Terpenoids and norlignans from Metasequoia glyptostroboides

Article abstract of DOI:10.1021/np100694k

Four new terpenoids, metaseglyptorin A (1), metasequoic acid C (2), 12α-hydroxy-8,15-isopimaradien-18-oic acid (3), and (-)-acora-2,4(14),8- trien-15-oic acid (4), and three new norlignans, metasequirins D-F (5-7), were isolated from Metasequoia glyptostr

Full text of DOI:10.1021/np100694k

ARTICLE
pubs.acs.org/jnp
Terpenoids and Norlignans from Metasequoia glyptostroboides
Liao-Bin Dong,†,‡ Juan He,† Yuan-Yuan Wang,† Xing-De Wu,†,‡ Xu Deng,†,‡ Zheng-Hong Pan,§ Gang Xu,†
Li-Yan Peng,† Yu Zhao,† Yan Li,† Xun Gong,† and Qin-Shi Zhao*,†
†State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy
of Science, Kunming 650204, People’s Republic of China
‡Graduate School of Chinese Academy of Sciences, Beijing 100039, People’s Republic of China
§Guangxi Institute of Botany, Chinese Academy of Science, Guilin 541006, People’s Republic of China
S Supporting Information
b
ABSTRACT: Four new terpenoids, metaseglyptorin A (1), metasequoic
acid C (2), 12R-hydroxy-8,15-isopimaradien-18-oic acid (3), and (-)-
acora-2,4(14),8-trien-15-oic acid (4), and three new norlignans, metase-
quirins D-F (5-7), were isolated from Metasequoia glyptostroboides,
together with 15 known compounds. Structures of the new compounds
were determined by analysis of their spectroscopic data, and the absolute
configuration of 7 was established by the modified Mosher method. All of the
compounds were evaluated for cytotoxicity against five human tumor cell lines.
etasequoia glyptostroboides Hu et Cheng (Taxodiaceae) is a
monotypic genus, endemic in China, which is well known
acid, 3-acetoxylabda-8(20),13-dien-15-oic acid,4 8R-hydroxylab-
da-13(16),14-dien-19-yl-cis-4-hydroxycinnamate,8 12S,13R-di-
hydroxylabda-8(17),14-dien-19-oic acid, 12S,13S-dihydroxy-
labda-8(17),14-dien-19-oic acid,9,10 15-norlabda-8(20),12E-diene-
14-carboxaldehyde-19-oic acid,11 15,16-bisnor-13-oxo-8(17),11E-
labdadien-19-oic acid,12 8β-hydroxy-isopimar-15-en-19-oic acid,13
sequosempervirin B, sequosempervirin D, sequosempervirin F,14
agatharesinol,15 agatharesinol acetonide,16 sequirin C,17 and
hinokiresinol.18
M
as a “living fossil” species. Since the first living M. glyptostroboides
was discovered in the south of China in the early 1940s, many
chemical investigations were carried out to get a better under-
standing of this genus. The earlier studies on this plant reported
flavonoids,1 norlignans,2 sesquiterpenoids,3 labdane-type
diterpenoids,4,5 abietane-type diterpenoids,6 and sterols.7
Our current investigation of the stems and leaves of M. glyptos-
troboides led to the isolation of four new terpenoids (1-4), three
new norlignans (5-7), and 15 known compounds. All com-
pounds were evaluated for cytotoxicity against five human
tumor cell lines (HL-60, SMMC-7721, A-549, MCF-7, and
SW480). This paper describes the isolation, structural char-
acterization, and cytotoxic activities of the compounds from
M. glyptostroboides.
’ RESULTS AND DISCUSSION
An EtOH extract of the stems and leaves of M. glyptostroboides
was partitioned between H2O and EtOAc. The EtOAc portion
was subjected to MCI, silica gel, Sephadex LH-20, and semipre-
parative HPLC to afford the new compounds metaseglyptorin A
(1), metasequoic acid C (2), 12R-hydroxy-8,15-isopimaradien-18-
oic acid (3), (-)-acora-2,4(14),8-trien-15-oic acid (4), metasequir-
ins D-F (5-7), and 15 known compounds. The known com-
pounds (see Supporting Information), compared with literature
data, were identified as 3-hydroxylabda-8(20),13-dien-15-oic
Compound 1 had the molecular formula C32H54O3, as
evidenced by the positive HRFABMS at m/z 487.4163 [M þ H]þ,
Received: September 27, 2010
Published: January 12, 2011
r
Copyright
2011 American Chemical Society and
234
dx.doi.org/10.1021/np100694k J. Nat. Prod. 2011, 74, 234–239
American Society of Pharmacognosy
|

Journal of Natural Products
ARTICLE
Table 1. 13C NMR and 1H NMR Spectroscopic Data for
Compound 1a in CDCl3 (125 and 500 MHz)
no.
δC, mult
δH (J in Hz)
no.
17
δC, mult
δH (J in Hz)
1a
1b
2a
2b
3
30.2, CH2
2.65, m
1.37, m
2.65, m
2.30, m
52.4, CH
1.53, m
32.0, CH2
179.5, qC
18
18.4, CH3 0.94, s
19a
19b
20
31.1, CH2 0.66, d (6.0)
0.55, d (6.0)
4
76.7, qC
45.1, CH
25.4, CH2
35.8, CH
1.35, m
5
1.88, m
21
18.1, CH3 0.84, d (6.0)
33.9, CH2 1.35, m
1.27, m
6a
6b
7a
7b
8
1.71, m
22a
22b
23
0.66, m
25.7, CH2
1.27, m
29.7, CH2 1.20, m
Figure 1. Key 2D NMR correlations of 1, 2, and 4.
0.98, d (11.5)
1.27, m
48.6, CH
22.7, qC
26.6, qC
24
49.5, CH
147.6, qC
1.85, m
Compound 2 had the molecular formula C20H32O3 (by HRE-
IMS), and the IR spectrum exhibited bands for OH (3440 cm-1
9
25
)
10
26a
26b
27
111.4, CH2 4.73, br s
4.64, br s
and R,β-unsaturated carbonyl groups (1692, 1641 cm-1). The
13C and DEPT NMR data (Table 2) showed the existence of
four methyls (δC 13.0, 13.5, 18.2, 24.1), six methylenes, three
methines, one quaternary carbon, a cyclohexane methylidene
(δC 147.3, 106.3), and an R,β-unsaturated carbonyl group
(δC 168.9, 160.8, 115.0, 73.4). The spectroscopic data of 2 were
very similar to those of 3-hydroxylabda-8(20),13-dien-15-oic
acid.4 Analysis of the 1D NMR spectra of the two compounds
revealed marked differences: signals for a singlet methyl and an
oxymethine in 3-hydroxylabda-8(20),13-dien-15-oic acid were
replaced by a doublet methyl (δH 0.84, d, J = 7.3 Hz, Me-18) and an
oxygenated quaternary carbon (δC 73.4, s, C-4) in 2. This
information suggested that one of the gem-dimethyl groups at
C-4 in 3-hydroxylabda-8(20),13-dien-15-oic acid was at C-3 in 2.
This was confirmed by the HMBC correlations of δH 0.84 (d,
J = 7.3 Hz, Me-18) with δC 26.4 (t, C-2), 39.6 (d, C-3), and 73.4
(s, C-4) and of δH 0.96 (s, Me-19) with δC 39.6 (d, C-3), 49.1
(d, C-5), and 73.4 (s, C-4) (Figure 1).
The relative configuration of compound 2 was determined
by a ROESY experiment (Figure 1). Biogenetically, Me-20 was
β-oriented and H-5 was R-oriented in labdane-type diterpenoids.4,5
The ROESY correlations of Me-20/Me-19 and H-5/Me-18
demonstrated that Me-19 was β-oriented and Me-18 was R-oriented.
Thus, compound 2 was established as 4R-hydroxy-methyl-18-
(4f3R)-abeolabda-8(17),E-13-dien-15-oic acid, designated as
metasequoic acid C.
11a
11b
12
26.6, CH2
2.11, m
1.15, m
17.7, CH3 1.56, s
33.1, CH2 1.65, d (8.8)
44.8, qC
28
19.5, CH3 0.90, s
26.0, CH3 1.22, s
31.7, CH3 1.24, s
26.6, CH2 1.31, m
1.12, m
13
29
14
48.8, qC
30
15a
15b
16a
16b
36.0, CH2
28.1, CH2
1.35, m
1.26, m
1.87, m
1.28, m
31a
31b
32
12.1, CH3 0.80, t (7.4)
a Assignments are based on 1D and 2D NMR experiments.
indicating six degrees of unsaturation. IR absorption bands at
3441, 1709, and 2959-2871 cm-1 indicated the presence of OH
and carbonyl functionalities. In the 1H NMR spectrum (Table 1),
seven methyls [δH 0.80 (3H, t, J = 7.4 Hz, Me-32), 0.84 (3H, d,
J = 6.0 Hz, Me-21), 0.90 (3H, s), 0.94 (3H, s), 1.22 (3H, s), 1.24
(3H, s), and 1.56 (3H, s)], a pair of upfield doublets [δH 0.66
(1H, d, J = 6.0 Hz, Ha-19) and 0.55 (1H, d, J = 6.0 Hz, Hb-19)],
and terminal olefinic protons at δH 4.73 (1H, br s, Ha-26) and
4.64 (1H, br s, Hb-26) suggested that 1 was a typical cycloartane
triterpenoid with a 24-ethyl side-chain. This was supported by
the 13C and DEPT NMR spectra, which exhibited 32 carbon
signals ascribable to seven methyls, 12 methylenes, five methines,
five quaternary carbons [including one oxygenated carbon (δC
76.7, s, C-4)], a terminal double bond (δC 147.6, s, C-25; 111.4, t,
C-26), and a carbonyl group (δC 179.5, s, C-3). In the HMBC
spectrum, correlations of δH 1.24 (3H, s, Me-30) with δC 76.7
(s, C-4), 45.1 (d, C-5), and 26.0 (q, C-29) and of δH 2.65 (1H, m,
Ha-2) and 2.30 (1H, m, Hb-2) with δC 179.5 (s, C-3) suggested
that the usual A ring of 1 was open between C-3 and C-4.
Compound 3 had the molecular formula C20H30O3, and the
IR spectrum indicated OH (3432 cm-1), carbonyl (1696 cm-1),
and olefinic (1640 cm-1) functionalities. 1D NMR experiments
showed that 3 had a carbonyl group (δC 182.4, qC, C-18), a
tetrasubstituted olefin (δC 134.1, s, C-9; 125.1, s, C-8), a terminal
double bond [δC 140.8, d, C-15; δH 5.88 (1H, dd, J = 17.4,
11.0 Hz, H-15); δC 114.7, t, C-16; δH 5.13 (1H, dd, J = 10.7,
1.2 Hz, Ha-16); 5.08 (1H, dd, J = 17.9, 1.4 Hz, Hb-16)], one
oxymethine group [δC 74.6, d, C-12; δH 3.53 (1H, dd, J = 9.2,
5.5 Hz, H-12)], three quaternary carbons, one tertiary carbon,
seven secondary carbons, and three methyl groups. The data
were similar to those of 8,15-isopimaradien-18-oic acid,21 except
that compound 3 had one more OH group (at C-12), as
1
Detailed 2D NMR (HSQC, H-1H COSY, HMBC, ROESY)
data analysis (Figure 1) indicated that the other parts of 1 were
the same as those of 24S-ethyl-4,4-dimethylphytosterol.19,20
However, since the crystals obtained could not meet the required
standard for a single-crystal X-ray experiment after many attempts,
the configuration at C-24 was left undetermined. Thus, the
structure of 1 was determined as 3,4-secocycloarta-4-hydroxy-
24-ethyl-25-en-3-oic acid, and it was named metaseglyptorin A.
1
evidenced by the H-1H COSY correlations of δH 3.53 (1H,
dd, J = 9.2, 5.5 Hz, H-12) with δH 1.90 (1H, m, Ha-11) and 1.81
(1H, m, Hb-11) and the HMBC correlation of δH 3.53 (1H, dd,
235
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Journal of Natural Products
ARTICLE
Table 2. 13C NMR and 1H NMR Spectroscopic Data for Compounds 2-4a in CDCl3 (100 and 400 MHz)
2b
3
4
no.
δC, mult
δH (J in Hz)
1.24, m
δC, mult
δH (J in Hz)
δC, mult
δHc (J in Hz)
1a
31.3, CH2
36.6, CH2
1.75, m
60.6, CH
2.31, m
1b
2a
1.17, m
1.59, m
1.27, m
1.60, m
1.03, m
26.4, CH2
39.6, CH
19.3, CH2
37.4, CH2
1.83, m
137.2, CH
133.5, CH
6.03, br d (7.0)
2b
3a
1.49, br d (14.1)
2.16, m
6.20, dd (7.0, 2.0)
3b
4
1.00, m
73.4, qC
49.1, CH
22.7, CH2
43.6, qC
53.1, CH
20.4, CH2
162.0, qC
45.6, qC
5
1.41, dd (10.9, 2.1)
1.68, br d (10.4)
1.18, m
1.35, br d (11.9)
1.90, m
6a
25.1, CH2
1.72, m
2.45, m
6b
7a
1.81, m
37.4, CH2
2.23, br d (13.0)
1.80, m
32.3, CH2
1.91, m
21.6, CH2
7b
8
147.3, qC
55.7, CH
39.9, qC
125.1, qC
134.1, qC
38.1, qC
129.0, qC
142.1, CH
40.2, CH2
9
1.50, br d (10.9)
7.13, m
10a
10b
11a
11b
12a
12b
13
2.42, br d (19.6)
2.19, ddd (19.6, 6.0, 2.9)
1.92, m
21.4, CH2
39.4, CH2
1.47, m
1.32, m
2.12, m
1.79, m
30.9, CH2
74.6, CH
1.90, m
27.7, CH
19.7, CH
1.81, m
3.53, dd (9.2, 5.5)
0.80, d (6.7)
160.8, qC
115.0, CH
40.3, qC
23.1, CH3
0.96, d (6.7)
4.82, br s
14a
14b
15
5.45, br s
1.94, br s
42.8, CH2
1.96, m
101.8, CH2
1.90, m
4.59, br s
168.9, qC
18.2, CH3
140.8, CH
114.7, CH2
5.88, dd (17.4, 11.0)
5.13, dd (10.7,1.2)
5.06, dd (17.9, 1.4)
1.09, s
172.4, qC
16a
16b
17a
17b
18
106.3, CH2
4.68, s
23.8, CH3
4.33, s
13.0, CH3
24.1, CH3
13.5, CH3
0.84, d (7.3)
0.96, s
182.4, qC
28.5, CH3
17.5, CH3
19
1.26, s
0.89, s
20
0.48, s
a Assignments are based on 1D and 2D NMR experiments. b In CDCl3-CD3OD. c 500 MHz.
J = 9.2, 5.5 Hz, H-12) with δC 40.3 (s, C-13). The ROESY
correlation of H-12 with Me-17 revealed β-orientation of H-12.
Thus, compound 3 was identified as 12R-hydroxy-8,15-isopi-
maradien-18-oic acid.
(1H, m, H-1) with δH 6.03 (1H, br d, J = 7.0 Hz, H-2). The
relative configuration of 4 was elucidated by the ROESY experi-
ment (Figure 1), and the correlations of H-11/H-6, H-11/H-7,
and H-12/H-6 placed these groups in close proximity. Thus,
compound 4 was determined to be (-)-acora-2,4(14),8-trien-
15-oic acid.
The HRESIMS of compound 4 showed a protonated molec-
ular ion at m/z 233.1538 [M þ H]þ, corresponding to the
molecular formula C15H20O2 with six degrees of unsaturation.
The 13C and DEPT NMR spectra showed that 4 had an R,β-
unsaturated carbonyl group (δC 172.4, 129.0, and 142.1), a
cyclopentane methylidene (δC 162.0 and 101.8), a disubstituted
olefin (δC 137.2 and 133.5), one quaternary carbon, two tertiary
Compound 5 had the molecular formula C19H22O6. The IR
spectrum showed the presence of OH (3433 cm-1) and aromatic
(1629 and 1514 cm-1) functionalities. The 1H NMR spectrum
(Table 3) revealed signals of two ABX systems of two benzene
rings [δH 6.99 (1H, d, J = 1.8 Hz, H-2), 6.75 (1H, d, J = 8.0 Hz,
H-5), 6.81 (1H, dd, J = 8.0, 2.0 Hz, H-6) and δH 7.03 (1H, d, J =
2.0 Hz, H-20), 6.73 (1H, d, J = 8.2 Hz, H-50), 6.83 (1H, d, J =
8.2 Hz, H-60)] together with signals of two aromatic O-methyl
groups at δH 3.83 (6H, s), indicating the presence of two 1,3,4-
trisubstituted aromatic rings. Correlations in the 1H-1H COSY
and HSQC spectra revealed the presence of a CH2(9)-CH-
(8)-CH(7)-CH(80)dCH(70) unit. The HMBC correlations
from H-7 at δH 3.49 (1H, t, J = 7.5 Hz) to C-1 (δC 134.2, s) and
H-70 at δH 6.35 (1H, d, J = 15.8 Hz) to C-10 (δC 130.7, s)
suggested that C-7 was connected to C-1 and C-70 was linked
1
carbons, three secondary carbons, and two methyls. In the H
NMR spectrum, the two methyls at δH 0.80 and 0.96 (each 3H, d,
J = 6.7 Hz, Me-12, 13) and a methine at δH 1.92 (1H, m, H-11)
suggested the presence of an isopropyl group. These data
suggested that 4 possessed a structure similar to that of
15-hydroxyacora-4(14),8-diene,22 except that C-15 was oxidized
to a carboxyl group and the presence of one more double bond
was located at C-2/C-3 in 4. These structural differences were
confirmed by HMBC correlation of δH 7.13 (1H, m, H-9) with
δC 172.4 (s, C-15) and the 1H-1H COSY correlation of δH 2.31
236
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Journal of Natural Products
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Table 3. 13C NMR and 1H NMR Spectroscopic Data for Compounds 5-7a in CD3COCD3 (100 and 400 MHz)
5
6
7
no.
δCb, mult
δH (J in Hz)
δC, mult
δH (J in Hz)
δC, mult
δH (J in Hz)
1
134.2, qC
113.2, CH
148.0, qC
145.8, qC
115.5, CH
122.1, CH
53.4, CH
75.7, CH
65.5,CH2
134.7, qC
112.0, CH
148.0, qC
145.6, qC
115.5, CH
120.0, CH
54.0, CH
79.2, CH
75.9,CH2
134.1, qC
129.2, CH
115.9, CH
156.5, qC
115.9, CH
129.2, CH
54.1, CH
79.7, CH
75.6,CH2
2
6.99, d (1.8)
6.36, d (2.0)
6.86, d (8.6)
6.67, d (8.6)
3
4
5
6.75, d (8.0)
6.65, d (8.0)
6.67, d (8.6)
6
6.81, dd (8.0, 2.0)
3.49, t (7.5)
6.51, dd (8.0, 2.0)
3.25, dd (4.8, 2.8)
4.23, br s
6.86, d (8.6)
7
3.25, dd (4.8, 3.0)
4.15, br s
8
3.97, m
9a
9b
10
20
30
40
50
60
70
80
3.61, dd (11.1,3.6)
3.45, dd (10.8, 7.2)
4.04, dd (9.2,4.4)
3.84, dd (9.2, 2.7)
4.04, dd (9.2,4.6)
3.81, dd (8.8, 3.2)
130.7, qC
109.9, CH
148.3, qC
146.9, qC
115.7, CH
120.3, CH
131.0, CH
129.6, CH
56.1, CH3
56.1, CH3
134.1, qC
110.8, CH
148.0, qC
146.4, qC
115.2, CH
119.9, CH
74.8, CH
91.2, CH
56.0, CH3
56.2, CH3
134.0, qC
110.9, CH
147.9, qC
146.3, qC
115.1, CH
120.0, CH
75.0, CH
90.8, CH
7.03, d (2.0)
7.02, d (1.6)
6.99, d (1.8)
6.73, d (8.2)
6.83, d (8.2)
6.35, d (15.8)
6.40, dd (15.8, 8.0)
3.83, s
6.75, d (8.0)
6.86, dd (8.2, 1.8)
4.88, br s
6.73, d (8.0)
6.82, dd (8.0, 2.0)
4.85, br s
4.18, dd (4.8, 3.5)
3.68, s
4.19, dd (4.8, 3.6)
3-OCH3
30-OCH3
3.83, s
3.78, s
56.1, CH3
3.77, s
a Assignments are based on 1D and 2D NMR experiments. b 500 MHz.
to C-10. The assignments of 7S, 8S of 5 were confirmed by
comparing its 1D NMR and specific rotation ([R]2D5 -40.5) data
with those of sequosempervirin B, whose structure was unam-
biguously established by a single-crystal X-ray experiment.14
Thus, compound 5 was named as metasequirin D.
Compound 6 gave a peak at 385.1272 [M þ Na]þ in the
HRESIMS, providing a molecular formula of C19H22O7, 16 mass
units higher than that of 5. The 1H NMR spectrum (Table 3) also
displayed signals of two 1,3,4-trisubstituted aromatic rings.
Compared to 5, signals for the C-70-C-80 double bond were
absent in 6 and were replaced by two oxymethine carbons at δC
74.8 (d, C-70) and 91.2 (d, C-80). On the basis of the molecular
formula, one ring was needed to meet the required degrees
of unsaturation. HMBC correlations were observed from H-9a
(δH 4.04, dd, J = 9.2, 4.4 Hz) and H-9b (δH 3.84, dd, J = 9.2,
2.7 Hz) to C-80 (δC 91.2, d), supporting the structure of
metasequirin E as 6.
Compound 7 was isolated as a colorless gum. The HRESIMS
displayed an [M þ Na]þ peak at m/z 355.1160 (C18H20O6), 30
mass units less than that of 6. According to the 1D NMR data
(Table 3), compound 7 was readily identified as an analogue of 6
in which the OMe at C-3 was replaced by H. Analysis of 2D NMR
data confirmed that the other parts of 7 were the same as those of
6. Thus, compound 7 was named metasequirin F.
The absolute configurations of 6 and 7 were deduced from
interpretation of ROESY experiments and utilization of the modified
Mosher method. The modified Mosher method23 was applied to
determine the absolute configurations of the secondary alcohols
in 7. From the values of Δδ (δS - δR) (Figure 2), the absolute
configurations at C-8 and C-70 were assigned as 8S and 70R,
respectively. In 6 and 7, the ROESY correlations of H-7/H-80,
H-7/H-8, H-8/H-9a, and H-6/H-9b as well as the small coupling
constants of H-7 (J = 4.8, 3.0 Hz)24 indicated H-7, H-8, and H-80
Figure 2. Determination of the absolute configuration of 7.
were on one side. Accordingly, the absolute configurations of 6
and 7 were established as 7R, 8S, 70R, 80S.
Structurally, compound 1 is a 24-ethyl cycloartane triterpenoid
with an opened A ring, which is also the first triterpenoid reported
from the genus Metasequoia. Compound 4 represents the first
example of an acorane-type sesquiterpenoid from the family
Taxodiaceae.
All of the compounds were evaluated for cytotoxicity against
five human tumor cell lines (HL-60, SMMC-7721, A-549, MCF-7,
and SW480) using the MTT method as reported previously.25
237
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Journal of Natural Products
ARTICLE
Cisplatin was used as the positive control. 8R-Hydroxylabda-
13(16),14-dien-19-yl-cis-4-hydroxycinnamate and sequirin C
showed moderate cytotoxicity against HL-60, with IC50 values
of 14.3 and 5.5 μM, respectively, while cisplatin gave IC50 values of
2.0 μM. The other compounds were inactive (IC50 values >40 μM).
Metaseglyptorin A (1): colorless plates (MeOH); mp 140-142 °C;
[R]2D5 þ29.6 (c 0.41, CHCl3); UV (CHCl3) λmax (log ε) 239 (2.49), 235
(2.35), 229 (2.44), 217 (2.64), 198 (2.84) nm; IR (KBr) νmax 3441,
2959, 2933, 2871, 1709, 1642, 1460, 1375, 1192, 1034 cm-1; 1H and 13
C
NMR data, see Table 1; positive FABMS m/z 487 [M þ H]þ; positive
HRFABMS m/z 487.4163 [M þ H]þ (calcd for C32H55O3, 487.4151).
Metasequoic Acid C (2): colorless gum; [R]2D5 þ20.5 (c 0.30,
CHCl3); UV (CHCl3) λmax (log ε) 240 (3.35), 222 (2.71), 217
(2.73) nm; IR (KBr) νmax 3440, 2930, 1692, 1641, 1463, 1384, 1247,
1157 cm-1; 1H and 13C NMR data, see Table 2; positive EIMS m/z 320
[M]þ; positive HREIMS m/z 320.2356 [M]þ (calcd for C20H32O3,
320.2351).
’ EXPERIMENTAL SECTION
General Experimental Procedures. Melting points were ob-
tained on an X-4 micro melting point apparatus. Optical rotations were
measured on a JASCO-20C digital polarimeter. IR spectra were obtained
on a Tensor 27 spectrometer with KBr pellets. UV spectra were recorded
using a Shimadzu UV-2401A spectrophotometer. 1D and 2D NMR
spectra were performed on Bruker AM-400, DRX-500, or AVANCE III-
600 spectrometers with TMS as an internal standard. Mass spectra were
taken on VG Auto Spec-3000 or API-Qstar-Pulsar instruments. Semi-
preparative HPLC was performed on an Agilent 1100 liquid chromato-
graph with a Zorbax SB-C18 (9.4 mm ꢀ 25 cm) column. Column chro-
matography (CC) was performed using silica gel (100-200 and 200-
300 mesh, Qingdao Marine Chemical Co. Ltd., Qingdao, People’s
Republic of China), MCI gel (75-150 μm; Mitsubishi Chemical Corpora-
tion, Japan), and Sephadex LH-20 (Amersham Pharmacia Biotech,
Sweden).
12R-Hydroxy-8,15-isopimaradien-18-oic acid (3): colorless gum;
[R]2D5 þ135.6 (c 0.09, CHCl3); UV (CHCl3) λmax (log ε) 240 (3.01),
227 (2.78), 204 (2.88), 198 (2.90) nm; IR (KBr) νmax 3432, 2957, 2927,
1696, 1640, 1466, 1377, 1252 cm-1; 1H and 13C NMR data, see Table 2;
positive ESIMS m/z 341 [M þ Na]þ; positive HRESIMS m/z 341.2091
[M þ Na]þ (calcd for C20H30O3Na, 341.2092).
(-)-Acora-2,4(14),8-trien-15-oic acid (4): colorless gum; [R]D25
-
59.2 (c 0.32, CHCl3); UV (CHCl3) λmax (log ε) 241 (3.60), 231 (3.11),
222 (3.14), 212 (3.10), 203 (3.10) nm; IR (KBr) νmax 3440, 2960, 1689,
1640, 1423, 1273 cm-1; 1H and 13C NMR data, see Table 2; positive
ESIMS m/z 233 [M þ H]þ; positive HRESIMS m/z 233.1538 [M þ H]þ
(calcd for C15H21O2, 233.1541).
Plant Material. Stems and leaves of M. glyptostroboides were
collected in the Kunming Botany Garden, Kunming, Yunnan Province,
People’s Republic of China, in May 2009, and were identified by one of
the authors (X.G.). A voucher specimen (200905M) was deposited with
the State Key Laboratory of Phytochemistry and Plant Resources in West
China, Kunming Institute of Botany, Chinese Academy of Sciences.
Extraction and Isolation. The air-dried and powdered stems and
leaves of M. glyptostroboides (19 kg) were extracted with 95% EtOH (3 ꢀ
70 L), each for 48 h, at room temperature, and concentrated in vacuo.
The crude extract was partitioned between H2O and EtOAc. The EtOAc
portion (436 g) was decolorized on MCI gel (eluted with 90% MeOH)
and then was chromatographed on a silica gel column (100-200 mesh)
eluting with a gradient of petroleum ether-acetone (1:0, 9:1, 8:2, 7:3,
3:2, and 0:1) to afford six fractions (A-F). Fraction C (21 g) was
fractionated by MPLC (MCI) eluting with MeOH-H2O (from 30% to
100%) to provide subfractions (C1-C3). Subfraction C2 was recrystal-
lized to afford 3-acetoxylabda-8(20),13-dien-15-oic acid (5 g). Subfrac-
tion C3 was chromatographyed over Sephadex LH-20 eluted with
MeOH and then chromatographed repeatedly over silica gel, then by
semipreparative HPLC (83% MeOH-H2O), to give 4 (6 mg), 8R-
hydroxylabda-13(16),14-dien-19-yl cis-4-hydroxycinnamate (30 mg),
15,16-bisnor-13-oxo-8(17),11E-labdadien-19-oic acid (7 mg), and 8β-
hydroxy-isopimar-15-en-19-oic acid (20 mg). Fraction D (40.5 g) was
chromatographed on MPLC (MCI gel) (0:1 f 1:0, MeOH-H2O)
to give subfractions D1-D3. Compounds 2 (5 mg), 3 (1.8 mg), 12S,13R-
dihydroxylabda-8(17),14-dien-19-oic acid (3 mg), 12S,13S-dihydroxylabda-
8(17),14-dien-19-oic acid (4 mg), and 15-norlabda-8(20),12E-diene-
14-carboxaldehyd-19-oic acid (6 mg) were isolated from subfraction D1
by repeated chromatography including silica gel, MCI, and Sephadex LH-20.
3-Hydroxylabda-8(20),13-dien-15-oic acid (2 g) was crystallized from
subfraction D2 directly. Subfraction D3 was subjected to repeated silica
gel CC eluted with petroleum ether-acetone (9:1 f 0:1) and then by
semipreparative HPLC (69% MeOH-H2O) to obtain sequosempervirin D
(20 mg), agatharesinol acetonide (30 mg), and hinokiresinol (15 mg). The
acetone-insoluble part of fraction D (2 g) was chromatographed over
Sephadex LH-20 eluting with CHCl3-MeOH (1:1) and then by semipre-
parative HPLC (94% MeOH-H2O) to afford 1 (20 mg). Fraction E
(36.6 g) was submitted to repeated chromatography and purified
by Sephadex LH-20 and semipreparative HPLC to afford 5 (35 mg),
6 (12 mg), 7 (50 mg), sequosempervirin B (60 mg), sequosempervirin
F (45 mg), agatharesinol (20 mg), and sequirin C (50 mg).
Metasequirin D (5): colorless gum; [R]2D5 -40.5 (c 0.17, MeOH);
UV (MeOH) λmax (log ε) 269 (4.03), 204 (4.41), 194 (4.11) nm; IR
(KBr) νmax 3433, 1629, 1514, 1272, 1032 cm-1; 1H and 13C NMR data,
see Table 3; positive ESIMS m/z 369 [M þ Na]þ; positive HRESIMS
m/z 369.1317 [M þ Na]þ (calcd for C19H22O6Na, 369.1314).
Metasequirin E (6): colorless gum; [R]2D5 þ22.2 (c 0.25, MeOH);
UV (MeOH) λmax (log ε) 281(3.49), 229 (3.84), 204 (4.41) nm; IR
(KBr) νmax 3430, 1612, 1517, 1273, 1033 cm-1; 1H and 13C NMR data,
see Table 3; positive ESIMS m/z 385 [M þ Na]þ; positive HRESIMS
m/z 385.1272 [Ma þ Na]þ (calcd for C19H22O7Na, 385.1263).
Metasequirin F (7): colorless gum; [R]2D5 þ8.7 (c 0.32, MeOH); UV
(MeOH) λmax (logε) 279 (3.18), 226 (3.71), 204 (3.91), 193 (3.58) nm; IR
(KBr) νmax 3441, 1620, 1516, 1271, 1033 cm-1; 1H and 13C NMR data, see
Table 3; positive ESIMS m/z 355 [M þ Na]þ; positive HRESIMS m/z
355.1160 [M þ Na]þ (calcd for C18H20O6Na, 355.1157).
Methylation of the Phenolic OH Groups of 7. Diazomethane
in ether was added to a solution of 7 (8 mg, 24 μmol) in anhydrous
tetrahydrofuran (1 mL), which was stirred under N2 at room tempera-
ture overnight. The reaction mixture was concentrated under reduced
pressure, and the residue was purified by silica gel CC (petroleum ether-
acetone, 7:3) to afford 8 (8 mg, 100%): 1H NMR (400 MHz,
CD3COCD3) δ 3.27 (1H, m, H-7), 3.71, 3.73, and 3.74 (each 3H, s, -
OCH3), 3.80 (1H, dd, J = 9.1, 3.0 Hz, H-9b), 4.04 (1H, dd, J = 9.1,
4.8 Hz, H-9a), 4.16 (1H, m, H-8), 4.20 (1H, t, J = 4.8 Hz, H-80), 4.86
(1H, br s, H-70), 6.74 (2H, d, J = 8.5 Hz, H-3/5), 6.81 (1H, d, J = 8.5 Hz,
H-60), 6.89 (1H, dd, J = 8.5, 1.8 Hz, H-50), 6.94 (2H, d, J = 8.5 Hz, H-2/6),
6.98 (1H, d, J = 1.8 Hz, H-20).
Preparation of the (S)- and (R)-MTPA Esters of 8. A mixture
of 8 (2 mg, 5.5 μmol), (R)-MTPA (5 mg, 21.4 μmol, 3.9 equiv), DCC
(4 mg, 19.4 μmol, 3.5 equiv), and DMAP (2 mg, 16.4 μmol, 3.0 equiv)
was dissolved in anhydrous CH2Cl2 (0.5 mL), which was stirred under
N2 at room temperature for 24 h. The reaction mixture was concentrated
under reduced pressure, and the residue was purified by silica gel
CC (petroleum ether-acetone, 9:1) to yield bis-(R)-MTPA ester 9a
(1.5 mg). Bis-(S)-MTPA ester 9b (1.2 mg) was prepared in the same
manner.
Bis-(R)-MTPA Ester of 8 (9a): 1H NMR (600 MHz, CDCl3) δ 3.33
(1H, dd, J = 6.2, 4.8 Hz, H-7), 3.79 (1H, dd, J = 10.2, 3.0 Hz, H-9b), 4.26
(1H, dd, J = 10.2, 5.4 Hz, H-9a), 4.42 (1H, t, J = 6.2 Hz, H-80), 5.34
(1H, m, H-8), 5.84 (1H, d, J = 6.2 Hz, H-70), 6.55 (1H, d, J = 1.8 Hz,
238
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Journal of Natural Products
ARTICLE
H-20), 6.68 (1H, dd, J = 8.2, 1.8 Hz, H-50), 6.71 (1H, d, J = 8.2 Hz, H-60),
6.81 (2H, d, J = 8.4 Hz, H-3/5), 7.01 (2H, d, J = 8.4 Hz, H-2/6).
Bis-(S)-MTPA Ester of 8 (9b): 1H NMR (600 MHz, CDCl3) δ 3.14
(1H, dd, J = 4.8, 3.0 Hz, H-7), 3.96 (1H, dd, J = 11.4, 3.0 Hz, H-9b), 4.25
(1H, dd, J = 11.4, 4.8 Hz, H-9a), 4.42 (1H, dd, J = 7.9, 4.8 Hz, H-80), 5.27
(1H, m, H-8), 5.77 (1H, d, J = 7.9 Hz, H-70), 6.74 (4H, br s, H-2/3/5/6),
6.80 (1H, s, H-20), 6.81 (1H, d, J = 8.2 Hz, H-50), 6.87 (1H, dd, J = 8.2,
1.8 Hz, H-60).
Cytotoxicity Assay. Cytotoxicity of all compounds against HL-
60, SMMC-7721, A-549, MCF-7, and SW480 cell lines was assessed
using the MTT method.25 Cells were plated in 96-well plates 12 h before
treatment and continuously exposed to different concentrations of
compounds. After 48 h, 20 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT) solution was added to each well, which
were incubated for another 4 h. Then 20% SDS (100 μL) was added to
each well. After 12 h at room temperature, the OD value of each well was
recorded at 595 nm. The IC50 value of each compound was calculated by
the Reed and Muench method.26
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’ ASSOCIATED CONTENT
(20) Desoky, E. K. Indian J. Chem., Sect. B Org. Chem. Incl. Med.
Chem. 1996, 35B, 1113–1115.
S
Supporting Information. 1D and 2D NMR spectra of 1,
b
2, 4, and 6, 1D NMR spectra of 3, 5, and 7, MS spectra of 1-7,
1H NMR spectra of 8, 9a, and 9b, and COSY NMR spectra of the
MTPA esters 9a and 9b are available free of charge via the
Internet at http://pubs.acs.org.
(21) ApSimon, J. W.; Craig, W. G.; Demarco, P. V.; Mathieson,
D. W.; Whalley, W. B. Chem. Commun. 1966, 361–363.
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’ AUTHOR INFORMATION
Corresponding Author
*To whom correspondence should be addressed. Tel: þ86-871-
5223058. Fax: þ86-871-5215783. E-mail: qinshizhao@mail.kib.ac.cn.
’ ACKNOWLEDGMENT
This work was financially supported by NSFC (No. U0932602),
the National Natural Science Foundation of China (No. 90813004
and No. 2009312311024), the 973 Program (No. 2009CB522300),
and the State Key Laboratory of Phytochemistry and Plant Resources
in West China (P2010-ZZ05).
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