Medicinal Flowers. IV. Marigold. (2): Structures of New Ionone and Sesquiterpene Glycosides from Egyptian Calendula officinalis.

Abstract

Following the characterization of hypoglycemic, gastric emptying inhibitory, and gastroprotective principles and the structure elucidation of calendasaponins A, B, C, and D, two new ionone glucosides (officinosides A and B), and two sesquiterpene oligoglycosides (officinosides C and D), were isolated from the flowers of Egyptian Calendula officinalis. The structures of the officinosides were elucidated on the basis of chemical and physicochemical evidence.

Full text

In the course of our studies on the bioactive constituents of
medicinal flowers,
1,2)
we have found that the methanolic ex-
tract and its 1-butanol-soluble fraction from the flowers of
Egyptian Calendula (C.) officinalis exhibited hypoglycemic,
gastric emptying inhibitory, and gastroprotective effects.
Thus far, we have isolated four oleanene-type triterpene oli-
goglycosides ( calendasaponins A—D), two new ionone glu-
cosides [officinosides A (1) and B (2)], and two new
sesquiterpene oligoglycosides [officinosides C (3) and D (4)],
from the 1-butanol-soluble fraction together with eight
known saponins, one sesquiterpene glucoside, and seven
flavonol glycosides. In the preceding paper,
1)
we reported the
isolation and structure elucidation of calendasaponins A—D.
Furthermore, we described the inhibitory activities of the
principle saponins from the flowers of C. officinalis on the in-
crease of serum glucose levels in oral glucose-loaded rats, on
gastric emptying in carboxymethyl cellulose sodium salt test
meal-loaded mice, and on ethanol- or indomethacin-induced
gastric mucosal lesions in rats and also discussed the struc-
ture requirements for these activities. This paper offers the
structure elucidation of officinosides A (1), B (2), C (3), and
D (4).
Structures of Ionone Glucosides, Officinosides A (1)
and B (2) Officinoside A (1) was isolated as a white pow-
der with positive optical rotation ([
a
]
D
22
113.0°). In the nega-
tive- and positive-ion FAB-MS of 1, quasimolecular ion
peaks were observed at m/z 387 (M2H)
2
and m/z 411
(M1Na)
1
, respectively. High-resolution MS analysis of the
quasimolecular ion peak (M1Na)
1
revealed the molecular
formula of 1 to be C
19
H
32
O
8
. The IR spectrum of 1 showed
absorption bands at 1671 cm
21
ascribable to olefin and strong
absorption bands at 3432, 1076, and 1038 cm
21
suggestive of
a glycosidic structure. Acid hydrolysis of 1 with 5% aqueous
sulfuric acid–1,4-dioxane liberated D-glucose, which was
identified by GLC analysis of the trimethylsilyl thiazolidine
derivative.
3)
Enzymatic hydrolysis of 1 with
b
-glucosidase
liberated (3S,5R,8S,9
x
)-5,8-epoxy-6-megastigmene-3,9-diol
(5). Since the stereostructure of 5 was tentatively presented,
4)
we investigated the total structure of 1 including the aglycone
moiety. The
1
H-NMR (1: pyridine-d
5
, 5: CDCl
3
) and
13
C-
NMR (Table 1) spectra of 1 and 5, which were assigned by
various NMR experiments,
5)
indicated the presence of three
singlet methyls [1:
d
1.13, 1.42, 1.92 (all s, 12, 11, 13-H
3
), 5:
d
1.19, 1.32, 1.61 (all s, 12, 11, 13-H
3
)], a doublet methyl [1:
d
1.43 (d, J56.6 Hz, 10-H
3
), 5:
d
1.17 (d, J56.4 Hz, 10-H
3
)],
three methines bearing an oxygen function [1:
d
3.94 (m, 9-
H), 4.49 (m, 3-H), 4.76 (br d, J5ca. 7 Hz, 8-H), 5:
d
3.81
(dd, J53.1, 6.4 Hz, 9-H), 4.24 (dd-like, 3-H), 4.71 (br d,
J5ca. 5 Hz, 8-H)], and an olefinic methine [1:
d
5.76 (br s,
7-H), 5:
d
5.37 (br s, 7-H)] together with a
b
-D-glucopyra-
nosyl moiety [
d
4.87 (d, J58.4 Hz, 19-H)] in 1. As shown in
Fig. 1, the planar structure of the aglycone and the position
974 Chem. Pharm. Bull. 49(8) 974—978 (2001) Vol. 49, No. 8
∗ To whom correspondence should be addressed. e-mail: shoyaku@mb.kyoto-phu.ac.jp © 2001 Pharmaceutical Society of Japan
Medicinal Flowers. IV.
1)
Marigold. (2): Structures of New Ionone and
Sesquiterpene Glycosides from Egyptian Calendula officinalis
Toshiyuki MARUKAMI, Akinobu KISHI, and Masayuki YOSHIKAWA*
Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607–8412, Japan.
Received February 20, 2001; accepted April 16, 2001
Following the characterization of hypoglycemic, gastric emptying inhibitory, and gastroprotective principles
and the structure elucidation of calendasaponins A, B, C, and D, two new ionone glucosides (officinosides A and
B), and two sesquiterpene oligoglycosides (officinosides C and D), were isolated from the flowers of Egyptian Cal-
endula officinalis. The structures of the officinosides were elucidated on the basis of chemical and physicochemi-
cal evidence.
Key words officinoside; Calendula officinalis; marigold; ionone glucoside; sesquiterpene oligoglycoside; Compositae
Chart 1

of a glycoside linkage in 1 were confirmed by
1
H–
1
H COSY
and HMBC experiments, which showed long-range correla-
tions between the following protons and carbons: 11, 12-H
3
and 1, 2, 6-C; 13-H
3
and 4, 5, 6-C; 10-H
3
and 8, 9-C; 7-H
and 1, 6, 8-C; 8-H and 7, 9-C; 19-H and 3-C. The relative
stereostructures of 1 and 5, except for the 9-position, was
confirmed by pNOESY experiment, in which NOE correla-
tions were observed between the following protons: 3-H and
4
a
, 19-H; 11-H
3
and 2
a
, 3, 8-H; 13-H
3
and 12-H
3
, 4
b
-H.
Officinoside B (2) was isolated as a white powder with
positive optical rotation ([
a
]
D
22
11.2°) and its IR spectrum
was similar to that of 1. The molecular formula C
19
H
32
O
8
of
2, which was the same as that of 1, was characterized from
the negative- and positive-ion FAB-MS [m/z 387 (M2H)
2
and m/z 411 (M1Na)
1
] and by high-resolution MS measure-
ment. Acid hydrolysis of 2 with 5% aqueous sulfuric acid–
1,4-dioxane furnished D-glucose,
3)
while a new aglycone
(6) was obtained by enzymatic hydrolysis. The
1
H-NMR
(CDCl
3
) and
13
C-NMR (Table 1) spectra
5)
of 6 showed sig-
nals assignable to three singlet methyls [
d
1.18, 1.33, 1.60
(all s, 12, 11, 13-H
3
)], a doublet methyl [
d
1.18 (d, J56.4 Hz,
10-H
3
)], two methylenes [
d
1.47 (m), 1.82 (dd, J53.7, 14.0
Hz) (2-H
2
), 1.75 (ddd, J51.8, 4.0, 14.0 Hz), 2.15 (ddd-like)
(4-H
2
)], three methines bearing an oxygen function [
d
3.57
(dd-like, 9-H), 4.22 (dd-like, 3-H), 4.52 (br d, J5ca. 6 Hz, 8-
H)], and an olefin methine [
d
5.30 (br s, 7-H)], while those
of 2 indicated the presence of an aglycone part and a
b
-D-
glucopyranosyl moiety [
d
4.90 (d, J58.4 Hz, 19-H)]. The
proton and carbon signals in the
1
H- and
13
C-NMR spectra of
2 and 6 were shown to be superimposable on those of 1 and
5, except for the signals due to the 7, 8, and 9-positions. The
HMBC experiment of 2 showed long-range correlations be-
tween the following protons and carbons: 11, 12-H
3
and 1, 2,
6-C; 13-H
3
and 4, 5, 6-C; 10-H
3
and 8, 9-C; 7-H and 1, 5, 6-
C; 8-H and 7, 9-C; 19-H and 3-C. On the basis of this evi-
dence and the
1
H–
1
H COSY data (Fig. 1), the planar struc-
ture of 2 and 6 were characterized. NOE correlations were
observed in the pNOESY experiment between the following
protons: 3-H and 4
a
, 19-H; 11-H
3
and 2
a
, 3-H; 13-H
3
and
12-H
3
, 8-H. This evidence led us to elucidate the relative
stereostructure of 2 to be as shown.
In order to clarify the absolute stereostructures of 1 and 2,
the aglycones, 5 and 6, were subjected to a modified Mosh-
er’s method.
6)
Namely, 5 and 6 were treated with (R)- and
(S)-
a
-methoxy-
a
-trifluoromethylphenyl acetate (MTPA) in
the presence of 1-ethyl-3-(3-dimethylaminopropyl)cardoi-
imide (EDC · HCl) and 4-dimethylaminopyridine (DMAP) to
give the 3,9-di-(R)-MTPA ester (5a, 6a) and the 3,9-di-(S)-
MTPA ester (5b, 6b), respectively. As shown in Fig. 3, the
signals due to protons attached to the 2, 7, 8, 11, and 12-po-
sitions in 5a and 6a were observed at lower fields (
Dd
: nega-
tive) as compared to those of 5b and 6b, while the signal due
to the 4 and 10-positions in 5a and 6a was observed at higher
fields (
Dd
: positive) as compared to those of 5b and 6b. On
the basis of the above evidence, the absolute stereostructures
of officinosides A and B were determined to be (3S,5R,8S,9R)-
5,8-epoxy-6-megastigmene-3,9-diol 3-O-
b
-D-glucopyranoside
(1) and (3S,5R,8R,9R)-5,8-epoxy-6-megastigmene-3,9-diol
August 2001 975
Fig. 1. H–H COSY and HMBC Correlations of 1—4
Fig. 2. NOE Correlations of 1—4
Table 1.
13
C-NMR Data of Officinosides A (1), B (2), C (3), and D (4), 5,
and 6
1
a)
2
a)
3
a)
4
a)
5
b)
6
b)
1
a)
2
a)
3
a)
4
a)
C-1 34.0 34.1 40.1 55.0 33.8 33.8 C-19 102.6 102.6 103.1 103.8
2 45.8 45.8 31.9 25.8 46.6 46.7 29 75.4 75.4 75.7 75.9
3 73.8 73.8 78.9 29.3 67.6 67.6 39 78.9 78.9 78.7 78.7
4 44.2 44.3 150.2 38.7 47.5 48.0 49 71.9 71.9 71.9 72.0
5 87.3 87.3 48.7 38.7 86.7 87.2 59 78.3 78.3 78.5 78.5
6 154.6 154.6 22.7 18.7 155.4 155.4 69 63.0 63.0 63.0 63.0
7 119.0 118.0 48.9 26.8 116.1 117.4 C-10 98.8 98.1
8 87.6 87.4 25.4 19.2 86.1 86.8 20 72.4 72.5
9 70.7 70.1 41.3 37.1 68.4 70.9 30 75.6 75.2
10 20.0 20.0 36.0 81.2 17.8 18.8 40 72.8 72.8
11 28.8 28.7 79.2 23.4 28.9 28.5 50 70.9 71.0
12 31.4 31.5 23.8 79.4 31.3 31.4 60 17.5 17.4
13 29.2 29.3 25.0 12.6 28.5 28.7
14 16.7 27.6
15 104.7 16.6
a) 125 MHz, pyridine-d
5
or b) CDCl
3
.

3-O-
b
-D-glucopyranoside (2).
Officinoside C (3) was isolated as a white powder with
negative optical rotation ([
a
]
D
27
27.0°), and its IR spectrum
showed absorption bands due to hydroxyl and exo-methylene
functions at 3432 and 1655 cm
21
. In the negative- and posi-
tive-ion FAB-MS of 3, quasimolecular ion peaks were ob-
served at m/z 545 (M2H)
2
and m/z 569 (M1Na)
1
and the
molecular formula, C
27
H
46
O
11
, of 3 was determined by high-
resolution MS measurement. Acid hydrolysis of 3 with 5%
aqueous H
2
SO
4
–1,4-dioxane liberated D-glucose and D-fu-
cose,
3)
while enzymatic hydrolysis of 3 with naringinase fur-
nished selin-4(15)-en-3
b
,11-diol (7).
7)
The
1
H-NMR (pyri-
dine-d
5
) and
13
C-NMR (Table 1) spectra
5)
of 3 showed sig-
nals due to a selin-4(15)-en-3
b
,11-diol moiety [
d
0.71, 1.39,
1.42 (s, 14, 12, 13-H
3
), 4.46 (m, 3-H), 4.79, 6.81 (br s, 15-
H
2
)], a
b
-D-glucopyranosyl moiety [
d
5.03 (d, J57.6 Hz, 19-
H)], and a
b
-D-fucopyranosyl moiety [
d
1.51 (d, J56.1 Hz,
60-H
3
), 4.81 (d, J57.4 Hz, 10-H)]. The planar structure and
the glycoside linkages of 3 were constructed on the basis of
1
H–
1
H COSY and HMBC. Thus, the
1
H–
1
H COSY experi-
ment for 3 indicated the presence of the partial structures
from the 1-position to the 3-position and from the 5-position
to the 9-position. In the HMBC experiment, long-range cor-
relations were observed between the 15-protons and the 3, 4,
5-carbons, between the 14-protons and the 1, 5, 9, 10-car-
bons, between the 12, 13-protons and the 11, 7-carbons, be-
tween the 19-proton and the 3-carbon, and between the 10-
proton and the 11-carbon. The relative stereostructure of 3
was characterized by pNOESY experiment, which showed
NOE correlations between the following protons: 14-H
3
and
2
b
, 6
b
, 8
b
, 9
b
-H; 2
a
-H and 3-H; 8
a
-H and 7, 9
a
-H. Since
the absolute stereostructure of 7 was not yet determined, 7
was subjected to a modified Mosher’s method.
6)
Conse-
quently, the signals due to protons attached to the 1 and 2-
carbons in the
1
H-NMR spectrum of the 3-(S)-MTPA ester
(7b) were observed at higher field as compared to those of
the 3-(R)-MTPA ester (7a) (
Dd
: negative), while the signal
due to protons on the 15-carbon of the 3-(S)-MTPA (7b) was
observed at lower field than those of the 3-(R)-MTPA (7a)
(
Dd
: positive). On the basis of the above evidence, the ab-
solute stereostructure of officinoside C was elucidated to be
12-O-
b
-D-fucopyranosyl selin-4(15)-en-3
b
,11-diol 3-O-
b
-D-
glucopyranoside (3).
Officinoside D (4), isolated as a white powder with nega-
tive optical rotation ([
a
]
D
26
214.5°), gave the quasimolecular
ion peaks at m/z 545 (M2H)
2
and m/z 569 (M1Na)
1
in the
negative- and positive-ion FAB-MS and the molecular for-
mula was defined as C
27
H
46
O
11
from the high-resolution MS
analysis. Acid hydrolysis of 4 with 5% aqueous H
2
SO
4
–1,4-
dioxane (1 : 1, v/v) liberated D-fucose and D-glucose,
3)
while
enzymatic hydrolysis of 4 with naringinase liberated flouren-
sadiol (8).
8)
The
1
H-NMR (pyridine-d
5
) and
13
C-NMR (Table
1) spectra
5)
of 4 showed a flourensadiol moiety [
d
0.31 (dd-
like, 6-H), 0.89 (d, J56.7 Hz, 15-H
3
), 1.07 (ddd-like, 7-H),
1.24, 1.36 (s, 13, 14-H
3
), 3.29, 4.16 (d, J510.1 Hz, 12-H
2
)], a
b
-D-fucopyranosyl moiety [
d
1.47 (d, J56.4 Hz, 69-H
3
), 4.80
(d, J57.7 Hz, 19-H)], and a
b
-D-glucopyranosyl moiety [
d
4.87 (d, J57.9 Hz, 10-H)]. In the HMBC experiment of 4,
long-range correlations were observed between the 19-proton
of the
b
-D-fucopyranosyl moiety and the 10-carbon and be-
tween the 10-proton of the
b
-D-glucopyranosyl moiety and
12-carbon. Furthermore, in the pNOESY experiment, NOE
correlations were observed between the following protons:
12-H
2
and 6, 7, 10-H; 13-H
3
and 8
b
, 5-H; 14-H
3
and 1, 8
b
,
9
b
-H; 15-H
3
and 6-H. On the basis of the above evidence, of-
ficinoside D was determined to be 12-O-
b
-D-fucopyranosyl
flourensadiol 10-O-
b
-D-glucopyranoside (4).
Experimental
The instruments used to obtain physical data and the experimental condi-
tions for chromatography were the same as described in our previous paper.
1)
Isolation of Officinosides A (1), B (2), C (3), and D (4) from the Dried
Flowers of C. officinalis L. Officinosides A (1), B (2), C (3), and D (4)
were isolated from the dried flowers of C. officinalis cultivated in Egypt, as
described earlier.
1)
Officinoside A (1): A white powder, [
a
]
D
22
113.0° (c50.6, MeOH). IR
(KBr): 3432, 1671, 1076, 1038 cm
21
. High-resolution positive-ion FAB-MS:
Calcd for C
19
H
32
O
8
Na (M1Na)
1
: 411.1995. Found: 411.2008.
1
H-NMR
(500 MHz, pyridine-d
5
)
d
: 1.13, 1.42, 1.92 (3H each, all s, 12, 11, 13-H
3
),
1.43 (3H, d, J56.6 Hz, 10-H
3
), 1.52 (1H, dd, J53.3, 13.8 Hz, 2
a
-H), 1.98
(1H, dd, J54.0, 13.8 Hz, 4
b
-H), 2.05 (1H, br d, J5ca. 14 Hz, 2
b
-H), 2.62
(1H, br d, J5ca. 14 Hz, 4
a
-H), 3.94 (1H, m, 9-H), 4.49 (1H, m, 3-H), 4.76
(1H, br d, J5ca. 7 Hz, 8-H), 4.87 (1H, d, J58.4 Hz, 19-H), 5.76 (1H, br s, 7-
H).
13
C-NMR (125 MHz, pyridine-d
5
)
d
C
: given in Table 1. Negative-ion
FAB-MS: m/z 387 (M2H)
2
, 225 (M2C
6
H
11
O
5
)
2
. Positive-ion FAB-MS:
m/z 411 (M1Na)
1
.
Officinoside B (2): A white powder, [
a
]
D
26
11.2° (c50.5, MeOH). IR
(KBr): 3432, 1670, 1078, 1036 cm
21
. High-resolution positive-ion FAB-MS:
Calcd for C
19
H
32
O
8
Na (M1Na)
1
: 411.1995. Found: 411.1981.
1
H-NMR
(500 MHz, pyridine-d
5
)
d
: 1.14, 1.42, 1.90 (3H each, all s, 12, 11, 13-H
3
),
1.43 (3H, d, J56.4 Hz, 10-H
3
), 1.52 (1H, dd, J53.6, 13.8 Hz, 2
a
-H), 1.97
(1H, dd, J54.3, 13.8 Hz, 4
b
-H), 2.06 (1H br d, J5ca. 14 Hz, 2
b
-H), 2.59
(1H, br d, J5ca. 14 Hz, 4
a
-H), 4.01 (1H, dd, J55.2, 6.4 Hz, 9-H), 4.48 (1H,
m, 3-H), 4.90 (1H, d, J58.4 Hz, 19-H), 4.94 (1H, br d, J5ca. 5 Hz, 8-H),
5.56 (1H, br s, 7-H).
13
C-NMR (125 MHz, pyridine-d
5
)
d
C
: given in Table 1.
Negative-ion FAB-MS: m/z 387 (M2H)
2
. Positive-ion FAB-MS: m/z 411
(M1Na)
1
.
Officinoside C (3): A white powder, [
a
]
D
27
27.0° (c50.7, MeOH). IR
(KBr): 3432, 1655, 1074, 1038 cm
21
. High-resolution positive-ion FAB-MS:
Calcd for C
27
H
46
O
11
Na (M1Na)
1
: 569.2938. Found: 569.2953.
1
H-NMR
(500 MHz, pyridine-d
5
)
d
: 0.71, 1.39, 1.42 (3H each, all s, 14, 12, 13-H
3
),
1.06 (1H, m, 9
a
-H), 1.09 (1H, m, 1
a
-H), 1.31 (1H, m, 1
b
-H), 1.34 (1H, m,
8
b
-H), 1.35 (1H, m, 6
a
-H), 1.42 (1H, m, 9
b
-H), 1.51 (3H, d, J56.1 Hz, 60-
H
3
), 1.57 (1H, m, 5-H), 1.59 (1H, m, 7-H), 1.77 (1H, m, 2
a
-H), 1.81 (1H,
m, 6
b
-H), 1.96 (1H, m, 8
a
-H), 2.11 (1H, m, 2
b
-H), 4.46 (1H, m, 3-H), 4.79,
6.81 (1H each, both br s, 15-H
2
), 4.81 (1H, d, J57.4 Hz, 10-H), 5.03 (1H, d,
J57.6 Hz, 19-H).
13
C-NMR (125 MHz, pyridine-d
5
)
d
C
: given in Table 1.
976 Vol. 49, No. 8
Fig. 3

Negative-ion FAB-MS: m/z 545 (M2H)
2
. Positive-ion FAB-MS: m/z 569
(M1Na)
1
.
Officinoside D (4): A white powder, [
a
]
D
26
214.5° (c50.3, MeOH). IR
(KBr): 3432, 2940, 1167, 1075 cm
21
. High-resolution positive-ion FAB-MS:
Calcd for C
27
H
46
O
11
Na (M1Na)
1
: 569.2938. Found: 569.2924.
1
H-NMR
(500 MHz, pyridine-d
5
)
d
: 0.31 (1H, dd-like, 6-H), 0.89 (3H, d, J56.7 Hz,
15-H
3
), 1.07 (1H, ddd-like, 7-H), 1.24, 1.36 (3H each, both s, 13, 14-H
3
),
1.25 (1H, m, 3
a
-H), 1.47 (3H, d, J56.4 Hz, 69-H
3
), 1.56 (2H, m, 2-H
2
), 1.62
(1H, m, 8
a
-H), 1.65 (1H, m, 9
b
-H), 1.72 (1H, m, 3
b
-H), 1.92 (1H, m, 9
a
-
H), 1.95 (1H, m, 4-H), 2.07 (1H, dd-like, 8
b
-H), 2.22 (1H, m, 5-H), 2.24
(1H, m, 1-H), 3.29, 4.16 (1H each, both d, J510.1 Hz, 12-H
2
), 4.80 (1H, d,
J57.7 Hz, 19-H), 4.87 (1H, d, J57.9 Hz, 10-H).
13
C-NMR (125 MHz, pyri-
dine-d
5
)
d
C
: given in Table 1. Negative-ion FAB-MS: m/z 545 (M2H)
2
, 383
(M2C
6
H
11
O
5
)
2
. Positive-ion FAB-MS: m/z 569 (M1Na)
1
.
Acid Hydrolysis of and Officinosides (1—4) A solution of 1—4 (5 mg
each) in 5% aq. H
2
SO
4
–1,4-dioxane (2 ml, 1 : 1, v/v) was heated under reflux
for 1 h. After cooling, the reaction mixture was neutralized with Amberlite
IRA-400 (OH- form) and the residue was removed by filtration. After re-
moval of the solvent from the filtrate in vacuo, the residue was transferred to
a Sep-Pak C18 cartridge with H
2
O and MeOH. The H
2
O eluate was concen-
trated and the residue was treated with L-cysteine methyl ester hydrochloride
(4 mg) in pyridine (0.5 ml) at 60 °C for 1 h. After reaction, the solution was
treated with N,O-bis(trimethylsilyl)trifluoroacetamide (0.2 ml) at 60 °C for 1
h. The supernatant was then subjected to GLC analysis to identify the deriv-
atives of D-glucose (i) from 1—4; D-fucose (ii) from 3 and 4; GLC condi-
tions: column: Supeluco STB
TM
-1, 30 m30.25 mm (i.d.) capillary column,
column temperature: 230 °C, He flow rate: 15 ml/min, t
R
: i: 24.2 min, ii: 17.2
min.
Enzymatic Hydrolysis of Officinoside A (1) A solution of 1 (12 mg) in
0.2 M acetate buffer (pH 4.4, 2.0 ml) was treated with
b
-glucosidase (Orien-
tal Yeast Co., 20 mg) and the whole mixture was stirred at 38 °C for 48 h.
The reaction mixture was poured into EtOH and removal of the solvent
under reduced pressure gave a product. The product was purified by normal-
phase silica gel column chromatography [1.0 g, CHCl
3
–MeOH–H
2
O (30:3:
1, lower layer, v/v)] to give (3S,5R,8S,9
x
)-5,8-epoxy-6-megastigmene-3,9-
diol (5, 4.3 mg, 61.5%), which was identified by comparison of the
1
H-NMR
data and [
a
]
D
with reported values.
4)
5: An amorphous powder, [
a
]
D
23
110.6 °C (c50.1, CHCl
3
). IR (film):
3453, 1632, 1078 cm
21
.
1
H-NMR (500 MHz, CDCl
3
)
d
: 1.17 (3H, d, J56.4
Hz, 10-H
3
), 1.19, 1.32, 1.61 (3H, all s, 12, 11, 13-H
3
), 1.50 (1H, m), 1.84
(1H, dd, J54.0, 13.6 Hz) (2-H
2
), 1.75 (1H, ddd, J52.0, 4.0, 13.6 Hz), 2.15
(1H, ddd-like) (4-H
2
), 3.81 (1H, dd, J53.1, 6.4 Hz, 9-H), 4.24 (1H, dd-like,
3-H), 4.71 (1H, br d, J5ca. 5 Hz, 8-H), 5.37 (1H, br s, 7-H).
13
C-NMR (125
MHz, CDCl
3
)
d
C
: given in Table 1.
Preparation of the (R)-MTPA Ester (5a) and the (S)-MTPA Ester (5b)
from (3S,5R,8S,9
xx
)-5,8-Epoxy-6-megastigmene-3,9-diol (5) A solution
of 5 (0.8 mg) in CH
2
Cl
2
(0.5 ml) was treated with (R)-MTPA (50 mg) in the
presence of EDC · HCl (50 mg) and 4-DMAP (20 mg), and the mixture was
stirred at 50 °C under an N
2
atmosphere for 2 h. It was poured into ice-water
and the whole was extracted with AcOEt. The AcOEt extract was succes-
sively washed with 5% aqueous HCl, aqueous saturated NaHCO
3
, and brine,
then dried over MgSO
4
and filtered. Removal of the solvent from the filtrate
under reduced pressure furnished a residue, which was purified on a normal-
phase silica gel column [0.6 g, n-hexane–AcOEt (5 : 1, v/v) to give 5a (1.1
mg, 47%). Through a similar procedure, 5b (0.9 mg, 44%) was prepared
from 5 (0.7 mg) by the use of (S)-MTPA (50 mg), EDC · HCl (50 mg), and 4-
DMAP (20 mg).
5a: A white powder.
1
H-NMR (500 MHz, CDCl
3
)
d
: 1.17, 1.23, 1.28 (3H
each, all s, 12, 13, 11-H
3
), 1.26 (3H, d, J55.8 Hz, 10-H
3
), 1.68 (1H, dd,
J53.8, 15.2 Hz), 2.05 (1H, dd-like) (2-H
2
), 1.82 (1H, dd, J53.8, 15.2 Hz),
2.38 (1H, dd-like) (4-H
2
), 3.54, 3.58 (3H each, both s, MTPA-OMex
2
), 5.54
(1H, dd-like, 8-H), 5.70 (1H, br s, 7-H), 7.40—7.55 (10H).
5b: A white powder.
1
H-NMR (500 MHz, CDCl
3
)
d
: 0.86, 1.20, 1.25 (3H
each, all s, 12, 11, 13-H
3
), 1.52 (3H, d, J55.8 Hz, 10-H
3
), 1.54 (1H, m), 1.93
(1H, dd-like) (2-H
2
), 1.89 (1H, dd, J54.1, 15.1 Hz), 2.49 (1H, dd-like) (4-
H
2
), 3.58 (6H, s, MTPA-OMex
2
), 5.52 (1H, dd-like, 8-H), 5.69 (1H, br s, 7-
H), 7.41—7.55 (10H).
Enzymatic Hydrolysis of Officinoside B (2) A solution of 2 (13 mg) in
0.2 M acetate buffer (pH 4.4, 2.0 ml) was treated with
b
-glucosidase (Orien-
tal Yeast Co., 20 mg) and the whole mixture was stirred at 38 °C for 48 h.
The reaction mixture was poured into EtOH and removal of the solvent
under reduced pressure gave a product. The product was purified by normal-
phase silica gel column chromatography [1.0 g, CHCl
3
–MeOH–H
2
O (30:3:
1, lower layer, v/v)] to give (3S,5R,8R,9
x
)-5,8-epoxy-6-megastigmene-3,9-
diol (6, 3.5 mg, 46.2%).
6: An amorphous powder, [
a
]
D
25
261.1° (c50.1, CHCl
3
). IR (film): 3453,
1632, 1028 cm
21
.
1
H-NMR (500 MHz, CDCl
3
)
d
: 1.18 (3H, d, J56.4 Hz,
10-H
3
), 1.18, 1.33, 1.60 (3H, all s, 12, 11, 13-H
3
), 1.47 (1H, m), 1.82 (1H,
dd, J53.7, 14.0 Hz) (2-H
2
), 1.75 (1H, ddd, J51.8, 4.0, 14.0 Hz), 2.15 (1H,
ddd-like) (4-H
2
), 3.57 (1H, dd-like, 9-H), 4.22 (1H, dd-like, 3-H), 4.52 (1H,
br d, J5ca. 6 Hz, 8-H), 5.30 (1H, br s, 7-H).
13
C-NMR (125 MHz, CDCl
3
)
d
C
: given in Table 1.
Preparation of the (R)-MTPA Ester (6a) and the (S)-MTPA Ester (6b)
from (3S,5R,8R,9
xx
)-5,8-Epoxy-6-megastigmene-3,9-diol (6) A solution
of 6 (1.0 mg) in CH
2
Cl
2
(0.5 ml) was treated with (R)-MTPA (50 mg) in the
presence of EDC · HCl (50 mg) and 4-DMAP (20 mg), and the mixture was
stirred at 50 °C under an N
2
atmosphere for 2 h. It was poured into ice-water
and the whole was extracted with AcOEt. The AcOEt extract was succes-
sively washed with 5% aqueous HCl, aqueous saturated NaHCO
3
, and brine,
then dried over MgSO
4
and filtered. Removal of the solvent from the filtrate
under reduced pressure furnished a residue, which was purified on a normal-
phase silica gel column [0.6 g, n-hexane–AcOEt (5 : 1, v/v) to give 6a (0.5
mg, 17%). Through a similar procedure, 6b (1.4 mg, 48%) was prepared
from 6 (1.0 mg) by the use of (S)-MTPA (50 mg), EDC · HCl (50 mg), and 4-
DMAP (20 mg).
6a: A white powder.
1
H-NMR (500 MHz, CDCl
3
)
d
: 1.01, 1.11, 1.14 (3H
each, all s, 11,12, 13-H
3
), 1.25 (3H, d, J55.8 Hz, 10-H
3
), 1.76 (1H, dd,
J53.8, 15.2 Hz), 1.80 (1H, dd-like) (2-H
2
), 1.76 (1H, dd, J53.8, 15.2 Hz),
1.99 (1H, dd-like) (4-H
2
), 3.53, 3.57 (3H each, both s, MTPA-OMex
2
), 5.38
(1H, dd-like, 8-H), 5.24 (1H, br s, 7-H), 7.38—7.55 (10H).
6b: A white powder.
1
H-NMR (500 MHz, CDCl
3
)
d
: 0.68, 0.82, 1.35 (3H
each, all s, 11, 12, 13-H
3
), 1.37 (3H, d, J55.8 Hz, 10-H
3
), 1.50 (1H, m), 1.64
(1H, dd-like) (2-H
2
), 1.96 (1H, dd, J54.1, 15.1 Hz), 2.11 (1H, dd-like) (4-
H
2
), 3.56 (6H, s, MTPA-OMex
2
), 5.06 (1H, br s, 7-H), 5.42 (1H, dd-like, 8-
H), 7.36—7.52 (10H).
Enzymatic Hydrolysis of Officinoside C (3) A solution of 3 (10 mg) in
0.2 M acetate buffer (pH 4.0, 2.0 ml) was treated with naringinase (Sigma
Co., Ltd., 20 mg) and the whole mixture was stirred at 40 °C for 24 h. The
reaction mixture was poured into EtOH and removal of the solvent under re-
duced pressure gave a product. The product was purified by normal-phase
silica gel column chromatography [1.0 g, CHCl
3
–MeOH–H
2
O (30:3:1,
lower layer, v/v)] to give selin-4(15)-en-3
b
,11-diol (7, 4.0 mg, 91.8%),
which were identified by comparison of their physical data ([
a
]
D
, IR,
1
H-
NMR,
13
C-NMR) with reported values.
7)
Preparation of the (R)-MTPA Ester (7a) and the (S)-MTPA Ester (7b)
from Selin-4(15)-en-3
bb
,11-diol (7) A solution of 7 (0.6 mg) in CH
2
Cl
2
(0.5 ml) was treated with (R)-MTPA (50 mg) in the presence of EDC · HCl
(50 mg) and 4-DMAP (20 mg), and the mixture was stirred at 50 °C under
an N
2
atmosphere for 3 h. It was poured into ice-water and the whole was ex-
tracted with AcOEt. The AcOEt extract was successively washed with 5%
aqueous HCl, aqueous saturated NaHCO
3
, and brine, then dried over MgSO
4
and filtered. Removal of the solvent from the filtrate under reduced pressure
furnished a residue, which was purified on a normal-phase silica gel column
[0.6 g, n-hexane–AcOEt (5 : 1, v/v) to give 7a (0.4 mg, 35%). Through a
similar procedure, 7b (0.4 mg, 19%) was prepared from 7 (1.1 mg) by the
use of (S)-MTPA (50 mg), EDC · HCl (50 mg), and 4-DMAP (20 mg).
7a: A white powder.
1
H-NMR (500 MHz, CDCl
3
)
d
: 0.72 (3H, s, 14-H
3
),
0.85 (2H, m, 9-H), 0.87 (2H, m, 1-H
2
), 1.20 (6H, s, 12, 13-H
3
), 1.64 (1H,
dd-like, 5-H), 1.66 (2H, m, 7-H), 1.81 (2H, m, 6-H
2
), 1.69 (1H, dd-like),
2.04 (1H, m) (2-H
2
), 3.61 (3H, s, MTPA-OMe), 4.51, 4.66 (1H each, both br
s, 15-H
2
), 5.40 (1H, m, 3-H), 7.38—7.58 (5H).
7b: A white powder.
1
H-NMR (500 MHz, CDCl
3
)
d
: 0.71 (3H, s, 14-H
3
),
0.85 (2H, m, 9-H), 0.86 (2H, m, 1-H
2
), 1.21 (6H, s, 12, 13-H
3
), 1.61 (1H,
dd-like, 5-H), 1.66 (2H, m, 7-H), 1.81 (2H, m, 6-H
2
), 1.66 (1H, dd-like),
2.02 (1H, m) (2-H
2
), 3.56 (3H, s, MTPA-OMe), 4.61, 4.95 (1H each, both br
s, 15-H
2
), 5.39 (1H, m, 3-H), 7.38—5.58 (5H).
Enzymatic Hydrolysis of Officinoside D (4) A solution of 4 (20 mg) in
0.2 M acetate buffer (pH 4.0, 2.0 ml) was treated with naringinase (40 mg)
and the whole mixture was stirred at 40 °C for 24 h. The reaction mixture
was poured into EtOH and removal of the solvent under reduced pressure
gave a product. The product was purified by normal-phase silica gel column
chromatography [1.0 g, CHCl
3
–MeOH–H
2
O (30:3:1, lower layer, v/v)] to
give flourensadiol (8, 7.6 mg, 87%), which was identified by comparison of
their physical data ([
a
]
D
, IR,
1
H-NMR,
13
C-NMR) with reported values.
8)
References and Notes
1) Part III: Yoshikawa M., Murakami T., Kishi A., Kageura T., Matsuda
H., Chem. Pharm. Bull., 49 (7), 863—870 (2001).
August 2001 977

2) a) Yoshikawa M., Morikawa T., Murakami T., Toguchida I., Harima S.,
Matsuda H., Chem. Pharm. Bull., 47, 340—345 (1999); b) Yoshikawa
M., Morikawa T., Toguchida I., Harima S., Matsuda H., ibid., 48,
651—656 (2000).
3) Hara S., Okabe H., Mihashi K., Chem. Pharm. Bull., 34, 1843—1845
(1986).
4) Behr D., Wahlberg I., Nishida T., Enzell C. R., Acta Chem. Scand. B,
33, 701—704 (1979).
5) The
1
H- and
13
C-NMR spectra of 1—8 were assigned on the basis of
homo- and hetero-correlation spectroscopy (
1
H–
1
H,
1
H–
13
C COSY),
homo- and hetero-nuclear Hartmann–Hahn spectroscopy (
1
H–
1
H,
1
H–
13
C HOHAHA), heteronucler multiple bond correlation (HMBC),
phase-sensitive nuclear Overhauser effect spectroscopy (pNOESY) ex-
periments.
6) Ohtani I., Kusumi T., Kashman H., Kakisawa H., J. Am. Chem. Soc.,
113, 4092—4096 (1991).
7) Pascual-T. De J., Bellido I. S., Gonzales M. S., Tetrahedron, 36, 371—
376 (1980).
8) Kingston D. G. I., Phytochemistry, 14, 2033—2037 (1975).
978 Vol. 49, No. 8