
4:
5:
-OH
O
CH2OH
CH2OH
COOH
8:
CH3
-OH
betulinic acid (9):
HO
CH2OH
H
H
H
H
H
H
O
O
HO
O
12
friedelin (13)
4-epifriedelin (14)
15
HOCH2
HO
H
R
H
R
R
H
H
HO
O
O
HO
R
ursolic acid (20): COOH
uvaol (21): CH2OH
COOH
CH2OH
oleanoic acid (18):
erythrodiol (19):
17
octandronol (16)
HO
H
H
O
HO
HO
22
Isoursenol (23)
3-dehydrohancokinol
Chart 2.

Y. Zhang et al. / Tetrahedron 64 (2008) 7347–7352
7349
Table 1
13C NMR data for 1–3 and related compound (1a)
OH
Position
1
1a
21.4
38.1
2
3
Position
18.1 16
1
1a
33.7
32.3
45.3
45.4
2
3
HO
1
2
3
4
5
6
7
8
18.1
27.7
76.3 215.4
40.8 50.0
142.0 142.7 142.0 142.0 20
122.0 121.3 122.0 122.0 21
23.7
44.6
35.5
50.0
34.2
32.7
36.6
37.8
29.2
18.1
27.7
76.3
33.7
34.6
34.7
27.8 17
76.3 18
40.8 19
32.3
45.3
45.5
124.2 124.2
140.7 140.7
70.8
29.0
25.5
17.5
15.2
20.6
32.9
29.9
30.0
31.6
44.6
38.4
29.6
76.9
31.6
44.4
38.6
29.7
77.0
i
ii
40.8
H
OH
PCC,CH2Cl2
0 °C, 1.5 hr
23.7
44.2
35.7
51.0
33.6
32.6
36.6
37.9
29.1
23.7
44.6
35.5
50.0
34.2
32.7
36.5
37.9
29.2
23.7 22
44.6 23
35.5 24
50.0 25
34.2 26
32.8 27
36.5 28
37.9 29
29.2 30
70.8 147.4 147.4
1
28.4
24.6
17.1
14.9
20.7
32.8
29.0
25.5
17.5
15.2
20.2
32.9
29.0
25.5
17.5
15.2
20.2
34.0
H
9
O
10
11
12
13
14
15
1a
Figure 2.
29.9 111.4 111.3
30.0 17.3 17.3
methines, and 5 quaternary carbons. The 1H–1H COSY experiment
on 2 indicated the presence of partial structure drawn in bold lines,
and in the HMBC experiment, long-range correlations were
observed between the following proton and carbon pairs: H-6 and
C-8, 10; H2-18 and C-14, 16, 19; H2-19 and C-16, 18; H-21 and C-19,
29, 30; H3-23 and C-3–5, 24; H3-24 and C-3–5, 23; H3-25 and C-
8–11; H3-26 and C-8, 13–15; H3-27 and C-12–14, 18; H3-28 and
C-16–19; H3-29 and C-21, 22, 30; and H3-30 and C-21, 22, 29. Fur-
thermore, the molecular formula C30H50O2 of 2 was determined
based on the results of the molecular ion peak at m/z 442 and
HREIMS measurement, and the fragment ion peaks were identical
to those of 1, which were observed at m/z 152 for C10H16O and 290
for C20H34O. In the NOESY experiment, the NOE correlations were
observed similar to those of 1 as shown in Figure 1, which suggested
that the stereochemistry of 2 was the same as that of 1 except for the
side chain. Finally, the (R)- and (S)-MTPA esters (2a and 2b) were
derived from 2 upon reaction with (R)- and (S)-MTPA in the
presence of EDC$HCl and 4-DMAP. The protons on the 29- and
30-position of the (S)-MTPA ester (2b) resonated at higher field than
those of the (R)-MTPA ester (2a) [Dd: negative], while the protons on
the 8-, 11-, 12-, 15-, 16-, 18–20-, and 26–28-position of 2b were
observed at lower fields compared to those of 2a [Dd: positive]
(Fig. 3). Consequently, the absolute stereostructure of the 21-position
in 2 was elucidated to be in Rorientation, and the stereostructure of 2
was determined to be (3S,8S,9R,10S,13S,14R,17S,21R)-D:B-friedo-
Measured in CDCl3 at 125 MHz.
1. The evidence mentioned above indicated that the steric config-
urations of the H-8, H-10, Me-4 (H3-23), Me-13 (H3-27), and C(19)
to be
-axial. Furthermore, the steric configuration of the H-3 was de-
termined to be -equatorial, which was also supported since the
a-axial, while those of Me-9 (H3-25) and Me-14 (H3-26) to be
b
a
proton and carbon signals at A and B rings (H-1–H-10 and H-23–25)
of 1 in the 1H and 13C NMR spectra were superimposable on those
of baruol31 with the steric configuration of H
a
(equatorial)-3,
Ha(axial)-10, and Meb(axial)-9 (H3-25). Finally, the oxidation of 1
with pyridinium chlorochromate (PCC) was carried out to give 1a as
the product (Fig. 2). The CD spectrum (MeOH) of 1a showed Cotton
effect at 298 nm (D3 ꢀ0.82) in MeOH and 300 nm (D3 ꢀ0.92) in
dioxane, carbonyl n/
p , which was very similar to that of 3-
*
dehydrohancokinol32 (Chart 2) [295 nm ([
q
] ꢀ4100) in dioxane]
reported by Lou et al. On the basis of the structural similarities of A-
and B-ring, the absolute stereostructure at the 10-position in 1a
was elucidated to be in S orientation. Consequently, 1 was de-
termined to be (3S,8S,9R,10S,13S,14R,17S)-D:B-friedobacchar-5,20-
dien-3b,22-diol.
Foliasalacin D2 (2) was isolated as a white powder with positive
29
optical rotation ([
absorption bands at 3422, 1636, and 1096 cmꢀ1 ascribable to
hydroxyl, double bond, and ether functions. The 1H (CDCl3) and 13
NMR (Table 1) spectra30 showed signals assignable to 7 methyls
0.86, 0.89, 0.96, 1.02, 1.05, 1.13, 1.70 (3H each, all s, H3-28, 25, 26,
23, 27, 24, 30)], 2 methines bearing an oxygen function [ 3.46 (1H,
s-like, H-3), 3.99 (1H, t-like, J¼6.2 Hz, H-21)], a terminal double
bond [ 4.83, 4.91 (1H each, both br s, H2-29)], and a tri-substituted
double bond [ 5.59 (1H, m, H-6)], together with 10 methylenes, 2
a]
þ17.1 in CHCl3). The IR spectrum of 2 showed
D
bacchar-5,22-dien-3b,21-diol.
Foliasalacin D3 (3) was isolated as a white powder with positive
C
29
optical rotation ([
C
a
]
þ20.8 in CHCl3). Its molecular formula
D
30H50O2 was determined based on the results of the molecular ion
[
d
peaks at m/z 442 and HREIMS measurement. The 1H (CDCl3) and 13
C
d
NMR (Table 1) spectra30 indicated that the stereostructures of A–D
rings and the planar structure of the side chain part were the same
as those of 2. Finally, the (R)- and (S)-MTPA esters (3a and 3b) were
d
d
OH
27
28
21
29
29
20
22
30
12
8
19
11
9
OH
1
14
10
30
16
15
3
26
HO
HO
6
2 and 3
24
23
1
27H H
1H–1H COSY :
NOESY :
19
18
HMBC :
H
10
H
H
H
HO
2
8
H
3
1−3
23
24
Figure 1. 1H–1H COSY, HMBC, and NOE correlations of 1–3.

7350
Y. Zhang et al. / Tetrahedron 64 (2008) 7347–7352
OR
3.3. Extraction and isolation
+0.13
+0.04
+0.01
+0.04
+0.10
+0.12 21
−0.05
−0.09
(R)- or (S)-MTPA
−0.05
+0.02
+0.03
.
R
EDC HCl
+0.05
+0.07
+0.05
The dried leaves of S. chinensis Linn. (5.8 kg) were finely cut and
extracted three times with methanol (MeOH) under reflux for 3 h.
Evaporation of the solvent under reduced pressure provided
a methanolic extract (756 g, 13.0%). The MeOH extract (712 g) was
partitioned into an EtOAc–H2O (1:1, v/v) mixture to furnish an
EtOAc-soluble fraction (222 g, 4.1%) and an aqueous phase. The
aqueous phase was further extracted with n-BuOH to give an
n-BuOH-soluble fraction (130 g, 2.4%) and an H2O-soluble fraction
(361 g, 6.6%). The EtOAc fraction (200 g) was subjected to ordinary-
phase silica gel column chromatography [3.8 kg, hexane–EtOAc
(40:1 to 10:1 to 5:1 to 1:1, v/v)–CHCl3–MeOH–H2O (10:3:1, v/v/v,
lower layer)–MeOH] to give 16 fractions [Fr. 1 (0.7 g), Fr. 2 (1.3 g), Fr.
3 (28.3 g), Fr. 4 (0.9 g), Fr. 5 (9.0 g), Fr. 6 (14.9 g), Fr. 7 (3.2 g), Fr. 8
(10.7 g), Fr. 9 (9.1 g), Fr. 10 (6.2 g), Fr. 11 (4.2 g), Fr. 12 (13.8 g), Fr. 13
(4.1 g), Fr. 14 (43.2 g), Fr. 15 (16.9 g), and Fr. 16 (26.3 g)]. Fraction 3
(28.3 g) was subjected to ordinary-phase silica gel column
chromatography [1.0 kg, hexane–hexane–EtOAc (200:1 to 150:1 to
100:1 to 50:1 to 20:1 to 10:1, v/v)–EtOAc] to afford 16 fractions [Fr.
3-1 (10 mg), Fr. 3-2 (5170 mg), Fr. 3-3 (7570 mg), Fr. 3-4 (790 mg),
Fr. 3-5 (1260 mg), Fr. 3-6 (3390 mg), Fr. 3-7 (1020 mg), Fr. 3-8
(3480 mg), Fr. 3-9 (250 mg), Fr. 3-10 (260 mg), Fr. 3-11 (300 mg), Fr.
3-12 (110 mg), Fr. 3-13 (350 mg), Fr. 3-14 (300 mg), Fr. 3-15
(420 mg), and Fr. 3-16 (1800 mg)]. Fr. 3-6 (3390 mg) was further
recrystallized with CHCl3–MeOH (1:1, v/v) to give friedelin (13,
1860.0 mg, 0.042%). Fraction 4 (0.9 g) was isolated with ordinary-
phase silica gel column chromatography [40 g, hexane–hexane–
EtOAc (200:1 to 150:1 to 75:1 to 50:1 to 10:1, v/v)–EtOAc] to furnish
13 fractions [Fr. 4-1 (19 mg), Fr. 4-2 (24 mg), Fr. 4-3 (84 mg), Fr. 4-4
(94 mg), Fr. 4-5 (34 mg), Fr. 4-6 (45 mg), Fr. 4-7 (100 mg), Fr. 4-8
(61 mg), Fr. 4-9 (78 mg), Fr. 4-10 (64 mg), Fr. 4-11 (126 mg), Fr. 4-12
(101 mg), and Fr. 4-13 (121 mg)]. Fr. 4-7 (100 mg) was recrystallized
with CHCl3–MeOH (1:1, v/v) to give 4-epifriedelin (14, 48.8 mg,
0.0011%). Fraction 10 (6.2 g) was subjected to Sephadex LH-20
column chromatography [200 g, MeOH–CHCl3 (1:1, v/v)] to give
two fractions [Fr. 10–1 (2400 mg) and Fr. 10–2 (3600 mg)]. Fr. 10-2
(3600 mg) was subjected to reversed-phase silica gel column
chromatography [120 g, CH3CN–H2O (75:25 to 85:15 to 90:10 to
100:5, v/v)[–CH3CN–CHCl3] to give nine fractions [Fr. 10-2-1
(150 mg), Fr. 10-2-2 (164 mg), Fr. 10-2-3 (192 mg), Fr. 10-2-4
(817 mg), Fr. 10-2-5 (204 mg), Fr. 10-2-6 (70 mg), Fr. 10-2-7 (49 mg),
Fr. 10-2-8 (159 mg), and Fr. 10-2-9 (583 mg)]. Fr. 10-2-2 (164 mg)
was further purified by HPLC [MeOH–H2O (92:8, v/v)] to furnish
betulinic acid (9, 28.0 mg, 0.00062%), Fr. 10-2-2-4 (12.8 mg), and Fr.
10-2-2-7 (5.5 mg). Fr. 10-2-2-4 (12.8 mg) was further purified by
HPLC [CH3CN–H2O (75:25, v/v)] to give lup-20(29)-en-3-on-28-ol
(8, 3.2 mg, 0.00007%). Fr. 10-2-2-7 (5.5 mg) was purified by HPLC
[MeOH–H2O (88:12, v/v)] to afford 29-norlupan-3,20-dione (5,
1.8 mg, 0.00004%). Fr. 10-2-3 (192 mg) was separated with HPLC
[MeOH–H2O (92:8, v/v)] and finally HPLC [MeOH–H2O (88:12, v/v)]
to furnish foliasalacin D2 (2, 2.3 mg, 0.00005%), lup-20(29)-en-
+0.05
+0.08
4-DMAP
CHCl3
60 °C, 6 h
+0.08
+0.04
2
−0.11
+0.05
+0.07
+0.02
HO
2a: R=(R)-MTPA
2b: R=(S)-MTPA
OR
−0.02
−0.07
−0.08
−0.12
−0.02
−0.03
+0.05
+0.09
21 +0.04
(R)- or (S)-MTPA
−0.01
−0.02
−0.06 S
−0.06
−0.06
.
EDC HCl
−0.05
−0.01
4-DMAP
CHCl3
60 °C, 6 h
−0.10
+0.13
3
−0.03
−0.06
−0.02
HO
3a: R=(R)-MTPA
3b: R=(S)-MTPA
R
=
S −
values in ppm (500 MHz)
Figure 3. Application of the modified Mosher’s method to 2 and 3.
derived from 3 upon reaction with (R)- and (S)-MTPA in the
presence of EDC$HCl and 4-DMAP. The protons on the 29- and
30-position of the (S)-MTPA ester (3b) resonated at lower field than
those of the (R)-MTPA ester (3a) [Dd: positive], while the protons on
the 8-, 11-, 12-, 15-, 16-, 18–20-, and 26–28-position of 3b were
observed at higher fields compared to those of 3a [Dd: negative]
(Fig. 3). Thus, 3 was elucidated to be the 21-isomer of 2 and the
absolute stereochemistry was characterized as shown.
3. Experimental
3.1. General
The following instruments were used to obtain physical data:
specific rotations, Horiba SEPA-300 digital polarimeter (l¼5 cm);
CD spectra, JASCO J-720WI spectrometer; UV spectra, Shimadzu
UV-1600 spectrometer; IR spectra, Shimadzu FTIR-8100 spec-
trometer; 1H NMR spectra, JEOL JNM-LA500 (500 MHz) spectrom-
eter; 13C NMR spectra, JEOL JNM-LA500 (125 MHz) spectrometer
with tetramethylsilane as an internal standard; EIMS and HREIMS,
JEOL JMS-GCMATE mass spectrometer; FABMS and HRFABMS, JEOL
JMS-SX 102A mass spectrometer; HPLC detector, Shimadzu RID-6A
refractive index and SPD-10A UV–vis detectors. HPLC column,
Cosmosil 5C18-MS-II (Nacalai Tesque Inc., 250ꢁ4.6 mm i.d. and
250ꢁ20 mm i.d.) columns were used for analytical and preparative
purposes, respectively.
The following experimental conditions were used for chroma-
tography: normal-phase silica gel column chromatography, silica
gel BW-200 (Fuji Silysia Chemical, Ltd., 150–350 mesh); reversed-
phase silica gel column chromatography, Chromatorex ODS
DM1020T (Fuji Silysia Chemical, Ltd., 100–200 mesh); Diaion HP-20
column chromatography (Nippon Rensui); TLC, pre-coated TLC
plates with silica gel 60F254 (Merck, 0.25 mm) (normal-phase) and
silica gel RP-18 F254S (Merck, 0.25 mm) (reversed-phase); detection
was achieved by spraying with 1% Ce(SO4)2–10% aqueous H2SO4,
followed by heating.
3b,15a-diol (6, 23.8 mg, 0.00053%), and 30-hydroxylup-20(29)-en-
3-one (11, 23.5 mg, 0.00052%). Fr. 10-2-4 (817 mg) was subjected to
HPLC [MeOH–H2O (92:8, v/v)] and HPLC [MeOH–H2O (88:12, v/v)]
to give foliasalacins D1 (1, 13.1 mg, 0.00029%), D2 (2, 8.8 mg,
0.00020%), and D3 (3, 10.7 mg, 0.00024%) together with 3
b-
hydroxy-20-oxo-30-norlupane (4, 55.2 mg, 0.0013%), 12 -hydroxy
b
D:A-friedooleanan-3-one (17, 1.8 mg, 0.00004%), and 30-hydroxy-
lup-20(29)-en-3-one (11, 588.8 mg, 0.013%). Fr. 10-2-5 (204 mg)
was purified with HPLC [MeOH–H2O (92:8, v/v)] and finally HPLC
3.2. Plant material
The dried leaves of S. chinensis Linn. were collected at Nakhon Si
Thammarat province, Thailand in 2006 and identified by Dr.
Pongpiriyadacha Y. (Rajamangala University of Technology
Srivijaya). A voucher of the plant is on file in our laboratory (2006.
Thai-06).
[MeOH–H2O (88:12, v/v)] to afford 3b-hydroxy-20-oxo-30-norlu-
pane (4, 16.9 mg, 0.00038%) and octandronol (16, 4.2 mg,
0.00009%). Fraction 11 (4.2 g) was subjected to reversed-phase
silica gel column chromatography [150 g, MeOH–H2O (70:30 to
80:20 to 90:10, v/v)–MeOH–CHCl3] to afford five fractions [Fr. 11-1

Y. Zhang et al. / Tetrahedron 64 (2008) 7347–7352
7351
(45 mg), Fr. 11-2 (54 mg), Fr. 11-3 (40 mg), Fr. 11-4 (799 mg), and Fr.
(500 MHz, CDCl3)
d
0.88, 0.91, 0.98, 1.04, 1.05, 1.11, 1.71 (3H each, all
11-5 (1960 mg)]. Fr. 11-4 (799 mg) was separated by HPLC [MeOH–
H2O (92:8, v/v)] to give 16 fractions [Fr. 11-4-1 (5.0 mg), Fr. 11-4-2
(9.1 mg), Fr. 11-4-3 (7.1 mg), Fr. 11-4-4 (55.9 mg), Fr. 11-4-5
(18.5 mg), Fr. 11-4-6 (107.7 mg), Fr. 11-4-7 (17.7 mg), Fr. 11-4-8
(29.2 mg), Fr. 11-4-9 (34.5 mg), Fr. 11-4-10 (54.7 mg), Fr. 11-4-11
(64.5 mg), Fr. 11-4-12 (78.6 mg), Fr. 11-4-13 (32.2 mg), Fr. 11-4-14
(15.7 mg), Fr. 11-4-15 (18.1 mg), and Fr. 11-4-16 (16.2 mg)]. Frs. 11-
4-4 and 11-4-10 were identified as betulin (7, 55.9 mg, 0.0012%)
s, H3-28, 25, 26, 23, 27, 24, 30), 1.06 (1H, d, J¼14.5 Hz, H-18
a
), 1.18,
), 1.43
1.59 (1H each, both m, H2-19), 1.25 (1H, d, J¼14.5 Hz, H-18
b
(1H, dd, J¼4.8, 12.4 Hz, H-8), 2.08 (1H, m, H-10), 3.46 (1H, s-like,
H-3), 3.99 (1H, t-like, J¼6.4 Hz, H-21), 4.83, 4.91 (1H each, both br s,
H2-29), 5.61 (1H, m, H-6). 13C NMR data (125 MHz, CDCl3) dC: given
in Table 1. EIMS: m/z 442 [M]þ (10), 424 (22), 406 (7), 391 (2), 343
(10), 325 (18), 290 (53), 275 (22), 257 (31), 203 (100), 152 (36), 134
(72).
and 3b-hydroxy-20-oxo-30-norlupane (4, 54.7 mg, 0.0012%). Fr. 11-
4-5 (18.5 mg) was purified by HPLC [MeOH–H2O (88:12, v/v)] to
give betulin (7, 1.8 mg, 0.00004%) together with betulinic acid (9,
11.9 mg, 0.00026%). Fr. 11-4-6 (107.7 mg) was isolated with HPLC
[CH3CN–MeOH–H2O (65:16:19, v/v/v)] to furnish oleanoic acid (18,
14.8 mg, 0.00033%), ursolic acid (20, 15.1 mg, 0.00034%), eryth-
rodiol (19, 16.5 mg, 0.00037%), uvaol (21, 16.2 mg, 0.00037%), and
foliasalacin D1 (1, 6.0 mg, 0.00014%). Fr. 11-4-7 (17.7 mg) was sep-
arated by HPLC [MeOH–H2O (88:12, v/v)] to afford isoursenol (23,
2.2 mg, 0.00005%). Fr. 11-4-11 (64.5 mg) was isolated with HPLC
[CH3CN–MeOH–H2O (65:16:19, v/v/v)] and finally HPLC [MeOH–
3.3.4. PCC oxidation of 1
A solution of 1 (1.9 mg) in dry CH2Cl2 was treated with
pyridinium chlorochromate (PCC, 5.0 mg) and the whole mixture
was stirred at 0 ꢂC for 1.5 h. The reaction mixture was poured into
ice-water and extracted with EtOAc. The EtOAc extract was washed
with saturated aqueous NaHCO3 and brine, then dried over MgSO4,
and filtrated. Removal of the solvent under reduced pressure gave
a crude product, which was purified by silica gel column chroma-
tography [500 mg, hexane–EtOAc (10:1 to 5:1, v/v)] to furnish 1a
(0.6 mg, 32%).
19
H2O (88:12, v/v)] to give lup-20(29)-ene-3
0.00014%). Fr. 11-4-12 (78.6 mg) was subjected to HPLC [CH3CN–
MeOH–H2O (65:16:19, v/v)] to furnish lup-20(29)-ene-3 ,30-diol
b
,30-diol (10, 6.4 mg,
Compound 1a. A white powder, [
a
]
þ36.6 (c 0.02, CHCl3). CD
D
[MeOH, nm, (D3)]: 298 (ꢀ0.82), CD [dioxane, nm, (D3)]: 300
(ꢀ0.92). High-resolution EIMS calcd for C30H50O2 (M)þ: 440.3654,
found: 440.3651. IR (KBr): 3456, 2928, 1713, 1466, 1377, 1228, 1176,
b
(10, 6.4 mg, 0.00014%) and 30-hydroxylup-20(29)-en-3-one (11,
38.2 mg, 0.00085%). Fr. 11-4-14 (15.7 mg) was isolated with HPLC
972, 756 cmꢀ1 1H NMR (500 MHz, CDCl3)
. d 0.87, 0.88, 0.95, 1.10,
[MeOH–H2O (88:12, v/v)] to give 19a(H)-taraxastane-3
(22, 3.0 mg, 0.00007%). Fr. 11-4-15 (18.1 mg) was further subjected
to HPLC [MeOH–H2O (88:12, v/v)] to afford friedelan-3-one-29-ol
(15, 3.8 mg, 0.00009%) and 3b,20-dihydroxy lupane (12, 4.0 mg,
b
,20
a
-diol
1.23, 1.24, 1.33, 1.33 (3H each, all s, H3-25, 28, 26, 27, 24, 23, 29, 30),
2.41–2.50 (2H, m, H2-2), 5.58 (1H, d, J¼15.6 Hz, H-21), 5.67 (1H, m,
H-6), 5.64 (1H, dd, J¼6.8, 15.6 Hz, H-20). 13C NMR data (125 MHz,
CDCl3) dC: given in Table 1. EIMS: m/z 440 (Mþ, 1), 422 (29), 406
(63), 341 (99), 340 (53), 290 (2), 191 (67), 150 (3), 189 (49), 177 (77),
82 (100).
0.00009%).
The known compounds were identified by comparison of their
physical data ([
reported values.
a
]D, 1H NMR, 13C NMR, and MS) with those of
3.3.5. Preparation of the (R)-MTPA ester (2a)
and (S)-MTPA ester (2b) from 2
3.3.1. Foliasalacin D1 (1)
A solution of 2 (3.6 mg) in dehydrated CHCl3 (1.0 mL) was
treated with (R)-2-methoxy-2-trifluoromethylphenylacetic acid
[(R)-MTPA, (31.5 mg)] in the presence of 1-ethyl-3-(3-dimethyla-
minopropyl)carbodiimide hydrochloride (EDC$HCl, 24.9 mg) and
4-dimethyl-aminopyridine (4-DMAP, 10.5 mg), and the mixture
was stirred under reflux at 60 ꢂC for 6 h. The reaction mixture was
poured into ice-water and extracted with EtOAc. The EtOAc extract
was washed successively with 5% HCl, NaHCO3–saturated H2O, and
brine, and dried over Na2SO4, and filtered. Removal of the solvent
from the filtrate under reduced pressure afforded the residue that
was subjected to normal-phase silica gel CC [500 mg, hexane–
EtOAc (20:1 to 10:1 to 5:1)] to give 2a (2.0 mg, 37%). Using a similar
procedure, (S)-MTPA ester (2b, 1.8 mg, 39%) was obtained from 2
(3.1 mg) with (S)-MTPA (30.2 mg), EDC$HCl (26.9 mg), and 4-DMAP
(8.8 mg).
23
A white powder, [
a
]
þ24.6 (c 0.60, CHCl3). High-resolution
D
EIMS calcd for C30H50O2 (M)þ: 442.3811, found: 442.3814. IR (KBr):
3420, 2932, 2870, 1640, 1456, 1385, 1215, 1096, 972, 756 cmꢀ1. 1H
NMR (500 MHz, CDCl3)
(3H each, all s, H3-28, 25, 26, 23, 27, 24, 29, 30), 1.07 (1H, d,
d 0.87, 0.91, 0.98, 1.04, 1.08, 1.14, 1.32, 1.32
J¼13.7 Hz, H-18
a
), 1.24 (1H, d, J¼13.7 Hz, H-18 ), 1.43 (1H, dd, J¼4.1,
b
11.6 Hz, H-8), 1.96, 2.36 (1H each, both dd, J¼6.9, 13.7 Hz, H2-19),
2.08 (1H, m, H-10), 3.47 (1H, t-like, J¼2.5 Hz, H-3), 5.58 (1H, d,
J¼15.8 Hz, H-21), 5.62 (1H, m, H-6), 5.64 (1H, dd-like, J¼6.9,15.8 Hz,
H-20). 13C NMR data (125 MHz, CDCl3) dC: given in Table 1. EIMS:
m/z 442 (Mþ, 2), 424 (10), 406 (63), 343 (27), 325 (100), 290 (8),
191 (67), 152 (18).
3.3.2. Foliasalacin D2 (2)
A white powder, [
a]
þ17.1 (c 0.43, CHCl3). High-resolution
Compound 2a. Colorless oil. 1H NMR (500 MHz, CDCl3)
d 0.81,
29
D
EIMS calcd for C30H50O2 (M)þ: 442.3811, found: 442.3806. IR (KBr):
3422, 3069, 3015, 2948, 1636, 1096, 897, 758 cmꢀ1 1H NMR
(500 MHz, CDCl3) 0.86, 0.89, 0.96, 1.02, 1.05, 1.13, 1.70 (3H each, all
s, H3-28, 25, 26, 23, 27, 24, 30), 1.05, 1.72 (1H each, both m, H2-19),
), 1.43
0.82, 0.90, 0.94, 1.07, 1.15, 1.73 (3H each, all s, H3-28, 27, 25, 26, 23,
24, 29), 1.51, 1.60 (1H each, both m, H2-1), 1.67, 1.86 (1H each, both
m, H2-2), 1.87 (2H, m, H2-7), 1.36 (1H, dd, J¼4.6, 11.6 Hz, H-8), 2.08
(1H, m, H-10), 1.47, 1.59 (1H each, both m, H2-11), 0.87, 1.54 (1H
each, both m, H2-12), 1.13, 1.27 (1H each, both m, H2-15), 1.19, 1.54
(1H each, both m, H2-16), 0.97, 1.19 (1H each, both d, J¼14.1 Hz,
H2-18), 1.00, 1.56 (1H each, both m, H2-19), 1.50, 1.72 (1H each, both
m, H2-20), 3.49 (1H, br s, H-3), 3.55 (3H, s, –COOCH3), 4.97, 5.05 (1H
each, both br s, H2-30), 5.37 (1H, dd, J¼5.2, 7.7 Hz, H-21), 5.64 (1H,
m, H-6), 7.37–7.52 (5H, m, Ph–H).
.
d
1.07 (1H, d, J¼14.5 Hz, H-18
a
), 1.24 (1H, d, J¼14.5 Hz, H-18
b
(1H, dd, J¼4.8, 12.4 Hz, H-8), 2.06 (1H, m, H-10), 3.46 (1H, s-like,
H-3), 3.99 (1H, t-like, J¼6.2 Hz, H-21), 4.83, 4.91 (1H each, both br s,
H2-29), 5.59 (1H, m, H-6). 13C NMR data (125 MHz, CDCl3) dC: given
in Table 1. EIMS: m/z 442 (Mþ, 6), 424 (23), 406 (89), 391 (33), 343
(10), 325 (58), 290 (71), 275 (23), 257 (37), 203 (33), 152 (40), 134
(100).
Compound 2b. Colorless oil. 1H NMR (500 MHz, CDCl3)
d 0.85,
0.90, 0.95, 0.96, 1.07, 1.15, 1.62 (3H each, all s, H3-28, 25, 27, 26, 23,
24, 29), 1.51, 1.60 (1H each, both m, H2-1), 1.67, 1.86 (1H each, both
m, H2-2), 1.87 (2H, m, H2-7), 1.40 (1H, dd, J¼4.6, 11.6 Hz, H-8), 2.08
(1H, m, H-10), 1.49, 1.62 (1H each, both m, H2-11), 0.91, 1.55 (1H
each, both m, H2-12), 1.20, 1.32 (1H each, both m, H2-15), 1.27, 1.59
3.3.3. Foliasalacin D3 (3)
29
A white powder, [
a
]
þ20.8 (c 0.53, CHCl3). High-resolution
D
EIMS calcd for C30H50O2 (M)þ: 442.3811, found: 442.3803. IR (KBr):
3422, 3069, 3015, 2948, 1636, 1094, 897, 758 cmꢀ1 1H NMR
.

7352
Y. Zhang et al. / Tetrahedron 64 (2008) 7347–7352
(1H each, both m, H2-16), 1.04, 1.24 (1H each, both d, J¼14.0 Hz,
H2-18), 1.10, 1.67 (1H each, both m, H2-19), 1.58, 1.77 (1H each, both
m, H2-20), 3.49 (1H, br s, H-3), 3.54 (3H, s, –COOCH3), 4.92, 4.96 (1H
each, both br s, H2-30), 5.32 (1H, dd, J¼6.4, 6.4 Hz, H-21), 5.64 (1H,
m, H-6), 7.37–7.52 (5H, m, Ph–H).
References and notes
1. Yoshikawa, M.; Murakami, T.; Shimada, H.; Matsuda, H.; Yamahara, J.; Tanabe,
G.; Muraoka, O. Tetrahedron Lett. 1997, 38, 8367–8370.
2. Yoshikawa, M.; Murakami, T.; Yashiro, K.; Matsuda, H. Chem. Pharm. Bull. 1998,
46, 1339–1340.
3. Matsuda, H.; Murakami, T.; Yashiro, K.; Yoshikawa, M. Chem. Pharm. Bull. 1999,
47, 1725–1729.
4. Yoshikawa, M.; Nishida, N.; Shimoda, H.; Takada, M.; Kawahara, Y.; Matsuda, H.
Yakugaku Zasshi 2001, 121, 371–378.
5. Yoshikawa, M.; Morikawa, T.; Matsuda, H.; Tanabe, G.; Muraoka, O. Bioorg. Med.
Chem. 2002, 10, 1547–1554.
6. Yoshikawa, M.; Ninomiya, K.; Shimoda, H.; Nishida, N.; Matsuda, H. Biol. Pharm.
Bull. 2002, 25, 72–76.
7. Yoshikawa, M.; Shimoda, H.; Nishida, N.; Takada, M.; Matsuda, H. J. Nutr. 2002,
132, 1819–1824.
8. Yoshikawa, M. FOOD Style 2002, 21, 72–78.
9. Morikawa, T.; Kishi, A.; Pongpiriyadacha, Y.; Matsuda, H.; Yoshikawa, M. J. Nat.
Prod. 2003, 66, 1191–1196.
3.3.6. Preparation of the (R)-MTPA ester (3a)
and (S)-MTPA ester (3b) from 3
A solution of 3 (2.1 mg) in dehydrated CHCl3 (1.0 mL) was
reacted with (R)-MTPA (20.0 mg) in the presence of EDC$HCl
(20.0 mg) and 4-DMAP (8.0 mg), and the mixture was stirred under
reflux at 60 ꢂC for 6 h. Workup of the reaction mixture as described
for 2 gave 3a (1.2 mg, 38%). Using a similar procedure, (S)-MTPA
ester (3b, 1.3 mg, 42%) was obtained from 3 (2.1 mg) with (S)-MTPA
(33.2 mg), EDC$HCl (27.1 mg), and 4-DMAP (11.0 mg).
Compound 3a. Colorless oil. 1H NMR (500 MHz, CDCl3)
d 0.85,
10. Kishi, A.; Morikawa, T.; Matsuda, H.; Yoshikawa, M. Chem. Pharm. Bull. 2003, 51,
1051–1055.
0.90, 0.97, 1.00,1.05,1.14,1.59 (3H each, all s, H3-28, 25, 26, 27, 23, 24,
29), 1.50, 1.59 (1H each, both m, H2-1), 1.67, 1.85 (1H each, both m,
H2-2), 1.88 (2H, m, H2-7), 1.42 (1H, dd, J¼4.6, 11.9 Hz, H-8), 2.08 (1H,
m, H-10),1.50,1.60 (1H each, both m, H2-11), 0.92,1.58 (1H each, both
m, H2-12), 1.14, 1.34 (1H each, both m, H2-15), 1.30, 1.60 (1H each,
both m, H2-16), 1.05, 1.28 (1H each, both m, H2-18), 1.18, 1.70 (1H
each, both m, H2-19), 1.62 (2H, m, H2-20), 3.49 (1H, br s, H-3), 3.56
(3H, s, –COOCH3), 4.91, 4.95 (1H each, both br s, H2-30), 5.30 (1H, dd,
J¼6.4, 6.4 Hz, H-21), 5.62 (1H, m, H-6), 7.39–7.52 (5H, m, Ph–H).
11. Yoshikawa, M.; Pongpiriyadacha, Y.; Kishi, A.; Kageura, T.; Wang, T.; Morikawa,
T.; Matsuda, H. Yakugaku Zasshi 2003, 123, 871–880.
12. Matsuda, H.; Yoshikawa, M.; Morikawa, T.; Tanabe, G.; Muraoka, O. J. Trad. Med.
2005, 22, 145–153.
13. Yoshikawa, M.; Xu, F.; Nakamura, S.; Wang, T.; Matsuda, H.; Tanabe, G.; Mur-
aoka, O. Heterocycles, in press.
14. Nakamura, S.; Zhang, Y.; Pongpiriyadacha, Y.; Wang, T.; Matsuda, H.; Yoshikawa,
M. Heterocycles 2007, 75, 131–143.
15. Zhang, Y.; Nakamura, S.; Pongpiriyadacha, Y.; Matsuda, H.; Yoshikawa, M. Chem.
Pharm. Bull. 2008, 56, 547–553.
16. Nakamura, S.; Zhang, Y.; Wang, T.; Matsuda, H.; Yosikawa, M. Heterocycles,
in press.
17. Yosikawa, M.; Zhang, Y.; Wang, T.; Nakamura, S., Matsuda, H. Chem. Pharm. Bull.,
in press.
18. Wu, T.; Cheng, Z.; Liu, H.; Li, Y.; Wang, Z. Zhongguo Yaoxue Zazhi 2005, 40,
1460–1462.
19. Cole, B. J. W.; Bentley, M. D.; Hua, Y.; Bu, L. J. Wood Chem. Tech. 1991, 11, 209–223.
20. Tanaka, R.; Masuda, K.; Matsunaga, S. Phytochemistry 1993, 32, 472–474.
21. Shashi, B. M.; Asish, P. K. Phytochemistry 1994, 37, 1517–1575.
22. Li, X.; Yao, Y.; Zheng, Y.; Lin, W.; Deng, Z. Zhongguo Tianran Yaowu 2006, 4,
188–191.
23. Wijeratne, D. B. T.; Kumar, V.; Sultanbawa, M.; Uvais, S. J. Chem. Soc., Perkin
Trans. 1 1981, 10, 2724–2726.
24. Akihisa, T.; Tokuda, H.; Ukiya, M.; Suzuki, T.; Enjo, F.; Koike, K.; Nikaido, T.;
Nishino, H. Chem. Pharm. Bull. 2004, 52, 153–156.
25. Chang, C.; Wu, T.; Hsieh, Y.; Kuo, S.; Chao, P. L. J. Nat. Prod. 1999, 62, 327–328.
26. Zhang, K.; Liu, J.; Wang, Y.; Huang, H.; Chen, Y. Zhongshan Daxue Xuebao 1998,
37, 85–88.
27. Wang, M.; Yang, L.; Tu, Y. Zhongguo Zhongyao Zazhi 2006, 31, 307–308.
28. Ames, T. R.; Beton, J. L.; Bowers, A.; Halsall, T. G.; Jones, E. R. H. J. Chem. Soc.
1954, 1905–1919.
29. Chivers, H.; Corbett, R. E.; Mitchell, R. E. M. J. Chem. Soc. C 1966, 20, 1814–1816.
30. The 1H and 13C NMR spectra of 1–3, 1a-3a, 2b, and 3b were assigned with the
aid of distortionless enhancement by polarization transfer (DEPT), homo-
correlation spectroscopy (1H–1H COSY), heteronuclear multiple-quantum
coherence (HMQC), and HMBC experiments.
Compound 3b. Colorless oil. 1H NMR (500 MHz, CDCl3)
d 0.78,
0.90, 0.95, 0.98, 1.05, 1.14, 1.72 (3H each, all s, H3-28, 25, 26, 27, 23,
24, 29), 1.50, 1.59 (1H each, both m, H2-1), 1.67, 1.85 (1H each, both
m, H2-2), 1.88 (2H, m, H2-7), 1.41 (1H, dd, J¼4.3, 12.3 Hz, H-8), 2.08
(1H, m, H-10), 1.48, 1.59 (1H each, both m, H2-11), 0.89, 1.56 (1H
each, both m, H2-12), 1.08, 1.31 (1H each, both m, H2-15), 1.20, 1.55
(1H each, both m, H2-16), 0.99, 1.22 (1H each, both m, H2-18), 1.06,
1.62 (1H each, both m, H2-19), 1.56 (2H, m, H2-20), 3.49 (1H, br s,
H-3), 3.54 (3H, s, –COOCH3), 4.96, 5.04 (1H each, both br s, H2-30),
5.34 (1H, dd, J¼6.9, 6.9 Hz, H-21), 5.62 (1H, m, H-6), 7.39–7.52 (5H,
m, Ph–H).
Acknowledgements
This research was supported by the 21st COE program and
Academic Frontier Project from the Ministry of Education, Culture,
Sports, Science and Technology of Japan.
Supplementary data
31. Nu´ n˜ez, M. J.; Lo´ pez, M. R.; Jime´nez, I. A.; Moujir, L. M.; Ravelo, A. G.; Bazzocchi,
I. L. Tetrahedron Lett. 2004, 45, 7367–7370.
32. Lou, H.; Li, X.; Onda, M.; Konda, Y.; Urano, M.; Harigaya, Y.; Takayanagi, H.;
Ogura, H. Chem. Pharm. Bull. 1991, 39, 2271–2276.
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.tet.2008.05.054.