Iodine-catalyzed etherification of morroniside

Article abstract of DOI:10.1248/cpb.57.112

In this study, we describe a highly selective etherification procedure of unprotected morroniside catalyzed by molecular iodine in acetone. The etherification reaction furnished 7-O-alkyl ether derivatives in reasonable yields within few hours under neutr

Full text of DOI:10.1248/cpb.57.112

112
Notes
Chem. Pharm. Bull. 57(1) 112—115 (2009)
Vol. 57, No. 1
Iodine-Catalyzed Etherification of Morroniside
a
b
b
,c
*
Fortunatus SUNGHWA, Hiroaki SAKURAI, Ikuo SAIKI, and Mamoru KOKETSU
a Department of Chemistry, Faculty of Science, University of Dar es salaam; Dar es salaam, Tanzania: b Division of
Pathogenic Biochemistry, Institute of Natural Medicine, University of Toyama; Toyama 930–0194, Japan: and c Division of
Instrumental Analysis, Life Science Research Center, Gifu University; Gifu 501–1193, Japan.
Received October 6, 2008; accepted October 28, 2008; published online October 29, 2008
In this study, we describe a highly selective etherification procedure of unprotected morroniside catalyzed by
molecular iodine in acetone. The etherification reaction furnished 7-O-alkyl ether derivatives in reasonable yields
within few hours under neutral conditions. Studies of the obtained products on cytotoxicity activity in colon 26-
L5 cell line were examined. Among the tested compounds, 7-O-dodecylmorroniside showed moderate cytotoxic
activity, having IC50 values equal to 20.9 mM.
Key words etherification; morroniside; cytotoxicity
The use of natural iridoid glycosides as starting materials study possibility of obtaining similar derivatives from com-
for the synthesis of biological active compounds received pound 1 for biological evaluations.
considerable attention. The importance of these compounds
When morroniside (1) was treated with few drops of
has been demonstrated in various synthetic approaches.1—10) methanol in the presence of iodine and acetone at room tem-
Recent synthetic applications have resulted into conversion perature, compound 3a was obtained (Chart 1). The control
of aucibin to aminoside antibiotic analogues.11)
experiments revealed that no formation of compound 3a oc-
Morroniside (1) has been found to have some applications curred, when iodine was omitted during the reaction or when
in pharmacology as well as in cosmetics. The compound can acetone was replaced completely with methanol. The posi-
be used as a-glucosidase inhibitor in drugs and healthy food tion of alkyl ethers formed was confirmed by heteronuclear
for preventing impaired glucose tolerance, diabetes and obe- multiple bond correlation (HMBC) experiments on com-
sity.12) Penta-acetate derivatives of 1 possessing melanin in- pound 3a. It was confirmed by HMBC experiments that the
hibitory effect can be used as one of the component in skin- one methoxy group at d 3.31 was attached to C-7 at d 95.6.
whitening agent.13) So far little is known about other biologi- In regard to these observations more attentions were there-
cally important derivatives derived from morroniside (1).
fore directed towards this reaction so as to obtain long chain
The use of iodine as a catalyst in organic synthesis is well alkyl ether derivatives.
known. Iodine has been employed in carbohydrate chemistry,
To determine the scope and selectivity of this reaction, dif-
as a catalyst for the isopropylidenation of some simple ferent alcohols (including long chain aliphatic alcohols) were
monosaccharides and sugar alcohol.14) It is now found that tested under the same conditions. As shown in Table 1, most
iodine can be used efficiently as catalyst in various syntheses
of carbohydrate derivatives.
Results and Discussion
Morroniside (1) was isolated from methanolic root extract
of the plant Strychnos cocculoides by repeated silica gel vac-
uum liquid chromatography using methanol/dichloromethane
(20 : 80) as solvents. Morroniside (1) was isolated as the
1
major compound and observed in H-NMR spectrum as a
mixture of anomeric forms.15,16) This compound has been
isolated for the first time from plant of the genus Strychnos.
It was found that some alditol long-chain alkyl ether deriv-
atives are interesting class of carbohydrate with important bi-
ological functions.17) Therefore, we found worthwhile to
Chart 1
Table 1. Synthesis of Morroniside Derivatives 3
ROH
2
Time
(h)
Yield (%)
ROH
2
Time
(h)
Yield (%)
Entry
Entry
3
3
1
2
3
4
5
6
7
Methanol
Ethanol
Propanol
Isopropanol
t-Butanol
n-Butanol
Cyclohexanol
4
4
4
4
4
4
4
84.2 (3a)
88.0 (3b)
74.4 (3c)
78.5 (3d)
21.0 (3e)
85.9 (3f)
70.3 (3g)
8
9
10
11
12
13
Benzyl alcohol
cis-2-Hexenol
1-Octanol
1-Dodecanol
1-Hexadecanol
1-Octadecanol
0.5
1
4
1
4
53.2 (3h)
74.7 (3i)
50.4 (3j)
68.5 (3k)
56.2 (3l)
54.4 (3m)a)
4
a) Reaction at 40 °C.
∗ To whom correspondence should be addressed. e-mail: koketsu@gifu-u.ac.jp
© 2009 Pharmaceutical Society of Japan

January 2009
113
of the selected alcohols gave the corresponding products in concentration IC50 values equal to 20.9 mM (Table 2).
reasonable yields (50.4—88.0%). The results indicated that
In conclusion, a convenient method based on iodine-cat-
the reaction with benzyl alcohol was completed by the short- alyzed etherification of iridoid glycoside has been developed.
est reaction time as compared to other selected alcohols. Low The reaction featured under mild and neutral conditions, high
yield of the corresponding product was obtained, when com- selectivity, and reasonable yields. Further structural modifi-
pound 1 was treated with t-butanol. The introduction of long- cations on compound 1 to introduce other functional groups
chain alkyl ether using 1-octanol and 1-dodecanol showed a are currently under investigations.
further possibility of introducing other long chain aliphatic
hydrocarbons. Therefore, compounds 3l and 3m were ob-
tained using similar approach. Due to low solubility of 1-oc-
Experimental
General Experimental Procedure IR spectra were recorded on a
JASCO FTIR-460 Plus spectrophotometer. Optical rotations were determined
tadecanol in acetone, etherification reaction was performed at
40 °C.
with a JASCO P-1010 polarimeter at specified temperature and concentra-
tions in the solvents indicated. 1H- (600 MHz) and 13C- (125 MHz) NMR
spectra were recorded on JEOL ECA 600 spectrometer with tetramethyl
silane (TMS) as an internal standard. MS were recorded using a JEOL JMS-
700/GI spectrometer. Column chromatography was performed on silica gel
Attempts to obtain alkyl ether derivatives using D-glucose,
methyl a-D-glucoside and loganic acid as substrates under
the same conditions were unsuccessful. Only starting materi-
als and some decomposed products were recovered. This
type of etherification reaction using alcohol in the presence
of iodine and acetone as a solvent has not yet been reported.
The mechanism of the reaction described herein is activated
by the presence of iodine. The sequences are initiated by the
formation of an acetone–iodine addition complex at hydroxyl
group (C-7),18) followed by attack of alcohol to form the
product.19) So far the present reaction can be unique and spe-
cific etherification on hydroxyl group (C-7) of unprotected
iridoid glucoside 1. The presence of acetone in this reaction
N-60 (40—50 mm) and, TLC on pre-coated plates of silica gel 60 F254
.
Extraction and Isolation Morroniside (1) was isolated from methanol
extract of root bark of Strychnos cocculoides using vacuum liquid chro-
matography followed by column chromatography (methanol/dichloromethane
ꢀ2/8).
Reaction Conditions Morroniside (1) (0.134 mmol), iodine (1.7 mg,
0.1 mmol) and alcohol 2 (1.5 mmol) in acetone (5 ml) were stirred at room
temperature for 0.5—4 h. The excess iodine was removed by addition of 5%
sodium thiosulphate until colourless, concentrated in vacuo, and the result-
ant products were purified by silica gel column chromatography (methanol/
dichloromethaneꢀ1/99).
Morroniside (1)16) White amorphous solid. 1H-NMR (CD3OD, 600
MHz) d: 1.31 (3H, d, Jꢀ7.2 Hz, H-10b), 1.38 (3H, d, Jꢀ7.2 Hz, H-10a),
therefore promotes etherification reaction rather than iso- 1.75 (1H, ddd, Jꢀ2.0, 4.0, 9.0 Hz, H-9b), 1.80 (1H, ddd, Jꢀ2.0, 4.0, 9.0 Hz,
propylidenation reaction.20,21)
H-9a), 1.88 (2H, m, H-6b), 2.00 (2H, m, H-6a), 2.82 (1H, dt, Jꢀ4.0,
13.0 Hz, H-5a), 3.12 (1H, dt, Jꢀ4.0, 13.0 Hz, H-5b), 3.35—3.41 (4H, m, H-
Some of the prepared alkyl ether derivatives have also
2ꢁ, H-3ꢁ, H-4ꢁ, H-5ꢁ), 3.70 (3H, s, OCH3), 3.87 (1H, dd, Jꢀ2.0, 7.0 Hz, H-
been isolated from natural sources. Compound 3b was iso-
8a), 3.93 (1H, dd, Jꢀ3.0, 7.0 Hz, H-8b), 4.24 (1H, dd, Jꢀ2.0, 12.0 Hz, H-
lated from ethanol extract of Fructus corni.22) The alkyl ether
6ꢁa), 4.44 (1H, dd, Jꢀ2.0, 12.0 Hz, H-6ꢁb), 4.78 (1H, d, Jꢀ4.0 Hz, H-7),
derivative 3f was isolated from Cornus officinalis,23) and its
4.80 (1H, d, Jꢀ8.0 Hz, H-1ꢁ), 5.82 (1H, d, Jꢀ8.6 Hz, H-1a), 5.86 (1H, d,
Jꢀ9.0 Hz, H-1b), 7.51 (1H, s, H-3); 13C-NMR (CD3OD, 150 MHz) d: 19.8
7R isomer was obtained from leaves of Hydrangea macro-
(C-10), 27.3 (C-5b), 31.9 (C-5a), 34.5 (C-6b), 37.1 (C-6a), 39.7 (C-9a),
phylla subsp. serrata.24)
40.4 (C-9b), 51.8 (OCH3), 62.6 (C-6ꢁ), 65.8 (C-8b), 71.4 (C-2ꢁ), 74.0 (C-
Recently, it is reported that incubation of cells with morro-
4ꢁ), 74.9 (C-8a), 77.8 (C-5ꢁ), 78.3 (C-3ꢁ), 95.5 (C-7), 96.9 (C-1), 99.9 (C-
1ꢁ), 110.7 (C-4), 154.4 (C-3), 168.7 (CꢀO). IR (KBr) cmꢂ1: 3423, 1699,
1638. FAB-MS m/z: 429.1 [MꢃNa]ꢃ.
niside led to a significant dose-dependent elevation of cellu-
lar GSH accompanied by a marked protection against H2O2-
mediated cytotoxicity. Morroniside (1) inhibited the forma-
tion of intracellular oxygen species and the activation of cas-
pase-3 and 9.25) This result stimulated us to prepare a series
of morroniside derivatives and to test their cytotoxicity. The
alkyl ether derivatives were tested for their cytotoxicity to-
ward colon 26-L5 cell line. Among them, compound 3k
showed the strongest cytotoxic activity, having inhibitory
7-O-Methylmorroniside (3a) White amorphous solid. 1H-NMR
(CD3OD, 600 MHz) d: 1.31 (3H, d, Jꢀ7.0 Hz, H-10b), 1.36 (3H, d, Jꢀ7.0
Hz, H-10a), 1.47 (1H, ddd, Jꢀ2.0, 4.0, 9.0 Hz, H-9b), 1.76 (1H, m, H-9a),
1.88 (2H, m, H-6b), 1.96 (2H, m, H-6a), 2.78 (1H, dt, Jꢀ4.0, 13.0 Hz, H-
5a), 3.00 (1H, dt, Jꢀ4.0, 13.0 Hz, H-5b), 3.26—3.45 (4H, m, H-2ꢁ, H-3ꢁ,
H-4ꢁ, H-5ꢁ), 3.32 (3H, s, OCH3), 3.65 (3H, s, OCH3), 3.85 (1H, dd, Jꢀ2.0,
7.0 Hz, H-8a), 3.90 (1H, dd, Jꢀ3.0, 7.0 Hz, H-8b), 4.44 (1H, dd, Jꢀ2.0,
12.0 Hz, H-6ꢁa), 4.44 (1H, dd, Jꢀ2.0, 12.0 Hz, H-6ꢁb), 4.70 (1H, d,
Jꢀ4.0 Hz, H-7), 4.75 (1H, d, Jꢀ8.0 Hz, H-1ꢁ), 5.76 (1H, d, Jꢀ9.0 Hz, H-
1a), 5.84 (1H, d, Jꢀ9.0 Hz, H-1b), 7.45 (1H, s, H-3); 13C-NMR (CD3OD,
150 MHz) d: 19.6 (C-10), 28.0 (C-5b), 31.8 (C-5a), 33.7 (C-6b), 35.6 (C-
6a), 40.2 (C-9a), 40.4 (C-9b), 51.7 (OCH3), 54.9 (OCH3), 62.8 (C-6ꢁ), 66.3
(C-8b), 71.7 (C-2ꢁ), 74.0 (C-4ꢁ), 75.0 (C-8a), 78.0 (C-5ꢁ), 78.5 (C-3ꢁ), 95.6
(C-7), 99.5 (C-1), 100.1 (C-1ꢁ), 110.7 (C-4), 154.4 (C-3), 168.6 (CꢀO). IR
(KBr) cmꢂ1: 3426, 1705, 1639. FAB-MS m/z: 443.2 [MꢃNa]ꢃ, 421.2
[Mꢃ1]ꢃ.
Table 2. Cytotoxic Activity of Morroniside Derivatives against the Colon
26-L5 Cell Line
Compound
R
IC50 (mM)
1
H
Methyl
Ethyl
Propyl
Isopropyl
t-Butyl
n-Butyl
969
1707
1248
1085
1031
1014
887
7-O-Ethylmorroniside (3b) White amorphous solid. 1H-NMR (CD3OD,
600 MHz) d: 1.22 (3H, t, Jꢀ7.0 Hz, CH3), 1.32 (3H, d, Jꢀ7.0 Hz, H-10b),
1.38 (3H, d, Jꢀ7.0 Hz, H-10a), 1.51 (1H, ddd, Jꢀ2.0, 4.0, 9.0 Hz, H-9b),
1.81 (1H, m, H-9a), 1.91 (2H, m, H-6b), 2.00 (2H, m, H-6a), 2.78 (1H, dt,
Jꢀ4.0, 13.0 Hz, H-5a), 3.00 (1H, dt, Jꢀ4.0, 13.0 Hz, H-5b), 3.20—3.47
(4H, m, H-2ꢁ, H-3ꢁ, H-4ꢁ, H-5ꢁ), 3.69 (3H, s, OCH3), 3.89 (1H, m, H-8a),
4.34 (1H, dd, Jꢀ2.0, 12.0 Hz, H-6ꢁa), 4.58 (1H, dd, Jꢀ2.0, 12.0 Hz, H-6ꢁb),
4.78 (1H, d, Jꢀ4.0 Hz, H-7), 4.86 (1H, d, Jꢀ8.0 Hz, H-1ꢁ), 5.80 (1H, d,
Jꢀ9.0 Hz, H-1a), 5.88 (1H, d, Jꢀ9.0 Hz, H-1b), 7.51 (1H, s, H-3); 13C-
NMR (CD3OD, 150 MHz) d: 15.4 (CH3), 19.6 (C-10), 28.0 (C-5b), 31.8 (C-
5a), 33.9 (C-6b), 35.8 (C-6a), 40.1 (C-9a), 40.4 (C-9b), 51.7 (OCH3), 62.9
(C-6ꢁ), 63.8 (OCH2), 66.3 (C-8b), 71.6 (C-2ꢁ), 74.1 (C-4ꢁ), 74.9 (C-8a),
77.9 (C-5ꢁ), 78.4 (C-3ꢁ), 95.6 (C-7), 98.1 (C-1), 100.0 (C-1ꢁ), 111.6 (C-4),
154.4 (C-3), 168.7 (CꢀO). IR (KBr) cmꢂ1: 3428, 1707, 1637. FAB-MS m/z:
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
Cyclohexyl
Benzyl
cis-2-Hexenyl
1-Octyl
1-Dodecyl
1-Hexadecyl
1-Octadecyl
921
1166
524
69.3
20.9
96.1
156
3m

114
Vol. 57, No. 1
457.1 [MꢃNa]ꢃ.
(C-5ꢁ), 77.3 (C-3ꢁ), 94.5 (C-7), 95.2 (C-1), 99.0 (C-1ꢁ), 110.6 (C-4), 153.4
7-O-Propylmorroniside (3c) White amorphous solid. 1H-NMR (C-3), 167.6 (CꢀO). IR (KBr) cmꢂ1: 3420, 1708, 1639. FAB-MS m/z: 511.2
(CD3OD, 600 MHz) d: 0.99 (3H, t, Jꢀ7.0 Hz, CH3), 1.29 (3H, d, Jꢀ7.0 Hz,
H-10b), 1.35 (3H, d, Jꢀ7.0 Hz, H-10a), 1.48 (1H, ddd, Jꢀ2.0, 4.0, 9.0 Hz,
H-9b), 1.66—1.53 (4H, m, 2ꢄCH2), 1.78 (1H, m, H-9a), 1.88 (2H, m, H-
6b), 2.00 (2H, m, H-6a), 2.77 (1H, dt, Jꢀ4.0, 13.0 Hz, H-5a), 3.04 (1H, dt, 1.94 (2H, m, H-6), 3.09 (1H, dt, Jꢀ4.0, 13.0 Hz, H-5b), 3.18—3.30 (6H, m,
Jꢀ4.0, 13.0 Hz, H-5b), 3.16—3.36 (4H, m, H-2ꢁ, H-3ꢁ, H-4ꢁ, H-5ꢁ), 3.65 H-2ꢁ, H-3ꢁ, H-4ꢁ, H-5ꢁ, H-6ꢁ), 3.64 (3H, s, OCH3), 3.83 (1H, m, H-8), 4.33
(3H, s, OCH3), 3.83 (1H, m, H-8), 4.30 (1H, dd, Jꢀ2.0, 12.0 Hz, H-6ꢁa),
4.54 (1H, dd, Jꢀ2.0, 12.0 Hz, H-6ꢁb), 4.75 (1H, d, Jꢀ4.0 Hz, H-7), 4.80
[MꢃNa]ꢃ, 489.3 [Mꢃ1]ꢃ.
7-O-Benzylmorroniside (3h) White amorphous solid. 1H-NMR
(CD3OD, 600 MHz) d: 1.26 (3H, d, Jꢀ7.0 Hz, H-10), 1.80 (1H, m, H-9),
(1H, dd, Jꢀ2.0, 12.0 Hz, H-6ꢁa), 4.50 (1H, d, Jꢀ12.0 Hz, CH2Ph), 4.64
(1H, d, Jꢀ12.0 Hz, CH2Ph), 4.75 (1H, d, Jꢀ4.0 Hz, H-7), 4.80 (1H, d,
(1H, d, Jꢀ8.0 Hz, H-1ꢁ), 5.75 (1H, d, Jꢀ9.0 Hz, H-1a), 5.83 (1H, d, Jꢀ8.0 Hz, H-1ꢁ), 5.80 (1H, d, Jꢀ9.0 Hz, H-1), 7.24—7.34 (5H, d, aromatic
Jꢀ9.0 Hz, H-1b), 7.47 (1H, s, H-3); 13C-NMR (CD3OD, 150 MHz) d: 15.4 H), 7.48 (1H, s, H-3); 13C-NMR (CD3OD, 150 MHz) d: 19.5 (C-10), 28.0
(CH3), 18.3 (C-10), 22.5 (CH2), 26.7 (C-5b), 30.5 (C-5a), 32.6 (C-6b), 34.4 (C-5b), 29.3 (C-5a), 31.8 (C-6b), 33.8 (C-6a), 40.4 (C-9), 51.7 (OCH3),
(C-6a), 38.8 (C-9a), 39.9 (C-9b), 51.7 (OCH3), 62.8 (C-6ꢁ), 66.3 (C-8b), 62.8 (C-6ꢁ), 66.6 (C-8b), 70.0 (OCH2), 71.7 (C-2ꢁ), 75.0 (C-4ꢁ), 78.0 (C-3ꢁ),
70.1 (OCH2), 71.7 (C-2ꢁ), 74.2 (C-4ꢁ), 75.0 (C-8a), 78.0 (C-5ꢁ), 78.5 (C-3ꢁ),
95.6 (C-7), 98.3 (C-1), 100.1 (C-1ꢁ), 111.7 (C-4), 154.5 (C-3), 168.7
(CꢀO). IR (KBr) cmꢂ1: 3423, 1707, 1638. FAB-MS m/z 471.3 [MꢃNa]ꢃ,
449.3 [Mꢃ1]ꢃ.
78.5 (C-5ꢁ), 95.6 (C-7), 97.6 (C-1), 100.0 (C-1ꢁ), 111.6 (C-4), 128.7—129.3
(aromatic C), 139.3 (aromatic C), 154.5 (C-3), 168.7 (CꢀO). IR (KBr)
cmꢂ1: 3419, 1706, 1638. FAB-MS m/z: 517.2 [MꢃNa]ꢃ, 495.2 [Mꢃ1]ꢃ.
7-O-cis-2-Hexenylmorroniside (3i) White amorphous solid. 1H-NMR
7-O-Isopropylmorroniside (3d) White amorphous solid. 1H-NMR (CD3OD, 600 MHz) d: 0.89 (3H, t, Jꢀ7.0 Hz, CH3), 1.35 (3H, d, Jꢀ7.0 Hz,
(CD3OD, 600 MHz) d: 1.12 (3H, d, Jꢀ7.0 Hz, CH3), 1.18 (3H, d, Jꢀ7.0 Hz,
H-10), 1.40—1.37 (4H, m, 2ꢄCH2), 1.78 (1H, m, H-9a), 2.04 (2H, m, H-6),
CH3), 1.29 (3H, d, Jꢀ7.0 Hz, H-10b), 1.35 (3H, d, Jꢀ7.0 Hz, H-10a), 1.48 2.79 (1H, dt, Jꢀ4.0, 13.0 Hz, H-5a), 3.05 (1H, dt, Jꢀ4.0, 13.0 Hz, H-5b),
(1H, ddd, Jꢀ2.0, 4.0, 9.0 Hz, H-9b), 1.66—1.53 (1H, m, CH), 1.78 (1H, m, 3.18—3.35 (4H, m, H-2ꢁ, H-3ꢁ, H-4ꢁ, H-5ꢁ), 3.69 (3H, s, OCH3), 3.83 (1H,
H-9a), 1.83 (2H, m, H-6b), 2.00 (2H, m, H-6a), 2.78 (1H, dt, Jꢀ4.0, m, H-8), 4.31 (1H, dd, Jꢀ2.0, 12.0 Hz, H-6ꢁa), 4.58 (1H, dd, Jꢀ2.0,
13.0 Hz, H-5a), 3.03 (1H, dt, Jꢀ4.0, 13.0 Hz, H-5b), 3.16—3.35 (4H, m, H- 12.0 Hz, H-6ꢁb), 4.74 (1H, d, Jꢀ4.1 Hz, H-7), 4.80 (1H, d, Jꢀ8.0 Hz, H-1ꢁ),
2ꢁ, H-3ꢁ, H-4ꢁ, H-5ꢁ), 3.65 (3H, s, OCH3), 3.84 (1H, m, H-8), 4.30 (1H, dd, 5.55 (2H, m, CHꢀCH), 5.77 (1H, d, Jꢀ9.0 Hz, H-1a), 5.85 (1H, d,
Jꢀ2.0, 12.0 Hz, H-6ꢁa), 4.53 (1H, dd, Jꢀ2.0, 12.0 Hz, H-6ꢁb), 4.73 (1H, d, Jꢀ9.0 Hz, H-1b), 7.47 (1H, s, H-3); 13C-NMR (CD3OD, 150 MHz) d: 14.0
Jꢀ4.0 Hz, H-7), 4.80 (1H, d, Jꢀ8.0 Hz, H-1ꢁ), 5.76 (1H, d, Jꢀ9.0 Hz, H-
(CH3), 19.6 (C-10), 23.7 (2ꢄCH2), 28.0 (C-5b), 30.5 (C-5a), 31.8 (C-6),
1a), 5.84 (1H, d, Jꢀ9.0 Hz, H-1b), 7.48 (1H, s, H-3); 13C-NMR (CD3OD, 40.4 (C-9), 51.7 (OCH3), 62.8 (C-6ꢁ), 65.3 (OCH2), 66.4 (C-8b), 71.6 (C-
150 MHz) d: 18.3 (C-10), 20.5, 22.4 [(CH3)2], 26.8 (C-5b), 30.7 (C-5a), 2ꢁ), 74.2 (C-4ꢁ), 75.0 (C-8a), 77.9 (C-3ꢁ), 78.5 (C-5ꢁ), 95.7 (C-7), 97.5 (C-
33.1 (C-6), 39.3 (C-9), 51.7 (OCH3), 62.6 (C-6ꢁ), 65.2 (C-8b), 68.8 (OCH),
70.4 (C-2ꢁ), 70.5 (C-4ꢁ), 73.8 (C-8a), 76.8 (C-5ꢁ), 77.3 (C-3ꢁ), 94.4 (C-7),
1), 100.0 (C-1ꢁ), 111.6 (C-4), 126.9, 134.6 (CHꢀCH), 154.5 (C-3), 168.7
(CꢀO). IR (KBr) cmꢂ1: 3422, 1703, 1638. FAB-MS m/z 511.2 [MꢃNa]ꢃ,
95.1 (C-1), 98.9 (C-1ꢁ), 110.5 (C-4), 153.3 (C-3), 167.5 (CꢀO). IR (KBr) 489.3 [Mꢃ1]ꢃ.
cmꢂ1: 3406, 1707, 1638. FAB-MS m/z: 471.2 [MꢃNa]ꢃ, 449.3 [Mꢃ1]ꢃ.
7-O-Octylmorroniside (3j) White amorphous solid. 1H-NMR (CD3OD,
7-O-t-Butylmorroniside (3e) White amorphous solid. 1H-NMR 600 MHz) d: 0.85 (3H, t, Jꢀ7.0 Hz, CH3), 1.26—1.61 (15H, m, 6ꢄCH2, H-
(CD3OD, 600 MHz) d: 1.27 [9H, s, (CH3)3], 1.29 (3H, d, Jꢀ7.0 Hz, H-10b), 10), 1.78 (1H, m, H-9a), 1.89 (2H, m, H-6), 3.00 (1H, dt, Jꢀ4.0, 13.0 Hz,
1.35 (3H, d, Jꢀ7.0 Hz, H-10a), 1.52 (1H, ddd, Jꢀ2.0, 4.0, 9.0 Hz, H-9b),
H-5a), 3.05 (1H, dt, Jꢀ4.0, 13.0 Hz, H-5b), 3.18—3.60 (6H, m, H-2ꢁ, H-3ꢁ,
1.81 (1H, m, H-9a), 1.83 (2H, m, H-6b), 2.00 (2H, m, H-6a), 2.83 (1H, dt, H-4ꢁ, H-5ꢁ, H-6ꢁ), 3.65 (3H, s, OCH3), 3.90 (1H, m, H-8), 4.28 (1H, dd,
Jꢀ4.0, 13.0 Hz, H-5a), 3.08 (1H, dt, Jꢀ4.0, 13.0 Hz, H-5b), 3.21—3.40 Jꢀ2.0, 12.0 Hz, H-6ꢁa), 4.53 (1H, dd, Jꢀ2.0, 12.0 Hz, H-6ꢁb), 4.75 (1H, d,
(4H, m, H-2ꢁ, H-3ꢁ, H-4ꢁ, H-5ꢁ), 3.70 (3H, s, OCH3), 3.87 (1H, m, H-8),
4.30 (1H, dd, Jꢀ2.0, 12.0 Hz, H-6ꢁa) 4.54 (1H, dd, Jꢀ2.0, 12.0 Hz, H-6ꢁb),
Jꢀ4.1 Hz, H-7), 4.81 (1H, d, Jꢀ8.0 Hz, H-1ꢁ), 5.84 (1H, d, Jꢀ9.0 Hz, H-1),
7.48 (1H, s, H-3); 13C-NMR (CD3OD, 150 MHz) d: 13.3 (CH3), 18.5 (C-10),
4.77 (1H, d, Jꢀ4.0 Hz, H-7), 4.79 (1H, d, Jꢀ8.0 Hz, H-1ꢁ), 5.84 (1H, d, 22.6—29.5 (aliphatic CH2), 26.7 (C-5b), 29.5 (C-5a), 31.9 (C-6), 39.3 (C-
Jꢀ9.0 Hz, H-1a), 5.88 (1H, d, Jꢀ9.0 Hz, H-1b), 7.50 (1H, s, H-3); 13C- 9), 50.6 (OCH3), 61.7 (C-6ꢁ), 65.2 (C-8b), 67.3 (OCH2), 70.5 (C-2ꢁ), 73.9
NMR (CD3OD, 150 MHz) d: 15.4 [(CH3)3], 18.3 (C-10), 26.7 (C-5b), 27.5 (C-4ꢁ), 76.8 (C-3ꢁ), 77.4 (C-5ꢁ), 94.5 (C-7), 97.2 (C-1), 99.0 (C-1ꢁ), 110.5
[C(CH3)3], 30.8 (C-5a), 32.5 (C-6), 39.0 (C-9), 50.4 (OCH3), 61.6 (C-6ꢁ),
(C-4), 153.4 (C-3), 167.6 (CꢀO). IR (KBr) cmꢂ1: 3428, 1708, 1638. FAB-
65.0 (C-8b), 70.4 (C-2ꢁ), 73.7 (C-4ꢁ), 74.1 (C-8a), 76.6 (C-5ꢁ), 77.2 (C-3ꢁ), MS m/z: 541.4 [MꢃNa]ꢃ, 517.3 [Mꢃ1]ꢃ.
94.3 (C-7), 98.2 (C-1), 98.8 (C-1ꢁ), 110.5 (C-4), 153.1 (C-3), 167.3 (CꢀO).
7-O-Dodecylmorroniside (3k) White amorphous solid. 1H-NMR
IR (KBr) cmꢂ1: 3440, 1704, 1639. FAB-MS m/z: 485.3 [MꢃNa]ꢃ, 463.3 (CD3OD, 600 MHz) d: 0.86 (3H, t, Jꢀ7.0 Hz, CH3), 1.25—1.41 (23H, m,
[Mꢃ1]ꢃ.
aliphatic H, H-10), 1.46 (1H, m, H-6), 1.57 (2H, m, OCH2), 1.77 (1H, m, H-
7-O-n-Butylmorroniside (3f) White amorphous solid. 1H-NMR 9a), 1.88 (1H, m, H-6), 3.00 (1H, m, H-5b), 3.24—3.65 (6H, m, H-2ꢁ, H-3ꢁ,
(CD3OD, 600 MHz) d: 0.91 (3H, d, Jꢀ7.0 Hz, CH3), 1.29 (3H, d, Jꢀ7.0 Hz,
H-10b), 1.35 (3H, d, Jꢀ7.0 Hz, H-10a), 1.37—1.58 (5H, m, aliphatic H, H-
H-4ꢁ, H-5ꢁ, H-6ꢁ), 3.66 (3H, s, OCH3), 3.85 (1H, m, H-8), 4.30 (1H, dd,
Jꢀ2.0, 12.0 Hz, H-6ꢁa), 4.53 (1H, dd, Jꢀ2.0, 12.0 Hz, H-6ꢁb), 4.74 (1H, d,
9b), 1.78 (1H, m, H-9a), 1.88 (2H, m, H-6b), 2.00 (2H, m, H-6a), 2.79 Jꢀ4.0 Hz, H-7), 4.80 (1H, d, Jꢀ8.0 Hz, H-1ꢁ), 5.85 (1H, d, Jꢀ9.0 Hz, H-1),
(1H, dt, Jꢀ4.0, 13.0 Hz, H-5a), 3.03 (1H, dt, Jꢀ4.0, 13.0 Hz, H-5b), 3.36—
3.16 (4H, m, H-2ꢁ, H-3ꢁ, H-4ꢁ, H-5ꢁ), 3.65 (3H, s, OCH3), 3.85 (1H, m, H-
7.48 (1H, s, H-3); 13C-NMR (CD3OD, 150 MHz) d: 13.2 (CH3), 18.4 (C-10),
22.5—31.8 (aliphatic C), 26.2 (C-5b), 29.4 (C-5a), 32.7 (C-6), 39.2 (C-9),
8), 4.30 (1H, dd, Jꢀ2.0, 12.0 Hz, H-6ꢁa), 4.54 (1H, dd, Jꢀ2.0, 12.0 Hz, H- 50.5 (OCH3), 61.6 (C-6ꢁ), 65.1 (C-8b), 67.2 (OCH2), 70.4 (C-2ꢁ), 73.8 (C-
6ꢁb), 4.75 (1H, d, Jꢀ4.0 Hz, H-7), 4.80 (1H, d, Jꢀ8.0 Hz, H-1ꢁ), 5.75 (1H, 4ꢁ), 76.7 (C-3ꢁ), 77.3 (C-5ꢁ), 94.4 (C-7), 97.2 (C-1), 98.9 (C-1ꢁ), 110.4 (C-
d, Jꢀ9.0 Hz, H-1a), 5.84 (1H, d, Jꢀ9.0 Hz, H-1b), 7.47 (1H, s, H-3); 13C-
NMR (CD3OD, 150 MHz) d: 13.1 (CH3), 18.5 (C-10), 19.2 (CH2), 26.9 (C-
5b), 30.7 (C-5a), 31.7 (CH2), 32.8 (C-6), 39.1 (C-9), 51.4 (OCH3), 61.7 (C-
4), 153.3 (C-3), 167.5 (CꢀO). IR (KBr) cmꢂ1: 3416, 1708, 1638. FAB-MS
m/z: 597.3 [MꢃNa]ꢃ, 575.3 [Mꢃ1]ꢃ.
7-O-Hexadecylmorroniside (3l) White amorphous solid. 1H-NMR
6ꢁ), 65.2 (C-8b), 67.0 (OCH2), 70.4 (C-2ꢁ), 73.1 (C-4ꢁ), 73.9 (C-8a), 76.8 (CDCl3, 600 MHz) d: 0.87 (3H, t, Jꢀ7.0 Hz, CH3), 1.25—1.43 (33H, m,
(C-5ꢁ), 77.4 (C-3ꢁ), 94.5 (C-7), 97.2 (C-1), 99.0 (C-1ꢁ), 110.5 (C-4), 154.4 aliphatic H, H-10), 1.43 (1H, m, H-6), 1.57 (2H, m, OCH2), 1.75 (1H, m, H-
(C-3), 167.6 (CꢀO). IR (KBr) cmꢂ1: 3424, 1708, 1638. FAB-MS m/z: 485.3 9a), 1.88 (1H, m, H-6), 3.00 (1H, m, H-5b), 3.24—3.65 (6H, m, H-2ꢁ, H-3ꢁ,
[MꢃNa]ꢃ, 463.3 [Mꢃ1]ꢃ.
H-4ꢁ, H-5ꢁ, H-6ꢁ), 3.69 (3H, s, OCH3), 3.85 (1H, m, H-8), 4.25 (1H, m, H-
7-O-Cylohexylmorroniside (3g) White amorphous solid. 1H-NMR 6ꢁ), 4.53 (1H, m, H-6ꢁ), 4.74 (1H, br d, H-7), 4.80 (1H, br d, H-1ꢁ), 5.75 (1H,
(CD3OD, 600 MHz) d: 1.31 (3H, d, Jꢀ7.0 Hz, H-10), 1.52 (2H, m, CH2),
1.79 (2H, m, CH2), 1.81 (1H, m, H-9a), 1.87 (2H, m, CH2), 1.92 (2H, m, H-
br d, H-1), 7.47 (1H, s, H-3); 13C-NMR (CDCl3, 150 MHz) d: 14.0 (CH3),
19.1 (C-10), 22.6—31.8 (aliphatic C), 26.3 (C-5), 32.6 (C-6), 38.9 (C-9),
6b), 2.00 (2H, m, H-6a), 2.82 (1H, dt, Jꢀ4.0, 13.0 Hz, H-5a), 3.05 (1H, dt, 51.4 (OCH3), 61.5 (C-6ꢁ), 64.7 (C-8b), 67.6 (OCH2), 69.8 (C-8a), 72.9 (C-
Jꢀ4.0, 13.0 Hz, H-5b), 3.20—3.40 (4H, m, H-2ꢁ, H-3ꢁ, H-4ꢁ, H-5ꢁ), 3.55 4ꢁ), 73.5 (C-2ꢁ), 76.0 (C-3ꢁ), 76.2 (C-5ꢁ), 95.2 (C-7), 97.9 (C-1), 99.8 (C-1ꢁ),
(2H, m, OCH), 3.69 (3H, s, OCH3), 3.87 (1H, m, H-8), 4.40 (1H, dd, Jꢀ2.0,
110.6 (C-4), 153.3 (C-3), 166.9 (CꢀO). IR (KBr) cmꢂ1: 3426, 1707, 1637.
12.0 Hz, H-6ꢁa), 4.64 (1H, dd, Jꢀ2.0, 12.0 Hz, H-6ꢁb), 4.72 (1H, d, FAB-MS m/z: 653.4 [MꢃNa]ꢃ, 631.4 [Mꢃ1]ꢃ.
Jꢀ4.0 Hz, H-7), 4.79 (1H, d, Jꢀ8.0 Hz, H-1ꢁ), 5.80 (1H, d, Jꢀ9.0 Hz, H-
7-O-Octadecylmorroniside (3m) White amorphous solid. 1H-NMR
1a), 5.86 (1H, d, Jꢀ9.0 Hz, H-1b), 7.50 (1H, s, H-3); 13C-NMR (CD3OD, (CDCl3, 600 MHz) d: 0.87 (3H, t, Jꢀ7.0 Hz, CH3), 1.25—1.41 (37H, m,
150 MHz) d: 18.4 (C-10), 24.0 (CH2), 24.2 (CH2), 25.7 (2ꢄCH2), 26.9 (C-
5b), 30.7 (C-5a), 31.6 (C-6), 33.3 (CH2), 39.3 (C-9), 50.6 (OCH3), 61.7 (C-
aliphatic H, H-10), 1.43 (1H, m, H-6), 1.57 (2H, m, OCH2), 1.75 (1H, m, H-
9a), 1.95 (1H, m, H-6), 3.08 (1H, m, H-5b), 3.33—3.70 (6H, m, H-2ꢁ, H-3ꢁ,
6ꢁ), 65.2 (C-8b), 65.3 (OCH), 70.5 (C-2ꢁ), 73.9 (C-4ꢁ), 75.1 (C-8a), 76.8 H-4ꢁ, H-5ꢁ, H-6ꢁ), 3.71 (3H, s, OCH3), 3.89 (1H, m, H-8), 4.27 (1H, m, H-

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6ꢁ), 4.50 (1H, m, H-6ꢁ), 4.74 (1H, br d, H-7), 4.83 (1H, br d, H-1ꢁ), 5.75 (1H,
br d, H-1), 7.46 (1H, s, H-3); 13C-NMR (CDCl3, 150 MHz) d: 14.0 (CH3),
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4ꢁ), 74.4 (C-2ꢁ), 76.0 (C-3ꢁ), 76.2 (C-5ꢁ), 95.1 (C-7), 97.9 (C-1), 99.8 (C-1ꢁ),
110.8 (C-4), 152.8 (C-3), 166.9 (CꢀO). IR (KBr) cmꢂ1: 3441, 1707, 1639.
FAB-MS m/z: 681.4 [MꢃNa]ꢃ, 659.5 [Mꢃ1]ꢃ.
Cytotoxicity Bioassay Murine metastatic colon carcinoma cells (colon
26-L5) were cultured in RPMI-1640 medium (Invitrogen, Carlsbad, CA,
U.S.A.) supplemented with 10% heat-inactivated fetal bovine serum (FBS),
and 2 mM of L-glutamine, 100 units/ml of penicillin and 100 mg/ml of strep-
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24 h. WST-1 solution (DOJINDO, Kumamoto, Japan) was added to each
well (10 ml/well) for 2 h before the end of the incubation. The activity was
assessed by measuring the absorbance of the culture at 450 nm using a plate
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