New cholestane glycosides from the leaves of Cordyline terminalis

Article abstract of DOI:10.1248/cpb.60.275

Four new cholestane glycosides (1-4) were isolated from the leaves of Cordyline terminalis (Agavaceae). The structures of the new compounds were determined on the basis of spectroscopic analysis and a few chemical transformations followed by chromatographic and spectroscopic analyses.

Full text of DOI:10.1248/cpb.60.275

—
February 2012
Note
Chem. Pharm. Bull. 60(2) 275 279 (2012)
275
New Cholestane Glycosides from the Leaves of Cordyline terminalis
Akihito Yokosuka,* Takeyuki Suzuki, and Yoshihiro Mimaki*
Laboratory of Medicinal Pharmacognosy, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences;
1432–1 Horinouchi, Hachioji, Tokyo 192–0392, Japan.
Received October 20, 2011; accepted November 29, 2011; published online November 30, 2011
—
Four new cholestane glycosides (1 4) were isolated from the leaves of Cordyline terminalis (Agavaceae).
The structures of the new compounds were determined on the basis of spectroscopic analysis and a few
chemical transformations followed by chromatographic and spectroscopic analyses.
Key words cholestane glycoside; Cordyline terminalis; Agavaceae
The genus Cordyline (Agavaceae), with about 20 species, is 6.30 (1H, brs), 6.11 (1H, brd, J=6.0Hz), 5.88 (1H, brd,
distributed in south-east Asia, Australia, and New Zealand.1) J=3.6Hz), and 5.65 (1H, brd, J=8.3Hz), which were re-
The occurrence of steroidal sapogenins in several Cordyline moved by the addition of HCl vapor, as well as signals for
species such as C. australis, C. neocaledonica, and C. can- five steroidal methyl groups at δ 1.36 (3H, s), 1.33 (3H, s),
nifolia has been documented.2) Previously, we studied the 1.23 (3H, d, J=7.0Hz), and 0.90 (3H×2, d, J=6.5Hz), and an
chemical components of the leaves of C. stricta and isolated olefinic proton at δ 5.97 (1H, brd, J=4.5Hz). Comparison of
1
seven new steroidal glycosides together with six known com- the H- and 13C-NMR assignments of 1a, which were estab-
1
pounds.3) C. terminalis (L.) KUNTH is a perennial plant that lished by analysis of the H–1H shift correlation spectroscopy
is found in Malaysia. Although the steroidal sapogenins of (COSY), 1H-detected heteronuclear multiple-quantum coher-
smilagenin and sarsasapogenin were isolated from C. termi- ence (HMQC), and 1H-detected heteronuclear multiple-bond
nalis var. petiolaris,2) there have been no reports concerning
the secondary metabolites of C. terminalis. As part of our
continuous chemical investigations of plants of the genus
Cordyline, we have now examined the leaves of C. terminalis,
resulting in the isolation of four new cholestane glycosides
—
(1 4). This is a report on the structure elucidation of the new
glycosides on the basis of spectroscopic analyses and a few
chemical transformations followed by chromatographic and
spectroscopic analysis. The cytotoxic activities of the isolated
glycosides and aglycones against HL-60 human leukemia cells
are also reported.
The leaves of C. terminalis (6.0kg) were extracted with
MeOH. After removal of solvent, the MeOH extract was
passed through a porous-polymer polystyrene resin (Diaion
HP-20) column eluted with 30% MeOH, 50% MeOH, MeOH,
EtOH, and EtOAc. The MeOH eluate-portion was repeatedly
subjected to silica gel and octadecylsilanized (ODS) silica gel
—
column chromatography to afford compounds 1 4.
Compound 1 was isolated as an amorphous solid and its
molecular formula was deduced to be C33H56O10 on the basis
of the high resolution (HR)-electrospray ionization (ESI)-
time-of-flight (TOF)-MS (m/z: 635.3756 [M+Na]+), 13C-NMR,
and distortionless enhancement by polarization transfer
1
(DEPT) spectral data. The H-NMR spectrum of 1 contained
signals for two tertiary methyl groups at δ 1.26 (3H, s) and
1.24 (3H, s), three secondary methy groups at δ 1.17 (3H, d,
J=7.0Hz) and 0.87 (3H×2, d, J=6.6Hz), and an olefinic pro-
ton at δ 5.89 (1H, brd, J=7.3Hz) characteristic of a steroidal
derivative, and an anomeric proton of a hexopyranosyl group
at δ 5.00 (1H, d, J=7.7Hz). Enzymatic hydrolysis of 1 with
naringinase yielded an aglycone (1a) and D-glucose. The iden-
tification of D-glucose, including the absolute configuration,
was carried out by direct HPLC analysis of the hydrolysate
using an optical rotation detector. The above data suggested
that 1 was a steroidal monoglucoside.
1
The H-NMR spectrum of 1a (C27H46O5) contained signals
for five exchangeable protons at δ 6.31 (1H, brd, J=6.0Hz),
*To whom correspondence should be addressed. e-mail: yokosuka@toyaku.ac.jp;
mimakiy@toyaku.ac.jp
© 2012 The Pharmaceutical Society of Japan

276
Vol. 60, No. 2
connectivity (HMBC) spectra, with those of the known poly-
oxysteroid of (22S)-cholest-5-ene-1β,3β,16β,22-tetrol,4) sug-
gested that the structures of the ring A, C, D portions, and
—
—
side chain (C-1 C-5, C-10 C-27) were identical to those
of the reference compound. However, significant differ-
ences were recognized in the signals for the ring B portion
1
—
(C-6 C-9). In the H-NMR spectrum of 1a, signals for five
oxymethine protons were observed at δ 4.87 (ddd, J=7.5, 7.0,
3.5Hz), 4.22 (m), 4.14 (brd, J=6.5Hz), 4.01 (m, W1/2=26.9Hz),
and 3.85 (dd, J=11.0, 4.5Hz), which were associated with the
one-bond coupled carbon signals at δ 71.8, 75.3, 72.2, 67.9,
and 78.0, respectively, by the HMQC spectrum. The proton
spin-coupling systems revealed by the 1H–1H COSY spec-
trum, and long-range correlations between δH 4.87 and δC 43.1
(C-13), δH 4.14 and δC 140.6 (C-5)/131.4 (C-6), δH 4.01 and
δC 140.6 (C-5), and between δH 3.85 and δC 49.8 (C-9)/43.2
(C-10) detected in the HMBC spectrum (Fig. 1), allowed the
five hydroxy groups to be located at C-1, C-3, C-7, C-16, and
C-22. Nuclear Overhauser effect (NOE) correlations between
δH 4.01 (H-3) and δH 3.85 (H-1), δH 4.14 (H-7) and δH 1.59
(H-9)/1.33 (H-14), and between δH 4.87 (H-16) and δH 1.71
(H-17) observed in the phase-sensitive nuclear Overhauser
effect spectroscopy (NOESY) spectrum (Fig. 2) and the pro-
ton spin-coupling constants indicated that 1a had the usual
steroid ring junctions as shown in Fig. 2 and the C-1β, C-3β,
C-7β, and C-16β configurations. The large J value between
H-17 and H-20 (J=11.5Hz) indicated that the H17–C17–C20–H20
part was preferably trans-oriented, and an NOE correlation
between H-20 and Me-18, Me-21 and H-12eq/H-17, and be-
tween H-22 and H-16 made it possible to assign the 17β and
20S configurations. The absolute configuration at the C-22
Fig. 1. HMBC Correlations of 1a
Bold lines indicate the 1H–1H couplings and arrows indicate 1H/13C long-range
correlations.
Fig. 2. Important NOE Correlations of 1a
chiral center was elucidated as S by application of advanced
5)
’
Mosher s method. Compound 1a was treated with (S)- and
(R)-α-methoxy-(trifluoromethyl)phenylacetic acids (MTPA)
under the presence of 1-[3-(dimethylamino)propyl]-3-ethylcar-
bodiimide hydrochloride (EDC·HCl) and 4-(dimethylamino)-
pyridine (4-DMAP) to yield C-24 (S)- and (R)-MTPA esters.
1
When the H-NMR spectrum of the (S)-MTPA ester (1b) was
compared with that of the (R)-MTPA ester (1c), the signals
assignable to H-16, H-17, Me-18, H-20, and Me-21 of 1b were
observed at lower fields than those of 1c, whereas the signals
for H2-23, H2-24, H-25, Me-26, and Me-27 of 1b appeared
at higher fields than those of 1c (Fig. 3). The structure of 1a
Fig. 3. Chemicals Shift Differences between (S)-MTPA Ester (1b) and
(R)-MTPA Ester (1c) of 1a
was assigned as (22S)-cholest-5-ene-1β,3β,7β,16β,22-pentol.
1
The H- and 13C-NMR spectra of 1 showed the presence of a able protons at δ 6.59 (1H, brs), 6.25 (1H, brs), 6.04 (1H, brd,
β-D-glucopyranosyl unit (Glc) [δH 5.00 (d, J=7.7Hz); δC 101.3, J=5.8Hz), 5.90 (1H, brs), and 5.80 (1H, brs), which were
75.4, 78.6, 72.3, 78.2, 63.5] in its molecule. An HMBC corre- removed by the addition of HCl vapor. Comparison of the
lation was observed between H-1 of Glc at δH 5.00 and C-1 of 1H- and 13C-NMR assignments of 2a with those of 1a showed
the aglycone moiety at δC 82.5. Accordingly, the structure of 1 that the C-7 oxymethine carbon signal, which was observed at
was characterized as (22S)-3β,7β,16β,22-tetrahydroxycholest-5- δ 72.2 in 1a, was displaced by a methylene carbon signal at
en-1β-yl β-D-glucopyranoside.
δ 32.2 in 2a, and the H2-7 methylene proton signals at δ 2.02
Compound 2 was shown to have a molecular formula of (1H, m) and δ 1.62 (1H, m) exhibited spin-couplings with the
C33H56O10 as determined by HR-ESI-TOF-MS analysis (m/z H-6 olefinic proton at δ 5.65 (1H, brd, J=5.4Hz) and the H-8
613.3987 [M+H]+). The 1H-NMR spectrum of 2 contained methine proton at δ 1.63 (1H, m). On the other hand, the C-25
signals for five steroidal methyl groups at δ 1.43 (3H×2, s), methine carbon signal, which was observed at δ 28.5 in 1a,
1.29 (3H, s), 1.19 (3H, s), 1.14 (3H, d, J=7.0Hz), an olefinic was displaced by a quaternary carbon signal at δ 69.7 in 2a,
proton at δ 5.59 (1H, brd, J=5.5Hz), and an anomeric proton and the methyl doublet signals at δ 0.90 (3H×2, d, J=6.5Hz)
at δ 5.00 (1H, d, J=7.5Hz). Enzymatic hydrolysis of 2 with in 1a were observed as singlet methyl signals at δ 1.44 (3H×2,
naringinase yielded an aglycone (2a; C27H46O5) and D-glucose. s) in 2a. The methyl signals at δ 1.44 (3H×2, s) exhibited
3
The molecular formula of 2a was the same as that of 1a. The 2JC–H correlations with the C-25 and JC–H correlations with
1H-NMR spectrum of 2a contained signals for five exchange- C-24 at δ 42.4 in the HMBC spectrum and were assigned to

February 2012
277
Me-26 and Me-27. All other signals appeared at almost the 60 cells with an IC50 value of 15.6μg/mL, while etoposide,
same positions between 1a and 2a. In the HMBC spectrum of used as a positive control, had an IC50 value of 0.26μg/mL.
—
2, a long-range correlation was observed between H-1 of Glc Compounds 1 4, and 2a were not cytotoxic to HL-60 cells at
at δ 5.00 and C-1 of the aglycone at δ 82.7. Thus, the structure sample concentrations of 20μg/mL.
of 2 was determined to be (22S)-3β,16β,22,25-tetrahydroxy-
cholest-5-en-1β-yl β-D-glucopyranoside.
Experimental
Compound 3 was deduced as C33H58O10 by HR-ESI-TOF-
Optical rotations were measured using a JASCO P-1030
1
MS (m/z: 637.3908 [M+Na]+). In the H-NMR spectrum, the (Tokyo, Japan) automatic digital polarimeter. IR spectra
signal due to five steroidal methyl groups [δH 1.43 (3H×2, were recorded on a JASCO FT-IR 620 spectrophotometer.
s), 1.13 (3H, s), 1.11 (3H, d, J=7.5Hz), 1.05 (3H, s)] and an NMR spectra were recorded on a Bruker DRX-500 (500MHz
anomeric proton [δH 5.06 (1H, d, J=7.1Hz)] were observed. for 1H-NMR, Karlsruhe, Germany) and a Bruker AV-600
1
Enzymatic hydrolysis of 3 with naringinase yielded an agly- (600MHz for H-NMR) spectrometer using standard Bruker
cone (3a, C27H48O5) and D-glucose. The molecular formula of pulse programs. Chemical shifts are given as δ values in
3a was higher than that of 2a by two hydrogen atoms. The reference to tetramethylsilane (TMS) as an internal standard.
spectral features of 3a showed close similarity to those of ESI-TOF-MS data were recorded on a Waters-Micromass
1
2a. Comparison of the H-NMR spectrum of 3a with that of LCT mass spectrometer (Manchester, U.K.). Diaion HP-20
2a, the olefinic proton signal due to H-6 was missing in 3a. (Mitsubishi Chemical, Tokyo, Japan), silica gel (Fuji Silysia
Furthermore, the olefinic carbon signals due to C-5 and C-6 Chemical, Aichi, Japan), and ODS silica gel (Nacalai Tesque,
in 2a were displaced by aliphatic carbon signals at δ 43.0 Kyoto, Japan) were used for column chromatography. TLC
(CH) and 29.1 (CH2) in 3a. These data indicated that 3a was was carried out on Silica gel 60 F254 (thickness: 0.25mm,
a 5,6-dihydro derivative of 2a. The 13C-NMR shift of Me-19 Merck, Darmstadt, Germany) and RP18 F254S plates (thickness:
at δ 7.6 and the NOE correlation between H-2ax and Me- 0.25 or 0.5mm, Merck), and spots were visualized by spray-
19 accounted for the A/B trans ring junction (H-5α). In the ing the plates with 10% H2SO4 aqueous solution, followed by
HMBC spectrum of 3, a long-range correlation was observed heating. HPLC was performed with a system composed of a
between H-1 of Glc at δ 5.06 and C-1 of the aglycone at δ CCPM pump (Tosoh, Tokyo, Japan), a CCP PX-8010 control-
81.1. On the basis of these data, the structure of 3 was found ler (Tosoh), an RI-8010 (Tosoh) or a Shodex OR-2 (Showa
to be (22S)-3β,16β,22,25-tetrahydroxy-5α-cholestan-1β-yl β-D- Denko, Tokyo, Japan) detector, and a Rheodyne injection port.
glucopyranoside.
A TSK-gel ODS-100Z column (10mm i.d.×250mm, 5μm,
Compound 4 had the molecular formula C39H66O13 by HR- Tosoh) was employed for preparative HPLC. The follow-
ESI-TOF-MS (m/z: 765.4446 [M+Na]+, Calcd for C39H66NaO13: ing materials and reagents were used for cell culture assay:
765.4401). The 1H-NMR spectrum contained signals for 96-well flat-bottom plate (Iwaki Glass, Chiba, Japan); RPMI
two anomeric protons at δ 6.39 (1H, brs) and 5.05 (1H, d, 1640 medium, etoposide, and 3-(4,5-dimethylthiazol-2-yl)-2,5-
J=7.5Hz), as well as five steroidal methyl groups [δ 1.44 diphenyl-2H-tetrazolium bromide (MTT) (Sigma-Aldrich, St.
(3H×2, s), 1.21 (3H, d, J=7.0Hz), 1.16 (3H, s), and 1.09 (3H, Louis, MO, U.S.A.); fetal bovine serum (FBS) (BioWhittaker,
s)] and an olefinic proton [δ 5.33 (1H, d, J=4.5Hz)]. Acid Walkersville, MO, U.S.A.); penicillin G sodium salt and strep-
hydrolysis of 4 gave L-rhamnose and D-glucose, while the tomycin sulfate (Gibco, Grand Island, NY, U.S.A.). All other
aglycone decomposed under acidic conditions. On comparison chemicals used were of biochemical reagent grade.
of the 13C-NMR spectrum of 4 with that of 2, the structures
Plant Material C. terminalis was purchased from Fuji
—
—
of the ring B D portions and side chain (C-6 C-27) for Green (Tokyo, Japan) in September 2006. A voucher specimen
the cholestane skeletone are identical to those of 2. However, has been deposited in our laboratory (voucher No. CT-2006-
differences were recognized in the signals for the ring A por- 001, Laboratory of Medicinal Pharmacognosy).
1
1
—
tion (C-1 C-5). Analysis of the H– H COSY, HMQC, and
Extraciton and Isolation The leaves of C. terminalis
HMBC spectra of 4 revealed that the aglycone moiety of 4 (fresh weight, 6.0kg) were extracted with MeOH (45L). The
was (22S)-cholest-5-ene-3β,16β,22,25-tetrol and that the sugar MeOH extract was concentrated under reduced pressure, and
moiety of 4 was composed of a terminal α-L-rhamnopyranosyl the viscous concentrate (420g) was passed through a Diaion
unit (Rha) [δH 6.39 (brs); δC 102.0, 72.6, 72.8, 74.2, 69.5, HP-20 column, successively eluted with 30% MeOH, 50%
18.7] and a C-2 substituted β-D-glucopyranosyl unit (Glc) [δH MeOH, MeOH, EtOH, and EtOAc. Column chromatography
5.05 (d, J=7.5Hz); δC 100.4, 77.8, 79.6, 71.8, 78.3, 62.7]. In (CC) of the MeOH-eluate portion (55g) on silica gel and elu-
the HMBC spectrum of 4, long-range correlations were ob- tion with a stepwise gradient mixture of CHCl3–MeOH–H2O
served between H-1 of Rha and C-2 of Glc and between H-1 (9:1:0, 40:10:1, 30:10:1, 20:10:1, 1:1:0), and finally with
—
of Glc and C-3 of the aglycone. The structure of 4 was shown MeOH alone, gave 6 fractions (I VI). Fraction IV was chro-
to be (22S)-16β,22,25-trihydroxycholest-5-en-3β-yl O-α-L- matographed on ODS silica gel eluted with MeCN–H2O (2:5)
—
rhamnopyranosyl-(1→2)-β-D-glucopyranoside.
to give 9 subfractions (IVa IVi). Fraction IVa was further
—
Compounds 1 4 are new cholestane glycosides and their separated by ODS silica gel CC eluted with MeOH–H2O
aglycones are new polyhydroxylated cholestane derivatives. (3:2), silica gel CC with CHCl3–MeOH (4:1, 3:1), and pre-
Compound 1 is a rare type of cholestane glycoside with a hy- parative HPLC using MeOH–H2O (3:1) to yield 1 (67.0mg)
droxy group at C-7β, as well as those at the C-1β, C-3β, C-16β, and 2 (24.8mg). Fraction IVe was chromatographed on ODS
and C-22S positions.
silica gel eluted with MeOH–H2O (1:1) to give 11 subfrac-
—
—
The new glycosides (1 4) and aglycones (1a, 2a) were tions (IVea IVek). Fraction IVej was separated by silica
evaluated for their cytotoxic activities against HL-60 cells. gel CC eluted with CHCl3–MeOH (5:1) to yield 3 (30.5mg).
Compound 1a exhibited weak cytotoxic activity against HL- Fraction IVg was separated by ODS silica gel CC eluted with

278
Vol. 60, No. 2
Table 1. 13C-NMR Data for 1, 1a, 2, 2a, 3, 3a, and 4 in C5D5N
was treated with naringinase (Sigma Aldrich, EC 232-962-
4, 90.3mg) in AcOH/AcOK buffer (pH 4.3, 2mL) at room
temperature for 72h. The reaction mixture was subjected
to silica gel CC eluted with CHCl3–MeOH (19:1) to yield
1a (17.2mg) and a sugar fraction (10.0mg), which was then
analyzed by HPLC under the following conditions: column,
Capcell Pak NH2, UG80 (4.6mm i.d×250mm, 5μm, Shiseido,
Tokyo, Japan); solvent, MeCNH2O (85:15); flow rate, 1.0mL/
min; detection, OR. Identification of D-glucose present in the
sugar fraction was carried out by comparison of its retention
time and optical rotation with those of an authentic sample. tR
(min): 13.5 (D-glucose, positive optical rotation).
1
1a
2
2a
3
3a
4
1
2
82.5
37.5
67.8
43.2
139.7
131.5
71.7
41.8
48.7
42.5
23.9
40.9
43.1
55.1
40.2
71.8
57.7
13.8
14.4
36.1
15.3
75.3
32.1
36.7
28.4
22.7
23.0
101.3
75.4
78.6
72.3
78.2
63.5
78.0
43.8
67.9
43.0
140.6
131.4
72.2
41.8
49.8
43.2
24.3
41.2
43.1
55.3
40.3
71.8
57.8
13.8
13.5
36.1
15.3
75.3
32.2
36.8
28.5
22.8
23.0
82.7
37.5
67.8
43.7
139.6
124.8
31.9
33.3
50.4
42.8
23.8
40.9
42.4
54.9
37.2
71.5
58.3
13.7
14.7
36.1
15.6
76.4
28.9
42.3
69.7
29.7
30.5
101.2
75.4
78.6
72.4
78.2
63.6
78.2
43.5
68.1
44.0
140.3
124.5
32.2
33.2
51.2
42.3
24.2
41.1
42.4
55.1
37.2
71.5
58.3
13.6
14.2
36.1
15.6
76.3
28.9
42.4
69.7
29.7
30.5
81.1
37.5
67.7
39.6
43.0
29.0
32.3
36.6
54.9
41.4
23.7
41.1
42.5
54.8
37.2
71.5
58.4
13.8
8.2
77.3
44.0
67.9
39.6
43.0
29.1
32.5
36.4
55.8
42.0
24.7
41.4
42.6
55.0
37.3
71.5
58.4
13.8
7.6
37.5
30.2
75.0
39.0
140.9
121.9
32.2
31.9
50.5
37.0
21.1
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
1′
2′
3′
4′
5′
6′
1″
2″
3″
4″
5″
6″
Compound 1a: Amorphous solid. [α]D25 −9.3 (c=0.07,
MeOH). HR-ESI-TOF-MS m/z: 473.3246 [M+Na]+ (Calcd for
C27H46NaO5: 473.3243). IR νmax (film) cm−1: 3293 (OH), 2955
40.4
42.7
54.9
37.0
71.6
58.1
13.4
19.4
36.0
15.6
76.3
28.9
42.3
69.7
29.7
30.5
100.4
77.8
79.6
71.8
78.3
62.7
102.0
72.6
72.8
74.2
69.5
18.7
1
(CH), 1097 (C–O). H-NMR (600MHz, C5D5N) δ: 5.97 (1H,
brd, J=4.5Hz, H-6), 4.87 (1H, ddd, J=7.5, 7.0, 3.5Hz, H-16),
4.22 (1H, m, H-22), 4.14 (1H, brd, J=6.5Hz, H-7), 4.01 (1H,
m, W1/2=26.9Hz, H-3), 3.85 (1H, dd, J=11.0, 4.5Hz, H-1), 2.64
(1H, m, H-20), 1.71 (1H, dd, J=11.5, 7.0Hz, H-17), 1.36 (3H,
s, Me-19), 1.33 (3H, s, Me-18), 1.23 (3H, d, J=7.0Hz, Me-21),
0.90 (3H×2, d, J=6.5Hz, Me-26, 27). 13C-NMR (150MHz,
C5D5N): see Table 1.
36.1
15.6
76.4
28.9
42.2
69.7
29.7
30.5
100.4
75.5
78.6
72.2
78.3
63.5
36.1
15.6
76.4
28.9
42.3
69.7
29.8
30.5
Preparation of 1b and 1c To a solution of 1a (4.0mg) in
CH2Cl2 (0.4mL), (R)-(−)-MTPA (21.3mg), EDC·HCl (13.8mg),
and 4-DMAP (3.8mg) were added, and the mixture was
stirred at room temperature for 48h. The reaction mixture was
poured into optimal H2O, which was extracted with EtOAc
(3mL×3). Evaporation of the solution followed by silica gel
CC eluted with hexane–acetone (4:1) gave 1b (8.4mg). Using
the same procedure, a mixture of 1a (4.0mg) and (S)-MTPA
(21.4mg) yielded 1b (7.7mg).
Compound 2: Amorphous solid. [α]D25 −39.3 (c=0.10,
MeOH). HR-ESI-TOF-MS m/z: 613.3987 [M+H]+ (Calcd for
C33H57O10: 613.3952). IR νmax (film) cm−1: 3224 (OH), 2940
1
(CH), 1158 and 1075 (C–O). H-NMR (500MHz, C5D5N) δ:
5.59 (1H, brd, J=5.5Hz, H-6), 5.00 (1H, d, J=7.5Hz, Glc-
1), 4.74 (1H, ddd, J=7.7, 7.0, 3.5Hz, H-16), 4.56 (1H, brd,
J=11.7Hz, Glc-6a), 4.35 (1H, dd, J=11.7, 4.2Hz, Glc-6b), 4.24
(1H, dd, J=8.8, 8.8Hz, Glc-3), 4.19 (1H, m, H-22), 4.15 (1H,
dd, J=8.8, 8.8Hz, Glc-4), 4.07 (1H, dd, J=8.8, 7.5Hz, Glc-2),
4.01 (1H, dd, J=11.6, 4.0Hz, H-1), 3.89 (1H, m, W1/2=20.8Hz,
H-3), 2.62 (1H, m, H-20), 1.62 (1H, dd, J=11.0, 7.0Hz, H-17),
MeOH–H2O (3:2), silica gel CC with CHCl3–MeOH (3:1), 1.43 (3H×2, s, Me-26, 27), 1.29 (3H, s, Me-19), 1.19 (3H, s,
and preparative HPLC using MeCN–H2O (10:23) to yield 4 Me-18), 1.14 (3H, d, J=7.0Hz, Me-21). 13C-NMR (125MHz,
(5.9mg).
Compound 1: Amorphous solid. [α]D25 −7.8 (c=0.10,
C5D5N): see Table 1.
Enzymatic Hydrolysis of 2 Compound 2 (1.7mg) was
MeOH). HR-ESI-TOF-MS m/z: 635.3756 [M+Na]+ (Calcd for treated with naringinase (20.4mg) in AcOH/AcOK buffer (pH
C33H56NaO10: 635.3771). IR νm1ax (film) cm−1: 3215 (OH), 2951 4.3, 1mL) at room temperature for 168h. The reaction mix-
(CH), 1170 and 1073 (C–O). H-NMR (500MHz, C5D5N) δ: ture was subjected to silica gel CC eluted with CHCl3–MeOH
5.89 (1H, brd, J=7.3Hz, H-6), 5.00 (1H, d, J=7.7Hz, Glc-1), (9:1) to yield 2a (0.7mg) and a sugar fraction (0.4mg). HPLC
4.81 (1H, m, H-16), 4.55 (1H, dd, J=11.4, 2.7Hz, Glc-6a), 4.34 analysis of the sugar fraction under the same conditions as in
(1H, dd, J=11.4, 5.8Hz, Glc-6b), 4.23 (1H, dd, J=8.5,8.5Hz, the case of 1 showed the presence of D-glucose tR (min): 13.7
Glc-3), 4.19 (1H, m, H-22), 4.13 (1H, dd, J=8.5, 8.5Hz, Glc-4), (D-glucose, positive optical rotation).
4.05 (1H, dd, J=8.5, 7.7Hz, Glc-2), 4.04 (1H, brd, J=8.0Hz,
Compound 2a: Amorphous solid. [α]D25 −14.9 (c=0.07,
H-7), 4.00 (1H, dd, J=11.4, 4.0Hz, H-1), 3.94 (1H, m, Glc-5), MeOH). HR-ESI-TOF-MS m/z: 473.3252 [M+Na]+ (Calcd for
3.89 (1H, m, W1/2=20.8Hz, H-3), 2.59 (1H, m, H-20), 1.69 (1H, C27H46NaO5: 473.3243). IR νma1x (film) cm−1: 3364 (OH), 2961
m, H-17), 1.26 (3H, s, Me-19), 1.24 (3H, s, Me-18), 1.17 (3H, d, and 2923 (CH), 1095 (C–O). H-NMR (600MHz, C5D5N) δ:
J=7.0Hz, Me-21), 0.87 (3H×2, d, J=6.6Hz, Me-26, Me-27). 5.65 (1H, brd, J=5.4Hz, H-6), 4.79 (1H, m, H-16), 4.21 (1H,
13C-NMR (125MHz, C5D5N): see Table 1.
Enzymatic Hydrolysis of Compound
m, H-22), 4.00 (1H, m, W1/2=23.5Hz, H-3), 3.85 (1H, dd,
1
1
(35.0mg) J=10.5, 6.0Hz, H-1), 2.66 (1H, m, H-20), 2.02 (1H, m, H-7a),

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1.65 (1H, dd, J=12.0, 5.5Hz, H-17), 1.63 (1H, m, H-8), 1.62 4.0Hz, Glc-6b), 4.33 (1H, dd, J=9.0, 9.0Hz, Rha-4), 4.29 (1H,
(1H, m, H-7b), 1.44 (3H×2, s, Me-26, 27), 1.39 (3H, s, Me-19), dd, J=9.0, 7.5Hz, Glc-2), 4.22 (1H, m, H-22), 4.28 (1H, dd,
1.26 (3H, s, Me-18), 1.20 (3H, d, J=7.5Hz, Me-21). 13C-NMR J=9.0, 9.0Hz, Glc-3), 4.18 (1H, dd, J=9.0, 9.0Hz, Glc-4), 3.95
(150MHz, C5D5N): see Table 1.
(1H, m, W1/2=22.5Hz, H-3), 2.65 (1H, m, H-20), 1.79 (3H, d,
Compound 3: Amorphous solid. [α]D25 −42.4 (c=0.10, J=6.0Hz, Rha-6), 1.64 (1H, dd, J=11.0, 7.0Hz, H-17), 1.44
MeOH). HR-ESI-TOF-MS m/z: 637.3908 [M+Na]+ (Calcd for (3H×2, s, Me-26, 27), 1.21 (3H, d, J=7.0Hz, Me-21), 1.16 (3H,
C33H58NaO10: 637.3928). IR νmax (film) cm−1: 3282 (OH), 2931 s, Me-18), 1.09 (3H, s, Me-19). 13C-NMR (125MHz, C5D5N):
1
(CH), 1161 and 1076 (C-O). H-NMR (500MHz, C5D5N) δ: see Table 1.
5.06 (1H, d, J=7.1Hz, Glc-1), 4.77 (1H, ddd, J=7.9, 7.0, 4.8Hz,
Acid Hydrolysis of 4 A solution of 4 (3.9mg) in 1M HCl
°
H-16), 4.58 (1H, dd, J=11.0, 2.0Hz, Glc-6a), 4.35 (1H, dd, (dioxane–H2O, 1:1, 2mL) was heated at 95 C for 2.5h under
J=11.0, 6.0Hz, Glc-6b), 4.23 (1H, dd, J=9.0, 9.0Hz, Glc-3), Ar atmosphere. After cooling, the reaction mixture was neu-
4.17 (1H, m, H-22), 4.14 (1H, dd, J=9.0, 9.0Hz, Glc-4), 4.06 tralized by passage through an Amberlite IRA-96SB (Organo,
(1H, dd, J=9.0, 7.1Hz, Glc-2), 4.02 (1H, dd, J=11.5, 4.0Hz, Tokyo, Japan). The neutralized mixture was subjected to silica
H-1), 3.89 (1H, m, W1/2=22.0Hz, H-3), 2.61 (1H, m, H-20), gel CC eluted with hexane–Me2CO (19:1) to yield a sugar
1.59 (1H, dd, J=10.0, 7.0Hz, H-17), 1.43 (3H×2, s, Me-26, 27), fraction (1.3mg). HPLC analysis of the sugar fraction under
1.13 (3H, s, Me-19), 1.11 (3H, d, J=7.5Hz, Me-21), 1.05 (3H, s, the same conditions as in the case of 1 showed the presence
Me-18). 13C-NMR (125MHz, C5D5N): see Table 1.
of D-glucose and L-rhamnose tR (min): 13.7 (D-glucose, positive
Enzymatic Hydrolysis of 3 Compound 3 (5.1mg) was optical rotation), 7.4 (L-rhamnose, negative optical rotation).
treated with naringinase (40.0mg) in AcOH/AcOK buffer (pH HL-60 Cell Culture and Assay The cell growth was
4.3, 2mL) at room temperature for 168h. The reaction mix- measured with a modified MTT reduction assay as described
ture was subjected to silica gel CC eluted with CHCl3–MeOH in a previous paper.6) Briefly, HL-60 cells were maintained
(9:1) to yield 3a (2.3mg) and a sugar fraction (1.0mg). HPLC in RPMI 1640 medium containing heat-inactivated 10% (v/v)
analysis of the sugar fraction under the same conditions as in FBS supplemented with L-glutamine, 100unit/mL penicillin
the case of 1 showed the presence of D-glucose tR (min): 14.4 G sodium salt, and 100μg/mL streptomycin sulfate. The cells
(D-glucose, positive optical rotation).
(4×104 cells/mL) were continuously treated with each com-
Compound 3a: Amorphous solid. [α]D25 −11.5 (c=0.115, pound for 72h, and cell growth was measured with an MTT
MeOH). HR-ESI-TOF-MS m/z: 453.3585 [M+H]+ (Calcd for reduction assay procedure. A dose–response curve was plotted
C27H49O5: 453.3580). IR νmax (film) cm−1: 3347 (OH), 2962 and for 1a and the concentration giving 50% inhibition (IC50) was
1
2926 (CH), 1093 (C-O). H-NMR (600MHz, C5D5N) δ: 4.79 calculated.
(1H, m, H-16), 4.21 (1H, m, H-22), 4.00 (1H, m, W1/2=22.0Hz,
H-3), 3.75 (1H, dd, J=10.5, 5.0Hz, H-1), 2.67 (1H, m, H-20),
1.63 (1H, dd, J=10.5, 7.0Hz, H-17), 1.44 (3H×2, s, Me-26,
27), 1.23 (3H, s, Me-18), 1.17 (3H, s, Me-19), 1.20 (3H, d,
J=7.0Hz, Me-21). 13C-NMR (150MHz, C5D5N) δ: see Table 1.
Compound 4: Amorphous solid. [α]D25 −65.6 (c=0.10,
MeOH). HR-ESI-TOF-MS m/z: 765.4446 [M+Na]+ (Calcd for
C39H66NaO13: 765.4401). IR νmax (film) cm−1: 3422 and 3399
References
“
”
1) The Grand Dictionary of Horticulture, Vol. 2, Tsukamoto Y. (ex-
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ecutive ed.), Shogakukan, Tokyo, 1988, pp. 301 303.
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1
(OH), 2935 (CH), 1127 and 1069 (C–O). H-NMR (500MHz,
—
2761 2763 (1991).
C5D5N) δ: 6.39 (1H, brs, Rha-1), 5.33 (1H, brd, J=4.5Hz,
H-6), 5.05 (1H, d, J=7.5Hz, Glc-1), 5.00 (1H, dq, J=9.0,
6.0Hz, Rha-5), 4.79 (1H, ddd, J=7.0, 7.0, 3.5Hz, H-16), 4.81
(1H, brd, J=3.0Hz, Rha-2), 4.64 (1H, dd, J=9.0, 3.0Hz, Rha-
3), 4.53 (1H, dd, J=11.5, 2.0Hz, Glc-6a), 4.37 (1H, dd, J=11.5,
5) Ohtani I., Kusumi T., Kashman Y., Kakisawa H., J. Am. Chem.
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Soc., 113, 4092 4096 (1991).
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6) Yokosuka A., Mimaki Y., Sashida Y., Phytochemistry, 61, 73 78
(2002).