New diarylheptanoids and diarylheptanoid glucosides from the rhizomes of Tacca chantrieri and their cytotoxic activity

Article abstract of DOI:10.1021/np010470m

Two new diarylheptanoids (1, 2) and seven new diarylheptanoid glucosides (3-9) were isolated from the rhizomes of Tacca chantrieri. Their structures were determined by spectroscopic analysis, including 2D NMR data, and the results of hydrolytic cleavage. The absolute configurations of the 3,5-dihydroxyheptane moieties of the new diarylheptanoids were determined to be 3R and 5R by the application of the CD exciton chirality method to the corresponding 3,5-bis-p-bromobenzoyl derivatives. The cytotoxic activities of the isolated compounds and some derivatives against HL-60 human promyelocytic leukemia cells, HSC-2 human oral squamous carcinoma cells, and normal human gingival fibroblasts (HGF) are reported.

Full text of DOI:10.1021/np010470m

J . Nat. Prod. 2002, 65, 283-289
283
New Dia r ylh ep ta n oid s a n d Dia r ylh ep ta n oid Glu cosid es fr om th e Rh izom es of
Ta cca ch a n tr ier i a n d Th eir Cytotoxic Activity
Akihito Yokosuka,† Yoshihiro Mimaki,*,† Hiroshi Sakagami,‡ and Yutaka Sashida†
Laboratory of Medicinal Plant Science, School of Pharmacy, Tokyo University of Pharmacy and Life Science,
1432-1, Horinouchi, Hachiouji, Tokyo 192-0392, J apan, and Department of Dental Pharmacology,
Meikai University School of Dentistry, 1-1, Keyaki-dai, Sakado, Saitama 350-0283, J apan
Received September 28, 2001
Two new diarylheptanoids (1, 2) and seven new diarylheptanoid glucosides (3-9) were isolated from the
rhizomes of Tacca chantrieri. Their structures were determined by spectroscopic analysis, including 2D
NMR data, and the results of hydrolytic cleavage. The absolute configurations of the 3,5-dihydroxyheptane
moieties of the new diarylheptanoids were determined to be 3R and 5R by the application of the CD
exciton chirality method to the corresponding 3,5-bis-p-bromobenzoyl derivatives. The cytotoxic activities
of the isolated compounds and some derivatives against HL-60 human promyelocytic leukemia cells, HSC-2
human oral squamous carcinoma cells, and normal human gingival fibroblasts (HGF) are reported.
The family Taccaceae is composed of two genera, Tacca
and Schizocapsa, and about 10 species, with most distrib-
uted in tropical regions of Asia, The Pacific Islands, and
Australia.1 Tacca chantrieri Andre´, the subject of this
report, is a perennial plant that occurs in the southeast
area of mainland China, and its rhizomes have been used
for the treatment of gastric ulcers, enteritis, and hepatitis
in Chinese folk medicine. According to a Chinese herbal
dictionary, T. plantaginea has also been used for the same
purpose as T. chantrieri.2 Although the chemical constitu-
ents of T. plantaginea were examined extensively and a
series of five-cyclic highly oxygenated steroids with a
γ-enol-lactone ring, named taccalonolids, were isolated as
its characteristic components,3 there has been only one
report of the secondary metabolites of T. chantrieri, in
which only a few sterols, such as stigmasterol and dau-
costerol, and a diosgenin glycoside were present.4 There-
fore, our attention was directed to the constituents of T.
chantrieri rhizomes, on which a detailed phytochemical
investigation has been carried out. As a result, two new
diarylheptanoids (1, 2) and seven new diarylheptanoid
glucosides (3-9) were isolated. In this paper, we describe
the structure elucidation of the new diarylheptanoids
(1-9) and the cytotoxic activities of 1-9 and some deriva-
tives against HL-60 human promyelocytic leukemia cells,
HSC-2 human oral squamous carcinoma cells, and normal
human gingival fibroblasts (HGF).
Resu lts a n d Discu ssion
The rhizomes of T. chantrieri (dry wt. of 7.3 kg) were
extracted with hot MeOH twice. After removal of solvent,
the extract was passed through a porous-polymer poly-
styrene resin (Diaion HP-20) column, eluting with MeOH-
H2O gradients, EtOH, and EtOAc. The 50% MeOH eluate
portion was subjected to Si gel and octadecylsilanized
(ODS) Si gel column chromatography to afford compounds
1-9.
Compound 1 was isolated as a viscous syrup, [R]D +1.7°
(MeOH). The HREIMS of 1 showed an [M]+ peak at m/z
332.1623, corresponding to the empirical molecular formula
of C19H24O5, which was also deduced by analysis of its 13C
* To whom correspondence should be addressed. Tel: +81-426-76-4577.
Fax: +81-426-76-4579. E-mail: mimakiy@ps.toyaku.ac.jp.
NMR and DEPT spectral data. The IR spectrum suggested
the presence of hydroxyl groups (3347 cm-1) and aromatic
† Tokyo University of Pharmacy and Life Science.
‡ Meikai University School of Dentistry.
10.1021/np010470m CCC: $22.00
© 2002 American Chemical Society and American Society of Pharmacognosy
Published on Web 02/09/2002

284 J ournal of Natural Products, 2002, Vol. 65, No. 3
Yokosuka et al.
Compound 3 was obtained as a pale yellow amorphous
solid. Its molecular formula was determined to be C25H34O10
by positive-ion FABMS, which showed a pseudomolecular
ion peak at m/z 517 [M + Na]+, and by elemental analysis.
The deduced molecular formula was higher by C6H10O5
than that of 1. The1H NMR spectrum showed signals for a
1,3,4-trisubstituted aromatic group [δ 6.65 (1H, d, J ) 8.2
Hz), 6.64 (1H, d, J ) 1.8 Hz), and 6.52 (1H, dd, J ) 8.2,
1.8 Hz)], a 1,4-disubstituted aromatic group [δ 7.00 (2H,
d, J ) 8.5 Hz) and 6.67 (2H, d, J ) 8.5 Hz)], and a 3,5-
dihydroxyheptane moiety. In addition, a signal due to an
anomeric proton was observed at δ 4.29 (d, J ) 7.8 Hz).
Enzymatic hydrolysis of 3 with naringinase gave 1 and
D-glucose. Identification of D-glucose, including its absolute
configuration, was carried out by direct HPLC analysis of
the hydrolysate. Thus, 3 was considered to be a monoglu-
coside of 1. A long-range correlation was observed from the
anomeric proton to the C-3 carbon (δ 76.8) in the HMBC
spectrum. Furthermore, a downfield shift of C-3 (8.1 ppm)
and upfield shifts of C-2 (2.3 ppm) and C-4 (1.2 ppm) were
detected when the13C NMR spectrum of 3 was compared
with that of 1. The structure of 3 was elucidated as (3R,5R)-
3,5-dihydroxy-1-(3,4-dihydroxyphenyl)-7-(4-hydroxyphenyl)-
heptane 3-O-â-D-glucopyranoside.
Compound 4 was shown to have a molecular formula of
C25H34O10 from its positive-ion FABMS, elemental analysis,
and 13C NMR spectral data, which was the same as that
of 3. However, slight differences between the two com-
pounds were recognized in the 1H and 13C NMR signals
arising from the 3,5-dihydroxyheptane moiety. Enzymatic
hydrolysis of 4 with â-D-glucosidase gave 1 and D-glucose.
In the HMBC spectrum, a long-range correlation was
observed from the anomeric proton signal (δ 4.30) of the
D-glucose to C-5 (δ 76.9) of the heptane moiety. Thus, 4
was revealed to be an isomer of 3 with regard to the
D-glucose linkage position, and the structure was formu-
lated as (3R,5R)-3,5-dihydroxy-1-(3,4-dihydroxyphenyl)-7-
(4-hydroxyphenyl)heptane 5-O-â-D-glucopyranoside.
F igu r e 1. HMBC correlations of 1.
rings (1611 and 1515 cm-1). The UV spectrum showed an
absorption maximum due to substituted aromatic rings
(281.4 nm). The 1H NMR spectrum contained signals for
1,3,4-trisubstituted aromatic protons [δ 6.65 (1H, d, J )
8.4 Hz), 6.62 (1H, d, J ) 2.0 Hz), and 6.50 (1H, dd, J )
8.4, 2.0 Hz)] and 1,4-disubstituted aromatic protons [δ 6.99
(2H, d, J ) 8.5 Hz) and 6.67 (2H, d, J ) 8.5 Hz)]. In the
13C NMR spectrum, three oxygenated sp2 carbons were
observed at δ 156.3, 146.1, and 144.2, and treatment of 1
with CH2N2 in MeOH gave a trimethyl derivative (1a ).
Thus, the presence of both a 3,4-dihydroxyphenyl group
and a 4-hydroxyphenyl group in 1 was evident. In addition,
the 13C NMR spectrum showed the presence of a seven sp3
carbon atom units in 1, with these carbons being five
1
methylenes and two oxymethines. Signals in the H NMR
spectrum that could be ascribed to the C7 unit included
spin systems due to two pairs of adjacent methylenes, an
isolated methylene, and two hydroxymethine groups. Each
structural fragment constituting the C7 unit was connected
1
by analysis of the H-1H COSY and 2D TOCSY spectra,
allowing the construction of the symmetrical structural
unit -C(1)H2C(2)H2-C(3)H(OH)-C(4)H2-C(5)H(OH)-C(6)
-
H2C(7)H2-. The two aromatic rings were consequently
placed at the terminal methylene groups. This was ascer-
tained by long-range correlations from δΗ 6.99 (H-2′′ and
H-6′′) to δC 32.1 (C-7), δΗ 6.62 (H-2′) to δC 32.4 (C-1), and
6.50 (1H, H-6′) to δC 32.4 (C-1) in the HMBC spectrum
(Figure 1). The planar structure of 1 was assigned as 3,5-
dihydroxy-1-(3,4-dihydroxyphenyl)-7-(4-hydroxyphenyl)-
heptane.
The absolute configurations of the 3,5-dihydroxyl groups
were determined by the application of the CD exciton
chirality method to acyclic 1,3-dibenzoates.5 The trimethyl
derivative (1a ) was converted to the corresponding 3,5-bis-
(p-bromobenzoate) (1b), and its CD spectrum exhibited
positive (237.4 nm, ∆ꢀ +29.9) and negative (253.3 nm,
∆ꢀ -20.0) Cotton effects, which was consistent with a
negative chirality. Thus, the absolute configurations were
determined as 3R and 5R. The structure of 1 was shown
to be (3R,5R)-3,5-dihydroxy-1-(3,4-dihydroxyphenyl)-7-
(4-hydroxyphenyl)heptane.
Compound 5 (C26H34O10) was obtained as an amorphous
solid. The 1H and 13C NMR spectral features of 5 were quite
similar to those of 3, except for the presence of a signal for
one methoxyl group [δΗ 3.82 (3H, s); δC 56.4]. The HMBC
spectrum showed a correlation between the methoxyl
proton and the C-3′ carbon (δ 148.8). Enzymatic hydrolysis
of 5 with naringinase gave an aglycon (5a ) and D-glucose,
and consecutive methylation of 5a with CH2N2 afforded 1a .
Thus, 5 was shown to be (3R,5R)-3,5-dihydroxy-1-(4-
hydroxy-3-methoxyphenyl)-7-(4-hydroxyphenyl)heptane 3-O-
â-D-glucopyranoside.
Compound 2, [R]D +4.0° (MeOH), was isolated as a
viscous syrup. Its molecular formula was deduced as
Compound 6 showed an accurate [M + Na]+ ion peak at
m/z 533.1975 in the HRFABMS, corresponding to a mo-
C
19H24O6 by HREIMS, 13C NMR, and DEPT spectral data.
1
lecular formula C25H34O11 (∆ -2.5 mmu of calcd). The H
The spectral properties of 2 were suggestive of a 3,5-
dihydroxydiarylheptanoid whose planar structure was
completely symmetrical. Each of the aromatic proton
signals at δ 6.65 (d, J ) 8.1 Hz), 6.62 (d, J ) 2.0 Hz), and
6.50 (dd, J ) 8.1, 2.0 Hz) was integrated into two protons,
being consistent with the presence of two 3,4-dihydroxy-
phenyl groups. These data and analysis of the 1H-1H
COSY, HMQC and HMBC spectra identified the planar
structure of 2 as 3,5-dihydroxy-1,7-bis(3,4-dihydroxy-
phenyl)heptane. Compound 2 was methylated to protect
the phenolic hydroxyl groups (2a ) and then converted to
the 3,5-bis(p-bromobenzoate) derivative (2b) as performed
for 1. The CD spectrum of 2b exhibited positive and
negative Cotton effects, similar to 1b. The structure of 2
was therefore formulated as (3R,5R)-3,5-dihydroxy-1,7-bis-
(3,4-dihydroxyphenyl)heptane.
and 13C NMR spectra of 6 were similar to those of 3 with
the exceptions of the signals for one phenolic part. Enzy-
matic hydrolysis of 6 with â-D-glucosidase gave 3 and
D-glucose. The linkage position of D-glucose was shown to
be at C-3 of the aglycon by detecting a correlation from
the anomeric proton at δ 4.25 to the C-3 carbon at δ 76.8
in the HMBC spectrum. All of these data were consistent
with the structure (3R,5R)-3,5-dihydroxy-1,7-bis(3,4-dihy-
droxyphenyl)heptane 3-O-â-D-glucopyranoside, which was
assigned to compound 6.
Compound 7 was obtained as an amorphous solid with
a molecular formula of C26H36O11. Comparison of the 1H
and 13C NMR data of 7 with 6 revealed that 7 was closely
related to 6 but contained a methoxyl group [δΗ 3.82 (3H,
s); δC 56.4]. By detecting a correlation from the methoxyl

New Diarylheptanoids from Tacca
J ournal of Natural Products, 2002, Vol. 65, No. 3 285
tometer using standard Bruker pulse programs. Chemical
shifts are given as δ values with reference to tetramethylsilane
(TMS) as internal standard. MS were recorded on a Finnigan
MAT TSQ-700 (San J ose, CA) mass spectrometer. Elemental
analysis was carried out using an Elementar Vario EL
elemental analyzer (Hanau, Germany). Diaion HP-20 (Mi-
tsubishi-Kasei, Tokyo, J apan), Si gel (Fuji-Silysia Chemical,
Aichi, J apan), and ODS Si gel (Nacalai Tesque, Kyoto, J apan)
were used for column chromatography. TLC was carried out
on precoated Kieselgel 60 F254 (0.25 mm thick, Merck, Darm-
stadt, Germany) and RP18 F254 S plates (0.25 mm thick, Merck),
and spots were visualized by UV irradiation (254 nm) and
spraying the plates with 10% H2SO4 solution, followed by
heating. HPLC was performed using a system composed of a
CCPM pump (Tosoh, Tokyo, J apan), a CCP PX-8010 controller
(Tosoh), a RI-8010 (Tosoh) or a Shodex OR-2 (Showa-Denko,
Tokyo, J apan) detector, and Rheodyne injection port. A
Kaseisorb LC-ODS-120-5 column (10 mm i.d. × 250 mm, ODS
5 µm, Tokyo-Kasei, Tokyo, J apan) was employed for prepara-
tive HPLC. The following reagents were obtained from the
indicated companies: RPMI 1640 medium and DMEM (GIB-
CO BRL, Gland Island, NY); FBS (J RH Biosciences, Lenexa,
KS); penicillin, streptomycin, MTT, and R-MEM (Sigma, St.
Louis, MO). All other chemicals used were of biochemical
reagent grade.
proton signal to the C-3′ carbon in the HMBC spectrum,
the methoxyl group was placed at C-3′. Enzymatic hydroly-
sis of 7 with â-D-glucosidase followed by methylation with
CH2N2 afforded 2a . The structure of 7 was concluded to
be (3R,5R)-3,5-dihydroxy-1-(4-hydroxy-3-methoxyphenyl)-
7-(3,4-dihydroxyphenyl)heptane 3-O-â-D-glucopyranoside.
Compound 8 (C27H38O11) was assigned as a diarylhep-
tanoid glucoside structurally related to 6. The 1H NMR
spectrum showed signals for two methoxyl groups at δ 3.82
(3H × 2, s) and two 1,3,4-trisubstituted aromatic rings at
δ 6.79 (1H, d, J ) 1.7 Hz), 6.76 (1H, d, J ) 1.7 Hz), 6.68
(2H, d, J ) 8.0 Hz), 6.64 (1H, dd, J ) 8.0, 1.7 Hz), and
6.62 (1H, dd, J ) 8.0, 1.7 Hz). An HMBC correlation was
observed from the δ 3.82 resonance to C-3′ and C-3′′, which
appeared at the same position. Enzymatic hydrolysis of 8
with â-D-glucosidase followed by methylation with CH2N2
furnished 2a . Thus, the structure of 8 was established as
(3R,5R)-3,5-dihydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)-
heptane 3-O-â-D-glucopyranoside.
Compound 9 (C25H34O9) exhibited eight aromatic proton
signals at δ 7.02 (2H, d, J ) 8.6 Hz), 7.00 (2H, d, J ) 8.6
1
Hz), and 6.67 (4H, d, J ) 8.6 Hz) in the H NMR spectrum,
suggesting the presence of two 4-hydroxyphenyl groups.
Enzymatic hydrolysis of 9 with naringinase gave an
aglycon (9a ) and D-glucose. The planar structure of 9a was
identified as 3,5-dihydroxy-1,7-bis(4-hydroxyphenyl)hep-
tane by comparison of its NMR data with literature values.6
The positive specific rotation of 9a , [R]D +4.0° (MeOH),
confirmed the 3R and 5R configurations, since the corre-
sponding 3S and 5S form was reported to show an opposite
[R]D value.7 The structure of 9 was established as (3R,5R)-
3,5-dihydroxy-1,7-bis(4-hydroxyphenyl)heptane 3-O-â-D-
glucopyranoside.
P la n t Ma ter ia l. The rhizomes of T. chantrieri were col-
lected in SiMao City, Yunnan Province, People’s Republic of
China, in October 1996, and identified by one of the authors
(Y.S.). A voucher of the plant is on file in our laboratory
(voucher no. TC-96-003, Laboratory of Medicinal Plant Sci-
ence).
Extr a ction a n d Isola tion . The plant material (dry wt, 7.3
kg) was extracted with hot MeOH twice (each 3 L). The MeOH
extract was concentrated under reduced pressure and then
passed through a Diaion HP-20 column, eluting with 30%
MeOH, 50% MeOH, MeOH, EtOH, and EtOAc. Column
chromatography of the 50% MeOH eluate portion on Si gel
and elution with a stepwise gradient mixture of CHCl3-MeOH
(9:1; 4:1; 3:1; 2:1; 1:1), and finally with MeOH alone, gave four
fractions (I-IV). Fraction I was chromatographed on Si gel
eluting with CHCl3-MeOH-H2O (60:10:1) and CHCl3-EtOAc
(1:1; 1:2; 1:3; 1:4) and ODS Si gel with CH3CN-H2O (2:7) to
give 1 (382 mg). Fraction II was subjected to column chroma-
tography on ODS Si gel eluting with MeOH-H2O (1:2) and Si
gel with CHCl3-MeOH-H2O (60:10:1; 40:10:1) to give 2 (1.46
g), 5 (145 mg), and 8 (52.9 mg). Fraction III was subjected to
column chromatography on Si gel eluting with CHCl3-
MeOH-H2O (60:10:1; 50:10:1; 40:10:1; 30:10:1) and ODS Si
gel with MeOH-H2O (1:2; 2:3) to give 7 (91.3 mg) and 9 (190
mg) in pure form and a mixture of 3 and 4. The mixture was
separated by preparative HPLC using MeOH-H2O (2:3) to
furnish 3 (105 mg) and 4 (19.9 mg). Fraction IV was chro-
matographed on Si gel eluting with CHCl3-MeOH-H2O
(40:10:1; 30:10:1; 20:10:1) and ODS Si gel with MeOH-H2O
(1:3; 2:5) to give 6 (158 mg).
Diarylheptanoids are known to occur only in a limited
number species of higher plants belonging to the families
Zingiberaceae,8-11 Betulaceae,12 and Aceraceae.13 This is
the first isolation of diarylheptanoids from the plant family
Taccaceae.
The isolated compounds and some derivatives, in which
9b prepared by treatment of 9 with CH2N2 was included,
were evaluated for their cytotoxic activities against HL-
60 leukemia cells, HSC-2 cells, and normal HGF, using a
modified MTT assay method (Table 2).14 The diarylhep-
tanoids 1, 2, and 7a , and the diarylheptanoid glucosides
3, 4, 6, and 7, which each have three or four phenolic
hydroxyl groups, showed moderate cytotoxic activity against
HL-60 cells with IC50 values ranging from 1.8 to 6.4 µg/
mL, while those possessing two phenolic hydroxyl groups
(5, 5a , 8, 8a , 9, 9a ) did not exhibit apparent cytotoxic
activity even at a sample concentration of 10 µg/mL. It is
noteworthy that the compounds whose phenolic hydroxyl
groups were all masked with methyl groups (1a , 2a , 9b)
were also cytotoxic. These observations suggest that the
number of phenolic hydroxyl groups contributes to the
resultant cytotoxicity. As for activity against HSC-2 cells,
1a , 2a , and 9b showed considerable cytotoxicity. They
showed much higher cytotoxic activities against HSC-2
cells than against normal HGF.
Com p ou n d 1: viscous syrup; [R]23 +1.7° (c 0.12, MeOH);
D
UV (MeOH) λmax 281.4 nm (log ꢀ 3.63); UV (MeOH + 1 M
NaOH) λmax 290.6 nm; IR (film) νmax 3347 (OH), 2941 (CH),
1611 and 1515 (aromatic rings), 1447, 1366, 1282, 1236, 1173,
1153, 1113, 1091, 1059, 957, 826 cm-1 1H NMR (CD3OD) δ
;
6.99 (2H, d, J ) 8.5 Hz, H-2′′, H-6′′), 6.67 (2H, d, J ) 8.5 Hz,
H-3′′, H-5′′), 6.65 (1H, d, J ) 8.4 Hz, H-5′), 6.62 (1H, d, J )
2.0 Hz, H-2′), 6.50 (1H, dd, J ) 8.4, 2.0 Hz, H-6′), 3.80 (2H, m,
H-3, H-5), 2.68-2.45 (4H, m, H2-1, H2-7), 1.70-1.63 (4H, m,
H2-2, H2-6), 1.53 (2H, dd, J ) 6.6, 5.9 Hz, H2-4); 13C NMR, see
Table 1; HREIMS m/z 332.1623 [M]+ (calcd for C19H24O5,
332.1624).
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Optical rotations
were measured using a J ASCO DIP-360 (Tokyo, J apan)
automatic digital polarimeter. IR spectra were recorded on a
J ASCO FT-IR 620 spectrophotometer, UV spectra on a J ASCO
V-520 spectrophotometer, and CD spectra on a J ASCO J -720
instrument. NMR spectra were recorded on a Bruker DRX-
500 (500 MHz for 1H NMR, Karlsruhe, Germany) spectropho-
Meth yla tion of 1. Compound 1 (66.4 mg) was dissolved in
MeOH (5 mL) and cooled at 0 °C. A large excess of CH2N2 in
Et2O was added to the sample solution, and it was allowed to
stand at room temperature for 12 h. The reaction mixture was
chromatographed on Si gel eluting with hexane-Me2CO (2:1)
to yield 1a (40.0 mg).

286 J ournal of Natural Products, 2002, Vol. 65, No. 3
Yokosuka et al.
Ta ble 1. 13C NMR Data for Compounds 1-9 and Their Derivatives (1a , 4a , 5a , 7a -9a , and 9b) in CD3OD
carbon
1
1a
2
2a
3
4
5
5a
1
2
32.4
41.3
32.6
41.2
32.4
41.3
32.6
41.2
31.7
39.0
32.5
40.9
32.0
39.2
32.6
41.3
3
68.7
68.7
68.8
68.6
76.8
67.6
76.9
68.7
4
45.6
45.6
45.6
45.6
44.4
44.3
44.4
45.6
5
68.7
68.7
68.8
68.6
67.4
76.9
67.5
68.7
6
41.4
41.2
41.3
41.2
41.0
39.1
41.0
41.4
7
32.1
32.1
32.4
32.6
32.3
31.5
32.3
32.1
1′
2′
3′
4′
5′
6′
1′′
2′′
3′′
4′′
5′′
6′′
1
2
135.3
116.6
146.1
144.2
116.3
120.6
134.5
130.3
116.1
156.3
116.1
130.3
136.8
113.7
150.4
148.6
113.3
121.7
135.7
130.3
114.8
159.3
114.8
130.3
135.3
116.3
146.1
144.1
116.6
120.7
135.3
116.3
146.1
144.1
116.6
120.7
136.7
116.3
146.1
144.1
116.6
121.7
136.7
116.3
146.1
144.1
116.6
121.7
135.3
116.6
146.1
144.1
116.3
120.7
134.6
116.0
116.1
156.2
116.0
130.4
103.7
75.2
135.5
116.7
146.0
144.1
116.2
120.8
134.5
130.3
116.1
156.3
116.1
130.3
103.7
75.2
135.3
113.2
148.8
145.4
116.1
121.8
134.6
130.4
116.0
156.2
116.0
130.4
103.8
75.3
135.3
113.2
148.8
145.5
116.1
121.8
134.5
130.3
116.1
156.3
116.1
130.3
Glc
3
78.1
78.2
78.2
4
72.4
72.4
72.4
5
77.6
77.7
77.7
6
63.3
63.3
63.3
3′-OMe
4′-OMe
3′′-OMe
4′′-OMe
56.4
56.6
56.4
56.6
56.4
56.6
56.4
56.4
55.6
carbon
6
7
7a
8
8a
9
9a
9b
1
2
31.7
39.0
32.0
39.1
32.6
41.4
32.0
39.1
32.6
41.3
31.5
39.2
32.2
41.4
32.1
41.3
3
76.8
76.9
68.7
76.9
68.7
76.9
68.7
68.7
4
44.3
44.3
45.6
44.4
45.6
44.4
45.7
45.6
5
67.6
67.6
68.8
67.5
68.7
67.5
68.7
68.7
6
40.9
40.9
41.4
41.0
41.3
41.0
41.4
41.3
7
32.5
32.5
32.4
32.8
32.6
32.3
32.2
32.1
1′
2′
3′
4′
5′
6′
1′′
2′′
3′′
4′′
5′′
6′′
1
2
135.3
116.6
146.0
144.1
116.2
120.7
135.5
116.7
146.1
144.2
116.3
120.8
103.7
75.2
135.5
113.3
148.8
145.4
116.1
121.8
135.3
116.7
146.1
144.1
116.2
120.8
103.8
75.3
135.3
113.2
148.8
145.5
116.1
121.8
135.3
116.6
146.1
144.2
116.3
120.6
135.3
113.2
148.8
145.4
116.0
121.8
135.4
113.3
148.8
145.4
116.1
121.9
103.8
75.3
135.3
113.2
148.8
145.5
116.1
121.8
135.3
113.3
148.8
145.5
116.1
121.8
134.5
130.3
116.0
156.3
116.1
130.3
134.4
130.4
116.1
156.3
116.1
130.4
103.8
75.3
134.5
130.3
116.1
156.3
116.1
130.3
134.5
130.3
116.1
156.3
116.1
130.3
135.7
130.3
114.8
159.3
114.8
130.3
135.7
130.3
114.8
159.3
114.8
130.3
Glc
3
78.2
78.2
78.2
78.2
4
72.4
72.4
72.4
72.4
5
77.7
77.7
77.7
77.7
6
63.3
63.3
63.3
63.3
3′-OMe
4′-OMe
3′′-OMe
4′′-OMe
56.4
56.4
56.4
56.4
56.4
55.6
55.6
56.4
Com p ou n d 1a : amorphous solid; [R]25 +2.7° (c 0.07,
was added dropwise p-bromobenzoyl chloride (40 mg) dissolved
in pyridine (3 mL), and the mixture was stirred at 70 °C for 9
h. The reaction mixture was diluted with H2O (20 mL) and
extracted with CHCl3 (10 mL × 3). After concentration of the
CHCl3-soluble phase, it was chromatographed on Si gel eluting
with hexane-Me2CO (4:1) to yield 1b (8.0 mg).
D
MeOH); UV (EtOH) λmax 278.6 nm (log ꢀ 3.59); IR (film) νmax
3407 (OH), 2936 (CH), 1611, 1590 and 1514 (aromatic rings),
1454, 1246, 1178, 1155, 1064, 1030, 848, 812, 764 cm-1 1H
;
NMR (CD3OD) δ 7.08 (2H, d, J ) 8.6 Hz, H-2′′, H-6′′), 6.80
(2H, d, J ) 8.6 Hz, H-3′′, H-5′′), 6.83 (1H, d, J ) 8.2 Hz, H-5′),
6.80 (1H, d, J ) 2.0 Hz, H-2′), 6.73 (1H, dd, J ) 8.2, 2.0 Hz,
H-6′), 3.81 (2H, m, H-3, H-5), 3.80 (3H, s, MeO-3′), 3.78 (3H,
s, MeO-4′), 3.74 (3H, s, MeO-4′′), 2.74-2.53 (4H, m, H2-1, H2-
7), 1.73-1.65 (4H, m, H2-2, H2-6), 1.54 (2H, dd, J ) 6.6, 5.7
Hz, H2-4); 13C NMR, see Table 1; HREIMS m/z 374.2104 [M]+
(calcd for C22H30O5, 374.2093).
Com p ou n d 1b: amorphous solid; [R]24 -28.8° (c 0.16,
D
EtOH); UV (EtOH) λmax 243.8 nm (log ꢀ 4.64); CD (EtOH) λmax
(∆ꢀ) 237.4 (+29.9), 253.3 (-20.0); IR (film) νmax 2930, 2854 and
2834 (CH), 1716 (CdO), 1589, 1513 and 1483 (aromatic rings),
1464, 1454, 1418, 1397, 1360, 1270, 1173, 1156, 1141, 1116,
1102, 1068, 1032, 1011, 848, 809, 755, 682 cm-1; 1H NMR (CD3-
OD) δ 7.65 (2H, d, J ) 8.5 Hz), 7.64 (2H, d, J ) 8.5 Hz), 7.50
(2H × 2, d, J ) 8.5 Hz), 7.04 (2H, d, J ) 8.6 Hz, H-2′′, H-6′′),
Bis-p-br om oben zoa te (1b) of 1a . To a solution of 1a (15.3
mg) in pyridine (1 mL) containing DMAP (19 mg) as catalyst

New Diarylheptanoids from Tacca
J ournal of Natural Products, 2002, Vol. 65, No. 3 287
Ta ble 2. Cytotoxic Activities of Compounds 1-9 and Their
Derivatives (1a , 4a , 5a , 7a -9a , and 9a ) and Etoposide against
HL-60, HSC-2, and HGFa
J ) 8.5 Hz, H-2′′, H-6′′), 6.67 (2H, d, J ) 8.5 Hz, H-3′′, H-5′′),
6.65 (1H, d, J ) 8.2 Hz, H-5′), 6.64 (1H, d, J ) 1.8 Hz, H-2′),
6.52 (1H, dd, J ) 8.2, 1.8 Hz, H-6′), 4.29 (1H, d, J ) 7.8 Hz,
Glc-1), 3.93 (1H, m, H-3), 3.92 (1H, m, H-5), 3.89 (1H, dd, J )
11.5, 2.2 Hz, Glc-6a), 3.56 (1H, dd, J ) 11.5, 7.6 Hz, Glc-6b),
3.35 (1H, dd, J ) 9.3, 9.0 Hz, Glc-3), 3.25 (1H, ddd, J ) 9.3,
7.6, 2.2 Hz, Glc-5), 3.17 (1H, dd, J ) 9.3, 9.3 Hz, Glc-4), 3.16
(1H, dd, J ) 9.0, 7.8 Hz, Glc-2), 2.69-2.52 (4H, m, H2-1, H2-
7), 1.88-1.68 (2H, m, H2-2), 1.68-1.55 (3H, m, H-4a, H2-6),
1.49 (1H, ddd, J ) 14.1, 10.3, 2.3 Hz, H-4b); 13C NMR, see
Table 1; FABMS (positive mode) m/z 517 [M + Na]+; anal. C
57.25%, H 7.05% (calcd for C25H34O10‚3/2H2O, C 57.57%, H
7.15%).
IC50 (µg/mL)
compound
HL-60
HSC-2
HGF
1
1a
2
2a
3
4
5
5a
2.1
5.5
1.8
4.9
6.2
54.0
3.9
54.0
6.6
158
155
160
b
92.0
209
b
162
176
>250
174
220
>250
>250
b
5.5
>10
>10
3.0
En zym a tic Hyd r olysis of 3. Compound 3 (10.5 mg) was
treated with naringinase (Sigma, 23.5 mg) in HOAc/KOAc
buffer (pH 4.3, 10 mL) at room temperature for 12 h. The
reaction mixture was chromatographed on Si gel eluting with
hexane-Me2CO (1:1) followed by MeOH to yield 1 (3.4 mg)
and a sugar fraction (2.0 mg). The sugar fraction was passed
through a Sep-Pak C18 cartridge (Waters, Milford, MA) and a
Toyopak IC-SP M cartridge (Tosoh), which was then analyzed
by HPLC under the following conditions: column, Capcell Pak
NH2 UG80 (4.6 mm i.d. × 250 mm, 5 µm, Shiseido, Tokyo,
J apan); solvent, MeCN-H2O (17:3); flow rate, 0.8 mL/min;
detection, RI and OR. Identification of D-glucose present in
the sugar fraction was carried out by comparison of its
retention time and optical rotation with those of an authentic
sample; tR (min) 17.10 (D-glucose, positive optical rotation).
6
7
7a
189
>250
b
4.5
4.1
8
8a
9
9a
>10
>10
>10
>10
6.4
198
b
157
231
23.0
24.0
>250
b
213
177
173
>200
9b
etoposide
0.2
a
Key: HL-60 (human promyelocytic leukemia cells); HSC-2
(human oral squamous carcinoma cells); and HGF (normal human
b
gingival fibroblasts). Not determined.
6.76 (1H, d, J ) 1.8 Hz, H-2′), 6.74 (1H, d, J ) 8.2 Hz, H-5′),
6.73 (2H, d, J ) 8.6 Hz, H-3′′, H-5′′), 6.70 (1H, dd, J ) 8.2, 1.8
Hz, H-6′), 5.19 (2H, m, H-3, H-5), 3.73 (3H × 2, s, OMe × 2),
3.70 (3H, s, OMe), 2.65-2.56 (4H, m, H2-1, H2-7), 2.18-1.88
(6H, m, H2-2, H2-4, H2-6); EIMS m/z 740 [M]+.
Com p ou n d 4: amorphous solid; [R]25 -12.0° (c 0.10,
D
MeOH); UV (MeOH) λmax 280.6 nm (log ꢀ 3.21); UV (MeOH +
1 M NaOH) λmax 294.6 nm; IR (film) νmax 3347 (OH), 2924 (CH),
1612 and 1515 (aromatic rings), 1444, 1363, 1281, 1231, 1171,
Com p ou n d 2: viscous syrup; [R]23 +4.0° (c 0.10, MeOH);
1
1077, 1055, 1034, 825 cm-1; H NMR (CD3OD) δ 7.02 (2H, d,
D
UV (MeOH) λmax 283.4 nm (log ꢀ 3.79); UV (MeOH + 1 M
NaOH) λmax 292.4 nm; IR (film) νmax 3444 (OH), 2941 (CH),
1605 and 1519 (aromatic rings), 1455, 1372, 1283, 1114, 1060,
957, 866, 815, 788 cm-1; 1H NMR (CD3OD) δ 6.65 (2H, d, J )
8.1 Hz, H-5′, H-5′′), 6.62 (1H, d, J ) 2.0 Hz, H-2′, H-2′′), 6.50
(1H, dd, J ) 8.1, 2.0 Hz, H-6′, H-6′′), 3.83-3.78 (2H, m, H-3,
H-5), 2.63-2.44 (4H, m, H2-1, H2-7), 1.69-1.63 (4H, m, H2-2,
H2-6), 1.52 (2H, t-like, J ) 5.8 Hz, H2-4); 13C NMR, see Table
1; HREIMS m/z 348.1570 [M]+ (calcd for C19H24O6, 348.1573).
Meth yla tion of 2. Compound 2 (56.7 mg) was methylated
by the same procedure as described for 1 to yield 2a (32.8 mg).
J ) 8.4 Hz, H-2′, H-6′), 6.67 (2H, d, J ) 8.4 Hz, H-3′, H-5′),
6.65 (1H, d, J ) 8.1 Hz, H-5′′), 6.63 (1H, d, J ) 2.0 Hz, H-2′′),
6.50 (1H, dd, J ) 8.1, 2.0 Hz, H-6′′), 4.29 (1H, d, J ) 7.8 Hz,
Glc-1), 3.94 (1H, m, H-3), 3.93 (1H, m, H-5), 3.89 (1H, dd, J )
11.6, 2.2 Hz, Glc-6a), 3.58 (1H, dd, J ) 11.6, 7.4 Hz, Glc-6b),
3.35 (1H, dd, J ) 9.1, 8.9 Hz, Glc-3), 3.25 (1H, ddd, J ) 9.5,
7.4, 2.2 Hz, Glc-5), 3.19 (1H, dd, J ) 9.5, 8.9 Hz, Glc-4), 3.17
(1H, dd, J ) 9.1, 7.8 Hz, Glc-2), 2.72-2.46 (4H, m, H2-1, H2-
7), 1.88-1.70 (2H, m, H2-2), 1.70-1.56 (3H, m, H-4a, H2-6),
1.49 (1H, ddd, J ) 14.2, 10.2, 2.5 Hz, H-4b); 13C NMR, see
Table 1; FABMS (positive mode) m/z 517 [M + Na]+; anal. C
57.69%, H 7.24% (calcd for C25H34O10‚3/2H2O, C 57.57%, H
7.15%).
Com p ou n d 2a : amorphous solid; [R]26 +18.0° (c 0.10,
D
CHCl3); UV (EtOH) λmax 277 nm (log ꢀ 3.77); IR (film) νmax 3289
(OH), 2936 and 2840 (CH), 1604, 1588 and 1518 (aromatic
rings), 1465, 1451, 1419, 1335, 1276, 1260, 1238, 1194, 1155,
1140, 1108, 1066, 1025, 938, 904, 853, 835, 817, 803, 787, 765
cm-1; 1H NMR (CD3OD) δ 6.83 (2H, d, J ) 8.2 Hz, H-5′, H-5′′),
6.80 (2H, d, J ) 1.9 Hz, H-2′, H-2′′), 6.72 (2H, dd, J ) 8.2, 1.9
Hz, H-6′, H-6′′), 3.80 (3H × 2, s, OMe × 2), 3.77 (3H × 2, s,
OMe × 2), 3.83-3.77 (2H, m, H-3, H-5), 2.72-2.54 (4H, m,
H2-1, H2-7), 1.74-1.67 (4H, m, H2-2, H2-6), 1.55 (2H, t-like, J
) 5.7 Hz, H2-4); 13C NMR, see Table 1; EIMS m/z 404 [M]+.
Bis-p-br om oben zoa te (2b) of 2a . Compound 2a (10.0 mg)
was treated with p-bromobenzoyl chloride (40 mg) in pyridine
to yield 2b (4.8 mg).
En zym a tic Hyd r olysis of 4. Compound 4 (4.7 mg) was
treated with naringinase (24.2 mg) in HOAc/KOAc buffer (pH
4.3, 10 mL) at room temperature for 16 h to yield 1 (1.0 mg)
and a sugar fraction (1.0 mg). HPLC analysis of the sugar
fraction under the same conditions as in the case of 3 showed
the presence of D-glucose; tR (min) 17.24 (D-glucose, positive
optical rotation).
Com p ou n d 5: amorphous solid; [R]25D -2.0° (c 0.10, MeOH);
UV (MeOH) λmax 281.4 nm (log ꢀ 3.54); UV (MeOH + 1 M
NaOH) λmax 289.8 nm; IR (film) νmax 3374 (OH), 2925 (CH),
1613, 1600 and 1516 (aromatic rings), 1454, 1433, 1366, 1261,
1233, 1154, 1080, 1033, 821 cm-1 1H NMR (CD3OD) δ 7.00
;
Com p ou n d 2b: amorphous solid; [R]23 -37.5° (c 0.03,
(2H, d, J ) 8.6 Hz, H-2′′, H-6′′), 6.79 (1H, d, J ) 1.7 Hz, H-2′),
6.68 (1H, d, J ) 8.0 Hz, H-6′), 6.67 (2H, d, J ) 8.5 Hz, H-3′′,
H-5′′), 6.64 (1H, dd, J ) 8.1, 1.7 Hz, H-5′), 4.30 (1H, d, J ) 7.8
Hz, Glc-1), 3.92 (1H, m, H-3), 3.91 (1H, m, H-5), 3.89 (1H, dd,
J ) 11.5, 2.3 Hz, Glc-6a), 3.82 (3H, s, OMe), 3.55 (1H, dd, J )
11.5, 7.5 Hz, Glc-6b), 3.35 (1H, dd, J ) 9.3, 9.0 Hz, Glc-3),
3.24 (1H, ddd, J ) 9.1, 7.5, 2.3 Hz, Glc-5), 3.17 (1H, dd, J )
9.1, 9.0 Hz, Glc-4), 3.17 (1H, dd, J ) 9.3, 7.8 Hz, Glc-2), 2.73-
2.51 (4H, m, H2-1, H2-7), 1.91-1.72 (2H, m, H2-2), 1.71-1.57
(3H, m, H-4a, H2-6), 1.50 (1H, ddd, J ) 14.3, 10.3, 2.6 Hz,
H-4b); 13C NMR, see Table 1; FABMS (positive mode) m/z 531
[M + Na]+; anal. C 59.08%, H 7.44% (calcd for C26H34O10‚H2O,
C 59.30%, H 7.27%).
D
EtOH); UV (EtOH) λmax 242.2 nm (log ꢀ 4.45); CD (EtOH) λmax
(∆ꢀ) 236.9 (+15.0), 252.8 (-16.7); IR (film) νmax 2926 and 2854
(CH), 1714 (CdO), 1590, 1516 and 1483 (aromatic rings), 1464,
1454, 1418, 1397, 1360, 1264, 1237, 1173, 1156, 1141, 1117,
1103, 1068, 1030, 1011, 848, 805, 756, 682 cm-1; 1H NMR (CD3-
OD) δ 7.63 (2H × 2, d, J ) 8.6 Hz), 7.50 (2H × 2, d, J ) 8.6
Hz), 6.76 (2H, d, J ) 1.8 Hz, H-2′, H-2′′), 6.73 (2H, d, J ) 8.2
Hz, H-5′, H-5′′), 6.67 (2H, dd, J ) 8.2, 1.8 Hz, H-6′, H-6′′), 3.73
(3H × 4, s, OMe × 4), 5.20 (2H, m, H-3, H-5), 2.67-2.58 (4H,
m, H2-1, H2-7), 2.15-1.90 (6H, m, H2-2, H2-4, H2-6); EIMS m/z
768 [M]+.
Com p ou n d 3: amorphous solid; [R]25 -12.0° (c 0.10,
D
MeOH); UV (MeOH) λmax 281.6 nm (log ꢀ 3.55); UV (MeOH +
1 M NaOH) λmax 294.4 nm; IR (film) νmax 3357 (OH), 2924 (CH),
1612 and 1516 (aromatic rings), 1444, 1365, 1282, 1232, 1172,
En zym a tic Hyd r olysis of 5. Compound 5 (19 mg) was
treated with naringinase (34.3 mg) in HOAc/KOAc buffer (pH
4.3, 10 mL) at room temperature for 8 h to yield 5a (6.7 mg)
and a sugar fraction (4.2 mg). HPLC analysis of the sugar
1
1078, 1055, 1033, 825 cm-1; H NMR (CD3OD) δ 7.00 (2H, d,

288 J ournal of Natural Products, 2002, Vol. 65, No. 3
Yokosuka et al.
fraction under the same conditions as in the case of 3 showed
the presence of D-glucose; tR (min) 16.92 (D-glucose, positive
optical rotation).
Meth yla tion of 7a . Compound 7a (4.0 mg) was methylated
by the same procedure as described for 1 to yield 2a (2.8 mg).
Com p ou n d 8: amorphous solid; [R]23 -18.0° (c 0.10,
D
Com p ou n d 5a : amorphous solid; [R]25 +2.2° (c 0.09,
MeOH); UV (MeOH) λmax 282.0 nm (log ꢀ 3.70); UV (MeOH +
1 M NaOH) λmax 290.0 nm; IR (film) νmax 3388 (OH), 2936 (CH),
1602 and 1516 (aromatic rings), 1455, 1429, 1365, 1270, 1234,
D
MeOH); UV (MeOH) λmax 279.8 nm (log ꢀ 3.49); UV (MeOH +
1 M NaOH) λmax 288.8 nm; IR (film) νmax 3347 (OH), 2938 (CH),
1613, 1601 and 1516 (aromatic rings), 1454, 1433, 1368, 1262,
1154, 1123, 1079, 1034, 818 cm-1 1H NMR (CD3OD) δ 6.79
;
1233, 1152, 1059, 1035, 824, 797 cm-1
;
1H NMR (CD3OD) δ
(1H, d, J ) 1.7 Hz, H-2′), 6.76 (1H, d, J ) 1.7 Hz, H-2′′), 6.68
(2H, d, J ) 8.0 Hz, H-5′, H-5′′), 6.64 (1H, dd, J ) 8.0, 1.7 Hz,
H-6′), 6.62 (1H, dd, J ) 8.0, 1.7 Hz, H-6′′), 4.30 (1H, d, J ) 7.8
Hz, Glc-1), 3.94 (1H, m, H-3), 3.94 (1H, m, H-5), 3.88 (1H, dd,
J ) 11.5, 2.3 Hz, Glc-6a), 3.82 (3H × 2, s, OMe × 2), 3.54 (1H,
dd, J ) 11.5, 7.6 Hz, Glc-6b), 3.35 (1H, dd, J ) 9.2, 9.0 Hz,
Glc-3), 3.24 (1H, ddd, J ) 9.5, 7.6, 2.3 Hz, Glc-5), 3.17 (1H,
dd, J ) 9.2, 7.8 Hz, Glc-2), 3.16 (1H, dd, J ) 9.5, 9.0 Hz, Glc-
4), 2.74-2.52 (4H, m, H2-1, H2-7), 1.90-1.73 (2H, m, H2-2),
1.73-1.58 (3H, m, H-4a, H2-6), 1.51 (1H, ddd, J ) 14.2, 10.2,
2.4 Hz, H-4b); 13C NMR, see Table 1; FABMS (positive mode)
m/z 561 [M + Na]+; anal. C 57.87%, H 7.40% (calcd for
6.99 (2H, d, J ) 8.4 Hz, H-2′′, H-6′′), 6.76 (1H, d, J ) 1.8 Hz,
H-2′), 6.68 (1H, d, J ) 8.0 Hz, H-5′), 6.67 (2H, d, J ) 8.4 Hz,
H-3′′, H-5′′), 6.61 (1H, dd, J ) 8.0, 1.7 Hz, H-6′), 3.81 (2H, m,
H-3, H-5), 3.81 (3H, s, OMe), 2.69-2.50 (4H, m, H2-1, H2-7),
1.72-1.64 (4H, m, H2-2, H2-6), 1.54 (2H, dd, J ) 6.5, 5.7 Hz,
H2-4); 13C NMR, see Table 1; HREIMS m/z 346.1764 [M]+
(calcd for C20H26O5, 346.1780).
Meth yla tion of 5a . Compound 5a (4.0 mg) was methylated
by the same procedure as described for 1 to yield 1a (2.5 mg).
Com p ou n d 6: amorphous solid; [R]23D -8.0° (c 0.10, MeOH);
UV (MeOH) λmax 283.6 nm (log ꢀ 3.70); UV (MeOH + 1 M
NaOH) λmax 293.2 nm; IR (film) νmax 3347 (OH), 2942 (CH),
1605 and 1520 (aromatic rings), 1445, 1369, 1284, 1113, 1077,
C
27H38O11‚H2O, C 58.26%, H 7.24%).
En zym a tic Hyd r olysis of 8. Compound 8 (11.8 mg) was
1
1024, 813 cm-1; H NMR (CD3OD) δ 6.60 (2H, d, J ) 7.9 Hz,
treated with â-D-glucosidase (39 mg) in HOAc/NaOAc buffer
(pH 5.0, 10 mL) at room temperature for 18 h to yield 8a (5.5
mg) and D-glucose.
H-5′, H-5′′), 6.60 (1H, d, J ) 1.8 Hz, H-2′), 6.58 (1H, d, J ) 1.9
Hz, H-2′′), 6.48 (1H, dd, J ) 7.9, 1.8 Hz, H-6′), 6.46 (1H, dd, J
) 7.9, 1.9 Hz, H-6′′), 4.25 (1H, d, J ) 7.8 Hz, Glc-1), 3.89 (1H,
m, H-3), 3.87 (1H, m, H-5), 3.84 (1H, dd, J ) 11.6, 2.3 Hz,
Glc-6a), 3.53 (1H, dd, J ) 11.6, 7.4 Hz, Glc-6b), 3.31 (1H, dd,
J ) 9.0, 8.9 Hz, Glc-3), 3.20 (1H, ddd, J ) 9.3, 7.4, 2.3 Hz,
Glc-5), 3.14 (1H, dd, J ) 9.3, 8.9 Hz, Glc-4), 3.12 (1H, dd, J )
9.0, 7.8 Hz, Glc-2), 2.60-2.42 (4H, m, H2-1, H2-7), 1.83-1.65
(2H, m, H2-2), 1.64-1.52 (3H, m, H-4a, H2-6), 1.47 (1H, ddd,
J ) 14.2, 10.2, 2.4 Hz, H-4b); 13C NMR, see Table 1; HR-
FABMS (positive mode) m/z 533.1975 [M + Na]+ (calcd for
Com p ou n d 8a : amorphous solid; [R]25 +4.7° (c 0.09,
D
MeOH); UV (MeOH) λmax 280.1 nm (log ꢀ 3.49); UV (MeOH +
1 M NaOH) λmax 288.6 nm; IR (film) νmax 3333 (OH), 2932 (CH),
1600 and 1517 (aromatic rings), 1456, 1427, 1264, 1152, 1035,
1
796 cm-1; H NMR (CD3OD) δ 6.76 (2H, d, J ) 1.7 Hz, H-2′,
H-2′′), 6.68 (2H, d, J ) 7.9 Hz, H-5′, H-5′′), 6.61 (2H, dd, J )
7.9, 1.7 Hz, H-6′, H-6′′), 3.86-3.79 (2H, m, H-3, H-5), 3.81 (3H
× 2, s, OMe × 2), 2.70-2.52 (4H, m, H2-1, H2-7), 1.72-1.67
(4H, m, H2-2, H2-6), 1.54 (2H, t-like, J ) 5.6 Hz, H2-4); 13C
NMR, see Table 1; EIMS m/z 376 [M]+.
C
25H34O11‚Na, 533.2000).
En zym a tic Hyd r olysis of 6. Compound 6 (27.5 mg) was
Meth yla tion of 8a . Compound 8a (4.0 mg) was methylated
by the same procedure as described for 1 to yield 2a (2.1 mg).
Com p ou n d 9: amorphous solid; [R]23D -8.0° (c 0.10, MeOH);
UV (MeOH) λmax 279.4 nm (log ꢀ 3.44); UV (MeOH + 1 M
NaOH) λmax 281.6 nm; IR (film) νmax 3357 (OH), 2926 (CH),
1613 and 1515 (aromatic rings), 1454, 1371, 1237, 1172, 1078,
treated with naringinase (50 mg) in HOAc/KOAc buffer (pH
4.3, 10 mL) at room temperature for 12 h to yield 2 (18.0 mg)
and a sugar fraction (6.5 mg). HPLC analysis of the sugar
fraction under the same conditions as in the case of 3 showed
the presence of D-glucose; tR (min) 16.97 (D-glucose, positive
optical rotation).
1
1031, 825 cm-1; H NMR (CD3OD) δ 7.02 (2H, d, J ) 8.6 Hz,
Com p ou n d 7: amorphous solid; [R]25 -16.0° (c 0.10,
H-2′, H-6′), 7.00 (2H, d, J ) 8.6 Hz, H-2′′, H-6′′), 6.67 (4H, d,
J ) 8.6 Hz, H-3′, H-5′, H-3′′, H-5′′), 4.29 (1H, d, J ) 7.8 Hz,
Glc-1), 3.94 (1H, m, H-3), 3.92 (1H, m, H-5), 3.89 (1H, dd, J )
11.6, 2.4 Hz, Glc-6a), 3.58 (1H, dd, J ) 11.6, 7.5 Hz, Glc-6b),
3.35 (1H, dd, J ) 9.1, 9.0 Hz, Glc-3), 3.25 (1H, ddd, J ) 9.3,
7.5, 2.3 Hz, Glc-5), 3.17 (1H, dd, J ) 9.3, 9.0 Hz, Glc-4), 3.16
(1H, dd, J ) 9.1, 7.8 Hz, Glc-2), 2.71-2.52 (4H, m, H2-1, H2-
7), 1.87-1.71 (2H, m, H2-2), 1.70-1.57 (3H, m, H-4a, H2-6),
1.50 (1H, ddd, J ) 14.2, 10.2, 2.5 Hz, H-4b); 13C NMR, see
Table 1; FABMS (positive mode) m/z 501 [M + Na]+; anal. C
59.44%, H 7.38% (calcd for C25H34O9‚3/2H2O, C 59.39%, H
7.38%).
D
MeOH); UV (MeOH) λmax 282.2 nm (log ꢀ 3.45); UV (MeOH +
1 M NaOH) λmax 289.2 nm; IR (film) νmax 3388 (OH), 2924 (CH),
1604 and 1516 (aromatic rings), 1451, 1432, 1364, 1271, 1232,
1078, 1033, 815 cm-1 1H NMR (CD3OD) δ 6.79 (1H, d, J )
;
1.7 Hz, H-2′), 6.68 (1H, d, J ) 8.1 Hz, H-5′), 6.65 (1H, d, J )
7.9 Hz, H-5′′), 6.64 (1H, dd, J ) 8.1, 1.7 Hz, H-6′), 6.63 (1H, d,
J ) 1.8 Hz, H-2′′), 6.50 (1H, dd, J ) 7.9, 1.8 Hz, H-6′′), 4.30
(1H, d, J ) 7.8 Hz, Glc-1), 3.94 (1H, m, H-3), 3.93 (1H, m, H-5),
3.88 (1H, dd, J ) 11.6, 2.3 Hz, Glc-6a), 3.82 (3H, s, OMe), 3.58
(1H, dd, J ) 11.6, 7.3 Hz, Glc-6b), 3.34 (1H, dd, J ) 9.0, 8.8
Hz, Glc-3), 3.24 (1H, ddd, J ) 9.6, 7.3, 2.3 Hz, Glc-5), 3.18
(1H, dd, J ) 9.6, 9.0 Hz, Glc-4), 3.17 (1H, dd, J ) 8.8, 7.8 Hz,
Glc-2), 2.73-2.46 (4H, m, H2-1, H2-7), 1.90-1.73 (2H, m, H2-
2), 1.71-1.58 (3H, m, H-4a, H2-6), 1.50 (1H, ddd, J ) 14.2,
10.4, 2.5 Hz, H-4b); 13C NMR, see Table 1; HRFABMS (positive
mode) m/z 547.2139 [M + Na]+ (calcd for C26H36O11‚Na,
547.2155).
En zym a tic Hyd r olysis of 9. Compound 9 (94.0 mg) was
treated with naringinase (140 mg) in HOAc/KOAc buffer (pH
4.3, 15 mL) at room temperature for 18 h to yield 9a (50.0
mg) and a sugar fraction (15 mg). HPLC analysis of the sugar
fraction under the same conditions as in the case of 3 showed
the presence of D-glucose; tR (min) 16.97 (D-glucose, positive
optical rotation).
En zym a tic Hyd r olysis of 7. Compound 7 (19.8 mg) was
treated with â-D-glucosidase (Sigma, 94 mg) in HOAc/NaOAc
buffer (pH 5.0, 10 mL) at room temperature for 18 h to yield
7a (6.0 mg) and D-glucose.
Com p ou n d 9a : amorphous solid; [R]26 +4.0° (c 0.10,
D
MeOH); UV (MeOH) λmax 279.8 nm (log ꢀ 3.50); UV (MeOH +
1 M NaOH) λmax 289.2 nm; IR (film) νmax 3279 (OH), 2933 (CH),
1613, 1598 and 1514 (aromatic rings), 1454, 1365, 1239, 1173,
Com p ou n d 7a : amorphous solid; [R]24 +3.2° (c 0.06,
D
1
MeOH); UV (MeOH) λmax 282.6 nm (log ꢀ 3.71); UV (MeOH +
1 M NaOH) λmax 293.2 nm; IR (film) νmax 3376 (OH), 2936 (CH),
1604 and 1516 (aromatic rings), 1454, 1367, 1261, 1151, 1033,
1056, 826 cm-1; H NMR (CD3OD) δ 6.99 (4H, d, J ) 8.4 Hz,
H-2′, H-6′, H-2′′, H-6′′), 6.67 (4H, d, J ) 8.4 Hz, H-3′, H-5′,
H-3′′, H-5′′), 3.78 (2H, m, H-3, H-5), 2.68-2.50 (4H, m, H2-1,
H2-7), 1.69-1.63 (4H, m, H2-2, H2-6), 1.53 (2H, dd, J ) 6.5,
5.8 Hz, H2-4); 13C NMR, see Table 1; EIMS m/z 298 [M]+.
Meth yla tion of 9a . Compound 9a (66.4 mg) was methy-
lated by the same procedure as described for 1 to yield 9b (40.0
mg).
1
797 cm-1; H NMR (CD3OD) δ 6.76 (1H, d, J ) 1.8 Hz, H-2′),
6.68 (1H, d, J ) 8.1 Hz, H-5′), 6.64 (1H, d, J ) 8.1 Hz, H-5′′),
6.62 (1H, d, J ) 2.0 Hz, H-2′′), 6.61 (1H, dd, J ) 8.1, 1.8 Hz,
H-6′), 6.50 (1H, dd, J ) 8.1, 2.0 Hz, H-6′′), 3.81 (2H, m, H-3,
H-5), 3.82 (3H, s, OMe), 2.70-2.45 (4H, m, H2-1, H2-7), 1.72-
1.61 (4H, m, H2-2, H2-6), 1.53 (2H, t-like, J ) 5.6 Hz, H2-4);
13C NMR, see Table 1; HREIMS m/z 362.1724 [M]+ (calcd for
Com p ou n d 9b: amorphous solid; [R]28 +6.9° (c 0.14,
D
EtOH); UV (MeOH) λmax 278.0 nm (log ꢀ 3.47); IR (film) νmax
C
20H26O6, 362.1729).
3301 (OH), 2937 and 2838 (CH), 1612, 1582 and 1514

New Diarylheptanoids from Tacca
J ournal of Natural Products, 2002, Vol. 65, No. 3 289
(aromatic rings), 1462, 1442, 1428, 1336, 1250, 1178, 1095,
1066, 1032, 908, 819, 810 cm-1; 1H NMR (CD3OD) δ 7.08 (4H,
d, J ) 8.6 Hz, H-2′, H-6′, H-2′′, H-6′′), 6.80 (4H, d, J ) 8.6 Hz,
H-3′, H-5′, H-3′′, H-5′′), 3.78 (2H, m, H-3, H-5), 3.74 (6H, s,
OMe × 2), 2.71-2.53 (4H, m, H2-1, H2-7), 1.70-1.65 (4H, m,
H2-2, H2-6), 1.54 (2H, dd, J ) 6.5, 5.8 Hz, H2-4); 13C NMR, see
Table 1; EIMS m/z 344 [M]+.
30% FBS and antibiotics. When outgrowth of the cells was
observed, the medium was replaced twice until the cells
reached confluence. The cells were detached from the mono-
layer by trypsinization and recultured in 100 cm2 tissue culture
flasks until confluent monolayers were again obtained. Cells
between the fifth and seventh passages were used. Cells were
trypsinized and inoculated at 6 × 103 per each 96-microwell
plate (Falcon, flat bottom, treated polystyrene, Becton Dick-
inson, San J ose, CA) and incubated for 24 h. After washing
once with PBS, they were treated for 24 h without or with
test compounds. They were washed once with PBS and
incubated for 4 h with 0.2 mg/mL MTT in DMEM supple-
mented with 10% FBS. After the medium was removed, the
cells were lysed with 0.1 mL DMSO and the relative viable
cell number was determined by measuring the absorbance at
540 nm of the cell lysate, using Labsystems Multiskan
(Biochromatic, Helsinki, Finland) connected to a Star/DOT
Matrix printer J L-10.
Cell Cu ltu r e a n d Assa y for Cytotoxic Activity a ga in st
HL-60 Cells. HL-60 cells, which were obtained from Human
Science Research Resources Bank (J CRB 0085, Osaka, J apan),
were maintained in RPMI 1640 medium containing 10% heat-
inactivated FBS and antibiotics (100 units/mL penicillin and
100 µg/mL streptomycin) in a 5% CO2 humidified incubator
at 37 °C. The cells were washed and resuspended in the
medium to 4 × 104 cells/mL, and 196 µL of this cell suspension
was divided into 96-well flat bottom plates (Iwaki Glass, Chiba,
J apan). The cells were incubated in 5% CO2/air for 24 h at 37
°C. After incubation, 4 µL of EtOH-H2O (1:1) solution
containing the sample was added to give the final concentra-
tions of 0.1-10 µg/mL, and 4 µL of EtOH-H2O (1:1) was added
into control wells. The cells were further incubated for 72 h
in the presence of each agent, and then cell growth was
evaluated using a modified MTT reduction assay.11 At the end
of incubation, 10 µL of 5 mg/mL MTT in phosphate-buffered
saline was added to every well, and the plate was further
incubated in 5% CO2/air for 4 h at 37 °C. Then the plate was
centrifuged at 1500g for 5 min to precipitate MTT formazan.
An aliquot of 150 µL of supernatant was removed from every
well, and 175 µL of DMSO was added to dissolve the MTT
formazan crystals. The plate was mixed on a microplate mixer
for 10 min and then read on a microplate reader (Spectra
Classic, Tecan, Salzburg, Austria) at 550 nm. Each assay was
done in triplicate, and cytotoxicity was expressed as IC50 value,
which reduces the viable cell number by 50%.
Cell Cu ltu r e a n d Assa y for Cytotoxic Activity a ga in st
HSC-2 Cells a n d HGF . HSC-2 cells were maintained as
monolayer cultures at 37 °C in DMEM supplemented with 10%
heat-inactivated FBS in a humidified 5% CO2 atmosphere.
HGF were isolated, as described previously.15 Briefly, gingival
tissues were obtained from healthy gingival biopsies from a
10-year-old girl, undergoing periodontal surgery. The tissue
were cut into 1 to 2 mm3 pieces, washed twice with phosphate-
buffered saline (PBS, 0.01 M phosphate buffer, 0.15 M NaCl,
pH 7.4) supplemented with 100 U/mL penicillin and 100 µg/
mL streptomycin, and placed into 25 cm2 tissue culture flasks.
The explants were incubated in R-MEM supplemented with
Refer en ces a n d Notes
(1) Tsukamoto, Y., Exct. Ed. The Grand Dictionary of Horticulture;
Shogakukan: Tokyo, 1989; Vol. 1, pp 148-149.
(2) Dictionary of Chinese Medicinal Materials; Shanghai Scientific and
Technological Press: Shanghai, 1977; Vol. 2, pp 1356-1357.
(3) Chen, Z. L.; Wang, B. D.; Chen, M. Q. Tetrahedron Lett. 1987, 28,
1673-1676.
(4) Zhou, J .; Chen, C.; Liu, R.; Yang, C. Zhiwu Xuebao 1983, 25, 568-
573.
(5) Harada, N.; Saito, A.; Ono, H.; Gawronski, J .; Gawronska, K.;
Sugioka, T.; Uda, H.; Kuriki, T. J . Am. Chem. Soc. 1991, 113, 3842-
3850.
(6) Nomura, M.; Tokoroyama, T.; Kubota, T. Phytochemistry 1981, 20,
1097-1104.
(7) Alegrio, L. V.; Braz-Filho, R.; Gottlieb, O. R. Phytochemistry 1989,
28, 2359-2362.
(8) Tezuka, Y.; Gewali, M. B.; Ali, M. S.; Banskota, A. H.; Kadota, S. J .
Nat. Prod. 2001, 64, 208-213.
(9) Ali, M. S.; Tezuka, Y.; Awale, S.; Banskota, A. H.; Kadota, S. J . Nat.
Prod. 2001, 64, 289-293.
(10) Ali, M. S.; Tezuka, Y.; Banskota, A. H.; Kadota, S. J . Nat. Prod. 2001,
64, 491-496.
(11) Itokawa, H.; Aiyama, R.; Ikuta, A. Phytochemistry 1981, 20, 769-
771.
(12) Ohta, S.; Aoki, T.; Hirata, T.; Suga, T. J . Chem. Soc., Perkin Trans.
1 1984, 1635-1642.
(13) Nagai M.; Kenmochi, N.; Fujita, M.; Furukawa, N.; Inoue, T. Chem.
Pharm. Bull. 1986, 34, 1056-1060.
(14) Sargent, J . M.; Taylor, C. G. Br. J . Cancer 1989, 60, 206-210.
(15) Fujisawa, S.; Kashiwagi, Y.; Atsumi, T.; Iwakura, I.; Ueha, T.; Hibino,
Y.; Yokoe, I. J . Dent. 1999, 27, 291-295.
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