Microtropins Q–W, ent-Labdane glucosides: Microtropiosides G–I, ursane-type triterpene diglucoside and flavonol glycoside from the leaves of microtropis japonica

Article abstract of DOI:10.1248/cpb.c17-00459

Microtropins Q–W, (2S,3R)-2-ethyl-2,3-dihydroxybutyrate of various glucosides and glucose, as well as three ent-labdane diterpenoid glucosides, named microtropiosides G, H and I, an ursane-type triterpene diglucoside and a flavonoid glycoside were isolated from the MeOH extract of the leaves of Microtropis japonica. The structure of microtropioside A, also isolated from the branches of M. japonica, was elucidated spectroscopically in a previous experiment and was found to possess a rare seven-membered oxyrane ring. Its structure was confirmed by X-ray crystallographic analysis of its pentaacetate.

Full text of DOI:10.1248/cpb.c17-00459

9
30
Chem. Pharm. Bull. 65, 930–939 (2017)
Vol. 65, No. 10
Regular Article
Highlighted Paper selected by Editor-in-Chief
Microtropins Q–W, ent-Labdane Glucosides: Microtropiosides G–I,
Ursane-Type Triterpene Diglucoside and Flavonol Glycoside from the
Leaves of Microtropis japonica
a
a
a
b
a
Saori Terazawa, Yuka Uemura, Yuka Koyama, Susumu Kawakami, Sachiko Sugimoto,
a
,a,b
c
d
Katsuyoshi Matsunami, Hideaki Otsuka,* Takakazu Shinzato, Masatoshi Kawahata, and
Kentaro Yamaguchi
d
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: Subtropical Field Science Center, Faculty of Agriculture, University of the Ryukyus; 1 Sembaru,
d
Nishihara-cho, Nakagami-gun, Okinawa 903–0213, Japan: and Faculty of Pharmaceutical Sciences, Tokushima
Bunri University, Kagawa Campus; 1314 Shido, Sanuki, Kagawa 769–2193, Japan.
Received June 6, 2017; accepted July 31, 2017
Microtropins Q–W, (2S,3R)-2-ethyl-2,3-dihydroxybutyrate of various glucosides and glucose, as well
as three ent-labdane diterpenoid glucosides, named microtropiosides G, H and I, an ursane-type triterpene
diglucoside and a flavonoid glycoside were isolated from the MeOH extract of the leaves of Microtropis ja-
ponica. The structure of microtropioside A, also isolated from the branches of M. japonica, was elucidated
spectroscopically in a previous experiment and was found to possess a rare seven-membered oxyrane ring.
Its structure was confirmed by X-ray crystallographic analysis of its pentaacetate.
Key words Microtropis japonica; Celastraceae; microtropin; microtropioside; ent-labdane glucoside; flavonol
glycoside
Celastraceous plants moved into the limelight, after a potent spectroscopic data with those reported in the literature.
2
1
antileukemic ansa-type macrolide, maytansine, was isolated
Microtropin Q (1), [α]D −20.4, was isolated as an amor-
from an Ethiopian shrub, Maytenus serrata (formerly M. ova- phous powder and its elemental composition was determined
tus). In ongoing research on subtropical resource plants, a to be C H O N by observation of a quasi-molecular ion peak
Celestraceous plant, Microtropis japonica, collected in Oki- ([M+Na] ) in high-resolution (HR)-electrospray ionization
nawa, attracted our attention and its constituents were inves- (ESI)-MS. In the IR spectrum, three typical absorption bands
1,2)
17
27 10
+
−1
tigated. Microtropis japonica HALLIER f. (Celastraceae) is an at 3384, 2225, and 1735cm , were assignable to hydroxy
evergreen tree of about 5m in height, and has a distinct distri- groups, triple bond and ester functional groups, respectively.
13
bution in restricted southern parts of Kanto, Kyushu, Okinawa The C-NMR spectrum exhibited five signals for an aglycone
3)
islands and Taiwan. In previous papers, we have reported the together with six signals for a 2-ethyl-2,3-dihydroxybutyrate
isolation of six new ent-labdane diterpenoid glucosides, named moiety and six for 6-substituted glucopyranoside, which were
5,6)
microtropiosides A–F, from the 1-BuOH-soluble fraction of a seen in microtropins A–P. The absolute structure of glucose
4)
MeOH extract of the leaves of Microtropis japonica and mi- was determined to be D by HPLC analysis of its hydrolyzate
5,6)
crotropins A–P from the branches of the title plant. Further using a chiral detector, and thus the mode of sugar linkage
extensive isolation work on the leaf extract afforded seven was β from the coupling constant of the anomeric proton.
esters of (2″S,3″R)-2″-ethyl-2″,3″-dihydroxybutyric acid, named The absolute configuration of 2-ethyl-2,3-dihydroxybutyrate
microtropins Q–W (1–7) and the acid itself (8) (Fig. 1), three was expected to be the same as that of microtropin A, whose
5
)
new ent-labdane glucosides, named microtropiosides G–I structure was confirmed by X-ray crystallographic analysis.
9–11), a triterpene glucoside (12), and a flavonol glycoside Signals for the aglycone comprised one sp signal (δC 118.7),
(
5
)
(
13) (Fig. 2) along with microtropin D (14), microtropiosides two primary alcohols and a trisubstituted double bond; these
4)
5)
A and F (15, 16), a flavonol glycoside, kaempferol 3-O-β- functionalities were the same as those of microtropin D (14),
D-(2″-O-β-D-glucopyranosyl)galactopyranoside (lilyn) (17)
and 2-ethyl-3-methylmaleimide N-β-D-glucopyranoside (18).
7
,8)
but these were obviously different compound judged from
the C-NMR chemical shifts (Table 1). Since, in the phase-
9)
13
This paper deals with the structural elucidation of these new sensitive (PS) nuclear Overhauser effect (NOE) spectroscopy,
compounds.
significant correlations were observed between H-4 (δH 4.41
and 4.51) and H -5 (δH 4.20), the structure of microtropin Q
2
Results and Discussion
(1) was elucidated to be a Z isomer of microtropin D (14), as
New compounds were isolated by a combination of various shown in Fig. 1.
types of chromatography. and the structures were elucidated Microtropin R (2), [α]D −90.2, was isolated as an amor-
mainly by spectroscopic evidence. The structures of known phous powder and its elemental composition was determined
2
4
5
)
compounds, microtropin D (14), microtropiosides A and F to be C H O by HR-ESI-MS. The NMR spectra indicated
18
32
9
4)
7,8)
(
15, 16), lilyn (17) and 2-ethyl-3-methylmaleimide N-β-D- that 2 was analogous compound to 1 with a different agly-
glucopyranoside (18) were identified by comparison of their cone. The aglycone moiety was revealed to comprise one trip-
9)
*
To whom correspondence should be addressed. e-mail: hotsuka@hiroshima-u.ac.jp; otsuka-h@yasuda-u.ac.jp
©
2017 The Pharmaceutical Society of Japan

Vol. 65, No. 10 (2017)
Chem. Pharm. Bull.
931
Fig. 1. The Structures of New Compounds (1–8) and Compounds Appeared in the Text (14, 18–20)
let methyl, two methylenes, one primary alcohol and a disub- to be R, while since the H-2 and H-3 appeared at the same
1
1
stituted double bond. H– H correlation spectroscopy (COSY) chemical shift (δ 3.67), there was not a clue to determine the
H
exhibited sequential proton signals from H -6 to H -1 and the threo- or erythro-relationship. Therefore, the structure of 3
3
2
geometry of the double bond was determined to be in a Z-form was tentatively elucidated to be 2R,3ξ-2,3-butandiol 2-O-β-D-
from the rather small coupling constant (J=10.9Hz) of olefinic glucopyranose 6′-O-(2″S,3″R)-2″-ethyl-2,″3″-dihydroxybutyric
1
protons in the H-NMR spectrum. Therefore, the structure of acid ester, as shown in Fig. 1. Microtropin G (19), isolated
2
was elucidated to be Z-hex-3-en-1-ol β-D-glucopyranoside from the same plant, also possessed 2,3-butanediol as the
5
)
13
6
′-O-(2″S,3″R)-2″-ethyl-2,″3″-dihydroxybutyric acid ester, as aglycone. Its C-NMR chemical shifts of the aglycone moi-
shown in Fig. 1. ety were obviously different from those of 3 (Table 1), indi-
2
4
Microtropin S (3), [α]D −12.5, was isolated as an amor- cating that their aglycones were stereoisomers each other, as
phous powder and its elemental composition was determined shown in Fig. 1.
to be C H O by HR-ESI-MS. From the spectroscopic data,
microtropin S (3) was found to be an analogous compound to phous powder and its elemental composition was determined
2
5
Microtropin T (4), [α]D −12.9, was isolated as an amor-
16
30 10
the aforementioned compounds with a different aglycone. The to be C H O by HR-ESI-MS. The NMR spectroscopic
19
36 10
aglycone constituted only four carbons, two methyls and two data indicated that 4 was also analogous compound with
13
secondary alcohols, namely, 2,3-butanediol. The C-NMR seven carbons as the aglycone moiety. Signals for two doublet
chemical shifts of meso- and rac-2,3-butanediol were almost methyls and two oxygenated methines were observed in the
identical (C-1 and C-4: δ 18.6 and 18.7 for meso- and rac-2,3- NMR spectra and both of the methyl signals were correlated
C
butanediols, respectively, and C-2 and C-3: δ 72.6 and 72.7, with the oxygenated methine protons in the COSY spectrum.
C
respectively. On β-D-glucopyranosylation to the aglycone of Thus, the aglycone was assigned to be 2,6-heptanediol
the 2-position, chemical shift of C-1 methyl shifted up by 2.5 2-O-β-D-glucopyranoside and the absolute configuration of
10)
or 2.6ppm, whereas that of C-3 was by 0.9 or 1.0ppm. Thus, the 2-postion was deduced by an application of the β-D-
the absolute configuration of the 2-position was determined glucopyranosylation-induced shift trend rule between 2-hexa-

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32
Chem. Pharm. Bull.
Vol. 65, No. 10 (2017)
Fig. 2. Structures of Diterpenoids, Triterpenoid and Flavonol Glycosides
1
3
Table 1.
C-NMR Spectroscopic Data for Microtropins Q–W (1–7), (2″S,3″R)-2″-Ethyl-2,″3″-dihydroxybutyric Acid (8) and Reference Compounds,
a)
a)
Microtropins D (14) and G (19) (100MHz, CD OD)
3
C
1
14
2
3
19
4
5
6
7
C
8
1
2
3
4
5
6
7
8
118.7
119.4
146.3
65.8
116.7
118.2
144.6
68.2
70.6
28.9
16.2
80.9
71.5
18.7
18.5
83.5
72.2
18.7
22.2
78.0
37.9
22.7
40.2
68.3
23.5
154.1
119.9
116.6
152.2
116.6
119.9
132.1
120.4
146.4
147.3
117.2
125.7
39.6
134.6
126.4
21.6
58.6
63.2
14.7
64.5
α
β
1
2
3
4
5
6
1
2
3
4
5
6
′
104.0
74.9
77.7
71.4
75.5
65.0
176.2
82.9
72.9
16.9
29.3
8.4
104.2
74.9
77.7
71.4
75.5
65.0
176.2
82.9
72.8
16.9
29.3
8.4
104.4
75.1
77.9
71.7
75.3
65.3
176.2
82.9
72.9
16.9
29.3
8.2
104.3
75.1
77.9
71.6
75.4
65.3
176.1
82.9
72.8
16.8
29.2
8.4
105.7
75.2
77.7
71.5
75.5
65.2
176.1
82.9
72.8
16.8
29.2
8.4
104.3
75.1
77.9
71.6
75.4
65.3
176.1
82.9
72.8
16.8
29.2
8.4
103.8
75.0
77.8
71.6
75.4
65.4
176.2
82.9
72.8
16.8
29.2
8.3
104.8
74.9
77.4
71.4
75.7
65.2
176.2
83.0
72.9
16.9
29.4
8.4
94.1
98.4
′
′
′
′
′
″
″
″
″
″
″
73.9
76.3
74.8
78.0
72.8
71.9
70.8
75.4
65.4
65.6
176.30
82.94
72.86
16.86
29.31
8.38
176.30
82.99
72.86
16.88
29.30
8.29
1
178.1
82.2
72.6
17.1
29.1
8.3
2
3
4
5
6
a) Data from ref. 5.
nol and the aglycone. On β-D-glucopyranosylation, the methyl cone moiety was a para-substituted benzene [δH 6.70 (2H,
signal shifted up by 1.2ppm, whereas, the methylene carbon at d, J=8.1Hz, H-3 and 5) and 6.95 (2H, d, J=8.1Hz, H-2 and
the 3-position shifted by 2.0ppm, indicating that the 2-position 6)], namely p-hydroquinone. Therefore, the structure of 5
10)
had the S configuration. Therefore, the structure of 4 was was elucidated to be arbutin, 6′-O-(2″S,3″R)-2″-ethyl-2,″3″-
elucidated to be 2S,6ξ-heptanediol 2-O-β-D-glucopyranoside dihydroxybutyric acid ester, as shown in Fig. 1.
2
1
6
′-O-(2″S,3″R)-2″-ethyl-2,″3″-dihydroxybutyric acid ester, as
Microtropin V (6), [α]D −68.9, was isolated as an amor-
shown in Fig. 1.
phous powder and its elemental composition was C H O .
2
0
30 11
2
4
Microtropin U (5), [α]D −26.9, was isolated as an amor- NMR spectroscopic data indicated the presence of three
phous powder and its elemental composition was determined aromatic protons coupled in an ABX system [6.77 (1H, d,
to be C H O . The NMR spectra indicated that the agly- J=8.2Hz, H-5), 6.81 (1H, dd, J=8.2, 2.0Hz, H-6) and 7.01
18
26 10

Vol. 65, No. 10 (2017)
Chem. Pharm. Bull.
933
13
(
1H, d, J=2.0Hz, H-2)], methylene and a primary alcohol. In C-6′ from its C-NMR signals at 65.4 (α) and 65.6 (β) and
the heteronuclear multiple bond connectivity (HMBC), cor- therefore its structure was elucidated to be D-glucose 6′-O-
relation of the anomeric proton (δH 4.74) with the primary (2″S,3″R)-2″-ethyl-2,″3″-dihydroxybutyric acid ester. From the
1
alcohol carbon (δC 64.5) established the linkage position of integrals of the anomeric signals in the H-NMR spectrum,
the sugar moiety. Other HMBC correlations of H-2 and the ratio of α- and β-isomers was calculated to be nearly
H-6 with C-7 revealed the 1,3,4-subsitution of the benzene equal. The single prime mark system was used according to
ring and, therefore, the structure of 6 was elucidated to be previous compounds for the reader’s convenience.
2
4
3
(
,4-dihydroxyphenylethanol 8-O-β-D-glucopyranoside 6′-O-
2″S,3″R)-2″-ethyl-2,″3″-dihydroxybutyric acid ester, as shown was isolated as a colorless syrup and its elemental composi-
in Fig. 1. tion was determined to be C H O by HR-ESI-MS. NMR
(2S,3R)-2-Ethyl-2,3-dihydroxybutyric acid (8), [α] −3.5,
D
6
12
4
2
4
Microtropin W (7), [α]D −90.2, was isolated as an spectra indicated that compound 8 comprised of the same
amorphous powder and its elemental composition was carbon framework as the acid moiety of microtropins and
C H O . The C-NMR spectrum implied the presence of the free acid form was isolated for the first time. Instead
13
12
22
9
a 2″-ethyl-2,″3″-dihydroxybutyric acid segment; however, all of a rearrangement of the ethyl group (C-5 and C-6) of (S)-
the signals appeared as close dual resonances. D-Glucose was 2-hydroxy-2-ethyl-3-oxobutanoic acid (20) to C-3 to proceed
detected by the sugar analysis and two typical anomeric sig- the biosynthesis of isoleucine, the ketone functional group at
nals observed at δH 5.08 (d, J=3.8Hz, H-1′α) on δ 94.1 and C-3 was directly reduced to the secondary alcohol to form 8
C
δH 4.48 (d, J=7.8Hz, H-1′β) on δ 98.4 were those of α- and and needed to be derailed from the leucine biosynthetic route.
C
2
4
β-anomers, respectively. The ester linkage was expected on
Microtropioside G (9), [α]D −12.6, was isolated as an amor-
1
3
1
13
Table 2.
(
C- and H-NMR Spectroscopic Data for Microtropiosides G–I (9–11) and C-NMR Spectroscopic Data for Microtropioside B (21)
1
3
1
C-NMR at 100MHz and H-NMR at 400MHz with Multiplicities and Coupling Constants in Hz, CD OD)
3
a)
9
10
11
21
C
H
C
H
C
H
1
2
3
38.5
0.97m
.67m
1.31m
.62m
43.3
1.41m
40.5
1.60 ddd 13.7, 6.3, 1.6
1.65m
38.4
24.1
85.8
1
1.88 dd 13.3 4.6
24.2
85.9
75.4
45.9
4.17qui-like ca. 5
19.3
37.9
0.97m
1
1.47m
3.28 dd 12.0, 2.4
1.54 dd 13.6, 4.6
0.96m
1
.86 dd 13.6, 6.6
—
1.83m
4
5
6
39.3
57.4
20.7
—
0.96m
33.6
55.3
21.4
39.0
58.8
21.7
—
39.3
57.5
20.9
1.03 brd 11.8
1.42m
1.07 dd 9.8, 2.1
1.27m
1.67 2H m
1
.68 brd 13.1
1.65m
7
44.8
1.32m
44.6
1.41 brd 11.9
45.5
1.30m
45.0
1
.74m
1.76 ddd 11.9, 2.8, 2.7
1.74m
8
9
0
1
76.7
58.5
37.9
16.2
—
76.7
57.9
38.7
15.9
—
1.40 dd 11.0, 5.7
—
76.8
57.2
38.3
15.7
—
76.6
56.7
38.0
15.7
1.30 dd 8.9, 2.8
1.38m
1
—
—
1
1.48m
1.63m
1.50m
2
.02 dddd 12.0, 6.4, 6.4, 6.4
1.67m
1.60m
1
2
32.8
1.56m
32.7
1.42m
33.0
1.39m
32.8
1
.80 ddd 12.4, 6.4, 2.5
—
2.25 ddd 14.0, 5.7, 5.5
—
2.18 ddd 13.8, 6.5, 5.9
—
13
14
15
76.8
77.8
16.1
76.8
77.3
64.3
76.7
77.6
64.3
76.8
77.8
64.3
3.37m
3.76 dd 8.3, 2.5
3.47 dd 11.2, 8.3
3.69m
1.29 3H d 6.4
3.31m
3
.95 dd 11.2, 2.5
3.85m
16
17
18
19
24.8
25.1
28.7
16.9
1.12 3H s
1.31 3H s
1.02 3H s
0.81 3H s
24.8
25.5
24.7
33.4
1.07 3H s
1.30 3H s
1.01 3H s
0.96 3H s
24.6
25.8
28.4
74.2
1.05 3H s
1.26 3H s
1.01 3H s
3.34 d 9.7
24.6
25.7
28.8
16.9
3
.97 d 9.7
2
0
1
2
3
4
5
6
16.4
102.0
75.2
78.3
72.0
77.8
63.1
0.85 3H s
18.6
102.9
75.4
78.4
71.8
77.9
62.9
1.10 3H s
16.3
105.0
75.3
78.4
71.8
77.8
62.8
0.85 3H s
16.0
102.0
75.2
78.3
72.0
77.8
63.1
′
′
′
′
′
′
4.32 d 7.8
4.35 d 7.8
4.14 d 7.8
3.16 dd 9.2, 7.8
3.37m
3.16 dd 9.0, 7.8
3.37 dd 9.0, 9.0
3.30 dd 9.5, 9.0
3.28 ddd 9.5, 5.9, 2.2
3.69 dd 11.9, 5.9
3.88 dd 11.9, 2.2
3.16 dd 8.8, 7.8
3.34m
3.35m
3.28m
3.23 ddd 8.6, 4.8, 2.6
3.67 dd 11.8, 4.8
3.23m
3.44 dd 11.2, 5.5
3.90 dd 11.2, 2.3
3
.85 dd 11.8, 2.6
a) Data from ref. 4.

9
34
Chem. Pharm. Bull.
Vol. 65, No. 10 (2017)
phous powder and its elemental composition was determined
to be C H O by HR-ESI-MS. The strong IR absorption band
2
6
46
8
−
1
at 3360cm indicated that 9 was a glycosidic compound and
13
D-glucose was detected by sugar analysis. In the C-NMR
spectrum, six signals assignable to those of β-glucopyranoside
were observed and, of the remaining 20 resonances, those
assignable to rings A, B and C showed indistinguishable simi-
larity to those of microtropioside B (21) (Table 2), except that
the primary alcohol signal (C-15) observed in 21 was replaced
4)
1
by a methyl group (δ 16.1). In the H-NMR spectrum, the
C
primary alcohol protons also disappeared and instead of those,
Fig. 3. Diagnostic Two Dimensional NMR Correlations of Microtropio-
side I (10)
a methyl proton signal [δ 1.29 (3H)] was newly observed. The
H
position and mode of sugar linkage were found to be the same
as those of 21. Therefore, the structure of 9 was elucidated as
shown in Fig. 2.
2
3
Microtropioside H (10), [α]D −26.7, was isolated as an
amorphous powder and its elemental composition was de-
13
termined to be C H O by HR-ESI-MS. In the C-NMR
2
6
46
9
spectrum, 26 resonances were observed, of which six were
assigned to those of glucopyranoside and, of the remaining
2
0 resonances, those assignable to ring C and the side chain
C-14 and C-15) were indistinguishable from those in 9. The
positions of the hydroxy group were assigned by HMBC and
(
1
1
H– H COSY experiments and a D-glucose moiety was found
to be attached to the hydroxy group at the 2-position in β
mode (Fig. 3). Judged from coupling constants of H-1a, H-3a
and H-3b protons together with that of H-2 itself, the glucopy-
ranoxy group must be in an axial orientation, which was con-
firmed by the difference NOE experiment. Upon irradiation
of the anomeric proton, significant signal enhancements were
observed in the H -19 and H -20 proton signals. Therefore, the
Fig. 4. Diagnostic Two Dimensional NMR Correlations of Microtro-
pioside J (11)
Dual arrow curves denote HMBC correlations were observed in the both direc-
tions.
3
3
structure of microtropioside H (10) was elucidated, as shown
in Fig. 2.
2
5
Microtropioside I (11), [α]D −30.6, was isolated as an amor-
phous powder and its elemental composition was determined
13
to be C H O by HR-ESI-MS. The C-NMR spectrum dis-
2
6
46
9
played 26 resonances, of which six were also assigned to those
of glucoyranoside and a slightly shielded chemical shift of the
anomeric carbon (δ 105.0) was indicative that the glucosidic Fig. 5. NMR Spectroscopic Data of C-18 and C-19 Region
C
linkage was on the primary alcohol. Of the remaining signals,
those assignable to rings B and C, and a side chain and a chemical shifts between a and b are able to discriminate two
methyl group at the 13-position were essentially the same as structures in a straightfoward manner. Therefore, the structure
1
the corresponding signals of 9, 10 and 21 (Table 2). In the H– of 11 is shown in Fig. 2.
1
H COSY spectrum, correlations of protons on three consecu-
The stereochemistry of C-14 of compounds 9, 10 and 11
tive methylene carbons (H -1, H -2 and H -3) were observed, was determined to be S by indistinguishable similarity of
2
2
2
13
and the methyl protons at C-20 (δH 0.85) showed correlation
C-NMR chemical shifts to that of 21 (Table 2). However,
cross peaks with one end (δC 40.5) of the methylene carbon similar compounds which have the opposite stereochemistry
and the methyl protons at C-18 (δH 1.01) and the primary to 21 were reported to have close chemical shifts to that of
14)
alcohol methylene protons (δH 3.34 and 3.97) correlated with 21. Therefore, the stereochemistry of C-14 of compounds 9,
the methylene carbon (δ 37.9) at the other end in the HMBC 10 and 11 was tentatively determined and further investiga-
C
spectrum, establishing the scaffold of ring A (Fig. 4). In a tions were required to draw the unambiguous conclusion.
2
2
general trend, oxidation of the equatorial methyl group of the
Compound (12), [α]D −2.8, was isolated as amorphous
geminal ones at the 4-position, followed by transglucosyaltion, powder and its elemental composition was determined to be
proceeded to form a glucopyranosyl group at the C-18 position C H O . The IR spectrum exhibited absorptions assign-
4
3
72 15
1
1)
−1
(
normal type). A related compound, sagittarioside a, which able to hydroxy groups (3362cm ), aliphatic C–H stretching
has a glucopyranoxyl group at the C-19 position, showed (2927 and 2874cm ) and a double bond (1651cm ). Of the 43
−1
−1
13
13
a close resemblance with the C-NMR chemical shifts of
C-NMR resonances observed, 12 were assignable to those of
the C-5, C-18 and C-19 positions with those of 11. Figure 5 two terminal glucopyranosides. The remaining 31 resonances
13
shows the C-NMR chemical shifts of C-5, C-18 and C-19 of included one methoxy, seven methyls, seven methylenes, five
labdanes (normal type) which have a primary alcohol at the methines and three oxygenated methines as well as six qua-
12,13)
axial (a) and equatorial (b) positions.
Differences in the ternary carbons along with a tetrasubstituted double bond and

Vol. 65, No. 10 (2017)
Chem. Pharm. Bull.
935
a primary alcohol. These functional groups were assigned by troscopy (NOESY) experiment in which a correlation peak
the aid of distortionless enhancement by polarization transfer between two axial methyls, H -24 and H -25, was observed.
3
3
(
DEPT) and two-dimensional NMR experiments. Two doublet From the HMBC correlations between H-11 and C-12, C-13
1
methyls (δ 0.94 and 0.95), observed in the H-NMR spectrum and the methoxy carbon, the methoxy group must be placed
H
(
Table 3), suggested that compound 12 had an ursane-type adjacent to the double bond (Fig. 6). Further HMBC correla-
skeleton. The positions of the oxygenated carbons were mainly tion from H-11 to C-10, indicated that the methoxy group was
examined by the HMBC experiment (Fig. 6). The methylene at the 11-position and the double bond at C-12 and C-13. C-12,
protons of the primary alcohol showed correlation peaks with one of carbons of the tetrasubstituted double bond, must have
C-3, C-5 and C-24, and the position of the primary alcohol an oxygen substituent from its highly deshielded chemical
was confirmed by the PS-nuclear Overhauser effect spec- shift value (δ 145.8); thus, one hydroxy group was placed on
C
C-12. This enol hydroxy hydrogen is probably stabilized by a
chelate formation with the methoxy oxygen atom. HMBC cor-
relations between H -28 and C-16, and H-27 and C-15 together
3
1
1
with the H– H COSY correlation between H-15 and H-16
placed the remaining hydroxy group at the 16-position. From
1
fairy large H-NMR coupling constants of H-3 (J=8.1Hz with
H-2ax), H-11 (J=9.7Hz with H-9ax) and H-16 (J=11.2Hz with
H-15ax), the hydrogens at those positions were determined
to be in an axial orientation, namely the substituents were in
an equatorial orientation. The absolute configuration of the
sugar was analyzed by HPLC analysis of its hydrolyzate using
a chiral detector and the mode of linkage was determined
to be β from the coupling constant of the anomeric protons.
The position of the sugar linkage were established by the
HMBC correlations shown in Fig. 6. Therefore, the structure
of 12 was elucidated to be 3β,12,16β,23-tetrahydroxy-11α-
methoxyurs-12-ene 3,16-di-O-β-D-glucopyranoside, as shown
Fig. 6. Diagnostic Two Dimensional NMR Correlations of Triterpenoid in Fig. 2. A similar compound, 3β,12,15α-trihydroxy-11α-
Diglucoside (12)
methoxyurs-12-ene, was isolated from the stems of Siphondon
13
1
Table 3.
C- and H-NMR Spectroscopic Data for Compound 12 (150MHz and 600MHz, Respectively, CD OD)
3
C
H
C
H
1
2
40.2
1.23 ddd 13.6, 9.6, 3.2
21
22
23
32.5
1.33m
2
.14 ddd 13.6, 3.0, 3.0
1.77 dddd 13.6, 3.2, 3.0, 2.4
.94 dddd 13.6, 9.6, 8.1, 3.0
1.44m
26.6
36.7
67.4
1.59m
1
1.72m
3
4
5
6
83.1
44.26
48.4
18.8
3.85 dd 8.1, 2.4
—
3.68m
3.83m
1.28m
24
25
13.6
17.62
18.9
0.74 3H s
1.16 3H s
1.11 3H s
1.29 3Hs
0.82 3H s
0.94 3H d 6.3
0.95 3H d 6.3
3.22 3H s
4.40 d 7.8
3.16m
1.36m
1
.51 brdd 12.6, 7.9
26
7
34.6
1.35m
27
25.2
1
.65 ddd 12.6, 10.6, 3.6
—
28
23.7
8
9
44.2
49.6
29
17.59
21.7
1.90 d 9.7
4.10 d 9.7
30
1
1
1
1
1
1
0
1
2
3
4
5
39.1
—
–OCH3
53.5
78.36
145.8
116.9
44.29
36.4
1′
2′
3′
4′
5′
6′
105.8
75.7
—
—
—
78.40
71.8
3.34m
3.31m
1.03 dd 13.7, 3.6
.36 dd 13.7, 11.2
77.6
3.26m
2
62.8
3.68m
16
17
18
19
20
78.30
39.7
50.9
42.1
41.0
4.19 dd 11.2, 3.6
3.84 dd 11.6, 2.1
4.32 d 7.8
3.15m
—
2.48 d 11.5
1.41m
1″
2″
3″
4″
106.1
75.68
78.29
71.6
3.33m
—
3.29m
5
″
″
77.8
3.28m
6
62.9
3.68m
3
.82 dd 11.6, 2.2
1
Letters after H-NMR data are multiplicities and coupling constants (J) in Hz.

9
36
Chem. Pharm. Bull.
Vol. 65, No. 10 (2017)
JEOL α-400 or Brucker Avance III 600 spectrometer at 400 or
00MHz, and 100 or 150MHz, respectively, with tetramethyl-
6
silane as an internal standard. Positive- and negative-ion
HR-MS were taken with an Applied Biosystem QSTAR XL
system ESI (Nano Spray)-MS.
A highly-porous synthetic resin (Diaion HP-20) was pur-
chased from Mitsubishi Kagaku (Tokyo, Japan). Silica gel
CC and reversed-phase [octadecylsilanized silica gel (ODS)]
open CC were performed on silica gel 60 (Merck, Darmstadt,
Germany) and Cosmosil 75C -OPN (Nacalai Tesque, Kyoto,
18
Japan), [Φ=40mm, L=25cm, linear gradient: MeOH–H O
2
(
1:9, 1L) → (9:1, 1L), 5g-fractions being collected], respec-
tively. The droplet counter-current chromatograph (DCCC)
(Tokyo Rikakikai, Tokyo, Japan) was equipped with 500 glass
columns (Φ=2mm, L=40cm), the lower and upper layers of a
solvent mixture of CHCl –MeOH–H O–n-PrOH (9:12:8:2)
Fig. 7. An ORTEP Drawing of Microtropioside A Pentaacetate (15a)
The structure has crystallographic numbering.
3
2
celastrineus, which belongs to the same family (Celastraceae) being used as the stationary and mobile phases, respectively.
as the title plant used in this experiment. The enol hydrogen Five-gram fractions were collected and numbered according
1
was reported to appear as a singlet at δ 4.67 in the H-NMR to their order of elution with the mobile phase. HPLC was
H
15)
spectrum (CDCl3).
performed on an ODS column [Inertsil ODS-3 (5µm), 6mm
Kaempferol 3-O-β-D-(2‴-O-β-D-xylopyranosyl)(2″-O-β-D- ×250mm (GL Science, Tokyo, Japan), flow rate: 1.6mL/min],
2
3
glucopyranosyl)galactopyranoside (13) [α]D −45.7, was and the eluate was monitored with a UV detector at 254nm
isolated as a pale yellow amorphous powder. Its elemental and a refractive index monitor. meso- and rac-Butane-2,3-
composition was determined to be C H O by HR-ESI-MS. diols, 2-hexanol were purchased from Wako Pure Chemical
32
38 20
The IR spectrum showed absorption bands at 3406, 1651 and Industries, Ltd. (Osaka, Japan).
−1
1
606 cm , assignable to hydroxy groups, ketone functional
Plant Material Leaves of M. japonica HALLIER f. (Celas-
groups and aromatic ring(s), respectively. The UV absorption traceae) were collected in Kunigami Village, Kumigami
bands at 265, 332 and 349nm also indicated the presence of County, Okinawa, Japan, in July 1997, and a voucher speci-
1
aromatic ring(s). In the H-NMR spectrum, two aromatic pro- men was deposited in the Herbarium of Pharmaceutical Sci-
ton signals coupled in an AA’BB’ system, two meta-coupled ences, Graduate School of Biomedical Sciences, Hiroshima
aromatic protons, and three anomeric protons were observed University (97-MJ-Okinawa-0716). The plant species was
(
Table 4). The sugar analysis revealed the presence of D- identified by one (T.S.) of the authors.
13
xylose, D-glucose and D-galactose and the C-NMR spectral
Extraction and Isolation Leaves of M. japonica (3.25kg)
resonances of the aromatic region and the inner most sugar were extracted three times with MeOH (4.5L×3) at room
showed a close resemblance to those of kaempferol 3-O-β- temperature for one week and then concentrated to 3L in
D-(2″-O-β-D-glucopyranosyl)galactopyranoside (lilyn) (17), vacuo. The concentrated extract was washed with n-hexane
7
)
isolated from this plant and originally from Lilium candilum
(3L, 17.9g), and then the MeOH layer was concentrated to
8)
and Trigonella foenum-graecum. Thus, the terminal sugar a gummy mass. The latter was suspended in water (3L) and
was found to be β-D-xylopyranose and the position of the link- then extracted with EtOAc (3L) to give 171g of an EtOAc-
age was deduced by cross peaks between H-1⁗ (δH 4.58) and soluble fraction. The aqueous layer was extracted with
C-2‴ (δ 84.9) in the HMBC and H-2‴ (δ 3.46) on C-2‴ was 1-BuOH (3L) to give a 1-BuOH-soluble fraction (32.1g), and
C
H
correlated with the anomeric proton of β-D-glucopyranoside the remaining water-layer was concentrated to furnish 136g of
H-1‴, δH 4.72) in the COSY spectrum. Therefore, the struc- a water-soluble fraction.
(
ture of 13 was elucidated to be lilyn 2‴-O-β-D-xylopyranoside,
as shown in Fig. 2.
The 1-BuOH-soluble fraction (32.0g) was subjected to
Diaion HP-20 CC (Φ=60mm, L=46cm), using H O–MeOH
2
Microtropioside A (15) was first isolated from the branches (4:1, 2L), (3:2, 2L), (2:3, 2L), and (1:4, 2L), and MeOH
of the title plant and also from the leaves as well in this ex- (2L), 500mL-fractions being collected. The residue (5.16g) in
periment. Its structure was spectroscopically elucidated in the fraction 3 was subjected to silica gel (75g) CC with increasing
previous paper and its pentaacetate (15a) was prepared to back amounts of MeOH in CHCl [CHCl (2L), and CHCl –MeOH
3
3
3
4)
up the structure. After we published the above results, the (49:1, 2L), (24:1, 2L), (23:2, 2L), (9:1, 1L), (17:3, 1L),
pentaacetate formed a good crystal suitable for X-ray analysis; (4:1, 1L), (3:1, 1L) and (7:3, 1L)], 500-mL fractions being
Fig. 7 shows the ORTEP drawing of the crystal structure. The collected. The residue (1.09mg) was subjected to ODS open
absolute structure was finally confirmed using D-glucose as a CC to give a residue (409mg) in fractions 19–35, which was
chiral clue. The structure deduced by the spectroscopic meth- purified by DCCC to give 77.7mg of 8 in fractions 14–19.
od was found to be correct and the diterpene was confirmed
to belong to an enatio series.
The reside (3.18g) in fractions 4–6 obtained on Diaion
HP-20 CC was subjected to silica gel (75g) CC with increas-
ing amounts of MeOH in CHCl3 [CHCl3 (1.2L), CHCl3–
MeOH (49:1, 600mL), (24:1, 600mL), (23:2, 600mL), (9:1,
Experimental
General Procedure Optical rotations were measured on a 600mL), (17:3, 600mL), (4:1, 600mL), (3:1, 600mL) and
JASCO P-1030 polarimeter and IR spectra on a Horiba FT-710 (7:3, 600mL), and MeOH (1L)], 100-mL fractions being col-
1
13
spectrophotometer. H- and C-NMR spectra were taken on a lected. The residue (334mg) in fractions 41–46 was subjected

Vol. 65, No. 10 (2017)
Chem. Pharm. Bull.
937
to ODS open CC to give the residue (47.3mg) in fractions fractions 167–180 and 181–216, respectively. The former was
3–47 which was then applied to DCCC. The residue (8.57mg) purified HPLC [H O–MeOH (7:3)] to give 1.12mg of 2 from
2
2
in fractions 50–54 was finally purified by HPLC [H O–MeOH the peak at 6min and the latter 3.09mg of 9 from the peak at
2
(
3:1)] to furnish 1.56mg of 3 from the peak at 13min. The 4min. The residue (891mg) in fractions 29–33 was subjected
residue (25.1mg) in fractions 55–57 was purified by HPLC ODS open CC. The residue (261mg) in fractions 194–275 was
H O–MeOH (3:1)] to yield 5.04mg of 17 and 5.98mg of 18 applied to DCCC and the residue (20.6m) in fractions 64–72
[
2
from the peaks at 15min and 18min, respectively. The residue was purified by HPLC [H O–MeOH (7:3)] to give 1.80mg of
2
(334mg) in fractions 41–46 obtained on silica gel CC was 10 and 0.93mg of 11 from the peaks at 23 and 35min, respec-
subjected to ODS open CC [Φ=40mm, L=25cm, linear gradi- tively. The residue (594mg) in fractions 39–41 was subjected
ent: MeOH–H O (1:9, 2L) → (9:1, 2L), 5g-fractions being to ODS open CC. The residue (13.5mg) in fractions 206–210
2
collected] to give the residue (90.2mg) in fractions 51–89) was purified by HPLC [H O–MeOH (13:7)] to give 2.30mg
2
was then applied to DCCC. The residue (41.1mg) in fractions of 12 from the peak at 8min. The residue (522mg) in fractions
2
4–30 was purified by HPLC [H O–MeOH (17:3)] to give 42–44 was subjected ODS open CC to give 7.17mg of 14 in
2
1
2
1.4mg of 1 and 7.50mg of 16 from the peaks at 22min and fractions 280–282. The residue (419mg) in fractions 45–48
8min, respectively. was subjected to ODS open CC. The residue (179mg) in frac-
The residue (1.92g) in fraction 7 obtained on Diaion-HP-20 tions 10–13 was applied to DCCC. The residue (68.1mg) in
CC was subjected to silica gel (75g) CC with increasing fractions 12–17 was purified by HPLC [H O–MeOH (3:2)] to
2
amounts of MeOH in CHCl3 [CHCl3 (1L), CHCl –MeOH give 30.6mg of 13 from the peak at 7min.
3
(
5
(
49:1, 500mL), (24:1, 500mL), (23:2, 500mL), (9:1,
00mL), (17:3, 500mL), (4:1, 500mL), (3:1, 500mL) and
7:3, 500mL), CHCl –MeOH–H O (35:15:2, 500mL) and νmax cm : 3384, 2977, 2225, 1735, 1647, 1454, 1243, 1171,
Microtropin Q (1)
2
1
An amorphous powder; [α] −20.4 (c=0.76, MeOH); IR
D
−1
3
2
1
MeOH (500mL)], 100-mL fractions being collected. The resi- 1080, 1019; H-NMR (600MHz; CD OD) δ: 6.66 (1H, dd,
3
due (452mg) in fractions 24–34 was subjected ODS open CC. J=6.3, 5.7, 0.7Hz, H-3), 4.60 (1H, dd, J=11.8, 5.2Hz, H-6′a),
The residue (45.8mg) in fractions 1–16 was purified by HPLC 4.51 (1H, dd, J=14.9, 5.7Hz, H-4a), 4.41 (1H, dd, J=14.9,
[
Inertsil, 6mm ×250mm, H O–MeOH (17:3), 1.6mL/min] to 6.3Hz, H-4b), 4.31 (1H, d, J=7.8Hz, H-1′), 4.27 (1H, dd,
2
give two peaks at 6min and 7min (4.04mg and 2.30mg, re- J=11.8, 2.0Hz, H-6′b), 4.20 (2H, s, H -5), 3.93 (1H, q,
2
spectively) which were interconvertible and the compound was J=6.4Hz, H-3″), 3.49 (1H, ddd, J=9.0, 5.2, 2.0Hz, H-5′), 3.37
assigned as 8. The residue (95.1mg) in fractions 36–72 was (1H, dd, J=9.4, 9.0Hz, H-3′), 3.34 (1H, dd, J=9.4, 9.0Hz,
applied to DCCC to give two resides (16.7mg and 34.0mg) in H-4′), 3.18 (1H, dd, J=9.9, 7.8Hz, H-2′), 1.74 (1H, dq, J=14.3,
fractions 35–46 and 65–82, respectively. The former was puri- 7.5Hz, H-5″a), 1.55 (1H, dq, J=14.3, 7.5Hz, H-5″b), 1.18 (3H,
1
3
fied by HPLC [Inertsil, 6mm ×250mm, H O–MeOH (3:1), d, J=6.4Hz, H -4″), 0.88 (3H, t, J=7.5Hz, H -6″); C-NMR
2
3
3
1
.6mL/min] to give 5.75mg of 5 from the peak at 6min. The (150MHz, CD OD): Table 1; HR-ESI-MS (positive-ion mode)
latter was also purified by HPLC to afford [H O–MeOH (4:1)] m/z: 428.1522 [M+Na] (Calcd for C H O NNa: 428.1527).
Microtropin R (2)
An amorphous powder; [α] −90.2 (c=0.08, MeOH); IR
HP-20 CC was subjected to silica gel (75g) CC with increas- νmax cm : 3351, 2932, 1735, 1651, 1457, 1370, 1241, 1168, 1081,
3
+
2
17 27 10
to give 9.80mg of 18 from the peak at 23min.
The residue (3.20g) in fractions 8–10 obtained on Diaion
2
4
D
−1
1
ing amounts of MeOH in CHCl [CHCl (1L), CHCl –MeOH 712; H-NMR (600MHz; CD OD) δ: 5.45 (1H, dtt, J=10.9, 7.3,
3
3
3
3
(
5
49:1, 500mL), (24:1, 500mL), (23:2, 500mL), (9:1, 1.2Hz, H-4), 5.37 (1H, dtt, J=10.9, 7.3, 1.2Hz, H-3), 4.63 (1H,
00mL), (17:3, 500mL), (4:1, 500mL), (3:1, 500mL) and dd, J=11.7, 2.3Hz, H-6′a), 4.60 (1H, dd, J=11.7, 2.3Hz, H-6′a),
7:3, 500mL), CHCl –MeOH–H O (35:15:2, 500mL) and 4.27 (1H, d, J=7.6Hz, H-1′), 4.23 (1H, dd, J=11.7, 5.5Hz,
(
3
2
MeOH (500mL)], 100-mL fractions being collected. The resi- H-6′b), 3.92 (1H, q, J=6.4Hz, H-3″), 3.80 (1H, dt, J=9.5,
due (640mg) in fractions 32–37 was subjected to ODS open 7.3Hz, H-1a), 3.53 (1H, dt, J=9.5, 7.1Hz, H-1b), 3.48 (1H, ddd,
CC. The residue (127mg) in fractions 58–100 was applied to J=9.7, 5.5, 2.3Hz, H-5′), 3.30–3.38 (2H, overlapped, H-3′ and
DCCC and the residue (12.1mg) in fractions 21–23 was puri- 4′), 3.17 (1H, dd, J=9.1, 7.8Hz, H-2′), 2.37 (2H, dddd, J=7.3,
fied by HPLC [H O–MeOH (4:1)] to give 1.60mg of 6 from 7.3, 7.1, 1.2Hz, H -2), 2.07 (2H, qdd, J=7.6, 7.3, 1.2Hz, H -5),
2
2
2
the peak at 57min. The residue (49.4mg) in fractions 144–146 1.73 (1H, dq, J=13.7, 7.5Hz, H-5″a), 1.17 (3H, d, J=6.4Hz,
was applied to DCCC and the residue (10.9mg) in fractions H -4″), 0.96 (2H, d, J=7.6Hz, H -6), 0.88 (3H, t, J=7.5Hz,
3
3
13
in 90–101 was purified by HPLC [H O–MeOH (3:2)] to give H -6″); C-NMR (100MHz, CD OD): Table 1; HR-ESI-
2
3
3
+
2
.51mg of 4 from the peak at 22min. The residue (578m) in MS (positive-ion mode) m/z: 415.1946 [M+Na] (Calcd for
fractions (41–47) was subjected ODS open CC. The residue C H O Na: 415.1944).
18
32
9
(
1
95.7mg) in fractions 144–161 was purified by DCCC to give
1.0mg of 15 in fractions 20–29.
Microtropin S (3)
An amorphous powder; [α] −12.5 (c=0.10, MeOH); IR
2
4
D
−1
The residue (6.16g) in fractions 11–14 obtained on Diaion
HP-20 CC was subjected silica gel (170g) CC with increas-
ν
cm : 3340, 2934, 1736, 1457, 1371, 1238, 1170, 1078;
max
1
H-NMR (400MHz; CD OD) δ: 4.60 (1H, dd, J=11.8, 2.0Hz,
3
ing amounts of MeOH in CHCl [CHCl (2L), CHCl –MeOH H-6′a), 4.34 (1H, d, J=7.8Hz, H-1′), 4.20 (1H, dd, J=11.8,
3
3
3
(49:1, 1L), (24:1, 1L), (23:2, 1L), (9:1, 1L), (17:3, 1L), (4:1, 5.8Hz, H-6′b), 3.94 (1H, q, J=6.4Hz, H-3″), 3.67 (2H, m, H-2
1
1
L), (3:1, 1L) and (7:3, 1L), CHCl –MeOH–H O (35:15:2, and 3), 3.50 (1H, ddd, J=9.5, 5.8, 2.0Hz, H-5′), 3.35 (1H, m,
L) and MeOH (1L)], 200-mL fractions being collected. The H-3′), 3.34 (1H, m, H-4′), 3.19 (1H, dd, J=9.0, 7.8Hz, H-2′),
3 2
residue (439mg) in fractions 25–28 was subjected to ODS 1.74 (1H, dq, J=13.8, 7.5Hz, H-5″a), 1.54 (1H, dq, J=13.8Hz,
open CC. The residue (42.5mg) in fractions 211–215 was H-5″b), 1.16 (3H, d, J=6.1Hz, H -1), 1.17 (3H, d, J=6.4Hz,
3
applied to DCCC to give two residues (4.85 and 9.17mg) in H -4″), 1.12 (3H, d, J=6.2Hz, H -4), 0.87 (3H, t, J=7.5Hz,
3
3

9
38
Chem. Pharm. Bull.
Vol. 65, No. 10 (2017)
1
3
+
H -6″); C-NMR (100MHz, CD OD): Table 1; HR-ESI- ESI-MS (positive-ion mode) m/z: 333.1160 [M+Na] (Calcd for
3
3
+
MS (positive-ion mode) m/z: 405.1730 [M+Na] (Calcd for C H O Na: 333.1156).
12
22
9
C H O Na: 405.1731).
(2S,3R)-2-Ethyl-2,3-dihydroxybutyric Acid (8)
16
30 10
2
4
−1
Microtropin T (4)
Colorless syrup; [α]D −3.5 (c=5.15, MeOH); IR νmax cm :
2
5
An amorphous powder; [α] −12.9 (c=0.55, MeOH); IR 3384, 2979, 2927, 2885, 1732, 1458, 1242, 1171, 1087, 991,
D
−1
1
νmax cm : 3340, 2934, 2880, 1736, 1651, 1457, 1371, 1237, 771; H-NMR (400MHz; CD OD) δ: 3.90 (1H, q, J=6.5Hz,
3
1
1
169, 1077, 1040; H-NMR (400MHz; CD OD) δ: 4.61 (1H, H-3), 1.71 (1H, dq, J=13.9, 7.5Hz, H-5a), 1.53 (1H, dq, J=13.9,
3
dd, J=11.8, 2.0Hz, H-6′a), 4.34 (1H, d, J=7.8Hz, H-1′), 4.20 7.5Hz, H-5b), 1.17 (3H, d, J=6.5Hz, H -4), 0.89 (3H, d,
3
1
3
(
3
5
1H, dd, J=11.8, 5.8Hz, H-6′b), 3.92 (1H, q, J=6.4Hz, H-3″), J=7.5Hz, H -6); C-NMR (100MHz, CD OD): Table 1; HR-
3
3
+
.76 (1H, m, H-6), 3.72 (1H, m, H-2), 3.46 (1H, ddd, J=9.1, ESI-MS (positive-ion mode) m/z: 171.0627 [M+Na] (Calcd
.8, 2.0Hz, H-5′), 3.34 (1H, m, H-3′), 3.32 (1H, m, H-4′), 3.15 for C H O Na: 171.0628).
6
12
4
(1H, dd, J=8.9, 7.8Hz, H-2′), 1.73 (1H, dq, J=14.0, 7.3Hz,
Microtropioside G (9)
An amorphous powder; [α] −12.6 (c=0.21, MeOH); IR
H-5″b), 1.48 (2H, m, H -3), 1.46 (1H, m, H-5a), 1.42 (1H, m, νmax cm : 3360, 2964, 2934, 2874, 1735, 1457, 1161, 1070,
H-3b), 1.37 (1H, m, H-5b), 1.21(3H, d, J=6.5Hz, H -1), 1.17 1040; H-NMR (400MHz, CD OD): Table 2; C-NMR
2
4
H-5″a), 1.65 (1H, m, H-3a), 1.54 (1H, dq, J=14.0, 7.3Hz,
D
−
1
2
1
13
3
3
(
(
1
3H, d, J=6.4Hz. H -4″), 1.15 (3H, d, J=6.4Hz, H -7), 0.88 (100MHz, CD OD): Table 2; HR-ESI-MS (positive-ion mode)
3 3 3
13
+
3H, t, J=7.3Hz, H -6″); C-NMR (100MHz, CD OD): Table m/z: 509.3098 [M+Na] (Calcd for C H O Na: 509.3084).
3 3 26 46 8
+
; HR-ESI-MS (positive-ion mode) m/z: 447.2197 [M+Na]
Microtropioside H (10)
An amorphous powder; [α] −26.7 (c=0.12, MeOH); IR
cm : 3362, 2931, 2359, 1650, 1370, 1161, 1075, 1020;
H-NMR (600MHz, CD OD): Table 2; C-NMR (150MHz,
2
D
3
(Calcd for C H O Na: 447.2200).
19
36 10
−
1
Microtropin U (5)
An amorphous powder; [α] −26.9 (c=0.38, MeOH); IR
ν
max
2
4
1
13
D
3
−1
νmax cm : 3389, 2978, 2936, 1736, 1511, 1217, 1075, 1013, CD OD): Table 2; HR-ESI-MS (positive-ion mode) m/z:
94, 834, 777; UV λmax (MeOH) nm (logε): 333 (3.01), 285 525.3029 [M+Na] (Calcd for C H O Na: 525.3034).
3
+
8
26
46
9
1
(3.35), 222 (3.74); H-NMR (400MHz; CD OD) δ: 6.70 (2H, d,
Microtropioside I (11)
An amorphous powder; [α]
3
2
5
J=8.1Hz, H-3 and 5), 6.95 (2H, d, J=8.1Hz, H-2 and 6), 4.71
(
4
−30.6 (c=0.06, MeOH);
1
D
−
1
1H, d, J=7.2Hz, H-1′), 4.65 (1H, dd, J=12.0, 1.8Hz, H-6′a), IR νmax cm : 3360, 2929, 2871, 1370, 1077, 1027; H-NMR
1
3
.18 (1H, dd, J=12.0, 6.1Hz, H-6′b), 3.92 (1H, q, J=6.5Hz, (600MHz, CD OD): Table 2; C-NMR (100MHz, CD OD):
3
3
H-3″), 3.57 (1H, ddd, J=9.4, 6.1, 1.8Hz, H-5′), 3.45 (1H, dd, Table 2; HR-ESI-MS (positive-ion mode) m/z: 525.3031
+
J=9.0. 8.6Hz, H-3′), 3.42 (1H, m, H-2′), 3.40 (1H, m, H-4′), [M+Na] (Calcd for C H O Na: 525.3034).
.71 (1H, dq, J=14.0, 7.0Hz, H-5″a), 1.54 (1H, dq, J=14.0,
2
6
46
9
1
3β,12,16β,23-Tetrahydroxy-11α-methoxyurs-12-ene 3,16-Di-
7
.0Hz, H-5″b), 1.17 (3H, d, J=6.5Hz, H -4″), 0.84 (3H, t, O-β-D-glucopyranoside (12)
3
13
22
J=7.0Hz, H -6″); C-NMR (100MHz, CD OD): Table 1; HR-
An amorphous powder; [α]D −2.8 (c=0.15, MeOH); IR ν
3
3
max
+
−1
1
ESI-MS (positive-ion mode) m/z: 425.1417 [M+Na] (Calcd for cm : 3362, 2927, 2874, 1651, 1512, 1457, 1077, 1020; H-NMR
1
3
C H O Na: 425.1418).
(600MHz, CD OD): Table 3; C-NMR (150MHz, CD OD):
18
26 10
3
3
Microtropin V (6)
Table 3; HR-ESI-MS (positive-ion mode) m/z: 851.4757
2
1
+
An amorphous powder; [α] −68.9 (c=0.11, MeOH); IR [M+Na] (Calcd for C H O Na: 851.4763
D
43 72 15
−1
νmax cm : 3362, 2934, 1736, 1650, 1457, 1279, 1170, 1071,
Kaempferol 3-O-β-D-(2‴-O-β-D-Xylopyranosyl)(2″-O-β-D-
1
015; UV λmax (MeOH) nm (logε): 275 (3.88), 225 (3.32); glucopyranosyl)galactopyranoside (13)
H-NMR (600MHz; CD OD) δ: 7.01 (1H, d, J=2.0Hz, H-2),
.81 (1H, dd, J=8.2, 2.0Hz, H-6), 6.77 (1H, d, J=8.2Hz, IR νmax (film) cm : 3406, 2921, 2901, 1651, 1606, 1362, 1176,
1
23
Pale yellow amorphous powder; [α] −45.7 (c 0.91, MeOH);
3
D
-1
6
H-5), 4.74 (1H, d, J=7.6Hz, H-1′), 4.65 (1H, dd, J=12.0, 1070, 1040, 894: UV λmax (MeOH) nm (logε): 349 (4.11), 332
1
2
.2Hz, H-6′a), 4.25 (1H, dd, J=12.0, 5.7Hz, H-6′b), 3.94 (1H, (4.12), 265 (4.21), 209 (4.28); H-NMR (400MHz, CD OD):
3
13
q, J=6.5Hz, H-3″), 3.70 (2H, t, J=7.1Hz, H -8), 3.62 (1H, Table 4; C-NMR (100MHz, CD OD): Table 4; HR-ESI-
m, H-5′), 3.48 (1H, m, H-1′), 3.47 (1H, m, H-3′), 3.42 (1H, MS (positive-ion mode) m/z: 765.1840 [M+Na] (Calcd for
m, H-4′), 2.72 (2H, t, J=7.1Hz, H -7), 1.72 (1H, dq, J=14.0, C H O Na, 765.1840).
2
3
+
2
32 38 20
7.5Hz, H-5″a), 1.53 (1H, dq, J=14.0, 7.5Hz, H-5″b), 1.17 (3H,
Sugar Analysis About 500µg each of microtropins Q–W
d, J=6.5Hz, H -4″), 0.85 (3H, t, J=7.5Hz, H -6″); C-NMR (1–7), microtropiosides G–I (9–11) and compound 12 was hy-
150MHz, CD OD): Table 1; HR-ESI-MS (positive-ion mode) drolyzed with 1M HCl (0.1mL) at 90°C for 2h. The reaction
mixtures were partitioned with an equal amount of EtOAc
(0.1mL) and the water layers were analyzed with HPLC on
An amorphous powder; [α] −90.2 (c=0.08, MeOH); IR an amino column [Shodex Asahipak NH P-5 4E (Showa
νmax cm : 3399, 2980, 2932, 2855, 1733, 1641, 1391, 1240, Denko K.K., Tokyo, Japan); CH CN–H O (3:1), Φ=4.6mm,
106, 1078, 1043, 670; H-NMR (400MHz; CD OD) δ: 5.08 L=250mm; flow rate: 1mL/min]. using a chiral detector
d, J=3.8Hz, H-1′α) 4.48 (d, J=7.8Hz, H-1′β), 4.45 (1H, m, (JASCO OR-2090plus) to give a sole peak with a positive sign,
H-6′aα and 6′aβ), 4.26 (1H, m, H-6′bα and 6-'bβ), 3.99 (m, which had an identical retention time with that of authentic D-
H-5′α), 3.96 (1H, m, H-3″), 3.68 (m, H-3′β), 3.67 (m, H-3′α), glucose at 9.5min.
Similar HPLC analysis [InertSustain NH2 (GL Sci-
H-4′α), 3.13 (dd, J=9.2, 7.8Hz, H-2′β), 1.72 and 1.73 (each dq, ence); Φ=6mm, L=250mm; CH CN–H O (4:1); flow rate:
13
3
3
(
3
+
m/z: 469.1680 [M+Na] (Calcd for C H O Na: 469.1680).
2
0
30 11
Microtropin W (7)
2
4
D
2
−1
3
2
1
1
(
3
3.48 (m, H-5′β), 3.37 (m, H-4′β), 3.33 (m, H-2′α), 3.32 (m,
3
2
J=14.1, 7.3Hz, H-2″a), 1.53 and 1.54 (each dq, J=14.1, 7.3Hz, 1.6mL/min] of the hydrolyzate of 13 gave three peaks, which
H-5″b), 1.172 and 1.174 (each d, J=6.5Hz, H-4″), 087 (3H, t, had identical retention times with those of authentic D-xylose,
13
J=7.3Hz, H-6″); C-NMR (100MHz, CD OD): Table 1; HR- D-glucose and D-galactose with positive signs at 7.7, 10.7 and
3

Vol. 65, No. 10 (2017)
Chem. Pharm. Bull.
939
1
3
1
2
Table 4.
C- and H-NMR Spectroscopic Data for Kaempferol Glyco- calculations. The final goodness-of-fit on F was 1.078, and
the final R indices were R =0.0427 and wR =0.0993 based on
sides (13) and 17 (150MHz and 600MHz, Respectively, CD OD)
3
1
2
I>2σ(I), and R =0.0809 and wR =0.1330 with all data. The
1
2
1
3
17
largest differences of the peak and the hole were 0.262 and
−
3
C
H
C
−
0.257 eÅ , respectively. The CCDC deposit contains sup-
plementary crystallographic data (No. 1553754). These data
can be obtained free of charge via http://www.ccdc.cam.ac.uk/
conts/retrieving.html, or from the Cambridge Crystallographic
Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax:
(+44) 1223 336 033; or e-mail: deposit@ccdc.cam.ac.uk.
2
3
4
5
6
7
8
9
0
1
158.6
134.7
179.7
163.1
100.3
167.2
95.0
—
—
—
—
158.9
134.9
179.8
163.1
99.9
6.17 1H d 2
—
165.9
94.7
6.38 1H d 2
—
Acknowledgments The authors are grateful for access to
the superconducting NMR instrument and the HR-ESI-MS at
the Analytical Center of Molecular Medicine and the Analysis
Center of Life Science, respectively, of the Graduate School
of Biomedical Sciences, Hiroshima University. This work
was supported in part by Grants-in-Aid from the Ministry of
Education, Culture, Sports, Science and Technology of Japan,
and the Japan Society for the Promotion of Science (Nos.
158.3
105.5
122.9
132.3
116.3
161.5
101.1
82.7
158.5
105.8
122.8
132.9
116.3
161.5
101.5
80.3
1
—
′
—
2
3
′,6′
′,5′
8.08 2H d 9
6.90 2H d 9
—
4′
1″
2″
3″
4″
5″
6″
5.49 1H d 8
3.95 1H m
3.73 1H dd 10, 3
3.89 1H dd 3, 2
3.45 1H m
3.54 1H m
22590006, 23590130 and 15H04651). Thanks are also due to
74.5
74.8
the Takeda Science Foundation for the financial support.
69.8
70.1
77.1
76.9
Conflict of Interest The authors declare no conflict of
interest.
62.1
62.0
3
.63 1H m
1
2
3
4
5
6
‴
‴
‴
‴
‴
‴
104.0
84.9
78.0
70.8
77.7
62.7
4.72 1H d 8
104.8
75.5
78.2
71.4
77.9
62.7
References
3.46 1H dd 9, 8
3.57 1H m
1)
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3.51 1H dd 10, 9
3.18 1H ddd 10, 5, 2
3.59 1H m
2)
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3
.64 1H m
3
)
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1⁗
2⁗
3⁗
4⁗
5⁗
106.7
75.8
77.5
70.8
67.2
4.58 1H d 8
3.28 1H dd 9, 8
3.38 1H dd 9, 9
3.51 1H ddd 10, 9, 2
3.25 1H dd 11, 10
4)
5) Uemura Y., Sugimoto S., Matsunami K., Otsuka H., Takeda Y.,
3
.93 1H dd 11, 2
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7
)
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1
1.5min, respectively.
3
9b, 1813–1815 (1984).
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X-Ray Analysis of Microtropioside A Pentaacetate (15a)
A suitable crystal (0.25×0.10×0.06mm) was used for analysis.
The data were measured using a Rigakur AFC-5 four circle
diffractometer, using MoKα graphite-monochromated radia-
tion (λ=0.71073Å). The structure was solved by a direct meth-
od using the program SHELXTL-97 (Sheldrick, 2008). The
8)
(
9)
(
1
refinement and all further calculations were carried out using 11) Yoshikawa M., Yoshizumi S., Murakami T., Matsuda H., Yamahara
1
6)
SHELXTL-97. The absorption correction was carried out
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chemistry, 16, 107–110 (1977).
14) Garcia-Alvarez M. C., Rodriguez B., J. Org. Chem., 46, 1915–1918
17)
1
1
utilizing the SADABS routine. The H atoms were included
at calculated positions and treated as riding atoms using the
SHELXTL default parameters. The non-H atoms were refined
2
anisotropically, using weighted full-matrix least-squares on F .
(
1981).
5) Kaweetripob W., Mahidol C., Prawat H., Ruchirawat S., Phytochem-
istry, 96, 404–417 (2013).
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Å , T=173K, Z=2, D =1.235 Mg/m , μ(MoKα)=0.770 mm , 17) Sheldrick G. M., “SADABS,” University of Göttingen, Germany,
Figure 5 was drawn with ORTEP32 (Farrugia, 1997). Crystal
1
data: C H O , M=708.82l, monoclinic, P2 , a=18.715(4) Å,
37
56 13
1
b=6.582(2) Å, c=15.495(4) Å, β=92.798(18)°, V=1906.4(9)
1
3
3
−1
c
F(000)=764, 3913 reflections were measured in the range of
θ≤136.1°, 3771 being unique (R =0.0363) and used in all
1996.
2
int