Bioactive neolignans and lignans from the bark of machilus robusta

Article abstract of DOI:10.1021/np2001896

Sixteen new neolignans and lignans (1 - 16), together with 12 known analogues, have been isolated from an ethanol extract of the bark of Machilus robusta. Compounds 1 and 2 showed activity against HIV-1 replication in vitro, with IC₅₀ values of

Full text of DOI:10.1021/np2001896

ARTICLE
pubs.acs.org/jnp
Bioactive Neolignans and Lignans from the Bark of Machilus robusta
Yanru Li, Wei Cheng, Chenggen Zhu,* Chunsuo Yao, Liang Xiong, Ye Tian, Sujuan Wang, Sheng Lin,
Jinfeng Hu, Yongchun Yang, Ying Guo, Ying Yang, Yan Li, Yuhe Yuan, Naihong Chen, and
Jiangong Shi*
State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, and Key Laboratory of Bioactive Substances and
Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Institute of Materia Medica, Chinese Academy of Medical
Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China
S Supporting Information
b
ABSTRACT: Sixteen new neolignans and lignans (1ꢀ16),
together with 12 known analogues, have been isolated from an
ethanol extract of the bark of Machilus robusta. Compounds 1
and 2 showed activity against HIV-1 replication in vitro, with
IC50 values of 2.52 and 2.01 μM, respectively. At 10 μM, 6, 8,
and 9 reduced DL-galactosamine-induced hepatocyte (WB-
F344 cells) damage, and 9 could additionally attenuate rote-
none-induced PC12 cell damage. The known compounds (ꢀ)-
pinoresinol (17) and (þ)-lyoniresinol (18) were active against
serum deprivation induced PC12 cell damage.
achilus robusta W. W. Sm. (Lauraceae) is a plant that is
widely distributed in southern China; however, no chemi-
C-800, and C-900; and H-800/C-40, in combination with their
chemical shifts, verified the presence of the three phenylpropyl
units, the connection of C-8 and C-80, and the oxygen bridge
between C-40 and C-800. In addition, HMBC correlations from
H-5 and OMe-3 to C-3, from H-20/60 and OMe-30/50 to C-30/50,
and from H-500 and OMe-300 to C-300 located the methoxy groups
at C-3, C-30, C-50, and C-300, respectively. This combined with
the coupling constants of the aromatic protons (Table 1)
assigned the hydroxy groups at C-4, C-40, and C-400, respectively.
In the 1H NMR spectrum, the small coupling constant between
H-700 and H-800 (2.4 Hz) indicated the 700,800-erythro configura-
tion.4 In the CD spectrum, a negative Cotton effect at 242 nm
(Δε ꢀ1.38) demonstrated that 1 had the 800R configuration.5 In
addition, based on the bulkiness rule for the secondary alcohol,6
a positive Cotton effect around 350 nm (the E band) in the
Rh2(OCOCF3)4-induced CD spectrum (Supporting Informa-
tion Figure S29) supported the 700S configuration, which was
consistent with that defined by the 700,800-erythro and 800R
M
cal or biological studies of this plant have been reported. As part
of a continuing effort to assess the chemical diversity and
biological activities of species of the genus Machilus,1 an ethanol
extract of the bark of M. robusta was investigated. We describe
herein the isolation, structural elucidation, and biological assays
of 16 neolignans and lignans (1ꢀ16), together with 12 known
analogues. On the basis of IUPAC recommendations for the
nomenclature of lignans and neolignans,2 compounds 1 and 2 are
categorized as uncommon 40,800-oxy-8,80-sesquineolignans, 3 is a
40,800-oxy-2,70-cyclo-8,80-sesquineolignan, and 4 and 5 are abnor-
mal 10,20,30,40,50,60-hexanor-2,70-cyclolignans.
’ RESULTS AND DISCUSSION
Compound 1 had the molecular formula C31H40O8, as
indicated by HRESIMS combined with the NMR data. Its IR
spectrum showed the presence of hydroxy (3490 cmꢀ1) and
aromatic (1603, 1589, 1515, and 1463 cmꢀ1) functionalities.
The NMR spectra of 1 displayed resonances (Table 1) attribu-
table to two trisubstituted and one symmetric tetrasubstitut-
ed aromatic rings, four methoxy groups, an oxymethine, two
methylenes, two methyls, respectively, attached to an aliphatic
methine, and a methyl attached to a second oxymethine group.
This suggested that compound 1 was a 40,800-oxy-8,80-sesqui-
neolignan with four O-methyl and three hydroxy substituents,3
which was refined by 2D NMR data analysis (Supporting
Information, Figures S8ꢀS10). In the HMBC spectrum of 1,
correlations of H2-7/C-1, C-2, C-6, C-8, C-80, and C-9; H2-70/
C-10, C-20, C-60, C-8, C-80, and C-90; H-700/C-100, C-200, C-600,
configuration. Oxidation of 1 by RuO2 2H2O in trifluoroacetic
3
acid and trifluoroacetic anhydride7 yielded the product 1a,
which had spectroscopic data (Supporting Information, Scheme
S1 and Figures S1 and S13ꢀS15) identical to those of the co-
occurring 8 (see below). The 70S,8S,80S configuration of 1a was
proved by the [R]D and CD data (Experimental Section).
Accordingly, compound 1 was determined as (þ)-(700S,8S,80R,-
800R)-4,400-dihydroxy-3,30,300,50-tetramethoxy-40,800-oxy-8,80-se-
squineolignan-700-ol.
Received: February 25, 2011
Published: May 31, 2011
r
Copyright
2011 American Chemical Society and
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|

Journal of Natural Products
ARTICLE
40-oxy-30-methoxyphenyl moiety in 3. This suggested that 3
was 4,400-dihydroxy-30,300,5-trimethoxy-40,800-oxy-2,70-cyclo-8,80-
sesquineolignan-700-ol. The suggestion was confirmed by 2D
NMR data analysis (Supporting Information, Figures S34ꢀS36),
which refined the assignments of the 1D NMR data of 3
(Table 1). Especially, HMBC correlations from H-70 to C-1,
C-10, C-20, C-3, C-60, C-8, and C-90, together with their chemical
shifts, proved 2,70-cyclization in 3. The coupling constants
0
0
(J7b,8 = 12.0 Hz and J7 ,8 = 10.2 Hz) and NOE enhancements
of H-8 and H3-90 upon irradiation of H-70 indicated that H-80 was
trans-oriented to both H-70 and H-8, and the 700,800-erythro
configuration was indicated by the coupling constant (2.4 Hz)3
between H-700 and H-800 (Table 1 and Supporting Information,
Figure S32). In the CD spectrum of 3, Cotton effects [positive at
274 (Δε þ1.81) and negative at 291 nm (Δε ꢀ3.12)] indicated
the 70S configuration,8 with a negative Cotton effect at 230 nm
(Δε ꢀ1.56) suggesting the 800R configuration.5 In addition, the
700S configuration defined by the 700,800-erythro orientation was
supported by the positive E band (around 350 nm)6 in the
Rh2(OCOCF3)4-induced CD spectrum of 3 (Supporting Infor-
mation, Figure S40). Therefore, compound 3 was defined as
(ꢀ)-(70S,700S,8R,80S,800R)-4,400-dihydroxy-30,300,5-trimethoxy-
40,800-oxy-2,70-cyclo-8,80-sesquineolignan-700-ol.
Compound 4, C13H16O3 (HRESIMS and the NMR data),
displayed absorption bands for hydroxy (3439 cmꢀ1), conjugated
carbonyl (1665 cmꢀ1), and aromatic (1613 and 1509 cmꢀ1
)
functionalities in the IR spectrum. The NMR data (Table 1)
indicated that it contained a 1,2,4,5-tetrasubstituted aromatic
ring, an aromatic methoxy, a hydroxy, and a carbonyl group, as
well as two methyls, a methylene, and two methines. On the basis
of the splitting patterns and coupling constants for the reso-
nances of the methyl, methylene, and methine protons, in
combination with chemical shifts of the proton and carbon
resonances of these units (Table 1), compound 4 was con-
structed to be 4-hydroxy-5-methoxy-10,20,30,40,50,60-hexanor-
2,70-cyclolignan-70-one or its 5-hydroxy-4-methoxy isomer. In
the HMBC spectrum of 4 (Supporting Information, Figure S48),
correlations from H-3 to C-1, C-4, C-5, and C-70, from OH to
C-3, C-4, and C-5, and from OMe to C-5, together with their
shifts, located the hydroxy and methoxy groups at C-4 and C-5,
The spectroscopic data of compound 2 (Table 1 and Experi-
mental Section) indicated that it was an isomer of 1. Comparison
of the NMR data of 2 and 1 demonstrated that H-600, H-700, and
H-800 and C-40, C-600, C-700, C-800, and C-900 in 2 were shifted by
ΔδH þ0.19, ꢀ0.18, and ꢀ0.42 and ΔδC þ2.3, þ1.9, þ6.3, þ6.4,
and þ4.8 ppm, respectively. In addition, the coupling constant
between H-700 and H-800 was changed from 2.4 Hz for 1 to 8.5 Hz
for 2. This suggested that 2 was the 700,800-threo isomer of 1,
which was proved by the 2D NMR data of 2 (Supporting
Information, Figures S21ꢀS23). The CD spectrum of 2 dis-
played a positive Cotton effect at 233 nm (Δε þ2.07), demon-
strating the 800S configuration.5 This was supported by a positive
Cotton effect around 350 nm (the E band) in the Rh2-
(OCOCF3)4-induced CD spectrum that suggested the 700S
configuration according to the bulkiness rule for the secondary
respectively. The coupling constants (J7a,8 = 4.0 and J7b,8
=
11.0 Hz) indicated that H-7b and H-8 occupied trans pseudo-
diaxial positions. The NOE enhancements of H-7b and H3-9
upon irradiation of H-80 demonstrated that these protons were
cofacial. On the basis of the octant rule for cyclohexenones,9 a
positive Cotton effect at 332 nm for the n f π* transition and a
negative Cotton effect at 301 nm for the π f π* transition in the
CD spectrum suggested that 4 had the 80R configuration
(Supporting Information, Figure S51), which was supported by
calculation of CDs and specific rotations of the enantiomers
(Supporting Information, Figure S177 and Table S2). Therefore,
compound 4 was determined as (þ)-(8S,80R)-4-hydroxy-5-
methoxy-10,20,30,40,50,60-hexanor-2,70-cyclolignan-70-one.
The spectroscopic data of compound 5 (Table 1 and Experi-
mental Section) indicated that it was an isomer of 4. However,
the NMR resonances for the cyclohexenone moiety in 5 were
significantly shifted as compared to 4 (Table 1). The 2D NMR
data of 5 (Supporting Information, Figures S57ꢀS59) revealed
that it had the same planar structure as 4. In the NOE difference
spectrum of 5, H-7b was enhanced upon irradiation of either H3-
9 or H3-90. This indicated that H3-9 and H3-90 were cofacial and
that H-7b and Me-90 occupied the pseudo-diaxial positions,
alcohol6 (Supporting Information, Figure S29). The RuO2 2
3
H2O oxidation of 2 yielded the product 2a, identical to 1a
(Supporting Information, Scheme S1 and Figures S1 and
S26ꢀS28), which confirmed the 8S,80R configuration for 2.
Compound 3, C30H36O7 (HRESIMS and the NMR data),
exhibited spectroscopic data (Table 1 and Experimental Section)
similar to 1. Comparison of the NMR data of 3 with those of
1 indicated that the major difference was replacement of the
C-70 methylene group (H2-70 and C-70) in 1 by a methine group
in 3. In addition, the 4-hydroxy-3-methoxyphenyl and 40-oxy-
30,50-dimethoxyphenyl moieties in 1 were substituted, respec-
tively, by a 2-substituted 4-hydroxy-5-methoxyphenyl and a
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Table 1. NMR Spectroscopic Data (δ) of Compounds 1ꢀ5a
1
2
3
4
5
no.
δH
δC
133.5
δH
δC
133.6
δH
δC
δH
δC
δH
δC
1
131.9
133.5
136.8
136.2
125.7
112.3
144.3
151.0
109.9
2
6.64 br s
111.4
146.4
143.6
113.8
121.7
6.64 br s
111.4
146.3
143.6
114.0
121.7
125.9
3
6.39 s
6.62 s
121.0 7.54 s
143.9
112.4 7.55 s
144.3
4
5
6.83 d (7.8)
6.67 br s d (7.8)
6.83 d (8.0)
149.2
150.9
6
6.67 br s d (8.0)
111.2 6.62 s
109.4 6.63 s
7a
2.73 dd (13.5, 5.1) 39.2
2.72 dd (13.5, 5.5) 39.2
2.83 dd (16.2, 4.8) 39.2 2.89 dd
(16.5, 4.0)
2.67 dd
(16.5, 11.0)
35.4 1.97 m
37.4 2.99 dd (16.5, 4.5) 35.2
7b
2.35 dd (13.5, 9.3)
2.35 dd (13.5, 9.5)
2.67 dd (16.2, 12.0)
2.73 dd (16.5, 7.0)
8
1.79 m
39.3
1.78 m
39.3
1.72 m
36.6 2.41 m
34.1
15.5
9
0.88 d (6.6)
16.0
0.87 d (7.0)
16.1
1.14 d (6.0)
20.0 1.14 d (6.5) 20.3 0.97 d (7.0)
10
20
30
40
50
60
70a
70b
80
138.1
105.9
153.2
132.6
153.2
105.9
137.8
105.9
152.5
134.9
152.5
105.9
138.6
111.3
146.4
144.0
114.0
122.6
6.38 br s
6.36 br s
6.56 d (1.8)
6.86 d (7.8)
6.38 br s
6.36 br s
6.67 dd (7.8, 1.8)
3.44 d (10.2)
2.78 dd (13.5, 4.5) 39.4
2.26 dd (13.5, 9.6)
2.77 dd (13.5, 4.5) 39.3
2.26 dd (13.5, 10.0)
54.1
199.3
200.2
1.79 m
38.8
1.78 m
38.8
1.56 m
43.8 2.18 m
48.9 2.66 m
46.4
11.3
90
0.86 d (4.8)
16.5
0.86 d (7.0)
16.4
0.93 d (6.0)
17.1 1.26 d (6.5) 12.8 1.14 d (7.0)
100
200
300
400
500
600
700
800
900
132.0
108.6
146.4
144.4
114.0
118.8
72.8
132.7
109.5
146.4
145.2
114.0
120.7
79.1
131.7
108.3
146.5
144.6
113.9
118.7
72.5
6.98 br s
6.88 br s
6.83 d (1.8)
6.83 d (7.8)
6.67 br s d (7.8)
4.78 d (2.4)
4.34 m
6.86 d (8.0)
6.86 br s d (8.0)
4.60 d (8.5)
3.92 m
6.81 d (7.8)
6.37 dd (7.8, 1.8)
4.65 d (2.4)
4.05 m
82.2
86.6
81.8
1.12 d (6.3)
3.89 s/
12.8
1.17 d (6.0)
3.87 s/
17.6
1.06 d (6.6)
/3.89 s
12.9
3/5-OMe
30/50-OMe
300-OMe
55.8/
55.9/
/55.8 /3.94 s
55.8/
/56.0 /3.95 s
5.47/
/56.0
3.85 s/3.85 s
3.86 s
56.1/56.1 3.84 s/3.84 s
55.9/55.9 3.89 s/
55.9 3.82 s
56.0
3.86 s
55.9
4/40/400-OH
4.95
5.48/
a Data (δ) were measured in CDCl3 for 1H at 300 MHz for 1, 500 MHz for 2, 4, and 5, and 600 MHz for 3; for 13C at 100 MHz for 1 and 2, 125 MHz for 4
1
and 5, and 150 MHz for 3. Proton coupling constants (J) in Hz are given in parentheses. The assignments were based on DEPT, 1Hꢀ H COSY, gHSQC,
and HMBC experiments.
whereas Me-9 was pseudoequatorial in 5 (Supporting Informa-
tion, Figure S60). The CD spectrum of 5 gave a negative Cotton
effect at 326 nm for the n f π* transition and a positive Cotton
effect at 302 nm for the π f π* transition, indicating the 80R
configuration based on the octant rule for cyclohexenones9
(Supporting Information, Figure S62). The 8R,80R configuration
of 5 was supported by calculation of CDs and specific rotations of
the enantiomers (Supporting Information, Figure S180 and
Table S4). Thus, compound 5 was the 8-epimer of 4.
Compound 6, C20H24O5 (HRESIMS and the NMR data),
exhibited spectroscopic data (Tables 2 and 3 and Experimental
Section) diagnostic for 2,70-cyclolignane analogues with two
methoxy and three hydroxy groups substituted at the aromatic
rings.10 HMBC correlations from H-3, H-6, and OMe-5 to C-5,
from H-6 to C-2, C-4, and C-7, from H-60 and OMe-50 to C-50,
and from H-70 to C-1, C-2, C-3, C-10, C-20, C-60, C-80, and C-90
(Supporting Information, Figure S70), together with the shifts of
these proton and carbon resonances, indicated that compound 6
was 30,4,40-trihydroxy-5,50-dimethoxy-2,70-cyclolignan. In the
NOE difference spectrum of 6, H-20, H-60, and H-7a were
enhanced by irradiation of either H-8 or H-80, while H3-9 and
H3-90 were enhanced when H-70 was irradiated. These enhance-
ments combined with the coupling constant (J7 ,8 = 6.5 Hz)
revealed that H-8 and H-80 were cis-oriented, whereas H-70 and
H-80 were trans-oriented.11 The CD spectrum of 6 showed
a negative Cotton effect at 287 nm (Δε ꢀ2.46) and a posi-
tive Cotton effect at 273 nm (Δε þ2.99), suggesting the
70S configuration7 (Supporting Information, Figure S2). Thus,
0
0
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Table 3. 13C NMR Spectroscopic Data (δ) of Compounds 6ꢀ16a
no.
6
7
8
9
10
11
11a
12
132.3
13
132.8
14
15
16
1
127.6 127.7 127.8
130.4 130.6 131.0
116.0 116.1 116.2
143.3 143.0 143.7
145.0 143.5 145.2
110.5 110.5 110.8
133.8
111.4
146.3
143.5
113.9
121.7
38.8
132.7
133.6 127.8
111.4 130.8
146.3 116.2
143.6 143.7
114.0 145.2
121.7 110.8
134.1 135.0
111.4 110.7
146.2 148.2
143.5 146.8
114.0 115.4
121.7 119.9
133.6
2
111.4
146.3
143.6
113.9
121.7
39.1
111.2
146.5
143.9
114.1
121.6
33.2
111.3
146.4
143.8
114.0
121.6
39.8
108.5
146.6
145.2
114.0
119.4
88.7
3
4
5
6
7
35.2
29.1
15.7
35.1
28.9
15.6
35.7
29.9
15.8
39.2
38.8
16.4
35.7
29.9
15.9
39.3
38.9
16.2
88.2
45.2
13.0
8
39.1
38.9
42.7
34.9
48.6
9
16.2
16.2
65.3
15.6
14.9
10
20
30
40
50
60
70
80
90
139.1 140.4 138.3
109.6 114.9 106.2
143.4 145.0 146.9
130.6 141.4 133.1
146.5 116.1 146.9
104.0 121.8 106.2
133.8
109.3
143.5
130.2
146.7
103.8
39.1
133.7
105.6
146.8
132.9
146.8
105.6
39.3
137.6 142.9
105.9 106.5
152.9 153.0
136.0 136.4
152.9 153.0
105.9 106.5
132.1
105.5
146.9
133.0
146.9
105.5
40.1
131.5
105.4
146.9
132.9
146.9
105.4
33.5
133.8 134.2
130.1 104.6
115.0 148.5
153.4 136.1
114.9 148.5
130.1 104.6
131.5
105.2
147.0
133.1
147.0
105.2
38.8
50.7
40.5
50.2
40.6
51.0
40.8
39.4
39.0
16.1
51.2
40.7
16.3
44.8
38.4
16.1
55.8
87.9
39.2
39.0
35.0
42.6
45.6
49.1
15.9
16.1
16.3
16.2
16.1
15.8
65.2
13.2
73.2
3/5-OMe
/55.8
/55.8
/56.0
55.8/
55.8/
56.2/56.2
55.8 /56.0
55.8
55.8
56.1
55.9
30/50-OMe /56.1
40-OMe
56.6/56.6 /56.1
/56.0
60.8
56.3/56.3
61.1
56.2/56.2
56.2/56.2
56.6/56.6
56.3/56.3
9/90-OAc
171.1, 21.0
171.1, 21.0
a Data (δ) were measured in CDCl3 for 13C at 125 MHz for 6ꢀ11, 11a, and 14, 150 MHz for 12, 13, and 16, and 125 MHz in acetone-d6 for 15. Proton
1
coupling constants (J) in Hz are given in parentheses. The assignments were based on DEPT, 1Hꢀ H COSY, gHSQC, and HMBC experiments.
compound 6 was (þ)-(70S,8S,80S)-30,4,40-trihydroxy-5,50-di-
methoxy-2,70-cyclolignan.
S2 and Figures S2 and S98ꢀS100). Therefore, the 8S,80R config-
uration was assigned for 9.
Compound 7 was the 50-demethoxy analogue of 6, as indi-
cated by the spectroscopic data (Tables 2 and 3 and Experimental
Section) and confirmed by comparison of the CD data of 7 and 6
and the reported analogues.8c,12
The spectroscopic data of compound 10 (Tables 2 and 3 and
Experimental Section) indicated that it was the 30-methoxy
analogue of 9, which was proved by the RuO2 2H2O oxidation7
3
of 10 that generated 8 (Supporting Information, Scheme S1 and
Figures S1 and S105ꢀS106).
The spectroscopic data of compound 8 (Tables 2 and 3 and
Experimental Section) demonstrated that it was the 30-methoxy
derivative of 6. This was verified by 2D NMR data analysis of 8
(Supporting Information, Figures S85ꢀS86), which supported
the 1D NMR data assignments (Tables 2 and 3). In the NOE
difference spectrum (Supporting Information, Figure S87), en-
hancements of both H3-9 and H3-90 upon irradiation of H-70
proved the relative configuration of 8, while a negative Cotton
effect at 287 nm (Δε ꢀ3.38) and a positive Cotton effect at
272 nm (Δε þ2.57) in the CD spectrum confirmed the 70S
configuration.8,10 Thus, compound 8 was identified as (þ)-
(70S,8S,80S)-4,40-dihydroxy-30,5,50-trimethoxy-2,70-cyclolignan.
Compound 9, C20H26O5 (HRESIMS and the NMR data),
showed spectroscopic data (Tables 2 and 3 and Experimental
Section) similar to those of 6. However, comparison of the NMR
data of 6 and 9 indicated substitution of the 1,2-disubstituted
4-hydroxy-5-methoxyphenyl unit in 6 by a 4-hydroxy-3-methox-
yphenyl moiety in 9. In addition, the methine group (CH-70) in 6
was replaced by a methylene moiety (CH2-70) in 9. This
indicated that 9 was 30,4,40-trihydroxy-5,50-dimethoxylignan,
which was confirmed by 2D NMR data of 9 (Supporting
Information, Figure S93ꢀS95). Oxidative ring closure of 9 by
using Ag2O in benzene and acetone (2:1)13 yielded a product
(9a) that possessed spectroscopic data including the [R]D and
CD data identical to those of 6 (Supporting Information, Scheme
Compound 11 was the 40-methoxy derivative of 9, as demon-
strated by its spectroscopic data (Tables 2 and 3 and Experi-
mental Section) and confirmed by the RuO2 2H2O oxidative
3
ring closure of 11 that produced 11a, having CD spectroscopic
features similar to those of 6ꢀ8 (Supporting Information,
Schemes S1 and S2 and Figures S1, S2, and S114ꢀS115).
The HRESIMS combined with the NMR data indicated that
compound 12 had the molecular formula C23H30O7. The NMR
data of 12 resembled those of 10 (Tables 2 and 3) except for
replacement of the resonances for Me-9 in 10 by those assignable
to a CH2OAc unit in 12. The presence of OAc was supported by
an absorption band at 1731 cmꢀ1 in the IR spectrum. The 2D
NMR data analysis verified that compound 12 was the 9-OAc
derivative of 10. Especially, HMBC correlations from H2-9 to
C-7, C-8, C-80, and the carbonyl carbon of OAc, from H3-90 to
C-70, C-8, and C-80, from H-20/60 to C-30/50, C-40, and C-70, and
from OMe-30/50 to C-30/50, in combination with shifts of these
proton and carbon resonances, proved the location of the sub-
stituents in 12. Oxidation of 12 using Ag2O yielded 12a, of which
the 1H NMR spectrum showed a coupling constant of
8.4 Hz for H-7b and H-8, indicating that the two protons were
trans pseudo-diaxially oriented. In addition, in the NOE differ-
ence spectrum of 12a, H3-90 was enhanced by irradiation of either
H-7b or H-70 (Supporting Information, Figure S133). This
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revealed a cis-relationship between H-8 and H-80. The CD spectrum
of 12a gave a negative Cotton effect at 289 nm (Δε ꢀ1.79) and a
positive Cotton effect at 272 nm (Δε þ2.49), suggesting the 70S
configuration. Accordingly, compound 12 was elucidated as
(þ)-(8S,80R)-9-acetoxy-4,40-dihydroxy-3,30,50-trimethoxylignan.
The spectroscopic data of compound 13 (Tables 2 and 3 and
Experimental Section) demonstrated that it was the 90-OAc
isomer of 12. This was confirmed by 2D NMR data, particularly,
by HMBC correlations from H2-70 to C-10, C-20/60, and C-90,
from OH-4 to C-3, C-4, and C-5, and from OH-40 to C-30/50 and
C-40, in combination with shifts of these proton and carbon
resonances. In addition, the similarity of the [R]D and CD data
of 13 and 12 supported that the two compounds had the
same 8S,80R configuration. Therefore, compound 13 was (þ)-
(8S,80R)-90-acetoxy-4,40-dihydroxy-3,30,50-trimethoxylignan.
Compound 14 was (þ)-(8S,80R)-4,40-dihydroxy-3-methoxy-
lignan, as indicated by its spectroscopic data (Tables 2 and 3 and
Experimental Section) and confirmed by 2D NMR data analysis
(Supporting Information, Figures S148ꢀS150), as well as by
comparison of the [R]D and CD data of 14 and 9ꢀ13. This was
also supported by comparison of the spectroscopic data of 14 and
threo-4,40-dihydroxy-3-methoxylignan (pycnantolol) with the
opposite [R]D value and undetermined absolute configuration.14
Compound 15 exhibited spectroscopic data (Tables 2 and 3
and Experimental Section) identical to those of fragransin C1, but
with opposite specific rotation.15 2D NMR data and NOESY data
analysis of 15 (Supporting Information, Figures S193ꢀS196)
proved that it had the same planar structure and relative config-
uration as fragransin C1. Although fragransin C1 was reported to
display no Cotton effect in its CD spectrum,16 the CD spectrum
of 15 showed a coupled CD curve, positive at 251 nm (Δε þ0.37)
and negative at 227 nm (Δε ꢀ1.09) (Supporting Information,
Figure S162). On the basis of the CD exciton chirality rule,17
compound 15 was assigned to have the 7S,70R,8S,80R configura-
tion. Thus, compound 15 was defined as (ꢀ)-(7S,70R,8S,80R)-
4,40-dihydroxy-3,30,50-trimethoxy-7,70-epoxylignan.
The spectroscopic data of compound 16 (Tables 2 and 3 and
Experimental Section) indicated that it was an isomer of 15.
Comparison of the NMR data of 16 and 15 indicated that
resonances assignable to the two oxymethylenes in 16 substi-
tuted those for OCH-70 and Me-90 in 15, respectively. This
indicated that 16 was 4,40-dihydroxy-3,30,50-trimethoxy-7,90-
epoxylignan, which was confirmed by the 2D NMR data
(Supporting Information, Figures S167ꢀS169). In the NOE
difference spectrum of 16, irradiation of H-7 enhanced H-80 and
H3-9 and irradiation of H-70b enhanced H-8. The enhancements
revealed that H-8 was trans-oriented to both H-7 and H-80 on the
tetrahydrofuran ring. The CD spectrum of 16 showed a coupled
Cotton effect, positive at 235 nm and negative at 219 nm,
indicating exciton coupling between the π f π* transitions of
the phenyl chromophores (Supporting Information, Figure
S174). The positive chirality revealed the 7R,8R,80R configura-
tion for 16 on the basis of the CD exciton chirality rule.18 Thus,
compound 16 was assigned as (ꢀ)-(7R,8R,80R)-4,40-dihydroxy-
3,30,50-trimethoxy-7,90-epoxylignan.
8R,80R)-4,40-dihydroxy-3,30,5,50-tetramethoxy-7,70-epoxylignan-9,90-
diol,17a and (þ)-(7R,8R,70E)-4-hydroxy-3-methoxy-7,40-epoxy-8,30-
neolignan-70-ene.25
In the preliminary in vitro assays, compounds 1 and 2 inhib-
ited HIV-1 replication with IC50 values of 2.52 and 2.01 μM,
respectively (the positive control efavirenz gave a 42.5 ( 13.1%
inhibition at 0.001 μM). At 10 μM, compounds 6, 8, and
9 reduced DL-galactosamine (GalN)-induced hepatocyte (WB-
F344 cells) damage with 77.2 ( 4.1%, 64.0 ( 5.4%, and 68.0 (
3.7% inhibition, respectively, while the positive control bicyclol
gave a 67.0 ( 4.6% inhibition.26 At the same concentration,
compound 9 attenuated rotenone-induced PC12 cell damage by
increasing the cell viability from 73.0 ( 9.6% to 96.2 ( 15.6%,
and (ꢀ)-syringaresinol (17) and (þ)-lyoniresinol (18) showed
activities against serum deprivation induced PC12 cell damage by
increasing the cell viability from 66.6 ( 9.2% to 73.2 ( 8.8% and
79.4 ( 4.5%, respectively. Other compounds were inactive in
these assays. In addition, the isolates were also assessed for
cytotoxicity against several human cancer cell lines27 and Fe2þ
-
cystine-induced rat liver microsomal lipid peroxidation,28 but
were inactive at a concentration of 1.0 μM.
’ EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were
measured on a Rudolph Research Autopol III automatic polarimeter,
and UV spectra were obtained on a Cary 300 spectrometer. CD spectra
were recorded on a JASCO J-815 CD spectrometer. IR spectra were
recorded on a Nicolet 5700 FT-IR microscope instrument (FT-IR
microscope transmission). 1D and 2D NMR spectra were acquired at
1
300, 500, or 600 MHz for H and 100, 125, or 150 MHz for 13C,
respectively, on Varian Mecury-300 MHz or INOVA 400, 500 MHz, or
SYS 600 MHz spectrometers, in CDCl3 or acetone-d6, 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 (CC) was performed
with silica gel (200ꢀ300 mesh, Qingdao Marine Chemical Inc. Qingdao,
People’s Republic of China), Pharmadex LH-20 (Amersham Bios-
ciences, Inc., Shanghai, China), and MCI gel (CHP20P). Preparative
TLC separation was performed with high-performance silica gel TLC
plates (HSGF254, glass precoated, Yantai Jiangyou Silica Gel Develop-
ment Co., Ltd., Yantai, China). 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 a Prevail (250 ꢁ
10 mm i.d.) preparative column packed with C18 (5 μM). TLC was
carried out with glass precoated silica gel GF254 plates. Spots were
visualized under UV light or by spraying with 7% H2SO4 in 95% EtOH
followed by heating.
Plant Material. The bark of M. robusta was collected in August 2006
at Dayao Mountain, Guangxi Province, People’s Republic of China. The
plant was identified by Mr. Guang-Ri Long (Guangxi Forest Adminis-
tration, Guangxi 545005, China). A voucher specimen (no. 041) was
deposited at the Herbarium of the Department of Medicinal Plants,
Institute of Materia Medica, Chinese Academy of Medical Sciences and
Peking Union Medical College, Beijing 100050, China.
The known compounds were identified by comparing the
spectroscopic data with reported data as meso-dihydro-
guaiaretic acid,19 (ꢀ)-(8R,80S)-3,30,4-trimethoxy-40-hydroxylignan,19
(þ)-(8R,80R)-30,4,40-trihydroxy-3-methoxylignan,20 henricine
B,21 (þ)-guaiacin,22 (ꢀ)-isoguaiacin,11 (ꢀ)-pinoresinol (17),23
(ꢀ)-syringaresinol,23 (þ)-lyoniresinol (18),24 (ꢀ)-(70R,8R,80R)-
4,40-dihydroxy-3,30,5-trimethoxy-2,70-cyclolignan,1b (ꢀ)-(7S,70S,
Extraction and Isolation. The air-dried bark of M. robusta (5.3 kg)
was powdered and extracted with 45 L of aqueous 95% EtOH at room
temperature for 3 ꢁ 48 h. The EtOH extract was evaporated under
reduced pressure to yield a dark brown residue (535 g). The residue was
suspended in H2O (2000 mL) and partitioned with EtOAc (8 ꢁ
2000 mL). After removal of solvent, the EtOAc extract (261.0 g) was
subjected to CC over silica gel, eluting with a gradient of increasing
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acetone (0ꢀ100%) in petroleum ether, to produce nine fractions (AꢀI)
on the basis of TLC analysis. Fraction B (17.5 g) was further fractionated
via silica gel CC, eluting with petroleum etherꢀEtOAc (100:5ꢀ100:20),
to yield B1ꢀB5. Fraction B4 was subjected to CC over Sephadex LH-20,
eluting with petroleum etherꢀCHCl3ꢀMeOH (5:5:1), to obtain 14
(0.5 mg). B5 was separated by normal silica gel CC, eluting with a
data, see Table 2; 13C NMR (CDCl3, 125 MHz) data, see Table 3; (þ)-
ESIMS m/z 221 [M þ H]þ, 243 [M þ Na]þ; (þ)-HRESIMS m/z
221.1174 [M þ H]þ (calcd forC13H17O3, 221.1172), 243.0994 [M þ
Na]þ (calcd for C13H16O3Na, 243.0992).
(þ)-(8R,80R)-4-Hydroxy-5-methoxy-10,20,30,40,50,60-hexanor-2,70-cy-
clolignan-70-one (5): white, amorphous solid; [R]20 þ90.0 (c 0.03,
D
CHCl3); UV (MeOH) λmax (log ε) 207 (4.72), 233 (4.62), 274 (4.55),
315 (4.17) nm; CD (MeOH) 302 (Δε þ0.18), 326 (Δε ꢀ0.03); IR νmax
3355, 2961, 2925, 2853, 1659, 1602, 1509, 1468, 1278, 1025 cmꢀ1; 1H
NMR (CDCl3, 500 MHz) data, see Table 2; 13C NMR (CDCl3,
125 MHz) data, see Table 3; (þ)-ESIMS m/z 221 [M þ H]þ, 243
[M þ Na]þ, 463 [2 M þ Na]þ, 479 [2 M þ K]þ; (þ)-HRESIMS m/z
221.1169 [M þ H]þ (calcd for C13H17O3, 221.1172), 243.0994 [M þ
Na]þ (calcd for C13H16O3Na, 243.0992).
gradient of increasing Me2CO in CHCl3 (0ꢀ100%), to yield B5-1ꢀB5-4
.
B5-2 was subjected to Sephadex LH-20, eluting with petroleum etherꢀ
CHCl3ꢀMeOH (5:5:1), to give B5-2-1ꢀB5-2-11, of which B5-2-5 was
purified by high-performance silica gel preparative TLC plates, using a
mobile phase of petroleum etherꢀMe2CO (1.3:1), to obtain 4 (1.5 mg)
and 5 (1.0 mg). Fraction D (2.0 g) was chromatographed over MCI gel,
eluting with a gradient of increasing MeOH (30ꢀ100%) in H2O, to give
D1ꢀD6. D3 (1.023 g) was chromatographed over Sephadex LH-20,
eluting with a gradient of petroleum etherꢀCHCl3ꢀMeOH (2:2:1).
Subsequent fractions were separated by RP-HPLC using a mobile phase
of MeOHꢀH2O (62:38 or 67:33) to afford 6 (12.6 mg), 7 (1.0 mg), 8
(6.2 mg), 10 (50.2 mg), and 15 (15.1 mg). Fraction E (3.0 g) was
chromatographed over MCI gel, eluting with a gradient of increasing
MeOH (30ꢀ100%) in H2O, to give nine subfractions (E1ꢀE9). E6 was
subjected to CC over Sephadex LH-20, eluting with petroleum
etherꢀCHCl3ꢀMeOH (2:2:1), to give a fraction that was resolved by
RP-HPLC using a mobile phase of MeOHꢀH2O (72:28) to afford 1
(25.0 mg), 2 (89.0 mg), and 3 (0.8 mg). E3 was subjected to CC over
Sephadex LH-20, eluting with petroleum etherꢀCHCl3ꢀMeOH
(2:2:1), to give 11 (21.2 mg) and fractions E3-1ꢀE3-8. Fraction E3-4
was separated by RP-HPLC using a mobile phase of MeOHꢀH2O
(59:41) to afford compounds 9 (4.1 mg), 12 (2.5 mg), and 13 (1.6 mg)
and subfractions E3-4-1ꢀE3-4-3. Fraction E3-4-2 was purified by RP-HPLC
by using a mobile phase of MeOHꢀH2O (55:45) to afford 16 (3.0 mg).
(þ)-(700S,8S,80R,800R)- 4,400-Dihydroxy-3,30,300,50-tetramethoxy-40,800-
(þ)-(70S,8S,80S)-30,4,40-Trihydroxy-5,50-dimethoxy-2,70-cyclolignan (6):
white, amorphous powder, [R]20D þ66.6 (c 0.12, CHCl3); UV (MeOH)
λmax (log ε) 204 (4.86), 237 (4.22, sh), 284 (3.63) nm; CD (MeOH) 273
(Δε þ2.99), 287 (Δε ꢀ2.46) nm; IR νmax 3410, 2960, 2875, 2842, 1613,
1513, 1464, 1456, 1245, 1208, 1024 cmꢀ1; 1H NMR (CDCl3, 500 MHz)
data, see Table 2; 13C NMR (CDCl3, 125 MHz) data, see Table 3; (þ)-
ESIMS m/z 345 [M þ H]þ, 367 [M þ Na]þ, 383 [M þ K]þ 711[2 M þ
Na]þ; (þ)-HRESIMS m/z 345.1706 [M þ H]þ (calcd for C20H25O5,
345.1697), 367.1521 [M þ Na]þ (calcd for C20H24O5Na, 367.1516).
(þ)-(70S,8S,80S)-30,4,40-Trihydroxy-5-methoxy-2,70-cyclolignan (7):
white, amorphous solid, [R]20D þ11.8 (c 0.07, CHCl3); UV (MeOH)
λmax (log ε) 204 (4.32), 232 (3.80, sh), 286 (3.46) nm; CD (MeOH)
274 (Δε þ1.55), 294 (Δε ꢀ2.26) nm; IR νmax 3374, 2957, 2932, 2876,
1594, 1511, 1451, 1372, 1353, 1275, 1249, 1205 cmꢀ1 1H NMR
;
(CDCl3, 500 MHz) data, see Table 2; 13C NMR (CDCl3, 125 MHz)
data, see Table 3; (þ)-ESIMS m/z 315 [M þ H]þ, 337 [M þ Na]þ, 353
[M þ K]þ; (þ)-HRESIMS m/z 315.1590 [M þ H]þ (calcd for
C19H23O4, 315.1591), 337.1412 [M þ Na]þ (calcd for C19H22O4Na,
337.1410).
oxy-8,80-sesquineolignan-700-ol (1): white, amorphous solid; [R]20
D
þ3.1 (c 0.08, CHCl3); UV (MeOH) λmax (log ε) 203 (5.05), 232
(4.37, sh), 280 (3.75) nm; CD (MeOH) 242 (Δε ꢀ1.38) nm; IR νmax
3490, 2958, 2935, 2873, 1603, 1589, 1515,1463, 1425, 1271, 1124,
(þ)-(70S,8S,80S)-4,40-Dihydroxy-30,5,50-trimethoxy-2,70-cyclolignan (8):
white, amorphous powder; [R]20D þ97 (c 0.1, CHCl3); UV (MeOH)
λmax (log ε) 205 (4.35), 283 (3.28) nm; CD (MeOH) 272 (Δε þ2.57),
287 (Δε ꢀ3.38) nm; IR νmax 3418, 2959, 2882, 2840, 1615, 1514, 1460,
1247, 1215, 1116 cmꢀ1; 1H NMR (CDCl3, 600 MHz) data, see Table 2;
13C NMR (CDCl3, 125 MHz) data, see Table 3; (þ)-ESIMS m/z 359
[M þ H]þ, 381 [M þ Na]þ, 397 [M þ K]þ, 739 [2 M þ Na]þ.
(þ)-(8S,80R)-30,4,40-Trihydroxy-5,50-dimethoxylignan (9): white,
amorphous powder; [R]20D þ22.5 (c 0.08, CHCl3); UV (MeOH) λmax
(log ε) 203 (4.94), 232 (4.14, sh), 281 (3.43) nm; CD (MeOH) 271
(Δε þ0.27), 291 (Δε ꢀ0.88) nm; IR νmax 3454, 2970, 2959, 2870, 2851,
1
1033 cmꢀ1; H NMR (CDCl3, 300 MHz) and 13C NMR (CDCl3,
100 MHz) data, see Table 1; (þ)-ESIMS m/z 563 [M þ Na]þ, 579[M þ
K]þ; (þ)-HRESIMSm/z 563.2628[M þ Na]þ (calcd. for C31H40O8Na,
563.2615).
(þ)-(700S,8S,80R,800S)-4,400-Dihydroxy-3,30,300,50-tetramethoxy-40,800-
oxy-8,80-sesquineolignan-700-ol (2): white, amorphous solid; [R]20D þ
44.1 (c 0.13, CHCl3); UV (MeOH) λmax (log ε) 203 (4.84), 232 (4.08,
sh), 280 (3.40) nm; CD (MeOH) 214 (Δε þ2.19), 233 (Δε þ
2.07) nm; IR νmax 3443, 2958, 2936, 2873, 1603, 1589, 1515, 1463,
1424, 1271, 1125, 1035, 754 cmꢀ1; 1H NMR (CDCl3, 500 MHz) and
13C NMR (CDCl3, 100 MHz) data, see Table 1; (þ)-ESIMS m/z 563
[M þ Na]þ, 579 [M þ K]þ; (þ)-HRESIMS m/z 563.2617 [M þ Na]þ
(calcd for C31H40O8Na, 563.2615).
1
1611, 1514, 1467, 1269, 1102, 1021, 932, 818, 796 cmꢀ1; H NMR
(CDCl3, 500 MHz) data, see Table 2; 13C NMR (CDCl3, 125 MHz)
data, see Table 3; (þ)-ESIMS m/z 347 [M þ H]þ, 369 [M þ Na]þ, 385
[M þ K]þ;(þ)-HRESIMSm/z345.1708 [M ꢀ H]ꢀ (calcd for C20H25O5,
345.1707).
(ꢀ)-(70S,700S,8R,80S,800R)-4,400-Dihydroxy-30,300,5-trimethoxy-40,800-
oxy-2,70-cyclo-8,80-sesquineolignan-700-ol (3): white, amorphous
solid; [R]20D ꢀ40.5 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 204
(4.43), 232 (3.52, sh), 280 (3.13) nm; CD (MeOH) 230 (Δε ꢀ1.56),
274 (Δε þ1.81), 291 (Δε ꢀ3.12) nm; IR νmax 3437, 2960, 2931, 2873,
1605, 1514, 1464, 1452, 1431, 1267, 1246, 1209, 1151, 1034 cmꢀ1; 1H
NMR (CDCl3, 600 MHz) and 13C NMR (CDCl3, 150 MHz) data, see
Table 1; (þ)-ESIMS m/z 531 [M þ Na]þ, 547 [M þ K]þ; (þ)-
HRESIMS m/z 531.2362 [M þ Na]þ (calcd. for C30H36O7Na,
531.2353).
(þ)-(8S,80R)-4,40-Dihydroxy-3,30,50-trimethoxylignan (10): white,
amorphous powder, [R]20D þ6.01 (c 0.98, CHCl3); UV (MeOH) λmax
(log ε) 205 (4.94), 232 (3.71), 283 (3.23) nm; CD (MeOH) 264 (Δε
þ0.03), 286 (Δε ꢀ0.01) nm; IR νmax 3383, 2962, 1611, 1513, 1456,
1429, 1271, 1231, 1119, 1029 cmꢀ1; 1H NMR (CDCl3, 500 MHz) data,
see Table 2; 13C NMR (CDCl3, 125 MHz) data, see Table 3; ESIMS m/
z 361 [M þ H]þ, 383 [M þ Na]þ, and 399 [M þ K]þ.
(þ)-(8S,80R)-4-Hydroxy-3,30,40,50-tetramethoxylignan (11): white,
amorphous powder; [R]20D þ4.4 (c 0.090 CHCl3); UV (MeOH) λmax
(log ε) 203 (4.68), 230 (3.99), 280 (3.35) nm; IR νmax 3434, 2957, 2934,
2840, 1590, 1513, 1460, 1423, 1270, 1239, 1128, 1035, 1011 cmꢀ1; 1H
NMR (CDCl3, 500 MHz) data, see Table 2; 13C NMR (CDCl3, 125
MHz) data, see Table 3; (þ)-ESIMS m/z 375 [M þ H]þ, 397 [M þ
Na]þ, and 413 [M þ K]þ; (þ)-HRESIMS m/z 397.2010 [M þ Na]þ
(calcd for C22H30O5Na, 397.1991).
(þ)-(8S,80R)-4-Hydroxy-5-methoxy-10,20,30,40,50,60 -hexanor-2,70-cy-
clolignan-70-one (4): white needles (CHCl3); mp 159ꢀ161 °C; [R]20
D
þ74.3 (c 0.01, CHCl3); UV (MeOH) λmax (log ε) 208 (4.28) nm,
233 (4.24) nm, 273 (4.05) nm, 317 (3.24) nm; CD (MeOH) 301
(Δε ꢀ0.24), 332 (Δε þ0.30); IR νmax 3439, 2969, 2929, 2874, 1665,
1613, 1509, 1452, 1315, 1270, 1020 cmꢀ1; 1H NMR (CDCl3, 500 MHz)
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Chemical Transformation of Compounds 1, 2, 10, and 11.
While a suspension of RuO2 2H2O (15 mg) in a mixture of anhydrous
see Table 2; 13C NMR (CDCl3, 125 MHz) data, see Table 3; (þ)-
ESIMS m/z 301 [M þ H]þ; (þ)-HRESIMS m/z 301.1794 [M þ H]þ
(calcd for C19H25O3, 301.1798); 323.1617 [M þ Na]þ (calcd for
C19H24O3Na, 323.1618).
3
CH2Cl2 (0.5 mL), TFA (0.75 mL), and TFAA (0.35 mL) was stirred
at ꢀ10 °C, a solution of 1 (15 mg) in CH2Cl2 (0.5 mL) was added
dropwise, followed immediately by adding BF3ꢀEt2O (37.5 μL). After
stirring at 18 °C ((2 °C) for 8 h, the reaction mixture was treated with a
saturated NaHCO3 solution (2 mL) at 0 °C and extracted with CHCl3
(ꢁ3). The CHCl3 extract was evaporated under reduced pressure, and
the residue was subjected to RP-HPLC using a mobile phase of
(ꢀ)-(7S,70R,8S,80R)-4,40-Dihydroxy-3,30,50-trimethoxy-7,70-epoxylignan
(15): white, amorphous powder, [R]20 ꢀ2.2 (c 0.31, MeOH); UV
D
(MeOH) λmax (log ε) 204 (4.55), 232 (3.83, sh), 280 (3.30) nm; CD
(MeOH) 227 (Δε ꢀ1.09), 251 (Δε þ0.37); IR νmax 3431, 2961, 2937,
2875, 2844, 1613, 1518, 1463, 1429, 1213, 1116 cmꢀ1 1H NMR
;
(CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) data, see Table 2
and Table 3; (þ)-ESIMS m/z 375 [M þ H]þ, 397 [M þ Na]þ, 413
[M þ K]þ, 771 [2 M þ Na]þ; HRESIMS m/z 375.1796 [M þ H]þ
(calcd for C21H27O6, 375.1802), 397.1614 [M þ Na]þ (calcd for
C21H26O6Na, 397.1622).
1
MeOHꢀH2O (75:25) to afford 1a (2.5 mg), of which the H NMR
and CD data were identical to those of the natural product 8 isolated
from the plant material (Supporting Information, Figures S3). By
following the same procedure, 2a, 10a, and 11a were obtained from 2,
10, and 11, respectively. 2a and 10a had spectroscopic data identical
to those of 1a and 8. Compound 11a was determined to be (þ)-
(70S,8S,80S)-4-hydroxy-30,40,5,50-tetramethoxy-2,70-cyclolignan on the
basis of the following data: [R]20D þ75 (c 0.1, CHCl3); UV (MeOH)
λmax (log ε) 205 (4.72), 284 (3.37) nm; CD (MeOH) 270 (Δε þ2.58),
288 (Δε ꢀ2.29) nm; 1H NMR (CDCl3, 600 MHz) data, see Table 2;
13C NMR (CDCl3, 125 MHz) data, see Table 3.
(ꢀ)-(7R,8R,80R)-4,40-Dihydroxy-3,30,50-trimethoxy-7,90-epoxylignan
(16): white, amorphous powder; [R]20 ꢀ25.6 (c 0.08, MeOH); UV
D
(MeOH) λmax (log ε) 204 (4.06), 232 (3.26, sh), 280 (2.70) nm; CD
(MeOH) 219 (Δε ꢀ1.13), 235 (Δε þ1.82); IR νmax 3418, 2956, 2936,
2872, 2843, 1612, 1517, 1462, 1274, 1215, 1116, 1034 cmꢀ1; 1H NMR
(CDCl3, 600 MHz) and 13C NMR (CDCl3, 150 MHz) data, see Table 2
and Table 3; (þ)-ESIMS m/z 375 [M þ H]þ, 397 [M þ Na]þ;
HRESIMS m/z 375.1809 [M þ H]þ (calcd for C21H27O6, 375.1802),
397.1632 [M þ Na]þ (calcd for C21H26O6Na, 397.1622).
Anti-HIV Activity Assay. See ref 29.
(þ)-(8S,80R)-9-Acetoxy-4,40-dihydroxy-3,30,50-trimethoxylignan (12):
white, amorphous powder; [R]20D þ7.3 (c 0.15, CHCl3); UV (MeOH)
λmax (log ε) 203 (5.00), 232 (4.34, sh), 281 (3.90) nm; CD (MeOH) 221
(Δε þ1.63), 268 (Δε þ0.52), 290 (Δε ꢀ0.32) nm; IR νmax 3432, 2955,
2935, 2852, 1732, 1611, 1515, 1460, 1369, 1240, 1116, 1035 cmꢀ1; 1H
NMR (CDCl3, 600 MHz) data, see Table 2; 13C NMR (CDCl3,
150 MHz) data, see Table 3; (þ)-ESIMS m/z 441 [M þ Na]þ, 457
[M þ K]þ; (þ)-HRESIMS m/z 441.1891 [M þ Na]þ (calcd for
C23H30O7Na, 441.1884).
Cells, Culture Conditions, and Cell Proliferation Assay. See
ref 29c.
Protective Effect of DL-Galactosamine-Induced WB-F344
Cell Damage. See ref 1b.
PC12 Cell Protection Assay. See ref 30. PC12 cells at a density of
5 ꢁ 103 cells per well in 96-well plates were cultured in DMEM media
(Hyclone) supplemented with 5% fetal bovine serum (FBS, Hyclone),
5% horse serum (Hyclone), and L-glutamine (2 mM). Cultures were
maintained at 37 °C in 5% CO2 in a humidified incubator. After
incubation for 48 h, compounds at concentrations of 10 and/or 4 μM
rotenone were added to the cells. After incubation for another 48 h,
10 μL of the 5 mg/mL MTT (Sigma) was added and maintained for 4 h.
Absorbance was measured at 570 nm using an Ultramark microplate
reader. Cell viability was evaluated.
Chemical Transformations of Compounds 9 and 12. A
solution of 9 (13.0 mg) in benzene (0.9 mL) and acetone (0.45 mL) was
reacted over Ag2O (11.6 mg) at room temperature for 3 h. The reaction
mixture was filtered, the filtrate was evaporated under reduced pressure,
and the residue was purified by RP-HPLC using a mobile phase of
1
MeOHꢀH2O (59:41), to give product 9a (4.3 mg), of which the H
NMR and CD data were identical to those of the natural product 6
isolated from the plant material (Supporting Information, Figures S17).
By following the same procedure, 12a (0.6 mg) was obtained from 12
(2.5 mg). Compound 12a was determined to be (þ)-(70S,8S,80S)-9-
acetoxy-4,40-dihydroxy-30,5,50-trimethoxy-2,70-cyclolignan on the basis
of the following data: white, amorphous powder; [R]20D þ73.6 (c 0.05,
CHCl3); CD (MeOH) 241 (Δε þ4.04), 272 (Δε þ2.49), 289 (Δε
ꢀ1.79) nm; 1H NMR (acetone-d6, 600 MHz) δ 7.22 (1H, s, OH), 6.98
(1H, s, OH), 6.69 (1H, s, H-5), 6.34 (3H, s, H-3, H-20, and H-60), 4.08
(1H, dd, J = 10.8 and 5.4 Hz, H-9a), 3.91 (1H, dd, J = 10.8 and 8.4 Hz,
H-9b), 3.81 (3H, s, OMe-5), 3.72 (6H, s, OMe-30/50), 3.67 (1H, d, J = 6.0
Hz, H-70), 2.87 (1H, dd, J = 16.2 and 5.4 Hz, H-7a), 2.59(1H, dd, J = 16.2
and 8.4 Hz, H-7b), 2.25 (1H, m, H-80), 2.17 (1H, m, H-8), 0.91 (3H, s,
H3-90); (þ)-ESIMS m/z 439 [M þ Na]þ, 455 [M þ K]þ; (ꢀ)-ESIMS
m/z 415 [M ꢀ H]ꢀ.
’ ASSOCIATED CONTENT
S
Supporting Information. Copies of spectra of com-
b
pounds 1ꢀ16. This material is available free of charge via the
Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Tel: 86-10-83154789. Fax: 86-10-63017757. E-mail:
zhuchenggen@imm.ac.cn and shijg@imm.ac.cn.
(þ)-(8S,80R)-90-Acetoxy-4,40-dihydroxy-3,30,50-trimethoxylignan(13):
white, amorphous powder, [R]20D þ13.5 (c 0.14, CHCl3); UV (MeOH)
λmax (log ε) 203 (4.86), 232 (4.05, sh), 281 (3.37) nm; CD (MeOH)
208.5 (Δεþ2.28), 239 (Δεþ1.58), 271 (Δεþ0.96), 290 (Δεꢀ0.79) nm;
IR νmax 3437, 2958, 2939, 2843, 1731, 1612, 1515, 1461, 1429, 1368, 1116,
’ ACKNOWLEDGMENT
The authors are grateful to Dr. L. Li and Professor Yikang Si for
calculation of CDs and specific rotations. Financial support from
the National Natural Sciences Foundation of China (NNSFC;
grant nos. 30825044 and 20932007) and the National Science and
Technology Project of China (nos. 2009ZX09311-004 and
2009ZX09301-003-4-1) is acknowledged.
1
1035 cmꢀ1; H NMR (CDCl3, 600 MHz) data, see Table 2; 13C NMR
(CDCl3, 150 MHz) data, see Table 3; (þ)-ESIMSm/z419 [M þ H]þ, 441
[M þ Na]þ, 457 [M þ K]þ; (þ)-HRESIMS m/z 441.1888 [M þ Na]þ
(calcd for C23H30O7Na, 441.1884).
(þ)-(8S,80R)-4,40-Dihydroxy-3-methoxylignan (14): white, amor-
phous powder; [R]20 þ8.7 (c 0.08, MeOH); UV (MeOH) λmax
D
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