Megastigmane glucosides and megastigmanes From the leaves of Meliosma lepidota ssp. squamulata

Article abstract of DOI:10.1248/cpb.c15-00350

From the leaves of Meliosma lepidota ssp. squamulata, megastigmane glucosides with spiro-structures and megastigmanes were isolated. Their structures were determined by X-ray crystallographic analyses and spectroscopic investigation. The absolute structures of the megastigmanes were determined by the modified Mosher's method.

Full text of DOI:10.1248/cpb.c15-00350

608
Chem. Pharm. Bull. 63, 608–616 (2015)
Vol. 63, No. 8
Regular Article
Megastigmane Glucosides and Megastigmanes from the Leaves of
Meliosma lepidota ssp. squamulata
Yuka Uemura,a Mio Iwami,a Susumu Kawakami,b Sachiko Sugimoto,a Katsuyoshi Matsunami,a
,a,b
Hideaki Otsuka,* Takakazu Shinzato,c Masatoshi Kawahata,d and Kentaro Yamaguchid
a Graduate School of Biomedical Sciences, Hiroshima University; 1–2–3 Kasumi, Minami-ku, Hiroshima 734–8553,
b
Japan: Faculty of Pharmacy, Yasuda Women’s University; 6–13–1 Yasuhigashi, Asaminami-ku, Hiroshima 731–0153,
c
Japan: Subtropical Field Science Center, Faculty of Agriculture, University of the Ryukyus; 1 Sembaru, Nishihara-
d
cho, Nakagami-gun, Okinawa 903–0213, Japan: and Faculty of Pharmaceutical Sciences, Tokushima Bunri
University, Kagawa Campus; 1314–1 Shido, Sanuki, Kagawa 769–2193, Japan.
Received April 21, 2015; accepted May 14, 2015
From the leaves of Meliosma lepidota ssp. squamulata, megastigmane glucosides with spiro-structures
and megastigmanes were isolated. Their structures were determined by X-ray crystallographic analyses and
spectroscopic investigation. The absolute structures of the megastigmanes were determined by the modified
Mosher’s method.
Key words Meliosma lepidota ssp. squamulata; Sabiaceae; megastigmane glucoside; megastigmane
Meliosma lepidota Blume ssp. squamulata (HANCE) proton (δH 4.29) resonance. The 13C-NMR exhibited six sig-
BEUSEKOM (syn. M. squamulata HANCE) is a tall evergreen tree nals assignable as those of the glucopyranose moiety, and the
belonging to the family Sabiaceae. However, the International remaining 13 signals comprised those of four methyls, three
Plantꢀ Namesꢀ Indexꢀ currentlyꢀ classifiesꢀ Meliosma species in methylenes, three oxymethines, two oxygenated tertiary car-
the Meliosmaceae.1) Whereas the latest taxonomic publication bons and one quaternary carbon (Table 1). From the above
observes the classical taxon Sabiaceae.2) The Sabiaceae com- evidence, 1 was expected to be a megastigmane glucoside
prisesꢀaboutꢀoneꢀhundredꢀspeciesꢀinꢀfiveꢀmajorꢀgenera,ꢀandꢀM. and due to three degrees of unsaturation, the aglycone must
1
lepidota ssp. squamulata grows wild in the Amami Islands, have a bicyclic system. The H–1H correlation spectroscopic
the Okinawa Islands, Taiwan and Southern China.3) This plant (COSY) spectrum indicated the presence of two proton–
isꢀalsoꢀknownꢀasꢀaꢀfeedingꢀplantꢀforꢀtheꢀlarvaeꢀofꢀaꢀbutterfly,ꢀ proton sequences, –C(2)H–C(3)H–C(4)H2– and –CH(7)2–
Dichorragia nesimachus.4) There has been no report on the CH(8)2–C(9)H–C(10)H3, and diagnostic heteronulear multiple
constituents of the title plant and its medicinal use is also bond correlation (HMBC) spectroscopy (Fig. 2) suggested a
uncertain. This paper deals with isolation work on the plant.
planar structure of 1, as shown in Fig. 1, and the position of
sugar attachment to be the hydroxy group at the 2-position.
However, the crucial HMBC correlation from H-9 (δH 4.10)
Results and Discussion
Chromatographic isolation work on the 1-butyl alcohol and C-6 (δC 92.9), which supports the presence of a spiro-ring
(BuOH)-soluble fraction of a methanol (MeOH) extract of was, not observed (Fig. 2). Accordingly, 1ꢀ wasꢀ subjectedꢀ toꢀ
theꢀ titleꢀ plantꢀ affordedꢀ fiveꢀ newꢀ megastigmaneꢀ glucosides,ꢀ X-ray crystallographic analysis and Fig. 3 shows an ORTEP
named melionosides A–E (1–5), and two megastigmanes, drawing of it. The absolute structure of the aglycone was
named meliosma-ionols A (6) and B (7), as well as four confirmedꢀbyꢀHPLCꢀanalysisꢀofꢀtheꢀglucose,ꢀwhichꢀwasꢀfoundꢀ
known megastigmane glucosides, scorospiroside (8),5) citro- to be of the D-series. Therefore, the structure of melionoside
side A (9),6) dihydrosyringin (10),7) and (Z)-hex-3-en-1-ol A (1) was elucidated to be (2R,3R,5R,6R,9R)-2,3,5-trihydroxy-
β-D-glucopyranoside (11)8) (Fig. 1). The structures of the new megastigman-6,9-epoxide 2-O-β-D-glucopyranoside, as shown
compounds were mainly elucidated by NMR spectroscopic in Fig. 1.
investigation. X-Ray crystallographic analysis was performed
Melionoside B (2), [α]D24 −25.3, was isolated as an amor-
for melionoside A (1),ꢀandꢀtheꢀmodifiedꢀMosher’sꢀmethodꢀwasꢀ phous powder and its elemental composition was the same as
applied for determination of the absolute stereochemistry of that of melionoside A (1). The 13C-NMR spectrum exhibited
meliosma-ionols (6, 7). Those of known glucosides were iden- the same functional groups as those of 1, and the coupling
tifiedꢀ byꢀ comparisonꢀ ofꢀ theirꢀ spectroscopicꢀ dataꢀ withꢀ thoseꢀ pattern of the protons on the six membered-ring was es-
reported in the literature.
sentially the same that of 1. Since other NMR spectroscopic
Melionoside A (1), [α]D25 −36.3, was isolated as colorless data showed good similarity to those of 1, melionoside B
needles and its elemental composition was determined to be (2) was expected to be an isomeric compound of 1 as to the
C19H34O9 by high-resolution (HR)-electrospray ionization 6-position and/or 9-position. A phase-sensitive (PS) nuclear
(ESI)-MS. The IR spectrum showed a strong absorption band Overhauser exchange spectroscopy (NOESY) correlation, i.e.,
at 3433cm−1 attributable to stretching signals for hydroxy between H-7b (δH 1.98) and H3-12 (δH 1.08) (Fig. 4b), as was
1
groups. The H-NMR spectrum exhibited three singlet methyl also seen for 1ꢀ(Fig.ꢀ4a),ꢀwhichꢀsuggestedꢀtheꢀconfigurationꢀatꢀ
(δH 1.07, 1.16 and 1.19), one doublet methyl (δH 1.20), three the 6-position was the same as that of 1, and that between H-9
oxymethine proton (δH 3.38, 3.88 and 4.10), and one anomeric and H3-11ꢀenabledꢀassignmentꢀofꢀtheꢀabsoluteꢀconfigurationꢀatꢀ
*ꢀToꢀwhomꢀcorrespondenceꢀshouldꢀbeꢀaddressed.ꢀ e-mail:ꢀhotsuka@hiroshima-u.ac.jp;ꢀotsuka-h@yasuda-u.ac.jp
© 2015 The Pharmaceutical Society of Japan

Vol. 63, No. 8 (2015)
Chem. Pharm. Bull.
609
Fig. 1. Structures of Compounds Isolated
the 9-position as S, which is the opposite to that of 1 (Fig. 3). hydroxy groups at the C-3, C-5 and C-9 positions were as-
1
Therefore, the structure of melionoside B (2) was established signed in the H-NMR spectrum for pyridine-d5, and HMBC
to be (2R,3R,5R,6R,9S)-2,3,5-trihydroxymegastiman-6,9-epox- correlations between 9-OH (δH 7.14) and C-8 (δC 40.8) and
ide 2-O-β-D-glucopyranoside, as shown in Fig. 1.
C-9 (δC 107.2), and H3-10 (δH 1.82) and C-8 and C-9 together
Melionoside C (3), [α]D27 −13.3, was isolated as an amor- with the COSY relationship between H2-7 and H2-8 clearly
phous powder and its elemental composition was determined demonstrated that the ketal carbon was at the 9-position (3a),
to be C19H34O10. Melionoside C (3)ꢀwasꢀfirstꢀisolatedꢀbyꢀHPLCꢀ such correlations being found in 3b, and PS-NOESY correla-
as two peaks, which were, however, found to be interconvert- tions between H3-10 (δH 1.81) and H3-13 (δH 1.47) in 3a and
ible. The NMR spectra revealed that the two closely related 9-OH (δH 7.00) and H3-13 (δH 1.47) in 3b substantiated that
compounds existed as minor (3a)ꢀ andꢀ majorꢀ (b) isomers in a the minor isomer had the 9Sꢀconfigurationꢀandꢀtheꢀmajorꢀoneꢀ
ratio of approximately 1:2 in CD3OD as well as in pyridine- the 9Rꢀ configurationꢀ (Fig.ꢀ 5).ꢀ Therefore,ꢀ theꢀ structureꢀ ofꢀ 3
d5. NMR spectra also revealed that the two isomers possessed was depicted as a mixture of two interconvertible isomers, 3a
glucopyranose as a sugar component, and the functionalities (minor) and bꢀ(major),ꢀasꢀshownꢀinꢀFig.ꢀ1.
of the aglycone moieties were similar to those of 1 and 2, ex-
Melionoside D (4), [α]D27 −11.8, was isolated as an amor-
cept for the presence of ketal carbons (δC 107.8 and 107.3) and phous powder and its elemental composition was determined
the absence of one oxygenated methine carbon. The degrees to be C19H32O9. A total of 19 signals was observed in the
of unsaturation also suggested that the aglycone moieties com- 13C-NMR spectrum, six of which were assignable as those
prised bicyclic ring systems. The hydrogen atoms of the three of a glucopyranose unit. The remaining 13 carbons com-

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Chem. Pharm. Bull.
Vol. 63, No. 8 (2015)
Table 1. 13C-NMR Spectroscopic Data for Melionosides A–E (1–5), and Meliosma-Ionols A and B (6 and 7) (150MHz, CD3OD)
C
1
2
3a
3b
4
5
6
7
1
46.0
91.3
67.8
44.2
77.3
92.9
28.7
36.0
78.2
21.2
23.7
19.9
27.6
105.9
75.5
78.1
71.4
78.0
62.4
47.7
92.4
67.8
43.4
76.3
92.6
29.0
36.2
78.8
21.1
23.7
19.2
27.7
106.1
75.6
78.3
71.5
78.1
62.5
47.3 (46.9)a)
91.5 (91.5)
67.9 (67.2)
44.2 (45.3)
76.7 (75.8)
95.1 (94.4)
27.7 (27.7)
40.8 (40.8)
107.8 (107.2)
28.3 (29.0)
24.0 (24.2)
20.0 (20.5)
28.5 (29.3)
106.0 (106.5)
75.6 (75.7)
78.2 (78.7)
71.4 (71.5)
78.1 (78.3)
62.4 (62.4)
46.0 (46.7)a)
92.0 (92.9)
67.8 (67.0)
43.3 (44.4)
76.8 (75.9)
95.2 (94.5)
27.4 (27.4)
40.2 (40.2)
107.3 (106.8)
28.0 (28.9)
24.8 (25.0)
19.8 (20.5)
28.2 (28.4)
105.9 (106.5)
75.5 (75.6)
78.2 (78.7)
71.4 (71.6)
78.1 (78.5)
62.5 (62.6)
46.6 (46.7)a)
90.8 (91.9)
67.2 (66.7)
41.7 (42.5)
80.3 (80.4)
73.8 (73.4)
30.6 (30.9)
97.6 (97.5)
149.6 (149.2)
19.9 (20.5)
22.4b)(23.0)
17.8 (18.2)
22.5b)(23.1)
106.1 (106.9)
75.5 (76.0)
78.2 (79.1)
71.4 (71.9)
78.2 (78.8)
62.5 (62.9)
44.0
92.5
43.5
74.9
71.0
73.5
127.4
143.0
26.3
40.0
69.1
23.3
20.8
26.0
18.2
46.3
79.2
71.7
38.1
35.4
78.0
33.9
35.6
69.9
23.7
17.7
21.4
16.4
2
3
67.5
4
40.8c)
124.4
137.9
25.8
5
6
7
8
40.7c)
9
69.1
10
11
12
13
23.3
22.9
25.6
19.6
1′
106.2
75.5
78.2d)
2′
3′
4′
5′
6′
71.4
78.1d)
62.5
a) Data for pyridine-d5. b)–d) Interchangeable.
Fig. 2. Diagnostic HMBC Correlations of 1
Fig. 4. PS-NOESY Correlations of 1 (a) and 2 (b)
Fig. 3. Computer Generated ORTEP Drawing of 1
The structure has crystallographic numbering.
aglycone indicated a bicyclic system. One of the double bond
prised those of four methyls, two methylenes, two oxygenated carbons was highly deshielded (δC 149.6) and was implied to
methines, two oxygenated tertiary carbons, one quaternary carry an electronegative substituent. The above mentioned
carbon and a trisubstituted double bond representing a ma- functionalities indicated a structure formed on dehydration
gastigmane skeleton, and three degrees of unsaturation in the between 9-OH and H-8 of 3a or b, and placed a double bond

Vol. 63, No. 8 (2015)
Chem. Pharm. Bull.
611
Fig. 7. COSY and HMBC Correlations for 5
Fig. 5. Two-Dimensional Correlations of 3a and 3b
Fig.ꢀ 8.ꢀ ResultsꢀwithꢀModifiedꢀMosher’sꢀMethodꢀforꢀ6 and 7
between H-2 and H-3 (J=10.0Hz) indicated these protons were
in a diaxial orientation and to examine the absolute structure
of the aglycone, 5 was enzymatically hydrolyzed to give
Fig. 6. COSY and HMBC Correlations for 4
between C-8 and C-9, giving probable compound 4a in Fig. 5a. The aglycone 5a was found to be a known compound,
6. However, some HMBC correlations in pyridine-d5 did not isolated from Acer trucatum9) and NMR spectroscopically
support this structure. In pyridine-d5, two hydrogens on the identical. Since the stereochemistry of the side chain remained
hydroxy groups appeared at δH 5.33 and 6.11, the former being uncertain,10) the absolute structure of 5a was examined by
assigned to 3-OH from HMBC correlations with C-2 and theꢀ modifiedꢀ Mosher’sꢀ method11) and it proved to be exactly
C-4. The latter showed correlation cross peaks with C-1 and the same compound as that isolated from A. trucatum (data
C-7, which should not be observed in the case of 4a (Fig. 6). notꢀshown).ꢀGlucopyranoseꢀwasꢀanalayzedꢀbyꢀHPLCꢀtoꢀbeꢀofꢀ
Therefore, the structure of 4 must be depicted as a bicyclic the D-series and the mode of linkage was determined to be β
4a,5,6,7,8,8a-hexahydro-4H-chromene system, instead of a from the coupling constant (J=7.6Hz) of the anomeric proton.
spiro skeleton (4a). The PS-NOESY correlation between 6-OH Therefore, the structure of melionoside E (5) was elucidated
and H-2ax placed 6-OH and H-2ax on the same side (Fig. 6). to be (2R,3R,9R)-2,3,9-trihydroxymegastigman-5-ene 2-O-β-D-
Therefore, the structure of 4 is as shown in Fig. 1.
glucopyranoside, as shown in Fig. 1.
Melionoside
E
(5), [α]D22 −60.0, was isolated as an
Meliosma-ionol A (6), [α]D22 −118, was isolated as a color-
amorphous powder and its elemental composition was de- less syrup and its elemental composition was determined to
termined to be C19H32O9. The 13C-NMR spectrum exhib- be C13H24O4. NMR spectroscopic data indicated that 6 was
ited 19 signals, six of which were assigned to those of a a non-glycosidic compound and 13 signals observed in the
glucopyranose unit. The remaining 13 signals comprised 13C-NMR spectrum comprised those of four methyls, two
those of four methyls, three oxymethines and one tetra- methylenes, four oxymethines, one quaternary carbon and a
substituted double bond, indicating a megastigmane skel- tetrasubstituted double bond. The COSY spectrum indicated
eton. The COSY spectrum indicated the presence of two that three oxymethines were connected in a series manner,
carbon sequences, such as –C(2)H–C(3)H–C(4)H2– and and two methylenes, one oxygenated methine and terminal
–C(7)H2–C(8)H2–C(9)H–C(10)H3, and the HMBC correlations methyl were found to form another sequence. HMBC correla-
shown in Fig. 7 connected these sequences and the double tions gave a megastigmane skeleton using these sequences to-
bond to establish the structure of 5. The coupling constants gether with the remaining three methyls and the double bond.

612
Chem. Pharm. Bull.
Vol. 63, No. 8 (2015)
Fig. 9. Possible Relationship of Compounds 1–4
The coupling constant (J=11.2Hz) of H-2 indicated H-2 and up as 4 through elimination of a water molecule. A dehydrated
H-3 were in trans diaxial positions, and from that (J=4.2Hz) compound (3c) derived from 3a and c may exist in the extract,
between H-3 and H-4, H-4 was in an equatorial orientation. although its isolation has not been successful so far. The for-
Finally,ꢀ theꢀ modifiedꢀ Mosher’sꢀ method11) (Fig. 8) was applied mation of melionoside D (4) seems to be reversible, since after
to 6 to establish its structure to be (2R,3R,4R,9R)-2,3,4,9- a week or so slight amounts of 3a and b were found in the
tetrahydroxymegastigman-5-ene, as shown in Fig. 1.
NMR sample solution of 4, residual water molecules probably
Meliosma-ionol B (7), [α]D22 −0.45, was isolated as a color- being picked up in pyridine-d5. Due to the instability of 3a,
less syrup and its elemental composition was determined to b and 4, their absolute stereochemistries were not examined.
be C13H26O4. NMR spectroscopic data indicated that 7 was However, since melionosides A and B (1, 2) may be deriva-
also a megastigmane derivative with two singlet methyls, two tives of 3a, b or c, and the 13C-NMR spectral data for the ring
doublet methyls, three methylenes and four hydroxy groups. regions of melionosides A–D (1–4) exhibited close resem-
The COSY spectrum established two proton sequences, C(2)H blance,ꢀtheyꢀprobablyꢀhaveꢀtheꢀsameꢀabsoluteꢀconfigurations.
to C(13)H3 and C(7)H2 to C(10)H3, which were connected
through a quaternary carbon, C-1, and an oxygenated tertiary
Experimental
one, C-6, as shown in Fig. 1. The coupling constant (J=9.8Hz)
General Experimental Procedure Melting points were
of H-2 indicated that H-2 and H-3 were in a trans diaxial ge- measured with a Yanagimoto micromelting point apparatus
ometry, and those of H-5 (J=12.6, 6.8, 4.2Hz) with H-4 placed and are uncorrected. Optical rotations were measured on a
the C-13 methyl group in an equatorial position. From the JASCO P-1030 polarimeter and IR spectra on a Horiba FT-710
1
PS-NOESY correlations, H-3 (δH 3.50) and H-5 (δH 1.98), H-5 spectrophotometer. H- and 13C-NMR spectra were taken on
and H3-11 (δH 0.86), and H2-7 (δH 1.64 and 1.67) and H3-11, aꢀ JEOLꢀ α-400 or Brucker Avance III 600 spectrometer at
H-3, H-5, the side chain and H3-11 were expected to be on the 400MHz or 600MHz, and 100MHz or 150MHz, respectively,
sameꢀ face.ꢀ Finally,ꢀ theꢀ modifiedꢀ Mosher’sꢀ method11) (Fig. 8) with tetramethylsilane as an internal standard. Positive-ion
established the absolute structure of 7 to be (2S,3S,5R,6R,9R)- HR-MSꢀ wereꢀ takenꢀ withꢀ anꢀ Appliedꢀ Biosystemꢀ QSTARꢀ XLꢀ
2,3,6-trihydroxymegastigmane, as shown in Fig. 1.
system ESI (Nano Spray)-MS.
From the leaves of M. lepidota ssp. squamulata,ꢀ fiveꢀ newꢀ
A highly-porous synthetic resin (Diaion HP-20) was pur-
megastigmane glucoside, named melionosides A–E (1–5), and chased from Mitsubishi Kagaku (Tokyo, Japan). Silica gel
meliosma-ionols A (6) and B (7) were isolated, along with CC and reversed-phase [octadecylsilanized silica gel (ODS)]
four known compounds. The structure of 1ꢀwasꢀconfirmedꢀbyꢀ open CC were performed on silica gel 60 (Merck, Darmstadt,
X-ray crystallographic analyses. Melionoside C (3) was ob- Germany) and Cosmosil 75C18-OPN (Nacalai Tesque, Kyoto,
tained as a mixture of interconvertible isomers, 3a and b, their Japan), respectively. The droplet counter-current chromato-
rate of conversion being fairly rapid, probably on an hour-time graph (DCCC) (Tokyo Rikakikai, Tokyo, Japan) was equipped
scale. The biosynthetic precursor of 3a and b must be the with 500 glass columns (Φ=2mm, L=40cm), the lower and
9-keto derivative, 12, in Fig. 9. The ring closure between the upper layers of a solvent mixture of CHCl3–MeOH–H2O–
6-OH group and the ketone group forms a megastigmane glu- n-PrOH (9:12:8:2) being used as the stationary and mobile
coside possessing a spiro-structure at the 6-position and an in- phases, respectively. Five-gram fractions were collected and
terconvertible hemiketal at the 9-position. The alternative ring numbered according to their order of elution with the mobile
closure, between the 5-OH group and the ketone gives with a phase.ꢀ HPLCꢀ wasꢀ performedꢀ onꢀ anꢀ ODSꢀ columnꢀ (Inertsil;ꢀ
hemiketalic six-membered oxyrane derivative, 13, which ends GLꢀ Science,ꢀ Tokyo,ꢀ Japan),ꢀ andꢀ theꢀ eluateꢀ wasꢀ monitoredꢀ

Vol. 63, No. 8 (2015)
Chem. Pharm. Bull.
613
with a UV detector at 254nm and a refractive index monitor. 5 from the peak at 28min. The residue (86.1mg) in fractions
β-Glucosidase from almond, and (R)- and (S)-α-methoxy-α- 157–175ꢀ wasꢀ purifiedꢀ byꢀ DCCCꢀ toꢀ giveꢀ 16.9ꢀmgꢀ ofꢀ 8 in frac-
trifluoromethylphenylꢀ aceticꢀ acidsꢀ (MTPA)ꢀ wereꢀ purchasedꢀ tions 53–60.
fromꢀWakoꢀPureꢀChemicalꢀIndustries,ꢀLtd.ꢀ(Osaka,ꢀJapan).
Melionoside A (1)
Plant Materialꢀ ꢀLeavesꢀ ofꢀ M. lepidota ssp. squamu-
Colorless needles; mp 223–225°C; [α]D25 −36.3 (c=0.51,
lata were collected in Kunigami-gun, Okinawa, Japan, in July MeOH); IR νmax (KBr) cm−1: 3433, 2974, 2925, 2882, 1647,
1
2006, and a voucher specimen was deposited in the Herbari- 1073, 1037, 1018; H-NMR (600MHz, CD3OD) δ: 4.29 (1H,
um of Pharmaceutical Sciences, Graduate School of Biomedi- d, J=7.7Hz, H-1′), 4.10 (1H, dqd, J=12.1, 11.9, 5.9Hz, H-9),
calꢀSciences,ꢀHiroshimaꢀUniversityꢀ(06-MLS-Okinawa-0703).
3.88 (1H, ddd, J=11.5, 9.5, 5.3Hz, H-3), 3.87 (1H, dd, J=11.7,
Extraction and Separationꢀ ꢀLeavesꢀ ofꢀ M. lepidota spp. 2.0Hz, H-6′a), 3.68 (1H, dd, J=11.7, 4.9Hz, H-6′b), 3.38 (1H,
squamulata (8.80kg) were extracted three times with MeOH d, J=9.5Hz, H-2), 3.33–3.40 (3H, m, H-3′, 4′ and 5′), 3.28 (1H,
(4.5ꢀL×3) at room temperature for one week and then concen- dd, J=8.9, 7.7Hz, H-2′), 2.10 (1H, ddd, J=10.4, 10.3, 9.9Hz,
tratedꢀ toꢀ 3ꢀLꢀ in vacuo. The concentrated extract was washed H-7a), 2.05 (1H, dddd, J=12.1, 10.4, 10.3, 1.6Hz, H-8a), 1.95
with n-hexaneꢀ (3ꢀL,ꢀ 41.0ꢀg),ꢀ andꢀ thenꢀ theꢀ MeOHꢀ layerꢀ wasꢀ (1H, ddd, J=10.3, 10.2. 1.6Hz, H-7b), 1.89 (1H, dd, J=13.3,
concentrated to a gummy mass. The latter was suspended in 5.3Hz, H-4a), 1.83 (1H, dd, J=13.3, 11.5Hz, H-4b), 1.48 (1H,
waterꢀ(3ꢀL)ꢀandꢀthenꢀextractedꢀwithꢀEtOAcꢀ(3ꢀL)ꢀtoꢀgiveꢀ104ꢀgꢀ dddd, J=11.9, 10.3, 10.2, 9.9Hz, H-8b), 1.20 (3H, d, J=5.9Hz,
of an EtOAc-soluble fraction. The aqueous layer was extracted H3-10), 1.19 (3H, s, H3-12), 1.16 (3H, s, H3-13), 1.07 (3H,
withꢀ1-BuOHꢀ(3ꢀL)ꢀtoꢀgiveꢀaꢀ1-BuOH-solubleꢀfractionꢀ(125ꢀg),ꢀ s, H3-11); 13C-NMR (150MHz, CD3OD): Table 1; HR-ESI-
and the remaining water-layer was concentrated to furnish MS (positive-ion mode) m/z: 429.2089 [M+Na]+ (Calcd for
198g of a water-soluble fraction.
C19H34O9Na: 429.2095).
Theꢀ 1-BuOH-solubleꢀ fractionꢀ (124ꢀg)ꢀ wasꢀ subjectedꢀ toꢀ Di-
aion HP-20 CC (Φ=60mm, L=46cm), using H2O–MeOH
Melionoside B (2)
Colorless needles; mp 220–222°C; [α]D24 −25.3 (c=0.39,
(4ꢀ:ꢀ1,ꢀ 4ꢀL),ꢀ (3ꢀ:ꢀ2,ꢀ 4ꢀL),ꢀ (2ꢀ:ꢀ3,ꢀ 4ꢀL),ꢀ andꢀ (1ꢀ:ꢀ4,ꢀ 4ꢀL),ꢀ andꢀ MeOHꢀ MeOH); IR νmaxꢀ (film)ꢀ cm−1: 3396, 2973, 2929, 2882, 1649,
1
(4ꢀL),ꢀ 1ꢀL-fractionsꢀ beingꢀ collected.ꢀ Theꢀ residueꢀ (18.0ꢀg)ꢀ inꢀ 1078, 1032, 1008; H-NMR (600MHz, CD3OD) δ: 4.32 (1H,
fractionsꢀ 4–7ꢀ wasꢀ subjectedꢀ toꢀ silicaꢀ gelꢀ (450ꢀg)ꢀ CCꢀ withꢀ d, J=7.8Hz, H-1′), 4.10 (1H, ddq, J=11.3, 10.9, 5.9Hz, H-9),
increasing amounts of MeOH in CHCl3 [CHCl3ꢀ (6ꢀL),ꢀ andꢀ 3.88 (1H, ddd, J=10.5, 9.5, 6.1Hz, H-3), 3.86 (1H, dd, J=11.9,
CHCl3–MeOHꢀ (49ꢀ:ꢀ1,ꢀ 3ꢀL),ꢀ (24ꢀ:ꢀ1,ꢀ 3ꢀL),ꢀ (23ꢀ:ꢀ2,ꢀ 3ꢀL),ꢀ (9ꢀ:ꢀ1,ꢀ 3ꢀL),ꢀ 1.3Hz, H-6′a), 3.68 (1H, dd, J=11.9, 5.2Hz, H-6′b), 3.46 (1H,
(17ꢀ:ꢀ3,ꢀ3ꢀL),ꢀ(4ꢀ:ꢀ1,ꢀ3ꢀL),ꢀ(3ꢀ:ꢀ1,ꢀ3ꢀL)ꢀandꢀ(7ꢀ:ꢀ3,ꢀ3ꢀL)],ꢀ500-mLꢀfrac- d, J=9.5Hz, H-2), 3.33–3.40 (3H, m, H-3′, 4′ and 5′), 3.27
tions being collected. The residue (706mg) in fractions 34–38 (1H, dd, J=9.0, 7.8Hz, H-2′), 2.11 (1H, ddd, J=9.7, 9.5, 3.3Hz,
wasꢀsubjectedꢀtoꢀODSꢀCCꢀ[Φ=40mm, L=25cm, linear gradi- H-7a), 1.98 (1H, ddd, J=9.5, 9.5, 8.9Hz, H-7b), 1.97 (1H, dddd,
ent: MeOH–H2Oꢀ (1ꢀ:ꢀ9,ꢀ 2ꢀL)ꢀ →ꢀ (9ꢀ:ꢀ1,ꢀ 2ꢀL),ꢀ 10ꢀg-fractionsꢀ beingꢀ J=10.9, 10.7, 9.7, 9.5Hz, H-8a), 1.77 (1H, dd, J=13.1, 6.1Hz,
collected] to give a residue (141mg) in fractions 96–108 which H-4a), 1.76 (1H, dd, J=13.1, 10.5Hz, H-4b), 1.47 (1H, br ddd,
wasꢀthenꢀpurifiedꢀbyꢀDCCCꢀtoꢀgiveꢀaꢀresidueꢀ(52.6ꢀmg)ꢀinꢀfrac- J=11.3, 10.7, 8.9Hz, H-8b), 1.19 (1H, d, J=5.9Hz, H3-10),
tionsꢀ 43–49.ꢀ Finalꢀ purificationꢀ byꢀ HPLCꢀ (ODS,ꢀ Φ=10mm, 1.13 (3H, s, H3-13), 1.11 (3H, s, H3-11), 1.08 (3H, s, H3-12);
L=25cm; H2O–MeOH,ꢀ 3ꢀ:ꢀ2;ꢀ flowꢀ rate:ꢀ 2.8ꢀmL/min)ꢀ gaveꢀ 6 13C-NMR (150MHz, CD3OD): Table 1; HR-ESI-MS (positive-
(35.0mg) and 7 (7.6mg) from the peaks at 16min and 18min, ion mode) m/z: 429.2094 [M+Na]+ (Calcd for C19H34O9Na:
respectively.
429.2095).
The residue (1.75g) in fractions 39–43 obtained on silica
gel CC was separated by ODS CC [Φ=40mm, L=25cm,
Melionoside C (3)
Amorphous powder, [α]D27 −13.3 (c=0.26, MeOH); IR νmax
linear gradient: MeOH–H2Oꢀ (1ꢀ:ꢀ9,ꢀ 1ꢀL)ꢀ →ꢀ (1ꢀ:ꢀ1,ꢀ 1ꢀL),ꢀ andꢀ (film)ꢀ cm−1: 3374, 2979, 2941, 1587, 1483, 1376, 1159, 1076,
1
thenꢀ (1ꢀ:ꢀ1,ꢀ 1ꢀL)ꢀ →ꢀ (9ꢀ:ꢀ1,ꢀ 1ꢀL),ꢀ 10ꢀg-fractionsꢀ beingꢀ collected]ꢀ 1029, 996; 3a: H-NMR (600MHz, CD3OD) δ: 4.28 (1H, d,
to give 318mg of 9 in fractions 82–92. The residue (84.3mg) J=7.9Hz, H-1′), 3.89 (1H, m, H-3), 3.37 (1H, m, H-2), 3.86
in fractions 53–69 was separated by DCCC to give a resi- (1H, dd, J=12.0, 2.1Hz, H-6′a), 3.68 (1H, dd, J=12.0, 5.5Hz,
dueꢀ (35.8ꢀmg)ꢀ inꢀ fractionsꢀ 10–36,ꢀ whichꢀ wasꢀ finallyꢀ purifiedꢀ H-6′b), 3.27 (1H, m, H-2′), 3.34–3.33 (3H, m, H-3′, 4′ and 5′),
byꢀ HPLCꢀ [Cosmosilꢀ Cholesterꢀ (Nakalaiꢀ Tesque),ꢀ Φ=10mm, 2.26 (1H, m, H-7a), 2.00 (3H, m, H-7b, 8a and 8b), 1.85 (1H,
L=25cm; H2O–MeOH,ꢀ7ꢀ:ꢀ3;ꢀflowꢀrate:ꢀ2.8ꢀmL/min]ꢀtoꢀyieldꢀ3 m, H-4a), 1.79 (1H, m, H-4b), 1.51 (3H, s, H3-10), 1.34 (3H,
(6.6mg) from the peaks at 13min and 16min, and 4 (2.8mg) s, H3-13), 1.17 (3H, s, H3-12), 1.10 (3H, s, H3-11); (600MHz,
from the peak at 47min. The residue (41.3mg) in fractions pyridine-d5) δ: 7.14 (1H, brs, 9-OH), 5.86 (1H, brs, 5-OH),
76–79ꢀwasꢀpurifiedꢀbyꢀDCCCꢀtoꢀgiveꢀ7.7ꢀmgꢀofꢀ10 in fractions 5.36 (1H, brs, 3-OH), 5.02 (1H, overlapped with H2O signal,
32–38.ꢀTheꢀresidueꢀ(80.6ꢀmg)ꢀinꢀfractionsꢀ107–112ꢀwasꢀpurifiedꢀ H-1′), 4.60 (1H, m, H-3), 4.51 (1H, dd, J=11.7, 2.6Hz, H-6′a),
by DCCC to give 20.2mg of 1 in fractions 25–27 as a crys- 4.36 (1H, m, H-6′b), 4.28 (1H, dd, J=9.1, 9.1Hz, H-4′), 4.23
talline state. The residue (101mg) in fractions 113–121 was (1H, dd, J=9.1, 8.7Hz, H-3′), 4.13 (1H, m, H-2′), 4.10 (1H, d,
purifiedꢀ byꢀ DCCCꢀ toꢀ giveꢀ 9.5ꢀmgꢀ ofꢀ 11 in fractions 53–60. J=10.2Hz, H-2), 4.00 (1H, ddd, J=9.1, 5.7, 2.6Hz, H-5′), 2.75
Theꢀ residueꢀ (111ꢀmg)ꢀ inꢀ fractionsꢀ 122–131ꢀ wasꢀ subjectedꢀ toꢀ (1H, m, H-7a), 2.50 (1H, m, H-4a), 2.49 (1H, m, H-4b), 2.34
DCCC and then the residue (40.2mg) in fractions 33–39 was (1H, m, H-8a), 2.25 (1H, m, H-7b), 2.15 (1H, m, H-8b), 1.92
purifiedꢀ byꢀ HPLCꢀ (Φ=6mm, L=25cm; H2O–MeOH, 13:7; (3H, s, H3-13), 1.82 (3H, s, H3-10), 1.77 (3H, s, H3-12), 1.67
flowꢀ rate:ꢀ 1.6ꢀmL/min)ꢀ toꢀ giveꢀ 5.9ꢀmgꢀ ofꢀ 2 from the peak at (3H, s, H3-11); 13C-NMR (150MHz, CD3OD and pyridine-
1
20min. The residue (86.1mg) in fractions 144–156 was sepa- d5): Table 1; 3b: H-NMR (600MHz, CD3OD) δ: 4.31 (1H,
rated by DCCC to give a residue (56.3mg) in fractions 39–45, d, J=7.9Hz, H-1′), 3.89 (1H, m, H-3), 3.86 (1H, dd, J=12.0,
whichꢀwasꢀtheꢀpurifiedꢀbyꢀHPLCꢀ(ODS,ꢀΦ=10mm, L=25cm; 2.1Hz, H-6′a), 3.68 (1H, dd, J=12.0, 5.5Hz, H-6′b), 3.38 (1H,
H2O–MeOH,ꢀ20ꢀ:ꢀ7;ꢀflowꢀrate:ꢀ2.8ꢀmL/min)ꢀtoꢀaffordꢀ26.3ꢀmgꢀofꢀ m, H-2), 3.34–3.33 (3H, m, H-3′, 4′ and 5′), 3.27 (1H, m, H-2′),

614
Chem. Pharm. Bull.
Vol. 63, No. 8 (2015)
2.13 (2H, m, H2-7), 1.93 (2H, m, H2-8), 1.77 (2H, m, H2-4), 5.3Hz, H-7b), 1.56 (1H, m, H-8a), 1.51 (1H, m, H-8b), 1.77
1.49 (3H, s, H3-13), 1.24 (3H, s, H3-11), 1.15 (3H, s, H3-10), (3H, s, H3-13), 1.17 (3H, d, J=6.4Hz, H3-10), 1.13 (3H, s,
1.12 (3H, s, H3-12); (600MHz, pyridine-d5) δ: 7.00 (1H, brs, H3-12), 0.94 (3H, s, H3-11); 13C-NMR (150MHz, CD3OD):
9-OH), 5.97 (1H, brs, 5-OH), 5.24 (1H, brs, 3-OH), 4.84 (1H, Table 1; HR-ESI-MS (positive-ion mode) m/z: 267.15670
d, J=7.9Hz, H-1′), 4.67 (1H, m, H-3), 4.58 (1H, dd, J=11.7, [M+Na]+ (Calcd for C13H24O4Na: 267.1567).
2.6Hz, H-6′a), 4.36 (1H, m, H-6′b), 4.24 (1H, dd, J=9.4,
9.1Hz, H-4′), 4.15 (1H, dd, J=9.1, 8.7Hz, H-3′), 4.10 (1H, m,
Meliosma-ionol B (7)
Colorless syrup, [α]D22 −0.45 (c=0.44, MeOH); IR νmaxꢀ(film)ꢀ
H-2′), 4.07 (1H, d, J=9.4Hz, H-2), 3.91 (1H, ddd, J=9.4, 5.7, cm−1: 3368, 2970, 1650, 1458, 1373, 1108, 1048, 1012, 967;
2.6Hz, H-5′), 2.57 (1H, m, H-7a), 2.55 (1H, m, H-7b), 2.44 (1H, 1H-NMR (600MHz, CD3OD) δ: 3.56 (1H, tq, J=6.8, 6.0Hz,
m, H-4a), 2.42 (1H, m, H-4b), 2.27 (1H, m, H-8a), 2.13 (1H, m, H-9), 3.50 (1H, ddd, J=11.7, 9.8, 5.3Hz, H-3), 3.41 (1H, d,
H-8b), 2.00 (3H, s, H3-11), 1.81 (3H, s, H3-10), 1.72 (3H, s, J=9.8Hz, H-2), 1.98 (1H, dqd, J=12.6, 6.8, 4.2Hz, H-5), 1.70
H3-12), 1.47 (3H, s, H3-13); 13C-NMR (150MHz, CD3OD and (1H, ddd, J=12.8, 5.3, 4.2Hz, H-4a), 1.67 (1H, dt, J=16.0,
pyridine-d5): Table 1; HR-ESI-MS (positive-ion mode) m/z: 7.8Hz, H-7a), 1.64 (1H, dt, J=16.0, 7.8Hz, H-7b), 1.54 (2H,
445.2044 [M+Na]+ (Calcd for C19H34O10Na: 445.2044).
Melionoside D (4)
dd, J=7.8, 6.8Hz, H2-8), 1.50 (1H, ddd, J=12.8, 12.6, 11.7Hz,
H-4b), 1.16 (3H, d, J=6.0Hz, H3-10), 1.05 (3H, s, H3-12),
Amorphous powder, [α]D27 −11.8 (c=0.13, MeOH); IR νmax 0.93 (3H, d, J=6.8Hz, H3-13), 0.86 (3H, s, H3-11); 13C-NMR
(film)ꢀ cm−1: 3382, 2980, 2927, 1587, 1379, 1328, 1159, 1052, (100MHz, CD3OD): Table 1; HR-ESI-MS (positive-ion mode)
1029, 1014; 1H-NMR (600MHz, CD3OD) δ: 4.47 (1H, ddq, m/z: 267.1722 [M+Na]+ (Calcd for C13H26O4Na: 267.1723).
J=4.9, 2.3, 1.1Hz, H-8), 4.29 (1H, d, J=7.8Hz, H-1′), 3.87
Sugar Analysis About 500µg each of melionosides A–E
(1H, dd, J=11.7, 2.3Hz, H-6′a), 3.85 (1H, ddd, J=13.6, 9.8, (1–5) was hydrolyzed with 1Mꢀ HClꢀ (0.1ꢀmL)ꢀ atꢀ 90°Cꢀ forꢀ 2ꢀh.ꢀ
5.3Hz, H-3), 3.68 (1H, dd, J=11.7, 5.1Hz, H-6′b), 3.50 (1H, The reaction mixtures were partitioned with an equal amount
d, J=9.8Hz, H-2), 3.20–3.38 (3H, m, H-3′, 4′ and 5′), 3.26 ofꢀ EtOAcꢀ (0.1ꢀmL),ꢀ andꢀ theꢀ waterꢀ layersꢀ wereꢀ analyzedꢀ withꢀ
(1H, dd, J=9.1, 7.8Hz, H-2′), 2.33 (1H, ddq, J=17.1, 4.9, a chiral detector (JASCO OR-2090plus) on an amino column
1.3Hz, H-7a), 1.99 (1H, dd, J=11.7, 5.3Hz, H-4a), 1.91 (1H, [Asahipak NH2P-5 4E; CH3CN–H2O,ꢀ 4ꢀ:ꢀ1;ꢀ flowꢀ rateꢀ 1ꢀmL/
ddq, J=17.1, 2.3, 2.3Hz, H-7b), 1.85 (1H, dd, J=13.6, 11.7Hz, min]. Melionosides A–E (1–5) each gave a peak for D-glucose
H-4b), 1.61 (3H, ddd, J=2.3, 1.3, 1.1Hz, H3-10), 1.23 (3H, s, at the retention time of 13.7min with a positive optical rota-
H3-11), 1.16 (3H, s, H3-12), 1.00 (3H, s, H3-13); (600MHz, pyr- tionꢀsign.ꢀTheꢀpeakꢀwasꢀidentifiedꢀbyꢀco-chromatographyꢀwithꢀ
idine-d5) δ: 6.11 (1H, brs, 6-OH), 5.33 (1H, brs, 3-OH), 4.97 authentic D-glucose.
(1H, overlapped with H2O signal, H-1′), 4.55 (1H, m, H-8),
X-Ray Analysis of Melionoside A (1) A suitable crystal
4.54 (1H, m, H-6′a), 4.51 (1H, ddd, J=10.2, 9.6, 6.0Hz, H-3), (0.42 mm×0.07mm×0.05mm) was used for analysis. The
4.36 (1H, dd, J=11.3, 5.2Hz, H-6′b), 4.27 (1H, d, J=9.6Hz, data were measured using a Bruker SMART 1000 CCD dif-
H-2), 4.25 (1H, dd, J=9.4, 9.1Hz, H-4′), 4.21 (1H, dd, J=9.1, fractometer, using MoKα graphite-monochromated radiation
8.7Hz, H-3′), 4.11 (1H, m, H-2′), 3.97 (1H, ddd, J=9.4, 5.2, (λ=0.71073Å). The structure was solved by a direct method
2.3Hz, H-5′), 2.57 (1H, br dd, 17.3, 3.6Hz, H-7a), 2.54 (1H, usingꢀ theꢀ programꢀ SHELXTL-97.12)ꢀ Theꢀ refinementꢀ andꢀ allꢀ
dd, 13.6, 6.0Hz, H-4a), 2.51 (1H, dd, J=13.6, 10.2Hz, H-4b), furtherꢀcalculationsꢀwereꢀcarriedꢀoutꢀusingꢀSHELXTL-97.ꢀTheꢀ
2.28 (1H, ddq, J=17.3, 2.3, 1.9Hz, H-7b), 1.79 (3H, s, H3-11), absorption correction was carried out utilizing the SADABS
1.70 (3H, brs, H3-10), 1.53 (3H, s, H3-13), 1.45 (3H, s, H3-12); routine.13) The H atoms were included at calculated positions
13C-NMR (150MHz, CD3OD and pyridine-d5): Table 1; HR- andꢀ treatedꢀ asꢀ ridingꢀ atomsꢀ usingꢀ theꢀ SHELXTLꢀ defaultꢀ pa-
ESI-MS (positive-ion mode) m/z: 427.1939 [M+Na]+ (Calcd rameters. Figure 3 was drawn with ORTEP32.14) Crystal data:
for C19H32O9Na: 427.1936).
Melionoside E (5)
C19H34O9, M=406.46g mol−1, monoclinic, C2, a=22.469(5) Å,
b=6.3195(13) Å, c=14.461(3) Å, β=102.844(3)°, V=2002.0(7)
Amorphous powder, [α]D22 −60.0 (c=0.58, MeOH); IR Å3, T=90 K, Z=4, Dc=1.349g cm−3, µ(MoKα)=0.106 mm−1,
νmaxꢀ (film)ꢀ cm−1: 3335, 2967, 1650, 1557, 1512, 1456, 1160, F(000)=880,ꢀ 6056ꢀ reflectionsꢀ wereꢀ measuredꢀ inꢀ theꢀ rangeꢀ ofꢀ
1
1078, 1035, 996; H-NMR (600MHz, CD3OD) δ: 4.35 (1H, 2θ<56.66°, 3851 being unique and used in all calculations.
d, J=7.6Hz, H-1′), 3.87 (1H, dd, J=12.1, 1.7Hz, H-6′a), 3.79 Theꢀfinalꢀgoodness-of-fitꢀonꢀF2ꢀwasꢀ1.066,ꢀandꢀtheꢀfinalꢀR in-
(1H, ddd, J=10.0, 9.7, 6.8Hz, H-3), 3.71 (1H, m, H-9), 3.68 dices were R1=0.0459 based on I>2σ(I), and wR2=0.1411 with
(1H, m, H-6′b), 3.36 (2H, m, H-3′ and 5′), 3.34 (1H, m, H-4′), all data. The largest differences between the peak and the hole
3.29 (1H, m, H-2′), 3.27 (1H, d, J=10.0Hz, H-2), 2.34 (1H, were 0.733 and −0.337 eÅ–3, respectively. The CCDC deposit
dd, J=17.0, 6.8Hz, H-4a), 2.23 (1H, m, H-7a), 2.05 (1H, dd, contains supplementary crystallographic data (No. 1033761)
J=17.0, 9.7Hz, H-4b), 1.63 (3H, s, H3-13), 1.22 (3H, s, H3-12), These data can be obtained free of charge via http://www.
1.91 (1H, ddd, J=15.1, 10.2, 6.8Hz, H-7b), 1.50 (2H, m, H2-8), ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge
1.17 (3H, d, J=6.4Hz, H3-10), 1.03 (3H, s, H3-11); 13C-NMR Crystallographic Data Centre, 12 Union Road, Cambridge
(150MHz, CD3OD): Table 1; HR-ESI-MS (positive-ion mode) CB2 1EZ, UK; fax: (+44) 1223 336 033; or e-mail: deposit@
m/z: 413.2140 [M+Na]+ (Calcd for C19H32O9Na: 413.2146).
Meliosma-ionol A (6)
ccdc.cam.ac.uk.
Enzymatic Hydrolysis 5 to 5a Melionoside E (5: 5.1mg)
Colorless syrup, [α]D22 −118 (c=0.58, MeOH); IR νmax was hydrolyzed with 5mg of β-glucosidaseꢀinꢀ1ꢀmLꢀofꢀacetateꢀ
(film)ꢀ cm−1: 3386, 2969, 1651, 1512, 1457, 1118, 1083, 1038, buffer (pH 5.0, 20mM) at 37°C for 11h. The reaction mixture
1
963; H-NMR (600MHz, CD3OD) δ: 3.87 (1H, d, J=4.2Hz, wasꢀsubjectedꢀtoꢀsilicaꢀgelꢀCCꢀ(Φ=2.5cm, L=20cm) with in-
H-4), 3.73 (1H, qdd, J=6.4, 6.1, 6.1Hz, H-9) 3.53 (1H, dd, creasing amounts of MeOH in CHCl3 [CHCl3ꢀ (200ꢀmL),ꢀ andꢀ
J=11.2, 4.2Hz, H-3), 3.48 (1H, d, J=11.2Hz, H-2), 2.25 (1H, CHCl3–MeOHꢀ(19ꢀ:ꢀ1,ꢀ100ꢀmL),ꢀ(9ꢀ:ꢀ1,ꢀ100ꢀmL),ꢀ(17ꢀ:ꢀ3,ꢀ100ꢀmL),ꢀ
ddd, J=13.2, 12.1, 5.7Hz, H-7a), 1.96 (1H, ddd, J=13.2, 12.5, (4ꢀ:ꢀ1,ꢀ 100ꢀmL),ꢀ (3ꢀ:ꢀ1,ꢀ 100ꢀmL),ꢀ andꢀ (7ꢀ:ꢀ3,ꢀ 100ꢀmL)]ꢀ andꢀ MeOHꢀ

Vol. 63, No. 8 (2015)
Chem. Pharm. Bull.
615
(200ꢀmL),ꢀ 5-gꢀ fractionsꢀ beingꢀ collected.ꢀ 5a (2.3mg) was ob- (10H, aromatic protons), 5.15 (1H, m, H-9), 5.10 (1H, m, H-3),
tained in fractions 114–119 and D-glucose in the MeOH elu- 4.09 (1H, m, H-4), 3.84 (1H, m, H-2), 3.50 (6H, s, –OCH3×2),
ate. 5a: Colorless syrup, [α]D21 −78.7 (c=0.15, CHCl3), {[α]D25 1.96 (1H, m, H-7a), 1.70 (2H, m, H2-8), 1.68 (3H, s, H3-13),
−66.3 (c=0.056, CHCl3)}9); 1H-NMR (600MHz, CDCl3) δ: 1.67 (1H, m, H-7b), 1.29 (3H, d, J=6.1Hz, H3-10), 1.12 (3H,
3.81 (1H, m, H-9), 3.77 (1H, ddd, J=9.8, 9.8, 6.4Hz, H-3), s, H3-11), 1.00 (3H, s, H3-12); HR-ESI-MS (positive-ion mode)
3.27 (1H, d, J=9.8Hz, H-2), 2.33 (1H, dd, J=16.6, 6.4Hz, m/z: 699.2365 [M+Na]+ (Calcd for C33H38O8F6Na: 699.2363).
1
H-4a), 2.22 (1H, m, H-7a), 2.06 (1H, dd, J=16.6, 9.8Hz, H-4b), 6b: Amorphous powder, H-NMR (600MHz, CDCl3) δ: 7.39–
1.91 (1H, m, H-7b), 1.63 (3H, s, H3-13), 1.52 (2H, m, H2-8), 7.62 (10H, aromatic protons), 5.15 (1H, m, H-9), 5.03 (1H,
1.22 (3H, d, J=6.1Hz, H3-10), 1.13 (3H, s, H3-11), 0.93 (3H, m, H-3), 4.17 (1H, m, H-4), 3.75 (1H, m, H-2), 3.50 (6H, s,
s, H3-12); 13C-NMR (150MHz, CDCl3): essentially identi- –OCH3×2), 1.85 (1H, m, H-7a), 1.64 (2H, m, H2-8), 1.63 (3H,
1
cal data with those reported in ref. 9; H-NMR (600MHz, s, H3-13), 1.62 (1H, m, H-7b), 1.38 (3H, d, J=6.1Hz, H3-10),
CD3OD) δ: 3.70 (1H, dq, J=6.5, 6.2Hz, H-9), 3.67 (1H, ddd, 1.06 (3H, s, H3-11), 0.97 (3H, s, H3-12); HR-ESI-MS (positive-
J=10.1, 9.9, 6.5Hz, H-3), 3.15 (1H, d, J=10.1Hz, H-2), 2.27 ion mode) m/z: 699.2366 [M+Na]+ (Calcd for C33H38O8F6Na:
(1H, dd, J=16.9, 6.5Hz, H-4a), 2.22 (1H, m, H-7a), 2.01 (1H, 699.2363).
dd, J=16.9, 9.9Hz, H-4b), 1.91 (1H, ddd, J=15.1, 10.2, 6.8Hz,
Preparation of (R)- and (S)-MTPA Diesters (7a and
H-7b), 1.62 (3H, s, H3-13), 1.50 (2H, m, H2-8), 1.17 (3H, d, b) from 7 Using a similar procedure to that used for the
J=6.2Hz, H3-10), 1.11 (3H, s, H3-12), 0.93 (3H, s, H3-11); preparation of 6a and b from 6, 7a (0.8mg) and b (0.3mg)
13C-NMR (150MHz, CD3OD) δ: 138.3 (C-6), 124.5 (C-5), 81.1 were prepared from 7 (0.6mg each) by use of the respective
(C-2), 69.2 (C-9), 68.7 (C-3), 43.2 (C-1), 41.3 (C-4), 40.7 (C-8), amounts of (R)- and (S)-MTPA (19.5mg and 25.4mg), EDC
26.9 (C-12), 26.05 (C-7), 23.3 (C-10), 22.0 (C-11), 19.7 (C-13).
(11.4mg and 9.0mg), and DMAP (15.7mg and 15.4mg). 7a:
1
Preparation of (R)- and (S)-MTPA Diesters (5b and c) Amorphous powder, H-NMR (600MHz, CDCl3) δ: 7.40–7.55
from 5a A solution of 5aꢀ (0.7ꢀmg)ꢀ inꢀ 1ꢀmLꢀ ofꢀ dehydratedꢀ (10H, aromatic protons), 5.07 (1H, m, H-9), 4.99 (1H, ddd,
CH2Cl2 was reacted with (R)-MTPA (11.1mg) in the presence J=11.7, 9.8, 5.3Hz, H-3), 3.69 (1H, dd, J=9.8, 4.2Hz, H-2),
of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochlo- 3.58 (3H, s, –OCH3), 3.50 (3H, s, –OCH3), 1.98 (1H, m,
ride (EDC) (11.1mg) and N,N′-dimethyl-4-aminopyridine H-5), 1.85 (1H, m, H-4a), 1.70 (2H, m, H2-8), 1.64 (1H, m,
(4-DMAP) (10.0mg), and then the mixture was occasionally H-4b), 1.63 (1H, m, H-7a), 1.57 (1H, m, H-7b), 1.30 (3H, d,
stirredꢀatꢀ37°Cꢀforꢀ12ꢀh.ꢀAfterꢀtheꢀadditionꢀofꢀ1ꢀmLꢀofꢀCH2Cl2, J=6.2Hz, H3-10), 1.09 (3H, s, H3-11), 0.90 (3H, s, H3-12), 0.88
the solution was washed with H2Oꢀ (1ꢀmL),ꢀ 1ꢀMꢀ HClꢀ (1ꢀmL),ꢀ (3H, s, H3-13); HR-ESI-MS (positive-ion mode) m/z: 701.2518
NaHCO3-saturated H2Oꢀ (1ꢀmL),ꢀ andꢀ thenꢀ brineꢀ (1ꢀmL),ꢀ suc- [M+Na]+ (Calcd for C33H40O8F6Na: 701.2520). 7b: Amorphous
1
cessively. The organic layer was dried over Na2SO4 and then powder; H-NMR (600MHz, CDCl3) δ: 7.40–7.55 (10H, aro-
evaporatedꢀ underꢀ reducedꢀ pressure.ꢀ Theꢀ residueꢀ wasꢀ purifiedꢀ matic protons), 5.07 (1H, m, H-9), 4.96 (1H, ddd, J=11.7, 10.2,
onꢀ aꢀ preparativeꢀ TLCꢀ [silicaꢀ gelꢀ (0.25ꢀmmꢀ thickness),ꢀ beingꢀ 5.3Hz, H-3), 3.71 (1H, dd, J=10.2, 5.7Hz, H-2), 3.58 (3H,
applied for 9cm, developed with CHCl3–(CH3)2CO (19:1) for s, –OCH3), 3.55 (3H, s, –OCH3), 1.91 (1H, m, H-5), 1.76 (1H,
9cm, and then eluted with CHCl3–MeOH (5:1)] to furnish an m, H-4a), 1.74 (1H, m, H-7a), 1.64 (2H, m, H2-8), 1.50 (1H, m,
ester, 5b (0.1mg). Through a similar procedure, 5c (0.4mg) H-4b), 1.47 (1H, m, H-7b), 1.36 (3H, d, J=6.2Hz, H3-10), 1.06
was prepared from 5b (0.7mg) using (S)-MTPA (13.0mg), (3H, s, H3-12), 0.97 (3H, s, H3-11), 0.75 (3H, s, H3-13); HR-
EDC (10.5mg), and 4-DMAP (15.7mg). 5b: Amorphous pow- ESI-MS (positive-ion mode) m/z: 701.2520 [M+Na]+ (Calcd
1
der, H-NMR (600MHz, CDCl3) δ: 7.40–7.57 (10H, aromatic for C33H38O8F6Na: 701.2520).
protons), 5.15 (1H, m, H-3), 5.13 (1H, m, H-9), 3.57 (3H, s,
–OCH3), 3.51 (3H, s, –OCH3), 3.51 (1H, m, H-2), 2.46 (1H, dd,
Acknowledgments The authors are grateful for access to
J=16.7, 6.9Hz, H-4a), 2.34 (1H, m, H-7a), 2.14 (1H, m, H-a), the superconducting NMR instrument and the HR-ESI-MS at
2.08 (1H, m, H-7b), 1.93 (1H, m, H-8a), 1.65 (1H, m, H-8b), the Analytical Center of Molecular Medicine and the Analysis
1.54 (3H, s, H3-13), 1.30 (3H, d, J=6.1Hz, H3-10), 1.09 (3H, Centerꢀ ofꢀ Lifeꢀ Science,ꢀ respectively,ꢀ ofꢀ theꢀ Graduateꢀ Schoolꢀ
s, H3-11), 0.99 (3H, s, H3-12); HR-ESI-MS (positive-ion mode) of Biomedical Sciences, Hiroshima University. This work
m/z: 684.2413 [M+Na]+ (Calcd for C33H38O7F6Na: 683.2414). was supported in part by Grants-in-Aid from the Ministry of
5c: Amorphous powder, 1H-NMR (600MHz, CDCl3) δ: Education, Sports, Culture, Science and Technology of Japan,
7.26–7.55 (1H, aromatic protons), 5.15 (1H, m, H-9), 5.12 (1H, and the Japan Society for the Promotion of Science (Nos.
m, H-3), 3.58 (3H, s, –OCH3), 3.45 (3H, m, –OCH3), 2.50 22590006, 23590130 and 15H04651). Thanks are also due to
(1H, dd, J=16.7, 6.1Hz, H-4a), 2.32 (1H, m, H-7a), 2.16 (1H, theꢀTakedaꢀScienceꢀFoundationꢀforꢀtheꢀfinancialꢀsupport.
m, H-4b), 2.02 (1H, m, H-7b), 1.84 (1H, m, H-8a), 1.62 (1H,
m, H-8b), 1.49 (3H, s, H3-13), 1.38 (3H, d, J=6.1Hz, H3-10),
Conflict of Interest Theꢀ authorsꢀ declareꢀ noꢀ conflictꢀ ofꢀ
1.02 (3H, s, H3-11), 0.94 (3H, s, H3-12); HR-ESI-MS (positive- interest.
ion mode) m/z: 684.2412 [M+Na]+ (Calcd for C33H38O7F6Na:
683.2414).
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Preparation of (R)- and (S)-MTPA Diesters (6a and
b) from 6 Using a similar procedure to that used for the
preparation of 5b and c from 5a, 6a (0.3mg) and b (0.4mg)
were prepared from 6 (0.4mg each) by use of the respective
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1
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