Three new lignan derivatives from Lindera glauca (Siebold et Zucc.) blume

Article abstract of DOI:10.1002/hlca.201500002

Two new aryl-tetralin lignan glycosides, linderanosides A and B (1 and 2, resp.), and a new dihydrobenzofuran neolignan glycoside, linderanoside C (3), together with five known lignan derivatives (4-8) were isolated from the trunk of Lindera glauca. The structures of these new compounds were determined through spectroscopic analyses, including extensive 2D-NMR data and acid hydrolysis. The absolute configurations of the compounds were clarified by circular dichroism (CD) spectroscopic studies. Compounds 1-8 were evaluated for their cytotoxicity against A549 (non-small cell lung adenocarcinoma), SK-OV-3 (ovarian cancer cells), A498 (human kidney epithelial cells), and HCT-15 (colon cancer cells) human tumor cell lines using sulforhodamine B assays in vitro.

Full text of DOI:10.1002/hlca.201500002

Helvetica Chimica Acta – Vol. 98 (2015)
1087
Three New Lignan Derivatives from Lindera glauca (Siebold et Zucc.) Blume
a
a
a
b
a
by Won Se Suh ), Ki Hyun Kim ), Ho Kyung Kim ), Sang Un Choi ), and Kang Ro Lee* )
a) Natural Products Laboratory, School of Pharmacy, Sungkyunkwan University, Suwon 440-746, Korea
(
phone: þ 82-31-2907710; fax: þ 82-31-2907730; e-mail: krlee@skku.ac.kr)
b) Korea Research Institute of Chemical Technology, Daejeon 305-600, Korea
Two new aryl-tetralin lignan glycosides, linderanosides A and B (1 and 2, resp.), and a new
dihydrobenzofuran neolignan glycoside, linderanoside C (3), together with five known lignan derivatives
(4 – 8) were isolated from the trunk of Lindera glauca. The structures of these new compounds were
determined through spectroscopic analyses, including extensive 2D-NMR data and acid hydrolysis. The
absolute configurations of the compounds were clarified by circular dichroism (CD) spectroscopic
studies. Compounds 1 – 8 were evaluated for their cytotoxicity against A549 (non-small cell lung
adenocarcinoma), SK-OV-3 (ovarian cancer cells), A498 (human kidney epithelial cells), and HCT-15
(
colon cancer cells) human tumor cell lines using sulforhodamine B assays in vitro.
Introduction. – Lindera glauca (Siebold et Zucc.) Blume is a deciduous shrub
belonging to the Lauraceae family and widely distributed in Korea, China, and Japan
[
1]. L. glauca has been used in Korean traditional medicine to treat diverse diseases
such as paralysis, pain, extravasation, and cancer without any side effect [2]. Previous
phytochemical investigations on L. glauca reported the isolation of alkaloids,
butanolides, terpenoids, and phenolic compounds [3 – 8].
The nitrogen-containing compounds and monoterpenes from L. glauca were
reported to have anti-tumor activities [9]. As part of our efforts to search for bioactive
constituents of Korean medicinal plants with anti-tumor activity, we found that the
MeOH extract of the twigs of L. glauca had excellent cytotoxic activity against human
cancer cells using the sulforhodamine B (SRB) bioassay.
Our earlier phytochemical investigation on L. glauca resulted in the isolation of
anti-inflammatory lignans from CHCl -soluble fraction [8]. In the process of our
3
continuing efforts to study this source, we further isolated eight lignans (1 – 8),
including three new lignan glycoside derivatives, named linderanosides A – C (1 – 3,
resp.) from the AcOEt soluble fraction. We describe the isolation, and structural
determination of compounds 1 – 8, and the cytotoxic activities of the isolates.
Results and Discussion. – A MeOH extract of twigs of L. glauca was partitioned
successively with hexane, CHCl , AcOEt, and BuOH. Repeated chromatographic
3
purification of the AcOEt-soluble fraction afforded three new lignan glycosides (1 – 3),
together with five known lignan derivatives (4 – 8; Fig. 1).
Compound 1 was obtained as an amorphous gum. The molecular formula of 1 was
þ
determined to be C H O on the basis of a [M þ Na] peak (m/z 575.2104
2
7
36 12
þ
(
C H NaO ; calc. 575.2104)) in positive-ion high-resolution fast-atom bombardment
27 36 12
ꢀ
2015 Verlag Helvetica Chimica Acta AG, Zürich

1
088
Helvetica Chimica Acta – Vol. 98 (2015)
Fig. 1. Chemical structures of compounds 1 – 8
1
mass spectrometry (HR-FAB-MS). The H-NMR spectrum showed three aromatic H-
atoms d(H) 6.61 (s, HÀC(6)), 6.40 (s, HÀC(2’,6’)) and four aromatic MeO groups d(H)
1
3
.38 (s, MeOÀC(3)), 3.76 (s, MeOÀC(3’,5’)), 3.89 (s, MeOÀC(5); Table). H- and
1
3
C-NMR spectral data of 1 were similar to those of lyoniside (4) [10][11] isolated from
this source, except the signals assigned to the sugar unit (1: d(H) 4.92 (d, J ¼ 1.7,
HÀC(1’’)); d(C) 109.8, 85.5, 83.9, 78.8, and 63.0; 4: d(H) 4.30 (d, J ¼ 7.5, HÀC(1’’));
d(C) 105.2, 78.2, 75.2, 71.4, and 67.2), indicating that 1 had an arabinofuranose moiety
[
12] instead of the xylopyranose moiety in 4. This structure was confirmed by analysis of
1
1
the H, H-COSY, HMQC, and HMBC spectra (Fig. 2). The coupling constant (J ¼ 1.7)
of HÀC(1’’) suggested the a-configuration of the arabinose [12][13]. The arabinose unit
was placed at C(9’) by the observation of an HMBC from HÀC(1’’) to C(9’). Acid
hydrolysis of 1 afforded the aglycone, lyoniresinol (5), which was identified by
1
comparison of its H-NMR data [14], together with l-arabinose, which was identified
by Co-TLC analysis with an authentic sample (R of arabinose (CHCl /MeOH/H O
f
3
2
6
:4 :1) 0.55), and GC analysis [15]. Finally, the positions of the four MeO groups were
confirmed to be at C(3), C(5), C(3’), and C(5’) respectively, by the HMBC cross-peaks
of MeOÀC(3)/C(3), MeOÀC(5)/C(5), MeOÀC(3’)/C(3’), and MeOÀC(5’)/C(5’)
(
Fig. 2).
The absolute configuration of 1 was established on the basis of the examination of
the CD spectrum of 1 in combination with the NOESY experiment. The observed
NOESY correlations (Fig. 3) of HÀC(7’)/HÀC(9’), HÀC(2’)/HÀC(8’) and HÀC(7’)/
HÀC(8) indicated the relative configuration as (7’S*,8R*,8’R*). The CD spectrum of 1
showed positive Cotton effects at 242 and 272 nm consistent with those of the reported
compound, (þ)-lyoniresinol 3a-O-b-d-glucopyranoside [15]. Consequently, the abso-
lute configuration of 1 was determined to be (7’S,8R,8’R). Thus, the structure of 1 was

Helvetica Chimica Acta – Vol. 98 (2015)
1089
1
1
Fig. 2. Key HMBC (H ! C) and H, H-COSY (—) correlations of 1, 2, and 3
established as (þ)-(7’S,8R,8’R)-lyociresinol 9’-O-a-l-arabinofuranoside, and named
linderanoside A.
Compound 2 was isolated as amorphous gum with the molecular formula of
þ
C H O based on the positive-ion HR-FAB-MS data (m/z 575.2104 ([M þ Na] ,
2
7
36 12
þ
12
1
C H NaO ; calc. 575.2104)). The H-NMR spectrum showed two sets of aromatic H-
2
7
36
atoms at d(H) 6.61 (s, HÀC(6)), 6.48 (s, HÀC(2’,6’)), and four aromatic MeO groups at
1
d(H) 3.22 (s, MeOÀC(3)), 3.78 (s, MeOÀC(3’,5’)), 3.87 (s, MeOÀC(5)) (Table). H-
1
3
and C-NMR spectral data of 2 closely resembled those of nudiposide (7) [10][11], but
with differences being the chemical shifts of C(1), C(2), C(8), C(1’), C(7’), and C(8’)
(2: d(C) 127.7, 128.1, 35.2, 135.1, 41.9, 42.6; 7: d(C) 126.7, 129.9, 40.6, 139.5, 43.4, 46.7,
resp.), indicating that compound 2 was a stereoisomer of 7 at C(7’), C(8), and C(8’).
1
1
The H, H-COSY, HMQC, and HMBC correlations confirmed the planar structure of
2
(Fig. 2). The coupling constant (J ¼ 7.5 Hz) of the HÀC(1’’) of d-xylose suggested
that it was the b-form [11][16]. Acid hydrolysis of 2 gave polystachyol (2a) by
comparison of its H-NMR spectrum data [17], as well as d-xylose, which was identified
1
by Co-TLC analysis with an authentic sample (R of xylose (CHCl /MeOH/H O 8 :5 :1)
f
3
2
0
.56), and GC analysis [14]. The absolute configuration of 2 was assigned on the basis
of the examination of the CD spectrum of 2 in combination with the NOESY
experiment. The small coupling constant (J ¼ 4.5) between HÀC(7’) and HÀC(8’), as
opposed to the large coupling constant (J ¼ 7.1) between HÀC(7’) and HÀC(8’) in 7,
established that HÀC(7’) and HÀC(8’) are in the same orientation [11][18]. Also, the

1
090
Helvetica Chimica Acta – Vol. 98 (2015)

Helvetica Chimica Acta – Vol. 98 (2015)
1091
Fig. 3. Key NOESY (H $ H) correlations of 1 and 2
NOESY correlations of HÀC(8)/HÀC(2’), HÀC(2’)/HÀC(9’), and HÀC(7’)/HÀC(8’)
confirmed the relative configuration of 2 to be (7’S*,8S*,8’S*) (Fig. 3). In the CD
spectrum, positive Cotton effects at 247 and 272 nm indicated that 2 had (7’S,8S,8’S)
configuration [11][15][19]. On the basis of above data, compound 2 was determined as
(
þ)-(7’S,8S,8’S)-lyociresinol 9’-O-b-d-xylopyranoside, and named linderanoside B.
Compound 3 was obtained as amorphous gum. The HR-FAB-MS displayed a
þ
þ
11
molecular ion peak (m/z 545.1998 ([M þ Na] , C H NaO ; calc. 545.1999)),
26
34
1
consistent with a molecular formula of C H O . The H-NMR spectrum showed
26
34 11
the presence of four aromatic H-atoms at d(H) 6.62 (s, HÀC(2,6)), 6.64 (s, HÀC(2’)),
.65 (s, HÀC(6’)), two CH groups at d(H) 2.52 (t, J ¼ 7.6, CH (7’)), and 1.74 – 1.68 (m,
6
2
2
CH (8’)), two CH O groups at d(H) 4.02 – 3.98 (m, H ÀC(9)), 3.70 – 3.67 (m,
2
2
a
H ÀC(9)), and 3.46 (t, J ¼ 6.5, HÀC(9’)), two CH H-atoms at d(H) 5.50 (d, J ¼ 6.2,
b
HÀC(7)), and 3.54 – 3.51 (m, HÀC(8)), and three aromatic MeO groups at d(H) 3.72 (s,
1
13
MeOÀC(3,5)), and 3.77 (MeOÀC(3’); Table). H- and C-NMR spectral data were
quite similar with data for rel-(7R,8S)-3,3’,5-trimethoxy-4’,7-epoxy-8,5’-neolignan-
4
[
1
,9,9’-triol 9-b-d-glucopyranoside, which was isolated from Selaginella moellendorffii
20], except for the signals attributable to a sugar unit (3: d(H) 4.22 (d, J ¼ 7.5); d(C)
05.6, 78.1, 75.2, 71.4, and 67.2), indicating that 3 had a xylopyranose moiety instead of
the glucopyranose moiety in rel-(7R,8S)-3,3’,5-trimethoxy-4’,7-epoxy-8,5’-neolignan-
,9,9’-triol 9-b-d-glucopyranoside. This structure was confirmed by analysis of the
4
1
1
H, H-COSY, HMQC, and HMBC spectra (Fig. 2). The xylose unit was linked at C(9’)
which was proved by the detection of an HMBC from HÀC(1’’) to C(9’). The coupling
constant (J ¼ 7.5) of the HÀC(1’’) of d-xylose suggested that it was the b-form [11][16].
Acid hydrolysis of 3 yielded the aglycone (3a) which was identified as (7R,8S)-3,3’,5-
1
trimethoxy-4’,7-epoxy-8,5’-neolignan-4,9,9’-triol [20] by comparing its H-NMR spec-
trum data, together with d-xylose, which was identified by Co-TLC with an authentic
sample (R of xylose (CHCl /MeOH/H O 8 :5 :1) 0.56), and GC analysis [14]. The
f
3
2
absolute configuration at C(7) and C(8) were identified to be (7R) and (8S),
respectively, based on the coupling constants (J ¼ 6.2) between HÀC(7) and HÀC(8) in
1
H-NMR spectrum of 3 [20] and the CD spectrum showing a positive Cotton effect at
2
16 nm and a negative Cotton effect at 233 nm [21]. Thus, the structure of 3 was

1
092
Helvetica Chimica Acta – Vol. 98 (2015)
determined to be (7R,8S)-3,3’,5-trimethoxy-4’,7-epoxy-8,5’-neolignan-4,9,9’-triol 9-b-d-
xylopyranoside, and named linderanoside C.
The five known lignans were identified as lyoniside (4) [10][11], lyoniresinol (5)
[
[
14], 5-methoxy-9-b-d-xylopyranosyl-(À)-isolariciresinol (6) [22], nudiposide (7)
10][11], and ssioriside (8) [23][24] by comparing their spectroscopic data with the
reported data in the literature.
To evaluate compounds 1 – 8 as cytotoxic agents, we evaluated their anti-
proliferative activities against the A549, SK-OV-3, A498, and HCT-15 cell lines using
the SRB bioassay [25]. Doxorubicin was used as a positive control. The cytotoxicities of
doxorubicin against the A549, SK-OV-3, A498, and HCT-15 cell lines showed IC50
values of 0.076 Æ 0.003, 0.114 Æ 0.026, 0.043 Æ 0.007, and 1.124 Æ 0.064 mm, respectively.
Compounds 1 – 4, and 7 had selective cytotoxicity against A498 cells, with IC values of
50
2
0.86 Æ 0.94, 21.85 Æ 0.61, 22.67 Æ 1.16, 18.95 Æ 0.55, and 28.42 Æ 0.80 mm, respectively.
However, both compounds were inactive against the other cell lines (IC > 30 mm).
50
The other compounds were inactive against the four tested cell lines (IC > 30 mm).
50
This research was supported by the Basic Science Research Program through the National Research
Foundation of Korea (NRF), funded by the Ministry of Education, Science and Technology
(
2013R1A1A2A10005315). We are thankful to the Korea Basic Science Institute (KBSI) for the
measurements of NMR and MS spectra.
Experimental Part
General. TLC: silica gel 60 F254 and RP-18 F254s silica gel plates (Merck, Germmay); detection under
UV light and by spraying with 10% aq. H SO soln., followed by heating at 1208 for 1 min. Column
2
4
chromatography (CC): silica gel (SiO , 230 – 400 mesh; Merck, Germany), Lichroprep RP gel (40 –
2
18
6
0 mm; Merck, Darmstadt, Germany), and Sephadex LH-20 (Amersham Pharmacia Biotech, UK).
HPLC: prep. HPLC Gilson 306 pump, Gilson-101 RI detector, Phenomenex-Luna-C18-(2) column
(
5 mm, 250 mm  10.00 mm i.d.); tR in min. Optical rotation: JASCO P-1020 polarimeter (JASCO,
Japan). UV Spectra: Shimadzu UV-1601 UV-Visible spectrophotometer (Shimadzu, Japan) using MeOH
as a solvent; lmax (log e) in nm. CD Spectra: Jasco J-715 spectropolarimeter (JASCO, Japan) using MeOH
À1
1
as a solvent; l
(De) in nm. IR Spectra: Bruker IFS-66/S FT-IR spectrometer; n˜in cm . H- and
max
13
C-NMR spectra: Bruker AVANCEIII 700 NMR spectrometer; d in ppm rel. to Me Si as internal
4
standard, J in Hz. FAB-MS and HR-FAB-MS: JEOL JMS-700 (Jeol, Japan) mass spectrometer; in m/z.
Plant Material. The twigs of L. glauca were purchased from Hongcheon, Chungcheongbuk-do,
Korea, in March 2010. The plant was identified by one of the authors (K. R. L.). A voucher specimen
(
SKKU 2010-3B) has been deposited with the herbarium of the School of Pharmacy, Sungkyunkwan
University, Suwon, Korea.
Extraction and Isolation. The twigs of L. glauca (6 kg) were extracted with 80% MeOH two times
under reflux (MeOH/H O 80 :20; 2 Â 10 l). The MeOH extract (120 g) was suspended in dist. H O (1 l)
2
2
and then successively partitioned with hexane (3 Â 800 ml), CHCl (3 Â 800 ml), AcOEt (3 Â 800 ml),
3
and BuOH (3 Â 800 ml), yielding 2.5, 13.3, 6.4, and 17.5 g of residues, resp. The AcOEt soluble fraction
(
6.0 g) was separated by CC (SiO (35 g), CHCl /MeOH 50 :1 ! 1:1 (300 ml each)) to give 13 fractions,
2
3
Frs. A – M. Fr. D (425 mg) was subjected to CC (Sephadex LH-20 (100 g); CH Cl /MeOH 1:1) and
2
2
further separated by semi-prep. HPLC (RP-C18 ; MeOH/H O 35 :65; 2 ml/min) to yield 5 (t
2
R
3
6.2 min;12 mg). Fr. G (417 mg) was subjected to CC (RP-C18 (15 g); MeOH/H O 40 :60): Frs. G1 –
2
G8. Fr. G3 (32 mg) was purified by prep. HPLC (RP-C ; MeOH/H O 85 :15; 2 ml/min): 1 (t 27.1 min;
18
2
R
5
4
4
mg) and 2 (tR 33.2 min; 5 mg). Fr. G6 (47 mg) was purified by prep. HPLC (RP-C ; MeOH/H O
18 2
0 :60; 2 ml/min): 3 (t 34.2 min; 3 mg). Fr. H (635 mg) was subjected to CC (RP-C (15 g); MeOH/H O
R
18
2
0 :60): Frs. H1 – H8. Fr. H2 (263 mg) was purified by prep. HPLC (RP-C ; MeOH/H O 38 :62; 2 ml/
18
2

Helvetica Chimica Acta – Vol. 98 (2015)
1093
min): 4 (t 19.0 min; 90 mg) and 7 (t 21.2 min; 73 mg). Fr. H4 (29 mg) was purified by prep. HPLC (RP-
R
R
C ; MeOH/H O 40 :60; 2 ml/min): 6 (t 19.2 min; 7 mg) and 8 (tR 26.1 min; 8 mg).
18
2
R
Linderanoside A (¼(þ)-(7’S,8R,8’R)-Lyociresinol 9’-O-a-l-Arabinofuranoside; ¼ [(1S,2R,3R)-
1
,2,3,4-Tetrahydro-7-hydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)-3-(hydroxymethyl)-6,8-dimethoxy-2-
25
naphthalenyl]methyl a-l-Arabinofuranoside; 1). Amorphous gum. [a]D ¼ þ20.0 (c ¼ 0.30, MeOH). UV
(
1
5
MeOH): 228 (4.1), 284 (3.2). CD (MeOH): 242 (þ 32.5), 272 (þ 5.9), 287 (À2.2). IR (KBr): 3385, 2924,
1 13
611, 1513, 1462, 1221, 1113, 670. H- (700 MHz) and C-NMR (175 MHz): see Table. HR-FAB-MS:
þ
þ
12
75.2104 ([M þ Na] , C H NaO ; calc. 575.2104).
27
36
Linderanoside B (¼(þ)-(7’S,8S,8’S)-Lyociresinol 9’-O-b-d-Xylopyranoside; ¼ [(1S,2S,3S)-1,2,3,4-
Tetrahydro-7-hydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)-3-(hydroxymethyl)-6,8-dimethoxy-2-naphtha-
lenyl]methyl b-d-Xylopyranoside; 2). Amorphous gum. [a]2 ¼ þ108.0 (c ¼ 0.35, MeOH). UV (MeOH):
5
D
2
1
(
25 (4.0), 284 (3.1). CD (MeOH): 247 (þ100.1), 272 (þ44.2), 287 (À4.8). IR (KBr): 3423, 2924, 1641,
1 13
548, 1501, 1218, 1113, 673. H- (700 MHz) and C-NMR (175 MHz): see Table. HR-FAB-MS: 575.2104
þ
þ
12
[M þ Na] , C H NaO ; calc. 575.2104).
27
36
Linderanoside C (¼(7R,8S)-3,3’,5-Trimethoxy-4’,7-epoxy-8,5’-neolignan-4,9,9’-triol 9-b-d-Xylopyr-
anoside; ¼ [(2R,3S)-2,3-Dihydro-2-(4-hydroxy-3,5-dimethoxyphenyl)-5-(3-hydroxypropyl)-7-methoxy-
5
3
(
1
-benzofuranyl]methyl b-d-Xylopyranoside; 3). Amorphous gum. [a]2 ¼ À6.0 (c ¼ 0.30, MeOH). UV
D
MeOH): 210 (4.3), 288 (3.2). CD (MeOH): 216 (þ6.1), 233 (À6.2), 291 (À2.9). IR (KBr): 3385, 2924,
1 13
611, 1548, 1501, 1462, 1216, 1117, 1033, 673. H- (700 MHz) and C-NMR (175 MHz): see Table. HR-
þ
þ
11
FAB-MS: 545.1998 ([M þ Na] , C H NaO ; calc. 545.1999).
2
6
34
Acid Hydrolysis of Compound 1 – 3. Compounds 1 – 3 (each 1 mg) were hydrolyzed by 1n HCl
(
dioxane/H O 1:1, 2 ml) under reflux for 2 h. After cooling, the mixture was diluted with H O and
2
2
extracted with CHCl . The CHCl3 was removed under reduced pressure to give lyoniresinol (5),
3
polystachyol (2a), and (7R,8S)-3,3’,5-trimethoxy-4’,7-epoxy-8,5’-neolignan-4,9,9’-triol (3a). The struc-
tures were identified by 1H-NMR and comparing these data with those reported in the literature
[
15][17][19].
Lyoniresinol (¼(6R,7R,8S)-5,6,7,8-Tetrahydro-8-(4-hydroxy-3,5-dimethoxyphenyl)-6,7-bis(hydroxy-
methyl)-1,3-dimethoxynaphthalen-2-ol; 5). Amorphous gum. 1H-NMR (CD OD, 700 MHz): 6.60 (s,
3
HÀC(2’)); 6.41 (s, HÀC(2,6)); 4.32 (d, J ¼ 5.5, CH (7)); 3.87 (s, MeOÀC(3’)); 3.75 (s, MeOÀC(3,5)); 3.61
2
(
2
(
dd, J ¼ 10.0, 5.0, H ÀC(9’)); 3.50 (overlap, H ÀC(9’)); 3.50 (d, J ¼ 5.0, CH (9)); 3.40 (s, MeOÀC(5’));
a
b
2
.72 (dd, J ¼ 15.0, 5.0, H ÀC(7’)); 2.59 (dd, J ¼ 15.0, 11.0, H ÀC(7’)); 2.00 – 1.98 (m, HÀC(8)); 1.66 – 1.62
a
b
m, HÀC(8’)).
Polystachyol (¼(6S,7S,8S)-5,6,7,8-Tetrahydro-8-(4-hydroxy-3,5-dimethoxyphenyl)-6,7-bis(hydroxy-
methyl)-1,3-dimethoxynaphthalen-2-ol; 2a). Amorphous gum. 1H-NMR (CD OD, 700 MHz): 6.60 (s,
3
HÀC(2’)); 6.41 (s, HÀC(2,6)); 4.60 (d, J ¼ 4.4, CH (7)); 3.87 (s, MeOÀC(3’)); 3.76 (s, MeOÀC(3,5)); 3.61
2
(
dd, J ¼ 10.0, 5.0, H ÀC(9’); 3.62 – 3.58 (m, H ÀC(9’)); 3.50 (d, J ¼ 5.0, CH (9)); 3.27 (s, MeOÀC(5’)); 3.00
a
b
2
(
dd, J ¼ 17.0, 5.7, H ÀC(7’)); 2.67 (dd, J ¼ 17.0, 11.3, H ÀC(7’)); 2.04 – 2.00 (m, HÀC(8)); 2.01 – 1.98 (m,
a
b
HÀC(8’)).
(
7R,8S)-3,3’,5-Trimethoxy-4’,7-epoxy-8,5’-neolignan-4,9,9’-triol (¼4-[(2R,3S)-2,3-Dihydro-3-(hy-
droxymethyl)-5-(3-hydroxypropyl)-7-methoxy-1-benzofuran-2-yl]-2,6-dimethoxyphenol; 3a). Amor-
1
phous gum. H-NMR (CD OD, 700 MHz): 6.76 (s, HÀC(6’)); 6.75 (s, HÀC(2’)); 6.70 (s, HÀC(2,6));
3
5
3
1
.53 (d, J ¼ 6.2, HÀC(7)); 3.89 (s, MeOÀC(3’)); 3.89 – 3.85 (m, H ÀC(9)); 3.84 (s, MeOÀC(3,5)); 3.85 –
a
.82 (m, H ÀC(9)) 3.59 (t, J ¼ 6.5, CH (9’)); 3.51 – 3.48 (m, HÀC(8)); 2.65 (t, J ¼ 7.7, CH (7’)); 1.86 –
b
2
2
.82 (m, CH (8’)).
2
Determination of the Sugars of Compounds 1 – 3. Each layer was neutralized by passage through an
Amberlite IRA-67 column and was evaporated under reduced pressure to give the sugar fraction. The
sugars obtained from hydrolysis were dissolved in anh. pyridine (0.5 ml) followed by adding of l-cysteine
methyl ester hydrochloride (2 mg; Sigma, St. Louis, MO). The mixture was stirred at 608 for 1.5 h. After
the mixture was dried in vacuo, the residue was trimethylsilylated with 1-trimethylsilylimidazole (0.1 ml;
Sigma, St. Louis, MO) for 2 h. The mixture was partitioned between hexane and H O (1 ml, each), and
2
the org. layer (1 ml) was analyzed by gas chromatography (GC) [26]. Identification of l-arabinose and d-
xylose for 1, 2, and 3 was performed in each case by co-injection of the hydrolysate with derivatized
standard sugars, giving single peaks at l-arabinose (5.39 min) for 1 and d-xylose for 2 and 3 (5.55 and

1
094
Helvetica Chimica Acta – Vol. 98 (2015)
5
.54 min, resp.). t Values of authentic d-xylose and l-arabinose samples that were treated in the same
R
way were 5.53 and 5.40 min., resp.
Cytotoxicity Assay. A sulforhodamine B bioassay (SRB) was used to determine the cytotoxicity of
each compound against four cultured human cancer cell lines [25]. The cell lines (National Cancer
Institute, Bethesda, MD, USA) used were A549 (non-small cell lung adenocarcinoma), SK-OV-3
(
ovarian cancer cells), A498 (human kidney epithelial cells), and HCT-15 (colon cancer cells).
Doxorubicin (Sigma Chemical Co., ꢀ98%) was used as a positive control. Tested compounds were
demonstrated to be pure as evidenced by NMR and HPLC analysis (purity ꢀ95%). All experiments
were performed in triplicate, and all the 50% cell growth inhibitory concentration (IC ) were expressed
50
as mean Æ SEM.
REFERENCES
[
[
1] T. B. Lee, ꢁColoured Flora of Koreaꢂ, Hyang-Moon Publishing Co., Seoul, Korea, 1998, p. 404.
2] B. S. Chung, M. K. Shin, ꢁDictionary of Korean Folk Medicineꢂ, Younglim Publishing Co., Seoul,
Korea, 1990, p. 456.
[
[
[
[
[
3] Y.-C. Chang, C.-Y. Chen, F.-R. Chang, Y.-C. Wu, J. Chin. Chem. Soc. 2001, 48, 811.
4] H. Nii, K. Furukawa, M. Iwakiri, T. Kubota, Nippon Nogei Kagaku Kaishi 1983, 57, 725.
5] K. Seki, T. Sasaki, K. Haga, R. Kaneko, Phytochemistry 1994, 36, 949.
6] K. Seki, T. Sasaki, S. Wano, K. Haga, R. Kaneko, Phytochemistry 1995, 40, 1175.
7] G.-W. Huh, J.-H. Park, S. Shrestha, Y.-H. Lee, E.-M. Ahn, H. C. Kang, Y.-B. Kim, N.-I. Baek,
Holzforschung 2012, 66, 585.
[
[
8] K. H. Kim, E. Moon, S. K. Ha, W. S. Suh, H. K. Kim, S. Y. Kim, S. U. Choi, K. R. Lee, Chem. Pharm.
Bull. 2014, 62, 1136.
9] R. Wang, S. Tang, H. Zhai, H. Duan, China J. Chin. Mater. Med. 2011, 36, 1032.
[
[
[
[
[
[
[
[
[
10] E. ƒmite, H. Pan, L. N. Lundgren, Phytochemistry 1995, 40, 341.
11] K. H. Kim, E. Moon, S. Y. Kim, S. U. Choi, K. R. Lee, Food. Chem. Toxicol. 2012, 50, 3680.
12] S. Latza, D. Ganber, R. G. Berger, Phytochemisty 1996, 43, 481.
13] M. Zahid, A. Ali, O. Ishurd, A, Ahmed, Z. Ali, V. U. Ahmed, Y. Pan, Helv. Chim. Acta 2002, 85, 689.
14] K. Ohashi, H. Watanabe, Y. Okumura, T. Uji, I. Kitagawa, Chem. Pharm. Bull. 1994, 42, 1924.
15] S.-Y. Lee, S.-U. Choi, K.-R. Lee, Bull. Korean. Chem. Soc. 2011, 32, 3813.
16] M. K. Lee, S. H. Sung, H. S. Lee, J. H. Cho, Y. C. Kim, Arch. Pharm. Res. 2001, 24, 198.
17] S. K. Sadhu, P. Phattanawasin, M. S. Choudhuri, T. Ohtsuki, M. Ishibashi, J. Nat. Med. 2006, 60, 258.
18] A. Jutiviboonsuk, H. Zhang, G. T. Tan, C. Ma, V. H. Nguyen, M. C. Nguyen, N. Bunyapraphatsara,
D. D Soejarto, H. H. S. Fong, Phytochemistry 2005, 66, 2745.
[
[
19] R. Wangteeraprasert, K. Likhitwitayawuid, Helv. Chim. Acta 2009, 92, 1217.
20] Y.-H. Wang, Q.-Y. Sun, F.-M. Yang, C.-L. Long, F.-W. Zhao, G.-H. Tang, H.-M. Niu, H. Wang, Q.-Q.
Huang, J.-J. Xu, L.-J. Ma, Helv. Chim. Acta 2010, 93, 2467.
[
[
[
[
[
21] N. Matsuda, H. Sato, Y. Yaoita, M. Kikuchi, Chem. Pharm. Bull. 1996, 44, 1122.
22] S. K. Sadhu, A. Khatun, P. Phattanawasin, T. Ohtsuki, M. Ishibashi, J. Nat. Med. 2007, 61, 480.
23] M. Kuroda, K. Sakurai, Y. Mimaki, J. Nat. Med. 2011, 65, 198.
24] K. Yoshinari, Y. Sashida, H. Shimomura, Chem. Pharm. Bull. 1989, 37, 3301.
25] P. Skehan, R. Stroreng, D. Scudiero, A. Monks, J. Mcmahon, D. Vistica, J. T. Warren, H. Bokesch, S.
Kenney, M. R. Boyd, J. Natl. Cancer Inst. 1990, 82, 1107.
[
26] W. Jiang, W. Li, L. Han, L. Liu, Q. Zhang, S. Zhang, T. Nikaido, K. Koike, J. Nat. Prod. 2006, 69, 1577.
Received January 6, 2015