The unusual canangafruticosides A-E: Five monoterpene glucosides, two monoterpenes and a monoterpene glucoside diester of the aryldihydronaphthalene lignan dicarboxylic acid from leaves of Cananga odorata var. fruticosa

Article abstract of DOI:10.1016/j.phytochem.2010.06.009

From the leaves of Cananga odorata var. fruticosa, five unusual monoterpene glucosides, named canangafruticosides A-E (1-5), along with two unusual non-glucosidic monoterpenes (6, 7) were isolated. An aryldihydronaphthalene-type lignan dicarboxylate (8) was also isolated, with two moles of canangafruticoside A (1) on its ester moiety. This lignan also showed strong blue fluorescence emission under basic conditions. The structures of these compounds were elucidated by means of spectroscopic methods, with their absolute configurations determined by application of the modified Mosher's method to a compound chemically derived from canangafruticoside E.

Full text of DOI:10.1016/j.phytochem.2010.06.009

Phytochemistry 71 (2010) 1564–1572
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
The unusual canangafruticosides A–E: Five monoterpene glucosides, two
monoterpenes and a monoterpene glucoside diester of the
aryldihydronaphthalene lignan dicarboxylic acid from leaves of
Cananga odorata var. fruticosa
b
Jiro Nagashima a, Katsuyoshi Matsunami a, Hideaki Otsuka a, , Duangporn Lhieochaiphant ,
*
Sorasak Lhieochaiphant b
a Department of Pharmacognosy, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan
b Department of Pharmaceutical Sciences, Faculty of Pharmacy, Chiang Mai University, 239 Huaw Kaew Road, Chiang Mai 50200, Thailand
a r t i c l e i n f o
a b s t r a c t
Article history:
From the leaves of Cananga odorata var. fruticosa, five unusual monoterpene glucosides, named canan-
gafruticosides A–E (1–5), along with two unusual non-glucosidic monoterpenes (6, 7) were isolated.
An aryldihydronaphthalene-type lignan dicarboxylate (8) was also isolated, with two moles of cananga-
fruticoside A (1) on its ester moiety. This lignan also showed strong blue fluorescence emission under
basic conditions. The structures of these compounds were elucidated by means of spectroscopic methods,
with their absolute configurations determined by application of the modified Mosher’s method to a com-
pound chemically derived from canangafruticoside E.
Received 18 February 2010
Received in revised form 1 May 2010
Available online 9 July 2010
Keywords:
Cananga odorata var. fruticosa
Annonaceae
Ó 2010 Elsevier Ltd. All rights reserved.
Shrubby cananga
Unusual monoterpene
Canangafruticoside
Aryldihydronaphthalene dicarboxylate
1. Introduction
of C. odorata. One is called shrubby cananga [C. odorata (Lamarck)
Hooker f. & Thomson var. fruticosa (Craib) Corner] and the other
Cananga odorata (Lamarck) Hooker f. & Thomson var. fruticosa
(Craib) Corner (Annonaceae) is known as ylang–ylang and grows
in tropical Asia. Its leaves are long, smooth and glossy. Its flowers
are greenish yellow and curly, each one resembling a starfish,
and they yield a highly fragrant essential oil. The latter evokes a
feeling of deep and languid calm that melts away anxiety, tension
and stress, and thus is used in the field of aromatherapy. Thus,
investigation of its essential oil has been reported (Katague and
Kirch, 1963; Gaydou et al., 1986; Olivero et al., 1997). The isolation
of some other components, such as a guaiapyridine sesquiterpene
alkaloid and eudesmane sesquiterpenes from its fruit (Hsieh et al.,
2001), and alkaloids from its stem bark, have also been reported
(Leboeuf et al., 1975; Leboeuf and Cavé, 1976; Rao et al., 1986).
This phytochemical investigation herein was thus intended to
examine the non-volatile constituents from the plant part, which
has not yet been used as a medicinal.
one, fragrant cananga or wild cananga [C. odorata (Lamarck) Hoo-
ker f. & Thomson var. odorata] (Porcher, 2004). These two varieties
are cultivated in the Botanical Gardens of the Faculty of Pharmacy,
Chiang Mai University, Chiang Mai, Thailand. The former is a small
shrubby tree of 5 m in height and the latter is a tall, longer, tree of
about 30 m in height. This paper deals with investigation of con-
stituents of the leaves of shrubby cananga.
2. Results and discussion
Air-dried leaves of C. odorata var. fruticosa were extracted with
MeOH three times and the concentrated MeOH extract was parti-
tioned with solvents of increasing polarity. The 1-BuOH-soluble
fraction was separated by means of various chromatographic pro-
cedures including column chromatography (CC) on a highly-por-
ous synthetic resin (Diaion HP-20), and then normal silica gel
and reversed-phase octadecyl silica gel (ODS) CC, droplet coun-
ter-current chromatography (DCCC), and high-performance liquid
chromatography (HPLC), respectively, to afford 10 compounds
(1–10). The details and yields are given in Section 4. The structures
of the new unusual monoterpene glucosides, named canangafruti-
Although the International Plant Names Index (IPNI) lists only
one species, C. odorata, there are obviously two different varieties
* Corresponding author. Tel./fax: +81 82 257 5335.
E-mail address: hotsuka@hiroshima-u.ac.jp (H. Otsuka).
0031-9422/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2010.06.009

J. Nagashima et al. / Phytochemistry 71 (2010) 1564–1572
1565
Fig. 1. Structures of interest.
cosides A–E (1–5), the unusual monoterpenes (6, 7), and the unu-
sual monoterpene diester of the lignan dicarboxylic acid (8) were
elucidated on the basis of spectroscopic evidence (Fig. 1), and the
known compounds were identified as corchoionoside C (9) (Yos-
hydroxyl groups (3363 and 1074 cmꢁ1) and a carbonyl functional
group (1713 cmꢁ1). In the 1H NMR spectrum, signals assignable
to an anomeric proton (dH 4.47), olefinic protons on a cis-double
bond [dH 6.44 (1H, d, J = 7 Hz, H-9) and 4.60 (1H, d, J = 7 Hz, H-8)]
and an aldehyde (dH 9.81) were observed. The 13C NMR spectrum
with the DEPT experiments showed six signals assignable to b-glu-
copyranose and 10 resonances to three methylenes, one oxymeth-
ylene, one methine, one oxymethine, one disubstituted double
bond, one carbonyl carbon (dC 207.1) with a hydrogen atom, and
one quaternary carbon (Table 1). A highly deshielded olefinic car-
bon signal (dC 146.1) implied that the double bond was involved
in an enol system. The absolute structure of glucose was deter-
mined to be of the D-series using a chiral detector. Analysis of
the 1H–1H COSY and HSQC correlation spectra established proton
sequences from the oxymethine proton (dH 3.80) on C-2 to methy-
hikawa et al., 1997) and (+)-syringaresinol 4-O-b-D-glucopyranoide
(10) (Shahat et al., 2004) by comparison of spectroscopic data with
those reported in the literature. From C. odorata f. genuina, an unu-
sual monoterpene, called canangone (11) has been isolated (Calo-
prisco et al., 2002) and its absolute configuration was determined
by synthesis of optically active (+)-canangone (Koripelly et al.,
2007).
Canangafruticoside A (1), ½a D25
ꢀ
+23.8, was isolated as an amor-
phous powder and its molecular formula was calculated to be
C16H26O9 from the results of high resolution (HR)-electrospray ion-
ization (ESI) MS. The IR spectrum exhibited strong absorptions for

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J. Nagashima et al. / Phytochemistry 71 (2010) 1564–1572
Table 1
abled the location of the hydroxyl group in the equatorial position,
and the cross-peak of H-2 with H-8 observed in the PS-NOESY
experiment location of the double bond was also in the equatorial
position. The C-10 primary alcohol was placed in the equatorial
orientation based on the coupling constants of H-4 (J = 12 Hz) with
adjacent axial protons. Therefore, the structure of canangafrutico-
13C NMR spectroscopic data for canangafruticosides A–E (1–5) and related com-
pounds (6 and 7) (CD3OD).
C
1a
2b
3b
4b
5b
6a
7a
1
2
3
4
5
6
7
8
56.6
76.2
36.1
40.5
26.3
31.6
207.1
110.4
146.1
68.0
104.3
74.7
78.6
71.4
78.0
62.7
56.3
75.7
35.9
37.3
26.1
31.1
206.8
110.0
146.2
69.7
104.2
74.6
78.5
71.3
78.0
62.6
56.3
75.8
35.9
37.4
26.2
31.1
206.9
110.1
146.2
69.5
104.3
74.6
78.5
71.3
78.0
62.7
56.3
75.7
35.9
37.3
26.2
31.1
206.8
110.1
146.2
69.4
104.3
74.6
78.6
71.3
78.0
62.7
56.3
75.8
35.9
37.4
26.1
31.1
206.8
110.0
146.2
69.3
104.2
74.6
78.5
71.3
78.0
62.6
49.6
76.2
34.7
40.4
25.2
34.5
181.3
36.5
67.2
68.1
51.2
76.2
35.6
37.0
27.4
32.1
107.1
45.5
105.2
69.6
*
*
*
side
(hydroxymethyl)-1-[(Z)-2-hydroxylvinyl]cyclohexanecarbaldehyde
9-O-b- -glucopyranoside, as shown in Fig. 1. This cis-geometry of
A (1) was elucidated to be (1S ,2S ,4S )-2-hydroxyl-4-
D
the C-8–C-9 double bond was finally confirmed by analysis of the
PS-NOESY spectrum, in which H-8 and H-9 protons showed a sig-
nificant correlation cross-peak. The absolute configuration of can-
angafruticoside A (1) will be discussed later.
9
10
10
20
Canangafruticoside B (2), [a] +15.0, was isolated as an amor-
D
30
phous powder, and its elemental composition was determined to
be C25H32O10 by HRESIMS. The IR spectrum indicated the presence
of an aromatic ring (1606 and 1512 cmꢁ1), and the UV spectrum
supported the above evidence. In the 1H NMR spectrum, all the
protons observed in that of canangafruticoside A (1), were simi-
larly present, and further five finely coupled aromatic protons
and two protons from a trans double bond [dH 7.68 (1H, d,
J = 16 Hz) and 6.50 (1H, d, J = 16 Hz)] were represented by extra
signals. The 13C NMR spectrum was also similar to that of 1, except
for the presence of seven resonances, comprising five sp2 signals
with a hydrogen atom, two of which were of double strength,
one without a hydrogen atom, and a carbonyl carbon. Thus, the
structure of the extra moiety was expected to be trans cinnamic
acid. The H2-10 (dH 4.06) and C-10 (dC 69.7) signals were obviously
shifted downfield, when compared with the corresponding reso-
nances of 1. Thus, the ester position of the acyl moiety was
presumed to be on the hydroxyl group at C-10 and this was con-
firmed by the correlation peak for H2-10 and the carbonyl carbon
signal (dC 168.5) in the HMBC spectrum. Therefore, the structure
of canangafruticoside B (2) was elucidated to be 10-O-E-cinnamoyl
canangafruticoside A, as shown in Fig. 1.
40
50
60
100 (10)
200 (20)
300 (30)
400 (40)
500 (50)
600 (60)
700 (70)
800 (80)
900 (90)
–OCH3
–OCH3
135.7
129.2
130.0
131.5
130.0
129.2
146.3
118.9
168.5
127.2
131.2
116.9
161.3
116.9
129.2
146.6
115.9
169.3
127.8
133.4
115.9
160.0
115.9
133.4
146.2
115.2
168.4
127.8
115.2
146.9
149.6
116.6
123.0
147.0
115.2
169.3
(127.8)
(115.2)
(146.8)
(149.6)
(116.6)
(122.9)
(146.9)
(115.2)
(169.3)
55.5
56.1
a
At 150 MHz.
At 100 MHz.
b
Judging from the NMR spectroscopic data, canangafruticosides
C (3), [
ilar to compound 2 with the respective elemental compositions of
25H32O11, C25H32O11, and C25H32O12. The structure of the acyl moi-
a]D +29.5, D (4), [a]D +19.1, and E (5), [a]D +17.6, were sim-
C
ety of canangafruticoside C (3) was established to be E-p-coumaric
acid from the spectroscopic evidence that one more oxygen atom
existed in it than in 2, and typical para-disubsituted patterns of
protons [7.45 (2H, d, J = 9 Hz) and 6.81 (2H, d, J = 9 Hz)] on the aro-
matic ring were observed in the NMR spectra. The structure of can-
angafruticoside D (4) was assigned as Z-p-coumaric acid from the
coupling constants of protons [6.87 (1H, d, J = 13 Hz) and 5.77
(1H, d, J = 13 Hz)] on the double bond. Canangafruticoside E (5)
possessed one more oxygen atom than 3 and 4, and three aromatic
protons were coupled in an ABX system [7.05 (1H, d, J = 2 Hz), 6.95
(1H, dd, J = 8, 2 Hz) and 6.79 (1H, d, J = 8 Hz)]. The acyl moiety of 5
was determined to be E-caffeic acid. Therefore, the structures of
canangafruticosides C (3), D (4), and E (5) were elucidated to be
10-O-E-p-coumaroyl, 10-O-Z-p-coumaroyl, and 10-O-E-caffeoyl
canangafruticoside A, respectively, as shown in Fig. 1.
Fig. 2. 1H–1H COSY and diagnostic HMBC correlations for 1. Dual arrowhead curves
denote that HMBC correlations were observed both ways.
lene protons (dH 2.39 and 1.33) on C-6, and from the oxymethylene
protons (dH 3.40 and 3.37) on C-10 to the methine proton (dH 1.60)
on C-4 (Fig. 2). HMBC correlation cross-peaks between H-2 (dH
3.80) and C-1 (dC 56.6), and H-6a (dH 2.39) and C-2 (dC 76.2) estab-
lished the formation of a six membered-ring through the quater-
nary carbon, to which, thus, the aldehyde and the double bond
were attached. The cross-peaks between H-7 (dH 9.81) and C-1,
and H-8 (dH 4.60) and C-1 confirmed the substituents on the qua-
ternary carbon. The enol was stabilized by a sugar linkage, which
was substantiated by cross-peaks between an anomeric proton
(dH 4.47) and the enol carbon (dC 146.1, C-9), and H-9 (dH 6.44)
and an anomeric carbon (dC 104.3) in the HMBC spectrum. A large
coupling constant for H-2 and H-3 in the 1H NMR spectrum en-
Compound 6, [
and its elemental composition was determined to be C10H16O4 by
HRESIMS. The IR spectrum indicated the presence of an -lactone
a
]D ꢁ10.3, was isolated as an amorphous powder
c
(1748 cmꢁ1). The 1H–1H COSY spectrum exhibited the same two
proton sequences from H-2 to H2-6 and from H2-10 to H-4 as the
same as in 1 (Fig. 3). The NMR spectroscopic data indicated that
the C-7–C-8 double bond in 1 was reduced to a single bond and
the aldehyde was oxidized to carboxylic acid. The HMBC correla-
tion cross-peaks between H2-9 (dH 4.32 and 4.25) and C-7 (dC
181.3) supported the formation of a spiro-lactone ring. Therefore,
the structure of compound 6 was elucidated to be as shown in
Fig. 1. Although the numbering for 6 is not in accord with the for-

J. Nagashima et al. / Phytochemistry 71 (2010) 1564–1572
1567
Fig. 3. 1H–1H COSY and diagnostic HMBC correlations for 6. Dual arrowhead curve
denotes HMBC correlations were observed both ways.
Fig. 4. Diagnostic HMBC correlations for 8. Dual arrowhead curve denotes HMBC
correlations were observed both ways.
mal one, for the readers’ convenience, the same numbering as for
the aforementioned compounds is used in Fig. 1 and Table 1.
A similar lignan, aryldihydronaphthalene dicarboxylic acid dies-
ter, to compound 8 was isolated from Trigonotis peduncularis and
its fluorescent nature was examined (Otsuka et al., 2008). The com-
pound isolated from T. peduncularis emitted yellow fluorescence at
526 nm on excitation with UV light at 435 nm, whereas compound
8 emitted blue fluorescence at 494 nm on excitation with UV light
at 375 nm. The functional groups on the aromatic rings varied with
the excitation and emission wavelengths. Similar to in the case of
lignan from T. peduncularis, in neutral and acidic buffers, a limited
fluorescence strength was observed, but under basic conditions,
the fluorescence strength drastically increased (Fig. 5). The quan-
tum yield (ux) of compound 8 in a carbonate buffer at pH 9 was
0.454. This value was comparable to that of well-known fluores-
cent reagent rhodamine B.
Compound 7, [
a
]D ꢁ59.4, was isolated as an amorphous powder
and its elemental composition was determined to be C21H28O8 by
HRESIMS. The NMR spectroscopic data indicated that the presence
of a caffeoyl moiety, two hemiacetals (dC 107.1 with dH 4.98 and dC
105.2 with dH 5.09) and two methoxyl groups. Two methoxyl pro-
tons (dH 3.40 and 3.39) were correlated with two hemiacetalic car-
bons (dC 105.2 and 107.1, respectively) in the HMBC spectrum. The
PS-NOESY correlation of the H-3 axial proton (dH 1.24) and H-7 (dH
4.98) placed the methoxyl group at the C-7 position in the
a-orien-
tation and the PS-NOESY correlation of H-8 (dH 2.10) and H-9 (dH
a
5.09) placed the methoxyl group at the C-9 position in the b-orien-
tation. The formation mechanism for the two hemiacetals was as-
sumed to comprise the hydrolysis of canangafruticoside E (5) in
plants yielding a corresponding dialdehyde, the dialdehyde then
being cyclized to give didemethyl compound 70. During extraction
with MeOH, compound 7 might be formed from didemethyl com-
pound 70 as an artifact.
To determine the absolute configuration of the monoterpene
moiety, compound 7 was expected to be the most plausible com-
pound for the modified Mosher’s method (Ohtani et al., 1991),
although it was probably an artifact. Thus, compound 7 was first
methylated with trimethylsilyldiazomethane, and then esterifica-
Compound 8, [
and its fairly large molecular formula was calculated to be
50H62O23 by HRESIMS. The IR spectrum exhibited two types of
a]D +78.3, was isolated as an amorphous powder
tion was attempted using (R)- and (S)-a-methoxy-a-trifluorometh-
ylphenylacetic acids (MTPA) in the presence of 1-ethyl-3-
(3-dimethylaminopropyl)cardodiimide hydrochloride (EDC) and
N,N-dimethyl-4-aminopyridine (4-DMAP). However, no reaction
occurred. A further attempt under different conditions using (S)-
and (R)-MTPA chlorides/pyridine was also unsuccessful, probably
due to steric hindrance of dimethoxyl dihemiacetalic spiro ring sys-
tem. In the next attempt, the most abundant canangafruticoside E
(5) was used as a candidate due to its quantity. On direct enzymatic
C
carbonyl functional groups (1710 and 1671 cmꢁ1), and the pres-
ence of aromatic ring(s) was indicated by the UV absorption at
330 nm. In the 13C NMR spectrum, some signals appeared as pairs
of two close resonances. These resonances showed good similarity
to those of the monoterpene glucoside moiety of canangafrutico-
side B (2). The remaining 16 signals from 18 carbons comprised
those of tetra- and para-substituted aromatic rings, one trisubsti-
tuted double bond, two methines and two carbonyl carbons. In
the 1H spectrum, two aromatic protons appeared as doublets with
an ortho coupling constant and two pairs of two aromatic protons
also as doublets with an ortho coupling constant. Two methine pro-
tons were coupled to each other with a small coupling constant
and an olefinic proton appeared at dH 7.64 as a singlet. From the
above evidence, 18 carbons were expected to form an aryldihydro-
naphthalene dicarboxylate skeleton. The diagnostic HMBC correla-
tion cross peaks between H-8 (dH 6.86) and C-1 (dC 140.4), H-1 (dH
7.64) and C-2a (dC 168.7) and C-3 (dC 48.7), H-4 (dH 5.00) and C-20
(dC 129.5), and H-4 and C-3a (dC 174.1) supported the structure
(Fig. 4). Thus, the structure of 8 was established to be as shown
in Fig. 1, and H2-1000 (dH 4.00) and H2-100000 (dH 3.89) clearly showed
HMBC cross-peaks with C-2a and C-3a, respectively. The small cou-
pling constant of H3–4 indicated that H-3 and H-4 were in a trans
relationship, and judging from the positive Cotton effect at
257 nm in the CD spectrum, the configurations at the 3- and 4-
positions were assigned as S and R, respectively (Nishizawa et al.,
1990).
900
800
700
pH 5.0 acetate
600
pH 6.0 phosphate
pH 7.0 phosphate
500
A.U.
pH 8.0 phosphate
pH 9.0 carbonate
pH 10.0 carbonate
400
300
200
100
0
300
400
500
nm
600
700
Fig. 5. Emission spectra for compound 5 at various pHs.

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J. Nagashima et al. / Phytochemistry 71 (2010) 1564–1572
Scheme 1. Conversion of canangafruticoside E (5) to its tripivaloyl MTPA derivatives (5f, 5g).
hydrolysis of 5, the free enol thus formed would be spontaneously
converted to an aldehyde, and the resulting dialdehyde undergo
cyclizationto form a spiro ring system. To avoid spiro ring formation,
a chain of reactions was initiated by catalytic reduction of the double
bonds, and the a sequence of reactions being shown in Scheme 1. The
final results with the modified Mosher0s method in Fig. 6 showed
that the unusual monoterpene moiety had the 1S,2S,4S configura-
tion. Although the absolute stereochemistry of the remaining re-
lated compounds was not confirmed one by one, they were
expected to have the same absolute stereochemistry as that of 5.
3. Conclusions
It is noteworthy that the established monoterpene moiety does
not observe the general isoprene rule. Before now, only two com-
pounds with this carbon skeleton have been isolated, as previously
stated, one from C. odorata f. genuina called canangone (11)
(Caloprisco et al., 2002) and one from Juniperus communis called
1,4-dimethylcyclohex-3-enyl methyl ketone (12) (Thomas, 1973).
Biosynthetic investigation of these unusual monoterpenes is a
Fig. 6. Results of modified Mosher’s method (DdS–dR).

J. Nagashima et al. / Phytochemistry 71 (2010) 1564–1572
1569
subject of deep interest. A larger molecule, compound 8, was estab-
lished to be a derivative of an aryldihydronephthalene-type lignan
with two carboxylic acid terminals, to which one molecule of can-
angafruticoside A (1), respectively, was attached through an ester
bond. It is also interesting that under basic conditions, compound
8 emits strong blue fluorescence on irradiation with UV light at
375 nm.
MeOH–H2O eluent was subjected to silica gel (160 g) CC, with elu-
tion with CHCl3 (1 L) and CHCl3–MeOH [(99:1, 1 L), (97:3, 1 L),
(19:1, 1 L), (37:3, 1 L), (9:1, 1 L), (7:1, 1 L), (17:3, 1 L), (33:7, 1 L),
(4:1, 1 L) and (7:3, 1 L)], 200 mL fractions being collected. Com-
bined fractions 22–25 (254 mg) were separated by DCCC and then
the residue (141 mg) in fractions 1–72 was purified by HPLC
(MeOH–H2O, 1:9) to give 6 (3.8 mg) from the peak at 38 min.
The residue (19.9 g in fractions 9–12) obtained on Diaion HP-20
CC of the 40–60% MeOH–H2O eluent was subjected to silica gel
(500 g) CC, with elution with CHCl3 (2 L) and CHCl3–MeOH
[(99:1, 3 L), (97:3, 3 L), (19:1, 3 L), (37:3, 3 L), (9:1, 3 L), (7:1, 3 L),
(17:3, 3 L), (33:7, 3 L), (4:1, 3 L) and (7:3, 6 L)], 500 mL fractions
being collected. Combined fractions 23–31 (606 mg) were sepa-
rated by ODS open CC to give 10 (30.6 mg) in fractions 123–128.
Combined fractions 32–39 (1.28 g) were separated by ODS open
CC to give 9 (8.0 mg) in fractions 82–86 and a residue (126 mg)
in fractions 164–170, which was then purified by DCCC to afford
a residue (37.0 mg) in fractions 46–53. This was finally purified
by HPLC (MeOH–H2O, 23:27) to give 4 (7.1 mg) from the peak at
32 min. An aliquot of combined fractions 40–48 (2.49 g out of
2.71 g) was separated by ODS open CC to give a residue (224 mg)
in fractions 247–255, which was then purified by DCCC to afford
4. Experimental
4.1. General experimental procedure
Optical rotations and CD spectra were measured on JASCO P-
1030 and JASCO J-720 polarimeters, respectively, whereas IR and
UV spectra were established using Horiba FT-710 and JASCO V-
520 UV/vis spectrophotometers, respectively. Measurement of
fluorescence spectra was performed on a Hitachi F-2500 fluores-
cence spectrophotometer. 1H and 13C NMR spectra were acquired
on JEOL JNM
a-400 and ECA-600 spectrometers at 400 and 600,
and 100 and 150 MHz, respectively, with tetramethylsilane as an
internal standard. Positive-ion HRESIMS was performed with an
Applied Biosystem QSTAR XL system ESI (Nano Spray)-TOF-MS.
A highly-porous synthetic resin (Diaion HP-20) was purchased
from Mitsubishi Kagaku (Tokyo, Japan). Silica gel CC and re-
versed-phase [octadecyl silica gel (ODS)] open CC were performed
3
(25.6 mg) in fractions 55–65. Combined fractions 49–54
(1.06 g) were separated by ODS open CC to give a residue
(176 mg) in fractions 161–167, which was then purified by DCCC
to afford 5 (93.0 mg) in fractions 31–39. An aliquot of combined
fractions 55–68 (2.50 g out of 9.19 g) was separated by ODS open
CC to give two residues in fractions 21–26 (25.7 mg) and 88–99
(688 mg). The former was purified by DCCC to give 1 (2.9 mg) in
fractions 18–20. The latter was purified by DCCC to give a residue
(121 mg) in fractions 26–31, which was then purified by HPLC
(MeOH–H2O, 1:1) to yield 8 (17.9 mg).
The residue (10.8 g in fractions 13–17) obtained on Diaion HP-
20 CC of the 60–80% MeOH–H2O eluent was subjected to silica gel
(160 g) CC, with elution with CHCl3 (1 L), CHCl3–MeOH [(99:1, 1 L),
(49:1, 1 L), (97:3, 1 L), (24:1, 1 L), (47:3 1 L), (19:1, 1 L), (23:2, 1 L),
(17:3, 1 L) and (7:3, 1 L)] and CHCl3–MeOH–H2O (35:15:2, 1 L),
250 mL fractions being collected. Combined fractions 20–23
(300 mg) were separated by ODC open CC to give 7 (16.5 mg) in
fractions 175–190. Combined fractions 37–41 (458 mg) were sep-
arated by ODS open CC to give 2 (29.6 mg) in fractions 217–223.
on silica gel 60 (Merck, Darmstadt, Germany) and Cosmosil 75C18
-
OPN (Nacalai Tesque, Kyoto, Japan) [ = 50 mm, L = 25 cm, linear
U
gradient: MeOH–H2O (1:9, 1.5 L) ? (3:1, 1.5 L), fractions of 10 g
being collected], respectively. Droplet counter-current chromatog-
raphy (DCCC) (Tokyo Rikakikai, Tokyo, Japan) was equipped with
500 glass columns (
U = 2 mm, L = 40 cm), the lower and upper lay-
ers of a solvent mixture of CHCl3–MeOH–H2O-n-PrOH (9:12:8:2)
being used as the stationary and mobile phases, respectively.
Five-gram fractions were collected and numbered according to
their order of elution with the mobile phase. HPLC was performed
on an ODS-3 column (Inertsil; GL Science, Tokyo, Japan;
U = 6 mm,
L = 25 cm), and the eluate was monitored with a UV detector at
254 nm and a refractive index monitor.
Hesperidinase was a gift from Tokyo Tanabe Pharmaceutical
Co., Ltd. (Tokyo, Japan). (R)- and (S)-MTPAs were the products of
Wako Pure Chemical Industry Co., Ltd. (Tokyo, Japan).
4.4. Canangafruticoside A (1)
4.2. Plant material
Amorphous powder; ½a D25
ꢀ
+23.8 (c 0.24, MeOH); IR (film) mmax
1H NMR (CD3OD,
3363, 2926, 1713, 1655, 1594, 1375, 1074 cmꢁ1
;
Leaves of C. odorata var. fruticosa (Annonaceae) were collected
in March, 2005, from cultivated species in the Botanical Garden,
Faculty of Pharmacy, Chiang Mai University, Chiang Mai, Thailand
and a voucher specimen (05-COF-CMU-0308) was deposited in the
Herbarium of Faculty of Pharmacy, Chiang Mai University.
600 MHz) d: 9.81 (1H, s, H-7), 6.44 (1H, d, J = 7 Hz, H-9), 4.60 (1H, d,
J = 7 Hz, H-8), 4.47 (1H, d, J = 8 Hz, H-10), 3.85 (1H, dd, J = 12, 2 Hz,
H-60a), 3.80 (1H, dd, J = 12, 4 Hz, H-2), 3.65 (1H, dd, J = 12, 5 Hz, H-
60b), 3.42–3.27 (3H, m, H-30, 40 and 50), 3.40 (1H, dd, J = 11, 6 Hz, H-
10a), 3.37 (1H, dd, J = 11, 6 Hz, H-10b), 3.23 (1H, dd, J = 9, 8 Hz, H-
20), 2.39 (1H, ddd, J = 14, 4, 4 Hz, H-6a), 1.96 (1H, ddd, J = 13, 4, 4,
2 Hz, H-3a), 1.60 (1H, dddddd, J = 13, 12, 6, 6, 4, 4 Hz, H-4), 1.57
(1H, ddddd, J = 13, 4, 4, 4, 2 Hz, H-5a), 1.33 (1H, ddd, J = 14, 13,
4 Hz, H-6b), 1.22 (1H, ddd, J = 13, 12, 12 Hz, H-3b), 1.20 (1H, dddd,
J = 13, 13, 12, 4 Hz, H-5b); for 13C NMR (CD3OD, 150 MHz) spectro-
scopic data, see Table 1; HRESIMS (positive-ion mode) m/z:
385.1461 [M+Na]+ (Calcd. for C16H26O9Na: 385.1469).
4.3. Extraction and isolation
Dried leaves of C. odorata var. fruticosa (1.17 kg) were extracted
with MeOH (4.5 ꢂ 3 L) at 25 °C for 1 week and then concentrated
to 3 L in vacuo. The extract was washed with n-hexane (3 L,
36.3 g) and then the MeOH layer was concentrated to a gummy
mass. The latter was suspended in H2O (3 L) and extracted with
EtOAc (3 L) to give an EtOAc-soluble fraction (23.5 g). The aqueous
layer was extracted with 1-BuOH (3 L) to give a 1-BuOH-soluble
fraction (75.4 g), and the remaining aqueous-layer was concen-
trated to furnish a water-soluble fraction (173 g).
4.5. Canangafruticoside B (2)
Amorphous powder; ½a D25
ꢀ
+15.0 (c 1.95, MeOH); IR (film) mmax
The 1-BuOH-soluble fraction was subjected to a Diaion HP-20
3388, 2934, 1706, 1634, 1606, 1512, 1450, 1277, 1074 cmꢁ1; UV
column (
U
= 50 mm, L = 50 cm) using H2O–MeOH [(4:1, 4 L), (2:3,
kmax (MeOH): 322sh (3.75), 276 (4.06), 213 (4.17) nm (log e
); 1H
4 L), (3:2, 4 L), and (1:4, 4 L)], and MeOH (4 L), 1 L fractions being
collected. The residue (6.36 g in fractions 6–8) of the 20–40%
NMR (CD3OD, 400 MHz) d: 9.82 (1H, s, H-7), 7.68 (1H, d,
J = 16 Hz, H-700), 7.59 (2H, m, H-200 and 600), 7.39 (3H, m, H-300, 400

1570
J. Nagashima et al. / Phytochemistry 71 (2010) 1564–1572
and 500), 6.50 (1H, d, J = 16 Hz, H-800), 4.38 (1H, d, J = 7 Hz, H-8), 6.45
(1H, d, J = 7 Hz, H-9), 4.48 (1H, d, J = 8 Hz, H-10), 4.06 (2H, s, H2-10),
3.84 (1H, dd, J = 11, 4 Hz, H-2), 3.84 (1H, dd, J = 12, 2 Hz, H-60a),
3.66 (1H, dd, J = 12, 5 Hz, H-60b), 3.41–3.31 (3H, m, H-30, 40 and
50), 3.25 (1H, dd, J = 9, 8 Hz, H-20), 2.41 (1H, dd, J = 10, 3 Hz, H-
6a), 1.98 (1H, m, H-3a), 1.88 (1H, m, H-4), 1.59 (1H, m, H-5a),
1.41–1.28 (3H, m, H-3b, 5b and 6b); for 13C NMR (CD3OD,
100 MHz) spectroscopic data, see Table 1; HRESIMS (positive-ion
mode) m/z: 515.1879 [M+Na]+ (Calcd. for C25H32O10Na: 515.1887).
4.9. Compound 6
Amorphous powder; ½a D25
ꢀ
ꢁ10.3 (c 0.25, MeOH); IR (film) mmax
1H NMR (CD3OD,
3395, 2926, 1748, 1454, 1382, 1182, 1031 cmꢁ1
;
600 MHz) d: 4.32 (1H, ddd, J = 12, 7, 6 Hz, H-9a), 4.25 (1H, ddd,
J = 12, 7, 5 Hz, H-9b), 3.59 (1H, dd, J = 10, 4 Hz, H-2), 3.42 (2H, m,
H2-10), 2.55 (1H, ddd, J = 11, 7, 5 Hz, H-8a), 2.03 (1H, ddd, J = 11,
7, 6 Hz, H-8b), 1.98 (1H, d, J = 13 Hz, H-6a), 1.82–1.71 (2H, m, H2-
3), 1.63–1.53 (3H, m, H-4 and H2-5), 1.52 (1H, dd, J = 13, 4 Hz, H-
6b); for 13C NMR (CD3OD, 150 MHz) spectroscopic data, see Table 1;
HRESIMS (positive-ion mode) m/z: 223.0931 [M+Na]+ (Calcd. for
4.6. Canangafruticoside C (3)
C10H16O4Na: 223.0940).
Amorphous powder; ½a D25
ꢀ
+29.5 (c 1.05, MeOH); IR (film) mmax
4.10. Compound 7
3367, 2931, 1711, 1603, 1512, 1273, 1168, 1072 cmꢁ1; UV kmax
e
); 1H NMR (CD3OD,
Amorphous powder; ½a D25
ꢀ
ꢁ59.4 (c 1.09, MeOH); IR (film) mmax
(MeOH): 310 (4.15), 211 (4.26) nm (log
400 MHz) d: 9.82 (1H, s, H-70), 7.61 (1H, d, J = 16 Hz, H-7), 7.45
(2H, d, J = 9 Hz, H-20 and 60), 6.81 (2H, d, J = 9 Hz, H-30 and 50),
6.45 (1H, d, J = 7 Hz, H-9), 6.31 (1H, d, J = 16 Hz, H-80), 4.48 (1H,
d, J = 8 Hz, H-10), 4.38 (1H, d, J = 7 Hz, H-8), 4.06 (2H, s, H2-10),
3.86 (1H, dd, J = 12, 2 Hz, H-60a), 3.84 (1H, dd, J = 11, 4 Hz, H-2),
3.66 (1H, dd, J = 12, 5 Hz, H-60b), 3.40–3.31 (3H, m, H-30, 40 and
50), 3.24 (1H, dd, J = 9, 8 Hz, H-20), 1.98 (1H, m, H-3a), 1.88 (1H,
m, H-4), 1.59 (1H, m, 5a), 2.41 (dd, J = 10, 3 Hz, H-6a), 1.41–1.28
(3H, m, H-3b, 5b and 6b); for 13C NMR (CD3OD, 100 MHz) spectro-
scopic data, see Table 1; HRESIMS (positive-ion mode) m/z:
531.1832 [M+Na]+ (Calcd. for C25H32O11Na: 531.1836).
3362, 2936, 1698, 1455, 1394, 1167, 1096, 1023 cmꢁ1; UV kmax
(MeOH): 325 (4.15), 297sh (4.09), 240sh (4.12), 217 (4.22) nm
1
(log
e
); H NMR (CD3OD, 600 MHz) d: 7.04 (1H, s, H-20), 6.94 (1H,
d, J = 8 Hz, H-60), 6.78 (1H, d, J = 8 Hz, H-50), 7.54 (1H, d, J = 16 Hz,
H-70), 6.25 (1H, d, J = 16 Hz, H-80), 5.09 (1H, dd, J = 6, 2 Hz, H-9),
4.98 (1H, s, H-7), 4.07 (1H, dd, J = 11, 7 Hz, H-10a), 4.03 (1H, dd,
J = 11, 7 Hz, H-10b), 3.60 (1H, dd, J = 11, 4 Hz, H-2), 3.40 (3H, s,
–OCH3 on C-9), 3.39 (3H, s, –OCH3 on C-7), 2.10 (1H, dd, J = 14,
6 Hz, H-8a), 2.00 (2H, m, H-3a and 6a), 1.90 (1H, dd, J = 14, 2 Hz,
H-8b), 1.84 (1H, m, H-4), 1.54 (1H, m, H-5a), 1.30 (1H, m, H-6b),
1.24 (1H, m, H-3b), 1.15 (1H, m, H-5b); for 13C NMR (CD3OD,
150 MHz) spectroscopic data, see Table 1; HRESIMS (positive-ion
mode) m/z: 431.1670 [M+Na]+ (Calcd. for C21H28O8Na: 431.1676).
4.7. Canangafruticoside D (4)
4.11. Compound 8
Amorphous powder; ½a D25
ꢀ
+19.1 (c 0.46, MeOH); IR (film) mmax
3367, 2931, 171, 1603, 1513, 1450, 1273, 1166, 1075 cmꢁ1; UV
Amorphous powder; ½a D25
ꢀ
+78.3 (c 1.13, MeOH); IR (film) mmax
kmax (MeOH): 310 (4.37), 227 (4.16) nm (log e
); 1H NMR (CD3OD,
3373, 2931, 2871, 1710, 1671, 1615, 1509, 1452, 1281, 1205,
400 MHz) d: 9.82 (1H, s, H-7), 7.58 (2H, d, J = 9 Hz, H-200 and 600),
6.87 (1H, d, J = 13 Hz, H-700), 6.75 (2H, d, J = 9 Hz, H-300and 500),
6.45 (1H, d, J = 7 Hz, H-9), 5.77 (1H, d, J = 13 Hz, H-800), 4.60 (1H,
d, J = 7 Hz, H-8), 4.47 (1H, d, J = 8 Hz, H-10), 4.06 (2H, s, H2-10),
3.86 (1H, dd, J = 12, 2 Hz, H-60a), 3.81 (1H, dd, J = 11, 4 Hz, H-2),
3.66 (1H, dd, J = 12, 5 Hz, H-60b), 3.38–3.28 (3H, m, H-30, 40 and
50), 3.24 (1H, dd, J = 9, 8 Hz, H-20), 2.41 (1H, dd, J = 10, 3 Hz, H-
6a), 1.98 (1H, m, H-3a), 1.88 (1H, m, H-4), 1.59 (1H, m, H-5a),
1.41–1.28 (3H, m, H-3b, 5b and 6b); for 13C NMR (CD3OD,
100 MHz) spectroscopic data, see Table 1; HRESIMS (positive-ion
mode) m/z: 531.1832 [M+Na]+ (Calcd. for C25H32O11Na: 531.1836).
1073 cmꢁ1; UV kmax (MeOH): 330 (4.20), 224 (4.40) nm (log
e);
1H NMR (CD3OD, 600 MHz) d: 9.81 (1H, s, H-70000), 9.79 (1H, s, H-
700), 7.64 (1H, s, H-1), 6.86 (1H, d, J = 8 Hz, H-8), 6.86 (2H, d,
J = 9 Hz, H-20 and 60), 6.76 (1H, d, J = 8 Hz, H-7), 6.60 (2H, d,
J = 9 Hz, H-30 and 50), 6.45 (1H, d, J = 7 Hz, H-90000), 6.43 (1H, d,
J = 7 Hz, H-900), 5.00 (1H, br d, J = 2 Hz, H-4), 4.61 (1H, d, J = 7 Hz,
H-80000), 4.52 (1H, d, J = 7 Hz, H-800), 4.48 (2H, d, J = 8 Hz, H-1000 and
100000), 4.00 (2H, m, H2-1000), 3.90 (1H, d, J = 2 Hz, H-3), 3.89 (2H,
m, H2-100000), 3.86 (2H, dd, J = 12, 2 Hz, H-6000a and 600000a), 3.81
(1H, dd, J = 11, 4 Hz, H-20000), 3.76 (1H, dd, J = 11, 4 Hz, H-200), 3.67
Table 2
13C NMR spectroscopic data for compound 8 (150 MHz, CD3OD).
4.8. Canangafruticoside E (5)
8
Amorphous powder; ½a D25
ꢀ
+17.6 (c 1.79, MeOH); IR (film) mmax
C
C
C
3371, 2938, 1699, 1602, 1521, 1450, 1278, 1180, 1074 cmꢁ1; UV
1
2
2a
3
3a
4
4a
5
6
7
8
8a
10
20
30
40
50
60
140.4
122.2
168.7
48.7
174.1
40.4
125.0
144.3
149.5
114.6
123.0
125.0
134.6
129.5
116.1
157.0
116.1
129.5
100
200
300
400
500
600
700
800
900
1000
56.32
75.8
35.7
37.35
26.0
31.3
206.89
110.17
146.1
69.8
10000
20000
30000
40000
50000
60000
70000
80000
90000
100000
56.32
75.5
35.9
37.39
26.1
31.4
206.94
110.21
146.2
70.2
kmax (MeOH): 324 (4.15), 300sh (4.07), 242sh (4.01), 215 (4.17)
nm (log e
); 1H NMR (CD3OD, 400 MHz) d: 9.82 (1H, s, H-7), 7.54
(1H, d, J = 16 Hz, H-700), 7.05 (1H, d, J = 2 Hz, H-200), 6.95 (1H, dd,
J = 8, 2 Hz, H-600), 6.79 (1H, d, J = 8 Hz, H-500), 6.45 (1H, d, J = 7 Hz,
H-9), 6.25 (1H, d, J = 16 Hz, H-800), 4.61 (1H, d, J = 7 Hz, H-8), 4.48
(1H, d, J = 8 Hz, H-10), 4.06 (1H, dd, J = 11, 6 Hz, H-10a), 4.02 (1H,
dd, J = 11, 6 Hz, H-10b), 3.86 (1H, dd, J = 12, 2 Hz, H-60a), 3.84
(1H, dd, J = 12, 4 Hz, H-2), 3.67 (1H, dd, J = 12, 5 Hz, H-60b), 3.36–
3.23 (3H, m, H-30, 40 and 50), 3.25 (1H, dd, J = 9, 8 Hz, H-20), 2.41
(1H, dd, J = 10, 3 Hz, H-6a), 1.97 (1H, ddd, J = 12, 4, 4 Hz, H-3a),
1.87 (1H, m, H-4), 1.58 (1H, m, H-5a), 1.38 (1H, m, H-5b), 1.36
(1H, d, J = 10 Hz, H-6b), 1.36 (1H, ddd, J = 12, 12, 12 Hz, H-3b); for
13C NMR (CD3OD, 100 MHz) spectroscopic data, see Table 1; HRE-
SIMS (positive-ion mode) m/z: 547.1793 [M+Na]+ (Calcd. for
1000
2000
3000
4000
5000
6000
104.3
74.6
78.6
71.3
78.0
62.7
100000
200000
300000
400000
500000
600000
104.3
74.6
78.6
71.3
78.0
62.7
C
25H32O12Na: 547.1785).

J. Nagashima et al. / Phytochemistry 71 (2010) 1564–1572
1571
(2H, dd, J = 12, 5 Hz, H-6000b and 600000b), 3.38–3.25 (6H, m, H-3000, 4000,
5000, 300000, 400000 and 500000), 3.26 (2H, dd, J = 9, 8 Hz, H-2000and 200000), 2.35
(2H, m, H-600a and 60000a), 1.83 (1H, m, H-400), 1.94–1.82 (2H, m,
H-300a and 30000a), 1.67 (1H, m, H-40000), 1.51 (1H, m, H-50000b), 1.37–
1.22 (6H, m, H-300b, 500a, 600b, 30000b, 50000a and 60000b), 1.10 (1H, m,
H-500b); for 13C NMR (CD3OD, 150 MHz) spectroscopic data, see
and 50), 3.16 (1H, dd, J = 8, 8 Hz, H-20), 1.93 (1H, ddd, J = 15, 7,
7 Hz, H-8a), 1.83 (1H, ddd, J = 15, 7, 7 Hz, H-8b), 1.82 (1H, m, H-
3a), 1.72 (1H, m, H-6a), 1.52 (1H, m, H-4), 1.23–1.26 (1H, m, H-
5a), 1.26 (1H, m, H-6b), 1.18 (1H, m, H-3b), 1.12 (1H, dddd, J = 13,
13, 13, 3 Hz, H-5b); HRESIMS (positive-ion mode) m/z: 389.1787
[M+Na]+ (Calcd. for C16H30O9Na: 389.1782).
Table 2; CD
D
e
(nm) ꢁ0.07 (361), +12.6 (309), +7.33 (257), ꢁ29.8
(229) (c 5.49 ꢂ 10ꢁ5 m, MeOH); HRESIMS (positive-ion mode) m/
4.12.4. Enzymatic hydrolysis of deacyldihydrocanangafruticoside A
7-ol (5c)
z: 1053.3579 [M+Na]+ (Calcd. for C50H62O23Na: 1053.3574).
Dihydrocanangafruticoside A 7-ol (5c) (2.2 mg) was dissolved in
20 mM acetate buffer (1.2 mL) and then hesperidinase (1 mg) was
added. The mixture was incubated for 18 h at 37 °C. The aglycone
(5d) (0.9 mg) and glucose were purified by prep. TLC (CHCl3–
MeOH–H2O, 15:6:1). Aglycone (5d): colorless oil; 1H NMR (CD3OD,
400 MHz) d: 3.84 (1H, d, J = 11 Hz, H-7a), 3.76 (1H, ddd, J = 11, 8,
6 Hz, H-9a), 3.69 (1H, ddd, J = 11, 6, 5 Hz, H-9b), 3.64 (1H, d,
J = 11 Hz, H-7b), 3.52 (1H, dd, J = 12, 5 Hz, H-2), 3.42 (1H, dd,
J = 11, 6 Hz, H-10a), 3.41 (1H, dd, J = 11, 6 Hz, H-10b), 1.85–1.78
(3H, m, H-6a, 8a and 8b), 1.69 (1H, ddd, J = 15, 5, 5 Hz, H-3a),
1.53 (1H, m, H-4), 1.32–1.22 (2H, m, H-5a and 6b), 1.15–1.07
(2H, m, H-3b and 5b); HRESIMS (positive-ion mode) m/z:
227.1257 [M+Na]+ (Calcd. for C10H20O4Na: 227.1253). Glucose
was analyzed with a chiral detector (JASCO OR-2090plus) on an
amino column [Asahipak NH2P-50 4E, CH3CN–H2O (4:1), 1 mL/
min], a peak at 14.5 min with a positive optical rotation sign being
observed. Peak was identified by co-chromatography with authen-
4.12. Modified Mosher0s method for canangafruticoside E (5)
4.12.1. Catalytic hydrogenation of canangafruticoside E (5)
Canangafruticoside E (5) (10.0 mg) was dissolved in 1 mL of
MeOH and then reduced with PtO2 (1 mg) under a H2 atmosphere
for 1 h. The catalyst was removed by filtration and the filtrate was
evaporated to dryness to give tetrahydrocanangafruticoside E (5a)
(10.1 mg). Tetrahydrocanangafruticoside E (5a): amorphous pow-
der; 1H NMR (CD3OD, 400 MHz) d: 9.82 (1H, s, H-7), 6.66 (1H, dd,
J = 8, 2 Hz, H-500), 6.61 (1H, d, J = 2 Hz, H-200), 6.50 (1H, d, J = 8 Hz,
H-600), 4.22 (1H, d, J = 8 Hz, H-10), 4.02 (1H, dd, J = 10, 6 Hz, H-
10a), 4.01 (1H, dd, J = 10, 6 Hz, H-10b), 4.00 (1H, ddd, J = 11, 6,
6 Hz, H-9a), 3.86 (1H, m, H-60a), 3.68 (1H, m, H-2), 3.66 (1H, m,
H-60b), 3.60 (1H, m, H-9b), 3.33 (1H, m, H-30), 3.33–3.25 (1H, m,
H-40), 3.25 (1H, m, H-50), 3.13 (1H, dd, J = 9, 8 Hz, H-20), 2.76 (2H,
t, J = 7 Hz, H2-700), 2.55 (2H, t, J = 7 Hz, H2-800), 2.26 (1H, ddd,
J = 14, 7, 6 Hz, H-8a), 2.07 (1H, ddd, J = 14, 3, 3 Hz, H-6a), 1.86
(1H, m, H-3a), 1.81 (2H, m, H-4 and 8b), 1.54 (1H, m, H-5a), 1.22
(2H, m, H-5b and 6b), 1.16 (1H, m, H-3b); HRESIMS (positive-ion
mode) m/z: 551.2091 [M+Na]+ (Calcd. for C25H36O12Na: 551.2098).
tic D-glucose.
4.12.5. Protection of the primary alcohols of aglycone (5d)
The aglycone (5d) (0.9 mg) was dissolved in pyridine (1 mL) and
pivaloyl chloride (8 lL) was added. The reaction mixture was stir-
4.12.2. NaBH4 reduction of tetrahydrocanangafruticoside E (5a)
Tetrahydrocanangafruticoside E (5a) (10.1 mg) was dissolved in
EtOH (1 mL) and then cooled on an ice-bath. NaBH4 (1 mg) in EtOH
red for 3 h at 25 °C. To the reaction mixture, H2O (1 mL) was added,
followed by extracted with EtOAc (2 mL ꢂ 3). The residue from the
dried (Na2SO4) organic layer was purified by prep. TLC [CHCl3–
(CH3)2CO, 20:1] to give tripivalate (1.2 mg) (5e). Tripivalate (5e):
colorless oil; 1H NMR (CDCl3, 400 MHz) d: 4.28 (1H, d, J = 12 Hz,
H-7a), 4.22 (2H, t, J = 7 Hz, H2–9), 4.18 (1H, d, J = 12 Hz, H-7b),
3.94 (1H, dd, J = 11, 6 Hz, H-10a), 3.91 (1H, dd, J = 11, 6 Hz, H-
10b), 3.60 (1H, dd, J = 12, 5 Hz, H-2), 1.90 (1H, ddd, J = 14, 7, 7 Hz,
H-8a), 1.85 (1H, m, H-3a), 1.84 (1H, ddd, J = 14, 7, 7 Hz, H-8b),
1.78 (1H, m, H-4), 1.76 (1H, m, H-6a), 1.35 (1H, m, H-3b), 1.35–
1.10 (2H, m, H2-5), 1.21 (1H, m, H-6b), 1.180, 1.184, 1.20 (each
9H, each s, CH3 ꢂ 3); HRESIMS (positive-ion mode) m/z: 479.2984
[M+Na]+ (Calcd. for C25H44O7Na: 479.2979).
(500
l
L) was added, followed by stirring for 30 min. Excess NaBH4
L of a 1% EtOH solution of
was quenched by the addition of 500
l
acetic AcOH. The reduced product was purified by prep. TLC
(CHCl3–MeOH–H2O, 15:6:1) to give tetrahydrocanangafruticoside
E 7-ol (5.6 mg) (5b). Tetrahydrocanangafruticoside E 7-ol (5b):
amorphous powder; 1H NMR (CD3OD, 400 MHz) d: 6.66 (1H, d,
J = 8 Hz, H-500), 6.62 (1H, d, J = 2 Hz, H-200), 6.51 (1H, dd, J = 8,
2 Hz, H-600), 4.29 (1H, d, J = 8 Hz. H-10), 4.10 (1H, ddd, J = 10, 7, 7,
Hz, H-9a), 3.92 (2H, d, J = 6 Hz, H2-10), 3.86 (1H, dd, J = 12, 1 Hz,
H-60a), 3.84 (1H, d, J = 11 Hz, H-7a), 3.76 (1H, ddd, J = 10, 7, 7 Hz,
H-9b), 3.66 (1H, m, H-60b), 3.60 (2H, m, H-2 and 7b), 3.35 (1H, m,
H-30), 3.33–3.25 (2H, m, H-40 and 50), 3.17 (1H, dd, J = 9, 8 Hz, H-
20), 2.77 (2H, t, J = 7 Hz, H2-700), 2.56 (2H, t, J = 7 Hz, H2-800), 1.92
(1H, m, H-8a), 1.83 (1H, ddd, J = 15, 7, 7 Hz, H-8b), 1.72 (1H, m,
H-6a), 1.71 (1H, ddd, J = 13, 3, 3 Hz, H-3a), 1.65 (1H, m, H-4), 1.37
(1H, m, H-5a), 1.31 (1H, m, H-6b), 1.16 (1H, m, H-3b), 1.11 (1H,
m, 5b); HRESIMS (positive-ion mode) m/z: 553.2258 [M+Na]+
(Calcd. for C25H38O12Na: 551.2255).
4.12.6. Preparation of (R)- and (S)-MTPA esters (5f and 5g) of
tripivalate (5e)
A solution of 5e (0.6 mg) in dry CH2Cl2 (1 mL) was reacted with
(R)-MTPA (50 mg) in the presence of EDC (40 mg) and 4-DMAP
(25 mg), and then the mixture was occasionally stirred at 25 °C
for 30 min and then at 40 °C for 5 min. After the addition of CH2Cl2
(1 mL), the solution was washed with H2O (1 mL), 4 N HCl (1 mL),
NaHCO3-saturated H2O, and then brine (1 mL), successively. The
organic layer was dried (Na2SO4) and then evaporated under re-
duced pressure. The residue was purified by prep. TLC [silica gel
(0.25 mm thickness), being applied for 18 cm, with development
with CHCl3–(CH3)2CO (19:1) for 9 cm and then elution with
CHCl3–MeOH (9:1)] to furnish an MTPA ester, 5f (0.5 mg). Through
a similar procedure, the (S)-MTPA ester (5g) of tripivalate (0.6 mg)
was prepared from 5e (0.6 mg) using (S)-MTPA (44 mg), EDC
(31 mg), and 4-DMAP (21 mg).
4.12.3. Removal of acyl group from tetrahydrocanangafruticoside E
7-ol (5b)
Tetrahydrocanangafruticoside E 7-ol (5b) (5.6 mg) was dis-
solved in methanolic solution of 0.1 M NaOCH3 (1 mL), followed
by stirring for 30 min at 25 °C. The reaction solvent was neutral-
ized with Amberlite IR-120B (H+) and dihydrocanangafruticoside
A 7-ol (5c) (2.2 mg) was purified by prep. TLC (CHCl3–MeOH–
H2O, 15:6:1). Dihydrocanangafruticoside A 7-ol (5c): amorphous
1
powder; H NMR (CD3OD, 400 MHz) d: 4.29 (1H, d, J = 8 Hz, H-10),
(R)-MTPA ester (5f): 1H NMR (CDCl3, 400 MHz) d: 7.43 (2H, m,
aromatic protons), 7.33–7.31 (3H, m, aromatic protons), 4.98 (1H,
dd, J = 11, 5 Hz, H-2), 4.43 (1H, J = 12 Hz, H-7a), 3.98 (2H, m, H2-
9), 3.87 (2H, m, H2-10), 3.68 (1H, d, J = 12 Hz, H-7b), 3.47 (3H, s,
–OCH3), 2.00 (1H, m, H-3a), 1.89 (1H, m H-6a), 1.83 (1H, m, H-4),
4.11 (1H, ddd, J = 10, 7, 7 Hz, H-9a), 3.87 (2H, br d, J = 12 Hz, H-7a
and 60a), 3.77 (1H, ddd, J = 10, 7, 7 Hz, H-9b), 3.66 (1H, dd, J = 12,
4 Hz, H-60b), 3.63 (1H, d, J = 12 Hz, H-7b), 3.62 (1H, m, H-2), 3.41
(2H, d, J = 6 Hz, H2-10), 3.34 (1H, m, H-30), 3.32–3.25 (2H, m, H-40

1572
J. Nagashima et al. / Phytochemistry 71 (2010) 1564–1572
1.66 (1H, m, H-8a), 1.51 (1H, m, H-5a), 1.46 (1H, m, H-8b), 1.40 (1H,
m, H-3b), 1.24 (1H, m, H-6b), 1.10 (1H, m, H-5b), 1.13 (9H, s,
–CH3 ꢂ 3), 1.01 (9H, s, –CH3 ꢂ 3), 1.00 (9H, s, –CH3 ꢂ 3); HRESIMS
(positive-ion mode) m/z 695.3372 [M+Na]+ (Calcd. for C35H51O9F3-
Na: 695.3377).
References
Caloprisco, E., Fourneron, J.-D., Faure, R., Demarne, F.-E., 2002. Unusual lactones
from Cananga odorata (Annonaceae). J. Agric. Food Chem. 50, 78–80.
Gaydou, E.M., Randriamiharisoa, R., Bianchini, J.P., 1986. Composition of the
essential oil of ylang–ylang (Cananga odorata Hook. Fil. et Thomson forma
genuina) from Madagascar. J. Agric. Food Chem. 34, 481–487.
Hsieh, T.-J., Chang, F.-R., Chia, Y.-C., Chen, C.-Yi., Chiu, H.-F., Wu, Y.-C., 2001.
Cytotoxic constituents of the fruits of Cananga odorata. J. Nat. Prod. 64, 616–
619.
IPNI, 2005. <http://www.ipni.org/ipni/plantnamesearchpage.do>.
Katague, D.B., Kirch, E.R., 1963. Analysis of the volatile components of ylang–ylang
oil by gas chromatography. J. Pharm. Sci. 52, 252–258.
Koripelly, D., Saak, W., Christoffers, J., 2007. Synthesis of optically active (+)-
canangone, its 6-epimer, and determination of absolute configuration. Eur. J.
Org. Chem. 5840–5841.
(S)-MTPA ester (5g): 1H NMR (CDCl3, 400 MHz) d: 7.42 (2H, m,
aromatic protons), 7.34–7.32 (3H, m, aromatic protons), 4.96 (1H,
dd, J = 11, 5 Hz, H-2), 4.39 (1H, J = 12 Hz, H-7a), 4.05 (2H, m, H2-
9), 3.86 (1H, dd, J = 11, 6 Hz, H-10a), 3.92 (1H, dd, J = 11, 6 Hz, H-
10b)3.70 (1H, d, J = 12 Hz, H-7b), 3.41 (3H, s, –OCH3), 1.95 (1H,
m, H-3a), 1.90 (1H, m H-6a), 1.80 (1H, m, H-4), 1.75 (1H, m, H-
8a), 1.50 (1H, m, H-5a), 1.55 (1H, m, H-8b), 1.30 (1H, m, H-3b),
1.25 (1H, m, H-6b), 1.09 (1H, m, H-5b), 1.122 (9H, s, –CH3 ꢂ 3),
1.119 (18H, s, –CH3 ꢂ 6); HRESIMS (positive-ion mode) m/z
696.3381 [M+Na]+ (Calcd. for C35H51O9F3Na: 695.3377).
Leboeuf, M., Cavé, A., 1976. Alkaloids of Annonaceae. XIV. Identification of
canangine and eupolauridine, new alkaloids with a naphthyridine nucleus. J.
Nat. Prod. (Lloydia) 39, 459–460.
Leboeuf, M., Streith, J., Cavé, A., 1975. Alkaloids of Annonaceae: alkaloids of the bark
of Cananga odorata Hook. F. and Thomson. Ann. Pharm. Fr. 33, 43–47.
Nishizawa, M., Tuda, M., Hayashi, K., 1990. Two caffeic acid tetramers having
enantiomeric phenyldihydronaphthalene moiety from Macrotomia euchroma.
Chem. Pharm. Bull. 29, 26452649.
Ohtani, I., Kusumi, T., Kashman, Y., Kakisawa, H., 1991. High-field FT NMR
application of Mosher0s method. The absolute configurations of marine
terpenoids. J. Am. Chem. Soc. 113, 40924096.
Olivero, J., Gracia, T., Payares, P., Vivas, R., Díaz, D., Daza, E., Geerlings, P., 1997.
Molecular structures and gas chromatographic retention behavior of the
components of ylang–ylang oil. J. Pharm. Sci. 86, 625630.
Otsuka, H., Kuwabara, H., Hoshiyama, H., 2008. Identification of sucrose diesters of
aryldihydronaphthalene-type lignan from Trigonotis peducularis and the nature
of their fluorescence. J. Nat. Prod. 71, 1178–1181.
4.13. Fluorescence measurement of compound 9
The buffer solutions, used, were as follows, 20 mM acetate buf-
fer for pH 5.0, 20 mM phosphate buffer for pH 6.0, 7.0 and 8.0,
20 mM borate buffer for pH 8.0 and 9.0, and carbonate buffer for
pH 9.0 and 10.0. The sample concentration was adjusted to
5.5 lM and measured at 25 °C. Maximum excitation wavelengths
were determined by measurement of emission spectra.
Acknowledgements
Porcher, M.H., 2004. 1995–2020, Sorting Cananga names. Multilingual Multiscript
Plant Name Database
– A Work in Progress. Institute for Land and Food
Resources, The University of Melbourne. <http://gmr.landfood.unimelb.edu.au/
Plantnames/Sorting/Cananga.html>.
Rao, J.U.M., Giri, G.S., Hanumaiah, T., Rao, K.V.J., 1986. Sampangine, a new alkaloid
from Canaga odorata. J. Nat. Prod. 49, 346–347.
The authors are grateful for access to the superconducting NMR
instrument (JEOL
a-400) at the Analytical Center of Molecular
Medicine and an Applied Biosystem QSTAR XL system ESI (Nano
Spray)-MS at the Analysis Center of Life Science of the Graduate
School of Biomedical Sciences, Hiroshima University. The authors
are also grateful for NMR (JEOL ECA-600) analyses at the Natural
Science Center for Basic Research and Development, Hiroshima
University. Thanks are also due to JSPS-NRCT Core University Pro-
gram on Natural Products. The authors thank Prof. Tohru Koike of
the Graduate School of Biomedical Sciences, Hiroshima University
for the assistance with fluorescence measurements.
Shahat, A.A., Abdel-Azim, N.S., Pieters, L., Vlietinck, A.J., 2004. Isolation and NMR
spectra of syringaresinol-b-D-glucoside from Cressa cretica. Fitoterapia 75, 771–
773.
Thomas, A.F., 1973. 1,4-Dimethylcyclohex-3-enyl methyl ketone, a monoterpenoid
with a novel skeleton. Helv. Chim. Acta 56, 1800–1802.
Yoshikawa, M., Shimada, H., Saka, M., Yoshimizu, S., Yamahara, J., Matsuda, H., 1997.
Medicinal foodstuffs. V. Moroheiya. (1): absolute stereostructures of
corchoionosides A, B, and C, histamine release inhibitors from the leaves of
Vietnamese Corchorus olitorius L. (Tiliaceae). Chem. Pharm. Bull. 45, 464–469.