Secoiridoid and iridoid glucosides from Syringa afghanica.

Article abstract of DOI:10.1016/S0031-9422(02)00024-9

Phytochemical investigation of the dried leaves of Syringa afghanica, has led to the isolation of nine secoiridoid glucosides, safghanosides A-H and 2"-epi-frameroside, as well as an iridoid glucoside, syringafghanoside along with nineteen known compounds. The structures were elucidated by spectroscopic and chemical means.

Full text of DOI:10.1016/S0031-9422(02)00024-9

Phytochemistry 59 (2002) 779–787
www.elsevier.com/locate/phytochem
Secoiridoid and iridoid glucosides from Syringa afghanica
Yukiko Takenakaa, Naoaki Okazakia, Takao Tanahashia,*,
Naotaka Nagakuraa, Toyoyuki Nishib
aKobe Pharmaceutical University, 4-19-1, Motoyamakita-machi, Higashinada-ku, Kobe 658-8558, Japan
bThe Nippon Shinyaku Institute for Botanical Research, 39, Sakanotsuji-cho, Oyake, Yamashina-ku, Kyoto 607-8182, Japan
Received 5 July 2001; received in revised form 29 November 2001
Abstract
Phytochemical investigation of the dried leaves of Syringa afghanica, has led to the isolation of nine secoiridoid glucosides, saf-
ghanosides A–H and 200-epi-frameroside, as well as an iridoid glucoside, syringafghanoside along with nineteen known compounds.
The structures were elucidated by spectroscopic and chemical means. # 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Syringa afghanica; Oleaceae; Secoiridoid glucosides; Iridoid glucoside; Oleoside; Safghanosides A–H; 200-Epi-frameroside; Syringafghanoside
1. Introduction
oleoside dimethyl ester (Kikuchi and Yamauchi, 1984),
methyl glucooleoside (Kuwajima et al., 1989), isoligustro-
side (Kikuchi et al., 1987), isooleuropein (Kikuchi et al.,
1987), ligstroside (Kikuchi and Yamauchi, 1984), oleur-
opein (Inoue et al., 1985), formoside (11) (Tanahashi et
al., 1993), 100-O-b-d-glucosylformoside (Tanahashi et
al., 1993), fraxiformoside (12) (Tanahashi et al., 1992),
1000-O-b-d-glucosylfraxiformoside (Tanahashi et al.,
1993), rhoifolin (Kaneo and Matsuda, 1978), and
luteolin (Markham et al., 1978).
Syringa afghanica C. K. Schneid. (=S. persica Aitch.)
is a shrub found in Afghanistan and Pakistan (Kita-
mura, 1960). The bark of this plant species has been
used as a tonic and antipyretic in folklore. In the course
of our chemical studies on the glycosides of oleaceous
plants (Tanahashi et al., 1999, Takenaka et al., 2000), we
investigated the constituents of the dried leaves of S.
afghanica and isolated seven monomeric and two dimeric
secoiridoid glucosides as well as an iridoid glucoside.
This paper deals with their structural determinations.
Compound 1, was obtained as a colorless amorphous
powder, analysed for C32H40O17 from its HR–SIMS. Its
UV and IR spectra suggested the presence of an enol-
ether system conjugated with a carbonyl group (241.5 nm,
and 1716, 1635 cmÀ1) that was typical of a secoiridoid
nucleus. In addition, absorptions due to an aromatic ring
2. Results and discussion
1
The n-BuOH soluble fraction of the methanolic extract
of the dried leaves of S. afghanica was fractionated by
column chromatography on ODS and then purified by
prep. HPLC, affording nine secoiridoid glucosides named
safghanosides A–H (1–8) and 200-epi-frameroside (9), and
an iridoid glucoside, syringafghanoside (10) together with
19 known compounds, cinnamic acid, 1-O-trans- and cis-
cinnamoyl-b-d-glucopyranose, syringin (Karasawa et al.,
1986), acteoside (Imakura et al., 1985), poliumoside
(Andary et al., 1985), lipedoside A-I (He et al., 1994),
(279.5 nm, 1448 cmÀ1) were observed. The H and 13C
NMR spectra of 1 exhibited typical signals of an oleo-
1
side 11-methyl ester (13) unit. Moreover, its H NMR
spectrum displayed resonances for an anomeric proton
at ꢀ 5.60 (d, J=8.0 Hz), a pair of trans-olefinic protons
at ꢀ 6.58 and 7.81 (each d, J=16.0 Hz), and five aro-
matic protons at ꢀ 7.41–7.63, suggesting the presence of
a 1-O-acyl-b-glucose moiety and a trans-cinnamic acid
moiety. The HMBC experiments with 1 revealed sig-
3
nificant J interactions between H-3 and C-11, between
OMe and C-11, between H2-6 and C-7, between H2-600
and C-7 and between H-100 and C-1000. These findings
suggested that in the structure of 1, the C-7 carboxyl
group of oleoside 11-methyl ester moiety was linked to
* Corresponding author. Tel.: +81-78-441-7546; fax: +81-78-441-
7547.
E-mail address: tanahash@kobepharma-u.ac.jp (T. Tanahashi).
0031-9422/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(02)00024-9

780
Y. Takenaka et al. / Phytochemistry 59 (2002) 779–787
the C-600 hydroxyl group of 1-O-trans-cinnamoyl-b-D-
glucopyranose. Thus, 1 was established as depicted and
given the trivial name safghanoside A.
Compound 3 was isolated as an amorphous powder
with the molecular formula C30H40O17. The NMR
spectra of 3 exhibited the signals assignable to an oleo-
side (14) moiety together with a p-hydroxyphenethyl
alcohol moiety and an additional b-glucose unit. The
ester linkages of oleoside with p-hydroxyphenethyl
alcohol and glucose units were determined by HMBC
experiments, which showed cross-peaks between H-100
and C-11 and between H-1000 and C-7. Consequently, the
structure of the new compound was represented by 3
and designated safghanoside C.
Safghanoside B (2), C32H40O17 was recognized to be
an isomer of 1. The only significant differences in their
1H NMR spectra were the chemical shifts and the cou-
pling constants of a pair of olefinic protons (2: ꢀ 6.04
and 7.09, each d, J=12.5 Hz), suggesting that 2 pos-
sessed a cis-cinnamoyl group instead of a trans-cinna-
moyl group as in 1. Accordingly, glucoside 2 was
formulated as shown.

Y. Takenaka et al. / Phytochemistry 59 (2002) 779–787
781
The 1H NMR spectrum (Table 1) of compound 4 was
similar to that of fraxiformoside (12) (Tanahashi et al.,
1992) except that the signals in the aromatic region were
observed as an AA0BB0 and an ABX spin systems in 4
instead of two AA0BB0 spin systems as in 12. The 13C
NMR spectral data (Table 2) of 4 were also super-
imposable on those of 12, except for the aromatic car-
bon signals, which were in good agreement with those
of the 3,4-dihydroxyphenyl moiety of oleuropein. These
finding indicated that the glucoside 4 possessed an
oleoside, a p-hydroxyphenethyl moiety and a 3,4-dihy-
droxyphenethyl moiety. The important HMBC correla-
tion observed between H2-100 and conjugated carbonyl
signal was indicative of an ester linkage of C-11 of an
oleoside (14) unit with an alcoholic hydroxyl group of
3,4-dihydroxyphenethyl alcohol. Attachment of the p-
hydroxyphenethyl alcohol unit at C-7 through a phenolic
hydroxyl group was supported by comparative studies on
the 13C NMR spectra of formoside (11) (Tanahashi et al.,
1993). Thus, compound 4 was formulated as shown and
designated safghanoside D.
between H2-100b and C-11a and between OMe and
C-11b. Furthermore, the chemical shifts of C-300b, C-
500b, C-700b and C-7b suggested that the C-600b hydroxyl
group was esterified with the C-7b carboxyl group. The
structural difference between 7 and 8 could be ascribed
to the point of ester linkage of another p-hydroxy-
phenethyl moiety, which was discriminated by a combi-
nation of 2D-NMR experiments and the chemical shifts
3
of carbon signals. The important J correlation in 7,
observed between H2-100a and C-7a, was indicative of an
ester linkage of C-7a of the oleoside unit with the C-100a
hydroxyl of the p-hydroxyphenethyl group. On the
other hand, the chemical shifts of the aromatic carbons
and C-7a in 8 suggested the attachment of the phenolic
hydroxyl group at C-600a to C-7a. Consequently, the glu-
cosides were represented by 7 and 8, and were designated
safghanosides G and H, respectively.
The 13C NMR spectrum of compound 9, C27H38O15
(HR–SIMS m/z 601.2155 [MÀH]À), indicated that 9
was composed of an oleoside 11-methyl ester unit and a
monoterpene unit. The combination of 2D-NMR stud-
ies suggested that the planar structure of 9 was the same
as frameroside (16) (Takenaka et al., 2000). The relative
configurations of the cyclopentane moiety in 9 were
suggested by its NOESY spectrum. The important NOE
interaction was observed between H3-600 and H-300 but
not between H3-600 and H-200, demonstrating that H-300
exists on the same b face as the methyl group (H3-600),
while H-200 has a orientation.
Glucoside 5, named safghanoside E, on HR–SIMS
exhibited a peak at m/z 807.2727 ([M–H]À) consistent
with a molecular formula of C38H48O19. The 1H and 13
C
NMR spectral (Tables 1 and 2) features of 5 were simi-
lar to those of 4, except for the presence of signals
assignable to an extra b-glucopyranosyl unit and for the
chemical shifts of the signals arising from the p-
hydroxyphenethyl unit. The glycosidation shifts of C-1000
(+7.4 ppm) and C-2000 (À3.0 ppm), when compared with
the corresponding signals of 4, suggested the attachment
of a glucose unit at the hydroxyl group on C-1000. This
was further confirmed by an HMBC experiment, which
showed cross-peaks between H-10000 at ꢀ 4.30 (d, J=8.0
Hz) and C-1000 at ꢀ 71.5 as well as between H2-100 and
C-11. Accordingly, the structure of the new compound
was represented by 5.
Safghanoside F (6), C38H48O18, was isolated as a col-
orless amorphous powder. The 1H NMR spectrum
(Table 1) of 6 revealed signals attributable to an oleo-
side unit and two p-hydroxyphenethyl moiety, with an
additional signal of an anomeric proton at ꢀ 4.28 (d,
J=8.0 Hz). Its 13C NMR spectrum (Table 2) was very
similar to that of nuezhenide (15), except for the
appearance of an additional set of signals assigned to a
p-hydroxyphenethyl moiety. This unit was determined
to be attached at C-11 by HMBC cross-peaks between
H2-100 and C-11 as well as between H2-6000 and C-7 and
between H-1000 and C-10000. Thus, compound 6 was for-
mulated as shown.
Methylation of 9 with CH2N2–Et2O yielded the
methylated derivative 9a, which exhibited 1H NMR
signals for two additional carbomethoxyl groups at ꢀ
3.61 and 3.66, supporting the presence in 9 of two car-
1
boxyl groups. The H NMR spectral data of 9a was
unexpectedly identical with those of a methylated com-
pound of frameroside (16). This fact suggested that 9
has the same stereochemistry as 16, apart from C-200,
which is epimerisable because of the adjacent carbo-
methoxyl group. Comparative studies of 1H and 13C
NMR spectra of 9a, 9 and 16 as well as the analysis of
the NOESY spectrum of 9a indicated that no epimer-
ization had taken place in preparation of 9a from 9. By
contrast, methylated compound of 16 was not the
expected 17 (Takenaka et al., 2000) but a C-200 epimer of
17, i.e. 9a. A similar epimerization has been reported for
ꢁ-(2-formyl-3-methylcyclopentyl)acrylaldehyde (Pagnoni
et al., 1976). Thus, compound 9 was determined to be
200-epi-frameroside.
Compound 10 was obtained as a colorless amorphous
powder. The HR–SIMS of 10 exhibited a strong
[MÀH]À at m/z 507.1866, indicating a molecular for-
mula of C25H32O11 for 10. It showed UV maxima at
216, 222, and 276 nm and IR bands at 3393, 1712, 1637,
and 1570 cmÀ1. The 1H NMR spectrum displayed,
besides the signals corresponding to a trans-cinnamic
acid and a b-glucose unit, signals for a secondary
Compounds 7 and 8 were recognized as isomers,
C49H60O23, from their HR–SIMS. The NMR spectral
features (Tables 1 and 2) demonstrated clearly the presence
of two oleoside units, two p-hydroxyphenethyl moieties
and a carbomethoxyl group in each compound. The
HMBC experiments with 7 and 8 showed interactions

Table 1
1H NMR spectroscopic data of compounds 4–8 in CD3OD
H
4
5
6
H
7
8
a Part
5.91 br s
b Part
a Part
6.01g brs
b Part
1
3
5
6
6.01 br s
7.52 s
6.01 br s
5.90 br s
7.46 s
1
3
5
6
6.02 br s
7.51c
6.03g br s
7.52
s
7.50c
s
s
7.51
s
7.57 s
4.07 dd
(9.0, 4.0)
4.07 dd
2.66 dd
2.87 dd
6.17 qd
1.75 dd
(9.0, 5.0)
(14.0, 9.0)
(14.0, 5.0)
(7.0, 1.0)
(7.0, 2.0)
(8.0)
3.97 dd
2.43 dd
2.69 dd
6.08 qd
1.72 dd
4.79 d
(9.0, 4.0)
(14.0, 9.0)
(14.0, 4.0)
(7.0, 1.0)
(7.0, 1.0)
(8.0)
3.94 dd
(9.0, 4.0) 4.07 dd (9.0, 4.0) 4.06 dd (9.0, 4.0) 4.11 dd (9.0, 4.0)
2.66 dd (14.0, 9.0)
2.87 dd (14.0, 4.0)
6.16 br q (7.0)
2.43 dd (14.0, 9.0) 2.66 dd (14.0, 9.0) 2.72 dd (14.0, 9.0) 2.65 dd (14.0, 9.0)
2.72 dd (14.0, 4.0) 2.87 dd (14.0, 4.0) 2.95 dd (14.0, 4.0) 2.85 dd (14.0, 4.0)
8
8
6.05 qd
1.59 dd
(7.0, 1.0) 6.16 qd (7.0, 1.0) 6.17 qd (7.0, 1.0) 6.17 qd (7.0, 1.0)
(7.0, 1.0) 1.74 dd (7.0, 1.0) 1.74 dd (7.0, 1.0) 1.74 dd (7.0, 1.0)
10
1.74 dd
(7.0, 2.0)
(8.0)
10
OMe
10
30
60
10, 10000 4.82 d
30, 30000 3.41 t
50, 50000
4.81
3.41
d
t
4.30
d
(8.0)
4.28 d
3.46 m
(8.0)
3.71
4.80d
3.41
s
d
t
3.72
4.82
3.41
s
d
t
(8.0)
(8.0)
3.40 t
(9.0)
4.82d
3.41
d
t
(8.0)
(8.5)
(8.0)
(8.5)
4.82
3.41
d
t
(8.0)
(8.5)
(8.0)
(8.5)
3.28 m
60, 60000 3.63 dd (12.0, 5.0)
3.63a dd
3.82b dd
(12.0, 5.0) 3.66a dd (12.0, 5.0) 3.66 dd
(12.0, 2.0) 3.86b dd (12.0, 2.0) 3.88 dd
3.76 dt (10.0, 7.0) 4.22 dt
(12.0, 5.5) 4.20 dd (12.0, 5.5)
3.63e dd (12.0, 5.5) 3.66e dd (12.0, 5.5) 3.63 dd (12.0, 5.5) 3.63 dd (12.0, 5.5)
3.83f dd (12.0, 2.0) 3.88f dd (12.0, 2.0) 3.82 dd (12.0, 2.0) 3.82 dd (12.0, 2.0)
3.83 dd (12.0, 1.0)
(12.0, 1.5) 4.35 dd (12.0, 2.0)
(14.0, 7.0) 3.68 ddd (9.5, 8.0, 6.5) 100
3.94 ddd (9.5, 8.0, 6.5)
100, 1000 4.27 dt (11.0, 6.5) 3.74 t (7.0) 4.28
m
4.16 dt (11.0, 6.5) 4.29 dt (11.0, 6.5) 3.74
4.29 dt (11.0, 6.5)
t
(7.0)
4.36
t
(6.5)
4.30 dt (11.0, 6.5)
4.09 dt (10.0, 7.0)
200, 2000 2.80 t
400, 4000 6.67 d
500, 5000
700, 7000 6.68 d
800, 8000 6.56 dd
(6.5)
(2.0)
2.81 t (7.0) 2.81
7.24 d (8.0) 6.67
6.99 d (8.0)
t
(7.0)
(2.0)
2.94
7.29
6.98
6.98
t
(7.0)
(8.0)
(8.0)
(8.0)
(8.0)
2.83 br t
7.03 d
6.68 d
6.68 d
7.03 d
(7.0)
(8.5)
(8.5)
(8.5)
(8.5)
2.81 m
7.05 d
6.70 d
6.70 d
7.05 d
200
400
500
700
800
2.86
7.06
6.70
6.70
7.06
t
(6.5)
(8.5)
(8.5)
(8.5)
(8.5)
2.94
7.28
7.02
7.02
7.28
t
(6.5)
(8.5)
(8.5)
(8.5)
(8.5)
2.82
7.25
6.99
6.99
7.25
t
(7.0)
(8.5)
(8.5)
(8.5)
(8.5)
2.98
7.29
7.01
7.01
7.29
t
(6.5)
(8.5)
(8.5)
(8.5)
(8.5)
d
d
d
d
d
(8.5)
(8.5)
(8.5)
(8.5)
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
(8.0)
6.99 d (8.0) 6.68
d
(8.0)
(8.0, 2.0) 7.24 d (8.0) 6.56 dd
(8.0, 2.0) 7.29
aÀg
Assignments may be interchangeable.

Y. Takenaka et al. / Phytochemistry 59 (2002) 779–787
783
Table 2
13C NMR spectroscopic data of compounds 4–8 in CD3OD
C
4
5
6
7
8
a Part
b Part
a Part
b Part
1
95.3
155.3
95.4
95.4 95.295.2 95.4
155.2155.3
109.6
95.4
j
3
155.3
109.5
31.8
155.2j
109.4
31.8
155.3
109.4
31.8
155.4
109.4
31.8
4
109.5
31.8
109.5
31.9
5
31.7
6
41.0
171.7
41.1
41.241.3
173.0
125.0
41.1
41.1
41.1
171.6
7
171.7
125.1
130.7
13.8
173.2171.6
125.0
130.4
171.7
8
125.1
130.6
125.2
130.6
13.9
125.2
130.7
13.9
125.2
130.6
13.9
9
10
130.7
13.8
13.8
1681.26186.281.768.2168.2 168.1
13.6
11
168.7
OMe
10, 10000
20, 20000
30, 30000
40, 40000
500, 50000
600, 60000
100, 1000
200, 2000
300, 3000
400, 4000
5 , 5000
600, 6000
700, 7000
800, 8000
52.0
100.9k
74.8
52.0
101.1
74.8
78.0
71.5
78.5n
62.7
66.0
35.5
101.0
74.8
77.9
71.4
78.4
62.7
101.1
74.8a
78.0
71.5b
78.2c
62.7d
66.4
104.4
75.2a
78.0
71.7b
78.4c
62.8d
71.5
100.9
74.8
78.0
71.5e
78.5
62.7
66.5
35.3
104.5
101.0k
74.8
78.0
101.1
74.8
78.0
71.5
78.4n
62.7
64.1
39.6
75.0
78.0
78.0
71.6l
71.7e
75.3
65.1
72.3
36.4
71.5l
78.5
78.5
62.7m
66.5
62.9m
66.5
66.4
35.5
64.1
39.6
35.6
36.6
35.4
35.3
131.0
116.4
146.3
144.9
117.0
121.3
138.2131.0
131.0
122.6
150.5
122.6
131.0
138.0
130.5
130.3
130.3
137.2 138.3
137.5
116.4
146.3
145.0
117.0
121.3
131.0
122.5
150.6
122.5
131.0
131.1
131.1
131.1
116.4
157.1
116.4
131.1
131.0
122.8
150.8
122.8
131.0
131.0o
122.6p
150.6
131.1o
122.7p
150.8
122.7q
131.1r
116.3f
156.8g
116.3h
131.1i
116.2f
157.1g
116.2h
131.0i
122.6q
131.0r
aÀr
Assignments may be interchangeable.
methyl group, three pairs of methylene protons and four
methine protons. The residual unit was formulated as the
at C-9. Oleoside-type glucosides with C6–C2 unit(s) such
as 4–8 represent common structures found in some
genera of the family. However, glucoside 9 is the first
example of oleoside-type secoiridoid glucoside esterified
with a cyclopentane monoterpene unit isolated from the
genus Syringa.
same cyclopentanoid monoterpene unit as in 9 by its 13
C
NMR, COSY, HMBC and NOESY spectra. The linkage
of these components were determined by an HMBC
experiment, which revealed cross-peaks between H2-9 and
H-10 and between H2-60 and C-100, suggesting the structure
of 10 as shown. Methylation of 10 with CH2N2–Et2O
yielded a methylate, whose spectral data were fully
consistent with the structure 10a. Finally, an (R)-PGME
amide (Ohtani et al., 1991) was prepared from 10 in
order to confirm the absolute configurations of the
monoterpene unit. Compound 10 was subjected to
alkaline hydrolysis and the resulting monoterpene lac-
tone 18 was converted to the (R)-PGME amide 19
which was identical with a (R)-PGME amide derived
from 16 (Takenaka et al., 2000). This unexpected result
could be accounted for by the fact that the asymmetric
center at C-9 was inverted in a similar manner as
observed for the formation of epi-nepetalactone (Trave
et al., 1968). Thus, the structure of the new compound
was represented by 10 with the absolute stereochemistry
1S, 2S, 3S and 8S and designated as syringafghanoside.
Glucosides 1–9 are secoiridoid glucosides of the oleo-
side type, which have so far been isolated only from the
oleaceous plants and structurally characterized by the
esterified C-7 carboxyl group and the ethylidene group
3. Experimental
3.1. General
1H (300 or 500 MHz) and 13C (75 or 125 MHz) NMR:
TMS as int. standard. SIMS: glycerol or 3-nitrobenzyl
alcohol as matrix. TLC: silica gel.
3.2. Plant material
Leaves of Syringa afghanica C. K. Schneid. were col-
lected at the Nippon Shinyaku Institute for Botanical
Research, Kyoto, Japan. A voucher specimen (KPU011)
is deposited in the laboratory of Kobe Pharmaceutical
University.
3.3. Isolation of glucosides
Dried leaves of S. afghanica (357.6 g) were extracted
with hot MeOH. After concentration, the extract (64.6 g)

784
Y. Takenaka et al. / Phytochemistry 59 (2002) 779–787
was suspended in H2O and filtered through a celite layer.
The filtrate and washings were combined and extracted
successively with CHCl3 and n-BuOH. The n-BuOH
extract (23.5 g) was subjected to Wakogel LP-40C18
(Wako Pure Chemical Industries, Ltd., Osaka, Japan)
chromatography. Elution with MeOH–H2O mixtures of
the indicated MeOH content gave 5 fractions I–V. Frac-
tion I (1–15% MeOH eluent, 4.40 g) was further purified
by chromatography on a Wakogel LP-40C18 column to
give 3 subfractions I-1–I-3. Fraction I-1 (1% MeOH elu-
ent, 380 mg) was further purified by preparative HPLC
(mBondasphere 5 m C18–100 A, H2O–MeOH, 3:2or 13:7)
and preparative TLC (CHCl3–MeOH, 7:3 or acetone–
CHCl3–H2O, 8:2:1), giving cinnamic acid (15 mg), 1-O-
trans-cinnamoyl-b-d-glucopyranose (10 mg), 1-O-cis-cin-
namoyl-b-d-glucopyranose (3.2mg), syringin (2mg),
acteoside (20 mg), oleoside dimethyl ester (15 mg) and
methyl glucooleoside (4.1 mg). Fr I-2(7–10% MeOH
eluent, 800 mg) was further purified by preparative HPLC
J=12.0, 5.0 Hz, H-600), 4.32(1H, dd, J=12.0, 2.0 Hz,
H-600), 4.81 (1H, d, J=8.0 Hz, H-10), 5.60 (1H, d, J=8.0
Hz, H-100), 5.91 (1H, br s, H-1), 6.09 (1H, qd, J=7.0, 1.0
Hz, H-8), 6.58 (1H, d, J=16.0 Hz, H-2), 7.41–7.63 (5H,
m, H-5000–9000), 7.51 (1H, s, H-3), 7.81 (1H, d, J=16.0 Hz,
H-3000); 13C NMR (CD3OD): ꢀ 13.7 (C-10), 31.8 (C-5),
41.2(C-6), 52.0 (OMe), 62.8 (C-6 0), 64.7 (C-600), 71.2(C-
40), 71.5 (C-400), 74.0 (C-20), 74.7 (C-200), 76.1 (C-500), 77.8
00
(C-50), 78.0 (C-30), 78.4 (C-300), 95.2(C-1), 95.8 (C-1 ),
100.9 (C-10), 109.4 (C-4), 118.3 (C-2000), 125.0 (C-8), 129.5
Â2(C-5 000, 9000), 130.1 Â2(C-6 000, 8000), 130.5 (C-9), 131.9 (C-
7000), 135.6 (C-4000), 147.8 (C-3000), 155.2(C-3), 167.0 (C-1 000),
168.8 (C-11), 173.0 (C-7); HMBC: H2-6!C-7, H-10!C-
1, OMe!C-11, H-100!C-1000, H2-600!C-7; HR–SIMS
found: 695.2183 [M– H]À; C32H39O17 requires 695.2188.
3.5. Safghanoside B (2)
26
Colourless amorphous powder, ½ꢁŠ À158ꢀ (c 0.33,
max
D
nm (log "): 217 sh (4.14), 225 (4.17),
MeOH
MeOH); UV l
240 (4.19), 277 (3.99); IR ꢂ
(H2O–MeOH, 11:9, 3:2or H O–MeCN, 4:1) and pre-
2
KBr
max
parative TLC (CHCl3–MeOH, 7:3, acetone–CHCl3–H2O,
8:2:1 or n-BuOH–AcOH–H2O, 8:2:1), giving isoligustro-
side (12mg), isooleuropein (11 mg) and safghanoside C ( 3)
(12 mg). Fr I-3 (20–50% MeOH eluent, 2.8 g) was purified
by preparative HPLC (H2O–MeOH, 9:11, 2:3, 3:7 or
H2O–MeCN, 3:1) and preparative TLC (CHCl3–MeOH–
AcOH, 7:3:0.1), giving rhoifolin (12.0 mg), poliumoside
(21.0 mg), luteolin (15.0 mg), lipedoside A-I (2.4 mg) and
200-epi-frameroside (9) (4.1 mg). The following fractions of
the initial column chromatography were also purified by
preparative HPLC (H2O–MeOH, 3:7, 1:1, 11:9, 3:2,
73:27, 3:1 or H2O–MeCN, 13:7, 7:3, 4:1) and preparative
TLC (CHCl3–MeOH, 7:3, acetone–CHCl3–H2O, 8:2:1 or
n-BuOH–AcOH–H2O, 8:2:1), Fr II (15–20% MeOH elu-
ent, 480 mg): ligstroside (14 mg), formoside (11) (49 mg),
100-O-b-d-glucosylformoside (6.8 mg) and safghanosode F
(6) (3.0 mg); fr III (20–40% MeOH eluent, 2.31 g): oleur-
opein (13 mg), 1000-O-b-d-glucosylfraxiformoside (41 mg),
safghanoside A (1) (39 mg), safghanoside B (2) (12 mg),
safghanoside D (4) (31 mg) and safghanoside E (5) (49
mg); fr IV (40–50% MeOH eluent, 3.85 g): fraxiformoside
(12) (4.2mg) and syringafghanoside ( 10) (78 mg); fr V (50–
60% MeOH eluent, 344 mg): safghanoside G (7) (10 mg)
and safghanoside H (8) (4.0 mg).
cmÀ1: 3393, 1733, 1716,
1
1635, 1437, 1074; H NMR (CD3OD): ꢀ 1.73 (3H, dd,
J=7.0, 1.5 Hz, H3-10), 2.51 (1H, dd, J=14.0, 8.0 Hz, H-
6), 2.74 (1H, dd, J=14.0, 5.0 Hz, H-6), 3.42(1H, t,
J=8.0 Hz, H-30), 3.67 (1H, dd, J=12.0, 5.5 Hz, H-60),
3.68 (3H, s, OMe), 3.87 (1H, dd, J=12.0, 1.5 Hz, H-60),
4.00 (1H, dd, J=8.0, 5.0 Hz, H-5), 4.23 (1H, dd, J=12.0,
5.0 Hz, H-600), 4.32(1H, dd, J=12.0, 2.0 Hz, H-600), 4.80
(1H, d, J=8.0 Hz, H-10), 5.53 (1H, d, J=8.0 Hz, H-100),
5.90 (1H, br s, H-1), 6.04 (1H, d, J=12.5 Hz, H-2000), 6.09
(1H, br q, J=7.0 Hz, H-8), 7.09 (1H, d, J=12.5 Hz, H-3000),
7.33–7.65 (5H, m, H-5000–9000), 7.50 (1H, s, H-3); 13C NMR
(CD3OD): ꢀ 13.7 (C-10), 31.7 (C-5), 41.2(C-6), 5.20
0
(OMe), 62.7 (C-60), 64.7 (C-600), 71.2(C-4 ), 71.5 (C-400),
73.9 (C-20), 74.7 (C-200), 76.1 (C-50), 77.9 (C-500), 78.0 (C-
30), 78.4 (C-300), 95.2(C-1), 95.6 (C-1 00), 100.9 (C-1000)0,
109.4 (C-4), 119.5 (C-2000), 125.0 (C-8), 129.1Â2(C-6
,
8000), 130.4 (C-7000), 130.5 (C-9000), 131.3Â2(C-5 000, 9000),
136.0 (C-4000), 146.3 (C-3000), 155.2(C-3), 166.0 (C-1 000),
168.7 (C-11), 173.0 (C-7); HMBC: H-10!C-1, H2-6!C-
7, OMe!C-11, H-100!C-1000, H2-600!C-7; HR–SIMS
found: 695.2198 [M– H]À; C32H39O17 requires 695.2188.
3.6. Safghanoside C (3)
27
Colourless amorphous powder, ½ꢁŠ À102ꢀ (c 0.83,
max
3.4. Safghanoside A (1)
D
nm (log "): 227 (4.18), 238 sh (4.11),
MeOH
MeOH); UV l
27
Colourless amorphous powder, ½ꢁŠ À143ꢀ (c 0.96,
max
KBr
277 (3.26), 285 sh (3.15); IR ꢂ
cmÀ1: 3393, 1740,
D
nm (log "): 218 (4.27), 223 (4.28),
max
MeOH
MeOH); UV l
241.5 (4.16), 279.5 (4.32); IR ꢂ
1716, 1635, 1517, 1076; 1H NMR (CD3OD): ꢀ 1.76 (3H,
dd, J=7.0, 1.0 Hz, H3-10), 2.54 (1H, dd, J=15.0, 9.0
Hz, H-6), 2.73 (1H, dd, J=15.0, 3.0 Hz, H-6), 2.86 (2H,
t, J=7.0 Hz, H2-200), 3.41 (1H, t, J=9.0 Hz, H-30), 3.66
(2H, dd, J=12.0, 5.5 Hz, H-60, 6000), 3.81 (1H, dd,
J=12.0, 1.0 Hz, H-60), 3.89 (1H, dd, J=12.0, 2.0 Hz, H-
6000), 3.98 (1H, dd, J=9.0, 3.0 Hz, H-5), 4.25, 4.28 (each
1H, dt, J=10.0, 7.0 Hz, H2-100), 4.80 (1H, d, J=8.0 Hz,
KBr
max
cmÀ1: 3403, 1716,
1
1635, 1448, 1076; H NMR (CD3OD): ꢀ 1.73 (3H, dd,
J=7.0, 2.0 Hz, H3-10), 2.50 (1H, dd, J=14.0, 9.0 Hz, H-
6), 2.75 (1H, dd, J=14.0, 5.0 Hz, H-6), 3.46 (1H, t,
J=8.0 Hz, H-30), 3.68 (1H, dd, J=12.0, 5.5 Hz, H-60),
3.71 (3H, s, OMe), 3.87 (1H, dd, J=12.0, 1.5 Hz, H-60),
4.00 (1H, dd, J=9.0, 5.0 Hz, H-5), 4.25 (1H, dd,

Y. Takenaka et al. / Phytochemistry 59 (2002) 779–787
785
KBr
H-10), 5.44 (1H, d, J=8.0 Hz, H-1000), 5.93 (1H, br s, H-1),
6.10 (1H, br q, J=7.0 Hz, H-8), 6.71 (2H, d, J=8.0 Hz, H-
500, 700), 7.07 (2H, d, J=8.0 Hz, H-400, 800), 7.45 (1H, s, H-3);
13C NMR (CD3OD): ꢀ 13.9 (C-10), 31.3 (C-5), 35.4 (C-
200), 40.5 (C-6), 62.4 (C-6000), 62.8 (C-60), 66.5 (C-100), 71.0
or 71.5 (C-40 or C-4000), 73.9 or 74.8 (C-20 or C-2000), 78.0 Â2
(C-50, 5000), 78.5 or 78.7 (C-30 or C-3000), 95.4 or 95.9 (C-1 or
C-1000), 100.9 (C-10), 109.4 (C-4), 116.3Â2(C-5 00, 700), 125.5
(C-8), 130.2Â2(C-3 00, 9), 131.0Â2(C-4 00, 800), 155.3 (C-3),
157.1 (C-600), 168.3 (C-11), 171.9 (C-7); HMBC: H-
10!C-1, H-3!C-11, H2-6!C-7, H-100 (ꢀ 4.27)!C-11,
H2-200!C-10, 400, 800, H-1000 (ꢀ 5.44)!C-7; HR–SIMS
found: 671.2194 [M–H]À; C30H39O17 requires 671.2194.
(4.24), 277 (3.64), 288 sh (3.54); IR ꢂ
cmÀ1: 3413,
max
1739, 1705, 1634, 1508, 1076; 1H and 13C NMR
(CD3OD): see Tables 1 and 2; HMBC: H-1a (ꢀ 5.91)
!C-10a (ꢀ 101.0), 9a (ꢀ 130.4), H-5a (ꢀ 3.94)!C-6a (ꢀ
41.3), 7a (ꢀ 173.2), H2-6a (ꢀ 2.43, 2.72)!C-4a (ꢀ 109.5),
7a (ꢀ 173.2), 9a (ꢀ 130.4), H-8a (ꢀ 6.05)!C-9a (ꢀ 130.4),
10a (ꢀ 13.6), H2-100a (ꢀ 4.16, 4.29)!C-7a (ꢀ 173.2), H2-100a
(ꢀ 4.29)!C-300a (ꢀ 130.3), H-400a and 800a (ꢀ 7.06)!C-
200a (ꢀ 35.4), 600a (ꢀ 157.1), H-500a and 700a (ꢀ 6.70)!C-
300a (ꢀ 130.3), 600a (ꢀ 157.1), H-1b (ꢀ 6.02)!C-10b (ꢀ
100.9), 9b (ꢀ 130.6), H-5b (ꢀ 4.07)!C-6b (ꢀ 41.1), H2-6b
(ꢀ 2.66, 2.87)!C-4b (ꢀ 109.4), 7b (ꢀ 171.6), 9b (ꢀ 130.6),
H-8b (ꢀ 6.16)!C-9b (ꢀ 130.6), 10b (ꢀ 13.9), H2-100b (ꢀ
4.29)!C-11a (ꢀ 168.2), 200b (ꢀ 35.3), H2-200b (ꢀ
2.94)!C-100b (ꢀ 66.5), 300b (ꢀ 137.2), H-400b and 800b (ꢀ
7.28)!C-600b (ꢀ 150.8), H-500b and 700b (ꢀ 7.02)!C-300b
(ꢀ 137.2), 600b (ꢀ 150.8), OMe (ꢀ 3.71)!C-11b (ꢀ 168.7);
HR–SIMS found: 1015.3454 [M–H]À; C49H59O23
requires 1015.3449.
3.7. Safghanoside D (4)
27
Colourless amorphous powder, ½ꢁŠ À129ꢀ (c 1.05,
max
D
nm (log "): 220 (4.26), 230 sh (4.24),
MeOH
MeOH); UV l
KBr
282.5 (3.49); IR ꢂ
cmÀ1: 3389, 1743, 1697, 1635, 1508,
max
1074; H and 13C NMR (CD3OD): see Tables 1 and 2;
HMBC: H-10!C-1, H-1!C-10, H-3!C-11, H2-6!C-7,
H-100 (ꢀ 4.27)!C-11, 300, H2-200!C-100, 300, H-400!C-200,
300, 500, H-700!C-600, H-800!C-600, H2-1000!C-2000, H2-
2000!C-1000, 4000, 8000, H-4000 and 8000 (ꢀ 7.24)!C-6000, H-5000
and 7000 (ꢀ 6.99)!C-6000; HR–SIMS found: 645.2172 [M–
H]À; C32H37O14 requires 645.2185.
1
3.11. Safghanoside H (8)
28
Colourless amorphous powder, ½ꢁŠ À142ꢀ (c 0.33,
max
D
nm (log "): 225.5 sh (4.22), 236
MeOH
MeOH); UV l
KBr
max
(4.28), 268 sh (3.22); ꢂ
cmÀ1: 3397, 1751, 1701, 1631,
1508, 1074; H and 13C NMR (CD3OD): see Tables 1
and 2; HMBC: H-10a (ꢀ 4.82)!C-1a (ꢀ 95.4), H-3a (ꢀ
7.51)!C-1a (ꢀ 95.4), 11a (ꢀ 168.1), H-5a (ꢀ 4.06)!C-3a
(ꢀ 155.3), 4a (ꢀ 109.4), 9a (ꢀ 130.7), 11a (ꢀ 168.1), H2-6a
(ꢀ 2.72, 2.95)!C-5a (ꢀ 31.8), 7a (ꢀ 171.6), H-8a (ꢀ
6.17)!C-9a (ꢀ 130.7), H3-10a (ꢀ 1.74)!C-9a (ꢀ 130.7),
H2-100a (ꢀ 3.74)!C-300a (ꢀ 138.3), H2-200a (ꢀ 2.82)!C-
100a (ꢀ 64.1), 300a (ꢀ 138.3), H-400a and 800a (ꢀ 7.25)!C-
200a (ꢀ 39.6), 600a (ꢀ 150.6), H-500a and 700a (ꢀ 6.99)!C-
300a (ꢀ 138.3), 600a (ꢀ 150.6), H-10b (ꢀ 4.82)!C-1b (ꢀ
95.4), H-3b (ꢀ 7.57)!C-1b (ꢀ 95.4), 11b (ꢀ 168.7), H-5b
(ꢀ 4.11)!C-3b (ꢀ 155.4), 4b (ꢀ 109.4), 9b (ꢀ 130.6), 11b
(ꢀ 168.7), H2-6b (ꢀ 2.65, 2.85)!C-5b (ꢀ 31.8), 7b (ꢀ
171.7), H-8b (ꢀ 6.17)!C-9b (ꢀ 130.6), H3-10b (ꢀ
1.74)!C-9b (ꢀ 130.6), H2-100b (ꢀ 4.36)!C-11a (ꢀ 168.1),
H2-200b (ꢀ 2.98)!C-100b (ꢀ 66.0), 300b (ꢀ 137.5), H-400b
and 800b (ꢀ 7.29)!C-200b (ꢀ 35.5), 600b (ꢀ 150.8), H-500b
and 700b (ꢀ 7.01)!C-300b (ꢀ 137.5), 600b (ꢀ 150.8), OMe (ꢀ
3.72)!C-11b (ꢀ 168.7); HR–SIMS found: 1015.3470
[M–H]À; C49H59O23 requires 1015.3449.
1
3.8. Safghanoside E (5)
26
Colourless amorphous powder, ½ꢁŠ À104ꢀ (c 0.69,
max
D
nm (log "): 220 (4.23), 243 sh (4.08),
MeOH
MeOH); UV l
KBr
282 sh (3.44); IR ꢂ
cmÀ1: 3383, 1740, 1697, 1522,
max
1074; H and 13C NMR (CD3OD): see Tables 1 and 2;
HMBC: H-10!C-1, H-3!C-11, H2-6!C-7, H2-100!C-
11, 200, H2-200!C-300, 400, 800, H-400!C-500, H-700!C-600, H2-
1000!C-10000, H2-2000!C-1000, 3000, 4000, 8000, H-4000 and 8000!C-
6000, H-5000 and 7000!C-3000, 6000, H-10000!C-1000; HR–SIMS
found: 807.2727 [M–H]À; C38H47O19 requires 807.2713.
1
3.9. Safghanoside F (6)
27
Colourless amorphous powder, ½ꢁŠ À101ꢀ (c 0.27,
max
D
nm (log "): 225.5 (4.27), 265 sh
MeOH
MeOH); UV l
KBr
(3.43), 286.5 sh (3.33); IR ꢂ
cmÀ1: 3407, 1749, 1734,
max
1635, 1508, 1078; 1H and 13C NMR (CD3OD): see
Tables 1 and 2; HMBC: H-3!C-11, H2-6!C-7, H-10
!C-1, H2-100!C-11, H2-200!C-100, 300, 400, 800, H-400 and
800!C-600, H-500 and 700!C-300, 600, H-1000!C-10000, H2-
10000!C-1000, H2-20000!C-10000, 30000 40000, 80000, H-40000 and
80000!C-60000, H-50000 and 70000!C-30000, 60000; HR–SIMS
found: 791.2770 [M–H]À; C38H47O18 requires 791.2764.
3.12. 200-Epi-frameroside (9)
24
Colourless amorphous powder, ½ꢁŠ À116ꢀ (c 0.22,
D
MeOH
KBr
MeOH); UV l
nm (log "): 237 (3.98); ꢂ
cmÀ1
:
max
max
3413, 1717, 1636, 1580, 1078; 1H NMR (CD3OD): ꢀ
1.09 (3H, d, J=7.0 Hz, H3-600), 1.23 (1H, m, H-500), 1.53
(1H, tt, J=12.0, 8.0 Hz, H-400), 1.74 (3H, dd, J=7.0, 1.0
Hz, H-10), 1.83 (1H, br dt, J=12.0, 6.0 Hz, H-400), 2.03
(1H, dt, J=12.0, 7.0 Hz, H-500), 2.29 (1H, m, H-100), 2.45
(1H, m, H- 200), 2.48 (1H, dd, J=14.0, 9.0 Hz, H-6), 2.59
3.10. Safghanoside G (7)
27
Colourless amorphous powder, ½ꢁŠ À182ꢀ (c 0.62,
max
D
nm (log "): 227 (4.35), 246.5 sh
MeOH
MeOH); UV l

786
Y. Takenaka et al. / Phytochemistry 59 (2002) 779–787
(1H, br dd, J=8.0, 4.5 Hz, H-300), 2.71 (1H, dd, J=14.0, 4.5
Hz, H-6), 2.90 (1H, m, H-800), 3.41 (1H, t, J=8.5 Hz, H-30),
3.71 (3H, s, OMe), 3.71 (1H, dd, J=12.0, 6.0 Hz, H-60),
3.90 (1H, dd, J=12.0, 2.0 Hz, H-60), 3.99 (1H, dd, J=9.0,
4.5 Hz, H-5), 4.27 (2H, m, H2-900), 4.81 (1H, d, J=8.0 Hz,
H-10), 5.92(1H, br s, H-1), 6.11 (1H, qd, J=7.0, 1.0 Hz, H-
8), 7.52(1H, s, H-3); 13C NMR (CD3OD): ꢀ 13.7 (C-10),
31.9 (C-5), 41.1 (C-6), 51.9 (OMe), 62.8 (C-60), 71.6 (C-40),
74.8 (C-20), 78.0 (C-50), 78.6 (C-30), 94.9 (C-1), 100.6 (C-10),
109.4 (C-4), 124.8 (C-8), 130.7 (C-9), 155.2 (C-3), 168.6 (C-
11), 172.9 (C-7). See Table 3 for carbon signals due to
cyclopentane monoterpene unit; HMBC: H-1!C-10, H3-
10!C-8, 9, H-3!C-1, 4, 5, 11, H-5!C-7, 9, H2-6!C-7,
H-8!C-5, 9, 10, H-10!C-1, OMe!C-11, H-100!C-700,
H-200!C-400, H-400 (ꢀ 1.83)!C-300, H-500 (ꢀ 1.23)!C-300,
600, H3-600!C-100, 200, 500, H-800!C-300, H2-900!C-7, 1000;
NOESY: H-300$H3-600, H2-900$H-400 (ꢀ 1.83); HR–SIMS
found: 601.2155 [M–H]À; C27H37O15 requires 601.2134.
3.14. Syringafghanoside (10)
28
Colourless amorphous powder, ½ꢁŠ À21ꢀ (c 1.01,
max
D
nm (log "): 216 (4.10), 222 sh (4.03),
cmÀ1: 3393, 1712, 1637, 1570, 1082;
MeOH
MeOH); UV l
KBr
276 (4.17); IR ꢂ
max
1H NMR (CD3OD): ꢀ 1.02(3H, d, J=7.0 Hz, H3-6),
1.14 (1H, m, H-5), 1.53 (1H, m, H-4), 1.85 (1H, m, H-4),
1.93 (1H, m, H-5), 2.25 (1H, m, H-1), 2.51 (2H, m, H-2,
3), 2.84 (1H, m, H-8), 3.35 (1H, t, J=8.0 Hz, H-30), 3.70
(1H, dd, J=10.0, 7.0 Hz, H-9), 4.12(1H, dd, J=10.0,
4.0 Hz, H-9), 4.25 (1H, d, J=8.0 Hz, H-10), 4.36 (1H,
dd, J=12.0, 6.0 Hz, H-60), 4.50 (1H, dd, J=12.0, 2.0 Hz,
H-60), 6.57 (1H, d, J=16.0 Hz, H-200), 7.35–7.44 (3H, m,
H-600, 700, 800), 7.55–7.62(2H, m, H-500, 900), 7.72(1H, d,
J=16.0 Hz, H-300); 13C NMR (CD3OD): ꢀ 64.9 (C-60),
71.9 (C-30), 74.9 (C-20), 75.4 (C-50), 77.8 (C-40), 105.0 (C-
10), 118.8 (C-200), 129.3Â2(C-5 00, 900), 130.0Â2(C-6 00, 800),
131.6 (C-700), 135.8 (C-400), 146.5 (C-300), 168.5 (C-100).
See Table 3 for carbon signals due to cyclopentane
monoterpene unit; HMBC: H-1!C-2, H-2!C-7, H-
3!C-2, 4, H-4 (ꢀ 1.85)!C-2, H2-4!C-3, H-5 (ꢀ 1.93)
!C-3, 4, H3-6!C-1, 2, 5, H2-9!C-3, 10, H-9 (ꢀ
3.70)!C-10, H-10!C-9, 50, H2-60!C-50, 100, H-200!C-
100, 400, H-300!C-100, 400; NOESY: H-1$H-4 (ꢀ 1.53), H-
3$H3-6, H-8$H-4 (ꢀ 1.53), H-9 (ꢀ 4.12)$H-4 (ꢀ 1.85);
HR–SIMS found: 507.1866 [M–H]À; C25H31O11
requires 507.1867.
3.13. Methylation of 9
A solution of 9 (2.0 mg) in MeOH was treated with
CH2N2–MeOH under ice-cooling. The reaction mixture
1
was evaporated in vacuo to give 9a (1.8 mg). H NMR
(CD3OD): ꢀ 1.07 (3H, d, J=6.5 Hz, H3-600), 1.24 (1H,
dddd, J=12.0, 11.5, 8.5, 6.5 Hz, H-500), 1.54 (1H, m, H-400),
1.73 (3H, dd, J=7.0, 1.5 Hz, H3-10), 1.87 (1H, m, H-400),
20.002 (1H, dtd, J=12.0, 7.5, 1.5 Hz, H-500), 2.24 (1H, m, H-
1 ), 2.47 (1H, dd, J=14.5, 9.0 Hz, H-6), 2.50 (2H, m, H-200,
300), 2.69 (1H, dd, J=14.5, 4.5 Hz, H-6), 2.79 (1H, ddd,
J=11.0, 6.5, 4.0 Hz, H-800), 3.61 (3H, s, OMe), 3.66 (3H, s,
OMe), 3.72(3H, s, OMe), 3.90 (1H, dd, J=12.0, 2.5 Hz, H-
60), 3.97 (1H, dd, J=9.0, 4.5 Hz, H-5), 4.21 (1H, dd,
J=11.0, 6.5 Hz, H-900), 4.26 (1H, dd, J=11.0, 4.0 Hz, H-
900), 4.81 (1H, d, J=8.0 Hz, H-10), 5.91 (1H, br s, H-1), 6.11
(1H, qd, J=7.0, 1.0 Hz, H-8), 7.52(1H, s, H-3). HR–SIMS
found: 629.2463 [M–H]À; C29H41O15 requires 629.2447.
This compound was identical with dimethyl ester derived
from frameroside.
3.15. Methylation of 10
A solution of 10 (9.0 mg) in MeOH was treated with
CH2N2–Et2O under ice-cooling. The reaction mixture
was evaporated in vacuo and the residue was subjected
to preparative HPLC (mBondasphere 5mC18–100 A,
MeCN–H2O, 9:11) to give 10a (2.7 mg). Colourless
26
amorphous powder, ½ꢁŠ À46ꢀ (c 0.18, MeOH); UV
max
D
nm (log "): 214 sh (3.93), 216 (3.99), 221 (3.92),
MeOH
l
KBr
276 (4.22); IR ꢂ
cmÀ1: 3419, 1732, 1710, 1637, 1521,
max
1083; 1H NMR (CD3OD): ꢀ 1.00 (3H, d, J=7.0 Hz, H3-
6), 1.12(1H, m, H-5), 1.51 (1H, m, H-4), 1.85 (1H, m,
H-4), 1.91 (1H, m, H-5), 2.19 (1H, m, H-1), 2.45 (1H, m,
H-2), 2.51 (1H, m, H-3), 2.74 (1H, ddd, J=10.0, 7.0, 4.0
Hz, H-8), 3.59 (3H, s, OMe), 3.63 (3H, s, OMe), 3.70
(1H, dd, J=10.0, 7.0 Hz, H-9), 4.04 (1H, dd, J=10.0,
4.0 Hz, H-9), 4.22 (1H, d, J=8.0 Hz, H-10), 4.35 (1H,
dd, J=12.0, 6.0 Hz, H-60), 4.50 (1H, dd, J=12.0, 2.0 Hz,
H-60), 6.58 (1H, d, J=16.0 Hz, H-200), 7.40–7.42(3H, m,
H-600, 700, 800), 7.61–7.63 (2H, m, H-500, 900), 7.73 (1H, d,
J=16.0 Hz, H-300); 13C NMR (CD3OD): ꢀ 64.9 (C-60),
71.8 (C-30), 74.9 (C-20), 75.4 (C-5), 77.9 (C-40), 104.9 (C-
10), 118.8 (C-200), 129.3Â2(C-5 00, 900), 130.1Â2(C-6 00, 800),
131.6 (C-700), 135.8 (C-400), 146.5 (C-300), 168.5 (C-100).
See Table 3 for carbon signals due to cyclopentane
monoterpene unit; HMBC: H-1!C-5, H-2!C-4, 7, H-
3!C-7, H-4 (ꢀ 1.85)!C-2, 3, H-4!C-5, H-5 (ꢀ
1.91)!C-1, H3-6!C-1, H-8!C-10, H2-9!C-8, 10, 10,
Table 3
13C NMR spectroscopic data of the cyclopentane monoterpene unit of
9, 9a, 10, 10a and 16 in CD3OD
C
9
9a
40.5
16
C
1
10
40.1
10a
40.2
100
40.3
36.5
55.3
200
55.254.8
41.8
30.9
57.2
54.9
300
42.1
43.9
4
3
41.9
31.2
34.5
42.4
400
31.230.6
34.8
21.4
30.8
500
35.0
21.9
33.8
22.8
180.0
48.8
66.6
180.0
5
34.7
21.5
600
6
22.0
700
179.0
47.3
66.0
175.0a
7
179.2177.4
49.248.7
71.3
800
47.8
65.6
177.3a
8
900
9
10
71.2
176.1
51.9
1000
OMe
OMe
177.0
178.1
51.9
52.5
OMe
OMe
52.2
a
Assignments may be interchangeable.

Y. Takenaka et al. / Phytochemistry 59 (2002) 779–787
787
H-10!C-9, 20, 30, 50, H-20!C-10, 40, H-30!C-20, 40, H-60
(ꢀ 4.35)!C-50, 100, H-200!C-100, 400, H-300!C-100, 200,
OMe (ꢀ 3.56)!C-7, OMe (ꢀ 3.63)!C-10; NOESY: H-
3$H3-6, H-3$H-9 (ꢀ 3.70), H-4 (ꢀ 1.85)$H-9 (ꢀ 3.70);
HR–SIMS Found: 537.2345 [M+H]+; C27H37O11
requires 537.2337.
C., 1985. Poliumoside, a caffeic glycoside ester from Teucrium
belion. Phytochemistry 24, 362–364.
He, Z.-D., Ueda, S., Akaji, M., Fujita, T., Inoue, K., Yang, C.-R.,
1994. Monoterpenoid and phenylethanoid glycosides from Ligus-
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Imakura, Y., Kobayashi, S., Mima, A., 1985. Bitter phenyl propanoid
glycosides from Campsis chinensis. Phytochemistry 24, 139–146.
Inoue, K., Tanahashi, T., Inouye, H., 1985. Two secoiridoid gluco-
sides from Jasminum mesnyi. Phytochemistry 24, 1299–1303.
Kanao, M., Matsuda, H., 1978. Studies on the constituents of the
leaves of Lespedeza thunbergii (DC.) Nakai. I. The structure of
miyaginin. Yakugaku Zasshi 98, 366–368.
3.16. Alkaline hydrolysis of 10 followed by preparation
of the (R)-PGME amide 18
A solution of 10 (10.4 mg) in 0.2M NaOH (1 ml) was
stirred for 2h at room temperature, neutralized with
Amberlite IR-120 (H+ form), following which the reac-
tion mixture was extracted with CHCl3. The CHCl3
layer was concentrated to give trans-cinnamic acid (2.3
mg). The H2O layer was concentrated to give a residue
(7.1 mg). This being hydrolysed with b-glucosidase (2
Karasawa, H., Kobayashi, H., Takizawa, N., Miyase, T., Fukushima,
S., 1986. Studies on the constituents of Cistanchis herba. VIII. Iso-
lation and structure of a new phenylethanoid glycoside, cistanoside
G. Yakugaku Zasshi 106, 721–724.
Kikuchi, M., Yamauchi, Y., Yanase, C., Nagaoka, I., 1987. Structures
of new secoiridoid glycosides from the leaves of Syringa vulgaris
Linn. Yakugaku Zasshi 107, 245–248.
Kikuchi, M., Yamauchi, Y., 1984. Studies on the constituents of
Ligustrum species. IX. On the components of the fruits of Ligustrum
obtusifolium Sieb. et Zucc. (3). Yakugaku Zasshi 104, 390–393.
Kitamura, S., 1955. Flora of Afghanistan. In: The Committee of
the Kyoto University Scientific Expedition to the Karakoram and
Hindukush. Results of the Kyoto University Scientific Expedition to
the Karakoram and Hindukush, Vol. II. Kyoto University, Kyoto,
p. 302.
ꢀ
mg), 37 C, pH 5.0, then added to Amberlite IR-120
(H+ form) and stirred for 3 h at room temperature. The
solution was applied to a Sephadex LH-20 column,
eluted with H2O. Evaporation of the eluate gave 18 (4.0
1
mg). 18: H NMR (CDCl3, 300 MHz): ꢀ 1.23 (3H, d,
J=6.0 Hz, H3-10), 1.29 (1H, m, H-7), 1.54 (1H, m, H-6),
1.97 (1H, m, H-6), 1.97 (1H, m, H-7), 2.06 (1H, m, H-8),
2.53 (1H, t, J=10.0 Hz, H-9), 2.96 (1H, m, H-5), 3.13
(1H, ddd, J=10.0, 6.0, 4.0 Hz, H-4), 4.40 (2H, m, H2-3).
To a solution of 18 (4.0 mg) in DMF (1 ml) were added
Kuwajima, H., Matsuuchi, K., Takaishi, K., Inoue, K., Fujita, T.,
Inouye, H., 1989. A secoiridoid glucoside from Ligustrum japoni-
cum. Phytochemistry 28, 1409–1411.
Markham, K.R., Ternai, B., Stanley, R., Geiger, H., Mabry, T.J.,
1978. Carbon-13 NMR studies of flavonoids-III. Naturally occur-
ring flavonoid glycosides and their acylated derivatives. Tetra-
hedron 34, 1389–1397.
.
(R)-PGME HCl (4.6 mg), PyBOP (11.8 mg), HBT (3.1
mg) and TEA (0.02ml), and whole was stirred at room
temperature for 2h. The reaction mixture was poured into
dil. HCl and extracted with CHCl3. The CHCl3 layer was
dried and concentrated in vacuo. The residue was purified
by preparative HPLC (H2O–CH3CN, 1:1), to give 19 (2.9
mg). This compound was identical with the PGME amide
derived from frameroside (1H NMR, SIMS).
Ohtani, I., Kusumi, T., Kashman, Y., Kakisawa, H., 1991. High-field
FT NMR application of Mosher’s method. The absolute config-
urations of marine terpenoids. Journal of the American Chemical
Society 113, 4092–4096.
Pagnoni, U.M., Pinetti, A., Trave, R., Garanti, L., 1976. Mono-
terpenes of Teucrium marum. Australian Journal of Chemistry 29,
1375–1381.
Tanahashi, T., Watanabe, H., Itoh, A., Nagakura, N., Inoue, K., Ono,
M., Fujita, T., Morita, M., Chen, C.-C., 1992. A secoiridoid gluco-
side from Fraxinus formosana. Phytochemistry 31, 2143–2145.
Tanahashi, T., Watanabe, H., Itoh, A., Nagakura, N., Inoue, K., Ono,
M., Fujita, T., Morita, M., Chen, C.-C., 1993. Five secoiridoid glu-
cosides from Fraxinus formosana. Phytochemistry 32, 133–136.
Tanahashi, T., Sakai, T., Takanaka, Y., Nagakura, N., Chen, C.-C.,
1999. Structure elucidation of two secoiridoid glucosides from Jas-
minum officinale L. var. grandiflorum (L.). Chemical and Pharma-
ceutical Bulletin 47, 1582–1586.
Acknowledgements
We are grateful to Dr. M. Sugiura (Kobe Pharma-
1
ceutical University) for H and 13C NMR spectra, and
Dr. K. Saiki (Kobe Pharmaceutical University) for mass
spectra measurements.
Takenaka, Y., Tanahashi, T., Shintaku, M., Sakai, T., Nagakura, N.,
Parida, X., 2000. Secoiridoid glucosides from Fraxinus americana.
Phytochemistry 55, 275–284.
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