Calysolins I-IV, resin glycosides from calystegia soldanella

Article abstract of DOI:10.1021/np2006378

Four new resin glycosides having intramolecular cyclic ester structures (jalapins), named calysolins I-IV (1-4), were isolated from the methanol extract of leaves, stems, and roots of Calystegia soldanella, along with one known jalapin (5) derivative. The structures of 1-4 were determined on the basis of spectroscopic data and chemical evidence. They fall into two types, one having a 22-membered ring (1 and 4) and the other with a 27-membered ring (2 and 3). The sugar moieties of 1-4 were partially acylated by some organic acids. Compound 4 is the first example of a hexaglycoside of jalapin.

Full text of DOI:10.1021/np2006378

Article
pubs.acs.org/jnp
Calysolins I−IV, Resin Glycosides from Calystegia soldanella
Ayako Takigawa,† Haruka Muto,† Kiyotaka Kabata,† Masafumi Okawa,‡ Junei Kinjo,‡
Hitoshi Yoshimitsu,§ Toshihiro Nohara,§ and Masateru Ono*,†
†School of Agriculture, Tokai University, 5435 Minamiaso, Aso, Kumamoto 869-1404, Japan
‡Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1 Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan
§Faculty of Pharmaceutical Sciences, Sojo University, 4-22-1 Ikeda, Kumamoto 860-0082, Japan
S
* Supporting Information
ABSTRACT: Four new resin glycosides having intramolecular cyclic ester structures (jalapins), named calysolins I−IV (1−4),
were isolated from the methanol extract of leaves, stems, and roots of Calystegia soldanella, along with one known jalapin (5)
derivative. The structures of 1−4 were determined on the basis of spectroscopic data and chemical evidence. They fall into two
types, one having a 22-membered ring (1 and 4) and the other with a 27-membered ring (2 and 3). The sugar moieties of 1−4
were partially acylated by some organic acids. Compound 4 is the first example of a hexaglycoside of jalapin.
RESULTS AND DISCUSSION
he so-called resin glycosides are well-known as purgatives and
■
1
Tcommonly found in plants of Convolvulaceae. Calystegia
Compound 1, named calysolin I, was obtained as an amorphous
powder. The alkaline hydrolysis product of 1 was fractionated into
an organic acid fraction and a glycosidic acid. GC of the organic
acid fraction revealed the presence of 2-methylbutyric acid and nilic
soldanella Roem. et Schult. (Convolvulaceae) is distributed widely
on sandy beaches of seas and lakes in temperate regions of the
world. The roots of this plant are used for the treatment of
arthritis.2 The isolation and structure elucidation were reported for
two genuine resin glycosides, soldanellines A and B, as chemical
constituents of the root.3,4 In a previous paper,5 we reported the
isolation and structure elucidation of four new glycosidic acids,
calysolic acids A−D, which were obtained along with a known
glycosidic acid, soldanellic acid B,4 and three organic acids,
2S-methylbutyric, tiglic, and 2S-methyl-3S-hydroxybutyric (2S,3S-
nilic) acids, upon alkaline hydrolysis of the crude resin glycoside
fraction of the leaves, stems, and roots of C. soldanella. As part
of an ongoing study of the resin glycosides from this plant, the
isolation and structure elucidation of four new genuine resin
glycosides (1−4) along with a known resin glycoside (5) are
reported herein.
4
1
acid. The later was identified in soldanellic acid B by H NMR
spectroscopic comparison with that of an authentic sample.5 In the
negative-ion FABMS, 1 exhibited a [M − H]− ion peak at m/z
1053 and fragment ion peaks at m/z 953 [1053 − 100 (niloyl unit
(Nla))]−, 825 [1053 − 144 (hexosyl unit − H2O) − 84 (2-methyl-
butyryl unit (Mba))]−, 807 [953 − 146 (methylpentosyl unit)]−,
725 [825 − 100]−, 579 [725 −146]−, 417 [579 − 162]−, and 271
[417 − 146]− (Figure S22, Supporting Information). Furthermore,
the molecular formula of 1 was analyzed as C50H86O23 by positive-
ion HRFABMS. The 1H NMR spectrum of 1 exhibited signals due
to H-2 (δ 2.41 (ddq, J = 7.0, 7.0, 7.0 Hz)) of Mba, H-2 (δ 3.00
(dq, J = 7.0, 7.0 Hz)) of Nla, two primary methyl groups (δ 0.89
(dd, J = 7.0, 7.0 Hz), 0.85 (t, J = 7.5 Hz)), five secondary methyl
groups (δ 1.75 (d, J = 6.5 Hz), 1.54 (d, J = 6.5 Hz), 1.52 (d, J = 7.0 Hz),
1.46 (d, J = 6.5 Hz), 1.11 (d, J = 7.0 Hz)), and four anomeric
protons (δ 5.79 (s), 5.57 (d, J = 8.0 Hz), 5.27 (d, J = 8.0 Hz),
4.74 (d, J = 7.5 Hz)). The 13C NMR spectrum showed signals
The flesh leaves, stems, and roots of C. soldanella were
extracted with methanol. This extract was suspended in
H2O and then extracted successively with ethyl acetate and
n-butyl alcohol. The ethyl acetate−soluble fraction was subjected
to silica gel and HPLC on ODS and silica gel to yield five resin
glycosides (1−5).
Received: July 30, 2011
Published: October 12, 2011
© 2011 American Chemical Society and
American Society of Pharmacognosy
2414
dx.doi.org/10.1021/np2006378|J. Nat. Prod. 2011, 74, 2414−2419

Journal of Natural Products
Article
assignable to three carboxyl carbons (δ 175.4, 175.3, 172.9) and
four anomeric carbons (δ 103.6, 101.7, 99.9, 98.1). These data
indicated that 1 is composed of 1 mol each of 2-methylbutyric
acid, nilic acid, and soldanellic acid B and that three ester
groups exist in the molecule; hence, the carboxyl group of the
aglycone 11S-hydroxyhexadecanoic (11S-jalapinolic) acid of 1
is also linked intramolecularly with a hydroxy group of the
sugar moiety to form a macrocyclic ester structure, as in already
known jalapins.6 The presence of the macrocyclic ester
structure was supported by the nonequivalent signals due to
H2-2 of the jalapinoloyl unit (Jla) of 1 observed at δ 2.71 (1H,
ddd, J = 2.5, 15.5, 15.5 Hz) and 2.61 (1H, ddd, J = 4.5, 4.5, 15.5
Hz), whereas the methyl ester5 of soldanellic acid B exhibited
the equivalent signal due to H2-2 of Jla at δ 2.32 (2H, t, J = 7.5
Glc′, respectively. Taking the J values of signals due to the
anomeric and methine protons of the sugar moiety into account,
the conformations of the quinovopyranosyl and glucopyranosyl
4
units were C1 and that of the rhamnopyranosyl unit was
1
concluded to be C4. The configurations of the component
2-methylbutyric acid and nilic acid units of the crude resin
glycoside fraction of this plant have been determined previously
as S and 2S,3S, respectively.5 Accordingly, the structure of 1 was
assigned as 11S-jalapinolic acid 11-O-(2-O-2S,3S-niloyl)-α-L-
rhamnopyranosyl-(1→2)-[O-(3-O-2S-methylbutyryl)-β-D-glu-
copyranosyl-(1→3)]-O-β-D-glucopyranosyl-(1→2)-β-D-qinovo-
pyranoside, intramolecular 1,2⁗-ester.
Compound 2 (calysolin II) was obtained as an amorphous
powder and furnished tiglic acid, 2-methylbutyric acid, nilic
7
5
1
1
Hz) in the H NMR spectrum.
acid, and calysolic acid A on alkaline hydrolysis. The H and
13C NMR spectra of 2 indicated that 2 is composed of 1 mol
each of tiglic acid, nilic acid, and calysolic acid A and 2 mol of
2-methylbutyric acid. Further, a [M − H]− ion peak observed
at m/z 1381 in the negative-ion FABMS of 2 and the
In order to clarify the positions of the ester linkages, the
1H NMR signals due to the sugar moiety of 1 were assigned on
the basis of 1H−1H COSY, HMQC, and HMBC spectra. On com-
parison of the chemical shifts of signals due to the sugar moieties
between 1 and soldanellic acid B methyl ester,5 downfield shifts
(Δδ = δ(1) − δ(soldanellic acid B methyl ester)) owing to
acylation were seen for signals due to H-2 (Δδ = 1.09) of
rhamnosyl unit (Rha), H-2 (Δδ = ca. 1.53) and H-3 (Δδ =
1.73) of the second glucosyl unit (Glc′) in 1. Thus, the ester
linkages could be located at the OH-2 of Rha, the OH-2 of Glc′,
and the OH-3 of Glc′. The positions of each ester linkage of Jla,
Nla, and Mba were determined by negative-ion FABMS and the
HMBC spectrum of 1 and the HRFABMS of the peracetate
(1a) of 1. The fragment ion peaks at m/z 825 and 807 in the
negative-ion FABMS suggested that the ester linkage of Nla
might be placed at the OH-2 of Rha (Figure S22, Supporting
Information). However, the sites of the ester linkages of Mba
and Jla, both of which are in the Glc′ unit, were not discernible.
In the HMBC spectrum, a key cross peak was observed
between H-2 of Glc′ and C-1 of Jla, while the counterparts of
H-2 of Rha and H-3 of Glc′ could not be defined because the
13C NMR signals due to C-1 of Mba and C-1 of Nla appeared
at almost the same chemical shifts (Figure S23, Supporting
Information). The HRFABMS of 1a gave a fragment ion peak
at m/z 373.1500, which was ascribable to the fragment ion of a
2-O-2-methyl-3-acetoxybutyryl-3,4-O-diacetylrhamnopyranosyl
unit. Therefore, the ester linkages of Nla, Jla, and Mba could be
located at the OH-2 of Rha, the OH-2 of Glc′, and the OH-3 of
1
nonequivalent signals due to H2-2 of Jla in the H NMR
spectrum suggested that 2, like 1, has an intramolecular
macrocyclic ester structure.
1
The H NMR spectrum of 2 showed, in comparison with
that of calysolic acid A methyl ester,5 acylation shifts (Δδ =
δ(2) − δ(calysolic acid A methyl ester)) of signals due to H-2
(Δδ = 1.19) and H-4 (Δδ = 1.47) of Rha, H-3 (Δδ = 1.47) and
H-4 (Δδ = ca. 1.52) of Glc′, and H-2 (Δδ = ca. 1.45) of the
third glucose unit (Glc′′). Thus, the ester linkages of 2 could be
located at OH-2 and OH-4 of Rha, OH-3 and OH-4 of Glc′, and
OH-2 of Glc′′. The sites of each ester linkage of the 2-methyl-
butyryl, tigloyl (Tig), Nla, and Jla units were determined using the
HMBC spectrum of 2, with key cross peaks observed between
H-2 of Rha and C-1 of Nla, H-4 of Rha and C-1 of Tig, H-3 of
Glc′ and C-1 of Mba, H-4 of Glc′ and C-1 of the second
2-methylbutyryl unit (Mba′), and H-2 of Glc′′ and C-1 of Jla
(Figure S23, Supporting Information). Therefore, Nla, Tig,
Mba, Mba′, and Jla were attached to OH-2 of Rha, OH-4 of Rha,
OH-3 of Glc′, OH-4 of Glc′, and OH-2 of Glc′′, respectively.
These inferences were supported by the negative-ion FABMS of
2, in which fragment ion peaks were observed at m/z 1237,
1053 [1381 − 100 − 82 (Tig) − 146]− and 907 [1237 − 162 −
84 × 2]− (Figure S22, Supporting Information). Accordingly, the
structure of 2 was assigned as 11S-jalapinolic acid 11-O-(2-O-2S,
2415
dx.doi.org/10.1021/np2006378|J. Nat. Prod. 2011, 74, 2414−2419

Journal of Natural Products
Article
a
1
Table 1. H NMR Spectroscopic Data (in pyridine-d5, 500 MHz) for 1 and 2
position
1
2
position
1
2
Qui-1
4.74 d (7.5)
4.72 d(7.0)
ca. 4.32
Rha-1
5.79 s
6.34 d (1.0)
2
4.17 dd (7.5, 9.0)
ca. 4.35
2
5.91 d (3.0)
5.99 dd (1.0, 3.5)
4.83 dd (3.5, 9.5)
5.74 dd (9.5, 9.5)
5.19 dq (9.5, 6.5)
1.61 d (6.5)
ca. 2.54
3
ca. 4.31
3
4.62 dd (3.0, 9.5)
4.12 dd (9.5, 9.5)
5.04 dq (9.5, 6.5)
1.75 d (6.5)
4
3.51 dd (9.0, 9.0)
3.65 m
3.56 dd (9.0, 9.0)
3.65 dq (9.0, 6.5)
1.60 d (6.5)
4
5
5
6
1.54 d (6.5)
6
Glc-1
5.57 d (8.0)
5.83 d (7.5)
Jla-2
2.71 ddd (2.5, 15.5, 15.5)
2.61 ddd (4.5, 4.5, 15.5)
ca. 3.72
2
4.11 dd (8.0, 9.0)
4.27 dd (9.0, 9.0)
3.81 dd (9.0, 9.0)
ca. 3.70
ca. 4.16
2
ca. 2.42
3
3.95 dd (9.0, 9.0)
3.90 dd (9.0, 9.0)
3.71 ddd (2.5, 5.5, 9.0)
4.46 dd (2.5, 12.0)
ca. 4.19
11
ca. 3.79
4
16
0.85 t (7.5)
0.86 t (7.0)
5
Mba-2
2.41 ddq (7.0, 7.0, 7.0)
ca. 1.77
2.42 ddq (7.0, 7.0, 7.0)
1.77 m
6
ca. 4.34
3
6
4.06 dd (5.0, 11.5)
5.27 d (8.0)
3
ca. 1.42
1.47 m
Glc′-1
4.90 d (8.0)
4
0.89 dd (7.0, 7.0)
1.11 d (7.0)
0.93 dd (7.5, 7.5)
1.16 d (7.0)
2.52 ddq (7.0, 7.0, 7.0)
1.87 m
2
5.54 dd (8.0, 9.5)
5.87 dd (9.5, 9.5)
4.19 dd (9.5, 9.5)
4.03 m
3.94 dd (8.0, 9.0)
5.57 dd (9.0, 9.0)
5.44 dd (9.0, 9.0)
3.88 m
5
3
Mba′-2
4
3
5
3
1.54 m
6
4.42 dd (2.0, 12.0)
ca. 4.32
4.09 dd (2.5, 12.0)
4.00 dd (4.5, 12.0)
4.94 d (8.0)
4
0.96 dd (7.5, 7.5)
1.22 d (7.0)
2.77 dq (7.0, 7.0)
ca. 4.28
6
5
Glc″-1
Nla-2
3.00 dq (7.0, 7.0)
ca. 4.36
2
3
4
5
6
6
5.46 dd (8.0, 9.5)
ca. 4.27
3
4
1.46 d (6.5)
1.52 d (7.0)
1.37 d (6.5)
1.32 d (7.0)
7.19 dq like (1.5, 6.5)
1.68 d (6.5)
1.85 br s
ca. 4.18
5
3.83 m
Tig-3
4.44 dd (2.0, 12.0)
ca. 4.31
4
5
a
Chemical shifts (δ) are in ppm relative to TMS. Coupling constants (J) in Hz are given in parentheses.
3S-niloyl,4-O-tigloyl)-α-L-rhamnopyranosyl-(1→2)-[O-β-D-gluco-
pyranosyl-(1→6)-O-(3,4-di-O-2S-methylbutyryl)-β-D-glucopyrano-
syl-(1→3)]-O-β-D-glucopyranosyl-(1→2)-β-D-quinovopyranoside,
intramolecular 1,2⁗-ester.
and a primary methyl group and six anomeric protons in the
1H NMR spectrum and exhibited signals due to three carboxyl
carbons and six anomeric carbons in the 13C NMR spectrum.
Thus, 4 was composed of 1 mol each of 2-methylbutyric acid,
tiglic acid, and calysolic acid D, and the carboxyl group of Jla of 4
was also linked intramolecularly with a hydroxy group of the sugar
moiety to form a macrocyclic ester structure.
Compound 3 (calysolin III) was obtained as an amorphous
powder, and its molecular formula was found to be the same as
that of 2. On alkaline hydrolysis, 3 furnished the same organic
acids and glycosidic acid as those obtained from 2. The
1H NMR spectrum of 3, which was superimposable on that of
2, indicated signals due to one unit each of Nla, Tig, and calysolic
1
The H NMR signals due to the sugar moiety of 4 were
compared with those of methyl ester5 of calysolic acid D, and
signals due to H-2 of Rha, H-2 of Glc′, and H-3 of Glc‴ showed
downfield shifts of 1.07, ca. 1.62, and ca. 1.59 ppm, respectively,
owing to acylation. In addition, the HMBC spectrum of 4
exhibited key correlations between H-2 of Glc′ and C-1 of Jla and
H-3 of Glc′′ and C-1 of Tig (Figure S23, Supporting Information).
Accordingly, the structure of 4 was proposed as 11S-jalapinolic
acid 11-O-β-D-glucopyranosyl-(1→4)-O-(3-O-tigloyl)-β-D-gluco-
pyranosyl-(1→3)-O-(2-O-2S-methylbutyryl)-α-L-rhamnopyrano-
syl-(1→2)-[O-β-D-glucopyranosyl-(1→3)]-O-β-D-glucopyranosyl-
(1→2)-β-D-quinovopyranoside, intramolecular 1,2⁗-ester.
Compound 5 was identified as soldanelline B on the basis of
its physical and spectroscopic data.4
1
acid A, and two 2-methybutyryl units. Comparison of the H
NMR signals due to the sugar moiety between 3 and calysolic acid
A methyl ester indicated acylation shifts (Δδ = δ(3) − δ(calysolic
acid A methyl ester)) of signals due to H-3 (Δδ = 1.45) and H-4
(Δδ = 1.89) of Rha, H-3 (Δδ = 1.60) and H-4 (Δδ = ca. 1.65) of
Glc′, and H-2 (Δδ = ca. 1.50) of Glc′′. In addition, the HMBC
spectrum of 3 showed cross peaks between H-3 of Rha and C-1 of
Nla, H-4 of Rha and C-1 of Tig, H-3 of Glc′ and C-1 of Mba, H-4
of Glc′ and C-1 of Mba′, and H-2 of Glc′′ and C-1 of Jla (Figure
S23, Supporting Information). Accordingly, 3 was concluded to be
a positional isomer of 2, in which the Nla unit was at the OH-3 of
Rha rather than at the OH-2 of Rha.
Compound 4 (calysolin IV) was obtained as an amorphous
powder and afforded tiglic acid, 2-methylbutyric acid, and calysolic
acid D5 on alkaline hydrolysis. Negative-ion FABMS of 4 indicated
a [M − H]− ion peak at m/z 1359 and fragment ion peaks at m/z
1277 [1359 − 82]−, 1215 [1359 − 144]−, 953 [1277 − 162 × 2]−,
723 [953 − 84 − 146]−, 579 [723 − 144]−, 417 [579 − 162]−,
and 271 [417 − 146]− (Figure S22, Supporting Information).
Compound 4 showed signals due to one unit each of Mba and
Tig, a nonequivalent methylene group assignable to H2-2 of Jla,
The calysolins in this study have an intramolecular cyclic
ester structure. Therefore, they can be classified as jalapin-type
compounds.6 Further, they fall into two types: one having a 22-
membered ring with the intramolecular ester group attached at
OH-2 of the second glucose unit (1 and 4) and the other with a
27-membered ring with an ester linkage at OH-2 of the third
glucose unit (2 and 3). Calysolin IV (4) is the first example of a
hexaglycoside of jalapin, and calysolines II (2) and III (3) are
the first representatives of the calysolic acid A as the component
glycosidic acid.
2416
dx.doi.org/10.1021/np2006378|J. Nat. Prod. 2011, 74, 2414−2419

Journal of Natural Products
Article
a
Table 2. 13C NMR Spectroscopic Data (in pyridine-d5, 125 MHz) for 1−4
position
1
2
3
4
position
1
2
3
4
Qui-1
103.6
80.4
78.8
76.9
72.2
18.4
101.7
77.0
84.7
70.1
77.0
63.1
99.9
72.9
76.1
70.0
78.3
61.5
103.6
79.5
79.0
76.7
72.6
18.4
100.8
75.3
90.3
70.7
77.4
63.0
104.1
72.5
75.9
69.0
73.3
66.5
102.0
75.0
76.0
71.6
79.0
62.2
103.2
79.1
78.9
76.8
72.5
18.5
101.4
76.7
90.4
70.6
77.3
63.2
104.6
72.5
76.3
69.1
73.1
66.1
102.0
75.0
76.1
71.7
79.1
62.3
103.4
80.9
78.7
76.9
72.4
18.3
101.9
76.4
85.3
70.2
77.0
63.2
100.4
75.0
75.0
72.6
78.8
62.2
Rha-1
98.1
73.1
70.8
74.2
69.3
18.3
172.9
33.9
81.8
14.2
175.4
41.5
26.8
11.7
16.7
97.8
73.1
68.2
75.9
66.7
18.5
173.1
34.4
82.6
14.2
175.8
41.1
26.9
11.8
16.6
175.7
41.2
26.7
11.8
16.3
175.4
48.3
69.8
21.1
13.3
167.5
128.8
138.0
14.3
12.4
101.2
69.5
73.4
72.3
67.1
18.5
173.0
34.4
82.2
14.2
176.1
41.1
26.8
11.6
16.6
175.7
41.1
26.7
11.8
16.4
174.8
48.1
68.4
19.8
11.6
167.5
128.6
138.5
14.4
12.5
97.5
72.2
79.5
73.1
69.0
18.2
173.3
33.9
81.5
14.2
176.4
41.0
27.3
11.6
16.3
2
2
3
3
4
4
5
5
6
6
Glc-1
Jla-1
2
2
3
11
4
16
b
5
Mba-1
6
2
Glc′-1
3
2
4
3
5
4
Mba′-1
5
2
6
3
Glc″-1
4
2
5
b
3
Nla-1
175.3
48.2
70.4
21.2
13.4
4
2
5
3
6
4
Glc‴-1
2
105.4
73.9
77.0
76.3
76.8
61.2
104.6
75.4
78.2
71.8
77.9
62.9
5
Tig-1
167.4
129.4
137.2
14.1
3
2
3
4
5
4
5
6
12.4
Glc⁗-1
2
3
4
5
6
a
b
Chemical shifts (δ) are in ppm relative to TMS. Assignments may be interchangeable.
(10 L) at room temperature for 1 month, and the solvent was removed
under reduced pressure to afford a MeOH extract (126.9 g). This
extract was suspended in H2O (3.5 L) and then successively extracted
with ethyl acetate (1.6 L) and n-butyl alcohol (0.6 L) to afford an ethyl
acetate soluble fraction (48.57 g) and an n-butyl alcohol soluble
fraction (8.15 g). A part (45.87 g) of the ethyl acetate soluble fraction
was chromatographed over a silica gel column using gradient mixtures
of CHCl3−MeOH−H2O (20/1/0, 10/2/0.1, 8/2/0.2, 7/3/0.5, 6/4/1,
0/1/0) as eluents to furnish fractions 1−17. HPLC (column 1) of
fraction 7 (2035 mg) eluted with 95% MeOH gave 2 (18 mg), 5
(97 mg), and fractions 7.1−7.10. Fraction 7.2 (71 mg) was subjected
to HPLC (column 1) with 90% MeOH as eluent to afford 1 (11 mg)
and 3 (39 mg). HPLC (column 1) of fraction 16 (3044 mg) using
90% MeOH as eluent gave fractions 16.1−16.8. Fraction 16.7 (53 mg)
was subjected to HPLC (column 2) with CHCl3−MeOH−H2O
(10/2/0.1) as eluent to afford 4 (17 mg).
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
determined with a JASCO P-1020 polarimeter. The 1H and 13C
NMR spectra were recorded by using a JEOL ECA-500 spectrometer,
and chemical shifts are given on a δ (ppm) scale with tetramethylsilane
(TMS) as an internal standard. MS data were collected using a JEOL
JMS-700 mass spectrometer. Analytical GC was carried out with a
Shimadzu GC-8A gas chromatograph with a flame-ionization detector.
Colum chromatography was carried out over silica gel 60 (Merck, Art.
No. 1.07734). HPLC separation was performed on a Shimadzu LC-
10AS micro pump with a Shimadzu RID-10A RI detector or Shimadzu
SPD-10A UV detector. For HPLC column chromatography,
COSMOSIL 5C18-AR-II (Nacalai Tesque, Inc., Kyoto, Japan, 20 mm
i.d. × 250 mm, column 1) and COSMOSIL SIL-06 (Nacalai Tesque Inc.,
20 mm i.d. × 250 mm, column 2) were used.
Plant Material. The flesh leaves, stems, and roots of C. soldanella
were collected in Mie Prefecture, Japan, in May 2009 and identified by
Dr. Hiroaki Setoguchi (Graduate School of Human and Environ-
mental Studies, Kyoto University). A voucher specimen (CSMI2009)
has been deposited at the laboratory of Natural Products Chemistry,
School of Agriculture, Tokai University.
Calysolin I (1): amorphous powder; mp 148−155 °C; [α]25D −39.2
(c 0.3, MeOH); 1H NMR, see Table 1; 13C NMR, see Table 2;
FABMS (positive mode) m/z 1077 [M + Na]+; FABMS (negative
mode) m/z 1053 [M − H]−, 953 [1053 − 100]−, 825 [1053 − 144 −
84]−, 807 [953 − 146]−, 725 [825 − 100]−, 579 [725 − 146]−, 417
[579 − 162]−, 271 [417 − 146]−; HRFABMS (positive mode) m/z
1077.5459 (calcd for C50H86O23Na, 1077.5458).
Extarction and Isolation. The cut fresh leaves, stems, and roots
of C. soldanella (916.9 g) were extracted with methanol (MeOH)
2417
dx.doi.org/10.1021/np2006378|J. Nat. Prod. 2011, 74, 2414−2419

Journal of Natural Products
Article
a
1
Table 3. H NMR Spectroscopic Data (in pyridine-d5, 500 MHz) for 3 and 4
position
3
4
position
3
4
Qui-1
4.75 d (7.5)
4.73 d (7.5)
ca. 4.12
Glc⁗-1
2
5.04 d (8.0)
2
ca. 4.30
3.83 dd (8.0, 8.5)
ca. 4.12
3
ca. 4.34
4.32 dd (9.0, 9.0)
3.51 dd (9.0 9.0)
ca. 3.67
3
4
3.54 dd (9.0, 9.0)
3.65 dq (9.0, 6.5)
1.57 d (6.5)
5.71 d (8.0)
4.13 dd (8.0, 9.0)
3.80 dd (9.0, 9.0)
3.89 dd (9.0, 9.0)
3.72 ddd (3.0, 7.0, 9.0)
ca. 4.47
4
ca. 4.12
5
5
ca. 3.76
6
1.53 d (6.5)
6
ca. 4.28
b
Glc-1
5.43
6
4.18 dd (4.5, 10.5)
5.84 d (1.5)
6.09 dd (1.5, 3.5)
4.86 dd(3.5, 9.5)
4.23 dd (9.5, 9.5)
5.09 d (9.5, 6.5)
1.73 d (6.5)
2
ca. 4.12
Rha-1
6.26 d (1.0)
3
ca. 4.12
2
4.96 dd (1.0, 3.0)
6.05 dd (3.0, 9.5)
6.16 dd (9.5, 9.5)
5.30 dq (9.5, 6.5)
1.62 d (6.5)
4
ca. 3.74
3
5
ca. 3.67
4
6
4.32 dd (5.0, 11.5)
4.03 dd (6.5, 11.5)
5.25 d (8.0)
5.58 dd (8.0, 9.5)
5
6
ca. 4.18
6
Glc′-1
4.89 d (7.5)
ca. 4.07
Jla-2
2.62 ddd (7.0, 7.0, 12.5)
ca. 2.45
2.70 ddd (3.0, 11.5, 16.0)
ca. 2.61
2
2
b
3
5.70 dd (9.5, 9.5)
5.57 dd (9.5, 9.5)
4.03 ddd (3.5, 3.5, 9.5)
ca. 4.18
4.59
11
ca. 3.80
ca. 3.73
4
ca. 4.15
16
0.86 t (7.0)
0.85 t (7.0)
ca. 2.62
5
ca. 4.15
Mba-2
2.39 ddq (7.0, 7.0, 7.0)
ca. 1.62
6
4.50 br d (11.5)
ca. 4.28
3
1.80 m
6
ca. 4.08
3
ca. 1.32
ca.1.54
Glc″-1
5.01 d (8.0)
5.51 dd (8.0, 9.5)
ca. 4.30
4
0.70 dd (7.5, 7.5)
1.14 d (7.0)
0.94 dd (7.5, 7.5)
1.25 d (7.0)
2
5
3
Mba′-2
2.47 ddq (7.0, 7.0, 7.0)
1.81 m
4
4.19 dd (9.5, 9.5)
ca. 3.87
3
5
3
1.50 m
6
ca. 4.47
4
0.95 dd (7.5, 7.5)
1.19 d (7.0)
6
ca. 4.30
5
Glc‴-1
5.31 d (7.5)
4.06 dd (7.5, 9.5)
5.78 dd (9.5, 9.5)
4.44 dd (9.5, 9.5)
ca. 3.65
Nla-2
2.75 dq (7.0, 7.0)
ca. 4.34
2
3
4
5
6
6
3
4
1.21 d (7.0)
5
1.10 d (7.0)
Tig-3
7.24 dq like (1.5, 7.0)
1.70 dd like (1.5, 7.0)
7.05 dq like (1.5, 6.5)
1.61 d (6.5)
ca. 4.38
4
5
ca. 4.37
1.95 br s
1.84 br s
a
b
Chemical shifts (δ) are in ppm relative to TMS. Coupling constants (J) in Hz are given in parentheses. Signals were deformed by virtual coupling.
Calysolin II (2): amorphous powder; mp 143−147 °C; [α]25
ether (3 × 5 mL). The ether layer was dried over MgSO4 and
evaporated to furnish an organic acid fraction, which was analyzed by
GC (Shimadzu GC-8A gas chromatograph with flame-ionization
detector; column, Unisole 30T (5%), 3.2 mm i.d. × 2 m glass column
(column 1); carrier gas N2, 1.0 kg/cm2; column temperature, 120 °C;
tR (min) = 4.42 (2-methylbutyric acid) for 1−4, 10.38 (tiglic acid) for
2−4). A part of the organic acid fraction was methylated with
trimethylsilyldiazomethane−ether, and then the reaction mixture was
analyzed by GC (column, column 1; column temperature, 100 °C;
carrier gas, N2 1.0 kg/cm2; tR (min) = 8.60 (methyl nilate) for 1−3).
The aqueous layer was desalted by MCI gel CHP 20 column
chromatography (H2O, acetone) to give a glycosidic acid as an
amorphous powder (3 mg from 1, 4 mg from 2, 4 mg from 3, 4 mg
from 4). The glycosidic acids derived from 1−4 were each identical
with soldanellic acid B, calysolic acid A, calysolic acid A, and calysolic
D
−33.0 (c 1.3, MeOH); H NMR, see Table 1; 13C NMR, see Table
2; FABMS (positive mode) m/z 1405 [M + Na]+; FABMS (negative
mode) m/z 1381 [M − H]−, 1299 [1381 − 82]−, 1281 [1381 − 100]−,
1237 [1381 − 144]−, 1199 [1281 − 82]−, 1053 [1199 − 146]−, 907 [1237
− 84 × 2 − 162]−, 579 [907 − 82 − 100 − 146]−, 417 [579 − 162]−;
HRFABMS (positive mode) m/z 1405.6981 (calcd for C66H110O30Na,
1405.6979).
1
Calysolin III (3): amorphous powder; mp 140−145 °C; [α]25
D
−27.4 (c 1.7, MeOH); 1H NMR, see Table 3; 13C NMR, see Table 2;
FABMS (positive mode) m/z 1405 [M + Na]+; FABMS (negative
mode) m/z 1381 [M − H]−, 1299 [1381 − 82]−, 1281 [1381 −
100]−, 1237 [1381 − 144]−, 1199 [1281 − 82]−, 1053 [1199 − 146]−,
907 [1237 − 84 × 2 − 162]−, 579 [907 − 82 − 100 − 146]−, 417 [579
− 162]−; HRFABMS (negative mode) m/z 1381.6995 (calcd for
C66H109O30, 1381.6704).
1
acid D, respectively, by comparison of H NMR spectra with those of
Calysolin IV (4): amorphous powder; mp 170−175 °C; [α]25
authentic samples.5
D
−18.2 (c 0.6, MeOH); 1H NMR, see Table 3; 13C NMR, see Table 2;
FABMS (negative mode) m/z 1359 [M − H]−, 1277 [1381 − 82]−,
1215 [1359 − 144]−, 953 [1277 − 162 × 2]−, 723 [953 − 84 − 146]−,
579 [723 − 144]−, 417 [579 − 162]−, 271 [417 − 146]−; HRFABMS
(negative mode) m/z 1359.6423 (calcd for C62H103O32, 1359.6432).
Alkaline Hydrolysis of 1−4. Solutions of 1 (5 mg), 2 (5 mg), 3
(5 mg), and 4 (5 mg) in 1,4-dioxane−1 M KOH (1/1, 2 mL) were
heated to 95 °C for 1 h. The reaction mixture was adjusted to pH 3
with 1 M HCl and then diluted with H2O (10 mL) and extracted with
Acetylation of 1. A solution of 1 (3 mg) in Ac2O−pyridine (1/1,
1 mL) was left to stand at room temperature overnight. The solvent
was removed under an N2 stream to give a residue. The residue was
partitioned between diethyl ether (0.5 mL) and H2O (0.5 mL). The
diethyl ether layer was concentrated under a N2 stream to afford 1a
(3 mg).
Peracetate of 1 (1a): amorphous powder; FABMS (positive mode)
m/z 1455 [M + Na]+, 373, 313, 83; HRFABMS (positive mode) m/z
373.1500 (calcd for C17H25O9, 373.1499).
2418
dx.doi.org/10.1021/np2006378|J. Nat. Prod. 2011, 74, 2414−2419

Journal of Natural Products
Article
ASSOCIATED CONTENT
* Supporting Information
■
S
Figures giving NMR spectra and other data for 1−4. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel :+81-9676-7-3947. Fax: +81-9676-7-3960. E-mail:
mono@agri.u-tokai.ac.jp.
■
ACKNOWLEDGMENTS
We express our appreciation to Mr. H. Harazono of Fukuoka
University for the measurement of the FABMS.
■
REFERENCES
(1) Pereda-Milanda, R.; Rosas-Ramirez, D.; Castaneda-Geo
■
́
mez, J. In
̃
Resin Glycosides from the Morning Glory Family: Progress in the
Chemistry of Organic Natural Products; Kinghorn, A. D., Falk, H.,
Kobayashi, J., Eds.; Springer: Vienna and New York, 2010; Vol. 92,
pp 77−153.
(2) Hotta, M., Ogata, K., Nitta, A., Hosikawa, K., Yanagi, M.,
Yamazaki, K., Eds. Useful Plants of the World; Heibonsha: Tokyo, 1989;
p 197.
(3) Gaspar, E. M. Tetrahedron Lett. 1999, 40, 6861−6864.
(4) Gaspar, E. M. J. Org. Chem. 2001, 369−373.
(5) Takigawa, A.; Setoguchi, H.; Okawa, M.; Kinjo, J.; Miyashita, H.;
Yokomizo, K.; Yoshimitsu, H.; Nohara, T.; Ono, M. Chem. Pharm.
Bull. 2011, 59, 1163−1168.
(6) Ono, M.; Nakagawa, K.; Kawasaki, T.; Miyahara, K. Chem.
Pharm. Bull. 1993, 41, 1925−1932.
(7) Noda, N.; Ono, M.; Miyahara, K.; Kawasaki, T. Tetrahedron
1987, 43, 3889−3902.
NOTE ADDED AFTER ASAP PUBLICATION
■
This paper was published on the Web on Oct 12, 2011, with an
error in the last value in column 2 of Table 1. The corrected
version was reposted on Oct 17, 2011.
2419
dx.doi.org/10.1021/np2006378|J. Nat. Prod. 2011, 74, 2414−2419