Ebenamariosides A-D: Triterpene glucosides and megastigmanes from the leaves of diospyros maritima

Article abstract of DOI:10.1248/cpb.c19-00803

The 1-BuOH-soluble fraction of the methanol (MeOH) extract of Diospyros maritima was separated by chromatographic techniques to give three new oleanane-type and one new ursane-type triterpene glucoside, named ebenamariosides A-D (1-4); two megastigmanes were also isolated. The structures of triterpene glucosides was elucidated with extensive investigation by one and two dimensional NMR spectroscopy and the structures were confirmed by partial enzymatic hydrolyses to give the corresponding mono-glucosides and aglycones. The structures of the megastigmanes, including their absolute stereochemistries, were elucidated by spectroscopic evidence and by the modified Mosher's method. Two megastigmanes were chemically correlated and their absolute structures were unambiguously determined. The cytotoxicity of the triterpene glucosides and their degradation products were assayed. They did not show any significant activity.

Full text of DOI:10.1248/cpb.c19-00803

Vol. 67, No. 12
Chem. Pharm. Bull. 67, 1337–1346 (2019)
1337
Regular Article
Ebenamariosides A–D: Triterpene Glucosides and Megastigmanes from
the Leaves of Diospyros maritima
Susumu Kawakami,a Erika Miura,a Ayaka Nobe,a Masanori Inagaki,a Motohiro Nishimura,a
,a
Katsuyoshi Matsunami,b Hideaki Otsuka,* and Mitsunori Aramotoc
a Department of Natural Product Chemistry, Faculty of Pharmacy, Yasuda Women’s University; 6–13–1 Yasuhigashi,
b
Asaminami-ku, Hiroshima 731–0153, Japan: Department of Pharmacognosy, Graduate School of Biomedical and
c
Health Sciences, Hiroshima University; 1–2–3 Kasumi, Minami-ku, Hiroshima 734–8553, Japan: and Iriomote
Station, Tropical Biosphere Research Center, University of the Ryukyus; 870 Aza Uehara, Taketomi-cho, Yaeyama-
gun, Okinawa 907–1541, Japan.
Received September 18, 2019; accepted October 10, 2019
The 1-BuOH-soluble fraction of the methanol (MeOH) extract of Diospyros maritima was separated by
chromatographic techniques to give three new oleanane-type and one new ursane-type triterpene glucoside,
named ebenamariosides A–D (1–4); two megastigmanes were also isolated. The structures of triterpene glu-
cosides was elucidated with extensive investigation by one and two dimensional NMR spectroscopy and the
structures were confirmed by partial enzymatic hydrolyses to give the corresponding mono-glucosides and
aglycones. The structures of the megastigmanes, including their absolute stereochemistries, were elucidated
by spectroscopic evidence and by the modified Mosher’s method. Two megastigmanes were chemically cor-
related and their absolute structures were unambiguously determined. The cytotoxicity of the triterpene glu-
cosides and their degradation products were assayed. They did not show any significant activity.
Key words Diospyros maritima; Ebenaceae; ebanamarioside; triterpene glucoside; megastigmane
Introduction
Diospyros maritima Blume (Ebenaceae) is a tall evergreen
tree with a height of about 10m, distributed in Okinawa,
Taiwan, Malaysia, Micronesia and Australia.1) In summer, it
bears green sap fruits of 2 to 3cm in diameter, which then
turn to a dark orange colour in autumn. It is known that the
fruits contain a toxic naphthoquinone derivative, plumbagin,
and their constituents have been extensively investigated
by Higa et al.2–4) Recently, from the leaves and branches of
a related Thai medicinal plant, D. mollis, the isolation of
naphthoquinone glycosides was reported.5) In our continu-
ing work on Okinawan resource plants, the constituents of
the leaves of D. maritima were investigated to give eight
ent-kaurane-type diterpenoid glycosides, called diosmari-
osides A–H.6) Further extensive work resulted in the isola-
tion of four new triterpene saponins, named ebenamariosides
A–D (1–4) and two megastigmanes (5, 6), along with two
known flavonol glycosides, kaempferol 3-O-β-D-(2″,6″-di-
O-α-L-rhamonopyranosyl)glucopyranoside (7)7) and 3-O-β-D-
(2″,6″-di-α-L-rhamonopyranosyl)galactopyranoside (8)8) (Fig.
1). The cytotoxicity of the triterpenoids was assayed using the
human lung adenocarcinoma cell line A549 and the parasitic
protozoan Leishmania major.
Results and Discussion
The leaves of D. maritima extracted with MeOH and the
MeOH extracts were separated by solvent partition accord-
ing to the polarity of the constituents. The relatively polar
1-BuOH-soluble fraction was separated by various kinds of
chromatography to afford four new triterpene saponins (1–4)
and two new megastigmanes (5, 6), together with two known
flavonol glycosides (7, 8). The structures of new triterpene
Fig. 1. Compounds Isolated and Related Ones
derivatives were elucidated using one- and two-dimensional
*To whom correspondence should be addressed. e-mail: otsuka-h@yasuda-u.ac.jp
© 2019 The Pharmaceutical Society of Japan

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Chem. Pharm. Bull.
Vol. 67, No. 12 (2019)
spectroscopies. The structures of megastigmanes were eluci- less amorphous powder and its elemental composition was the
dated by NMR and circular dichroism (CD) spectroscopies, same as that of 1. NMR spectra were similar to those of 1.
and as well as by the modified Mosher method. The biological Two anomeric protons and carbons (δH 4.79 on δC 105.5 and
1
activity of four new triterpene saponins and their derivatives δH 5.16 on δC 105.9) were also observed in the H-, 13C- and
was assayed against the human lung adenocarcinoma cell line heteronuclear single quantum correlation NMR spectra and D-
A549 and the parasitic protozoan Leishmania major.
glucose was a sole sugar unit. The distinct difference between
Ebenamarioside A (1), [α]D25 +15.1, was isolated as a 1 and 2 in the NMR signal was the carboxy carbons (C-28),
colorless amorphous powder and its molecular formula such as δC 176.4 in 1 was shifted down to δC 180.2 in 2 and
was determined to be C42H68O14 by observation of a quasi- the anomeric carbon signal from an ester linkage (δC 95.8)
molecular ion peak ([M+Na]+) in the high-resolution (HR) appeared in 1 was not observed in 2. While, in the HMBC
electrospray-ionisation (ESI) mass spectrometry. The IR spectrum, one of the anomeric proton at δH 5.16 was corre-
spectrum showed strong absorption bands assignable to hy- lated with C-6′ (δC 70.2) and then the other anomeric proton
droxy (3439cm−1) and ester carbonyl (1745cm−1) functional at δH 4.79 with C-29 (δC 81.4). Thus, the structure of 2 was
1
groups. In the H-NMR spectrum, signals assignable to six 3β,20-dihydroxyolean-12-en-28-oic acid 29-O-β-D-(6′-O-β-D-
singlet methyls, oxymethylene protons (δH 3.40 and 3.91), glucopyranosyl)glucopyranoside, namely mesembryanthenoi-
oxymethine proton (δH 3.45), olefinic proton (δH 5.43) and digenic acid 29-O-β-gentiobioside, as shown in Fig. 1.
two anomeric protons [δH 6.35 (d, J=8.3Hz) and 4.85 (d,
Ebenamarioside C (3), [α]D27 +14.0, was isolated as a color-
J=7.8Hz)] were observed (Table 1). Since HPLC analysis of less amorphous powder and its elemental composition wad
the hydrolysate of 1 revealed the presence of D-glucose as a determined to be C42H68O15, with one more oxygen atom than
sole sugar component, two D-glucose molecules were expected those of 1 and 2. NMR spectroscopic data for the C, D and
to be present in 1. In the 13C-NMR spectrum, other than 12 E-rings were essentially the same as those of 1 and 2. In the
signals assignable to those of glucopyranose units, 30 signals 1H-NMR spectrum, two oxymethine protons (δH 3.39 on δC
observed comprised of six methyls, eleven methylenes includ- 83.8 and δH 4.10 on δC 68.6) as well as oxymethylene protons
ing one oxymethylene, four methines with an oxygenated one, (δH 3.39 and 3.89) were observed (Table 1). The position of
six quaternary carbons, one trisubstituted double bond and a the oxymethine protons were placed at the vicinal positions
1
carboxyl functional group. From the above evidence and the from the H–1H correlation spectroscopy correlation (COSY)
degrees of unsaturation (Δ=7), ebenamarioside A (1) was (Fig. 3) and from the evidence of HMBC correlations, namely,
assumed to be an oleanolic acid derivative with a primary H3-23 (δH 1.25) and 24 (δH 1.08) and C-3 (δC 83.8) (Fig. 3).
hydroxy group. In the heteronuclear multiple bond connectiv- From the PS-NOESY correlations between H-2 (δH 4.10) and
ity (HMBC) spectrum, geminal oxymethylene protons showed H3-25 (axial) (δH 1.02), H-3 (δH 3.39) and H3-23 (axial), and
correlation with a methyl carbon (C-30, δC 19.7), methylene H3-23 and H-5 (axial) (δH 1.00) (Fig. 3), the hydroxy groups
carbons, C-19 (δC 41.1) and 21 (δC 29.2) as well as a quater- at the 2- and 3-positions were placed in equatorial positions,
nary carbon at δC 35.5 (Fig. 2). The significant correlation which were further confirmed by the large coupling constant
in the phase sensitive-nulear Overhauser effect spectroscopy of the vicinal protons (J=9.2Hz). Two oxymethines were
1
(PS-NOESY) spectrum between H-18 (δH 3.27) and H3-30 (δH found to be coupled each other from the H–1H-COSY spec-
1.10) on C-30 enabled us to place the oxymethylene carbon at troscopic evidence. The positions of the oxymethylene protons
the C-29 position (Fig. 2). Similarly, the oxymethine proton and sugar linkages were assigned by the similar manner used
was placed at the 3-position from diagnostic HMBC between for ebenamarioside A (1). Therefore, the structure of 3 was
H-3 and C-4, C-23 and C-24 (Fig. 2). From the axial (10.2Hz) elucidated to be 2α,3β,29-trihydroxyolean-12-en-28-oic acid
and equatorial (4.6Hz) coupling constants of H-3, the hydroxy 28-O-β-D-glucopyranosyl ester 29-O-β-D-glucopyranoside, as
group at the 3-position was in a β equatorial orientation. The shown in Fig. 1. On enzymatic hydrolysis of 3 using crude
positions of the sugar linkages were established to be on the naringinase, 28-O-β-D-glucopyranosyl ester (3a) was obtained.
carboxyl group at the C-28 and the hydroxy group at C-29 Glucoside 3a is a known compound, isolated from the stem
from HMBC correlations H-1′ (δH 6.35) on δC 95.8 and C-28 bark of Terminalia superba (=3a′)12); however, its 13C-NMR
(δC 176.4), and H-1″ (δH 4.85) on δC 105.5 and C-29 (δC 81.4), data for MeOH-d4 were slightly different from those of 3 to
respectively. The mode of linkage was assigned to be β from confirm the structure (Table 2). 13C-NMR data of 3a for pyri-
the coupling constants of the anomeric protons. Therefore, the dine-d5 and dimethyl sulfoxide (DMSO)-d6 were also slightly
structure of ebenamarioside A (1) was elucidated to be 3β,29- different from those of 3a′ (Table 2). Furthermore, the optical
dihydroxyolean-12-en-28-oic acid 28-O-β-D-glucopyranosyl rotation value reported for 3a′ was [α]D −18.1 (c 0.1, MeOH)
ester 29-O-β-D-glucopyranoside, as shown in Fig. 1. Partial which showed an opposite sign to that of 3a. In our report,
enzymatic hydrolysis of 1 using crude β-glucosidase liberated the structure of 3a was carefully elucidated with a highly de-
two monoglucosidic compounds (1a and 1b) and an aglycone tailed survey of the one- and two-dimensional NMR spectra.
(1c). The structure of compound 1a was elucidated to be me- On the other hand, enzymatic hydrolysis of 3 using crude
sembryanthenoidigenic acid 28-O-β-D-glucopyranosyl ester, β-glucosidase gave 2α,3β,29-trihydroxyolean-12-en-28-oic acid
isolated from Salicornia europaea,9) whereas that of 1b me- 29-O-β-D-glucopyranoside (3b) and an aglycone (3c). Glu-
sembryanthenoidigenic acid 29-O-β-D-glucopyranoide, whose coside 3b has not been isolated as a natural product and the
isolation have not yet been reported. The aglycone (1c) was aglycone (3c) was a known one, isolated from the pericarps of
spectroscopically identified with mesembryanthenoidigenic Akebia trifoliata.13)
acid, isolated from a South American cactus, Rhipsalis me-
Ebenamarioside D (4), [α]D26 −5.6, was isolated as a color-
sembryanthemoides.10,11)
less amorphous powder and its elemental composition was
Ebenamarioside B (2), [α]D27 –5.6, was isolated as a color- determined to be C42H68O15, which was the same as that of

Vol. 67, No. 12 (2019)
Chem. Pharm. Bull.
1339
Table 1. 1H-NMR Spectroscopic Data for Ebenamariosides A–D (1–4) (600MHz, Pyridine-d5)
H
1
1
2
3
4
0.99 ddd 12.7, 12.7, 4.1
1.55m
1.04m
1.57m
1.85m
1.28m
1.25m
2.24 dd 11.9, 4.2
4.10 ddd 11.9, 9.2, 4.2
3.39 d 9.2
2.24 dd 12.3, 4.0
4.10 ddd 12.3, 9.3, 4.0
3.38 d 9.3
0.99m
2
3
5
6
1.85 2H m
3.45 dd 10.2, 4.6
0.85 brd 11.8
1.37m
3.47 dd 10.2, 5.7
0.86 brd 12.0
1.37m
1.00m
1.40m
1.38m
1.55m
1.57m
1.54m
1.52m
7
1.37m
1.32m
1.38m
1.38m
1.49 ddd 12.8, 12.8, 3.4
1.64 dd 11.1, 6.7
1.95 2H m
1.50m
1.49m
1.54m
9
1.66 dd 8.9, 8.9
1.95m
1.74 dd 10.2, 7.3
1.95m
1.70m
11
2.02 2H m
2.17m
2.09 brdd 13.4, 10.2
5.39 brs
12
15
5.43 dd 3.5 3.5
1.15m
5.48 brs
1.19m
5.42 brs
1.12m
1.15m
2.37 ddd 13.7, 13.7, 3.6
1.95m
2.17m
2.35 ddd 13.4, 13.4. 3.5
1.98 2Hm
2.43 ddd 13.5, 13.5, 4.7
1.93m
16
1.98 2Hm
2.10 ddd 13.7, 13.7, 3.6
3.27 dd 13.8, 4.2
1.43 dd 13.6, 4.2
2.01 dd 13.8, 13.6
—
2.03m
18
19
3.36 dd 13.7, 3.8
1.50m
3.25 dd 13.6, 3.6
1.41m
2.54 d 11.3
1.70m
2.09m
2.00m
—
20
21
—
—
1.26m
1.35m
1.43m
1.33m
1.56m
1.67 ddd 13.7, 13.7, 4.1
1.82m
1.79m
1.65 ddd 13.9, 13.9, 3.6
1.79 brd 13.4
1.87 ddd 13.9, 13.9, 3.6
1.25 3H s
1.93m
22
1.85m
1.73m
1.87m
2.06m
1.96m
23
24
25
26
27
29
1.23 3H s
1.25 3H s
1.04 3H s
0.90 3H s
1.03 3H s
1.27 3H s
3.41 d 9.2
4.04 d 9.2
1.22 3H s
1.25 3H s
1.07 3H s
1.02 3H s
1.15 3H s
1.12 3H s
0.93 3H d 6.3
1.04 3H s
1.08 3H s
0.93 3H s
1.02 3H s
1.14 3H, s
1.12 3H s
1.21 3H s
1.19 3H s
3.40 d 9.2
3.39 d 9.0
3.91 d 9.2
3.89 d 9.0
30
1.10 3H s
1.09 3H s
3.84 dd 9.4, 3.1
4.00m
1′
6.35 d 8.3
4.79 d 7.7
6.32 d 8.2
6.25 d 8.2
2′
3′
4′
5′
6′
4.22 dd 8.8, 8.3
4.38 dd 9.0, 8.8
4.25 dd 9.2, 9.0
3.99 ddd 9.2, 4.6, 2.3
4.43 dd 12.0, 4.6
4.48 dd 12.0, 2.3
4.85 d 7.8
4.02 dd 8.6, 7.7
4.21 dd 8.9, 8.6
4.16 dd 9.2, 8.9
4.21m
4.20 dd 8.8, 8.2
4.28 dd 8.9, 8.8
4.36 dd 9.1, 8.9
4.03m
4.19 dd 8.6, 8.2
4.27m
4.34 dd 9.3, 9.1
4.00m
4.37 dd 11.4, 5.8
4.88 brd 11.4
5.16 d 7.9
4.42 dd 11.0, 5.4
4.46 brd 11.0
4.83 d 7.7
4.38 dd 12.0, 4.3
4.44 dd 12.0, 2.1
4.85 d 7.7
1″
2″
3″
4″
5″
6″
4.07 dd 8.2, 7.8
4.30 dd 8.6, 8.2
4.42 dd 8.9, 8.6
4.05 ddd 8.9, 5.4, 2.3
4.43 dd 11.8, 5.4
4.59 dd 11.8, 2.3
4.07m
4.05m
4.04 dd 8.2. 7.7
4.27m
4.25m
4.24m
4.26m
4.24m
4.23 dd 9.1, 8.9
3.99m
3.95m
3.98m
4.39 dd 11.9, 5.2
4.53 brd 11.9
4.41 dd 11.4, 5.5
4.58 brd 11.4
4.41 dd 12.1, 5.7
4.58 dd 12.1, 2.2
1
3. The H-NMR spectrum showed resonances for five singlet ity was compared with that of 3. 13C-NMR chemical shifts
methyls and one doublet methyl, two oxygenated methines, of the A and B rings were essentially the same as those of
two anomeric protons and one olefinic proton along with two 3 and the presence of a double bond between C-12 and C-13
oxymethylene protons coupled in a geminal system, but also was also similar to aforementioned triterpene aglycones.
coupled with one more proton [δH 3.84 (dd, J=9.4, 3.1Hz) The proton spin-spin coupling sequences from the doublet
and 4.00 (m)]. Although six quaternary carbons were observed methyl signal (δH 0.93) to oxymethylene protons via two me-
1
in the 13C-NMR spectra of aforementioned oleanane-type thine signals (δH 1.70 and 1.26) were observed in the H–1H
aglycones, only five quaternary ones were present in the COSY spectrum (Fig. 4). The HMBC correlations between
molecule, and two more methine (C-19 and -20) and one less the doublet methyl proton and C-18 (δC 53.3), C-19 (δC 34.4)
methylene carbons were observed in 4, when the functional- and C-20 (δC 44.6), and the oxymethylene protons and C-19,

1340
Chem. Pharm. Bull.
Vol. 67, No. 12 (2019)
Fig. 3. Two Dimensional NMR Correlations of Ebenamarioside C (3)
Fig. 2. Two Dimensional NMR Correlations of Ebenamarioside A (1)
gested these substituents were in the same face and those be-
C-20 and C-21 (δC 25.6) established the scaffold of the ring E tween H-11b (δH 3.68) and H-2eq (δH 1.80), H-3 (δH 4.09) and
to possess the ursane-type carbon frame work (Fig. 4). This H-4eq (δH 2.00) these were in the same face and the opposite
was also supported by the significant PS-NOESY correla- face to the side chain (Fig. 5b). A related compound (9) which
tions H-18 (δH 2.54) and H3-29 (δH 0.93), and H-18 and H-20 showed superimposable NMR spectra with those of 5 was
(δH 1.26). The sugar linkages were assigned by the similar isolated from Asclepias fruticosa and the absolute structure of
manner used for ebenamariosides A and C (1, 3). Therefore, 9 was determined by the modified Mosher’s method15) to have
the structure of ebenamarioside D (4) was elucidated to 1R,3S,5R,6S,9R configurations.16) Compound 5 was also sub-
be 2α,3β,30-trihydroxyursan-12-en-28-oic acid 28-O-β-D- jected to the modified Mosher method and, as a result, 5 was
glucopyranosyl ester 30-O-β-D-glucopyranoside, as shown in found to have 1R,3S,5R,6S,9S configurations (Fig. 6). There-
Fig. 1. Enzymatic hydrolysis using crude β-glucosidase gave fore, 5 has the opposite configuration at the 9-position to that
a mixture of monoglucosidic compounds (4a and 4b) and of 9 and thus it was found to be a new compound in nature.
2α,3β,30-trihydroxyurs-12-en-28-oic acid as an aglycone (4c),
Compound 6, [α]D25 −0.74, was isolated as a colorless syrup
which is known as a microbial transformation product from and its elemental composition was determined to be C13H20O4
corosolic acid by Streptomyces asparaginoviolaceus.14) The which was two hydrogen fewer than that of 5. 13C-NMR
mixture of monoglucosidic compounds was separated by silica spectrum also displayed 13 signals including three methyls,
gel CC and HPLC to give 2α,3β,30-trihydroxyurs-12-en-28-oic three methylenes, one oxygenated methine, two oxygen-
acid 28-O-β-D-glucopyranosyl ester (4a) and 2α,3β,30- ated tertiary carbons, one quaternary carbon, one ketone,
trihydroxyurs-12-en-28-oic acid 30-O-β-D-glucopyranoside instead of oxygenated methine and one disubstituted double
(4b). These two monoglucosidic compounds were first de- bond, whose NMR chemical shifts for CDCl3 were almost
scribed in this experiment.
superimposable to those of drummondol [6a, [α]D23 −21.0
Compound 5, [α]D26 +10.2, was isolated as a colorless powder (MeOH)] isolated from Sesbania drummondii by Powell and
and its elemental composition was determined to be C13H22O4. Smith, Jr.,17) whose geometry at the 9-position and the abso-
1
In the H-NMR spectrum, two singlet (δH 0.88 and 1.12) and lute configuration of bicyclo[3,2,1]octane region remains to
one doublet (δH 1.28, J=6.4Hz) methyls, two olefinic protons be determined. Meanwhile, Çaliş et al. isolated drummondol
[δH 6.03 (dd, J=15.5, 4.5Hz) and 6.06 (d, J=15.5Hz)] coupled 9-O-β-D-glucopyranoside from Capparis spinosa and the
in a trans geometry, two sets of methylene protons (δH 1.64 absolute configuration of the aglycone (6b) of the 9-position
and 1.80, and 1.75 and 2.00), two oxygenated methylene pro- was determined to be 9S by the modified Mosher method.16,18)
tons (δH 3.68 and 3.77) and two oxygenated methine (δH 4.09 That of the ring region was discussed using the Cotton effects
and 4.35) protons were found (Table 3). The 13C-NMR spec- in the CD spectrum of 6b, compared with those of (+)-(S)-
trum displayed 13 signals including three methyls, three meth- abscisic acid metabolites. Compound 6 showed similar Cotton
ylenes, two methines with oxygen atoms, two olefinic carbons, effects [∆ε (nm): +0.64 (241), –0.30 (296)] to those of 6b18)
two oxygenated tertiary and one quaternary carbon (Table 3). and hence the absolute stereochemistries of 6 and 6b were ex-
The number of carbons and degrees of unsaturation suggested pected to be the same at the 6-position. NaBH4 reduction of 6
that compound 5 was a megastigmane with a bicyclic scaffold. gave two products (6c and 6d). Hydride was introduced from
Two 1H–1H COSY correlations [–C(2)H2-C(3)HOH-C(4)H2– the less hindered 3si face to form the major compound 6d and
and –C(7)H=C(8)H-C(9)HOH-C(10)H3] and HMBC cor- the minor compound 6c was obtained by the reduction of 6
1
relations between H2-11 (δH 3.68 and 3.77) and C-5 (δC 87.5) from the 3re face. The H-NMR signal of the H-3 proton of 6d
and other diagnostic correlations shown in Fig. 5a suggested was appeared as a doublet of a doublet, J=5.7, 5.7Hz, indicat-
5
was 5,11-eopxy-3,6,9-trihydroxymegastigman-7-ene. The ing that the α-hydroxy group formed at the 3-position was in
relative stereochemistry was established by the PS-NOESY the pseudo axial orientation due to steric hindrance toward the
spectrum. Correlations between H-7 (δH 6.06) and H-2ax (δH epoxide ring.19,20) Meanwhile, the NMR spectroscopic data of
1.64), H-4ax (δH 1.75), H3-12 (δH 0.88) and H3-13 (δH 1.12) sug- the minor one (6c) were identical with those of 5 and similarly

Vol. 67, No. 12 (2019)
Chem. Pharm. Bull.
1341
Table 2. 13C-NMR Spectroscopic Data for Ebenamariosides A–D (1–4) and Their Derivatives (150MHz, Pyridine-d5)
C
1
1b
2
3
3a
3aa)
3a′b)
3ac)
3b
4
4a
4b
1
39.0
28.1
78.1
39.4
55.8
18.8
33.2
40.0
48.1
37.4
23.8
123.0
144.1
42.1
28.3
23.5
47.4
41.0
41.1
35.5
29.2
31.8
28.8
16.6
15.7
17.6
26.1
176.4
81.4
19.7
95.8
74.2
79.0
71.1
79.4
62.2
105.5
75.3
78.7
71.7
78.6
62.9
39.0
28.1
78.1
39.4
55.8
18.8
33.3
39.8
48.1
37.4
23.8
122.7
144.8
42.2
28.4
23.8
47.1
41.2
41.3
35.8
29.4
32.5
28.8
16.6
15.6
17.5
26.2
180.2
81.6
19.8
39.0
28.1
78.5
39.4
55.8
18.8
33.3
39.8
48.1
37.4
23.8
122.6
144.8
42.2
28.3
23.8
47.1
41.2
41.2
35.7
39.3
32.5
28.8
16.6
15.6
17.5
26.2
180.2
81.4
19.8
105.5
75.2
78.5
71.7
77.3
70.2
105.9
75.2
78.6
71.7
78.1
62.7
47.8
68.6
83.8
40.0
55.9
18.8
33.1
39.8
48.1
38.5
23.5
122.9
144.0
42.1
28.2
23.9
47.2
40.9
41.0
35.5
29.2
31.7
29.3
17.6
16.9
17.5
26.0
176.4
81.3
19.7
95.8
74.1
78.9
71.1
79.3
62.2
105.4
75.2
78.6
71.7
78.5
62.9
47.9
68.6
83.8
40.0
55.9
18.9
33.1
39.9
48.2
38.6
23.5
122.8
144.4
42.2
28.3
24.0
47.5
41.2
41.0
36.4
28.9
32.1
29.3
17.7
17.0
17.6
26.1
176.5
73.7
19.7
95.8
74.2
79.0
71.2
79.4
62.2
48.2
69.5
84.5
40.4
56.7
19.6
32.4
40.5
49.1
39.3
24.0
123.7
145.0
42.9
28.9
24.7
48.3
41.9
41.4
36.9
29.3
33.9
29.3
17.8
17.2
17.7
26.3
178.0
74.3
19.6
95.8
74.0
78.7
71.1
78.3
62.4
47.0
66.8
81.9
39.1
55.1
18.2
32.2
39.0
47.4
36.9
23.5
122.7
143.8
41.5
28.0
23.2
46.4
42.2
41.7
35.3
31.8
33.7
28.8
17.3
16.0
16.9
25.7
175.9
74.7
19.9
95.8
74.1
79.1
71.3
78.6
62.2
46.8
67.0
82.1
38.9
54.7
17.9
32.1
38.8
47.0
37.5
22.4
121.4
143.5
41.2
27.1
22.9
46.2
39.9
39.9
35.3
27.8
30.9
28.7
17.0
16.3
16.6
25.5
175.1
72.1
19.0
94.0
72.3
77.6
69.4
76.6
60.6
47.8
68.6
83.8
39.9
55.9
18.9
33.2
39.8
48.1
38.5
23.8
122.5
144.8
42.2
28.3
23.9
47.0
41.2
41.3
35.7
29.4
32.5
29.3
17.7
16.9
17.5
26.2
180.3
81.6
19.8
48.1
68.6
83.8
39.8
55.9
18.8
33.5
40.2
48.1
38.4
23.8
126.4
138.1
42.5
28.6
24.7
48.2
53.3
34.4
44.6
25.6
36.5
29.4
17.7
17.1
17.7
23.7
176.2
17.1
73.2
95.7
74.0
78.8
71.2
79.1
62.3
104.8
75.2
78.6
71.7
78.5
62.9
48.1
68.6
83.8
39.8
55.9
18.9
33.5
40.3
48.1
38.5
23.9
126.2
138.5
42.6
28.7
24.7
48.4
53.4
33.7
47.2
25.5
36.8
29.4
17.8
17.1
17.7
23.8
176.3
17.1
65.0
95.8
74.1
79.0
71.2
79.3
62.3
48.0
68.6
83.8
39.9
55.9
18.8
33.5
40.0
48.1
38.5
23.7
125.9
139.0
42.5
28.6
25.0
47.9
53.6
34.6
45.0
25.9
37.2
29.4
17.7
17.0
17.5
23.9
179.9
17.3
73.5
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
1′
2′
3′
4′
5′
6′
1″
2″
3″
4″
5″
6″
105.5
75.4
78.7
71.8
78.6
62.9
105.5
75.4
78.7
71.8
78.6
62.9
105.1
75.3
78.7
71.8
78.6
63.0
a) Data for CD3OD. b) Data were taken from ref. 12 (CD3OD). c) Data for DMSO-d6.
6c was subjected to the modified Mosher’s method to give respectively, where that of the positive control etoposide was
(R)- and (S)-α-methoxy-α-trifluoromethylphenylacetic acid 23.3 4.3µM, while other compounds did not show any ac-
(MTPA) esters of 6c, which were the identical compounds tivity at 100µg/mL. Unfortunately, none of compounds were
with 5a and 5b from 5. Therefore, the absolute configurations active toward L. major at 100µg/mL.
of drummondol (6) isolated in this experiment was confirmed
Closing Remarks From the leaves of Diospyros maritima,
to be 1R,5R,6S,9S, which was the same as the aglycone of four triterpene saponins, named ebenamariosides A–D (1–4)
(9S)-drummondol 9-O-β-D-glucopyranoside from C. spinosa and two megastigmanes (5, 6) were isolated. The structures
(Fig. 1) and 6 is expected to be a new compound as a non-glu- of ebenamariosides were carefully elucidated by interpreta-
cosidic form; however, a direct correlation with the original tion of one- and two-dimensional NMR spectroscopies and
drummondol (6a) was precluded.
enzymatic hydrolysis of 1, 3 and 4 using crude β-glucosidase
The cytotoxic activity of isolated ebenamariosides (1–4), and naringinase gave corresponding monoglucosides and agly-
their derivatives (1a, 1b, 1c, 3a, 3b, 3c, 4a 4b and 4c), and cones. The structures of megastigmanes were confirmed by
compounds 5 and 6 was assayed using the human lung ad- the modified Mosher’s method and the Cotton effect in the CD
enocarcinoma cell line A549. Compounds 1c and 4c showed spectrum. Assays of inhibitory activities for triterpene deriva-
slight activity with IC50 values of 174 16 µM and 107 8µM, tives toward human lung adenocarcinoma cell line, A549 and

1342
Chem. Pharm. Bull.
Vol. 67, No. 12 (2019)
Leishmania major did not show any significant activity.
Experimental
General Experimental Procedures Optical rotations
were measured on a JASCO P-2200 digital polarimeter. IR
spectra were measured on JASCO FT/IR-6100 spectropho-
1
tometers. H- and 13C-NMR spectra were taken on a Bruker
Avance III at 600MHz and 150MHz, respectively, with
tetramethylsilane as an internal standard. CD spectra were
obtained with a JASCO J-720 spectropolarimeter. Positive
and negative-ion HR-ESI-MS were performed with a Thermo
Fisher Scientific LTQ Orbitrap XL. Silica gel column chroma-
tography (CC) was performed on silica gel 60 (70–230 mesh)
(E. Merck, Darmstadt, Germany) and reversed-phase octadec-
ylsilanized (ODS) open CC on Cosmosil 75C18-OPN (Nacalai
Tesque, Kyoto, Japan) (Φ=50mm, L=25cm). HPLC was per-
formed on an ODS column [Inertsil ODS-3 (GL Science Inc.,
Tokyo, Japan; Φ=10mm, L=25cm, 4.0mL/min), Cosmosil
Fig. 5. a) 1H–1H COSY and HMBC Correlations of 5; b) PS-NOESY
Correlations of 5
a) Significant long range 1H–1H correlations due to formation of W-figure were
Fig. 4. Two Dimensional NMR Correlations of Ebenamarioside D (4)
observed between H-2ax and H-12a and H-2 eq and H-4 eq.
Table 3. NMR Spectroscopic Data for Megastimane Derivatives (5, 6) (C: 150MHz, H: 600MHz, CD3OD)
5
6
C
H
C
H
1
2
48.8
44.4
—
1.64 (ddd, 13.6, 10.5, 2.1)
1.80 (ddd, 13.6, 7.2, 1.4)
4.09 (dddd, 10.5, 10.5, 7.2, 7.2)
1.75 (dd, 13.6, 10.5)
2.00 (ddd, 13.6, 7.2, 1.4)
—
49.5
53.2
—
2.35 (dd, 18.1, 2.6)
2.65 (dd, 18.1, 2.9)
—
3
4
66.0
45.8
211.4
53.9
2.43 (dd, 18.1, 2.6)
2.78 (d, 18.1)
—
5
6
87.5
82.6
87.5
82.4
—
—
7
127.0
139.6
69.2
6.06 (d, 15.5)
125.7
140.7
68.9
6.02 (dd, 15.4, 1.5)
6.17 (dd, 15.4, 5.5)
4.38 (dqd, 6.4, 5.5, 1.5)
1.28 (3H, d, 6.4)
1.18 (3H, s)
8
6.03 (d, 15.5, 4.5)
4.35 (qd, 6.4, 4.5)
1.28 (d, 6.4)
9
10
11
12
24.0
24.0
16.3
0.88 (3H, s)
19.2
77.1
3.68 (d, 7.4)
78.4
3.65 (d, 7.5)
3.77 (dd, 7.4, 2.1)
1.12 (3H, s)
3.91 (dd, 7.5, 2.9)
1.18 (3H, s)
13
19.5
19.2
Multiplicities and coupling constants in Hz are in the parentheses.

Vol. 67, No. 12 (2019)
Chem. Pharm. Bull.
1343
(160mg) in fractions 223–234 was purified by HPLC (Inertsil
ODS-3, H2O–MeOH, 1:1) to give 36.4mg of 3 from the peak
at 10.2min. The residue (88.1mg) in fractions 240–249 was
purified by HPLC (Cosmosil πNAP, H2O–MeOH, 2:3) to give
32.8mg of 4 from the peak at 10.2min. The residue (2.81g)
in fractions 66–71 of silica gel CC was separated by ODS
CC (Φ=50mm, L=25cm), and eluted with a linear gradient
solvent system from MeOH–H2O (1:9, 2L) to MeOH–H2O
(9:1, 2L), 10g-fractions being collected. The residue (136mg)
in fractions 106–129 was purified by HPLC (Cosmosil PBr,
H2O–MeOH, 3:2) to give 15.0mg of 7 and 19.9mg of 8 from
the peaks at 25.0min and 26.4min, respectively.
Fig. 6. Results of the Modified Mosher’s Method of Compound 5
Figures are δ5b–δ5a in ppm.
The residue (64.5g) in fractions 5–19 of a Diaion HP-20
πNAP (Nacalai Tesque; Φ=10mm, L=25cm, 4.0mL/min) CC was subjected to silica gel CC (Φ=80mm, L=40cm),
and Cosmosil PBr (Nacalai Tesque; Φ=10mm, L=25cm, and eluted with CHCl3 (6L), CHCl3–MeOH (99:1, 6L), (49:1,
4.0mL/min)], and the eluate was monitored with photodiode 6L), (97:3, 6L), (19:1, 6L), (37:3, 6L), (9:1, 6L), (7:1, 6L),
arrtay (200–400nm) and refractive index monitors. Crude (17:3, 6L), (33:7, 6L), (4:1, 6L), (3:1, 6L), and (7:3, 6L),
β-glucosidase (Sumizyme BGA) was a generous gift from 1L-fractions being collected. The residue (3.00g out of 12.3g)
Shin Nihon Chemical Co., Ltd. (Anjo, Aichi, Japan) (Lot No. in fractions 53–62 was separated by ODS CC (Φ=50mm,
0930708-03). Crude naringinase was from Amano Enzyme L=25cm), and eluted with a linear gradient solvent system
Inc. (Nagoya, Aichi, Japan) as a gift (Lot No. NAG1252306). from MeOH–H2O (1:9, 2L) to MeOH–H2O (9:1, 2L), 10g-
MTPAs were purchased from FUJIFILM Wako Pure Chemi- fractions being collected. The residue (63.0mg) in fractions
cal Corporation (Osaka, Japan).
243–246 was purified by HPLC (Inertsil ODS-3, H2O–MeOH,
Plant Material Leaves of D. maritima were collected in 3:7) to give 10.8mg of 1 from the peak at 5.9min. The
Taketomi-cho, Yaeyama-gun, Okinawa, Japan, in November, residue (196mg) in fractions 247–263 was purified by HPLC
2003 and a voucher specimen was deposited in the Herbarium (Inertsil ODS-3, H2O–MeOH–CH3COOH, 7:13:0.1) to give
of Pharmaceutical Sciences, Graduate School of Biomedical 3.8mg of 2 from the peak at 18.4min.
and Health Sciences, Hiroshima University (03-DM-Oki-
Ebenamarioside A (1) Colorless amorphous powder, [α]D25
nawa-1105). The plant was identified by one of the authors +15.1 (c=0.72, MeOH); IR νmax (film) cm−1: 3439, 2928, 2871,
(M.A.).
1745, 1636, 1458, 1072; 1H-NMR (600MHz, pyridine-d5):
Extraction and Isolation Air-dried leaves of D. maritima Table 1; 13C-NMR (150MHz, pyridine-d5): Table 2; HR-ESI-
(7.80kg) were extracted with MeOH (45L) three times. The MS (positive-ion mode): m/z: 819.4499 [M+Na]+ (Calcd for
MeOH extract was concentrated to 6L and then washed with C42H68O14Na: 819.4501).
n-hexane (6L, 245g). The methanolic layer was concentrated
Ebenamarioside B (2) Colorless amorphous powder,
to a viscous gum. The gummy mass was suspended in H2O [α]D27 −5.6 (c=0.43, MeOH); IR νmax (film) cm−1: 3393, 2936,
1
(6L), and then partitioned with ethyl acetate (EtOAc) (6L) and 2872, 1686, 1043; H-NMR (600MHz, pyridine-d5): Table 1;
1-butanol (1-BuOH) (6L), successively, to give 397g and 216g 13C-NMR (150MHz, pyridine-d5): Table 2; HR-ESI-MS (neg-
of EtOAc and 1-BuOH-soluble fractions. The remaining water- ative-ion mode): m/z: 795.4529 [M−H]− (Calcd for C42H67O14:
layer was concentrated to give a H2O-soluble fraction (245g). 795.4525).
The 1-BuOH-soluble fraction was subjected to a Diaion HP-20
Ebenamarioside C (3) Colorless amorphous powder,
CC (Φ=80mm, L=50cm), and eluted with H2O–MeOH [α]D27 +14.0 (c=0.10, pyridine); IR νmax (film) cm−1: 3353,
(4:1, 5L), (3:2, 5L), (2:3, 5L), and (1:4, 5L), and MeOH 2927, 2876, 1705, 1045; 1H-NMR (600MHz, pyridine-d5):
(5L), 1L-fractions being collected.
Table 1; 13C-NMR (150MHz, pyridine-d5): Table 2; HR-ESI-
The residue (17.5g) in fractions 4–7 of a Diaion HP-20 CC MS (positive-ion mode): m/z: 835.4447 [M+Na]+ (Calcd for
was subjected to silica gel CC (Φ=40mm, L=55cm), and C42H68O15Na: 835.4450).
eluted with CHCl3 (3L), CHCl3–MeOH (99:1, 3L), (49:1,
Ebenamarioside D (4) Colorless amorphous powder,
3L), (97:3, 3L), (19:1, 3L), (37:3, 3L), (9:1, 3L), (7:1, 3L), [α]D26 −5.6 (c=0.86, MeOH); IR νmax (film) cm−1: 3400, 2932,
1
(17:3, 3L), (33:7, 3L), (4:1, 3L), (3:1, 3L), and (7:3, 3L), 2877, 1740, 1071; H-NMR (600MHz, pyridine-d5): Table 1;
500mL-fractions being collected. Compounds 6 (266mg) 13C-NMR (150MHz, pyridine-d5): Table 2; HR-ESI-MS (posi-
and 5 (437mg) were obtained in fractions 18–19 and 23–26, tive-ion mode): m/z: 835.4449 [M+Na]+ (Calcd C42H68O15Na:
respectively.
835.4450).
The residue (29.2g) in fractions 11–14 of a Diaion HP-20
CC was subjected to silica gel CC (Φ=50mm, L=54.5cm),
Compound 5
Colorless amorphous powder, [α]D26 +10.2 (c=0.33, MeOH);
and eluted with CHCl3 (3L), CHCl3–MeOH (99:1, 3L), (49:1, IR νmax (film) cm−1: 3379, 2930, 2876, 1450, 1375, 1135, 1043;
3L), (97:3, 3L), (19:1, 3L), (37:3, 3L), (9:1, 3L), (7:1, 3L), 1H-NMR (600MHz, CD3OD): Table 3; 13C-NMR (150MHz,
(17:3, 3L), (33:7, 3L), (4:1, 3L), (3:1, 3L), and (7:3, 3L), CD3OD): Table 3; HR-ESI-MS (positive-ion mode): m/z:
500mL-fractions being collected. The residue (3.00g out of 265.1411 [M+Na]+ (Calcd C13H22O4Na: 265.1410).
6.74g) in fractions 57–65 of silica gel CC was separated by
Compound 6
ODS CC (Φ=50mm, L=25cm), and eluted with a linear gra-
Colorless syrup, [α]D26 approx. 0.00 (c=0.81, MeOH); IR
dient solvent system from MeOH–H2O (1:9, 2L) to MeOH– νmax (film) cm−1: 3414, 2932, 2877, 1715, 1455, 1242, 1042;
H2O (9:1, 2L), 10g-fractions being collected. The residue 1H-NMR (600MHz, CD3OD): Table 3; (CDCl3) δ: 6.22 (1H,

1344
Chem. Pharm. Bull.
Vol. 67, No. 12 (2019)
dd, J=15.4, 5.4Hz, H-8), 5.91 (1H, dd, J=15.4, 1.2Hz, H-7), MS (positive-ion mode) m/z: 633.4000 [M−H]− (Calcd for
4.45 (1H, qd, J=6.4, 5.4Hz, H-9), 3.91 (1H, dd, J=8.3, C36H57O9: 633.3997); mesembryanthenoidigenic acid (1c): [α]D25
3.0Hz, H-12a), 3.75 (1H, d, J=8.3Hz, H-12b), 2.65 (1H, d, +24.9 (c=0.14, MeOH), HR-ESI-MS (positive-ion mode) m/z:
J=18.3Hz, H-4a), 2.60 (1H, dd, J=18.3, 2.0Hz. H-4b), 2.55 471.3473 [M −H]− (Calcd for C30H47O4: 471.3469).10,11)
(1H, dd, J=18.1, 3.0Hz, H-2a), 2.42 (1H, dd, J=18.1, 2.0Hz,
Ebenamarioside C (3) (9.5mg) in 1mL of H2O was hy-
H-2b), 1.33 (3H, d, J=6.4Hz, H3-10), 1.21 (3H, s, H3-13); 0.99 drolyzed with crude naringinase (15mg) at 37°C for 72h.
(3H, s, H3-11); 13C-NMR (150MHz, CD3OD): Table 3; (CDCl3) The reaction mixture was subjected silica gel CC (Φ=2cm,
δ: 208.6 (C-3), 139.8 (C-8), 123.7 (C-7), 85.5 (C-5), 81.6 (C-6), L=15cm) with increasing amounts of MeOH in CHCl3
77.2 (C-12), 68.1 (C-9), 52.6 (C-4), 52.5 (C-2), 47.7 (C-1), [CHCl3-MeOH (9:1, 50mL), (9:1, 100mL to 7:3, 100mL,
24.0 (C-10), 18.7 (C-13), 15.6 (C-11); CD (c=6.46×10−5 M, linear gradient), (7:3, 50mL) and (1:1, 100mL)], 10-mL
MeOH) ∆ε (nm): +0.64 (241), −0.30 (296); HR-ESI-MS (pos- fractions being collected. 29-O-β-D-glucopyranosyl estre (3a)
itive-ion mode): m/z: 263.1254 [M+Na]+ (Calcd C13H20O4Na: was obtained in fractions 11–13. Similarly ebenamarioside
263.1254).
C (3) (15mg) in 1mL of H2O was hydrolyzed with crude
Sugar Analysis About 500µg each of 1–4 was hydrolyzed β-glucosidase and silica gel CC with the same condition as
with 1M HCl (0.1mL) at 90°C for 2h. The reaction mixtures above gave 5.2mg of an aglycone (3c) and 5.6mg of 29-O-β-
were partitioned with an equal amount of EtOAc (0.1mL), and D-glucopyranoside (3b) in fractions 5–8 and 11–13, respec-
the water layers were analyzed by HPLC with a chiral detec- tively. 2α,3β,29-Trihydroxyolean-12-en-28-oic acid 28-O-β-
tor (JASCO OR-4090) on an amino column [InertSustain NH2, D-glucopyranosyl ester (3a): Amorphous powder, [α]D24 +18.7
4.6×250mm (GL Science Inc.), CH3CN–H2O (4:1), flow rate: (c=0.08, MeOH), 13C-NMR (150MHz, pyridine-d5, MeOH-d4
1mL/min]. All the hydrolyzates gave a peak for D-glucose at and DMSO-d6): Table 1, HR-ESI-MS (positive-ion mode)
10.9min with positive optical rotation signs. The peaks were m/z: 673.3922 [M+Na]+ (Calcd for C36H58O10Na: 673.3922).
identified by co-chromatography with an authentic sample.
2α,3β,29-Trihydroxyolean-12-en-28-oic
acid
29-O-β-D-
Enzymatic Hydrolysis of 1, 3 and 4 to 1a, 1b and 1c, 3a, glucopyranoside (3b): Amorphous powder; [α]D24 +9.6
3b and 3c, and 4a, 4b and 4c, Respectively Ebenamari- (c=0.28, MeOH); IR νmax (film) cm−1: 3379, 2934, 2872, 1687,
oside A (1) (9.8mg) in 1mL of H2O hydrolyzed with 15mg 1459, 1050; H-NMR (600MHz, pyridine-d5) δ: 5.43 (1H, dd,
1
of crude glucosidase at 37°C for 72h. The reaction mixture J=3.3, 3.3Hz, H-12), 4.87 (1H, d, J=7.7Hz, H-1″), 4.60 (1H,
was subjected to silica gel CC (Φ=2cm, L=15cm) with dd, J=11.8, 2.2Hz, H-6″a), 4.44 (1H, dd, J=11.8, 5.3Hz,
increasing amounts of MeOH in CHCl3 [CHCl3–MeOH H-6″b), 4.28 (2H, m, H-3″ and 4″), 4.10 (2H, m, H-2 and 2″),
(9:1, 50mL), (9:1, 100mL to 7:3, 100mL, linear gradient), 4.00 (1H, m, H-5″), 3.94 (1H, d, J=9.1Hz, H-29a), 3.43 (1H,
(7:3, 50mL) and (1:1, 100mL)], 10-mL fractions being col- d, J=9.1Hz, H-29b), 3.40 (1H, d, J=9.6Hz, H-3), 3.35 (1H,
lected. An aglycone (1c) (2.8mg) was obtained in fractions dd, J=13.3, 3.1Hz, H-18), 2.25 (1H, dd, J=12.4, 4.2Hz,
5–6 and the monosaccharide mixture (1a and 1b) (5.5mg) in H-1a), 2.18 (1H, ddd, J=13.3, 13.0, 3.1Hz, H-15a), 2.10 (1H,
fractions 9–12. The mixture fraction was purified by HPLC m, H-11a), 2.05 (1H, m, H-22a), 2.01 (1H, m, H-19a), 1.98 (2H,
(ODS-3, H2O–MeOH, 4:1) to give 1.9mg of 1a and 3.0mg of m, H2-16), 1.95 (1H, m, H-11b), 1.84 (1H, brd, J=13.6Hz,
1b from the peaks at 4.8min and 8.7min, respectively. Me- H-22b), 1.76 (1H, m, H-9), 1.75 (1H, m, H-21a), 1.56 (1H,
sembryanthenoidigenic acid 28-O-β-D-glucopyranosyl ester m, H-6a), 1.51 (1H, m, H-7a), 1.43 (2H, m, H-19b and 21b),
(1a): Amorphous powder, [α]D25 +31.4 (c=0.09, MeOH), HR- 1.38 (1H, m, H-6b), 1.31 (1H, m, H-7b), 1.28 (1H, m, H-1b),
ESI-MS (positive-ion mode) m/z: 657.3975 [M+Na]+ (Calcd 1.27 (3H, s, H3-23), 1.23 (3H, s, H3-27), 1.21 (3H, s, H3-30),
for C36H58O9Na: 657.3973)9); mesembryanthenoidigenic acid 1.20 (1H, m, H-15b), 1.08 (3H, s, H3-24), 1.02 (3H, s, H3-26),
30-O-β-D-glucopyranoside (1b): Amorphous powder, [α]D25 1.01 (1H, m, H-5), 0.99 (3H, s, H3-25, 13C-NMR (150MHz,
+14.5 (c=0.15, MeOH), IR νmax (film) cm−1: 3370, 2929, 2867, pyridine-d5): Table 2; HR-ESI-MS (positive-ion mode) m/z:
1
1686, 1636, 1457, 1077; H-NMR (600MHz, pyridine-d5) δ: 649.3951 [M −H]− (Calcd for C36H57O10: 649.3946). 2α,3β,29-
5.47 (1H, dd, J=3.3, 3.3Hz, H-12), 4.61 (1H, dd, J=11.7, Trihydroxyolean-12-en-28-oic acid (3c): Amorphous powder;
2.1Hz, H-6″a), 4.44 (1H, dd, J=11.7, 5.3Hz, H-6″b), 4.87 (1H, [α]D24 +34.6 (c=0.26, MeOH); HR-ESI-MS (positive-ion
d, J=7.7Hz, H-1″), 4.28 (1H, dd, J=8.7, 8.4Hz, H-4″), 4.27 mode) m/z: 487.3422 [M−H]− (Calcd for C30H47O5: 487.3418).
(1H, dd, J=8.4, 8.3Hz, H-3″), 4.10 (1H, dd, J=8.3, 7.7Hz,
Ebenamarioside C (4) (16.6mg) in 1mL of H2O was hy-
H-2″), 4.00 (1H, m, H-5″), 3.95 (1H, d, J=9.3Hz, H-29a), drolyzed with crude β-glucosidase 37°C for 72h. The reac-
3.46 (1H, dd, J=10.2, 5.6Hz, H-3), 3.44 (1H, d, J=9.3Hz, tion mixture was separated by silica gel CC with the same
H-29b), 3.37 (1h, dd, J=13.6, 3.9Hz, H-18), 2.19 (1H, ddd, condition as above to give 4.1mg of an aglycone (4c) and
J=13.2, 13.0, 3.6Hz, H-15a), 2.12 (1H, ddd, J=13.0, 13.0, 9.4mg of a mixture of two monoglucosidic compounds (4a
3.0Hz, H-16a), 2.08 (1H, ddd, J=14.0, 14.0, 4.0Hz, H-22a), and 4b) in fractions 5–8 and 10–15, respectively. The mixture
2.04 (1H, dd, J=13.9, 13.8Hz, H-19a), 1.93 (3H, m, H2-11 and was purified by HPLC (ODS-3, H2O–MeOH, 4:1) to afford
H-16b), 1.84 (3H, m, H2-2 and H-22b), 1.75 (1H, ddd, J=13.6, 6.3mg of 28-O-β-D-glucopyranosyl ester (4a) and 1.9mg of
13.3, 3.6Hz, H-21a), 1.66 (1H, dd, J=11.0, 6.7Hz, H-9), 1.57 30-O-β-D-glucopyranoside (4b) from the peaks at 3.9min and
(1H, m. H-6a), 1.56 (1H, m, H-1a), 1.51 (1H, ddd, J=12.6, 9.2min, respectively. 2α,3β,30-Trihydroxyurs-12-en-28-oic
12.6, 3.5Hz, H-7a), 1.45 (1H, dd, J=13.6, 4.2Hz, H-19b), 1.43 acid 28-O-β-D-glucopyranosyl ester (4a): Amorphous powder;
(1H, m, H-21b), 1.38 (1H, m, H-6b), 1.33 (1H, m, H-7b), 1.26 [α]D24 +17.9 (c 0.31, MeOH); IR νmax (film) cm−1: 3373, 2925,
1
(3H, s, H3-27), 1.25 (3H, s, H3-23), 1.21 (3H, s, H3-30), 1.18 2877, 1732, 1456, 1073; H-NMR (600MHz, pyridine-d5) δ:
(1H, m, H-15b), 1.04 (3H, s, H3-24), 1.03 (3H, s, H3-26), 1.01 6.31 (1H, d, J=8.1Hz, H-1′), 5.48 (1H, dd, J=3.4, 3.4Hz,
(1H, m, H-1b), 0.91 (3H, s, H3-25), 0.87 (1H, brd, J=10.4Hz, H-12), 4.47 (1H, dd, J=11.8, 2.4Hz, H-6′a), 4.41 (1H, dd,
H-5); 13C-NMR (150MHz, pyridine-d5): Table 2, HR-ESI- J=11.8, 4.4Hz, H-6′b), 4.39 (1H, dd, J=9.5, 8.9Hz, H-4′),

Vol. 67, No. 12 (2019)
Chem. Pharm. Bull.
1345
4.31 (1H, dd, J=8.9, 8.7Hz, H-3′), 4.23 (1H, dd, J=8.7,
(R)-MTPA ester of 5 (5a): amorphous powder; 1H-NMR
8.1Hz, H-2′), 4.11 (1H, ddd, J=11.1, 9.6, 4.3Hz, H-2), 4.04 (600MHz, CDCl3) δ: 7.35–7.55 (5H, m, aromatic protons), 6.05
(1H, ddd, J=9.5, 4.4, 2.4Hz, H-5′), 3.93 (1H, dd, J=10.7, (1H, d, J=15.2Hz, H-7), 6.02 (1H, dd, J=15.2, 4.9Hz, H-8),
2.9Hz, H-30a), 3.88 (1H, dd, J=10.7, 5.6Hz, H-30b), 3.39 5.66 (1H, qd, J=6.5, 4.9Hz, H-9), 5.44 (1H, dddd, J=10.5,
(1H, d, J=9.4Hz, H-3), 2.65 (1H, d, J=11.5Hz, H-18), 2.49 10.5, 7.2, 7.2Hz, H-3), 3.87 (1H, d, J=8.2Hz, H-12a), 3.77
(1H, ddd, J=13.8, 13.7, 4.4Hz, H-15a), 2.26 (1H, dd, J=12.5, (1H, dd, J=8.2, 2.0Hz, H-12b), 3.53 (3H, s, –OMe), 3.51 (3H,
4.4Hz, H-1a), 2.21 (1H, ddd, J=13.5, 13.4, 4.2Hz, H-16a), s, –OMe), 2.18 (1H, ddd, J=13.6, 7.2, 1.5Hz, H-4), 2.03 (1H,
2.07 (1H, m, H-22a), 2.06 (1H, m, H-16b), 2.05 (2H, m, H2-11), ddd, J=13.6, 7.2, 1.5Hz, H-2), 1.73 (1H, dd, J=13.6, 10.5Hz,
2.01 (1H, m, H-19), 1.86 (2H, m, H2-21), 1.84 (1H, m, H-22b), H-4), 1.70 (1H, ddd, J=13.6, 10.5, 2.0Hz, H-2), 1.45 (3H, d,
1.74 (1H, dd, J=10.0, 7.4Hz, H-9), 1.54 (1H, m, H-7a), 1.52 J=6.5Hz, H3-10), 1.08 (3H, s, H3-13), 0.90 (3H, s, H3-11); HR-
(1H, m, H-6a), 1.40 (1H, m, H-7b), 1.38 (1H, m, H-6b), 1.27 ESI-MS (positive-ion mode) m/z: 697.2206 [M+Na]+ (Calcd
(1H, m, H-1b), 1.26 (3H, s, H3-23), 1.20 (3H, s, H3-27), 1.19 for C33H36O8F6Na: 697.2207).
(3H, s, H3-26), 1.18 (1H, m, H-15b), 1.16 (1H, m, H-20), 1.11
(S)-MTPA ester of 5 (5b): amorphous powder; 1H-NMR
(3H, d, J=6.4Hz, H3-29), 1.09 (3H, s, H3-24), 1.04 (3H, s, (600MHz, CDCl3) δ: 7.35–7.55 (10H, m, aromatic protons),
H3-25), 1.01 (1H, d, J=12.1Hz, H-5); 13C-NMR (150MHz, 6.11 (1H, d, J=15.5Hz, H-7), 6.08 (1H, dd, J=15.5, 5.5Hz,
pyridine-d5): Table 2; HR-ESI-MS (positive-ion mode) m/z: H-8), 5.63 (1H, qd, J=6.5, 5.5Hz, H-9), 5.44 (1H, dddd,
673.3925 [M +Na]+ (Calcd for C36H58O10Na: 673.3922).
J=10.7, 10.7, 7.1, 7.1Hz, H-3), 3.88 (1H, d, J=8.3Hz, H-12a),
30-O-β-D- 3.78 (1H, dd, J=8.3, 2.1Hz, H-11b), 3.52 (3H, s, –OMe), 3.49
2α,3β,30-Trihydroxyurs-12-en-28-oic
acid
glucopyranoside (4b): Amorphous powder; [α]D25 +5.5 (3H, s, –OMe), 2.25 (1H, ddd, J=13.6, 7.1, 1.5Hz, H-4), 1.99
(c=0.10, MeOH); IR νmax (film) cm−1: 3370, 2930, 2871, (1H, ddd, J=13.6, 7.1, 1.5Hz, H-2), 1.85 (1H, ddd, J=13.6,
1686, 1457, 1050; 1H-NMR (600MHz, pyridine-d5) δ: 5.45 10.7Hz, H-4), 1.62 (1H, ddd, J=13.6, 10.7, 2.1Hz, H-2),
(1H, dd, J=3.1, 3.1Hz, H-12), 4.90 (1H, d, J=7.8Hz, H-1″), 1.40 (3H, d, J=6.5Hz, H3-10), 1.10 (3H, s, H3-13), 0.91 (3H,
4.62 (1H, dd, J=11.7, 2.2Hz, H-6″a), 4.45 (1H, dd, J=11.7, s, H3-11); HR-ESI-MS (positive-ion mode) m/z: 697.2206
5.3Hz, H-6″b), 4.30 (1H, dd, J=8.8, 8.9Hz, H-3″), 4.27 (1H, [M+Na]+ (Calcd for C33H36O8F6Na: 697.2207).
dd, J=8.9, 8.9Hz, H-4″), 4.10 (1H, m, H-2), 4.09 (1H, m,
NaBH4 Reduction of 6 To a solution of 6 (11.0mg) in
H-2″), 4.07 (1H, m, H-30a), 4.03 (1H, m, H-5″), 3.89 (1H, dd, MeOH (1mL) was added 8.2mg of NaBH4 and the reaction
J=9.5, 3.5Hz, H-30b), 3.40 (1H, d, J=9.4Hz, H-3), 2.65 (1H, mixture was stirred for 5min at 25°C. Excess NaBH4 was
d, J=11.3Hz, H-18), 2.32 (1H, ddd, J=13.8, 13.8, 4.4Hz, quenched by the addition of 1mL of acetone and then the
H-15a), 2.24 (1H, dd, J=12.5, 4.4Hz, H-1a), 2.07 (1H, m, reaction mixture was evaporated to dryness. The resultant
H-16a), 2.04 (1H, m, H-21a), 2.03 (1H, m, H-22a), 2.02 (2H, residue was purified by HPLC [Inertsil ODS-3, 6×250mm,
m, H2-11), 1.96 (1H, m, H-16b), 1.94 (1H, m, H-22b), 1.75 H2O–MeOH (1:4), flow rate: 1.6mL/min] to give 3.1mg of
(2H, m, H-9 and 19), 1.66 (1H, m, H-21b), 1.55 (1H, m, H-6a), 6c (=5) and 5.8mg of 6d from the peaks at 10.9min and
1.54 (1H, m, H-7a), 1.39 (1H, m, H-20), 1.38 (1H, m, H-6b), 13.3min. respectively.
1.37 (1H, m, H-7b), 1.28 (3H, s, H3-23), 1.28 (1H, m, H-1b),
Compound 6c: amorphous powder; [α]D24 +7.1 (c=0.31,
1.17 (3H, s, H3-27), 1.16 (1H, m, H-15b), 1.08 (3H, s, H3-24), MeOH); HR-ESI-MS (positive-ion mode) m/z: 265.1409
1.04 (3H, s, H3-26), 1.03 (1H, m, H-5), 1.01 (3H, d, J=6.4Hz, [M+Na]+ (Calcd for C13H22O4Na: 265.1410).
H3-29), 0.98 (3H, s, H3-25); 13C-NMR (150MHz, pyridine-
Compound 6d: amorphous powder; [α]D24 −5.5 (c=0.29,
d5): Table 2; HR-ESI-MS (positive-ion mode) m/z: 649.3948 MeOH); IR νmax (film) cm−1: 3402, 2929, 2889, 1604, 1453,
[M−Na]− (Calcd for C30H57O10: 649.3946).
1381, 1101, 1053; H-NMR (600MHz, CD3OD) δ: 6.02 (1H,
1
2α,3β,30-Trihydroxyurs-12-en-28-oic acid (4c): Amorphous dd, J=15.5, 5.6Hz, H-8), 5.82 (1H, dd, J=15.5, 1.0Hz, H-7),
powder; [α]D23 +17.7 (c=0.13, EtOH); HR-ESI-MS (positive- 4.33 (1H, qd, J=6.4, 5.6Hz, H-9), 4.12 (1H, d, J=6.9Hz,
ion mode) m/z: 487.3423 [M−H]− (Calcd for C30H47O5: H-12a), 4.02 (1H, dd, J=5.7, 5.7Hz, H-3), 3.76 (1H, dd,
487.3418).
J=6.9, 2.3Hz, H-12b), 2.13 (1H, dd, J=15.5, 5.7Hz, H-4a),
Preparation of (R)- and (S)-MTPA Esters (5a and 5b) 2.02 (1H, ddd, J=15.3, 5.7, 2.2Hz, H-2a), 1.84 (1H, dd,
from 5 A solution of 5 (1.0mg) in 0.5mL of dry CH2Cl2 J=15.5, 2.2Hz, H-4b), 1.73 (1H, dd, J=15.3, 2.3Hz, H-2b),
were reacted with (R)-MTPA (21.5mg) in the presence of 1.25 (3H, d, J=6.4Hz, H3-10), 1.13 (3H, s, H3-13), 0.86 (3H,
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochlo- s, H3-11); 13C-NMR (150MHz, CD3OD) δ: 139.4 (C-8), 126.8
ride (EDC) (13.4mg) and N,N-dimethyl-4-aminopyridine (C-7), 87.2 (C-5), 82.5 (C-6), 76.3 (C-12), 69.1 (C-9), 66.0
(4-DMAP) (16.1mg). The mixture was then occasionally (C-3), 48.0 (C-1), 45.1 (C-4), 44.9 (C-2), 24.1 (C-10), 19.7
stirred at 37°C for 24h. After the addition of CHCl3 (1.5mL), (C-13), 16.3 (C-11); HR-ESI-MS (positive-ion mode) m/z:
the reaction mixture was successively washed with H2O 265.1408 [M+Na]+ (Calcd for C13H22O4Na: 265.1410).
(1mL), 1M HCl (1mL), NaHCO3-saturated H2O (1mL), and
Cytotoxic Activity toward Human Lung Adenocarci-
brine (1mL). The organic layer was dried with Na2SO4 and noma, A549 Cells Cytotoxic activity toward lung adeno-
evaporated under reduced pressure. The residue was purified carcinoma cells was determined by colorimetric cell viability
by preparative TLC [silica gel (0.25mm thickness), being ap- assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
plied for 8cm width, with development with CHCl3–MeOH lium bromide (MTT). Lung adenocarcinoma cell line A549
(19:1) for 9cm and then eluting with CHCl3–MeOH (1:1)] was purchased from the JCRB Cell Bank, Japan. A549
to furnish an ester 5a (0.5mg) from the band at Rf =0.67. cells were cultured in Dulbecco’s modified Eagle’s medium
Through the same procedure, 5b (0.6mg, Rf =0.59) were supplemented with 10% heat inactivated FCS, and kanamycin
prepared from 5 (1.0mg) using (S)-MTPA (25.4mg), EDC (100 µg/mL) and amphotericin B (5.6µg/mL). In a 96-well
(15.8mg), and 4-DMAP (15.5mg), respectively.
plate, 1µL aliquots of sample solutions and the cancer cells

1346
Chem. Pharm. Bull.
Vol. 67, No. 12 (2019)
(5 ×103 cells/well) in 100µL medium were added to each Japan Society for the Promotion of Science (Nos. 22590006,
well, and then the plate incubated at 37°C under a 5% CO2 23590130, 25860078, 15H04651, 17K08336 and 18K06740).
atmosphere for 72h. A solution (100µL) of MTT (0.5mg/mL)
was then added to each well and the incubation was continued
Conflict of Interest The authors declare no conflict of
for a further 1h. The absorbance of each well was measured interest.
at 540nm using a Molecular Devices Versamax tunable mi-
croplate reader. DMSO was used as a negative control and
Supplementary Materials The online version of this ar-
doxorubicin as a positive control. The cytotoxic activity was ticle contains supplementary materials.
calculated as:
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