Aliphatic glucoside, zanthoionic acid and megastigmane glucosides: Zanthoionosides A–E from the leaves of zanthoxylum ailanthoides

Article abstract of DOI:10.1248/cpb.c17-00211

From the leaves of Zanthoxylum ailanthoides, 4-O-p-E-coumaric acid esters of 2-propanol β-D-glucopyranoside, megastigmane and megastigmane glucosides were isolated. Their structures were elucidated by spectroscopic evidence. The absolute configurations of the megastigmane and aglycone of megastigmane glucosides were determined by the octant rule and modified Mosher’s method after protection of carboxylic acids by p-bromophenacyl esters and primary alcohols by pivaloyl esters.

Full text of DOI:10.1248/cpb.c17-00211

7
54
Chem. Pharm. Bull. 65, 754–761 (2017)
Vol. 65, No. 8
Regular Article
Aliphatic Glucoside, Zanthoionic Acid and Megastigmane Glucosides:
Zanthoionosides A–E from the Leaves of Zanthoxylum ailanthoides
a
b
a
a
Serika Teshima, Susumu Kawakami, Sachiko Sugimoto, Katsuyoshi Matsunami,
,
a,b
c
Hideaki Otsuka,* and Takakazu Shinzato
a
Graduate School of Biomedical and Health Sciences, Hiroshima University; 1–2–3 Kasumi, Minami-ku, Hiroshima
b
7
7
34–8553, Japan: Faculty of Pharmacy, Yasuda Women’s University; 6–13–1 Yasuhigashi, Asaminami-ku, Hiroshima
c
31–0153, Japan: and Subtropical Field Science Center, Faculty of Agriculture, University of the Ryukyus; 1
Senbaru, Nishihara-cho, Nakagami-gun, Okinawa 903–0312, Japan.
Received March 10, 2017; accepted April 19, 2017
From the leaves of Zanthoxylum ailanthoides, 4ꢀ-O-p-E-coumaric acid esters of 2-propanol β-D-
glucopyranoside, megastigmane and megastigmane glucosides were isolated. Their structures were elucidated
by spectroscopic evidence. The absolute configurations of the megastigmane and aglycone of megastigmane
glucosides were determined by the octant rule and modified Mosher’s method after protection of carboxylic
acids by p-bromophenacyl esters and primary alcohols by pivaloyl esters.
Key words Zanthoxylum ailanthoides; Rutaceae; megastigmane; zanthoionoside
−1
−1
The genus Zanthoxylum belonging to the family Rutaceae (1701 cm ) and an aromatic ring (1512cm ). The UV absorp-
comprises about 200 species that are found in tropical and tion band also suggested the presence of an aromatic ring. In
1
moderate areas of both hemispheres. Z. ailanthoides SIEBOLD the H-NMR spectrum, two non-equivalent methyl signals (δ
H
et ZUCCARINI is a tall, deciduous tree that grows in Japan 1.23 and 1.27) were observed and these methyl groups were
1
1
(
Honshu and Kyushu), Okinawa, Taiwan, China and the Phil- connected through an oxymethine signal (δH 4.07) in H– H
1)
ippines. Some less polar compounds such as amides and ben- correlation spectrometry (COSY). The NMR spectra exhib-
zenoids, nor-lignan and phenyl propanoids, and pheophorbides ited anomeric proton (δH 4.41) and carbon (δC 102.6) signals
with antiproliferative and apoptosis-inducing activities against (Table 1), and HPLC analysis of the hydrolyzate of 1 revealed
human leukemia cells, HL-60 and WEHI-3 have been isolated the presence of D-glucose as a sugar unit. Thus, the core
from the stem bark, stem wood and leaves of Z. ailanthoides, unit of 1 was established as 2-propanol D-glucopyranoside
2
–4)
respectively.
In the course of continuing investigation, Z. and the mode of sugar linkage was inferred as β from the
ailanthoides was chosen for phytochemical study. The leaves coupling constant (J=7.7Hz) of the anomeric proton. The
13
of Z. ailanthoides are known to be used for a carminative and remaining seven C signals accounted for nine carbons, due
5
)
the fruits for a stomachic. This paper deals with the isolation to a symmetrically substituted benzene ring (Table 1), along
of polar compounds from the leaves of Z. ailanthoides.
with proton signals of a trans double bond [δH 7.68 (1H, d,
J=16.0Hz, H-7″) and 6.39 (1H, d, J=16.0Hz, H-8″)], and they
were assigned as those of a p-E-coumaroyl group. COSY con-
Results and Discussion
Air-dried leaves of Z. ailanthoides were extracted with nectivity from H-1′ to H-4′ and heteronuclear multiple-bond
MeOH three times and the concentrated MeOH extract was correlation (HMBC) between deshielded H-4′ (δH 4.84) and
partitioned with solvents of increasing polarity. The 1-BuOH- C-9″ (δ 168.6) substantiated the structure of 1 as 2-propanol
C
soluble fraction was separated by means of various chromato- β-D-glucopyranoside 4′-O-p-E-coumarate, as shown in Fig. 2.
graphic procedures including column chromatography (CC) on Non-equivalent upfield shifts of methyl groups of 2-propanol
a highly porous synthetic resin (Diaion HP-20), normal silica on β-D-glucopyranosylation are well discussed in the litera-
1
2)
gel and reversed-phase octadecyl silica gel (ODS) CC, droplet ture.
counter-current chromatography (DCCC), and HPLC to afford
Zanthoionic acid (2) was isolated as an amorphous powder
2
-propanol β-D-glucopyranoside 4′-O-p-E-coumaric acid ester and its elemental composition was determined to be C H O
13 20 4
(1), a new megastigmane and five megastigmane glucosides, by HR-ESI-MS. The IR spectrum exhibited absorptions for
named zanthoionic acid (2) and zanthoionosides A–E (3–7), hydroxy groups, two types of carbonyl groups (1701 and
−
1
1
respectively. These novel compounds were isolated along with 1713 cm ) and a double bond. In the H-NMR spectrum,
6
)
the known compounds dendranthemoside B (8), kiwiionoside signals for two singlet methyl, one doublet methyl and one
9), icariside B (10), citroside A (11), linalool 3-O-α- trans double bond were observed and the presence of two
7
)
8)
9)
(
1
10)
L-arabinopyranosyl-(1″→6′)-β-D-glucopyranoside (12)
and methylene, one oxymethine, two methine, carbonyl, carboxyl
11)
13
sibiricose A (13) (Fig. 1).
and quaternary carbons was confirmed by C-NMR and from
5
Compound 1 was isolated as an amorphous powder and distortionless enhancement by polarization transfer spectra.
1
1
its elemental composition was determined as C H O by Two H– H COSY correlations from H -4 through H-9, and
18
24
8
2
high-resolution (HR)-electrospray ionization (ESI)-MS. The H-2eq and H-4eq by long-range coupling, as well as HMBC
IR spectrum showed absorption bands ascribable to hy- correlations between H -11 and H -12 and C-2 and C-6, H -2
3
3
2
−1
droxy groups (3307cm ), an ester carbonyl functional group and H -4 and C-3, H-6 and C-1, H-8 and C-10, and other di-
2
*
To whom correspondence should be addressed. e-mail: otsuka-h@yasuda-u.ac.jp; hotsuka@hiroshima-u.ac.jp
©
2017 The Pharmaceutical Society of Japan

Vol. 65, No. 8 (2017)
Chem. Pharm. Bull.
755
Fig. 2. Structures of New Compounds Isolated
Fig. 1. Structures of Known Compounds Isolated
13
Table 1.
C
C-NMR Spectroscopic Data for 1–7 (150MHz, CD OD)
3
1
2
3
4
5
6
7
1
2
3
4
5
6
7
8
9
22.1
72.8
23.8
39.6
56.8
34.1
46.1
71.4
78.3
33.9
58.0
133.2
136.0
78.0
24.1
24.1
32.8
17.2
102.3
75.4
78.2
71.4
77.9
62.5
34.4
46.4
75.5
39.5
27.3
59.9
134.7
132.8
74.9
67.8
23.9
32.8
21.8
102.8
75.4
78.5
71.9
77.9
63.0
33.6
45.2
70.3
88.7
32.8
58.2
133.6
133.3
74.3
67.6
32.3
23.8
17.2
105.7
75.2
77.9
71.2
77.6
62.3
34.1
45.7
82.6
78.4
32.9
58.2
133.8
133.3
74.4
67.7
32.3
23.8
17.2
107.0
76.0
78.3
71.8
78.0
63.0
34.0
46.1
71.3
78.3
33.8
58.2
134.3
132.1
72.8
75.5
32.5
24.0
17.2
104.9
75.3
78.0
71.6
77.9
62.7
214.2
50.1
35.0
58.0
131.0
134.5
74.0
1
1
Fig. 3. H– H COSY and HMBC Correlations of Zanthoionic Acid (2)
Dual Arrow Curves Indicate HMBC Correlations Were Observed for
Both Directions
10
11
12
13
178.7
21.4
31.0
21.8
14)
(
2a) (Fig. 3). Therefore, the structure of 2 was elucidated
as 5R,6R,7E,9R-megastigman-3-on-7-en-9-ol-10-oic acid as
shown in Fig. 2.
Zanthoionoside A (3) was isolated as an amorphous powder
and its elemental composition was determined to be C H O
8
by HR-ESI-MS. A strong IR absorption band at 3305cm
indicated 3 was a glycosidic compound. In the H-NMR spec-
trum, signals for two singlet methyls, two doublet methyls and
two olefinic protons coupled in trans geometry were observed.
The C-NMR spectrum exhibited six signals assignable to
glucopyranose and HPLC analysis of the enzymatic hydro-
lyzate revealed the presence of D-glucose. The remaining 13
signals were indicative that 3 was a derivative of megastig-
mane and comprised of four methyls, one methylene, three
oxymethines, two methines, one disubstituted double bond
1′
2′
3′
4′
5′
6′
1″
102.6
75.3
75.9
72.7
76.1
19 34
−1
62.6
1
127.2
131.3
116.9
161.4
147.2
114.9
168.6
2
3
″,6″
″,5″
13
4
7
8
9
″
″
″
″
1
1
agnostic correlations as shown in Fig. 3 established the scaf- and a quaternary carbon. The H– H COSY spectrum showed
fold of 2 to have a megastigmane skeleton with a carboxylic a proton chain from H -2 to H -10 and geminal dimethyl
2
3
acid at the end of the side chain. The coupling constant of protons were correlated with methylene carbon in the HMBC
H-6 (J=10.7Hz) confirmed that H-5 and H-6 were in axial spectrum. Thus, two hydroxy groups were placed on C-3
13)
positions and by application of the octant rule, a positive and C-4. Considering the coupling constants, J2–3=2.9 and
Cotton effect at 298nm enabled the assignment of the absolute 3.3Hz, suggested that H-3 was in the equatorial position, and
configuration of C-5 as R and thus C-6 also as R. The absolute the hydroxy group at the 3-position was in an axial position;
configuration at the 9-position was determined to be R by the J4–5=10.4Hz also suggested that H-4 and H-5 were in axial
modified Mosher’s method, using 2 p-bromophenacyl ester positions. From the coupling constant, J5–6=10.8Hz, it could

7
56
Chem. Pharm. Bull.
Vol. 65, No. 8 (2017)
be inferred that the side chain was equatorially linked at the
-position. The mode of sugar linkage was β, evidenced by
6
the coupling constant of the anomeric proton, which showed
a cross peak with the C-9 carbon in the HMBC spectrum.
Therefore, the structure of 3 was elucidated as megasitgam-
7-ene-4,5,9-triol 9-O-β-D-glucopyranose. The absolute con-
figurations were assigned by the application of the modified
Mosher’s method to the aglycone of 3 (3a). From MS spectral
data, two units of α-methoxy-α-trifluoromethylphenylacetic
acid (MTPA) were introduced into 3a and the positions of
esterification were deduced to be on the hydroxy groups at the
4
- and 9-positions from the obvious downfield shifts (δH 4.74
and 5.54, respectively for 3b and δ 4.69 and 5.54, respective-
H
ly for 3c) of H-4 and H-9 compared with the spectra of 3 and
3a. The coupling pattern and coupling constants of H-4 of 3b
and 3c were the same as those of 3 and 3a, suggesting that the
esterification occurred on the hydroxy group at the 4-position.
From this evidence, the absolute configurations were assigned
as 3S, 4R, 5S, 6S, and 9R (Fig. 4).
Zanthoionoside B (4) was isolated as an amorphous powder
and its elemental composition was determined to be C H O
8
19
34
by HR-ESI-MS. NMR spectroscopic data indicated that 4 was
also a derivative of megastigmane with one primary and two
1
1
secondary hydroxyl groups. From the H– H COSY correla-
tion from H -2 to H -10, the primary alcohol was placed at
2
2
13
the terminal carbon, C-10. The C-NMR spectrum exhibited
six signals assignable to those of glucopyranose, and HPLC
analysis of the enzymatic hydrolyzate revealed the presence
of D-glucose. The mode of sugar linkage was β based on the
coupling constant of the anomeric proton, and the position of
the sugar linkage was determined to be on the hydroxy group
Fig. 4. Results of Modifed Mosher’s Method
at the 3-position based on the HMBC correlation between glucopyranoside, as shown in Fig. 2.
the anomeric proton and C-3. The coupling constants of H-3 Zanthoionosides D and E (6 and 7) were isolated as amor-
J=3.7, 2.8, 2.8 and 2.2Hz) indicated that the hydroxy group at phous powders and their elemental composition was deter-
the 3-position was in the axial orientation and as J5–6=10.3Hz, mined as C H O by HR-ESI-MS, which was the same as
(
19
34
9
the C-13 methyl group and the side chain were in the equato- that of 5. NMR spectroscopic data showed strong similarity to
rial positions. Enzymatic hydrolysis of 4 gave an aglycone (4a) those of 5. On enzymatic hydrolysis, 6 and 7 gave D-glucose
whose primary alcohol was protected as pivaloyl ester (4b). and a common aglycone, whose physico-chemical properties
Application of the modified Mosher’s method to 4b revealed were the same as those of 5a. In the HMBC spectra of 6 and
the absolute configuration at C-3, C-5, C-6 and C-9 to be R, 7, anomeric protons showed cross peaks with C-3 and C-10,
R, R and S, respectively (Fig. 4). Therefore, the structure of 4 respectively. Therefore, the structures of 6 and 7 were eluci-
was elucidated as 3R,5R,6R,7E,9S-megastigman-7-ene-3,9,10- dated as 3S,4R,5S,6S,7E,9R-megastigman-7-ene-3,4,9,10-tetrols
triol 3-O-β-D-glucopyranoside, as shown in Fig. 2.
Zanthoionoside C (5) was isolated as an amorphous powder spectively, as shown in Fig. 2.
and its elemental composition was determined as C H O by From the leaves of Z. ailanthoides, 2-propanol β-D-
3-O-β-D-glucopyranoside and 10-O-β-D-glucopyranoside, re-
19
34
9
HR-ESI-MS. NMR spectroscopic data indicated that 5 was glucopyranoside 4′-O-p-E-coumarate (1), zanthoionic acid
a similar compound to 4 with one additional hydroxy group. (2) and zanthoionosides A–E (3–7) were isolated. Their
Methylene protons from C-2 through C-10 were correlated structures were elucidated mainly by the use of NMR spec-
1
1
by the H– H COSY spectrum and geminal dimethyl protons troscopic evidence. The absolute configuration of the cyclo-
showed cross peaks with C-2 in the HMBC spectrum. Thus, hexanone moiety of 2 was determined using the octant rule
a 3,4,9,10-tetrol structure was proposed for 5 and the position and that of the side chain was determined using the modified
of the sugar linkage was determined to be on the hydroxy Mosher’s method (Fig. 4). The absolute configurations of
group at C-4 by HMBC. The coupling constants, J2–3=3.0 and zanthoionosides A–E (3–7) were determined using the modi-
3.1Hz indicated that H-3 was in the equatorial position, and fied Mosher’s method. The isolation of a megastigmane with
J4–5=10.7Hz and J5–6=10.9Hz indicated substituents at C-4, a carboxylic acid functional group like 2 was a relatively rare
C-5 and C-6 in the equatorial positions. Enzymatic hydroly- example from a natural source. In general, megastimanes and
sis of 5 gave an aglycone (5a) and the primary alcohol was their glycosides do not have remarkable biological activity.
15,16)
protected as a pivaloyl ester (5b). (R)- and (S)-MTPA were However, they are known as constituents of wine flavor
prepared from the pivaloyl ester to give 4,9-diesters (5c and and a structurally related abscisic acid is a plant dormancy
17)
d) (Fig. 4). As a result, the structure of 5 was elucidated as hormone.
Inhibitory activity of histamine release from
3
S,4R,5S,6S,7E,9R-megastigman-7-ene-3,4,9,10-tetrol 4-O-β-D- antigen-stimulated RBL-2H3 cells was also reported for some

Vol. 65, No. 8 (2017)
Chem. Pharm. Bull.
757
18)
megastigmane glycosides.
of 8 from the peaks at 8 and 12min, respectively. The residue
1.67g) in fractions 44–49 was subjected to open ODS CC.
The residue (303mg) in fractions 78–92 was applied to DCCC
(
Experimental
General Experimental Procedure Optical rotations and to give a residue (155mg) in fractions 31–38, which was then
circular dichroism (CD) spectra were measured on JASCO purified by HPLC (H O–MeOH, 1:3) to give 18.0mg of 13
2
P-1030 and JASCO J-720 spectropolarimeters, respectively. IR from the peak at 28min. The residue (151mg) in fractions
and UV spectra were measured on Horiba FT-710 and JASCO 93–100 was again subjected to silica gel CC (70g) with in-
1
V-520 UV/Vis spectrophotometers, respectively. H- and creasing amounts of MeOH in CHCl [CHCl (400mL), and
3
3
13
C-NMR spectra were taken on a Bruker Avance III spec- CHCl –MeOH (19:1, 400mL), (9:1, 400mL), (17:3, 400mL),
3
trometer at 600 and 150MHz, respectively, with tetramethyl- (4:1, 400mL), (3:1, 400mL), (7:3, 400mL), (13:7, 400mL),
silane as an internal standard. Positive-ion HR-ESI-MS was (3:2, 400mL) and (1:1, 400mL)], 10-g fractions being col-
performed with an Applied Biosystems QSTAR XL system lected to afford 7.5mg of 10, 14.3mg of 7, and 84.7mg of 5
ESI (Nano Spray)-TOF-MS.
in fractions 182–190, 211–223, and 224–260, respectively. The
A highly porous synthetic resin (Diaion HP-20) was pur- residue (148mg) in fractions 101–107 was again subjected to
chased from Mitsubishi Kagaku (Tokyo, Japan). Silica gel CC silica gel CC (50g) with the same solvent system as above to
and reversed-phase (ODS) open CC were performed on silica yield 21.1mg of 11 and 78.1mg of 6 in fractions 185–206 and
gel 60 (Merck, Darmstadt, Germany) and Cosmosil 75C - 207–257, respectively.
18
OPN (Nacalai Tesque, Kyoto, Japan) [Φ=50mm, L=20cm,
The residue (30.5g) in fractions 9–15 obtained on Diaion
linear gradient: MeOH–H O (1:9, 1L)→(1:1, 1L) and then HP-20 CC was subjected to silica gel (700g) CC with increas-
2
(
1:1 500mL)→(3:7, 500mL), fractions of 10g being col- ing amounts of MeOH in CHCl [CHCl (3L), and CHCl –
3
3
3
lected], respectively. DCCC (Tokyo Rikakikai, Tokyo, Japan) MeOH (49:1, 3L), (24:1, 3L), (23:2, 3L), (9:1, 3L), (7:1,
was equipped with 500 glass columns (Φ=2mm, L=40cm), 3L), (17:3, 3L), (33:7, 3L), (4:1, 3L), (3:1, 3L), (7:3, 3L)
the lower and upper layers of a solvent mixture of CHCl3– and (3:2, 3L)], 500-mL fractions being collected. The residue
MeOH–H O–n-PrOH (9:12:8:2) being used as the stationary (1.83g out of 7.05g) in fractions 33–41 was subjected to open
2
and mobile phases, respectively. Five-gram fractions were col- ODS CC. The residue (253mg) in fractions 117–127 was puri-
lected and numbered according to their order of elution with fied by DCCC to give 16.1mg of 1 in fractions 63–68. The
the mobile phase. HPLC was performed on an ODS-3 column residue (2.06 out of 5.04g) in fractions 42–47 was subjected
(
Inertsil; GL Science, Tokyo, Japan; Φ=6mm, L=25cm), and ODS CC. The residue (112mg) in fractions 91–99 was puri-
the eluate was monitored with a UV detector at 254nm and a fied by DCCC to give 11.2mg of 3 in fractions 39–41. The
refractive index monitor. residue (239mg) in fractions 116–125 was subjected to DCCC
R)- and (S)-MTPA, and β-glucosidase (Almond, 10000U/ and the residue (54.4mg) in fractions 29–38 was purified by
70.3mg) were products of Wako Pure Chemical Industries, HPLC (H O–MeOH, 13:7) to give 10.8mg of 4 from the peak
(
2
2
Ltd. (Osaka, Japan).
Plant Material Leaves of Z. ailanthoides were collected
at 18min.
The residue (16.5g) in fractions 30–38 obtained on HP-20
in Kunigami-son, Kunigami-gun, Okinawa in July 2008 and CC was subjected to silica gel CC (400g) with increasing
a voucher specimen was deposited in the Herbarium of Phar- amounts of MeOH in CHCl [CHCl (2L), and CHCl –MeOH
3
3
3
maceutical Sciences, Graduate School of Biomedical Sciences, (49:1, 2L), (24:1, 2L), (47:3, 2L), (23:2, 2L), (9:1, 2L),
Hiroshima University (08-ZA-Okinawa-0708).
(7:1, 2L), (17:3, 2L), (33:7, 2L), (4:1, 2L), (3:1, 2L) and
Isolation and Extraction Leaves of Z. ailanthoides (7:3, 2L)], 300-mL fractions being collected. The residue
(
10.6kg) were extracted three times with MeOH (45L×3) (2.12g) in fractions 46–52 was separated by open ODS CC to
at room temperature for one week and then concentrated give 409mg of 2 in fractions 52–64. The residue (238mg) in
to 6L in vacuo. The concentrated extract was washed with fractions 221–233 was purified by DCCC to give 134mg of 12
n-hexane (6L, 161g), and then the MeOH layer was concen- in fractions 82–111.
trated to a gummy mass. The latter was suspended in water
(
2-Propanol β-D-Glucopyranoside 4′-O-E-Coumaric Acid
25
6L) and then extracted with EtOAc (6L) to give 177g of an Ester (1) Colorless amorphous powder, [α] –18.5 (c=1.07,
D
−1
EtOAc-soluble fraction. The aqueous layer was extracted with MeOH); IR νmax (film) cm : 3307, 1701, 1512, 1161, 1028;
-BuOH (6L) to give a 1-BuOH-soluble fraction (134g), and UV λmax (MeOH) nm (logε): 306 (3.62); H-NMR (CD OD,
1
1
3
the remaining water layer was concentrated to furnish 577g of 600MHz) δ: 7.68 (1H, d, J=16.0Hz, H-7″), 7.47 (2H, d,
a water-soluble fraction.
J=8.2Hz, H-2″ and 6″), 6.81 (2H, d, J=8.2Hz, H-3″ and 5″),
The 1-BuOH-soluble fraction (133g) was subjected to Di- 6.39 (1H, d, J=16.0Hz, H-8″), 4.84 (1H, overlapped by DHO
aion HP-20 CC (Φ=50mm, L=50cm), using H O–MeOH signal, H-4′), 4.41 (1H, d, J=7.7Hz, H-1′), 4.07 (1H, septet,
2
(
4:1, 3L), (3:2, 3L), (2:3, 3L), and (1:4, 3L), and MeOH J=6.2Hz, H-2), 3.64 (1H, dd, J=9.3, 9.2Hz, H-3′), 3.62 (1H,
(
3L), 1L-fractions being collected. The residue (23.3g) in dd, J=12.2, 2.0Hz, H-6′a), 3.43 (1H, ddd, J=8.3, 5.9, 2.0Hz,
fractions 4–8 was subjected to silica gel (500g) CC with H-5′), 3.54 (1H, dd, J=12.2, 5.9Hz, H-6′b), 3.26 (1H, dd,
increasing amounts of MeOH in CHCl3 [CHCl3 (3L), and J=9.3, 7.7Hz, H-2′), 1.27 (3H, d, J=6.2Hz, H -3, pro-R), 1.23
3
13
CHCl –MeOH (49:1, 3L), (24:1, 3L), (23:2, 3L), (9:1, 3L), (3H, d, J=6.2Hz, H -1, pro-S); C-NMR (CD OD, 150MHz):
3
3
3
(7:1, 3L), (17:3, 3L), (4:1, 3L), (3:1, 3L), (7:3, 3L) and Table 1; HR-ESI-MS (positive-ion mode): m/z: 391.1368
+
(
3:2, 3L)], 500-mL fractions being collected. The residue [M+Na] (Calcd C H O Na: 391.1363).
18 24 8
(4.25g) in fractions 26–33 was subjected to open ODS CC to
Zanthoionic Acid (2) Colorless amorphous powder,
2
5
−1
give a residue (179mg) in fractions 73–82 which was purified [α] –3.4 (c=0.50, MeOH); IR ν
by HPLC (H O–MeOH, 3:1) to give 6.9mg of 9 and 10.0mg 2931, 1713, 1701, 1650; H-NMR (CD OD, 600MHz) δ: 5.74
(film) cm : 3332, 2959,
D
max
1
2
3

7
58
Chem. Pharm. Bull.
Vol. 65, No. 8 (2017)
1
(
1H, dd, J=15.2, 5.4Hz, H-8), 5.56 (1H, dd, J=15.2, 9.8Hz, 2924, 2873, 1604, 1508, 1456, 1074, 1029; H-NMR (CD OD,
3
H-7), 4.43 (1H, d, J=5.4Hz, H-9), 2.42 (1H, d, J=13.2Hz, 600MHz) δ: 5.47 (1H, ddd, J=15.5, 9.7, 0.8Hz, H-7), 5.39 (1H,
H-2ax), 2.30 (1H, ddd, J=13.7, 4.4, 2.3Hz, H-4eq), 2.15 (1H, dd, J=15.5, 6.2Hz, H-8), 4.39 (1H, d, J=7.8Hz, H-1′), 4.11
dd, J=13.7, 11.9Hz, H-4ax), 1.96 (1H, dd, J=10.7, 9.8Hz, H-6), (1H, m, H-9), 3.91 (1H, m, H-3), 3.87 (1H, dd, J=11.8, 1.5Hz,
1
.90 (1H, overlapped, H-5), 2.02 (1H, dd, J=13.2, 2.3Hz, H-6′a), 3.68 (1H, dd, J=11.8, 4.5 H-6′b), 3.50 (1H, dd, J=11.1,
H-2eq), 0.99 (3H, s, H -12), 0.94 (3H, d, J=6.2Hz, H -13), 4.5Hz, H-10a), 3.43 (1H, dd, J=11.1, 7.4Hz, H-10b), 3.27 (3H,
3
3
1
3
0
.82 (3H, s, H -11); C-NMR (CD OD, 150MHz): Table overlapped, H-3′, 4′ and 5′), 3.23 (1H, dd, J=9.2, 7.8Hz, H-2′),
3 3
5
−
1
; CD [θ] (nm): +307 (298) (c=4.17×10 M, MeOH); HR- 3.06 (1H, dd, J=10.7, 3.3Hz, H-4), 2.06 (1H, dd, J=14.5,
+
ESI-MS (positive-ion mode): m/z: 263.1254 [M+Na] (Calcd 3.3Hz, H-2eq), 1.80 (1H, m, H-5), 1.46 (1H, dd, J=10.8,
C H O Na: 263.1254).
9.7Hz, H-6), 1.39 (1H, dd, J=14.5, 2.8Hz, H-2ax), 1.01 (3H,
1
3
20
4
Zanthoionoside
A
(3) Colorless amorphous powder, s, H -12), 0.91 (3H, d, J=6.4Hz, H -13), 0.82 (3H, s, H -11);
3
3
3
2
D
5
−1
13
[
1
6
α] –0.54 (c=0.75); IR ν
(film) cm : 3305, 2931, 2880,
C-NMR (CD OD, 150MHz): Table 1; HR-ESI-MS (positive-
max
3
1
+
457, 1368, 1267, 1228, 1070, 1032; H-NMR (CD OD, ion mode): m/z: 429.2093 [M+Na] (Calcd C H O Na:
3
19 34
9
00MHz) δ: 5.50 (1H, dd, J=15.6, 6.4Hz, H-8), 5.40 (1H, dd, 429.2095).
J=15.6, 9.9Hz, H-7), 4.36 (1H, quintet, J=6.4Hz, H-9), 4.35
Zanthoionoside
E
(7) Colorless amorphous powder,
2
1
−1
(1H, d, J=7.7Hz, H-1′), 3.91 (1H, m, H-3), 3.84 (1H, over- [α] –15.2 (c=0.65, MeOH); IR ν
(film) cm : 3335, 2964,
D
max
1
lapped, H-6′a), 3.68 (1H, dd, J=12.1, 4.8Hz, H-6′b), 3.17 (1H, 2932, 2873, 1684, 1508, 1457, 1075, 1033; H-NMR (CD OD,
3
overlapped, H-2′), 3.35–3.23 (3H, overlapped, H-3′, 4′ and 5′), 600MHz) δ: 5.51 (1H, ddd, J=15.5, 10.1, 1.0Hz, H-7), 5.38
3.06 (1H, dd, J=10.4, 3.3Hz, H-4), 1.77 (1H, m, H-5), 1.75 (1H, (1H, dd, J=15.5, 6.3Hz, H-8), 4.32 (1H, m, H-9), 4.30 (1H,
dd, J=14.6, 3.3Hz, H-2eq), 1.47 (1H, dd, J=10.8, 9.9Hz, H-6), d, J=7.7Hz, H-1′), 3.93 (1H, dd, J=10.3, 3.1Hz, H-10a),
.41 (1H, dd, J=14.6, 2.9Hz, H-2ax), 1.29 (3H, d, J=6.4Hz, 3.91 (1H, ddd, J=3.3, 3.1, 3.1Hz, H-3), 3.86 (1H, dd, J=11.9,
H -10), 1.04 (3H, s, H -11), 0.89 (3H, d, J=6.4Hz, H -13), 0.83 1.7Hz, H-6′a), 3.66 (1H, dd, J=11.9, 5.6Hz, H-6′b), 3.41 (1H,
1
3
3
3
13
(
3H, s, H -12); C-NMR (CD OD, 150MHz): Table 1; HR- dd, J=10.3, 8.8Hz, H-10b), 3.39–3.28 (3H, overlapped, H-3′,
3
3
+
ESI-MS (positive-ion mode): m/z: 413.2153 [M+Na] (Calcd 4′ and 5′), 3.22 (1H, dd, J=9.3, 7.7Hz, H-2′), 3.06 (1H, dd,
C H O Na: 413.2146).
J=10.4, 3.3Hz, H-4), 1.78 (1H, m, H-5), 1.74 (1H, dd, J=14.7,
19
34
8
Zanthoionoside
α] –31.4 (c=0.69, MeOH); IR ν
558, 1456, 1368, 1076, 1030; H-NMR (CD OD, 600MHz) J=6.6Hz, H -13), 0.82 (3H, s, H -11); C-NMR (CD OD,
B
(4) Colorless amorphous powder, 3.1Hz, H-2eq), 1.47 (1H, dd, J=10.8, 10.1Hz, H-6), 1.40 (1H,
2
1
−1
[
1
(film) cm : 3362, 2943, dd, J=14.7, 3.1Hz, H-2ax), 1.04 (3H, s, H -12), 0.90 (3H, d,
max 3
13
D
1
3 3 3 3
δ: 5.52 (1H, ddd, J=15.4, 9.9, 0.7Hz, H-7), 5.40 (1H, dd, 150MHz): Table 1; HR-ESI-MS (positive-ion mode): m/z:
+
J=15.4, 6.4Hz, H-8), 4.30 (1H, d, J=7.9Hz, H-1′), 4.11 (1H, 429.2094 [M+Na] (Calcd C H O Na: 429.2095).
19
34
9
dddd, J=7.3, 6.4, 4.5, 0.7Hz, H-9), 4.04 (1H, dddd, J=3.7, 2.8,
.8, 2.2Hz, H-3), 3.85 (1H, dd, J=11.8, 2.4Hz, H-6′a), 3.66 solution of 2 (3.0mg) in 1mL of EtOH was neutralized by the
1H, dd, J=11.8, 5.3Hz, H-6′b), 3.50 (1H, dd, J=11.1, 4.5Hz, addition of 0.1M NaOH using phenolphthalein as an indicator.
p-Bromophenacyl Ester (2a) of Zanthoionic Acid (2) A
2
(
H-10a), 3.43 (1H, dd, J=11.1, 7.3Hz, H-10b), 3.36 (1H, dd, To the neutralized solution was added zanthoionic acid (2)
J=8.8, 8.8Hz, H-3′), 3.26 (1H, overlapped, H-4′), 3.24 (1H, (0.3mg) to make it slightly acidic. p-Bromophenacylbromide
overlapped, H-5′), 3.16 (1H, dd, J=8.8, 7.9Hz, H-2′), 2.00 (1H, (6mg) in 1mL of EtOH was added and the reaction mixture
dddd, J=14.7, 2.8, 2.8, 2.4Hz, H-4eq), 1.89 (1H, m, H-5), 1.82 was kept at 60°C for 12h. The ethanol was evaporated and
(
9
1H, ddd, J=14.5, 2.4, 2.2Hz, H-2eq), 1.39 (1H, dd, J=10.3, the resulting residue was purified by prep. TLC (silica gel,
.9Hz, H-6), 1.37 (1H, dd, J=14.5, 3.7Hz, H-2ax), 1.05 (1H, CHCl ) to give 1.2mg of zanthoionic acid p-bromophenacyl
3
m, H-4ax), 1.03 (3H, s, H -11), 0.84 (3H, s, H -12), 0.79 (3H, d, ester (2a). Zanthoionic acid p-bromophenacyl ester (2a): Col-
J=6.6Hz, H -13); C-NMR (CD OD, 150MHz): Table 1; HR- orless amorphous powder, [α] –26.3 (c=0.08, CHCl ); IR
ESI-MS (positive-ion mode): m/z: 413.2142 [M+Na] (Calcd
C H O Na: 413.2146).
3
3
13
26
3
3
D
3
+
−1
ν
(film) cm : 3380, 2958, 2871, 1760, 1707, 1067, 669;
H-NMR (CDCl , 600MHz) δ: 7.77 (2H, d, J=8.4Hz, H-3′ and
max
1
1
9
34
8
3
Zanthoionoside
C
(5) Colorless amorphous powder, 5′), 7.65 (2H, d, J=8.4Hz, H-2′ and 6′), 5.75 (1H, overlapped,
2
D
1
−1
[
2
α] –3.08 (c=3.41, MeOH); IR ν
(film) cm : 3381, 2967, H-8), 5.73 (1H, overlapped, H-7), 5.43 (1H, d, J=16.2Hz,
max
1
936, 2878, 1652, 1508, 1456, 1072, 1011; H-NMR (CD OD, H-8′a), 5.38 (1H, d, J=16.2Hz, H-8′b), 4.90 (1H, d, J=9.3Hz,
3
6
00MHz) δ: 5.47 (1H, dd, J=15.6, 9.5Hz, H-7), 5.40 (1H, H-9), 2.40 (1H, ddd, J=13.7, 3.2, 2.5Hz, H-4a), 2.29 (1H, d,
dd, J=15.6, 6.2Hz, H-8), 4.36 (1H, d, J=7.8Hz, H-1′), 4.17 J=13.4Hz, H-2a), 2.12 (1H, dd, J=13.4, 2.5Hz, H-2b), 2.04
1H, ddd, J=3.1, 3.1, 3.0Hz, H-3), 4.12 (1H, ddd, J=7.5, 6.2, (1H, dd, J=13.7, 12.7Hz, H-4b), 1.97 (2H, overlapped, H-5
.3Hz, H-9), 3.81 (1H, dd, J=12.0, 2.0Hz, H-6′a), 3.69 (1H, and 6), 0.98 (3H, s, H -12), 0.97 (3H, d, J=5.8Hz, H -13), 0.85
(
4
3
3
13
dd, J=12.0, 5.0Hz, H-6′b), 3.51 (1H, dd, J=11.1, 4.3Hz, (3H, s, H -11); C-NMR (CDCl , 150MHz): 210.7 (C-3), 190.0
3
3
H-10a), 3.44 (1H, dd, J=11.1, 7.5Hz, H-10b), 3.26 (1H, dd, (C-7′), 173.1 (C-10), 133.8 (C-8), 132.6 (C-4′), 132.4 (C-2′ and
J=8.8, 7.8Hz, H-2′), 3.37–3.35 (3H, overlapped, H-3′, 4′ and 6′), 129.5 (C-7 and 1′), 129.2 (C-3′ and 5′), 71.3 (C-9), 66.7
5
′), 3.18 (1H, dd, J=10.7, 3.1Hz, H-4), 1.93 (1H, m, H-5), 1.74 (C-8′), 57.0 (C-6), 56.0 (C-2), 49.3 (C-4), 38.5 (C-1), 33.4 (C-5),
(
1H, dd, J=14.7, 3.1Hz, H-2eq), 1.51 (1H, dd, J=10.9, 9.5Hz, 30.5 (C-12), 21.3 (C-11), 21.1 (C-13); HR-ESI-MS (positive-
H-6), 1.43 (1H, dd, J=14.7, 3.0Hz, H-2ax), 1.04 (3H, s, H -12), ion mode): m/z: 459.0774 [M+Na]+ (Calcd C H O BrNa:
79
5
3
21 25
13
0
.97 (3H, d, J=6.4Hz, H -13), 0.82 (3H, s, H -11); C-NMR 459.0778).
3
3
(CD OD, 150MHz): Table 1; HR-ESI-MS (positive-ion mode):
Preparation of (R)- and (S)-MTPA Esters (2b and c)
of Xanthoionic Acid p-Bromophenacyl Ester (2a)
(6) Colorless amorphous powder, solution of 2a (0.6mg) in 1mL of dehydrated CH Cl was
3
+
m/z: 429.2093 [M+Na] (Calcd C H O Na: 429.2095).
A
19
34
9
Zanthoionoside
D
2
2
2
D
1
−1
[α] +11.7 (c=4.84, MeOH); IR ν
(film) cm : 3364, 2957, reacted with (R)-MTPA (36mg) in the presence of 1-ethyl-3-
max

Vol. 65, No. 8 (2017)
Chem. Pharm. Bull.
759
(3-dimethylaminopropyl)carbodiimide (EDC) (39mg) and of 2b and c, 3b (0.1mg) and c (0.4mg) were prepared from
4,4-dimethylaminipyridine (DMAP) (20mg), and then the 3a (0.8mg each) using respective amounts of (R)- and (S)-
mixture was occasionally stirred at 25°C 12h. After the ad- MTPA (28 and 26mg), EDC (30 and 28mg) and 4,4-DMAP
dition of 1mL of CH Cl , the solution was washed with H O (19 and 22mg). (R)-MTPA diester (3b): Amorphous powder,
2
2
2
1
(
1mL), 4N HCl (1mL), NaHCO -saturated H O, and then brine
H-NMR (CDCl , 600MHz) δ: 7.43–7.33 (10 H, overlapped,
3
2
3
(
1mL), successively. The organic layer was dried over Na SO4 aromatic protons), 5.54 (1H, quintet, J=6.5Hz, H-9), 5.50
2
and then evaporated under reduced pressure. The residue was (2H, overlapped, H-7 and 8), 4.74 (1H, dd, J=11.4, 2.8Hz,
purified by prep. TLC [silica gel, CHCl –(CH ) CO (20:1)] H-4), 4.14 (1H, brs, H-3), 3.57 (6H, s, –OCH ×2), 2.09
3
3 2
3
to furnish an (R)-MTPA ester, 2b (0.3mg). Through a similar (1H, m, H-5), 1.81 (1H, dd, J=15.1, 2.8Hz, H-2a), 1.52 (1H,
procedure, the (S)-MTPA ester (2c) (0.2mg) was prepared overlapped, H-6), 1.49 (1H, overlapped, H-2b), 1.35 (3H, d,
from 2a (0.6mg) using (S)-MTPA (44mg), EDC (36mg) and J=6.5Hz, H -10), 1.02 (3H, s, H -11), 0.79 (3H, s, H -12), 0.67
3
3
3
4
-DMAP (20mg). Xanthoionic acid p-bromophenacyl ester (3H, d, J=6.5Hz, H -13); HR-ESI-MS (positive-ion mode):
3
1
+
(
R)-MTPA ester (2b): Colorless amorphous powder; H-NMR m/z: 683.2414 [M+Na] (Calcd C H O F Na: 683.2414). (S)-
33 38 7 6
1
(
(
CDCl , 600MHz) δ: 7.76 (2H, d, J=8.4Hz, H-3′ and 5′), 7.65 MTPA diester (3c): Amorphous powder, H-NMR (CDCl3,
3
2H, d, J=8.4Hz, H-2′ and 6′), 7.41–7.39 (5H, overlapped, 600MHz) δ: 7.44–7.39 (10H, overlapped, aromatic protons),
aromatic protons), 5.78 (2H, overlapped, H-7 and 8), 5.65 (1H, 5.54 (1H, quintet, J=6.5Hz, H-9), 5.41 (2H, overlapped, H-7
d, J=6.3Hz, H-9), 5.50 (1H, d, J=16.2Hz, H-8′a), 5.30 (1H, d, and -8), 4.69 (1H, dd, J=11.2, 3.0Hz, H-4), 4.04 (1H, brs,
J=16.2Hz, H-8′b), 3.57 (3H, s, –OCH ), 2.40 (1H, ddd, J=13.7, H-3), 3.57 (6H, s, –OCH ×2), 2.05 (1H, m, H-5), 1.78 (1H, dd,
3
3
3.1, 2.5Hz, H-4a), 2.29 (1H, d, J=13.5Hz, H-2a), 2.11 (1H, dd, J=14.9, 3.3Hz, H-2a), 1.56 (1H, overlapped, H-6), 1.44 (1H,
J=13.5, 2.5Hz, H-2b), 2.04 (1H, overlapped, H-4b), 1.94 (1H, overlapped, H-2b), 1.39 (3H, d, J=6.5Hz, H -10), 0.95 (3H, s,
3
m, H-5), 1.93 (1H, m, H-6), 0.96 (3H, s, H -12), 0.88 (3H, d, H -11), 0.75 (3H, d, J=6.5Hz, H -13), 0.74 (3H, s, H -12); HR-
3
3
3
3
+
J=5.9Hz, H -13), 0.79 (3H, s, H -11); HR-ESI-MS (positive- ESI-MS (positive-ion mode): m/z: 683.2416 [M+Na] (Calcd
3
3
+
79
7
ion mode): m/z: 675.1172 [M+Na] (Calcd C H O BrF Na: C H O F Na: 683.2414).
31
32
3
33 38
7 6
6
75.1176). Xanthoionic acid p-bromophenacyl ester (S)-MTPA
Enzymatic Hydrolysis of Zanthoionoside B (4) Zan-
ester (2c): Colorless amorphous powder; H-NMR (CDCl3, thoionoside B (4) (6.4mg) was hydrolyzed in a similar manner
00MHz) δ: 7.74 (2H, d, J=8.6Hz, H-3′ and 5′), 7.64 (2H, to zanthoionoside A (3) and the hydrolyzate was purified by
1
6
d, J=8.6Hz, H-2′ and 6′), 7.43–7.38 (5H, overlapped, aro- silica gel CC (silica gel 70g, Φ=3cm, L=20cm). Aglycone
matic protons), 5.86 (2H, overlapped, H-7 and -8), 5.68 (1H, (4a) (3.0mg) was recovered in fractions 84–91 and glucose
d, J=6.8Hz, H-9), 5.44 (1H, d, J=16.1Hz, H-8′a), 5.28 (1H, d, was recovered in fractions 121–140. Aglycone (4a): color-
2
1
−1
J=16.1Hz, H-8′b), 3.57 (3H, s, –OCH ), 2.41 (1H, ddd, J=13.7, less syrup, [α] –11.0 (c=0.28, MeOH); IR ν (film) cm :
3
D
max
1
3
.6, 2.4Hz, H-4a), 2.29 (1H, d, J=13.5Hz, H-2a), 2.13 (1H, dd, 3360, 2948, 1622, 1557, 1455, 1365, 1073; H-NMR (CDCl ,
3
J=13.5, 2.4Hz, H-2b), 2.04 (1H, overlapped, H-4b), 1.98 (2H, 600MHz) δ: 5.51 (1H, dd, J=15.5, 9.6Hz, H-7), 5.40 (1H, dd,
m, H-5 and -6), 0.98 (3H, s, H -12), 0.93 (3H, d, J=5.8Hz, J=15.5, 6.3Hz, H-8), 4.11 (1H, m, H-9), 4.03 (1H, dddd, J=3.1,
3
H -13), 0.83 (3H, s, H -11); HR-ESI-MS (positive-ion mode): 3.1, 3.0, 2.8Hz, H-3), 3.49 (1H, dd, J=11.1, 4.6Hz, H-10a),
3
3
+
79
7
m/z: 675.1171 [M+Na] (Calcd C H O BrF Na: 675.1176).
3.43 (1H, dd, J=11.1, 7.3Hz, H-10b), 1.82 (1H, m, H-5), 1.81
31
32
3
Enzymatic Hydrolysis of Zanthoionoside A (3) Zan- (1H, m, H-2a), 1.65 (1H, dt, J=14.0, 2.8Hz, H-2b), 1.38 (1H,
thoionoside A (3) (5.6mg) was dissolved in 1mL of 20mM dd, J=10.4, 9.6Hz, H-6), 1.35 (1H, dd, J=14.6, 3.1Hz, H-4a),
acetate buffer and then 15mg of β-glucosidase was added. The 1.19 (1H, ddd, J=14.0, 11.9, 3.1Hz, H-4b), 1.05 (3H, s, H -11),
3
13
mixture was incubated for 18h at 37°C. The reaction mix- 0.84 (3H, s, H -12), 0.79 (3H, d, J=6.5Hz, H -13); C-NMR
3
3
ture was concentrated and then subjected to silica gel (50g, (CDCl , 150MHz) δ: 134.5 (C-7), 132.9 (C-8), 74.5 (C-9),
3
Φ=2cm, L=36cm) CC with CHCl (100mL), CHCl –MeOH 68.2 (C-3), 67.7 (C-10), 59.7 (C-6), 47.4 (C-2), 42.8 (C-4), 34.3
3
3
[
3
19:1 (100mL), 9:1 (100mL), 17:3 (100mL), 4:1 (100mL), (C-1), 32.8 (C-12), 27.2 (C-6), 23.9 (C-11), 21.7 (C-13); HR-
:1 (100mL), 7:3 (100mL), 13:7 (100mL) and 3:2 (100mL)] ESI-MS (positive-ion mode): m/z: 251.1619 [M+Na] (Calcd
+
and MeOH (100mL), fractions of 5g being collected. The C H O Na: 251.1618).
13
24
3
aglycone (3a) (1.9mg) was obtained in fractions 116–127 and
Preparation of Pivaloyl Ester (4b) from 4a The aglycone
glucose was recovered in fractions 181–200. Aglycone (3a): (4a) (3.0mg) was dissolved in 1mL of pyridine and 27.5µL
2
4
Colorless syrup, [α] +33.1 (c=0.13, CHCl ); IR ν (film) of pivaloyl chloride was added. The reaction mixture was
max
D
3
−1
1
cm : 3309, 2928, 2861, 1512, 1456, 1272, 1228; H-NMR stirred for 2h at 25°C. To the reaction mixture, 1mL of H O
2
(
5
CDCl , 600MHz) δ: 5.48 (1H, dd, J=15.4, 6.5Hz, H-8), was added, followed by extraction with 2mL of CHCl three
3
3
.37 (1H, ddd, J=15.4, 9.8, 0.8Hz, H-7), 4.32 (1H, quintet, times. The residue from the dried (Na SO ) organic layer was
2 4
J=6.5Hz, H-9), 4.00 (1H, ddd, J=3.4, 3.2, 3.2Hz, H-3), 3.15 purified by prep. TLC [silica gel, CHCl –MeOH (9:1)] to give
3
(
1H, dd, J=10.9, 9.8Hz, H-6), 1.81 (1H, dd, J=15.0, 3.2Hz, 2.2mg of pivaloyl ester (4b). Pivaloyl ester (4b): colorless
21
−1
H-2a), 1.76 (1H, m, H-5), 1.44 (1H, dd, J=10.9, 9.8Hz, H-6), syrup, [α] +5.33 (c=0.15, CHCl ); IR ν
1
s, H -13), 0.82 (3H, s, H -12); C-NMR (CDCl , 150MHz) 600MHz) δ: 5.58 (1H, dd, J=15.5, 9.8Hz, H-7), 5.42 (1H, dd,
(film) cm : 3397,
D
3
max
1
.28 (3H, d, J=6.4Hz, H -10), 1.04 (3H, s, H -11), 0.91 (3H, 2961, 1714, 1650, 1554, 1457, 1364, 1162; H-NMR (CDCl ,
3
3
3
13
3
3
3
δ: 137.2 (C-8), 130.1 (C-7), 77.3 (C-4), 70.1 (C-3), 68.9 (C-9), J=15.5, 6.5Hz, H-8), 4.38 (1H, m, H-9), 4.14 (1H, dd, J=11.4,
6.3 (C-6), 44.8 (C-2), 34.3 (C-1), 33.0 (C-5), 31.8 (C-12), 23.7 3.8Hz, H-10a), 4.11 (1H, m, H-3), 4.03 (1H, dd, J=11.4, 7.3Hz,
5
(
C-10), 23.4 (C-12), 16.5 (C-13); HR-ESI-MS (positive-ion H-10b), 1.88 (1H, m, H-5), 1.82 (1H, overlapped, H-2a), 1.63
+
mode): m/z: 251.1619 [M+Na] (Calcd C H O Na: 251.1618).
(1H, ddd, J=14.4, 2.7, 2.7Hz, H-4a), 1.40 (1H, overlapped,
13
24
3
Preparation of (R)- and (S)-MTPA Diesters (3b and c) -6), 1.38 (1H, overlapped, H-2b), 1.22 (9H, s, H -3′, -4′ and
3
of 3a In a similar manner to that used for the preparation 5′), 1.20 (1H, overlapped, H-4b), 1.06 (3H, s, H -11), 0.83 (3H,
3

7
60
Chem. Pharm. Bull.
Vol. 65, No. 8 (2017)
1
3
s, H -12), 0.79 (3H, d, J=6.5Hz, H -13); C-NMR (CDCl3, as that used for 4a, 1.6mg of pivaloyl ester (5b) was ob-
3
3
2
3
1
50MHz) δ: 178.7 (C-1′), 134.4 (C-7), 130.4 (C-8), 71.0 (C-9), tained. Pivaloyl ester (5b): colorless syrup: [α] +19.9 (c=0.10,
D
1
−
6
3
2
8.4 (C-10), 67.6 (C-3), 58.3 (C-6), 46.7 (C-2), 42.1 (C-4), MeOH); IR νmax (film) cm : 3309, 2977, 2871, 1733, 1653,
1
8.9 (C-2′), 33.3 (C-1), 32.2 (C-12), 27.2 (C-3′, -4′ and -5′), 1557, 1508, 1107; H-NMR (CDCl , 600MHz) δ: 5.55 (1H,
5.9 (C-5), 23.6 (C-11), 21.2 (C-13); HR-ESI-MS (positive-ion ddd, J=15.5, 10.0, 1.1Hz, H-7), 5.41 (1H, dd, J=15.5, 6.2Hz,
3
+
mode): m/z: 335.2195 [M+Na] (Calcd C H O Na: 335.2198). H-8), 4.39 (1H, m, H-9), 4.15 (1H, dd, J=11.4, 3.7Hz, H-10a),
18
32
4
Preparation of (R)- and (S)-MTPA Diesters (4c and 4.03 (1H, dd, J=11.4, 7.2Hz, H-10b), 4.00 (1H, ddd, J=3.3,
d) of Pivaloyl Ester (4b) In similar preparations as those 3.2, 3.2Hz, H-3), 3.15 (1H, dd, J=10.3, 3.3Hz, H-4), 1.82 (1H,
used for 2b and c, 4c (0.2mg) and d (0.2mg) were prepared dd, J=14.9, 3.2Hz, H-2a), 1.77 (1H, m, H-5), 1.48 (1H, dd,
from 4b (1.1mg each) using respective amounts of (R)- and J=10.9, 10.0Hz, H-6), 1.41 (1H, dd, J=14.9, 3.2Hz, H-2b),
(
4
S)-MTPA (20mg and 21mg), EDC (39mg and 35mg) and 1.23 (9H, s, H -3′, -4′ and -5′), 1.05 (3H, s, H -11), 0.92 (3H,
3
3
13
,4-DMAP (20mg and 22mg). (R)-MTPA diester of 4b (4c): d, J=6.5Hz, H -13), 0.82 (3H, s, H -12); C-NMR (CDCl3,
3 3
1
colorless syrup; H-NMR (CDCl , 600MHz) δ: 7.43–7.33 150MHz) δ: 178.7 (C-1′), 133.4 (C-7), 130.8 (C-8), 77.2 (C-4),
3
(
3
10H, overlapped, aromatic protons); 5.64 (1H, ddd, J=7.7, 7.7, 70.9 (C-9), 70.0 (C-3), 68.4 (C-10), 56.5 (C-6), 44.7 (C-2), 38.9
.2Hz, H-9), 5.56 (1H, dd, J=15.5, 9.5Hz, H-7), 5.38 (1H, dd, (C-2′), 32.9 (C-1), 32.7 (C-5), 31.8 (C-12), 27.2 (C-3′, -4′ and
J=15.5, 7.7Hz, H-8), 5.33 (1H, m, H-3), 4.24 (1H, dd, J=12.1, -5′), 23.4 (C-11), 16.6 (C-13); HR-ESI-MS (positive-ion mode):
+
3
.2Hz, H-10a), 4.06 (1H, dd, J=12.1, 7.7Hz, H-10b), 3.55 (6H, m/z: 351.2144 [M+Na] (Calcd C H O Na: 351.2141).
18
32
5
s, –OCH ×2), 1.86 (1H, dddd, J=14.0, 2.6, 2.6, 2.6Hz, H-4a),
1
(
1
Preparation of (R)- and (S)-MTPA Diesters (5c and d)
3
.71 (1H, dd, J=15.0, 2.6Hz, H-2a), 1.56 (1H, m, H-5), 1.36 from Pivaloyl Ester (5b) In a similar manner to that used
2H, overlapped, H-2b and -6), 1.24 (1H, overlapped, H-4b), for the preparation of 2b and c, 5c (0.1mg) and d (0.1mg)
.13 (9H, s, H -3′, -4′ and -5′), 0.75 (3H, H -11), 0.71 (3H, s, were prepared from 5b (0.8mg each) using respective amounts
3
3
H -12), 0.65 (3H, d, J=6.1Hz, H -13); HR-ESI-MS (positive- of (R)- and (S)-MTPA (24mg and 36mg), EDC (30mg and
3
3
+
ion mode): m/z: 767.2983 [M+Na] (Calcd C H O F Na: 39mg) and 4,4-DMAP (18mg and 25mg). (R)-MTPA dies-
67.2989). (S)-MTPA diester of 4b (4d): colorless syrup; ter of 5b (5c): colorless syrup; H-NMR (CDCl , 600MHz)
H-NMR (CDCl , 600MHz) δ: 7.44–7.31 (10H, overlapped, δ: 7.36–7.27 (10 H, overlapped, aromatic protons), 5.59 (1H,
38
46
8 6
1
7
3
1
3
aromatic protons); 5.68 (1H, ddd, J=7.7, 7.7, 3.0Hz, H-9), 5.48 ddd, J=7.7, 7.7, 3.5Hz, H-9), 5.57 (1H, dd, J=15.6, 10.1Hz,
(
1H, dd, J=15.4, 9.7Hz, H-7), 5.32 (1H, m, H-3), 5.30 (1H, dd, H-7), 5.36 (1H, dd, J=15.6, 7.7Hz, H-8), 4.66 (1H, dd, J=11.1,
J=15.4, 7.7Hz, H-8), 4.33 (1H, dd, J=12.4, 3.0Hz, H-10a), 3.1Hz, H-4), 4.19 (1H, dd, J=12.1, 3.5Hz, H-10a), 4.07 (1H,
.06 (1H, dd, J=12.4, 7.7Hz, H-10b), 3.56 (6H, s, –OCH ×2), m, H-3), 4.01 (1H, dd, J=12.1, 7.7Hz, H-10b), 3.58 (6H, brs,
4
3
1
.93 (1H, dddd, J=14.5, 3.2, 3.2, 3.2Hz, H-4a), 1.68 (1H, over- –OCH ×2), 1.77 (1H, m, H-5), 1.74 (1H, dd, J=15.0, 3.3Hz,
3
lapped, H-2a), 1.56 (1H, m, H-5), 1.37 (2H, overlapped, H-2b H-2a), 1.53 (1H, overlapped, H-6), 1.45 (1H, overlapped,
and 6), 1.24 (1H, overlapped, H-4b), 1.17 (9H, s, H -3′, -4′ and H-2b), 1.06 (9H, s, H -3′, -4′ and -5′), 0.95 (3H, s, H -11),
3
3
3
-
5′), 0.72 (3H, d, J=6.5Hz, H -13), 0.66 (3H, s, H -11), 0.52 0.71 (3H, s, H -12), 0.58 (3H, d, J=6.5Hz, H -13); HR-ESI-
3
3
3
3
+
(3H, s, H -12); HR-ESI-MS (positive-ion mode): m/z: 767.2988 MS (positive-ion mode): m/z: 783.2945 [M+Na] (Calcd
3
+
[
M+Na] (Calcd C H O F Na: 767.2989).
C H O F Na: 783.2943). (S)-MTPA diester of 5b (5d): col-
38 46 9 6
38
46
8
6
1
Enzymatic Hydrolysis of Zanthoionoside C (5) Zan- orless syrup; H-NMR (CDCl , 600MHz) δ: 7.36–7.21 (10H,
3
thoionoside C (5) (6.8mg) was hydrolyzed in a similar man- overlapped, aromatic protons), 5.64 (1H, ddd, J=7.5, 7.5,
ner to zanthoionoside A (3) and the hydrolyzate was purified 3.2Hz, H-9), 5.46 (1H, dd, J=15.9, 9.8Hz, H-7), 5.27 (1H, dd,
by silica gel CC {silica gel 50g, Φ=3cm, L=15.3cm with J=15.9, 7.5Hz, H-8), 4.61 (1H, dd, J=11.4, 3.4Hz, H-4), 4.28
CHCl3 (300mL) and CHCl –MeOH [19:1 (300mL), 9:1 (1H, dd, J=12.2, 3.2Hz, H-10a), 4.01 (1H, dd, J=12.2, 7.5Hz,
3
(
7
300mL), 17:3 (300mL), 4:1 (300mL), 3:1 (300mL) and H-10b), 3.96 (1H, m, H-3), 3.58 (6H, brs, –OCH ×2), 1.77 (1H,
:3 (300mL)], fractions of 10g being collected}. Aglycone m. H-5), 1.72 (1H, dd, J=14.5, 3.2Hz, H-2a), 1.55 (1H, over-
3
(5a) (2.0mg) was recovered in fractions 91–120 and glu- lapped, H-6), 1.45 (1H, overlapped, H-2b), 1.01 (9H, s, H -3′,
3
cose in fractions 151–180. Aglycone (5a): colorless syrup; -4′ and -5′), 0.87 (3H, s, H -11), 0.66 (3H, s, H -12), 0.66 (3H,
3
3
2
2
−1
[
2
5
6
α] +25.4 (c=0.13, MeOH); IR ν
(film) cm : 3395, 2961, d, J=6.5Hz, H -13); HR-ESI-MS (positive-ion mode): m/z:
D
max
3
1
+
925, 1654, 1558, 1508, 1456; H-NMR (CDCl , 600MHz) δ: 783.2946 [M+Na] (Calcd C H O F Na: 783.2943).
3
38 46
9 6
.52 (1H, ddd, J=15.5, 9.9, 0.9Hz, H-7), 5.42 (1H, dd, J=15.5,
.3Hz, H-8), 4.26 (1H, m, H-9), 4.00 (1H, ddd, J=3.2, 3.1, (7) Zanthoionosides D (6) (4.2mg) and E (7) (3.4mg) were
.1Hz, H-3), 3.67 (1H, dd, J=11.1, 3.2Hz, H-10a), 3.51 (1H, enzymatically hydrolyzed with 45mg of β-glucosidase in the
dd, J=11.1, 7.6Hz, H-10b), 3.15 (1H, dd, J=10.8, 3.1Hz, H-4), manner described for 5. Using a similar procedure as for
.82 (1H, dd, J=14.8, 3.1Hz, H-2a), 1.77 (1H, m, H-5), 1.47 zanthoionosides C (5), aglycones, 6a (1.4mg) and 7a (1.3mg)
1H, dd, J=10.8, 9.9Hz, H-6), 1.41 (1H, dd, J=14.8, 3.1Hz, were obtained in fractions 99–115 and 91–111, respectively
H-2b), 1.05 (3H, s, H -11), 0.91 (3H, d, J=6.5Hz, H -13), 0.82 and glucose was obtained in fractions 181–210. The aglycones,
Enzymatic Hydrolysis of Zanthoionosides D (6) and E
3
1
(
3
3
13
(
1
3H, s, H -12); C-NMR (CDCl , 150MHz) δ: 133.4 (C-7), 6a and 7a were identical with that (5a) of zanthoionoside C
31.2 (C-8), 77.1 (C-4), 73.1 (C-9), 70.0 (C-3), 66.8 (C-10), ([α]D, H- and C-NMR, and HR-ESI-MS).
3
3
1
13
56.5 (C-6), 44.7 (C-2), 32.9 (C-1), 32.7 (C-5), 31.8 (C-13), 23.4
Sugar Analysis of Compound 1 and Zanthoionosides
(
2
C-11), 16.5 (C-13); HR-ESI-MS (positive-ion mode): m/z: A–E (3–7) Compound 1 (0.8mg) was hydrolyzed with 1M
+
67.1565 [M+Na] (Calcd C H O Na: 267.1567).
HCl (0.1mL) at 80°C for 2h. The reaction mixture was parti-
13
24
4
Preparation of Pivaloyl Ester (5b) from 5a The agly- tioned with an equal amount of EtOAc (0.1mL), and the water
cone (5a) (2.0mg) was dissolved in 1mL of pyridine and layer was analyzed by HPLC. Glucose fractions obtained
5µL of pivaloyl chloride was added. In a similar work up from enzymatic hydrolysis of zanthoionosides A–E (3–7)
6

Vol. 65, No. 8 (2017)
Chem. Pharm. Bull.
761
3
)
Chen J.-J., Chung C.-Y., Hwang T.-L., Chen J.-F., J. Nat. Prod., 72,
07–111 (2009).
Chou S.-T., Chan H.-H., Peng H.-Y., Liou M.-J., Wu T.-S., Phyto-
medicine, 18, 344–348 (2011).
http://www2.odn.ne.jp/had26900/wild_plant/wp2_f/karasuzanshou.
htm.
Otsuka H., Takeda Ya., Yamasaki K., Takeda Y., Planta Med., 58,
were also analyzed by HPLC with a chiral detector (JASCO
1
OR-2090plus) on an amino column [Asahipak NH P-50 4E,
2
4)
CH CN–H O (3:1), 1mL/min]. The hydrolyzate and glucose
3
2
in the sugar fractions gave a peak for D-glucose at 9.5min
with a positive optical rotation sign. Each peak was identified
by co-chromatography with an authentic sample.
5
6
)
)
373–375 (1992).
Acknowledgments The authors are grateful for access to
the superconducting NMR instrument (Brucker Avance III
7) Liu C., Zou K., Guo Z., Zhao Y., Cheng F., Zhang H., China J. Chi-
nese Materia Med., 35, 1432–1434 (2010).
6
00) at the Analytical Center of Molecular Medicine of the
8) Hisamoto M., Kikuzaki H., Nakatani N., J. Agric. Food Chem., 52,
4
45–450 (2004).
Osorio C., Duque C., Fujimoto Y., J. Agric. Food Chem., 47, 1641–
645 (1999).
0) Watanabe N., Nakajima R., Watanabe S., Moon J.-H., Inagaki J.,
Sakata K., Yagi A., Ina K., Phytochemistry, 37, 457–459 (1994).
1) Miyase T., Noguchi H., Chen X.-M., J. Nat. Prod., 62, 993–996
1999).
2) Kasai R., Suzuo M., Asakawa J., Tanaka O., Tetrahedron Lett., 18,
75–178 (1977).
due to the Research Foundation for Pharmaceutical Sciences 13) Moffitt W., Woodward R. B., Moscowitz A., Klyne W., Djerassi C.,
Hiroshima University Faculty of Medicine, and for access to
an Applied Biosystems QSTAR XL system ESI (Nano Spray)-
MS at the Analysis Center of Life Science of the Graduate
School of Biomedical Sciences, Hiroshima University. This
work was supported in part by Grants-in-Aid from the Min-
istry of Education, Culture, Sports, Science and Technology
of Japan, and the Japan Society for the Promotion of Science
9)
1
1
1
(
1
(
Nos. 22590006, 23590130 and 15H04651). Thanks are also
1
and the Takeda Science Foundation for their financial support.
J. Am. Chem. Soc., 83, 4013–4018 (1961).
14) Ohtani I., Kusumi T., Kashman Y., Kakisawa H., J. Am. Chem.
Soc., 113, 4092–4096 (1991).
Conflict of Interest The authors declare no conflict of
interest.
1
5) Muñoz-González C., Sémon E., Martín-Álvarez P. J., Guichard E.,
Moreno-Arribas M. V., Feron G., Pozo-Bayón M. A., Aust. J. Grape
Wine Res., 21, 367–375 (2015).
References
1
6) Camara J. S., Alves M. A., Marques J. C., Food Chem., 101, 475–
84 (2007).
7) Eagles C. F., Wareing P. F., Nature (London), 199, 874–875 (1963).
8) Goda Y., Hoshino K., Akiyama H., Ishikawa T., Abe Y., Nakamura
1
)
Hatusima S., “Flora of the Ryukyus (Including Amami Islands,
Okinawa Islands and Sakishima Archipelago) (Added and Cor-
rected),” Biological Society of Okinawa, Naha, Japan, 1975, pp.
4
1
1
3
52–353.
Sheen W.-S., Tsai I.-L., Teng C.-M., Chen I.-S., Phytochemistry, 36,
13–215 (1994).
T., Otsuka H., Takeda Y., Tanimura A., Toyoda M., Biol. Pharm.
Bull., 22, 1319–1326 (1999).
2
)
2