Structure and bioassay of triterpenoids and steroids isolated from sinocalamus affinis

Article abstract of DOI:10.1021/np300259s

Five triterpenoids with a new 25-norfern carbon skeleton (1-5), a lupane triterpenoid (6), and four 20-hydroxyprogesterone acyl esters (7-10), together with 23 known compounds, were isolated from the stem (with skin removed) of Sinocalamus af f inis. The absolute configuration of compound 1 was confirmed by single-crystal X-ray crystallographic analysis using anomalous scattering of Cu Kα radiation. Compounds 1-5 exhibited inhibitory activity against protein tyrosine phosphatase 1B.

Full text of DOI:10.1021/np300259s

Article
pubs.acs.org/jnp
Structure and Bioassay of Triterpenoids and Steroids Isolated from
Sinocalamus affinis
Liang Xiong, Mei Zhu, Chenggeng Zhu, Sheng Lin, Yongchun Yang, and Jiangong Shi*
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of
Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China
S
* Supporting Information
ABSTRACT: Five triterpenoids with a new 25-norfern
carbon skeleton (1−5), a lupane triterpenoid (6), and four
20-hydroxyprogesterone acyl esters (7−10), together with 23
known compounds, were isolated from the stem (with skin
removed) of Sinocalamus affinis. The absolute configuration of
compound 1 was confirmed by single-crystal X-ray crystallo-
graphic analysis using anomalous scattering of Cu Kα
radiation. Compounds 1−5 exhibited inhibitory activity against protein tyrosine phosphatase 1B.
Sinocalamus affinis (Rendle) McClure (Poaceae) is widely
distributed and cultivated in southwestern China.1 Slices of the
stem (with skin removed), named “ci zhu ru” in Chinese, are
commonly used to treat various symptoms such as cough and
phlegm.1,2 Our previous study on the EtOAc-soluble portion of
an EtOH extract of “ci zhu ru” reported 36 lignans and
neolignans and their absolute configurations.3 During the
continued examination of the same extract, six triterpenoids
(1−6) and four 20-hydroxyprogesterone acyl esters (7−10),
together with 23 known compounds, were characterized.
Compounds 1−5 are triterpenoids with a new 25-norfern
carbon skeleton. This paper describes the isolation, structure
elucidation, and bioassay of these isolates.
pentacyclic nortriterpenediene with substitution of four
hydroxy groups; this conjecture was confirmed by 2D NMR
data analysis. The gHSQC spectrum of 1 furnished assignments
of the proton-bearing carbon and corresponding proton
1
resonances in the NMR spectra (Table 1). In the H−1H
gCOSY spectrum of 1, the homonuclear coupling correlations
of H2-1/H2-2/H-3; H2-6/H-7/H-8; H-11/H2-12; H2-15/H2-
16; H-18/H-19/H2-20/H-21/H-22/H3-29; and H-22/H3-30
revealed the presence of structural units containing the vicinally
coupled protons. In the HMBC spectrum, two- and three-bond
correlations of H2-1/C-3 and C-5; H2-6/C-4, C-5, C-7, C-8,
and C-10; H-11/C-8, C-10, C-12, and C-13; H3-23 and H3-24/
C-3, C-4, and C-5; H3-26/C-12, C-13, C-14, and C-18; H3-27/
C-8, C-13, C-14, and C-15; H2-28/C-16, C-17, C-18, and C-21;
and H3-29 and H3-30/C-21 and C-22, in combination with the
shifts of these proton and carbon resonances, indicated a gross
structure of 25-norfern-5(10),9(11)-diene-3,7,19,28-tetraol for
1. In the ROESY spectrum of 1, correlations of H2-28/H-19,
H3-26, and H3-30 and of H3-26/H-8 and H-19 indicated that
these protons were cofacial. ROESY correlations of H3-27/H-
6a, H-7, and H-18 and H3-23/H-3 and H-6a demonstrated that
these protons were located on the opposite side of the ring
system. This result was corroborated by the splitting patterns
and coupling constants of H-3, H-7, H-18, and H-19, indicating
that these protons had pseudoaxial orientations. The electronic
circular dichroism (ECD) spectrum of 1 displayed a positive
Cotton effect at 235 nm (Δε +3.66), which corresponded to
the π−π* transition of the conjugated diene chromophore. On
the basis of the allylic axial chirality rule for conjugated s-trans
dienes,4 the 8S,13R,14S configuration was assigned to 1. This
was confirmed by single-crystal X-ray crystallographic analysis
using the anomalous scattering of Cu Kα radiation. An ORTEP
drawing, with the atom numbering indicated, is shown in
RESULTS AND DISCUSSION
■
Compound 1 showed IR absorptions for hydroxy (3627, 3472,
and 3406 cm−1) and olefinic (3043 and 1466 cm−1)
functionalities. The molecular formula C29H46O4 of 1, with
seven hydrogen deficiencies, was indicated by HRESIMS and
1
NMR data. The H NMR spectrum of 1 displayed resonances
attributable to (a) four tertiary [δH 1.01 (H3-24 and H3-27),
1.04 (H3-23), and 1.06 (H3-26)] and two secondary [δH 0.83
(d, J = 6.6 Hz, H3-30) and 0.95 (d, J = 6.6 Hz, H3-29)] methyl
groups; (b) an isolated oxymethylene group [δH 3.82 (brd, J =
11.4 Hz, H-28a) and 3.72 (d, J = 11.4 Hz, H-28b)]; (c) three
oxymethines [δH 3.46 (dd, J = 7.8 and 2.4 Hz, H-3), 3.70 (dt, J
= 4.8 and 10.2 Hz, H-7), and 4.40 (dt, J = 3.0 and 10.2 Hz, H-
19)]; and (d) an olefinic methine group [δH 5.60 (dd, J = 6.0
and 3.0 Hz, H-11)]. In addition, it showed resonances
assignable to four exchangeable hydroxy protons (Table 1)
and partially overlapped resonances ascribable to several
aliphatic methylenes and methines between δH 1.20 and 2.50.
The 13C NMR and DEPT spectra of 1 revealed 29 carbon
resonances (Table 2) corresponding to the above protonated
units and seven quaternary carbons (three olefinic, δC 127.3,
136.4, and 137.6). These data suggested that 1 was an unusual
Received: April 7, 2012
Published: June 12, 2012
© 2012 American Chemical Society and
American Society of Pharmacognosy
1160
dx.doi.org/10.1021/np300259s | J. Nat. Prod. 2012, 75, 1160−1166

Journal of Natural Products
Article
a
1
Table 1. H NMR Data for Compounds 1−6
b
no.
1a
1
2
3
4
5
6
2.16 m
2.16 m
1.80 m
1.70 m
2.16 m
2.16 m
1.81 m
1.70 m
2.17 m
2.17 m
1.82 m
1.71 m
2.18 m
2.18 m
1.81 m
1.67 m
5.56 dd (3.0, 2.4)
1.64 m
0.98 m
1.63 m
1.57 m
1b
2a
2.14 m
2b
1.91 m
3
3.46 dd (7.8, 2.4)
3.46 dd (7.8, 2.4)
3.46 dd (7.8, 2.4)
3.88 dd (10.2, 3.0)
3.34 dd (11.2, 5.4)
1.86 m
4.47 dd (11.0, 5.5)
0.79 brd (10.0)
1.51 m
5
6a
2.42 dd (16.2, 4.8)
2.10 dd (16.2, 10.2)
3.70 dt (4.8, 10.2)
2.43 dd (16.2, 4.8)
2.12 dd (16.2, 10.2)
3.70 dt (4.8, 10.2)
2.43 dd (16.8, 4.8)
2.07 dd (16.8, 10.2)
3.69 dt (4.8, 10.2)
2.43 dd (16.2, 4.8)
2.13 dd (16.2, 10.2)
3.75 dt (4.8, 10.2)
2.01 dt (12.0, 3.6)
1.33 m
6b
1.40 m
7a
3.70 m
1.38 m
7b
1.04 m
8
2.02 d (10.2)
2.02 d (10.2)
2.01 d (10.2)
2.01 d (10.2)
1.85 d (10.2)
9
1.31 m
1.40 m
1.20 m
1.61 m
1.06 m
1.62 m
1.69 m
1.07 m
1.92 m
1.30 m
1.59 m
2.39 m
1.94 m
1.42 m
1.86 m
1.04 m
11a
11b
12a
12b
13
5.60 dd (6.0, 3.0)
5.60 dd (5.4, 2.4)
5.61 dd (6.0, 2.4)
5.58 dd (6.0, 3.0)
5.69 dt (5.4, 2.4)
2.15 dd (18.0, 3.0)
2.05 dd (18.0, 6.0)
2.08 dd (18.0, 2.4)
1.98 dd (18.0, 5.4)
2.79 dd (18.0, 6.0)
1.84 dd (18.0, 2.4)
2.13 dd (18.0, 3.0)
2.05 dd (18.0, 6.0)
2.15 m
2.02 m
15a
15b
16a
16b
18
2.37 dt (14.4, 3.6)
1.57 dt (3.6, 14.4)
1.70 dt (14.4, 3.6)
1.39 dt (3.6, 14.4)
1.85 d (10.2)
4.40 dt (3.0, 10.2)
2.12 m
2.39 dt (13.8, 3.6)
1.61 dt (3.6, 13.8)
1.52 dt (13.8, 3.6)
1.44 dt (13.8, 3.6)
1.75 d (10.8)
2.46 dt (14.4, 3.6)
2.14 dt (3.6, 14.4)
1.64 m
2.35 dt (14.4, 3.6)
1.55 dt (3.6, 14.4)
1.72 dt (14.4, 3.6)
1.39 dt (3.6, 14.4)
1.86 d (10.2)
4.41 dt (3.0, 10.2)
2.13 m
2.27 dt (14.4, 3.6)
1.51 dt (3.6, 14.4)
1.70 dt (13.2, 3.0)
1.36 dt (13.2, 3.0)
1.85 d (9.6)
1.64 m
2.23 s
19
4.19 brd (10.8)
4.39 brt (9.6)
2.11 m
20a
20b
21a
21b
22
3.83 dd (7.2, 1.8)
2.15 m
2.08 m
1.54 m
1.61 m
1.60 m
1.60 m
1.32 m
1.25 t (7.2)
1.32 m
1.30 m
1.78 m
1.04 s
1.89 m
1.04 s
1.90 m
1.04 s
1.78 m
1.78 m
0.99 s
23a
23b
24
3.67 brd (10.2)
3.50 dd (10.2)
0.95 s
0.84 s
1.01 s
1.02 s
1.01 s
0.72 s
0.84 s
25
0.85 s
26
1.06 s
1.11 s
1.15 s
1.07 s
1.04 s
1.02 s
27
1.01 s
1.02 s
1.01 s
1.01 s
1.03 s
0.98 s
28a
28b
29a
29b
30
3.82 brd (11.4)
3.72 brd (11.4)
0.95 d (6.6)
3.94 brd (11.4)
3.52 brd (11.4)
0.98 d (6.6)
4.02 brd (11.4)
3.76 brd (11.4)
1.00 d (6.6)
3.83 brd (11.4)
3.74 brd (11.4)
0.94 d (6.6)
3.80 (11.4, 4.2)
3.72 (11.4, 3.6)
0.93 d (6.6)
3.80 d (10.5)
3.33 d (10.5)
4.68 brs
4.58 brs
1.69 s
0.83 d (6.6)
3.48 d (6.0)
3.38 d (6.0)
3.07 d (6.0)
0.94 d (6.6)
3.53 brs
0.91 d (7.2)
3.64 brs
0.83 d (6.6)
3.52 d (4.2)
3.34 d (6.0)
3.08 d (6.6)
0.82 d (6.6)
3.56 brs
OH-3
OH-7
OH-19
OH-20
OH-23
OH-28
3.45 brs
3.46 brs
3.35 d (6.0)
3.09 brd (6.0)
3.52 brs
3.83 brs
3.55 t (5.4)
3.27 t (4.2)
3.27 t (4.2)
4.75 brs
3.29 brs
3.27 brt (4.2)
a
Data were measured at 600 MHz for 1−5 in acetone-d6 and 500 MHz for 6 in CDCl3. Coupling constants (J) in Hz are given in parentheses, and
coupling constants with hydroxy proton were ignored for the OH geminated protons. The assignments were based on DEPT, H−1H COSY,
1
b
HSQC, and HMBC experiments. Data for the myristoyl unit in 6: δ 2.29 (2H, t, J = 7.5 Hz), 1.62 (2H, m), 1.40 (2H, m) 1.28−1.25 (18H, m), 0.88
(3H, t, J = 7.5 Hz).
Figure 1. Therefore, compound 1 was deduced to be
(+)-(3S,7S,8S,13R,14S,17R,18R,19R,21S)-25-norfern-5(10),9-
(11)-diene-3,7,19,28-tetraol.
Compound 2 had spectroscopic data similar to those of 1.
The HRESIMS data indicated that it had the molecular formula
C29H46O5 with one more oxygen atom than 1. Comparison of
the NMR data of 2 and 1 indicated replacement of a methylene
group in 1 by a hydoxymethine [δH 3.83 (dd, J = 7.2 and 1.8
Hz, H-20) and 3.83 (brs, OH-20) and δC 82.8] functionality in
2. In addition, the H-28a and C-19 and C-21 resonances in 2
were deshielded by ΔδH +0.12 and ΔδC +10.2 and +3.5 ppm,
respectively, unlike those of 1, whereas the H-19 and H-28b
and C-18 and C-22 resonances were shielded by ΔδH −0.21
and −0.20 and ΔδC −2.5 and −5.0 ppm, respectively. This
revealed that 2 was the 20-OH analogue of 1, which was proved
by 2D NMR experiments on 2 that amended the assignments
of the NMR data. In the ROESY spectrum of 2, correlations of
H-18 with H-20, H-21, and H3-27 demonstrated that the 20-
OH group was β-oriented. The ECD spectrum of 2 showed a
positive Cotton effect at 245 nm (Δε +1.94), similar to that of
1161
dx.doi.org/10.1021/np300259s | J. Nat. Prod. 2012, 75, 1160−1166

Journal of Natural Products
Article
a
Table 2. 13C NMR Data for Compounds 1−6
b
no.
1
2
3
4
5
6
1
24.0
27.5
74.8
40.0
136.4
38.2
70.6
50.9
137.6
127.3
120.8
38.5
38.2
40.1
32.2
33.5
49.2
59.8
70.9
43.3
58.3
31.0
26.7
22.2
24.0
27.5
74.8
40.0
136.5
38.2
70.6
51.0
137.5
127.3
120.7
38.2
38.0
40.3
32.6
35.1
49.1
57.3
81.1
82.8
61.8
26.0
26.7
22.2
24.0
27.4
74.7
40.0
136.6
38.1
70.5
50.1
137.8
127.2
120.1
36.0
37.3
39.6
31.9
33.3
47.2
61.6
213.0
43.9
55.8
30.6
26.7
22.2
25.7
27.8
70.1
45.5
134.5
37.6
70.2
50.9
137.8
130.0
121.1
38.5
38.1
40.1
32.2
33.5
49.2
59.8
70.9
43.4
58.2
31.0
67.2
16.7
118.3
38.7
74.3
37.2
46.2
37.8
71.3
52.9
139.5
141.5
124.1
33.1
38.6
40.1
32.7
33.4
49.2
59.9
70.9
43.3
58.3
31.0
25.0
14.4
38.4
23.7
80.6
37.8
55.4
18.2
34.2
40.9
50.3
37.1
20.8
25.2
37.3
42.7
27.0
29.2
47.8
48.7
47.8
29.7
34.0
150.5
28.0
16.0
16.6
16.2
14.7
60.6
109.7
19.1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
The spectroscopic data of 3 (Tables 1 and 2 and
Experimental Section) showed that it was another analogue
of 1 with the molecular formula C29H44O4, as indicated by
HRESIMS data. Comparison of the NMR data of 3 with those
of 1 indicated replacement of one hydroxymethine unit in 1 by
a carbonyl group (δC 213.0) in 3. In addition, the H-18 doublet
in 1 was deshielded and changed into a singlet (δH 2.23) in 3,
and the C-18 and C-20 resonances in 3 were deshielded by ΔδC
+1.8 and +0.6, respectively. In contrast, the C-13, C-17, and C-
21 resonances were shielded by ΔδC −0.9, −2.0, and −2.5. This
revealed that 3 was the 19-oxo derivative of 1, a conclusion that
was supported by the presence of a carbonyl absorption at 1728
cm−1 in the IR spectrum of 3 and confirmed by 2D NMR and
ECD data. In particular, the ECD spectrum of 3 showed
Cotton effects, positive at 241 nm and negative at 286 nm,
arising from the π→π* transition of the conjugated s-trans
diene chromophore and the n→π* transition of the cyclo-
pentone chromophore, respectively. Applying the allylic axial
chirality rule to the conjugated s-trans diene chromophore4 and
the octant rule to the cyclopentone chromophore,5 the
observed Cotton effects predicted that the absolute config-
uration of 3 was consistent with that of 1. Therefore,
compound 3 was assigned as (+)-(3S,7S,8S,13R,14S,17R,-
18R,21S)-25-norfern-5(10),9(11)-diene-19-oxo-3,7,28-triol.
The spectroscopic data of compound 4 indicated that it was
an isomer of 2. Comparison of the NMR data of 4 and 2
demonstrated replacement of the secondary 20-OH group in 2
by a primary OH group in 4. In addition, the H-3 and C-4 and
C-10 resonances in 4 were deshielded by ΔδH +0.42 and ΔδC
+5.5 and +2.7, respectively. In contrast, the C-3, C-5, and C-24
resonances were shielded by ΔδC −4.7, −2.0, and −5.5. This
indicated that the primary OH group was located at C-23 in 4,
which was supported by the HMBC correlations of H3-24/C-3,
C-4, C-5, and C-23, in combination with the shifts of these
proton and carbon resonances. This regiochemistry was
confirmed by the correlations of H-7 (pseudoaxial)/H-6a
(pseudoequatorial)/H2-23 in the NOESY spectrum of 4. The
ECD spectrum of 4 displayed a positive Cotton effect at 235
nm (Δε +6.50), similar to that of 2, suggesting that the
absolute configuration around the s-trans diene chromophore of
17.5
15.2
63.3
23.4
23.6
17.0
15.2
63.7
22.7
23.2
16.9
15.2
63.7
23.1
23.3
17.4
15.3
63.3
23.4
23.6
16.8
16.2
63.3
23.4
23.6
a
13C NMR data (δ) were measured at 125 MHz for 1−5 in acetone-d6
and for 6 in CDCl3. The assignments were based on DEPT, H−1H
COSY, HSQC, and HMBC experiments. Data for the myristoyl unit
in 6: δ 173.7 (C-1′), 34.9 (C-2′), 31.9 (C-13′), 29.7−29.2 (C-4′−C-
11′), 25.2 (C-3′), 22.7 (C-12′), 14.1 (C-14′).
1
b
Figure 1. ORTEP diagram of compound 1 cocrystallizing with
acetone.
1, which indicated that the absolute configuration around the s-
trans diene chromophore of 2 was identical to that of 1.
Therefore, compound 2 was determined to be
(+)-(3S,7S,8S,13R,14S,17R,18R,19S,20S,21S)-25-norfern-5-
(10),9(11)-diene-3,7,19,20,28-pentaol.
1162
dx.doi.org/10.1021/np300259s | J. Nat. Prod. 2012, 75, 1160−1166

Journal of Natural Products
Article
4 was identical with that of 2. Therefore, compound 4 was
determined as (+)-(3S,4R,7S,8S,13R,14S,17R,18R,19R,21S)-25-
norfern-5(10),9(11)-diene-3,7,19,23,28-pentaol.
Compound 10 was (+)-(20S)-20-O-capryloylpregn-4-en-3-
one, as determined by HRESIMS at m/z 443.3564 [M + H]+
(calculated for C29H47O3, 443.3520) and as indicated by NMR
and CD data.
Compound 5 was an isomer of 1, as indicated by
spectroscopic data. Comparison of the NMR data of 5 and 1
indicated the presence of a trisubstituted double bond [δH 5.56
(dd, J = 3.0 and 2.4 Hz, H-1), δC 118.3 (C-1) and 141.5 (C-
10)] and a methine group [δH 1.86 (m, H-5), δC 46.2 (C-5)] in
5, replacing the tetrasubstituted double bond and one
methylene group (CH2-1) in 1, respectively. This suggested
that the C-5−C-10 double bond in 1 shifted to C-1 and C-10 in
5. The suggestion was confirmed by the 2D NMR data,
particularly by HMBC correlations from H-1 to C-3, C-5, and
C-9, combined with their shifts. In the NOESY spectrum,
correlations of H-3/H3-23/H-5/H-7/H3-27/H-18/H-21 dem-
onstrated an α-orientation of H-5. Therefore, compound 5 was
assigned as (+)-(3S,5S,7S,8S,13R,14S,17R,18R,19R,21S)-25-
norfern-1(10),9(11)-diene-3,7,19,28-tetraol, and the absolute
configuration was supported by a positive π→π* Cotton effect
at 236 nm in the ECD spectrum, based on the s-cis diene allylic
axial chirality rule.4,6
The known compounds were identified by comparison of
their spectroscopic data with reported data. They were
friedelin,12 maytensifolin B,13 3,21-dioxofriedelane,14 epifriede-
lanol,15 29-norlupan-3,20-dione,16 3-O-palmitoylbetulin,7 3-O-
lauroylbetulin, 28-O-lauroylbetulin,17 trans-phytol,18 α-tocoqui-
none,19 2,3-epoxy-α-tocoquinone,20 (E)-4-oxo-β-ionone,21 (E)-
4-oxo-β-dihydroionone,22 (3S,5R,6S)-3-acetoxy-5,6-epoxy-5,6-
dihydro-β-ionone,23 (22E)-ergosta-6,9,22-triene-3β,5α,8α-
triol,24 (22E)-ergosta-6,22-diene-3β,5α,8α-triol,25 (22E)-ergo-
sta-7,22-dien-3β-ol,26 ergosta-7,24(241)-dien-3β-ol,27 (24R)-
5α-stigmast-3,6-dione,28 (24R)-stigmast-4-en-3-one,29 cholest-
4-ene-3,24-dione,30 β-sitosterol,31 and β-daucosterol.32
In the in vitro bioassays, compounds 1−5 showed inhibitory
activity against protein tyrosine phosphatase 1B (PTP1B)33
with IC50 values of 6.8−16.6 μM. The positive control in this
assay was oleanolic acid (IC50, 5.6 μM). However, compounds
1−10 and the other known compounds, at a concentration of
10 μM, were inactive in the assays against nitric oxide
production in mouse peritoneal macrophages,3 HIV-1 repli-
cation,34 Fe2+-cystine-induced rat liver microsomal lipid
peroxidation,35 and DL-galactosamine-induced WB-F344 cell
damage,36 as well as cytotoxicity against several human cancer
cell lines.37
Compound 6 possessed the molecular formula C44H76O3, as
indicated by spectroscopic data. The NMR data of 6 showed
that it was an analogue of the co-occurring 3-O-palmitoylbe-
tulin7 except for substitution of the palmitoyl unit by a
myristoyl unit. This was proven by the 2D NMR analysis and
alkaline hydrolysis of 6 to afford betulin.8 Specifically, the
location of the myristoyl unit at C-3 was confirmed by a
correlation of H-3/C-1′ in the HMBC spectrum of 6. Thus,
compound 6 was defined as 3-O-myristoylbetulin.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured on a Rudolph Research Autopol III automatic polarimeter.
UV spectra were measured on a Cary 300 spectrometer. ECD spectra
were recorded on a JASCO J-815 ECD spectrometer. IR spectra were
recorded on a Nicolet 5700 FT-IR microscope instrument (FT-IR
microscope transmission). NMR spectra were obtained at 300, 500, or
Compound 7 showed IR absorptions for OH (3435 cm−1)
and carbonyl (1730 and 1669 cm−1) groups. The molecular
formula, C35H58O3, was indicated by HRESIMS and NMR data.
The NMR spectra of 7 displayed resonances characteristic of a
sterol ester (Table 2). Alkaline hydrolysis of 7 liberated 7a,
1
1
600 MHz for H, and 125 or 150 MHz for 13C, on a Varian Mecury-
having H NMR and EIMS data consistent with those of 20-
hydroxypregn-4-en-3-one.9 This revealed that 7 was 20-O-
myristoylpregn-4-en-3-one, which was confirmed by the
HMBC correlations of H3-21/C-17 and C-20 and H-20/C-1′.
The ECD spectrum of 7 showed Cotton effects at 235 (Δε
+5.78) and 322 (Δε −1.21) nm, corresponding to the π→π*
and n→π* transitions of the conjugated 4-en-3-one chromo-
phore, indicating that the tetracyclic nucleus of 7 possessed the
same absolute configuration as the common pregn-4-en-3-one
analogues on the basis of the octant rule.10 Comparison of the
1H NMR data of 7a with those of the 20S and 20R epimers of
20-hydroxypregn-4-en-3-one,11 especially the shifts of H3-19
and H3-21, demonstrated that the data of 7a were in agreement
with those of the 20S epimer. This suggested that 7 had a 20S
configuration. Therefore, compound 7 was defined as
(+)-(20S)-20-O-myristoylpregn-4-en-3-one.
Compound 8 possessed the molecular formula C33H54O3,
with two fewer CH2 units than 7, as indicated by HRESIMS
and NMR data. Comparison of the NMR data between 8 and 7
indicated that the myristoyl unit in 7 was substituted by a
lauroyl unit in 8. Thus, compound 8 was assigned as (+)-(20S)-
20-O-lauroylpregn-4-en-3-one. This conclusion was confirmed
by alkaline hydrolysis, 2D NMR, and ECD experiments.
The spectroscopic data of compound 9 were similar to those
of 8, except that the HRESIMS of 9 indicated the molecular
formula C31H50O3 for 9 with two fewer CH2 units than 8. Thus,
compound 9 was determined as (+)-(20S)-20-O-caprinoyl-
pregn-4-en-3-one.
300 MHz or INOVA 500 MHz or SYS 600 MHz spectrometer with
solvent peaks used as references. ESIMS data were measured with a Q-
Trap LC/MS/MS (turbo ionspray source) spectrometer. HRESIMS
data were measured using an Agilent Technologies 6520 Accurate
Mass Q-ToF LC/MS spectrometer. Column chromatography was
performed using silica gel (200−300 mesh, Qingdao Marine Chemical
Inc., China) and Sephadex LH-20 (Pharmacia Biotech AB, Uppsala
Sweden). HPLC separation was performed on an instrument
consisting of a Waters 600 controller, a Waters 600 pump, and a
Waters 2487 dual λ absorbance detector with an Alltima (250 × 10
mm) preparative column packed with C18 (5 μm). TLC was carried
out on precoated silica gel GF254 plates. Spots were visualized under
UV light (254 or 356 nm) or by spraying with 7% H2SO4 in 95%
EtOH followed by heating.
Plant Material. The skin-removed stems of Sinocalamus affinis
were collected at Pingle Town, Sichuan Province, China, in August
2008. Plant identification was verified by Dr. Yan Ren (Chengdu
University of TCM, Sichuan 610075, China). A voucher specimen
(No. ID-S-2326) was deposited at the Herbarium of the Department
of Medicinal Plants, Institute of Materia Medica, Beijing 100050,
China.
Extraction and Isolation. Air-dried slices of the skin-removed
stem of S. affinis (6 kg) were powdered and extracted with 95% EtOH
(3 × 40 L) at rt for 3 × 72 h. The EtOH extract was evaporated under
reduced pressure to yield a dark brown residue (330 g). The residue
was suspended in H2O (2500 mL) and then partitioned with EtOAc
(6 × 2500 mL). After the removal of the solvent, the EtOAc fraction
(120 g) was applied to a silica gel column. Successive elution with a
gradient of increasing acetone (0−100%) in petroleum ether afforded
10 fractions (F1−F10) based on TLC analysis. F2 was recrystallized in
1163
dx.doi.org/10.1021/np300259s | J. Nat. Prod. 2012, 75, 1160−1166

Journal of Natural Products
Article
a
Table 3. NMR Data (δ) for Compounds 7−10 in CDCl3
b
c
d
e
7
8
9
10
no.
δH
δC
δH
δC
δH
δC
δH
δC
1a
2.02 m
1.71 m
2.44 m
2.34 m
35.7
2.02 m
1.71 m
2.43 m
2.33 m
35.7
2.02 m
1.71 m
2.43 m
2.34 m
35.7
2.03 m
1.71 m
2.44 m
2.34 m
35.7
1b
2a
33.9
34.0
34.0
34.0
2b
3
199.6
123.8
171.3
32.9
199.6
123.8
171.3
32.8
199.6
123.8
171.3
32.9
199.5
123.9
171.2
32.9
4
5.73 s
5.73 s
5.73 s
5.73 s
5
6a
2.39 m
2.29 m
1.83 m
1.03 m
1.55 m
0.95 m
2.39 m
2.29 m
1.83 m
1.03 m
1.54 m
0.95 m
2.39 m
2.29 m
1.83 m
1.03 m
1.55 m
0.95 m
2.40 m
2.29 m
1.84 m
1.03 m
1.54 m
0.95 m
6b
7a
32.0
31.9
32.0
32.0
7b
8
35.3
53.7
38.6
20.7
35.3
53.7
38.6
20.7
35.3
53.7
38.6
20.7
35.3
53.8
38.6
20.7
9
10
11a
11b
12a
12b
13
14
15a
15b
16a
16b
17
18
19
20
21
1.55 m
1.42 m
1.91 m
1.17 m
1.55 m
1.42 m
1.91 m
1.18 m
1.56 m
1.42 m
1.91 m
1.16 m
1.54 m
1.41 m
1.92 m
1.18 m
38.6
38.6
38.6
38.6
41.7
55.7
24.0
41.7
55.7
24.0
41.7
55.7
24.0
41.7
55.7
24.0
1.05 m
1.68 m
1.17 m
1.83 m
1.48 m
1.56 m
1.18 s
1.05 m
1.69 m
1.17 m
1.83 m
1.48 m
1.55 m
1.18 s
1.06 m
1.69 m
1.18 m
1.83 m
1.47 m
1.57 m
1.18 s
1.08 m
1.68 m
1.17 m
1.84 m
1.47 m
1.58 m
1.19 s
25.5
25.5
25.5
25.5
55.5
17.4
12.5
72.6
20.6
55.5
17.4
12.5
72.6
20.6
55.5
17.4
12.5
72.6
20.6
55.5
17.4
12.5
72.6
20.6
0.72 s
0.73 s
0.72 s
0.73 s
4.95 m
1.22 d (6.5)
4.95 m
1.22 d (6.0)
4.95 m
1.22 d (6.0)
4.95 m
1.22 d (6.0)
a
1
Data were measured at 500 for H and 125 for 13C, respectively. Coupling constants (J) in Hz are given in parentheses. The assignments were
b
based on 1H−1H COSY, HSQC, and HMBC experiments of 7 and 8. Data for the myristoyl unit in 7: δ 2.26 (2H, t, J = 7.5 Hz), 1.59 (2H, m), 1.30
(2H, m), 1.28−1.25 (18H, m), 0.88 (3H, t, J = 7.5 Hz); δC 173.3 (C-1′), 34.9 (C-2′), 31.9 (C-13′), 29.6−29.1 (C-4′−C-11′), 25.1 (C-3′), 22.7 (C-
c
12′), 14.1 (C-14′). Data for the lauroyl unit in 8: δ 2.25 (2H, t, J = 7.0 Hz), 1.60 (2H, m), 1.30 (2H, m), 1.28−1.25 (14H, m), 0.88 (3H, t, J = 7.5
d
Hz); δC 173.3 (C-1′), 34.9 (C-2′), 31.9 (C-11′), 29.6−29.1 (C-4′−C-9′), 25.1 (C-3′), 22.7 (C-10′), 14.1 (C-12′). Data for the caprinoyl unit in 9: δ
2.24 (2H, t, J = 7.0 Hz), 1.57 (2H, m), 1.30 (2H, m), 1.28−1.25 (10H, m), 0.88 (3H, t, J = 7.5 Hz); δC 173.3 (C-1′), 34.9 (C-2′), 31.8 (C-9′), 29.7−
e
29.2 (C-4′−C-7′), 25.1 (C-3′), 22.7 (C-8′), 14.1 (C-10′). Data for the capryloyl unit in 10: δ 2.25 (2H, t, J = 7.5 Hz), 1.54 (2H, m), 1.30 (2H, m),
1.29−1.26 (8H, m), 0.89 (3H, t, J = 7.5 Hz); δC 173.3 (C-1′), 34.9 (C-2′), 31.8 (C-7′), 29.4−29.2 (C-4′−C-5′), 25.1 (C-3′), 22.7 (C-6′), 14.1 (C-8′).
petroleum ether−Me2CO (5:1) to yield friedelin (2.3 g). β-Sitosterol
(2.1 g) was crystallized from F3 in petroleum ether−Me2CO (5:1).
The remaining mixture of F3 (15.0 g) was subjected to CC over silica
gel with a gradient of increasing EtOAc (0−50%) in petroleum ether,
to yield subfractions F3‑1−F3‑6. F3‑1 was chromatographed over
Sephadex LH-20 (petroleum ether−CHCl3−MeOH, 5:5:1), followed
by recrystallization in petroleum ether−Me2CO (5:1), to give
epifriedelanol (438 mg). F3‑2 (0.8 g) was repeatedly chromatographed
over silica gel (petroleum ether−EtOAc, 50:1−10:1) to yield trans-
phytol (45.0 mg). The successive separation of F3‑3 (2.1 g) with
Sephadex LH-20 (petroleum ether−CHCl3−MeOH, 5:5:1) and with
RP semipreparative HPLC (98% MeOH in H2O) yielded 6 (10.0 mg),
(24R)-stigmast-4-en-3-one (36 mg), 3-O-palmitoylbetulin (3.0 mg), 3-
O-lauroylbetulin (2.5 mg), and 28-O-lauroylbetulin (1.6 mg). F3‑4 was
separated with Sephadex LH-20 (petroleum ether−CHCl3−MeOH,
5:5:1) to give F3‑4‑1−F3‑4‑5. Separation of F3‑4‑3 with semipreparative
HPLC (MeOH−H2O, 96:4) gave maytensifolin B (3.5 mg), 3,21-
dioxofriedelane (2.0 mg), and 29-norlupan-3,20-dione (4.1 mg).
(24R)-5α-Stigmast-3,6-dione (1.4 g) was crystallized from F3‑4‑4 in
petroleum ether−Me2CO (5:1), while the remaining mixture was
isolated with CC over silica gel (petroleum ether−EtOAc, 50:1−10:1),
followed by RP semipreparative HPLC purification (MeOH−H2O,
95:5), to yield α-tocoquinone (86.5 mg), 2,3-epoxy-α-tocoquinone
(3.1 mg), (22E)-ergosta-7,22-dien-3β-ol (3.6 mg), and ergosta-
7,24(241)-dien-3β-ol (2.2 mg). F3‑5 (2.5 g) was fractioned via
Sephadex LH-20 (petroleum ether−CHCl3−MeOH, 5:5:1) followed
by RP semipreparative HPLC (96% MeOH in H2O) purification to
yield 7 (5.5 mg), 8 (3.7 mg), 9 (2.0 mg), 10 (1.6 mg), and cholest-4-
ene-3,24-dione (0.8 mg). F3‑6 (1.6 g) was repeatedly separated by CC
over silica gel (petroleum ether−EtOAc, 50:1−3:1), followed by RP
semipreparative HPLC (94% MeOH in H2O) purification to yield
(E)-4-oxo-β-dihydroionone (1.8 mg), (3S,5R,6S)-3-acetoxy-5,6-epoxy-
5,6-dihydro-β-ionone (1.5 mg), (22E)-ergosta-6,9,22-triene-3β,5α,8α-
triol (3.6 mg), and (22E)-ergosta-6,22-diene-3β,5α,8α-triol (5.0 mg).
F6 (21.0 g) was separated by flash chromatography over MCI gel, to
give F6‑1−F6‑11. Separation of F6‑7 (3.6 g) by chromatography over
Sephadex LH-20 (petroleum ether−CHCl3−MeOH, 2:2:1) and RP
semipreparative HPLC (60% MeOH in H2O) yielded 1 (31.9 mg), 2
(1.8 mg), 3 (2.0 mg), 4 (4.1 mg), and 5 (3.4 mg). β-Daucosterol (23.2
mg) was precipitated as a white, amorphous powder from F6‑8 in
MeOH−CHCl3 (10:1).
(+)-(3S,7S,8S,13R,14S,17R,18R,19R,21S)-25-Norfern-5(10),9(11)-
diene-3,7,19,28-tetraol (1): colorless needles, mp 234−236 °C
(acetone); [α]20 +23 (c 0.5, MeOH); UV (MeOH) λmax (log ε)
D
244 (1.46) nm; ECD (MeOH) 235 (Δε +3.66) nm; IR νmax 3472,
3406, 2927, 1466, 1433, 1380, 1286, 1241, 1186, 1125, 1094, 1062,
1007, 963, 932, 889, 811, 794 cm−1; 1H NMR (acetone-d6, 600 MHz)
data, see Table 1; 13C NMR (acetone-d6, 125 MHz) data, see Table 2;
1164
dx.doi.org/10.1021/np300259s | J. Nat. Prod. 2012, 75, 1160−1166

Journal of Natural Products
Article
(+)-ESIMS m/z 481 [M + Na]+; (+)-HRESIMS m/z 481.3292 [M +
Na]+ (calcd for C29H46O4Na, 481.3288).
X-ray Crystallography of Compound 1. C29H46O4, M = 458.68,
monoclinic, P21, a = 14.374(6) Å, b = 7.464(6) Å, c = 15.053(7) Å, β =
116.02(1)°, V = 1451.3(2) Å3, Z = 2, Dcalcd = 1.183 g·cm−3, 4310
reflections independent, 3717 reflections observed (|F|2 ≥ 2σ|F|2), R1 =
0.0489, wR2 = 0.1312, S = 1.060.
The data were collected on a Rigaku MicroMax 002+ diffractometer
with Cu Kα radiation using the ω and κ scan technique to a maximum
2θ value of 144.56°. The crystal structures were solved by direct
methods using SHELXS-97, and all non-hydrogen atoms were refined
anisotropically using the least-squares method. All hydrogen atoms
were positioned by geometrical calculations and a difference Fourier
overlapping calculation. The absolute configuration was determined on
the basis of the Flack parameter of 0.0(3). Crystallographic data for the
structure of 1 have been deposited with the Cambridge Crystallo-
graphic Data Centre as supplementary publication CCDC 842894.
Copies of these data can be obtained free of charge at www.ccdc.cam.
ac.uk/conts/retrieving.html (or from the Cambridge Crystallographic
Data Centre, 12 Union Road, Cambridge CB21EZ, UK; fax: (+44)
1223-336-033; or e-mail: deposit@ccdc.cam.ac.uk).
549 [M + Na]+; (+)-HRESIMS m/z 527.4484 [M + H]+ (calcd for
C35H59O3, 527.4459).
(+)-(20S)-20-O-Lauroylpregn-4-en-3-one (8): colorless gum;
[α]20 +38.2 (c 0.03, CHCl3); UV (MeOH) λmax (log ε) 204
D
(2.28), 240 (1.67) nm; ECD (MeOH) 233 (Δε +5.77), 323 (Δε
−1.14) nm; 1H NMR (CDCl3, 500 MHz) data, see Table 3; 13C NMR
(CDCl3, 125 MHz) data, see Table 3; (+)-ESIMS m/z 499 [M + H]+,
521 [M + Na]+; (+)-HRESIMS m/z 499.4164 [M + H]+ (calcd for
C33H55O3, 499.4146).
(+)-(20S)-20-O-Caprinoylpregn-4-en-3-one (9): colorless gum;
[α]20 +38.6 (c 0.06, CHCl3); UV (MeOH) λmax (log ε) 210
D
(2.57), 240 (1.64) nm; ECD (MeOH) 232 (Δε +5.24), 325 (Δε
−1.07) nm; 1H NMR (CDCl3, 500 MHz) data, see Table 3; 13C NMR
(CDCl3, 125 MHz) data, see Table 3; (+)-ESIMS m/z 471 [M + H]+,
493 [M + Na]+; (+)-HRESIMS m/z 471.3903 [M + H]+ (calcd for
C31H51O3, 471.3833).
(+)-(20S)-20-O-Capryloylpregn-4-en-3-one (10): colorless gum;
[α]20 +38.2 (c 0.06, CHCl3); UV (MeOH) λmax (log ε) 210 (2.61),
D
240 (1.66) nm; ECD (MeOH) 235 (Δε +5.37), 323 (Δε −1.09) nm;
1H NMR (CDCl3, 500 MHz) data, see Table 3; 13C NMR (CDCl3,
125 MHz) data, see Table 3; (+)-ESIMS m/z 443 [M + H]+, 465 [M
+ Na]+; (+)-HRESIMS m/z 443.3564 [M + H]+ (calcd for C29H47O3,
443.3520).
Hydrolysis of 6−10. Compound 6 (7.0 mg) was stirred with KOH
(10 mg) in methanol (3 mL) for 2 h. The reaction solution was
partitioned between H2O (25 mL) and CHCl3 (25 mL). The CHCl3
phase was evaporated under reduced pressure to give a residue that
(+)-(3S,7S,8S,13R,14S,17R,18R,19S,20S,21S)-25-Norfern-5(10),9-
(11)-diene-3,7,19,20,28-pentaol (2): white, amorphous powder;
[α]20 +59.4 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 244 (1.49)
D
nm; ECD (MeOH) 245 (Δε +1.94) nm; IR νmax 3397, 2924, 2855,
1
1655, 1595, 1464, 1378, 1279, 1211, 1178. 1129, 1060, 973 cm−1; H
NMR (acetone-d6, 600 MHz) data, see Table 1; 13C NMR (acetone-
d6, 125 MHz) data, see Table 2; (+)-ESIMS m/z 497 [M + Na]+;
(+)-HRESIMS m/z 497.3231 [M + Na]+ (calcd for C29H46O5Na,
497.3237).
was separated by PTLC using petroleum ether−EtOAc (5:1) to afford
1
betulin with [α]20 +18.5 (c 0.06, CHCl3); H NMR (CDCl3, 500
D
MHz) and (+)-ESIMS data were identical with reported data.8
Similarly, compounds 8−10 were hydrolyzed to afford (20S)-20-
hydroxypregn-4-en-3-one with [α]20D +99.5 (c 0.2, CHCl3); 1H NMR
(CDCl3, 500 MHz) data were identical with reported data.11
(+)-(3S,7S,8S,13R,14S,17R,18R,21S)-25-Norfern-5(10),9(11)-diene-
19-oxo-3,7,28-triol (3): white, amorphous powder; [α]20 +55.6 (c
D
0.08, MeOH); UV (MeOH) λmax (log ε) 245 (1.52) nm; ECD
(MeOH) 241 (Δε +3.38), 286 (Δε −2.19) nm; IR νmax 3441, 2934,
2890, 1728, 1631, 1469, 1443, 1384, 1375, 1267, 1228, 1188, 1102,
1063, 1032, 999, 967 cm−1; 1H NMR (acetone-d6, 600 MHz) data, see
Table 1; 13C NMR (acetone-d6, 125 MHz) data, see Table 2;
(+)-ESIMS m/z 457 [M + H]+; (+)-HRESIMS m/z 457.3302 [M +
H]+ (calcd for C29H45O4, 457.3312), 479.3118 [M + Na]+ (calcd for
C29H44O4Na, 479.3132).
ASSOCIATED CONTENT
* Supporting Information
■
S
Copies of IR, MS, and 1D and/or 2D NMR for compounds 1−
10 and ECD spectra for 1−5 and 7−10. This can be accessed
free of charge via the Internet at http://pubs.acs.org.
(+)-(3S,4R,7S,8S,13R,14S,17R,18R,19R,21S)-25-Norfern-5(10),9-
AUTHOR INFORMATION
Corresponding Author
*Tel: 86-10-83154789. Fax: 86-10-63017757. E-mail: shijg@
imm.ac.cn.
■
(11)-diene-3,7,19,23,28-pentaol (4): white, amorphous powder;
[α]20 +32.5 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 202 (1.93),
D
241 (2.05) nm; ECD (MeOH) 235 (Δε +6.50) nm; IR νmax 3389,
2935, 1643, 1458, 1443, 1376, 1283, 1186, 1085, 1040, 968, 947, 887
1
cm−1; H NMR (acetone-d6, 600 MHz) data, see Table 1; 13C NMR
Notes
(acetone-d6, 125 MHz) data, see Table 2; (+)-HRESIMS m/z 497 [M
+ Na]+; (+)-HRESIMS m/z 497.3253 [M + Na]+ (calcd for
C29H46O5Na, 497.3237).
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
(+)-(3S,5S,7S,8S,13R,14S,17R,18R,19R,21S)-25-Norfern-1(10),9-
■
(11)-diene-3,7,19,28-tetraol (5): white, amorphous powder; [α]20
D
Financial support from the National Natural Sciences
Foundation of China (NNSFC; grant nos. 30825044 and
20932007), the Program for Changjiang Scholars and
Innovative Research Team in University (PCSIRT, grant no.
IRT1007), and the National Science and Technology Project of
China (no. 2011ZX09307-002-01) is acknowledged.
+28 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 236 (1.48) nm; ECD
(MeOH) 236 (Δε +2.69) nm; IR νmax 3192, 2921, 2850, 1647, 1470,
1
1421, 1380, 1127, 1037 cm−1; H NMR (acetone-d6, 600 MHz) data,
see Table 1; 13C NMR (acetone-d6, 125 MHz) data, see Table 2;
(+)-ESIMS m/z 481 [M + Na]+; (+)-HRESIMS m/z 481.3300 [M +
Na]+ (calcd for C29H46O4Na, 481.3288).
3-O-Myristoylbetulin (6): white, amorphous powder; [α]20 +31.5
D
REFERENCES
(c 0.08, CHCl3); IR νmax 2926, 2854, 1731, 1466, 1375, 1248, 1178,
■
1
1035, 980, 881, 722 cm−1; H NMR (CDCl3, 500 MHz) data, see
(1) Jiangsu New Medical College. Dictionary of Traditional Chinese
Medicine; Shanghai Science and Technology Publishing House:
Shanghai, 1986; p 2514.
(2) Jia, H. H.; Wu, X. D. Zhong Yao Cai 1992, 15, 35−36.
(3) Xiong, L.; Zhu, C. G.; Li, Y. R.; Tian, Y.; Lin, S.; Yuan, S. P.; Hu,
J. F.; Hou, Q.; Chen, N. H.; Yang, Y. C.; Shi, J. G. J. Nat. Prod. 2011,
74, 1188−1200.
(4) Gawronski, J. K.; Walborsky, H. M. In Circular Dichroism
Principles and Applications; Nakanishi, K.; Berova, N.; Woody, R. W.,
Ed.; Wiley-VCH, Inc.: New York, 1994; Chapter 11, pp 301−330.
Table 1; 13C NMR (CDCl3, 125 MHz) data, see Table 2; EIMS m/z
652 [M]+•; HREIMS m/z 652.5783 [M]+• (calcd for C44H76O3
652.5794).
(+)-(20S)-20-O-Myristoylpregn-4-en-3-one (7): colorless gum;
[α]20 +38 (c 0.03, CHCl3); UV (MeOH) λmax (log ε) 211 (2.42),
D
240 (1.65) nm; ECD (MeOH) 235 (Δε +5.78), 322 (Δε −1.21) nm;
IR νmax 2927, 2854, 1730, 1669, 1459, 1379, 1175, 1076, 948, 866
1
cm−1; H NMR (CDCl3, 500 MHz) data, see Table 3; 13C NMR
(CDCl3, 125 MHz) data, see Table 3; (+)-ESIMS m/z 527 [M + H]+,
1165
dx.doi.org/10.1021/np300259s | J. Nat. Prod. 2012, 75, 1160−1166

Journal of Natural Products
Article
(5) Lightner, D. A. In Circular Dichroism Principles and Applications;
Nakanishi, K.; Berova, N.; Woody, R. W., Ed.; Wiley-VCH, Inc.: New
York, 1994; Chapter 10, pp 259−299.
(6) Burgstahler, A. W.; Barkhurst, R. C. J. Am. Chem. Soc. 1970, 92,
7601−7603.
(7) Toriumi, Y.; Kakuda, R.; Kikuchi, M.; Yaoita, Y.; Kikuchi, M.
Chem. Pharm. Bull. 2003, 51, 89−91.
(8) Alakurtti, S.; Heiska, T.; Kiriazis, A.; Sierra, N. S.; Jaffe, C. L.;
Kauhaluoma, J. Y. Bioorg. Med. Chem. 2010, 18, 1573−1582.
(9) Monsalve, L. N.; Rada, M. Y. M.; Ghini, A. A.; Baldessari, A.
Tetrahedron 2008, 64, 1721−1730.
(10) Snatzke, G.; Laurent, H.; Wiechert, R. Tetrahedron 1969, 25,
761−769.
(11) (a) Kirk, D. N.; Toms, H. C.; Douglas, C.; White, K. A. J. Chem.
Soc., Perkin Trans. 1990, 2, 1567−1594. (b) Farooq, A.; Hanson, J. R.;
Iqbal, Z. Phytochemistry 1994, 37, 723−726.
(12) Akihisa, T.; Yamamoto, K. Chem. Pharm. Bull. 1992, 40, 789−
791.
(13) Nozaki, H.; Suzuki, H.; Hirayama, T.; Kasai, R.; Wu, R. Y.; Lee,
K. H. Phytochemistry 1986, 25, 479−485.
(14) Patra, A.; Mukhopadhyay, A. K.; Mitra, A. K. Org. Magn. Reson.
1981, 17, 166−168.
(15) Li, T. L.; Yin, X. H.; Pang, W. D.; Yang, J.; Liang, G. Y. Zhong
Yao Cai 2010, 33, 55−57.
(16) Cole, B. W.; Bentley, M. D.; Hua, Y.; Bu, L. J. Wood Chem.
Technol. 1991, 11, 209−223.
(17) Era, V.; Jaaskelainen, P. J. Am. Oil Chem. Soc. 1981, 58, 20−23.
(18) Brown, G. D. Phytochemistry 1994, 36, 1553−1554.
(19) Ling, T. J.; Ling, W. W.; Chen, Y. J.; Wan, X. C.; Xia, T.; Du, X.
F.; Zhang, Z. Z. Molecules 2010, 15, 8469−8477.
(20) Liebler, D. C.; Baker, F. P.; Kaysen, K. L. J. Am. Chem. Soc.
1990, 112, 6995−7000.
(21) Becher, E.; Albrecht, R.; Bernhard, K.; Leuenberger, H. G. W.;
Mayer, H.; Muller, R. K.; Schuep, W.; Wagner, H. P. Helv. Chim. Acta
̈
̈
1981, 64, 2419−2435.
(22) Rosenberger, M.; McDougal, P.; Bahr, J. J. Org. Chem. 1982, 47,
2130−2134.
(23) Buchecker, R.; Marti, U.; Eugster, C. H. Helv. Chim. Acta 1984,
67, 2043−2056.
(24) Ponce, M. A.; Ramirez, J. A.; Galagovsky, L. R.; Gros, E. G.;
Erra-Balsells, R. Photochem. Photobiol. Sci. 2002, 1, 749−756.
(25) Rivera, A.; Benavides, O. L.; Rios-Motta, J. Nat. Prod. Res. 2009,
23, 293−300.
(26) Keller, A. C.; Maillard, M. P.; Hostettmann, K. Phytochemistry
1996, 41, 1041−1046.
(27) Shirane, N.; Takenaka, H.; Ueda, K.; Hashimoto, Y.; Katoh, K.;
Ishii, H. Phytochemistry 1996, 41, 1301−1308.
(28) Lim, J. C.; Park, J. H.; Budesinsky, M.; Kasal, A; Han, Y. H.;
Koo, B. S.; Lee, S. I.; Lee, D. U. Chem. Pharm. Bull. 2005, 53, 561−
564.
(29) Gaspar, E. M.; Neves, H. J. Phytochemistry 1993, 34, 523−527.
(30) Ochi, K.; Matsunaga, I.; Shindo, M.; Kaneko, C. Chem. Pharm.
Bull. 1978, 26, 2386−2390.
(31) Zhang, X.; Geoffroy, P.; Miesh, M.; Julien-David, D.; Raul, F.;
Aoude-Werner, D.; Marchioni, E. Steroids 2005, 70, 886−895.
(32) Bayoumi, S. A. L.; Rowan, M. G.; Beeching, J. R.; Blagbrough, I.
S. Phytochemistry 2010, 71, 598−604.
(33) Xu, W.; Zhu, C.; Cheng, W.; Fan, X.; Chen, X.; Yang, S.; Guo,
Y.; Ye, F.; Shi, J. J. Nat. Prod. 2009, 72, 1620−1626.
(34) Fan, X. N.; Zi, J. C.; Zhu, C. G.; Xu, W. D.; Cheng, W.; Yang, S.;
Guo, Y.; Shi, J. G. J. Nat. Prod. 2009, 72, 1184−1190.
(35) Wang, Y.; Shang, X. Y.; Wang, S. J.; Mo, S. Y.; Li, S.; Yang, Y. C.;
Ye, F.; Shi, J. G.; He, L. J. Nat. Prod. 2007, 70, 296−299.
(36) Cheng, W.; Zhu, C. G.; Xu, W. D.; Fan, X. N.; Yang, Y. C.; Li,
Y.; Chen, X. G.; Wang, W. J.; Shi, J. G. J. Nat. Prod. 2009, 72, 2145−
2152.
(37) Mo, S. Y.; Wang, S. J.; Zhou, G. X.; Yang, Y. C.; Li, Y.; Chen, X.
G.; Shi, J. G. J. Nat. Prod. 2004, 67, 823−828.
1166
dx.doi.org/10.1021/np300259s | J. Nat. Prod. 2012, 75, 1160−1166