Wilsonols A - L, megastigmane sesquiterpenoids from the leaves of Cinnamomum wilsonii

Article abstract of DOI:10.1021/np4002493

Twelve new megastigmane sesquiterpenoids, wilsonols A-L (1-12), were isolated from the leaves of Cinnamomum wilsonii, along with seven known analogues (13-19). The structures of compounds 1-12 were established by spectroscopic analyses. The absolute configurations of 1-5 were determined by single-crystal X-ray diffraction analysis with Cu Kα irradiation, and the absolute configurations of 6-12 were determined by the modified Mosher's method. Compounds 1-9 and 13-19 were evaluated for in vitro cytotoxicity against five human cancer cell lines, HL-60, SMMC-7721, A-549, MCF-7, and SW-480, and compared against the Beas-2B immortalized (noncancerous) human bronchial epithelial cell line. Compound 13 exhibited IC₅₀ values ranging from 2.5 to 12 μM and selectivity indices of >10 against SMMC-7721, A-549, and MCF-7 cell lines. Selected compounds were evaluated for in vitro immunomodulatory activity.

Full text of DOI:10.1021/np4002493

Article
pubs.acs.org/jnp
Wilsonols A−L, Megastigmane Sesquiterpenoids from the Leaves of
Cinnamomum wilsonii
Penghua Shu,† Xialan Wei,† Yongbo Xue,† Weijie Li,† Jinwen Zhang,‡ Ming Xiang,† Mengke Zhang,†
Zengwei Luo,† Yan Li,§ Guangmin Yao,*,† and Yonghui Zhang*,†
†Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, School of Pharmacy, Tongji Medical College,
Huazhong University of Science and Technology, Wuhan 430030, People’s Republic of China
‡Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People’s
Republic of China
§State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of
Sciences, Kunming 650204, People’s Republic of China
S
* Supporting Information
ABSTRACT: Twelve new megastigmane sesquiterpenoids, wilsonols A−L (1−12), were isolated from the leaves of
Cinnamomum wilsonii, along with seven known analogues (13−19). The structures of compounds 1−12 were established by
spectroscopic analyses. The absolute configurations of 1−5 were determined by single-crystal X-ray diffraction analysis with Cu
Kα irradiation, and the absolute configurations of 6−12 were determined by the modified Mosher’s method. Compounds 1−9
and 13−19 were evaluated for in vitro cytotoxicity against five human cancer cell lines, HL-60, SMMC-7721, A-549, MCF-7, and
SW-480, and compared against the Beas-2B immortalized (noncancerous) human bronchial epithelial cell line. Compound 13
exhibited IC50 values ranging from 2.5 to 12 μM and selectivity indices of >10 against SMMC-7721, A-549, and MCF-7 cell lines.
Selected compounds were evaluated for in vitro immunomodulatory activity.
analogues (13−19), including two (14 and 18) not previously
reported as natural products, were isolated from the leaves of C.
wilsonii. Herein, we report the isolation and structure
determination of compounds 1−19, as well as the cytotoxicity
and immunomodulatory activities of selected compounds.
lants of the family Lauraceae comprise about 45 genera
Pand 2500 species that are distributed in tropical and
subtropical regions, mostly in southeastern Asia and America.
These species are economically important as sources of
medicine, timber, edible fruits, spices, and perfumes.1
Cinnamomum is one of the largest genera of Lauraceae, with
about 250 species. Bioactive diterpenoids,2 sesquiterpenoids,3
monoterpenoids,4 butanolides,3,5 dibenzocycloheptanoids,6 fla-
vonoids,7 phenolic compounds,8 and alkaloids9 have been
reported from the Cinnamomum species. In the course of
investigating the chemical constituents of traditional Chinese
medicinal and edible plants, our attention was drawn to
Cinnamomum wilsonii Gamble (Lauraceae), an evergreen tree of
medium height that is endemic to China and distributed in
Guangdong, Guangxi, Hubei, Hunan, Jiangxi, Shaanxi, and
Sichuan Provinces. Its leaves, branches, and bark contain high
levels of volatile oil and are an excellent source of soap and food
flavoring. Its edible leaves, known as ‘‘Yu-Xiang-Ye’’, have been
used as a spice to cook fish, mutton, beef, poultry, duck, and
chicken for flavoring and to remove fishy odors. The dried bark
has been used medicinally to treat traumatic injuries and
abdominal pain.1 However, there are no previous reports of
phytochemical studies on C. wilsonii. In the current study, 12
new megastigmane sesquiterpenoids (1−12) and seven known
RESULTS AND DISCUSSION
■
A 95% EtOH extract of the leaves of C. wilsonii was suspended
in H2O and partitioned successively with petroleum ether,
EtOAc, and n-BuOH. Repeated column chromatography of the
EtOAc and n-BuOH portions on silica gel, RP (reversed-phase)
C18, and Sephadex LH-20 columns, followed by semi-
preparative RP C18 HPLC, yielded 12 new (1−12) and seven
known (13−19) megastigmane sesquiterpenoids.
Wilsonol A (1) was obtained as colorless needle crystals (mp
101−102 °C, [α]25 −38). Its molecular formula was
D
established as C13H24O4 based on its quasi-molecular ion
peak at m/z 267.1553 [M + Na]+ (calcd for C13H24O4Na,
267.1572) in the HRESIMS spectrum, implying two degrees of
1
unsaturation. The H NMR spectrum (Table 1) of 1 exhibited
signals for four methyl groups [δH 0.83 (s, H3-12), 1.03 (d, H3-
Received: April 3, 2013
Published: July 3, 2013
© 2013 American Chemical Society and
American Society of Pharmacognosy
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2.8, 2.5 Hz, H-4β), 3.91 (ddd, J = 3.2, 2.8, 2.5 Hz, H-3α), and
4.29 (dq, J = 6.4, 5.9 Hz, H-9)], and two olefinic protons [δH
5.55 (d, J = 15.7 Hz, H-7) and 5.68 (dd, J = 15.7, 5.9 Hz, H-8)].
The 13C NMR and DEPT spectra (Table 3) showed 13 carbon
signals ascribable to four methyls (δC 13.4, C-13; 24.3, C-10;
26.7, C-12; 27.6, C-11), a methylene (δC 38.4, C-2), four
methines (δC 32.7, C-5; 69.4, C-9; 72.4, C-3; 77.1, C-4)
including three oxygenated ones (C-3, C-4, and C-9), two sp2
methines (δC 132.7, C-7; 135.2, C-8), and two quaternary
carbons (δC 39.3, C-1; 81.6, C-6). The double bond accounted
for one degree of unsaturation, implying the presence of a ring
in 1 to explain the second degree of unsaturation.
1
Interpretation of the H−1H COSY and HSQC spectra of 1
revealed the presence of two partial structures, “C-2−C-3−C-
4−C-5−C-13” and “C-7C-8−C-9−C-10” (Figure 1). These
two partial structures were connected through an oxygenated
quaternary carbon (C-6) on the basis of HMBC correlations of
H-4, H-5, H3-13, H-7, and H-8 to C-6. HMBC correlations
from H3-11 and H3-12 to C-1, C-2, and C-6 indicated that C-
11, C-12, C-2, and C-6 were all connected with C-1, and C-2
was connected to C-6 through C-1. HMBC correlations of H2-2
to C-1 and C-6 confirmed this connection. The relative
configuration of 1 was deduced from analysis of coupling
constants and the NOESY spectrum (Figure 1), which were
both consistent with a chair conformation for the cyclohexane
ring. The strong NOESY correlation between H-5 and H3-11
indicated that H-5 and H3-11 were axial bonds and assigned as
β-oriented. The 3-hydroxy-1-butenyl side chain at C-6 was
determined to be β-oriented based on the strong NOESY
correlations of H-7 to H-5β and H3-11β. The NOESY
correlation of H-4 and H-5β and their small coupling constant
J = 2.5 Hz suggested that H-4 was β-oriented. The α-
orientation of H-3 was deduced from the NOESY correlations
of H-3 to H-2α and H-2β, as well as the smaller coupling
constants (J = 3.2, 2.8, 2.5 Hz) between H-3 and H-2α, H-2β,
13), 1.13 (s, H3-11), and 1.24 (d, H3-10)], a pair of methylene
protons [δH 1.36 (br d, J = 15.0 Hz, H-2β) and 2.00 (dd, J =
15.0, 3.2 Hz, H-2α)], a methine proton [δH 2.23 (dq, J = 7.2,
2.5 Hz, H-5β)], three oxygenated methines [δH 3.64 (dd, J =
1
Table 1. H NMR [δ, mult, (J in Hz)] Data for Compounds 1−6 (400 MHz)
a
a
b
a
a
a
a
position
1
2
3
4
5a
5b
6
2a
2.00 dd (15.0,
3.2)
2.00 dd (15.0,
3.2)
1.85 dd (14.5,
2.8)
1.12 dd (12.0, 12.0)
1.39 dd (14.8,
3.2)
1.39 dd (14.8,
3.2)
1.66 overlap
2β
3a
3β
4α
1.36 br d (15.0)
1.36 br d (15.0)
1.23 dd (14.5,
2.8)
1.70 ddd (12.0, 3.8, 2.1) 1.67 dd (14.8,
3.2)
1.67 dd (14.8,
3.2)
1.31 ddd (12.0, 4.5, 2.3)
3.91 ddd (3.2, 2.8, 3.91 ddd (3.2, 2.8, 3.77 ddd (5.2, 2.8,
2.5) 2.5) 2.8)
3.79 ddd (3.2, 3.2, 3.83 ddd (3.2, 3.2,
3.2) 3.2)
3.74 dddd (12.0, 12.0,
4.5, 3.8)
3.74 dddd (11.6, 11.6,
4.5, 4.5)
1.02 ddd (12.0, 12.0,
12.0)
1.40 ddd (12.3, 12.2,
11.6)
4β
5β
6α
7a
7b
8a
3.64 dd (2.8, 2.5) 3.64 dd (2.8, 2.5) 3.54 dd (5.2, 2.2) 2.18 ddd (12.0, 4.5, 2.1) 3.50 dd (3.2, 2.4) 3.57 dd (3.2, 2.4) 1.59 overlap
2.23 dq (7.2, 2.5) 2.23 dq (7.2, 2.5) 2.27 dq (7.1, 2.2) 1.59 m
1.53 dd (11.0, 9.1)
1.90 m
1.17 m
1.57m
1.90 m
1.17 m
1.57m
1.88 m
5.55 d (15.7)
5.55 d (15.7)
6.74 d (15.8)
5.33 dd (15.4, 9.1)
1.67 overlap
1.54 overlap
1.65 overlap
1.07 m
1.55 m
1.07 m
1.55 m
5.68 dd (15.7,
5.9)
5.68 dd (15.7,
5.9)
6.12 d (15.8)
5.49 dd (15.4, 6.0)
8b
9
1.43 m
1.43 m
1.53 overlap
4.29 dq (6.4, 5.9) 4.29 dq (6.4, 5.9) 4.22 dq (6.5, 6.0) 3.68 m
3.70 m
3.63 m
10
11
12
13a
13b
1.24 d (6.4)
1.13 s
1.24 d (6.4)
1.14 s
2.21 s
1.22 d (6.5)
0.88 s
1.15 d (6.2)
0.98 s
1.18 d (6.2)
0.99 s
1.16 d (6.2)
0.99 s
1.11 s
0.83 s
0.86 s
0.73 s
0.91 s
0.90 s
0.91 s
0.98 s
1.03 d (7.2)
1.00 d (7.2)
0.86 d (7.1)
3.57 dd (10.8, 2.9)
3.23 dd (10.8, 6.9)
1.04 d (7.1)
1.05 d (7.1)
0.95 d (6.6)
a
b
Recorded in CD3OD. Recorded in DMSO-d6.
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1
Table 2. H NMR [δ, mult (J in Hz)] Data for Compounds 7−12 in CD3OD (400 MHz)
position
7
8
9
10
11
12
1.41 dd (12.0, 12.0)
2a
2β
3a
3.65 dd (12.5, 4.0)
1.44 dd (12.5, 4.6) 1.41 dd (12.0, 12.0)
1.41 dd (12.0, 12.0)
1.41 dd (12.0, 12.0)
1.77 dd (12.5, 12.5) 1.70 ddd (12.0, 3.6, 2.0) 1.70 ddd (12.0, 3.6, 2.0) 1.75 ddd (12.0, 3.6, 2.1) 1.75 ddd (12.0, 3.6, 2.1)
2.15 ddd (12.5, 12.5, 3.82 ddd (12.5, 9.5, 3.87 dddd (12.0, 10.0,
3.87 dddd (12.0, 10.0,
5.5, 3.6)
3.91 dddd (12.0, 9.9,
5.4, 3.6)
3.91 dddd (12.0, 9.9,
5.4, 3.6)
2.8)
4.6)
5.5, 3.6)
3β
1.69 ddd (12.5, 4.0,
2.9)
4α
4β
6β
7a
1.95 dd (16.2, 10.0)
1.95 dd (16.2, 10.0)
2.04 ddd (16.6, 9.9, 2.1) 2.04 ddd (16.7, 9.9, 2.1)
3.58 dd (2.9, 2.8)
1.86 d (10.4)
3.42. d (9.5)
2.49 ddd (16.2, 5.5, 2.0) 2.49 ddd (16.2,5.5, 2.0)
2.57 dd (16.6, 5.4)
2.57 dd (16.7, 5.4)
5.78 ddd (15.4, 10.4, 6.08 dd (15.9, 1.2) 2.17 ddd (13.2, 13.2, 5.0) 2.30 ddd (13.2, 13.2, 5.0) 6.05 d (15.8)
0.9)
6.04 d (15.8)
7b
8a
2.10 ddd (13.2, 13.2, 5.0) 1.97 ddd (13.2, 13.2, 5.0)
5.47 dd (15.4, 6.6)
5.76 dd (15.9, 6.2) 1.53 dddd (13.2, 13.2,
6.0, 5.0)
1.50 dddd (13.2, 13.2,
6.0, 5.0)
5.53 dd (15.8, 6.0)
5.53 dd (15.8, 6.1)
4.30 dq (6.4, 6.1)
8b
9
1.44 dddd (13.2, 13.2,
6.0, 5.0)
1.49 dddd (13.2, 13.2,
6.0, 5.0)
4.28 dq (6.6, 6.4)
4.33 ddq (6.4, 6.2,
1.2)
3.71 m
3.71 m
4.30 dq (6.4, 6.0)
10
1.26 d (6.4)
0.97 s
1.27 d (6.4)
0.83 s
1.17 d (6.3)
1.09 s
1.17 d (6.3)
1.09 s
1.27 d (6.4)
1.06 s
1.27 d (6.4)
1.07 s
11
12
0.91 s
1.19 s
1.08 s
1.08 s
1.06 s
1.05 s
13a
13b
1.05 s
1.20 s
4.11 d (11.8)
3.99 d (11.8)
4.11 d (11.8)
3.99 d (11.8)
4.12 d (11.6)
4.09 d (11.6)
4.11 d (11.6)
4.08 d (11.6)
Table 3. 13C NMR Data (δ) for Compounds 1−12 (100 MHz)
a
a
b
a
a
a
a
a
a
a
a
a
a
position
1
2
3
4
5a
5b
6
7
8
9
10
11
12
1
39.3
38.4
72.4
77.1
32.7
81.6
132.7
135.2
69.4
24.3
27.6
26.7
13.4
39.3
38.4
72.4
77.1
32.7
81.6
132.7
135.2
69.4
24.3
27.6
26.7
13.4
38.3
37.0
69.6
74.8
30.4
79.5
151.2
129.9
197.9
27.2
26.9
26.4
13.1
35.9
51.3
67.5
39.9
40.0
52.9
130.4
138.9
69.3
24.1
21.8
31.8
66.5
35.3
43.0
72.6
76.6
34.7
46.5
26.5
42.6
69.4
23.5
23.7
32.1
17.3
35.2
43.0
72.6
75.4
34.8
46.3
26.4
42.3
69.1
23.5
23.7
32.1
17.3
41.7
47.5
67.6
40.9
36.2
76.5
33.2
36.0
70.0
23.8
26.4
25.2
16.8
40.1
73.7
34.9
76.5
74.4
54.0
127.8
139.9
69.8
24.1
15.6
28.7
27.8
40.3
44.3
70.1
79.0
79.5
80.8
131.5
135.9
69.7
24.4
27.8
27.3
26.3
39.2
49.5
65.9
38.3
129.6
141.9
25.1
42.4
69.2
23.4
29.1
30.1
63.0
39.2
49.5
65.9
38.3
129.6
141.9
25.1
42.4
69.1
23.4
29.1
30.1
63.0
38.0
49.0
38.0
49.0
2
3
65.8
65.8
4
38.4
38.4
5
130.7
141.9
126.2
140.6
69.4
130.7
141.9
126.1
140.7
69.5
6
7
8
9
10
11
12
13
24.0
24.0
28.9
28.9
30.4
30.4
64.2
64.2
a
b
Recorded in CD3OD. Recorded in DMSO-d6.
1
Figure 1. Key H−1H COSY, HMBC, and NOESY of 1.
and H-4. The larger coupling constant (J = 15.7 Hz) between
H-7 and H-8 indicates that the geometry of the Δ7(8) double
bond is E. In order to determine its absolute configuration, a
suitable crystal of 1 was obtained and subjected to single-crystal
X-ray diffraction analysis using the anomalous scattering of Cu
Kα radiation. The final refinement of the given coordinate
(Figure 2) resulted in a Flack parameter of −0.04(11), which
allowed unambiguous assignment of the absolute configuration
of 1 as 3S,4S,5R,6R,9S.
Figure 2. X-ray ORTEP drawing of compound 1.
compound 1, according to the HRESIMS data (m/z
1
Wilsonol B (2) was obtained as colorless block crystals and
had the same molecular formula, C13H24O4, as that of
267.1560 [M + Na]+). The H and 13C NMR spectra (Tables
1 and 3) of 2 were nearly identical to those of 1, while the main
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Figure 3. ΔδH(S−R) values (in ppm) for the MTPA esters of 2 and 6−12.
differences were a slight upfield shift of CH3-13 (0.03 ppm) and
a downfield shift of CH3-11 and CH3-12 (0.01 and 0.03 ppm,
respectively) in 2 compared with those in 1. Extensive 2D
The 1H and 13C NMR spectra of 3 (Tables 1 and 3) were very
similar to those of 1, except that a ketone carbonyl (δC 197.9,
C-9) in 3 replaced the oxygenated methine (δH 4.29, H-9; δC
69.4, C-9) of 1. HMBC correlations between CH3-10 and both
C-8 and C-9 and from H-8 to both C-9 and C-10 in the HMBC
spectrum of 3 established connection of the ketone carbonyl C-
9 to C-8 and C-10. Detailed 1D and 2D NMR analysis
established the planar structure and the relative configuration of
3. The absolute configuration of 3 was determined to be
3S,4S,5R,6R by single-crystal X-ray diffraction analysis with Cu
Kα irradiation (Figure 5).
1
NMR analyses, including H−1H COSY, HSQC, HMBC, and
NOESY, showed that 2 had the same gross structure as that of
1, and both compounds had similar optical rotations.
Compound 2 should be the C-9 epimer of 1. In order to
determine the absolute configuration of C-9 in 2, (S)- and (R)-
MTPA esters (2a and 2b) were prepared.11 Significant Δδ
values (Δδ = δS‑MTPA‑ester − δR‑MTPA‑ester) were observed for
proton signals adjacent to C-9, as shown in Figure 3. According
to the rule of the modified Mosher’s method,10 the absolute
configuration of C-9 in 2 was determined to be R, contrasting
with the S configuration of C-9 in 1. The absolute configuration
of 2 was confirmed as 3S,4S,5R,6R,9R by single-crystal X-ray
diffraction analysis with Cu Kα irradiation (Figure 4).
The molecular formula of wilsonol C (3) was C13H22O4 by
HRESIMS, indicating three degrees of unsaturation. The UV
absorption maximum at 231 nm suggested the presence of an
α,β-unsaturated ketone, and the IR spectrum displayed
absorption bands for hydroxyl groups (3467 cm−1), a double
bond (1641 cm−1), and a conjugated carbonyl (1670 cm−1).
Figure 5. X-ray ORTEP drawing of compound 3.
Wilsonol D (4) had the molecular formula C13H24O3 by
HRESIMS, indicating two degrees of unsaturation. Inspection
of 1D and 2D NMR data (Tables 1 and 3) indicated that 4 was
a megastigmane derivative. Interpretation of the 1H−1H COSY
and HSQC data of 4 constructed the partial structure of “C-2−
C-3−C-4−C-5(C-13)−C-6−C-7−C-8−C-9−C-10”. HMBC
correlations from H3-11 and H3-12 to C-1, C-2, and C-6
indicated that C-11, C-12, C-2, and C-6 were all connected to
C-1, which completed a megastigmane skeleton. Therefore, 4
was designated as 3,9,13-trihydroxymegastigman-7-ene. The
Figure 4. X-ray ORTEP drawing of compound 2.
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larger coupling constants J = 12.0 Hz between H-3 and both H-
2 and H-4 and the strong NOESY correlations between H3-11/
H-3, H3-11/H-5, and H-3/H-5 suggested that H-3, H-5, and
CH3-11 occupied the axial bonds of the chair conformation of
the cyclohexane unit, which were assigned β-orientations. The
α-orientation of H-6 was deduced from the larger coupling
constants J = 11.0 Hz of H-6 with H-5 and the NOESY
correlations between H-7 and H-5, H3-11, H3-12, and H3-13
and between H-6 and H-2α and H-4α. The larger coupling
constant (J = 15.4 Hz) between H-7 and H-8 indicated the E-
geometry of the double bond. Finally, single-crystal X-ray
diffraction analysis with Cu Kα irradiation (Figure 6)
determined the absolute configuration of 4 as 3S,5R,6S,9R.
indicated that the absolute configurations of 5a and 5b were
3S,4S,5S,6S,9S and 3S,4S,5S,6S,9R, respectively.
The HRESIMS spectrum of wilsonol F (6) revealed the
molecular formula C13H26O3, requiring one degree of
unsaturation. 1D NMR (Tables 1 and 3) and 2D NMR data
of 6 established the planar structure to be 3,6,9-trihydrox-
ymegastigmane. The cross-peaks of H-3/H3-11 and H-3/H-5 in
the NOESY spectrum of 6 indicated the syn-relationship of
these groups, which were all assigned β-orientations. The
NOESY correlation of H-7 to H3-11 suggested the side chain
was β-oriented. The absolute configurations of C-3 and C-9 in
6 were elucidated to be 3S and 9R by applying the modified
Mosher’s method (Figure 3). Therefore, the absolute
configurations of C-5 and C-6 in 6 were determined as R.
Wilsonol G (7) was isolated as an amorphous powder, and
its molecular formula was determined to be C13H24O4. The
planar structure of 7 was assigned as 2,4,5,9-tetrahydroxyme-
gastigman-7-ene on the basis of the 1D (Tables 2 and 3) and
2D NMR analysis. The relative configuration of 7, except for C-
9, was characterized by the coupling constants and the NOESY
experiment. The larger coupling constant J = 12.5 Hz of H-2 (δ
3.65, dd) with one of the H-3a protons (δ 2.15, ddd) indicated
H-2 was axial and α-oriented. The smaller constants of H-4 (δ
3.58, dd, J = 2.9, 2.8 Hz) with H2-3 protons suggested H-4
should be equatorial and therefore β-oriented. The NOESY
correlations of H-6 and H-2α suggested that H-6 was also axial,
which established its α-orientation. The α-orientation of CH3-
13 was deduced from the cross-peak of H3-13 and H-6α in the
NOESY spectrum. Finally, the absolute configurations of C-2
and C-9 in 7 were assigned 2R and 9R by the modified
Mosher’s method (Figure 3). In consideration of the relative
configuration, the absolute configurations of C-4, C-5, and C-6
in 7 were assigned 4R, 5R, and 6R, respectively. The larger
coupling constant (J = 15.4 Hz) between H-7 and H-8
indicated the E geometry of the double bond.
Figure 6. X-ray ORTEP drawing of compound 4.
Wilsonol E (5) had the molecular formula C13H26O3. The
NMR spectra (Tables 1 and 3) indicated that compound 5 was
isolated as a mixture (ca. 3:2) of two epimers (5a and 5b) at C-
9. The planar structure of 5 was deduced to be 3,4,9-
trihydroxymegastigmane from analysis of 2D NMR data.
Similar to those of 1, the relative configuration of 5 was
deduced to be H-3α, H-4β, H-5β, and H-6α from the NOESY
correlations H-5/H3-11, H-7/H3-11, H-5/H-7, H-4/H-5, and
H-3/H2-2, as well as the smaller coupling constants J = 3.2 Hz
for H-3 with both H2-2 and H-4. Single-crystal X-ray diffraction
analysis of 5 using Cu Kα irradiation confirmed the relative
configuration. In the crystal structure (Figure 7), the ratio of
the two epimers 5a and 5b was determined as 60:40. The Flack
coefficient obtained [0.2(3)] for the given coordinates
Wilsonol H (8) had the molecular formula C13H24O5, as
1
deduced from HRESIMS data. The H and 13C NMR spectra
(Tables 2 and 3) of 8 closely resembled those of 2. The major
difference was the appearance of an oxygen-bearing quaternary
carbon (δC 79.5, C-5) in 8 that replaced the methine (δH 2.23,
dq, J = 7.2, 2.5 Hz, H-5; δC 32.7, C-5) in 2. This was supported
by HMBC correlations from H-3, H-4, H-7, and H3-13 to the
oxygenated quaternary carbon C-5. These data indicated that 8
was a 5-OH derivative of 2. The planar structure of 8 was
confirmed by comprehensive analysis of 1H−1H COSY, HSQC,
and HMBC spectra (Figure 8). The geometry of the side chain
double bond was assigned as E, based on the large coupling
constant J = 15.9 Hz between H-7 and H-8. The relative
configuration of 8 was determined from the analysis of NOESY
correlations (Figure 8) and coupling constants. The coupling
constant between H-4 and H-3 (J = 9.5 Hz) suggested a trans-
1
Figure 7. X-ray ORTEP drawing of compound 5.
Figure 8. Key H−1H COSY, HMBC, and NOESY correlations of 8.
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relationship of H-4 and H-3 and axial orientation. The strong
NOESY correlation of H-4β and H3-13 established the β-
orientation of H3-13. The α-orientation of the side chain was
deduced from the strong NOESY correlations between H-7 and
H3-11, H3-12, and H3-13 and lack of NOESY correlations
between H-7 and H-2β and H-4β. The absolute configurations
of C-3 and C-9 in 8 were determined to be S and R,
respectively, by the modified Mosher’s method (Figure 3).
With the relative configuration assigned, the absolute
configuration of 8 is therefore 3S,4R,5R,6R,9R.
configuration, compound 16 was recrystallized in MeOH
containing a small amount of CHCl3, and the crystal was
subjected to single-crystal X-ray diffraction analysis with Cu Kα
radiation. The Flack parameter, associated with the absolute
configuration, was calculated to be 0.01(15), which confirmed
the previously reported absolute configuration of compound 16
(Figure 9).
Wilsonol I (9) displayed an [M + Na]+ ion peak at m/z
251.1603, suggesting a molecular formula of C13H24O3, the
same as that of 4. The NMR data of 9 (Tables 2 and 3)
indicated that 9 had structural fragments similar to those of 4,
except for the presence of a double bond in the six-membered
ring of 9 (Δ5) and the absence of the side chain double bond in
4 (Δ7). HMBC correlations from H-3 and H3-13 to C-5 and
from H3-11 and H3-12 to C-6 in 9 supported this deduction.
The absolute configuration of 9 was determined to be 3S and
9S by the modified Mosher’s method (Figure 3). Therefore, 9
was established as (3S,9S)-3,9,13-trihydroxymegstigman-5-ene.
The HRESIMS of wilsonol J (10) gave a pseudomolecular
ion peak at m/z 251.1607 [M + Na]+, indicating, like that of 9,
a molecular formula of C13H24O3 (calcd for C13H24O3Na,
251.1623). The NMR data of 10 were similar to those of 9, and
Figure 9. X-ray ORTEP drawing of compound 16.
1
HSQC, H−1H COSY, and HMBC spectra revealed that 10
had the same planar structure and was a stereoisomer of 9. The
chemical shift values of H2-2, H-3, H2-4, C-2, C-3, and C-4 in
10 were nearly identical to those of 9, suggesting the 3S
configuration in 10. Thus, 10 must differ from 9 in the
configuration at C-9. This was proven by NMR analysis of
prepared (S)- and (R)-MTPA esters of 10 (10a and 10b),
which established the absolute configuration of 10 as 3S,9R
(Figure 3).
Compounds 1−9 and 13−19 were evaluated for cytotoxicity
against five human cancer cell lines, including myeloid leukemia
HL-60, hepatocellular carcinoma SMMC-7721, lung cancer A-
549, breast cancer MCF-7, and colon cancer SW-480 cells, as
well as the immortalized noncancerous Beas-2B human
bronchial epithelial cell line. The results (Table S1) were
notable for compound 13, which exhibited IC50 values of 5.0,
3.1, 2.5, 3.4, and 12 μM, respectively, higher potency than cis-
platin. Compound 13 was nontoxic to the Beas-2B cell line at
the highest concentration tested, resulting in selectivity indices
as high as >16 against the cancer-derived cells. The other
compounds tested showed no significant cytotoxicity (IC50 >10
μM). Lauroside B,18 a megastigmane glycoside isolated from
the leaves of Laurus nobilis (Lauraceae), was reported to
suppress the proliferation of human melanoma cell lines by
inducing apoptosis, and the mechanism is the inhibition of NF-
κB activation. The mechanism by which 13 exhibits cytotoxicity
is likely similar to that of lauroside B, due to their structural
similarity.
Selected compounds were also tested for in vitro
immunomodulation. Immune cells play a pivotal role in the
pathogenesis of cell-mediated autoimmune diseases and chronic
inflammatory disorders such as rheumatoid arthritis and
multiple sclerosis. Concanavalin A (ConA) and lipopolysac-
charide (LPS) are specific stimuli for T and B cells, respectively.
Compounds 1−4, 6, 9, 13−17, and 19 were evaluated in the
ConA/LPS-induced splenocyte proliferation assay.19 (Due to
the exhaustion of supply by the modified Mosher’s analysis,
compounds 5, 7, 8, 10−12, and 18 were not tested.) Results
(Tables S2−4) showed that compounds 1, 2, 9, and 17
inhibited the proliferation of ConA-induced murine T cells, and
compounds 9, 14, and 17 inhibited the proliferation of LPS-
induced murine B cells. Interestingly, compound 19 signifi-
cantly promoted the proliferation of both ConA-induced
murine T cells and LPS-induced murine B cells. The cytotoxity
assay (Tables S2−4) demonstrated that these active com-
Wilsonols K (11) and L (12) were isolated as white,
amorphous powders. Their HRESIMS [m/z 249.1453,
249.1456 ([M + Na]+), respectively] indicated the molecular
1
formula C13H22O3 for both 11 and 12. The H and 13C NMR
data revealed that 11 and 12 were structurally similar to 9 and
10 except for an additional C-7/C-8 double bond in 11 and 12,
in keeping with one additional degree of unsaturation
compared with 9 and 10. Analysis of the 1H−1H COSY,
HSQC, and HMBC spectra confirmed the planar structures of
11 and 12 as the 7,8-dehydro analogues of 9 and 10,
respectively. Similarly, the absolute configurations of 11 and
12 were assigned as 3S,9S and 3S,9R, respectively, following
application of the modified Mosher’s method (Figure 3).
Compounds 13−19 were identified as (3R,9S)-megastigman-
5-ene-3,9-diol 3-O-β-D-glucopyranoside (13),11 (3S,4R,9R)-
3,4,9-trihydroxymegastigman-5-ene (14),12 (3S,4S,5S,6S,9S)-
3,4-dihydroxy-5,6-dihydro-β-ionol (15),13 lasianthionoside A
(16),14 (3S,5S,6S,9R)-3,6-dihydroxy-5,6-dihydro-β-ionol
(17),15 apocynol A (18),16 and (+)-(6S,7E,9Z)-abscisic ester
(19),17 respectively, on the basis of the spectroscopic data
analysis and comparison with data reported in the literature.
Compounds 14 and 18 were previously reported as hydrolysis
products of (3S,4R,9R)-3,4,6-trihydroxymegastigman-5-ene 3-
O-β-D-glucopyranoside12 and apocynoside I,16 respectively, but
have not been reported previously as naturally occurring.
The absolute configuration of lasianthionoside A (16) has
been reported previously only by comparisons of optical
rotation and 13C NMR data with lasianthionoside C,14 without
further evidence. In order to determine its absolute
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MeOH) in CHCl3 containing a small amount of MeOH yielded 5 (7.2
mg). Subfraction A2-2 (eluted by 40% MeOH) was further purified by
HPLC (MeOH−H2O, 30:70) to afford 17 (7.2 mg) and 14 (9.3 mg).
Fraction A5 (eluted with CHCl3−MeOH, 20:1) was passed through
an RP C18 column eluted with MeOH−H2O (20:80 to 100:0) to give
five subfractions (A5-1 to A5-5). Repeated crystallization of
subfraction A5-1 yielded 1 (56 mg). Subfraction A5-2 was chromato-
graphed on a Sephadex LH-20 column (MeOH) to give compounds 2
(7.3 mg) and 6 (6.6 mg). Subfraction A5-3 was further purified by
semipreparative HPLC (MeOH−H2O, 30:70) to afford 15 (7.8 mg)
and 4 (4.7 mg). Subfraction A5-4 was further purified by semi-
preparative HPLC (CH3CN−H2O, 16:84) to afford 9 (6.2 mg), 12
(2.6 mg), 10 (8.1 mg), and 11 (2.6 mg). The n-BuOH extract (300 g)
was divided into five fractions (B1−B5) by silica gel CC eluted with
CHCl3−MeOH (30:1 to 2:1). Fraction B2 (eluted with CHCl3−
MeOH, 20:1) was further separated using RP-C18 CC (MeOH−H2O,
10:90 to 100:0) to afford seven subfractions (B2-1 to B2-7). Repeated
crystallization of B2-1 yielded 16 (23.0 mg). Subfraction B2-3 was
separated on Sephadex LH-20 (MeOH) to yield 8 (5.4 mg) and 13
(7.5 mg). Subfraction B2-6 was further purified on Sephadex LH-20
(MeOH) to furnish 3 (27.0 mg) and 7 (5.6 mg).
pounds are nontoxic to murine lymphocytes. Therefore, the
antiproliferative activities against ConA-induced murine T cells
and LPS-induced murine B cells of 1, 2, 9, 14, and 17 are not
involved with the general cytotoxity.
Megastigmane sesquiterpenoids are degradation products of
β-carotene in the biosynthetic pathway and are reported to
show antielastase activity,20 cytotoxicity against human
melanoma cell lines,18 mushroom tyrosinase inhibitory
activity,21 phytotoxicity on lettuce and tomato,22 and
promotion activity on HO-1 and SIRT1.23 Although there are
2500 species in the family of Lauraceae, there are only four
reports of the isolation of megastigmane sesquiterpenoids from
this family.13,18,24,25 This study implies that megastigmane
derivatives may be more prevalent in Lauraceae species. These
megastigmane compounds and the plant material C. wilsonii
may have value as a source of a targeted compound library for
investigating structure−activity relationships of megastigmane
pharmacophores for potential molecular drug targets that may
emerge from ongoing and future biological screening of
megastigmane sesquiterpenoids.
Wilsonol A (1): colorless needle crystals; mp 101−102 °C; [α]25
D
1
−38 (c 1.87, MeOH); H NMR data see Table 1, and 13C NMR data
see Table 3; HRESIMS m/z 267.1553 [M + Na]+ (calcd for
EXPERIMENTAL SECTION
C13H24O4Na, 267.1572).
■
Wilsonol B (2): colorless block crystals; mp 102−103 °C; [α]25
General Experimental Procedures. Melting points (uncor-
rected) were measured on a Beijing Tech X-5 microscopic melting
point apparatus. Optical rotations were determined in MeOH, at
concentrations indicated, on a Perkin-Elmer 341 polarimeter. UV and
FT-IR spectra were determined using Varian Cary 50 and Bruker
Vertex 70 instruments, respectively. NMR spectra were recorded on a
D
1
−34 (c 0.24, MeOH); H NMR data see Table 1, and 13C NMR data
see Table 3; HRESIMS m/z 267.1560 [M + Na]+ (calcd for
C13H24O4Na, 267.1572).
Wilsonol C (3): colorless block crystals; mp 171−172 °C; [α]25
D
−57 (c 0.55, MeOH); UV (MeOH) λmax (log ε) 231 (4.97) nm; IR
(KBr) νmax 3467, 1670, 1641, 1385, 1270, 1100, 1058 cm−1; 1H NMR
data see Table 1, and 13C NMR data see Table 3; HRESIMS m/z
265.1406 [M + Na]+ (calcd for C13H22O4Na, 265.1416).
Bruker AM-400 spectrometer, and the H and 13C NMR chemical
1
shifts were referenced to the solvent or solvent impurity peaks for
CDCl3 at δH 7.24 and δC 77.23, for DMSO-d6 at δH 2.50 and δC 39.51,
and for CD3OD at δH 3.31 and δC 49.15. High-resolution electrospray
ionization mass spectra (HRESIMS) were carried out in the positive-
ion mode on a Thermo Fisher LC-LTQ-Orbitrap XL spectrometer.
The X-ray diffraction experiments were carried out on a Bruker
SMART APEX-II CCD diffractometer equipped with graphite-
monochromatized Cu Kα radiation (λ = 1.541 78 Å). HPLC was
carried out on an Agilent 1200 quaternary system with a UV detector
using a reversed-phased C18 column (5 μm, 10 × 250 mm, YMC-pack
ODS-A). Column chromatography was performed using silica gel
(Qingdao Marine Chemical Inc., China), ODS (50 μm, YMC Co. Ltd.,
Japan), and Sephadex LH-20 (GE Healthcare Bio-Sciences AB,
Sweden). Thin-layer chromatography (TLC) was performed with
silica gel 60 F254 (Yantai Chemical Industry Research Institute) and
RP-C18 F254 plates (Merck, Germany).
Plant Material. The leaves of C. wilsonii were collected at Wuxi,
Sichuan Province, People’s Republic of China, in July 2010. The plant
material was identified by Prof. Changgong Zhang of the School of
Pharmacy, Tongji Medical College, Huazhong University of Science
and Technology. A voucher specimen (No. 2010-0701) was deposited
in the herbarium of Hubei Key Laboratory of Natural Medicinal
Chemistry and Resource Evaluation, Tongji Medical College,
Huazhong University of Science and Technology.
Extraction and Isolation. The air-dried leaves of C. wilsonii (50.6
kg) were extracted with 95% EtOH at room temperature (3 × 200 L)
and afforded a crude extract of 1.5 kg after evaporation of the solvent
under vacuum. The extract was suspended in H2O (10 L) and
partitioned sequentially with petroleum ether (6 × 15 L), EtOAc (6 ×
15 L), and n-BuOH (6 × 15 L). The EtOAc-soluble portion (102 g)
was fractionated via silica gel column chromatography (CC) eluting
with CHCl3−MeOH (55:1 to 5:1) to yield six fractions, A1−A6, on
the basis of TLC analysis. Fraction A1 (eluted by CHCl3−MeOH,
55:1) was chromatographed on a Sephadex LH-20 column (MeOH)
to give compounds 18 (2.4 mg) and 19 (4.8 mg). Fraction A2 (eluted
by CHCl3−MeOH, 45:1) was further separated using RP-C18 CC
(MeOH−H2O, 30:70 to 100:0) to afford four subfractions (A2-1 to
A2-4). Repeated crystallization of subfraction A2-1 (eluted by 30%
Wilsonol D (4): colorless block crystals; mp 153−155 °C; [α]25
D
1
−30 (c 0.07, MeOH); H NMR data see Table 1, and 13C NMR data
see Table 3; HRESIMS m/z 251.1611 [M + Na]+ (calcd for
C13H24O3Na, 251.1623).
Wilsonol E (5): colorless needle crystals; mp 164−165 °C, [α]25
D
1
−12 (c 0.15, MeOH); H NMR data see Table 1, and 13C NMR data
see Table 3; HRESIMS m/z 253.1767 [M + Na]+ (calcd for
C13H26O3Na, 253.1780).
1
Wilsonol F (6): white powder; [α]25 −13 (c 0.80, MeOH); H
D
NMR data see Table 1, and 13C NMR data see Table 3; HRESIMS m/
z 253.1770 [M + Na]+ (calcd for C13H26O3Na, 253.1780).
Wilsonol G (7): white powder; [α]25 −5 (c 0.29, MeOH); H
1
D
NMR data see Table 2, and 13C NMR data see Table 3; HRESIMS m/
z 267.1562 [M + Na]+ (calcd for C13H24O4Na, 267.1572).
Wilsonol H (8): white powder; [α]25 −19 (c 0.47, MeOH); H
1
D
NMR data see Table 2, and 13C NMR data see Table 3; HRESIMS m/
z 283.1501 [M + Na]+ (calcd for C13H24O5Na, 283.1521).
Wilsonol I (9): white powder; [α]25 −33 (c 0.15, MeOH); H
1
D
NMR data see Table 2, and 13C NMR data see Table 3; HRESIMS m/
z 251.1603 [M + Na]+ (calcd for C13H24O3Na, 251.1623).
Wilsonol J (10): white powder; [α]25 −4 (c 0.27, MeOH); H
1
D
NMR data see Table 2, and 13C NMR data see Table 3; HRESIMS m/
z 251.1607 [M + Na]+ (calcd for C13H24O3Na, 251.1623).
Wilsonol K (11): white powder; [α]25 −52 (c 0.06, MeOH); UV
D
1
(MeOH) λmax (log ε) 233 (3.59), 282 (3.51) nm; H NMR data see
Table 2, and 13C NMR data see Table 3; HRESIMS m/z 249.1453 [M
+ Na]+ (calcd for C13H22O3Na, 249.1467).
Wilsonol L (12): white powder; [α]25 −64 (c 0.05, MeOH); UV
D
1
(MeOH) λmax (log ε) 226 (3.62), 280 (3.64) nm; H NMR data see
Table 2, and 13C NMR data see Table 3; HRESIMS m/z 249.1456 [M
+ Na]+ (calcd for C13H22O3Na, 249.1467).
Preparation of (S)- and (R)-MTPA Ester Derivatives of 2 (ref
10). Compound 2 (1.0 mg) was dissolved in 1 mL of anhydrous
CH2Cl2. Dimethylaminopyridine (30 mg), 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride (37 mg) and (S)-
MTPA (46 mg) were then added in sequence. The reaction mixture
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was stirred at room temperature for 2 h. After addition of CH2Cl2 (1
mL), the solution was successively washed with H2O (1 mL), 5% HCl
(1 mL), NaHCO3-saturated H2O (1 mL), and brine (1 mL). The
organic layer was dried with Na2SO4 and evaporated under reduced
pressure. The residue was passed through a small silica gel column
using CHCl3−MeOH (50:1) as the eluent to furnish the (S)-MTPA
ester of 2 (2a, 1.4 mg). The (R)-MTPA derivative (2b, 1.3 mg) was
prepared with (R)-MTPA and purified in the same manner.
15.6, 6.7 Hz, H-8), 4.92 (1H, dd, J = 3.0, 2.9 Hz, H-4β), 4.81(1H, dd, J
= 12.5, 4.0 Hz, H-2α), 3.55 (3H, s, OCH3), 3.49 (3H, s, OCH3), 3.46
(3H, s, OCH3), 2.23 (1H, ddd, J = 12.5, 12.5,2.9 Hz, H-3α), 2.12 (1H,
ddd, J = 12.5, 4.0, 3.0 Hz, H-3β), 1.75 (1H, d, J = 10.2 Hz, H-6), 1.35
(3H, d, J = 6.5 Hz, H-10), 1.00 (3H, s, H-13), 0.93 (3H, s, H-11), 0.68
(3H, s, H-12); HRESIMS m/z 915.2735 [M + Na]+ (calcd for
C43H45O10F9Na, 915.2767).
1
(S)-MTPA derivative of 8 (8a): H NMR (MeOD, 400 MHz) δ
1
(S)-MTPA derivative of 2 (2a): H NMR (CDCl3, 400 MHz) δ
7.64−7.39 (10H, overlap, aromatic protons), 6.28 (1H, d, J = 15.6 Hz,
H-7), 5.76 (1H, dd, J = 15.6, 7.1 Hz, H-8), 5.68 (1H, dq, J = 7.1, 6.4
Hz, H-9), 5.38 (1H, ddd, J = 12.5, 9.5, 4.6 Hz, H-3α), 3.66 (1H, d, J =
9.5 Hz, H-4β), 3.61 (3H, s, OCH3), 3.55 (3H, s, OCH3), 1.75 (1H, dd,
J = 12.5, 12.5 Hz, H-2β), 1.49 (1H, dd, J = 12.5, 4.6 Hz, H-2α), 1.47
(3H, d, J = 6.4 Hz, H-10), 1.24 (3H, s, H-11), 1.15 (3H, s, H-13), 0.77
(3H, s, H-12); HRESIMS m/z 715.2287 [M + Na]+ (calcd for
C33H38O9F6Na, 715.2318).
7.53−7.34 (5H, overlap, aromatic protons), 5.65 (1H, d, J = 15.7 Hz,
H-7), 5.57 (1H, dd, J = 15.7, 6.0 Hz, H-8), 5.61 (1H, dq, J = 6.3, 6.0
Hz, H-9), 4.03 (1H, ddd, J = 3.2, 2.8, 1.2 Hz, H-3α), 3.64 (1H, dd, J =
2.8, 2.5 Hz, H-4β), 3.55 (3H, s, OCH3), 2.21 (1H, dq, J = 7.2, 2.5 Hz,
H-5β), 2.04 (1H, dd, J = 15.0, 3.2 Hz, H-2α), 1.42 (3H, d, J = 6.3 Hz,
H-10), 1.35 (1H, dd, J = 15.0, 1.2 Hz, H-2β), 1.07 (3H, s, H-11), 0.95
(3H, d, J = 7.2 Hz, H-13), 0.76 (3H, s, H-12); HRESIMS m/z
483.1953 [M + Na]+ (calcd for C23H31O6F3Na, 483.1970).
1
(R)-MTPA derivative of 8 (8b): H NMR (MeOD, 400 MHz) δ
1
7.60−7.39 (10H, overlap, aromatic protons), 6.36 (1H, d, J = 15.6 Hz,
H-7), 5.86 (1H, dd, J = 15.6, 7.1 Hz, H-8), 5.69 (1H, dq, J = 7.1, 6.4
Hz, H-9), 5.44 (1H, ddd, J = 12.5, 9.5, 4.6 Hz, H-3α), 3.42 (1H, d, J =
9.5 Hz, H-4β), 3.54 (3H, s, OCH3), 3.53 (3H, s, OCH3), 1.97 (1H, dd,
J = 12.5, 12.5 Hz, H-2β), 1.61 (1H, dd, J = 12.5, 4.6 Hz, H-2α), 1.40
(3H, d, J = 6.4 Hz, H-10), 1.29 (3H, s, H-11), 1.16 (3H, s, H-13), 0.86
(3H, s, H-12); HRESIMS m/z 715.2278 [M + Na]+ (calcd for
C33H38O9F6Na, 715.2318).
(R)-MTPA derivative of 2 (2b): H NMR (CDCl3, 400 MHz) δ
7.53−7.34 (5H, overlap, aromatic protons), 5.77 (1H, d, J = 15.7 Hz,
H-7), 5.67 (1H, dd, J = 15.7, 6.0 Hz, H-8), 5.61 (1H, dq, J = 6.3, 6.0
Hz, H-9), 4.04 (1H, ddd, J = 3.2, 2.8, 1.2 Hz, H-3α), 3.66 (1H, dd, J =
2.8, 2.5 Hz, H-4β), 3.51 (3H, s, OCH3), 2.26 (1H, dq, J = 7.2, 2.5 Hz,
H-5β), 2.06 (1H, dd, J = 15.0, 3.2 Hz, H-2α), 1.37 (3H, d, J = 6.3 Hz,
H-10),1.36 (1H, dd, J = 15.0, 1.2 Hz, H-2β), 1.12 (3H, s, H-11), 0.98
(3H, d, J = 7.2 Hz, H-13), 0.79 (3H, s, H-12); HRESIMS m/z
483.1947 [M + Na]+ (calcd for C23H31O6F3Na, 483.1970).
1
(S)-MTPA derivative of 9 (9a): H NMR (MeOD, 400 MHz) δ
7.56−7.36 (15H, overlap, aromatic protons), 5.14 (1H, m, H-3α), 5.12
(1H, m, H-9), 4.72 (2H, overlap, H-13), 3.53 (3H, s, OCH3), 3.50
(3H, s, OCH3), 3.46 (3H, s, OCH3), 2.31 (1H, ddd, J = 16.0, 5.3, 2.4
Hz, H-4α), 2.15 (2H, overlap, H-7), 2.06 (1H, dd, J = 16.0, 10.0 Hz,
H-4β), 1.74 (1H, ddd, J = 12.0, 3.6, 2.4 Hz, H-2β), 1.63 (2H, overlap,
H-8), 1.48 (1H, dd, J = 12.0, 12.0 Hz, H-2α), 1.28 (3H, d, J = 6.3 Hz,
H-10),1.07 (3H, s, H-11), 0.98 (3H, s, H-12); HRESIMS m/z
899.2800 [M + Na]+ (calcd for C43H45O9F9Na, 899.2818).
Preparation of (S)- and (R)-MTPA Ester Derivatives of 6−12
(ref 10). Wilsonol F (6) (1.0 mg) was dissolved in 2 mL of anhydrous
CH2Cl2. Dimethylaminopyridine (32 mg), triethylamine, and (R)-
MTPA chloride (26 μL) were then added in sequence. The reaction
mixture was stirred for 1 h at room temperature and then quenched by
the addition of 0.5 mL of aqueous MeOH. The solvents were removed
under vacuum, and the residue was passed through a small silica gel
column using CHCl3−MeOH (100:1) as the eluent to provide the
(S)-MTPA ester of 6 (6a, 1.4 mg). The (R)-MTPA derivative (6b, 1.6
mg) was prepared with (S)-MTPA chloride and purified in the same
manner. The same procedure was used to prepare the MTPA esters of
compounds 7 (7a, 7b), 8 (8a, 8b), 9 (9a, 9b), 10 (10a, 10b), 11 (11a,
11b), and 12 (12a, 12b).
1
(R)-MTPA derivative of 9 (9b): H NMR (MeOD, 400 MHz) δ
7.55−7.38 (15H, overlap, aromatic protons), 5.16 (1H, m, H-3α), 5.09
(1H, m, H-9), 4.72 (2H, overlap, H-13), 3.58 (3H, s, OCH3), 3.50
(3H, s, OCH3), 3.49 (3H, s, OCH3), 2.33 (1H, ddd, J = 16.0, 5.3, 2.4
Hz, H-4α), 2.12 (2H, overlap, H-7), 1.92 (1H, dd, J = 16.0, 10.0 Hz,
H-4β), 1.79 (1H, ddd, J = 12.0, 3.6, 2.4 Hz, H-2β), 1.57 (1H, dd, J =
12.0, 12.0 Hz, H-2α), 1.55 (2H, overlap, H-8), 1.33 (3H, d, J = 6.3 Hz,
H-10), 0.94 (3H, s, H-11), 0.93 (3H, s, H-12); HRESIMS m/z
899.2784 [M + Na]+ (calcd for C43H45O9F9Na, 899.2818).
1
(S)-MTPA derivative of 6 (6a): H NMR (MeOD, 400 MHz) δ
7.55−7.40 (10H, overlap, aromatic protons), 5.13 (1H, dddd, J = 12.1,
12.1, 4.6, 4.5 Hz, H-3β), 5.07 (1H, m, H-9), 3.57 (3H, s, OCH3), 3.52
(3H, s, OCH3), 1.88 (1H, m, H-5β), 1.83 (1H, dd, J = 12.1, 12.1 Hz,
H-2α), 1.67 (2H, overlap, H-8), 1.62 (1H, m, H-4β), 1.48 (2H,
overlap, H-7), 1.47 (1H, overlap, H-4α), 1.42 (1H, ddd, J = 12.1, 4.6,
2.1 Hz, H-2β), 1.36 (3H, d, J = 6.3 Hz, H-10), 0.91 (3H, s, H-11), 0.88
(3H, s, H-12), 0.84 (3H, d, J = 7.1 Hz, H-13); HRESIMS m/z
685.2549 [M + Na]+ (calcd for C33H40O7F6Na, 685.2576).
1
(S)-MTPA derivative of 10 (10a): H NMR (CDCl3, 400 MHz) δ
7.53−7.32 (15H, overlap, aromatic protons), 5.15 (1H, m, H-3α), 5.09
(1H, m, H-9), 4.74 (1H, d, J = 12.0 Hz, H-13a), 4.54 (1H, d, J = 12.0
Hz, H-13b), 3.54 (3H, s, OCH3), 3.51 (3H, s, OCH3), 3.47 (3H, s,
OCH3), 2.28 (1H, ddd, J = 16.1, 5.2, 2.4 Hz, H-4α), 2.16 (1H, m, H-
7a), 2.04 (1H, dd, J = 16.1, 10.0 Hz, H-4β), 1.94 (1H, m, H-7b), 1.72
(1H, ddd, J = 12.0, 3.6, 2.4 Hz, H-2α), 1.56 (2H, overlap, H-8), 1.47
(1H, dd, J = 12.0, 12.0 Hz, H-2β), 1.32 (3H, d, J = 6.3 Hz, H-10), 1.03
(3H, s, H-11), 0.91 (3H, s, H-12); HRESIMS m/z 899.2800 [M +
Na]+ (calcd for C43H45O9F9Na, 899.2818).
1
(R)-MTPA derivative of 6 (6b): H NMR (MeOD, 400 MHz) δ
7.53−7.40 (10H, overlap, aromatic protons), 5.17 (1H, dddd, J = 12.1,
12.1, 4.6, 4.5 Hz, H-3β), 5.07 (1H, m, H-9), 3.53 (3H, s, OCH3), 3.52
(3H, s, OCH3), 1.98 (1H, m, H-5β), 1.77 (2H, overlap, H-8), 1.76
(1H, dd, J = 12.1, 12.1 Hz, H-2α), 1.73 (1H, m, H-4β), 1.65 (1H,
overlap, H-4α), 1.64 (2H, overlap, H-7), 1.37 (1H, ddd, J = 12.1, 4.6,
2.1 Hz, H-2β), 1.27 (3H, d, J = 6.3 Hz, H-10), 1.04 (3H, s, H-11), 0.95
(3H, s, H-12), 0.94 (3H, d, J = 7.1 Hz, H-13); HRESIMS m/z
685.2545 [M + Na]+ (calcd for C33H40O7F6Na, 685.2576).
1
(R)-MTPA derivative of 10 (10b): H NMR (CDCl3, 400 MHz) δ
7.53−7.30 (15H, overlap, aromatic protons), 5.17 (1H, m, H-3α), 5.07
(1H, m, H-9), 4.68 (2H, overlap, H-13), 3.50 (3H, s, OCH3), 3.49
(3H, s, OCH3), 3.47 (3H, s, OCH3), 2.33 (1H, ddd, J = 16.1, 5.2, 2.4
Hz, H-4α), 2.23 (1H, m, H-7a), 2.03 (1H, m, H-7b), 1.96 (1H, dd, J =
16.1, 10.0 Hz, H-4β), 1.80 (1H, ddd, J = 12.0, 3.6, 2.4 Hz, H-2α), 1.59
(1H, dd, J = 12.0, 12.0 Hz, H-2β), 1.58 (2H, overlap, H-8), 1.24 (3H,
d, J = 6.3 Hz, H-10), 1.06 (3H, s, H-11), 1.00 (3H, s, H-12);
HRESIMS m/z 899.2784 [M + Na]+ (calcd for C43H45O9F9Na,
899.2818).
1
(S)-MTPA derivative of 7 (7a): H NMR (CDCl3, 400 MHz) δ
7.62−7.29 (15H, overlap, aromatic protons), 5.68 (1H, dd, J = 15.6,
10.2 Hz, H-7), 5.53 (1H, dq, J = 6.7, 6.5 Hz, H-9), 5.34 (1H, dd, J =
15.6, 6.7 Hz, H-8), 5.03 (1H, dd, J = 3.0, 2.9 Hz, H-4β), 4.87 (1H, dd,
J = 12.5, 4.0 Hz, H-2α), 3.59 (3H, s, OCH3), 3.53 (3H, s, OCH3), 3.52
(3H, s, OCH3), 2.36 (1H, ddd, J = 12.5, 12.5, 2.9 Hz, H-3α), 2.16 (1H,
ddd, J = 12.5, 4.0, 3.0 Hz, H-3β), 1.68 (1H, d, J = 10.2 Hz, H-6β), 1.38
(3H, d, J = 6.5 Hz, H-10), 0.90 (3H, s, H-13), 0.82 (3H, s, H-11), 0.57
(3H, s, H-12); HRESIMS m/z 915.2731 [M + Na]+ (calcd for
C43H45O10F9Na, 915.2767).
1
(S)-MTPA derivative of 11 (11a): H NMR (MeOD, 400 MHz) δ
7.53−7.34 (15H, overlap, aromatic protons), 6.30 (1H, d, J = 15.6 Hz,
H-7), 5.60 (1H, dq, J = 7.5, 6.4 Hz, H-9), 5.50 (1H, dd, J = 15.6, 7.5
Hz, H-8), 5.23 (1H, m, H-3α), 4.94 (1H, d, J = 11.3 Hz, H-13a), 4.77
(1H, d, J = 11.3 Hz, H-13b), 3.52 (3H, s, OCH3), 3.51 (3H, s, OCH3),
3.48 (3H, s, OCH3), 2.49 (1H, dd, J = 17.0, 5.3 Hz, H-4β), 2.19 (1H,
ddd, J = 17.0, 9.3, 2.1 Hz, H-4α), 1.81 (1H, ddd, J = 12.0, 3.6, 2.1 Hz,
1
(R)-MTPA derivative of 7 (7b): H NMR (CDCl3, 400 MHz) δ
7.55−7.33 (15H, overlap, aromatic protons), 5.83 (1H, dd, J = 15.6,
10.2 Hz, H-7), 5.54 (1H, dq, J = 6.7, 6.5 Hz, H-9), 5.47 (1H, dd, J =
1310
dx.doi.org/10.1021/np4002493 | J. Nat. Prod. 2013, 76, 1303−1312

Journal of Natural Products
Article
H-2α), 1.54 (1H, dd, J = 12.0, 12.0 Hz, H-2β), 1.36 (3H, d, J = 6.4 Hz,
H-10), 1.10 (3H, s, H-11), 0.95 (3H, s, H-12); HRESIMS m/z
897.2614 [M + Na]+ (calcd for C43H43O9F9Na, 897.2661).
Crystallographic data of 5: C13H26O3, M = 230.34, monoclinic, P2,
a = 6.7617(3) Å, b = 7.8646(3) Å, c = 12.6936(5) Å, α = γ = 90°, β =
102.866(2)°, V = 658.07(5) Å3, T = 298(2) K, Z = 2, Dcalcd = 1.162
Mg/m3, crystal size 0.20 × 0.10 × 0.10 mm3, F(000) = 256. The final
R1 value is 0.0398 (wR2 = 0.1108) for 5901 reflections [I > 2σ(I)].
Flack structure parameter: 0.2(3).
Crystallographic data of 16: one independent molecule of 16 and
two H2O molecules in the asymmetric unit, C19H32O9·2H2O, M =
440.48, monoclinic, P21, a = 11.2174(2) Å, b = 5.98460(10) Å, c =
16.7692(4) Å, α = γ = 90°, β = 98.3540(10)°, V = 1113.80(4) Å3, T =
298(2) K, Z = 2, Dcalcd = 1.313 Mg/m3, crystal size 0.20 × 0.10 × 0.10
mm3, F(000) = 476. The final R1 value is 0.0286 (wR2 = 0.0741) for
9701 reflections [I > 2σ(I)]. Flack structure parameter: 0.01(15).
Crystallographic data for the structures of 1−5 and 16 have been
deposited with the Cambridge Crystallographic Data Centre (deposit
number CCDC 927650, 927651, 927652, 927653, 927654, and
927655). Copies of the data can be obtained free of charge at www.
ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge Crys-
tallographic Data Centre, 12 Union Road, Cambridge CB21EZ, UK;
fax: (+44) 1223-336-033; or e-mail: deposit@ccdc.cam.ac.uk).
Cytotoxicity Assay. Five human cancer cell lines (HL-60, SMMC-
7721, A-549, MCF-7, and SW-480), together with one noncancerous
cell line, Beas-2B human bronchial epithelial, were used in the
cytotoxic activity assay. All cells were cultured in DMEM or RPMI-
1640 medium (Hyclone, Logan, UT, USA), supplemented with 10%
fetal bovine serum (Hyclone) at 37 °C in a humidified atmosphere
with 5% CO2. Cell viability was assessed by conducting colorimetric
measurements of the amount of insoluble formazan formed in living
cells based on the reduction of MTS (3-(4,5-dimethylthiazol-2-yl)-5-
(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)
(Sigma, St. Louis, MO, USA).26 Briefly, 100 μL of adherent cells was
seeded into each well of the 96-well culture plates and allowed to
adhere for 12 h before addition of test compounds, while suspended
cells were seeded just before the addition of the drug with initial
density of 1 × 105 cells/mL. Each tumor cell line was exposed to the
test compound at concentrations of 0.0625, 0.32, 1.6, 8, and 40 μM in
triplicate for 48 h, with DDP (cis-platin, Sigma) and Taxol (Sigma) as
positive controls. After incubation, 20 μL of MTS (5 mg/mL) was
added to each well, and the incubation continued for 4 h at 37 °C. The
cells were next lysed with 200 μL of 10% SDS after removing the
medium. The optical density of the lysate was measured at 595 nm in a
96-well microtiter plate reader (Bio-Rad 680). The IC50 value of each
compound was calculated using the method of Reed and Muench.27
Lymphocyte Proliferation Test. Splenic lymphocytes were
prepared as previously described by Kawaguchi et al.28 with a slight
modification. The prepared spleen cells (2.5 × 106 cells) were seeded
into each well of a 96-well microplate, and various concentrations of
compounds and 5 μg/mL of concanavalin A (Con A, from Canavalia
ensiformis Type III, Sigma), for selective stimuli on T cells, or
lipopolysaccharide (LPS, from Escherichia coli, Sigma), for selective
stimuli on B cells, were added. The plates were cultured at 37 °C with
5% CO2 in a humidified atmosphere for 48 h. For the last 4 h, 20 μL of
CCK-8 (Beyotime Institute of Biotechnology, Haimen, Jiangsu, P. R.
China) was added according to the technical manual. Absorbance at
450 nm with a 600 nm reference was measured with a microtiter plate
reader (Bio-Rad 680). All optical density (OD) values shown are the
mean of triplicate sample SD. Controls with and without ConA and
LPS were used to establish the baseline proliferation for stimulated and
unstimulated cells. Proliferation was assessed in terms of optical
density [(compounds (OD450) − positive control (OD450)/(positive
control (OD450)] × 100. The inhibitory or enhancement rate of >10%
is acceptable and considered active. In order to control for potential
compound toxicity to splenocytes, the assay was repeated (at cell
densities of 5 × 106 cells/200 μL/well) in the absence of both ConA
and LPS. Cell viability (%) was determined as [compounds (OD450)/
background (OD450)] × 100. All OD values shown are the mean of
1
(R)-MTPA derivative of 11 (11b): H NMR (MeOD, 400 MHz) δ
7.54−7.34 (15H, overlap, aromatic protons), 6.13 (1H, d, J = 15.6 Hz,
H-7), 5.61 (1H, dq, J = 7.5, 6.4 Hz, H-9), 5.35 (1H, dd, J = 15.6, 7.5
Hz, H-8), 5.22 (1H, m, H-3α), 4.79 (1H, d, J = 11.3 Hz, H-13a), 4.69
(1H, d, J = 11.3 Hz, H-13b), 3.56 (3H, s, OCH3), 3.50 (3H, s, OCH3),
3.48 (3H, s, OCH3), 2.44 (1H, dd, J = 17.0, 5.3 Hz, H-4β), 2.04 (1H,
ddd, J = 17.0, 9.3, 2.1 Hz, H-4α), 1.87 (1H, ddd, J = 12.0, 3.6, 2.1 Hz,
H-2α), 1.65 (1H, dd, J = 12.0, 12.0 Hz, H-2β), 1.42 (3H, d, J = 6.4 Hz,
H-10), 1.05 (3H, s, H-11), 0.96 (3H, s, H-12); HRESIMS m/z
897.2623 [M + Na]+ (calcd for C43H43O9F9Na, 897.2661).
1
(S)-MTPA derivative of 12 (12a): H NMR (CDCl3, 400 MHz) δ
7.52−7.32 (15H, overlap, aromatic protons), 6.08 (1H, d, J = 15.7 Hz,
H-7), 5.57 (1H, dq, J = 6.9, 6.5 Hz, H-9), 5.34 (1H, dd, J = 15.7, 6.9
Hz, H-8), 5.19 (1H, m, H-3α), 4.77 (1H, d, J = 11.6 Hz, H-13a), 4.70
(1H, d, J = 11.6 Hz, H-13b), 3.53 (3H, s, OCH3), 3.49 (3H, s, OCH3),
3.48 (3H, s, OCH3), 2.44 (1H, dd, J = 16.8, 5.7 Hz, H-4β), 2.14 (1H,
ddd, J = 16.8, 9.3, 2.1 Hz, H-4α), 1.77 (1H, ddd, J = 12.3, 3.0, 2.1 Hz,
H-2α), 1.51 (1H, dd, J = 10.3, 10.3 Hz, H-2β), 1.39 (3H, d, J = 6.5 Hz,
H-10), 1.00 (3H, s, H-12), 0.93 (3H, s, H-11); HRESIMS m/z
897.2623 [M + Na]+ (calcd for C43H43O9F9Na, 897.2661).
1
(R)-MTPA derivative of 12 (12b): H NMR (CDCl3, 400 MHz) δ
7.51−7.30 (15H, overlap, aromatic protons), 6.19 (1H, d, J = 15.7 Hz,
H-7), 5.56 (1H, dq, J = 6.9, 6.5 Hz, H-9), 5.38 (1H, dd, J = 15.7, 6.9
Hz, H-8), 5.21 (1H, m, H-3α), 4.70 (1H, d, J = 11.6 Hz, H-13a), 4.66
(1H, d, J = 11.6 Hz, H-13b), 3.51 (3H, s, OCH3), 3.50 (3H, s, OCH3),
3.47 (3H, s, OCH3), 2.43 (1H, dd, J = 16.8, 5.7 Hz, H-4β), 2.03 (1H,
ddd, J = 16.8, 9.3, 2.1 Hz, H-4α), 1.85 (1H, ddd, J = 12.3, 3.0, 2.1 Hz,
H-2α), 1.61 (1H, dd, J = 10.3, 10.3 Hz, H-2β), 1.34 (3H, d, J = 6.5 Hz,
H-10), 1.04 (3H, s, H-12), 1.00 (3H, s, H-11); HRESIMS m/z
897.2626 [M + Na]+ (calcd for C43H43O9F9Na, 897.2661).
Single-Crystal X-ray Diffraction Analysis and Crystallo-
graphic Data of 1−5 and 16. The intensity data for 1−5 and 16
were collected on a Bruker SMART APEX-II CCD diffractometer
equipped with graphite-monochromatized Cu Kα radiation (λ =
1.54178 Å). Data reduction was subsequently performed with Bruker
SAINT. Structure solution and refinement were performed with
SHELXS-97, and all non-hydrogen atoms were refined anisotropically
using the full-matrix least-squares method. All hydrogen atoms were
fixed at calculated positions. The final structures of 1−5 and 16 are
shown in Figures 2, 4−7, and 9, respectively.
Crystallographic data of 1: two independent molecules of 1 and
two H2O molecules in the asymmetric unit, C13H24O4·H2O, M =
262.34, monoclinic, P21, a = 6.0818(2) Å, b = 19.4319(7) Å, c =
12.9792(4) Å, α = γ = 90°, β = 90.883(2)°, V = 1533.71(9) Å3, T =
296(2) K, Z = 4, Dcalcd = 1.136 Mg/m3, crystal size 0.12 × 0.10 × 0.10
mm3, F(000) = 576. The final R1 value is 0.0324 (wR2 = 0.0907) for
36 185 reflections [I > 2σ(I)]. Flack structure parameter: −0.04(11).
Crystallographic data of 2: one independent molecule of 2 and
half a H2O molecule in the asymmetric unit, C13H24O4·0.5H2O, M =
253.33, orthorhombic, P21212, a = 13.0378(15) Å, b = 17.163(3) Å, c
= 6.2782(9) Å, α = γ = β = 90°, V = 1404.9(3) Å3, T = 296(2) K, Z =
4, Dcalcd = 1.198 Mg/m3, crystal size 0.12 × 0.10 × 0.10 mm3, F(000) =
556. The final R1 value is 0.0495 (wR2 = 0.1256) for 5027 reflections [I
> 2σ(I)]. Flack structure parameter: −0.3(4).
Crystallographic data of 3: C13H22O4, M = 242.31, orthorhombic,
P212121, a = 8.0315(2) Å, b = 10.5320(2) Å, c = 15.8116(3) Å, α = γ =
β = 90°, V = 1337.47(5) Å3, T = 296(2) K, Z = 4, Dcalcd = 1.203 Mg/
m3, crystal size 0.23 × 0.20 × 0.20 mm3, F(000) = 528. The final R1
value is 0.0386 (wR2 = 0.1000) for 7922 reflections [I > 2σ(I)]. Flack
structure parameter: 0.0(2).
Crystallographic data of 4: C13H24O3, M = 228.32, orthorhombic,
P212121, a = 8.6241(2) Å, b = 9.7034(2) Å, c = 15.9697(3) Å, α = γ =
β = 90°, V = 1336.39(5) Å3, T = 296(2) K, Z = 4, Dcalcd = 1.135 Mg/
m3, crystal size 0.15 × 0.12 × 0.10 mm3, F(000) = 504. The final R1
value is 0.0299 (wR2 = 0.0858) for 12 226 reflections [I > 2σ(I)]. Flack
structure parameter: 0.07(17).
triplicate sample
SD. Statistical analysis was carried out by the
Student t-test.
1311
dx.doi.org/10.1021/np4002493 | J. Nat. Prod. 2013, 76, 1303−1312

Journal of Natural Products
Article
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ASSOCIATED CONTENT
■
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* Supporting Information
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HRESIMS and 1D and 2D NMR spectra for compounds 1−12
and the MTPA ester derivatives of 2 and 6−12, IR spectrum of
3, UV spectra of 3, 11, and 12, crystal packing of compounds
1−5 and 16, X-ray crystallographic data (CIF files) for
compounds 1−5 and 16, cytotoxicity of compounds 1−9 and
13−19 against five cancer cell lines and one noncancerous
human Beas-2B cell line, cytotoxicity against murine
lymphocytes and immunomodulatory activity on murine
lymphocyte proliferation induced by concanavalin A (5 μg/
mL) or lipopolysaccharide (10 μg/mL) of compounds 1−4, 6,
9, 13−17, and 19. This material is available free of charge via
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AUTHOR INFORMATION
■
Corresponding Author
*Tel: 86-27-83691784. Fax: 86-27-83692762. E-mail: gyap@
mail.hust.edu.cn (G.Y.). Tel: 86-27-83692311. Fax: 86-27-
83692762. E-mail: zhangyh@mails.tjmu.edu.cn (Y.Z.).
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
We thank Prof. F. D. Horgen at Hawaii Pacific University for
his critical reading of the manuscript, and we are grateful to
Prof. C.-G. Zhang at Huazhong University of Science and
Technology for the authentification of the plant material and
Dr. X.-G. Meng at Central China Normal University for the
single-crystal X-ray data collection and analysis. This work was
financially supported by Scientific Research Foundation for the
Returned Oversea Chinese Scholars, State Education Ministry
of China (2010-1561, 40th, to G.Y.), Program for Youth
Chutian Scholar of Hubei Province of China (2011-6, to G.Y.),
and Program for New Century Excellent Talents in University,
State Education Ministry of China (NCET-2008-0224, to Y.Z.).
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