Cholestane glycosides from the bulbs of Ornithogalum thyrsoides and their cytotoxic activity against HL-60 leukemia cells

Article abstract of DOI:10.1021/np020114j

Twelve bisdesmosidic cholestane glycosides (1-12), including nine new ones (1-9), were isolated from the bulbs of Ornithogalum thyrsoides by monitoring the cytotoxic activity on HL-60 leukemia cells. The structural assignment of the new compounds was carried out by spectroscopic analysis and the results of hydrolytic cleavage. The 3-O-monoglucosides with an aromatic acyl group at the C-16 diglycoside moiety (1, 12) were extremely cytotoxic, with respective IC₅₀ values of 0.00016 and 0.00013 μg/mL, and the other compounds, except for 2, 5, and 8, also showed cytotoxic activity as potent as etoposide (IC₅₀ 0.30 μg/mL), used as a positive control. These cholestanes were concluded to contribute to the potent cytotoxicity of the crude O. thyrsoides bulb extract.

Full text of DOI:10.1021/np020114j

J . Nat. Prod. 2002, 65, 1417-1423
1417
Ch olesta n e Glycosid es fr om th e Bu lbs of Or n ith oga lu m th yr soid es a n d Th eir
Cytotoxic Activity a ga in st HL-60 Leu k em ia Cells
Minpei Kuroda, Yoshihiro Mimaki,* Akihito Yokosuka, Fusako Hasegawa, and Yutaka Sashida
Laboratory of Medicinal Plant Science, School of Pharmacy, Tokyo University of Pharmacy and Life Science,
1432-1, Horinouchi, Hachioji, Tokyo 192-0392, J apan
Received March 20, 2002
Twelve bisdesmosidic cholestane glycosides (1-12), including nine new ones (1-9), were isolated from
the bulbs of Ornithogalum thyrsoides by monitoring the cytotoxic activity on HL-60 leukemia cells. The
structural assignment of the new compounds was carried out by spectroscopic analysis and the results of
hydrolytic cleavage. The 3-O-monoglucosides with an aromatic acyl group at the C-16 diglycoside moiety
(1, 12) were extremely cytotoxic, with respective IC50 values of 0.00016 and 0.00013 µg/mL, and the other
compounds, except for 2, 5, and 8, also showed cytotoxic activity as potent as etoposide (IC50 0.30 µg/mL),
used as a positive control. These cholestanes were concluded to contribute to the potent cytotoxicity of
the crude O. thyrsoides bulb extract.
An acylated cholestane diglycoside, 17R-hydroxy-16â-[(O-
(2-O-p-methoxybenzoyl-â-D-xylopyranosyl)-(1f3)-2-O-acetyl-
R-L-arabinopyranosyl)oxy]cholest-5-en-22-one (13), isolated
by us from the bulbs of Ornithogalum saundersiae, has
received scientific attention because of its strong cytotox-
icity against a variety of tumor cell culture lines and
experimental animal tumors.1 Recently, we have reported
several 13-related compounds and their cytotoxic activity
against HL-60 human promyelocytic leukemia cells.2 Fur-
thermore, two novel cytotoxic cholestane glycosides, named
galtonioside A3 and candicanoside A,4 along with several
13 derivatives5 and polyoxygenated cholestane bisdesmo-
sides,6 have been isolated from Galtonia candicans, a
Liliaceae plant taxonomically related to O. saundersiae.
Compound 13, galtonioside A, and candicanoside A were
considered to have potential as new anticancer agents with
a new mode of action because they displayed differential
cytotoxicities in the J apanese Foundation for Cancer Re-
serch 38 cell line assay,7 and their cytotoxic profiles in the
mean graphs were not correlated to those shown by any of
the other compounds including currently used anticancer
drugs. During our ongoing project, which focused on higher-
plant antineoplastic constituents, a phytochemical analysis
was conducted with the MeOH extract of the bulbs of
Ornithogalum thyrsoides since it exhibited potent cytotoxic
activity against HL-60 cells. A cytotoxicity-guided fraction-
ation procedure of the MeOH extract has resulted in the
isolation of 12 cholestane glycosides (1-12), including nine
new ones (1-9). This paper deals with the structural
assignment of the new cholestane glycosides based on
spectroscopic analysis and the results of hydrolytic cleavage
and with the cytotoxicity of the isolated compounds against
HL-60 cells.
Resu lts a n d Discu ssion
The fresh bulbs of O. thyrsoides were extracted with hot
MeOH. The concentrated MeOH extract, which showed
cytotoxic activity against HL-60 cells with an IC50 value of
0.79 µg/mL, was passed through a porous-polymer poly-
styrene resin (Diaion HP-20) column and successively
* To whom correspondence should be addressed. Tel: +81-426-76-4577.
Fax: +81-426-76-4579. E-mail: mimakiy@ps.toyaku.ac.jp.
10.1021/np020114j CCC: $22.00
© 2002 American Chemical Society and American Society of Pharmacognosy
Published on Web 08/28/2002

1418 J ournal of Natural Products, 2002, Vol. 65, No. 10
Kuroda et al.
eluted with 30% MeOH, 50% MeOH, and EtOH. The EtOH
eluate fraction exhibited a highly cytotoxic activity against
HL-60 cells (IC50 0.028 µg/mL). Then, the EtOH fraction
was repeatedly subjected to column chromatography on
silica gel and on octadecylsilanized (ODS) silica gel and to
reversed-phase HPLC to furnish compounds 1 (35.6 mg),
2 (130 mg), 3 (61.1 mg), 4 (84.5 mg), 5 (894 mg), 6 (140
mg), 7 (128 mg), 8 (90.2 mg), 9 (22.1 mg), 10 (23.6 mg), 11
(18.9 mg), and 12 (68.1 mg).
the sugar moieties. The assigned 1H and 13C NMR shifts
were indicative of a terminal â-D-glucopyranosyl unit and
a substituted â-D-glucopyranosyl residue glycosylated at
C-6, as well as the O-â-D-xylopyranosyl-(1f3)-2-O-acetyl-
R-L-arabinopyranosyl group attached at C-16 of the aglycon
as in 11. The anomeric proton signal of the terminal
glucosyl residue at δ 5.15 gave an HMBC correlation with
the δ 70.1 resonance assignable to C-6 of the inner glucosyl
group, whose anomeric proton at δ 4.96, in turn, was
correlated with C-3 of the aglycon at δ 78.4. Thus, the
structure of 2 was defined as 3â-[(O-â-D-glucopyranosyl-
(1f6)-â-D-glucopyranosyl)oxy)]-17R-hydroxy-16â-[(O-â-D-
xylopyranosyl-(1f3)-2-O-acetyl-R-L-arabinopyranosyl)oxy]-
cholest-5-en-22-one.
Compounds 10-12 were identified as 16â-[(R-L-ara-
binopyranosyl)oxy]-3â-[(â-D-glucopyranosyl)oxy)]-17R-hy-
droxycholest-5-en-22-one (10),5 3â-[(â-D-glucopyranosyl)-
oxy)]-17R-hydroxy-16â-[(O-â-D-xylopyranosyl-(1f3)-2-O-
acetyl-R-L-arabinopyranosyl)oxy]cholest-5-en-22-one (11),5
and 3â-[(â-D-glucopyranosyl)oxy)]-17R-hydroxy-16â-[(O-(2-
O-3,4-dimethoxybenzoyl-â-D-xylopyranosyl)-(1f3)-2-O-acetyl-
R-L-arabinopyranosyl)oxy]cholest-5-en-22-one (12),5 respec-
tively.
Compound 3 was shown to have the molecular formula
C60H90O26 on the basis of the positive-ion FABMS (m/z 1249
[M + Na]+), 13C NMR data (60 carbon signals), and
1
elemental analysis. The H and 13C NMR spectral data of
3 were essentially analogous with those of 2 and suggestive
of a cholestane glycoside of the same type. The existence
of a 3,4-dimethoxybenzoyl group in addition to an acetyl
group in the molecule was indicated by the IR [1694 cm-1
(CdO), 1600, 1515 and 1456 cm-1 (aromatic ring)], UV [λmax
All the new compounds were revealed to be based upon
3â,16â,17R-trihydroxycholest-5-en-22-one by analysis of
their spectral data and differed from each other with regard
to the structures of the sugar moieties and the acyl groups
attached at the C-16 sugar residue.
1
292 nm (log ꢀ 3.74), 263 nm (log ꢀ 4.02)], H NMR [δ 8.06
Compound 1 was obtained as an amorphous solid with
a molecular formula C55H82O22, as determined by the data
of the positive-ion FABMS, showing an [M + Na]+ ion at
m/z 1117, the 13C NMR spectrum with a total of 55 carbon
signals, and elemental analysis. Analysis of the 1H and 13C
NMR spectra of 1 and comparison with those of 12 implied
that 1 differed from 12 only in terms of the aromatic acid
constituent. Instead of the signals for a 3,4-dimethoxyben-
zoyl group, those assignable to a 3,4,5-trimethoxybenzoyl
residue were observed at δH 7.71 (2H, s), 3.97 (3H, s), and
3.81 (3H × 2, s); δC 126.3 (C), 108.1 (CH) × 2, 153.7 (C) ×
2, 143.2 (C), 165.4 (CdO), 60.7 (OMe), and 56.2 (OMe ×
2). Alkaline hydrolysis of 1 with 0.4% KOH in EtOH gave
3,4,5-trimethoxybenzoic acid and 11. The ester linkage
position of the 3,4,5-trimethoxybenzoyl group at C-2 of the
xylosyl group in 4 was ascertained by a long-range cor-
relation from the xylose H-2 proton at δ 5.79 (dd, J ) 8.9,
7.6 Hz) to the carbonyl resonance of the 3,4,5-trimethoxy-
benzoyl moiety at δ 165.4 in the HMBC spectrum of 1.
Thus, the structure of 1 was shown to be 3â-[(â-D-glucopy-
ranosyl)oxy)]-17R-hydroxy-16â-[(O-(2-O-3,4,5-trimethoxy-
benzoyl-â-D-xylopyranosyl)-(1f3)-2-O-acetyl-R-L-arabinopy-
ranosyl)oxy]cholest-5-en-22-one.
(1H, dd, J ) 8.6, 1.9 Hz), 7.30 (1H, d, J ) 1.9 Hz), 7.07
(1H, d, J ) 8.6 Hz), 3.82 (3H, s), and 3.81 (3H, s)], and 13C
NMR [δ 123.1 (C), 113.5 (CH), 145.9 (C), 154.0 (C), 111.2
(CH), 124.4 (CH), 165.6 (CdO), 55.9 (OMe), and 55.8
(OMe)] spectra. Alkaline hydrolysis of 3 with 0.4% KOH
in EtOH yielded 2, 2a , and 3,4-dimethoxybenzoic acid. An
HMBC correlation from the xylose H-2 resonance at δ 5.71
(dd, J ) 9.0, 7.8 Hz) to the conjugated carbonyl carbon
signal at δ 165.6 gave evidence for the ester linkage
position of the 3,4-dimethoxybenzoyl moiety at C-2 of the
xylosyl residue. The structure of 3 was revealed to be 3â-
[(O-â-D-glucopyranosyl-(1f6)-â-D-glucopyranosyl)oxy)]-17R-
hydroxy-16â-[(O-(2-O-3,4-dimethoxybenzoyl-â-D-xylopyra-
nosyl)-(1f3)-2-O-acetyl-R-L-arabinopyranosyl)oxy]cholest-
5-en-22-one.
The 1H and 13C NMR spectral data of 4 (C61H92O27) were
superimposable on those of 3, except for the aromatic region
signals due to the substituted benzoyl moiety. The aromatic
acid linked to C-2 of the xylosyl residue was suggested to
1
be 3,4,5-trimethoxybenzoic acid by the UV, H NMR, and
13C NMR spectra. Alkaline hydrolysis of 4 with 0.4% KOH
in EtOH gave 3,4,5-trimethoxybenzoic acid, 2, and 2a .
Thus, 4 was characterized as 3â-[(O-â-D-glucopyranosyl-
(1f6)-â-D-glucopyranosyl)oxy)]-17R-hydroxy-16â-[(O-(2-O-
3,4,5-trimethoxybenzoyl-â-D-xylopyranosyl)-(1f3)-2-O-acetyl-
R-L-arabinopyranosyl)oxy]cholest-5-en-22-one.
Compound 2 was obtained as an amorphous solid. The
positive-ion FABMS (m/z 1085 [M + Na]+), 13C NMR data
(51 carbon signals), and elemental analysis allowed the
determination of the molecular formula of 2 as C51H82O23
,
which was higher by C6H10O5 than that of 11. The 1H NMR
spectrum of 2 showed signals for four anomeric protons at
δ 5.15 (d, J ) 7.8 Hz), 4.96 (d, J ) 7.7 Hz), 4.91 (d, J ) 7.5
Hz), and 4.66 (d, J ) 6.5 Hz), along with signals for five
steroid methyl groups and an acetyl group. Acid hydrolysis
of 2 with 1 M HCl in dioxane-H2O (1:1) liberated L-
arabinose, D-xylose, and D-glucose as the carbohydrate
moieties, and several degradation products from the agly-
con, whereas alkaline treatment with 3% NaOMe in MeOH
yielded a deacetyl derivative (2a : C49H80O22). The identi-
fication of the monosaccharides, including their absolute
configurations, was established by direct HPLC analysis
of the sugar fraction of the acid hydrolysate using a
combination of RI and optical rotary (OR) detectors.
Analysis of the 1H-1H COSY spectrum, starting from each
anomeric proton, and of the HMQC spectrum of 2 led to
the assignment of all the proton and carbon signals due to
Compound 5 was deduced as C57H92O28 from the positive-
ion FABMS (m/z 1247 [M + Na]+), 13C NMR spectrum (57
carbon signals), and elemental analysis data. The 1H NMR
spectrum of 5 contained signals for five anomeric protons
at δ 5.17 (d, J ) 7.9 Hz), 5.08 (d, J ) 7.8 Hz), 4.96 (d, J )
7.7 Hz), 4.91 (d, J ) 7.5 Hz), and 4.65 (d, J ) 6.5 Hz), along
with signals for five steroid methyl groups and an acetyl
group. Acid hydrolysis of 5 with 1 M HCl in dioxane-H2O
(1:1) gave L-arabinose, D-xylose, and D-glucose, whereas
alkaline treatment with 3% NaOMe in MeOH yielded a
deacetyl derivative (5a : C55H90O27). The above data and
inspection of the 13C NMR spectrum of 5 assumed that 5
structurally corresponded to 2 with one more â-D-glucopy-
ranosyl unit present and that the acetyl diglycosyl group
linked to C-16 of the aglycon was identical to that of 2 and
11. The additional glucosyl unit was supposed to be located
at C-4 of the terminal glucosyl part in 2 since a glycosy-

Cholestane Glycosides from Ornithogalum
J ournal of Natural Products, 2002, Vol. 65, No. 10 1419
lation shift could be detected around it when the 13C NMR
0.00013 µg/mL, and the other compounds, except for 2, 5,
and 8, also showed cytotoxic activity as potent as etoposide
(IC50 0.30 µg/mL), used as a positive control. These choles-
tanes were concluded to contribute to the potent cytotox-
icity of the crude O. thyrsoides bulb extract. Compound 11
is the corresponding deacyl derivative of 1 and 12 and was
less cytotoxic compared with 1 and 12. Compounds 2 and
5, which are the corresponding deacyl cholestanes of 3 and
4, and 6 and 7, respectively, did not show any apparent
cytotoxic activity even at the sample concentration of 10
µg/mL. These facts were consistent with the aromatic acid
ester group attached at the C-16 glycoside moiety playing
an important role for the appearance of the strong cytotoxic
activity, as observed in the related cholestanes.1,2,5 The
cytotoxic activity of 3 and 4, having an additional glucosyl
unit at C-6 of the terminal glucosyl moiety of 12 and 1,
respectively, was far less potent than that of 12 and 1 by
about 3 orders of magnitude. However, further glycosyla-
tion of the C-4 hydroxy group of the terminal glucosyl
moiety of 3 and 4 resulted in no discernible effects on the
activity.
spectrum of 5 was compared with that of 2. This was
3
confirmed by a J C,H correlation from the anomeric proton
signal of the terminal glucosyl residue at δ 5.17 to the δ
81.0 resonance assignable to C-4 of the inner glucosyl
group, whose anomeric proton at δ 5.08 had an HMBC
correlation with the δ 70.1 signal due to C-6 of the glucosyl
unit attached at C-3 of the aglycon. Accordingly, the
structure of 5 was elucidated as 3â-[(O-â-D-glucopyranosyl-
(1f4)-O-â-D-glucopyranosyl-(1f6)-â-D-glucopyranosyl)oxy)]-
17R-hydroxy-16â-[(O-â-D-xylopyranosyl-(1f3)-2-O-acetyl-
R-L-arabinopyranosyl)oxy]cholest-5-en-22-one.
Compound 6 was analyzed for C66H100O31 by combined
negative-ion FABMS (m/z 1387 [M - H]-), 13C NMR
spectrum (66 carbon signals), and elemental analysis.
Comparison of the 1H and 13C NMR spectra of 6 with those
of 5 showed their considerable structural similarity and
confirmed that 6 differed from 5 in the presence of a 3,4-
dimethoxybenzoyl group. Alkaline hydrolysis of 6 with 0.4%
KOH in EtOH gave 3,4-dimethoxybenzoic acid, 5, and 5a .
The ester linkage at the xylose C-2 position in 6 was formed
from 3,4-dimethoxybenzoic acid, as was evident from an
HMBC correlation between the signals of the xylose H-2
proton at δ 5.71 (dd, J ) 8.4, 7.7 Hz) and the carbonyl
carbon at δ 165.6. The structure of 6 was elucidated as 3â-
[(O-â-D-glucopyranosyl-(1f4)-O-â-D-glucopyranosyl-(1f6)-
â-D-glucopyranosyl)oxy)]-17R-hydroxy-16â-[(O-(2-O-3,4-
dimethoxybenzoyl-â-D-xylopyranosyl)-(1f3)-2-O-acetyl-R-
L-arabinopyranosyl)oxy]cholest-5-en-22-one.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Optical rotation was
measured by using a J ASCO DIP-360 (Tokyo, J apan) auto-
matic digital polarimeter. IR spectra were recorded on a
J ASCO FT-IR 620 spectrophotometer. NMR spectra were
1
recorded on a Bruker DPX-400 spectrometer (400 MHz for H
NMR, Karlsruhe, Germany) or on a Bruker DRX-500 spec-
1
The 1H and 13C NMR spectra of 7 (C67H102O32) and 8
(C65H98O31) were identical to those of 6, except for the
aromatic region signals due to the substituted benzoyl
moiety. The aromatic acid linked to C-2 of xylosyl residue
was suggested to be 3,4,5-trimethoxybenzoic acid in 7 and
4-hydroxy-3-methoxybenzoic acid in 8 by the UV, 1H NMR,
and 13C NMR spectra. On alkaline hydrolysis of 7 and 8
with 0.4% KOH, 3,4,5-trimethoxybenzoic acid, 5, and 5a
were obtained from 7, and 4-hydroxy-3-methoxybenzoic
acid, 5, and 5a from 8. The structures of 7 and 8 were
assigned as 3â-[(O-â-D-glucopyranosyl-(1f4)-O-â-D-glu-
copyranosyl-(1f6)-â-D-glucopyranosyl)oxy)]-17R-hydroxy-
16â-[(O-(2-O-3,4,5-trimethoxybenzoyl-â-D-xylopyranosyl)-
(1f3)-2-O-acetyl-R-L-arabinopyranosyl)oxy]cholest-5-en-22-
one and 3â-[(O-â-D-glucopyranosyl-(1f4)-O-â-D-glucopyran-
osyl-(1f6)-â-D-glucopyranosyl)oxy)]-17R-hydroxy-16â-[(O-
(2-O-4-hydroxy-3-methoxybenzoyl-â-D-xylopyranosyl)-(1f3)-
2-O-acetyl-R-L-arabinopyranosyl)oxy]cholest-5-en-22-one,
respectively.
trometer (500 MHz for H NMR) using standard Bruker pulse
programs. Chemical shifts are given as δ values with reference
to tetramethylsilane (TMS) as internal standard. MS were
recorded on a Finnigan MAT TSQ-700 (San J ose, CA) mass
spectrometer, using a dithiothreitol and dithioerythritol (3:1)
matrix. Elemental analysis was carried out using an Elemental
Vario EL (Hanau, Germany) elemental analyzer. Silica gel
(Fuji-Silysia Chemical, Aichi, J apan), ODS silica gel (Nacalai
Tesque, Kyoto, J apan), and Diaion HP-20 (Mitsubishi-Kasei,
Tokyo, J apan) were used for column chromatography. TLC was
carried out on precoated Kieselgel 60 F254 (0.25 mm thick,
Merck, Darmstadt, Germany) and RP-18 F254S (0.25 mm thick,
Merck) plates, and spots were visualized by spraying the plates
with 10% H2SO4 solution, followed by heating. HPLC was
performed by using a system comprised of a CCPM pump
(Tosoh, Tokyo, J apan), a CCP PX-8010 controller (Tosoh), an
RI-8010 detector (Tosoh), a Shodex OR-2 detector (Showa-
Denko, Tokyo, J apan), and a Rheodyne injection port with a
20 µL sample loop. A Kaseisorb NH2-60-5 column (4.6 mm i.d.
× 250 mm, 5 µm, Tokyo-Kasei, Tokyo, J apan) was employed
for HPLC analysis. The following reagents were obtained from
the indicated companies: RPMI 1640 medium (Gibco, Gland
Island, NY); FBS (Bio-Whittaker, Walkersville, MD); MTT
(Sigma, St. Louis, MO); penicillin and streptomycin (Meiji-
Seika, Tokyo, J apan). All other chemicals used were of
biochemical reagent grade.
P la n t Ma ter ia l. The bulbs of O. thyrsoides were purchased
from a nursery in Heiwaen, Nara, J apan. The bulbs were
cultivated, and the flowered plant was identified by one of the
authors (Y.S). A voucher specimen has been deposited in our
laboratory (voucher No. OT-99-004, Laboratory of Medicinal
Plant Science).
Extr a ction a n d Isola tion . The plant material (fresh
weight, 15.4 kg) was extracted with hot MeOH (twice). The
MeOH extract was concentrated under reduced pressure, and
the viscous concentrate (702 g) was passed through a Diaion
HP-20 column, successively eluting with 30% MeOH, 50%
MeOH, and EtOH. The EtOH eluate fraction exhibited potent
cytotoxic activity against HL-60 cells (IC50 0.028 µg/mL), while
the other fractions did not show apparent cytotoxicity (30%
MeOH and 50% MeOH: IC50 >20 µg/mL). Column chroma-
Compound 9 (C66H100O31) was presumed to be an isomer
of 6 with regard to the location of the 3,4-dimethoxybenzoyl
group linked to the xylosyl moiety. Alkaline hydrolysis of
9 gave 5, 5a , and 3,4-dimethoxybenzoic acid. In the HMBC
spectrum of 9, the downfield-shifted proton signal at δ 5.95
(dd, J ) 9.0, 8.8 Hz) attributable to H-3 of the xylosyl
moiety was correlated to the ester carbonyl carbon signal
at δ 166.4. The structure of 9 was characterized as 3â-[(O-
â-D-glucopyranosyl-(1f4)-O-â-D-glucopyranosyl-(1f6)-â-D-
glucopyranosyl)oxy)]-17R-hydroxy-16â-[(O-(3-O-3,4-dimeth-
oxybenzoyl-â-D-xylopyranosyl)-(1f3)-2-O-acetyl-R-L-ara-
binopyranosyl)oxy]cholest-5-en-22-one.
The isolated compounds were evaluated for their cyto-
toxic activity against HL-60 cells. The cells were continu-
ously treated with each sample for 72 h, and the cell growth
was measured by an MTT reduction assay procedure (Table
2).8 The 3-O-monoglucosides with an aromatic acyl group
at the C-16 diglycoside moiety (1, 12) were extremely
cytotoxic, with respective IC50 values of 0.00016 and

1420 J ournal of Natural Products, 2002, Vol. 65, No. 10
Kuroda et al.
Ta ble 1. 13C NMR Spectral Data for Compounds 1, 2, 2a , 3-5, 5a , and 6-9 in Pyridine-d5
carbon
1
2
2a
3
4
5
5a
6
7
8
9
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
37.4
30.2
78.1
39.3
140.8
121.8
32.2
32.0
50.0
36.9
20.9
32.7
46.5
48.5
34.6
88.4
85.7
13.6
19.4
46.3
11.9
218.9
32.7
39.3
27.7
22.8
22.4
102.5
75.6
78.6
71.7
78.5
62.9
37.4
30.4
78.4
39.5
141.0
121.7
32.2
32.0
49.9
36.9
20.8
32.7
46.5
48.4
35.0
88.3
85.7
13.5
19.4
46.4
11.9
219.0
32.8
39.5
27.9
22.8
22.5
103.0
75.1
78.5
71.7
77.2
70.1
105.4
75.2
78.5
71.6
78.7
62.8
37.4
30.4
78.4
39.4
140.9
121.8
32.2
32.0
49.9
36.8
20.9
32.5
46.4
48.5
36.1
88.9
86.2
13.6
19.4
46.1
12.1
219.6
32.6
39.5
27.9
23.0
22.5
103.0
75.1
78.4
71.7
77.2
70.1
105.5
75.2
78.5
71.6
78.7
62.8
37.4
30.4
78.4
39.5
140.9
121.7
32.2
31.9
49.9
36.9
20.8
32.7
46.5
48.4
34.6
88.3
85.7
13.6
19.4
46.3
11.9
218.9
32.6
39.2
27.7
22.8
22.4
102.9
75.1
78.5
71.7
77.2
70.0
105.4
75.2
78.5
71.6
78.6
62.8
37.4
30.4
78.4
39.5
140.9
121.6
32.1
31.9
49.9
36.9
20.8
32.7
46.5
48.4
34.5
88.4
85.7
13.6
19.4
46.3
11.9
218.9
32.6
39.2
27.7
22.8
22.4
102.9
75.1
78.5
71.7
77.2
70.0
105.4
75.2
78.5
71.6
78.6
62.8
37.4
30.4
78.5
39.5
140.9
121.8
32.1
32.0
49.9
36.9
20.8
32.7
46.5
48.4
35.0
88.3
85.8
13.5
19.4
46.4
11.9
218.9
32.8
39.4
27.9
22.8
22.5
102.8
75.1
78.4
71.5
77.1
70.1
104.9
74.7
76.7
81.0
76.5
62.0
104.8
74.6
78.4
71.6
78.2
62.4
101.4
72.2
80.0
68.6
66.5
106.7
74.2
78.2
70.9
67.2
37.4
30.4
78.4
39.4
140.8
121.9
32.2
32.0
49.9
36.9
20.9
32.5
46.4
48.5
36.1
88.9
86.2
13.6
19.5
46.1
12.1
219.5
32.6
39.4
27.9
23.0
22.5
102.8
75.1
78.4
71.5
77.1
70.0
104.9
74.7
76.7
80.9
76.5
62.0
104.9
74.6
78.5
71.6
78.2
62.4
105.5
71.7
83.8
68.9
67.3
106.9
75.1
78.2
71.0
67.1
37.4
30.4
78.4
39.4
140.9
121.8
32.1
31.9
49.9
36.9
20.8
32.7
46.5
48.4
34.6
88.3
85.7
13.6
19.4
46.3
11.9
218.8
32.7
39.2
27.7
22.8
22.4
102.7
75.1
78.4
71.5
77.1
70.0
104.9
74.7
76.6
80.9
76.5
62.0
104.9
74.6
78.4
71.5
78.2
62.4
100.8
72.1
81.0
67.7
65.4
103.7
75.2
76.3
70.7
67.0
123.1
113.5
149.5
154.0
111.2
124.4
165.6
55.9
55.8
169.3
20.9
37.4
30.4
78.4
39.4
140.9
121.8
32.1
32.0
49.9
36.9
20.8
32.7
46.5
48.4
34.5
88.4
85.7
13.6
19.4
46.3
11.9
218.8
32.7
39.2
27.7
22.8
22.4
102.7
75.2
78.5
71.6
77.1
70.1
104.9
74.7
76.7
80.9
76.5
62.0
104.9
74.6
78.5
71.5
78.2
62.4
100.8
72.2
81.0
67.8
65.5
103.5
75.6
76.2
70.7
67.0
126.3
108.1
153.6
143.2
153.6
108.1
165.4
60.7
56.2 × 2
169.3
20.9
37.4
30.4
78.4
39.4
140.9
121.8
32.1
31.9
49.9
36.9
20.8
32.7
46.5
48.4
34.7
88.3
85.8
13.6
19.4
46.3
11.9
218.9
32.7
39.2
27.7
22.8
22.4
102.7
75.2
78.5
71.6
77.1
70.1
104.9
74.7
76.7
81.0
76.5
62.0
105.0
74.6
78.5
71.5
78.2
62.4
100.8
72.0
81.0
67.6
65.3
103.8
75.0
76.4
70.7
67.0
122.1
113.9
148.3
153.2
116.1
125.0
165.7
55.8
37.4
30.4
78.4
39.4
140.9
121.8
32.1
32.0
49.9
36.9
20.8
32.7
46.5
48.4
35.0
88.2
85.7
13.5
19.4
46.4
11.8
218.8
32.9
39.5
27.9
22.8
22.5
102.8
75.2
78.4
71.5
77.1
70.1
104.9
74.7
76.7
81.0
76.5
62.0
104.9
74.6
78.5
71.6
78.2
62.4
101.3
72.1
80.5
68.8
68.9
106.4
71.9
79.3
66.7
66.7
123.1
113.2
149.5
153.8
111.2
124.2
166.4
55.8
55.7
170.0
21.5
25
26
27
Glc 1
2
3
4
5
6
Glc′ 1
2
3
4
5
6
Glc′′ 1
2
3
4
5
6
Ara 1
100.8
72.2
80.9
67.8
65.5
103.6
75.4
76.3
70.7
67.1
126.3
108.1
153.7
143.2
153.7
108.1
165.4
60.7
101.4
72.2
80.0
68.6
66.5
106.7
74.2
78.2
70.9
67.2
105.4
71.7
83.8
68.9
67.3
106.9
75.1
78.3
71.0
67.1
100.8
72.1
80.9
67.7
65.4
103.7
75.2
76.3
70.7
67.0
123.1
113.5
149.5
154.0
111.2
124.4
165.6
55.9
100.8
72.1
80.9
67.7
65.5
103.5
75.6
76.2
70.7
67.0
126.3
108.1
153.6
143.2
153.6
108.1
165.4
60.7
2
3
4
5
Xyl 1
2
3
4
5
Ar 1
2
3
4
5
6
7
OMe
56.2 × 2
169.3
20.9
55.8
169.3
20.9
56.2 × 2
169.3
20.9
Ac
170.0
21.5
170.0
21.5
169.3
20.9
tography of the EtOH eluate portion on silica gel and elution
with a stepwise gradient mixture of CHCl3-MeOH-H2O (90:
10:0; 40:10:1; 20:10:1), and finally with MeOH alone, gave four
fractions (I-IV). Fraction II was subjected to a silica gel
column eluting with CHCl3-MeOH-H2O (60:10:1) to collect
two additional fractions (IIa, IIb). Fraction IIa was purified
by silica gel column chromatography eluting with CHCl3-
MeOH-H2O (70:10:1; 80:10:1) and ODS silica gel column
chromatography with MeOH-H2O (2:1) and MeCN-H2O (1:
2; 2:3) and by preparative HPLC using MeCN-H2O (4:5) to
yield 1 (35.6 mg), 10 (23.6 mg), and 12 (68.1 mg). Fraction IIb
was subjected to an ODS silica gel column with MeCN-H2O
(1:2) to give 11 (18.9 mg). Fraction III was separated by silica
gel column chromatography eluting with CHCl3-MeOH-H2O
(40:10:1) and ODS silica gel column chromatography with
MeCN-H2O (1:2) to give four fractions (IIIa-IIId). Fraction

Cholestane Glycosides from Ornithogalum
J ournal of Natural Products, 2002, Vol. 65, No. 10 1421
Ta ble 2. 1H and 13C NMR Spectral Data for the Triglucoside
Moiety of 5 in Pyridine-d5
Alk a lin e Hyd r olysis of 1. Compound 1 (10.4 mg) was
treated with 0.4% KOH in EtOH (9 mL) at room temperature
for 1 h. The reaction mixture was neutralized by passage
through an Amberlite IR-120B (Organo, Tokyo, J apan) and
chromatographed on silica gel eluting with CHCl3-MeOH-
H2O (40:10:1) to give 11 (3.6 mg) and 3,4,5-trimethoxybenzoic
acid (0.5 mg).
position
1H
J (Hz)
13C
Glc 1
4.96 d
7.7
102.8
75.1
78.4
71.5
77.1
70.1
2
3
4
5
6
3.99 dd
4.21 dd
4.22 dd
4.10 ddd
4.80 dd
4.34 dd
5.08 d
4.05 dd
4.25 dd
4.32 dd
3.86 ddd
4.54 dd
4.44 dd
5.17 d
4.09 dd
4.26 dd
4.22 dd
3.97 ddd
4.53 dd
4.29 dd
8.3, 7.7
8.9, 8.3
9.2, 8.9
9.2, 8.1, 2.1
11.4, 2.1
11.4, 8.1
7.8
8.5, 7.8
8.6, 8.5
9.3, 8.6
9.3, 6.6, 2.4
11.9, 6.6
11.9, 2.4
7.9
8.8, 7.9
8.8, 8.9
10.9, 8.9
10.9, 5.8, 2.7
11.5, 2.7
11.5, 5.8
25
Com p ou n d 2: amorphous solid; [R]D -64.0° (c 0.10,
MeOH); IR (film) νmax 3386 (OH), 2935 and 2875 (CH), 1737
and 1691 (CdO), 1646, 1455, 1407, 1373, 1244, 1166, 1048
1
cm-1; H NMR (pyridine-d5) δ 5.82 (1H, dd, J ) 8.5, 6.5 Hz,
Glc′ 1
104.9
74.7
76.7
81.0
76.5
62.0
H-2 of Ara), 5.30 (1H, br d, J ) 4.8 Hz, H-6), 5.15 (1H, d, J )
7.8 Hz, H-1 of Glc′), 4.96 (1H, d, J ) 7.7 Hz, H-1 of Glc), 4.91
(1H, d, J ) 7.5 Hz, H-1 of Xyl), 4.66 (1H, d, J ) 6.5 Hz, H-1 of
Ara), 3.94 (1H, m, overlapping, H-3), 3.86 (1H, dd, J ) 8.2,
7.5 Hz, H-2 of Xyl), 3.33 (1H, q, J ) 7.4 Hz, H-20), 2.35 (3H,
s, Ac), 1.32 (3H, d, J ) 7.4 Hz, Me-21), 0.96 (3H, s, Me-18),
0.95 (3H, s, Me-19), 0.95 (3H, d, J ) 6.2 Hz, Me-26 or Me-27),
0.92 (3H, d, J ) 6.2 Hz, Me-26 or Me-27); 13C NMR, see Table
1; FABMS (positive-mode) m/z 1085 [M + Na]+; anal. C
55.10%, H 8.13% (calcd for C51H82O23‚3H2O, C 54.82%, H
7.94%).
2
3
4
5
6
Glc′′ 1
104.8
74.6
78.4
71.6
78.2
62.4
2
3
4
5
6
Acid Hyd r olysis of 2. A solution of 2 (5.0 mg) in 1 M HCl
(dioxane-H2O, 1:1, 2 mL) was heated at 92 °C for 1 h under
an Ar atmosphere. After cooling, the reaction mixture was
neutralized by passage through an Amberlite IRA-93ZU
(Organo, Tokyo, J apan) column and chromatographed on
Diaion HP-20 eluting with H2O-MeOH (3:2), followed by Me2-
CO-EtOH (1:1), to give a sugar fraction (2.1 mg). The sugar
fraction was passed through a Sep-Pak C18 cartridge (Waters,
Milford, MA) and a Toyopak IC-SP M cartridge (Tosoh), which
was then analyzed by HPLC under the following conditions:
column, Capcell Pak NH2 UG80 (4.6 mm i.d. × 250 mm, 5 µm,
Shiseido, Tokyo, J apan); solvent, MeCN-H2O (17:3); flow rate,
0.9 mL/min; detection, RI and OR. The identification of
L-arabinose, D-xylose, and D-glucose present in the sugar
fraction was carried out by comparison of their retention times
and polarities with those of authentic samples. tR (min): 11.42
(L-arabinose, positive polarity), 11.98 (D-xylose, positive polar-
ity), 19.98 (D-glucose, positive polarity).
Ta ble 3. Cytotoxic Acitivity of Compounds 1-12 and
Etoposide against HL-60 Cells
compound
IC50 (µg/mL)
1
2
3
0.00016
>10
0.81
4
0.70
5
6
>10
0.73
7
0.77
8
6.6
9
0.46
10
0.12
11
12
etoposide
0.013
0.00013
0.30
Alk a lin e Hyd r olysis of 2. Compound 2 (39.0 mg) was
treated with 3% NaOMe in MeOH (3 mL) at room temperature
for 2 h. The reaction mixture was neutralized by passage
through an Amberlite IR-120B column and then chromato-
graphed eluting with CHCl3-MeOH-H2O (30:10:1) to yield
2a (20.0 mg).
IIIb was subjected to a silica gel column eluting with CHCl3-
MeOH-H2O (60:10:1) and preparative HPLC using MeCN-
H2O (4:5) to give 3 (61.1 mg) and 4 (84.5 mg). Fraction IIIc
was chromatographed on silica gel eluting with CHCl3-
MeOH-H2O (30:10:1) and ODS silica gel with MeCN-H2O (2:
5) to give 2 (130 mg) and a mixture of 6 and 7, which was
separated by preparative HPLC using MeCN-H2O (2:3) to
furnish 6 (140 mg) and 7 (128 mg). Compound 9 (22.1 mg)
was isolated from fraction IIId by subjecting it to preparative
HPLC using MeCN-H2O (2:3). Fraction IV was subjected to
a silica gel column eluting with CHCl3-MeOH-H2O (40:10:
1; 30:10:1) and an ODS silica gel column with MeCN-H2O
(2:5) and to preparative HPLC using MeCN-H2O (2:3) to give
5 (894 mg) and 8 (90.2 mg).
25
Com p ou n d 2a : amorphous powder; [R]D -50.0° (c 0.10,
MeOH); IR (film) νmax 3376 (OH), 2934, 2902, and 2871 (CH),
1685 (CdO), 1072, 1044 cm-1 1H NMR (pyridine-d5) δ 5.28
;
(1H, br s, H-6), 5.19 (1H, d, J ) 7.6 Hz, H-1 of Xyl), 5.16 (1H,
d, J ) 7.8 Hz, H-1 of Glc′), 4.96 (1H, d, J ) 7.7 Hz, H-1 of
Glc), 4.66 (1H, d, J ) 7.7 Hz, H-1 of Ara), 3.92 (1H, m,
overlapping, H-3), 3.86 (1H, dd, J ) 8.2, 7.5 Hz, H-2 of Xyl),
3.39 (1H, q, J ) 7.4 Hz, H-20), 1.32 (3H, d, J ) 7.4 Hz, Me-
21), 0.94 (3H, s, Me-19), 0.94 (3H, d, J ) 6.4 Hz, Me-26 or
Me-27), 0.89 (3H, s, Me-18), 0.87 (3H, d, J ) 6.4 Hz, Me-26 or
Me-27); 13C NMR, see Table 1; FAB-MS (positive-mode) m/z
1043 [M + Na]+; anal. C 53.14%, H 7.93% (calcd for C49H80O22
‚
25
Com p ou n d 1: amorphous solid; [R]D -34.0° (c 0.10,
4H2O, C 53.14%, H 8.11%).
MeOH); IR (film) νmax 3418 (OH), 2937 and 2871 (CH), 1732,
1716, and 1696 (CdO), 1590, 1504, and 1457 (aromatic ring),
25
Com p ou n d 3: amorphous solid; [R]D -72.0° (c 0.10,
MeOH); IR (film) νmax 3365 (OH), 2932 and 2871 (CH), 1715
and 1694 (CdO), 1600, 1515, and 1456 (aromatic ring), 1416,
1370, 1270, 1226, 1174, 1131, 1067, 1043 cm-1; UV (MeOH)
1416, 1371, 1338, 1255, 1228, 1173, 1127, 1070, 1042 cm-1
;
UV (MeOH) λmax 268 nm (log ꢀ 4.07); 1H NMR (pyridine-d5) δ
7.71 (2H, s, H-2, H-6 of Ar), 5.79 (1H, dd, J ) 8.9, 7.6 Hz, H-2
of Xyl), 5.56 (1H, dd, J ) 7.4, 5.9 Hz, H-2 of Ara), 5.29 (1H, br
d, J ) 4.6 Hz, H-6), 5.16 (1H, d, J ) 7.6 Hz, H-1 of Xyl), 5.05
(1H, d, J ) 7.7 Hz, H-1 of Glc), 4.61 (1H, d, J ) 5.6 Hz, H-1 of
Ara), 3.97 (3H, s, OMe), 3.93 (1H, m, W1/2 ) 22.6 Hz, H-3),
3.81 (3H × 2, s, OMe × 2), 3.21 (1H, q, J ) 7.4 Hz, H-20), 2.01
(3H, s, Ac), 1.31 (3H, d, J ) 7.4 Hz, Me-21), 1.00 (3H, s, Me-
18), 0.98 (3H, s, Me-19), 0.86 (3H, d, J ) 6.0 Hz, Me-26 or
Me-27), 0.88 (3H, d, J ) 6.0 Hz, Me-26 or Me-27); 13C NMR,
see Table 1; FABMS (positive mode) m/z 1117 [M + Na]+; anal.
C 58.59%; H 7.68% (calcd for C55H82O22‚3/2H2O, C 58.86%, H
7.63%).
λ
max 292 nm (log ꢀ 3.74), 263 nm (log ꢀ 4.02); 1H NMR (pyridine-
d5) δ 8.06 (1H, dd, J ) 8.6, 1.9 Hz, H-6 of Ar), 7.30 (1H, d, J
) 1.9 Hz, H-2 of Ar), 7.07 (1H, d, J ) 8.6 Hz, H-5 of Ar), 5.71
(1H, dd, J ) 9.0, 7.8 Hz, H-2 of Xyl), 5.55 (1H, dd, J ) 7.4, 6.2
Hz, H-2 of Ara), 5.29 (1H, br d, J ) 4.7 Hz, H-6), 5.15 (1H, d,
J ) 7.8 Hz, H-1 of Glc′), 5.15 (1H, d, J ) 7.8 Hz, H-1 of Xyl),
4.95 (1H, d, J ) 7.7 Hz, H-1 of Glc), 4.60 (1H, d, J ) 5.8 Hz,
H-1 of Ara), 3.94 (1H, m, W1/2 ) 20.0 Hz, H-3), 3.82 and 3.81
(each 3H, s, OMe × 2), 3.20 (1H, q, J ) 7.4 Hz, H-20), 2.00
(3H, s, Ac), 1.30 (3H, d, J ) 7.4 Hz, Me-21), 0.99 (3H, s, Me-
18), 0.95 (3H, s, Me-19), 0.88 (3H, d, J ) 6.1 Hz, Me-26 or
Me-27), 0.86 (3H, d, J ) 6.1 Hz, Me-26 or Me-27); 13C NMR,

1422 J ournal of Natural Products, 2002, Vol. 65, No. 10
Kuroda et al.
see Table 1; FABMS (positive-mode) m/z 1249 [M + Na]+; anal.
C 56.55%, H 7.80% (calcd for C60H90O26‚H2O, C 56.64%; H
7.53%).
H-2 of Ar), 7.06 (1H, d, J ) 8.5 Hz, H-5 of Ar), 5.71 (1H, dd, J
) 8.4, 7.7 Hz, H-2 of Xyl), 5.54 (1H, dd, J ) 7.2, 5.8 Hz, H-2
of Ara), 5.29 (1H, br s, H-6), 5.16 (1H, d, J ) 8,0 Hz, H-1 of
Glc′′), 5.14 (1Η, d, J ) 7.7 Hz, H-1 of Xyl), 5.08 (1H, d, J ) 7.8
Hz, H-1 of Glc′), 4.96 (1H, d, J ) 7.7 Hz, H-1 of Glc), 4.59 (1H,
d, J ) 5.8 Hz, H-1 of Ara), 3.94 (1H, m, overlapping, H-3),
3.82 and 3.80 (each 3H, s, OMe), 3.19 (1H, q, J ) 7.3 Hz, H-20),
2.00 (3H, s, Ac), 1.29 (3H, d, J ) 7.3 Hz, Me-21), 0.99 (3H, s,
Me-18), 0.94 (3H, s, Me-19), 0.87 (3H, d, J ) 6.0 Hz, Me-26 or
Me-27), 0.85 (3H, d, J ) 6.0 Hz, Me-26 or Me-27); 13C NMR,
25
Com p ou n d 4: amorphous solid; [R]D -58.0° (c 0.10,
MeOH); IR (film) νmax 3363 (OH), 2936, 2904, and 2871 (CH),
1731, 1716, and 1694 (CdO), 1589, 1504, and 1457 (aromatic
ring), 1416, 1370, 1337, 1228, 1172, 1126, 1067, 1043 cm-1
;
UV (MeOH) λmax 266 nm (log ꢀ 4.01); 1H NMR (pyridine-d5) δ
7.56 (2H, s, H-2, H-6 of Ar), 5.71 (1H, dd, J ) 9.1, 7.6 Hz, H-2
of Xyl), 5.55 (1H, dd, J ) 7.5, 5.9 Hz, H-2 of Ara), 5.28 (1H, br
d, J ) 4.7 Hz, H-6), 5.16 (1H, d, J ) 7.6 Hz, H-1 of Xyl), 5.14
(1H, d, J ) 7.6 Hz, H-1 of Glc′), 4.95 (1H, d, J ) 7.7 Hz, H-1
of Glc), 4.60 (1H, d, J ) 5.9 Hz, H-1 of Ara), 4.00 (3H, s, OMe),
3.94 (1H, m, overlapping, H-3), 3.81 (3H × 2, s, OMe), 3.21
(1H, q, J ) 7.4 Hz, H-20), 2.01 (3H, s, Ac), 1.31 (3H, d, J ) 7.4
Hz, Me-21), 0.98 (3H, s, Me-18), 0.94 (3H, s, Me-19), 0.88 (3H,
d, J ) 6.1 Hz, Me-26 or Me-27), 0.86 (3H, d, J ) 6.1 Hz, Me-
26 or Me-27); 13C NMR, see Table 1; FABMS (positive-mode)
m/z 1279 [M + Na]+; anal. C 55.84%, H 7.66% (calcd for
see Table 1; anal. C 51.79%, H 7.31% (calcd for C66H100O31
‚
15/2H2O, C 51.99%, H 7.60%).
25
Com p ou n d 7: amorphous solid; [R]D -44.0° (c 0.10,
MeOH); IR (film) νmax 3386 (OH), 2938 and 2905 (CH), 1727
and 1695 (CdO), 1590, 1502, and 1460 (aromatic ring), 1416,
1370, 1338, 1229, 1166, 1125, 1066, 1045 cm-1; UV (MeOH)
1
λmax 266 nm (log ꢀ 3.98); H NMR (pyridine-d5) δ 7.58 (2H, s,
H-2, H-6 of Ar), 5.71 (1H, dd, J ) 9.1, 7.4 Hz, H-2 of Xyl), 5.56
(1H, dd, J ) 7.4, 5.8 Hz, H-2 of Ara), 5.30 (1H, br d, J ) 4.5
Hz, H-6), 5.17 (1H, d, J ) 7.8 Hz, H-1 of Glc′′), 5.16 (1H, d, J
) 7.5 Hz, H-1 of Xyl), 5.08 (1H, d, J ) 7.8 Hz, H-1 of Glc′),
4.97 (1H, d, J ) 7.7 Hz, H-1 of Glc), 4.60 (1H, d, J ) 5.8 Hz,
H-1 of Ara), 3.97 (3H, s, OMe), 3.94 (1H, m, overlapping, H-3),
3.81 (3H × 2, s, OMe × 2), 3.20 (1H, q, J ) 7.4 Hz, H-20), 1.89
(3H, s, Ac), 1.30 (3H, d, J ) 7.4 Hz, Me-21), 0.99 (3H, s, Me-
18), 0.96 (3H, s, Me-19), 0.88 (3H, d, J ) 6.0 Hz, Me-26 or
Me-27), 0.86 (3H, d, J ) 6.0 Hz, Me-26 or Me-27); 13C NMR,
see Table 1; FABMS (positive-mode) m/z 1441 [M + Na]+; anal.
C 54.28%, H 7.60% (calcd for C67H102O32‚3H2O, C 54.61%, H
7.39%).
C
61H92O27‚3H2O, C 55.87%, H 7.53%).
Alk a lin e Hyd r olysis of 3 a n d 4. Compounds 3 and 4 were
treated separately (each 20.0 mg) with 0.4% KOH in EtOH
(each 12 mL) at room temperature for 90 min. After neutral-
ization by passage through an Amberlite IR-120B column, each
reaction mixture was chromatographed on silica gel using
CHCl3-MeOH-H2O (30:10:1). Compound 3 gave 2 (4.5 mg),
2a (2.5 mg), and 3,4-dimethoxybenzoic acid (0.8 mg). Com-
pound 4 gave 2 (3.8 mg), 2a (2.2 mg), and 3,4,5-trimethoxy-
benzoic acid (0.4 mg).
25
Com p ou n d 5: amorphous solid; [R]D -46.0° (c 0.10,
25
MeOH); IR (film) νmax 3381 (OH), 2935 and 2904 (CH), 1737
Com p ou n d 8: amorphous solid; [R]D -60.0° (c 0.10,
and 1690 (CdO), 1372, 1244, 1163, 1048 cm-1
;
1H NMR
MeOH); IR (film) νmax 3392 (OH), 2939 and 2904 (CH), 1738
and 1695 (CdO), 1606, 1515, and 1458 (aromatic ring), 1428,
1372, 1277, 1230, 1165, 1066, 1047 cm-1; UV (MeOH) λmax 292
(pyridine-d5) δ 5.81 (1H, dd, J ) 8.3, 6.5 Hz, H-2 of Ara), 5.31
(1H, br d, J ) 4.5 Hz, H-6), 5.17 (1H, d, J ) 7.9 Hz, H-1 of
Glc′′), 5.08 (1H, d, J ) 7.8 Hz, H-1 of Glc′), 4.96 (1H, d, J )
7.7 Hz, H-1 of Glc), 4.91 (1H, d, J ) 7.5 Hz, H-1 of Xyl), 4.65
(1H, d, J ) 6.5 Hz, H-1 of Ara), 3.94 (1H, m, W1/2 ) 22.9 Hz,
H-3), 3.86 (1H, dd, J ) 8.2, 7.5 Hz, H-2 of Xyl), 3.32 (1H, q, J
) 7.4 Hz, H-20), 2.34 (3H, s, Ac), 1.30 (3H, d, J ) 7.4 Hz, Me-
21), 0.96 (3H, s, Me-19), 0.95 (3H, d, J ) 6.1 Hz, Me-26 or
Me-27), 0.94 (3H, s, Me-18), 0.92 (3H, d, J ) 6.1 Hz, Me-26 or
Me-27); 13C NMR, see Table 1; FABMS (positive-mode) m/z
1
nm (log ꢀ 3.77), 263 nm (log ꢀ 4.09); H NMR (pyridine-d5) δ
8.04 (1H, dd, J ) 8.3, 1.9 Hz, H-6 of Ar), 7.99 (1H, d, J ) 1.9
Hz, H-2 of Ar), 7.28 (1H, d, J ) 8.3 Hz, H-5 of Ar), 5.71 (1H,
dd, J ) 8.9, 7.6 Hz, H-2 of Xyl), 5.55 (1H, dd, J ) 7.2, 5.8 Hz,
H-2 of Ara), 5.30 (1H, br d, J ) 4.4 Hz, H-6), 5.17 (1H, d, J )
7.9 Hz, H-1 of Glc′′), 5.15 (1H, d, J ) 7.6 Hz, H-1 of Xyl), 5.08
(1H, d, J ) 7.8 Hz, H-1 of Glc′), 4.96 (1H, d, J ) 7.7 Hz, H-1
of Glc), 4.59 (1H, d, J ) 5.8 Hz, H-1 of Ara), 3.94 (1H, m, W1/2
) 20.5 Hz, H-3), 3.79 (3H, s, OMe), 3.19 (1H, q, J ) 7.3 Hz,
H-20), 2.00 (3H, s, Ac), 1.29 (3H, d, J ) 7.3 Hz, Me-21), 0.99
(3H, s, Me-18), 0.97 (3H, s, Me-19), 0.90 (3H, d, J ) 6.1 Hz,
Me-26 or Me-27), 0. 87 (3H, d, J ) 6.1 Hz, Me-26 or Me-27);
13C NMR, see Table 1; FABMS (negative-mode) m/z 1373 [M
- H]-; anal. C 54.65%, H 7.54% (calcd for C65H98O31‚3/2H2O,
C 54.96%, H 7.55%.
1247 [M + Na]+; anal. C 53.90%, H 8.01% (calcd for C57H92O28
5/2H2O, C 53.51%, H 7.72%).
‚
Acid Hyd r olysis of 5. Compound 5 (8.0 mg) was subjected
to acid hydrolysis as described for 5 to give a sugar fraction
(2.3 mg). HPLC analysis of the sugar fraction under the same
conditions as in the case of that of 2 showed the presence of
L-arabinose, D-xylose, and D-glucose.
25
Alk a lin e Hyd r olysis of 5. Compound 5 (50.2 mg) was
treated with 3% NaOMe in MeOH at room temperature for 3
h. The reaction mixture was neutralized by passage through
an Amberlite IR-120B column and chromatographed on silica
gel eluting with CHCl3-MeOH-H2O (20:10:1) to yield 5a (39.7
mg).
Com p ou n d 9: amorphous solid; [R]D -68.0° (c 0.10,
MeOH); IR (film) νmax 3388 (OH), 2934 and 2872 (CH), 1716
and 1694 (CdO), 1600, 1515, 1459, 1417, 1371, 1270, 1227,
1040 cm-1; UV (MeOH) λmax 292 nm (log ꢀ 3.77), 262 nm (log
ꢀ 4.09); 1H NMR (pyridine-d5) δ 7.94 (1H, dd, J ) 8.5, 1.9 Hz,
H-6 of Ar), 7.78 (1H, d, J ) 1.9 Hz, H-2 of Ar), 6.93 (1H, d, J
) 8.5 Hz, H-5 of Ar), 5.95 (1H, dd, J ) 9.0, 8.8 Hz, H-3 of Xyl),
5.83 (1H, dd, J ) 8.4, 6.7 Hz, H-2 of Ara), 5.29 (1H, br d, J )
4.7 Hz, H-6), 5.17 (1H, d, J ) 7.8 Hz, H-1 of Glc′′), 5.14 (1H,
d, J ) 7.7 Hz, H-1 of Xyl), 5.08 (1H, d, J ) 7.8 Hz, H-1 of
Glc′), 4.96 (1H, d, J ) 7.7 Hz, H-1 of Glc), 4.65 (1H, d, J ) 6.7
Hz, H-1 of Ara), 3.94 (1H, m, overlapping, H-3), 3.75 and 3.68
(each 3H, s, OMe), 3.28 (1H, q, J ) 7.4 Hz, H-20), 2.29 (3H, s,
Ac), 1.29 (3H, d, J ) 7.4 Hz, Me-21), 0.96 (3H, s, Me-19), 0.95
(3H, d, J ) 6.2 Hz, Me-26 or Me-27), 0.93 (3H, s, Me-18), 0.92
(3H, d, J ) 6.2 Hz, Me-26 or Me-27); 13C NMR, see Table 2;
FABMS (positive-mode) m/z 1411 [M + Na]+; anal. C 52.18%,
H 7.56% (calcd for C66H100O31‚7H2O, C 52.30%, H 7.58%).
Alk a lin e Hyd r olysis of 9-12. Compounds 6 (25.0 mg), 7
(5.0 mg), 8 (5.1 mg), and 9 (5.3 mg) were treated with 0.4%
KOH in EtOH. Compound 6 gave 5 (5.1 mg), 5a (5.0 mg), and
3,4-dimethoxybenzoic acid (1.8 mg); 7 gave 5 (1.1 mg), 5a (1.4
mg), and 3,4,5-trimethoxybenzoic acid (0.2 mg); 8 gave 5 (1.0
mg), 5a (1.1 mg), and 4-hydroxy-3-methoxybenzoic acid (0.3
mg); 9 gave 5 (0.7 mg), 5a (1.0 mg), and 3,4-dimethoxybenzoic
acid (0.2 mg).
25
Com p ou n d 5a : amorphous powder; [R]D -66.0° (c 0.10,
MeOH); IR (film) νmax 3376 (OH), 2934, 2902, and 2871 (CH),
1685 (CdO), 1375, 1162, 1071, 1045 cm-1; 1H NMR (pyridine-
d5) δ 5.29 (1H, br d, J ) 4.2 Hz, H-6), 5.19 (1H, d, J ) 7.6 Hz,
H-1 of Xyl), 5.16 (1H, d, J ) 7.8 Hz, H-1 of Glc′′), 5.09 (1H, d,
J ) 7.8 Hz, H-1 of Glc′), 4.97 (1H, d, J ) 7.7 Hz, H-1 of Glc),
4.52 (1H, d, J ) 7.7 Hz, H-1 of Ara), 3.94 (1H, m, W1/2 ) 21.1
Hz, H-3), 3.38 (1H, q, J ) 7.4 Hz, H-20), 1.29 (3H, d, J ) 7.4
Hz, Me-21), 0.94 (3H, s, Me-19), 0.92 (3H, d, J ) 6.4 Hz, Me-
26 or Me-27), 0.90 (3H, s, Me-18), 0.86 (3H, d, J ) 6.1 Hz,
Me-26 or Me-27); 13C NMR, see Table 1; FABMS (negative-
mode) m/z 1181 [M - H]-; anal. C 53.18%, H 8.11% (calcd for
C
55H90O27‚3H2O, C 53.39%, H 7.82%).
25
Com p ou n d 6: amorphous solid; [R]D -60.0° (c 0.10,
MeOH); FABMS (negative-mode) m/z 1387 [M - H]-; IR (film)
νmax 3394 (OH), 2938 and 2907 (CH), 1736, 1714, and 1697
(CdO), 1600, 1515, and 1459 (aromatic ring), 1417, 1371, 1270,
1227, 1173, 1131, 1065, 1044 cm-1; UV (MeOH) λmax 293 nm
(log ꢀ 3.81), 262 nm (log ꢀ 4.09); H NMR (pyridine-d5) δ 8.06
(1H, dd, J ) 8.5, 1.6 Hz, H-6 of Ar), 7.93 (1H, d, J ) 1.6 Hz,
1

Cholestane Glycosides from Ornithogalum
J ournal of Natural Products, 2002, Vol. 65, No. 10 1423
Cell Cu ltu r e Assa y. HL-60 cells, which were obtained from
Human Science Research Resources Bank (J CRB 0085, Osaka,
J apan), were maintained in RPMI 1640 medium containing
heat-inactivated 10% FBS supplemented with L-glutamine,
100 units/mL penicillin, and 100 µg/mL streptomycin. The
leukemia cells were washed and resuspended in the above
medium to 4 × 104 cells/mL, and 196 µL of this cell suspension
was placed in each well of a 96-well flat-bottom plate (Iwaki
Glass, Chiba, J apan). The cells were incubated in 5% CO2/air
for 24 h at 37 °C. After incubation, 4 µL of EtOH-H2O (1:1)
solution containing the sample was added to give the final
concentrations of 0.00001-10 µg/mL; 4 µL of EtOH-H2O (1:
1) was added into control wells. The cells were further
incubated for 72 h in the presence of each agent, and then
cell growth was evaluated by an MTT assay procedure. At the
end of incubation, 10 µL of 5 mg/mL MTT in phosphate-
buffered saline was added to every well, and the plate was
further incubated in 5% CO2/air for 4 h at 37 °C. The plate
was then centrifuged at 1500g for 5 min to precipitate cells
and formazan. An aliquot of 150 µL of the supernatant was
removed from every well, and 175 µL of DMSO was added to
dissolve the MTT formazan crystals. The plate was mixed on
a microshaker for 10 min and then read on a microplate reader
(Spectra Classic, Tecan, Salzburg, Austria) at 550 nm. Each
assay was done in triplicate, and cytotoxicity was expressed
as IC50 value, which reduced the viable cell number by 50%.
Ack n ow led gm en t. We are grateful to Dr. Y. Shida and
Mr. H. Fukaya, Tokyo University of Pharmacy and Life
Science, for the measurements of the mass spectra and
elemental analysis.
Refer en ces a n d Notes
(1) Mimaki, Y.; Kuroda, M.; Kameyama, A.; Sashida, Y.; Hirano, T.; Oka,
K.; Maekawa, R.; Wada, T.; Sugita, K.; Beutler, J . A. Bioorg. Med.
Chem. Lett. 1997, 7, 633-636.
(2) Kuroda, M.; Mimaki, Y.; Yokosuka, A.; Sashida, Y.; Beutler, J . A. J .
Nat. Prod. 2001, 64, 88-91.
(3) Kuroda, M.; Mimaki, Y.; Sashida, Y.; Yamori, T.; Tsuruo, T. Tetra-
hedron Lett. 2000, 41, 251-255.
(4) Mimaki, Y.; Kuroda, M.; Sashida, Y.; Yamori, T.; Tsuruo, T. Helv.
Chim. Acta 2000, 83, 2698-2704.
(5) Kuroda, M.; Mimaki, Y.; Yokosuka, A.; Sashida, Y. Chem. Pharm.
Bull. 2001, 49, 1042-1046.
(6) Mimaki, Y.; Kuroda, M.; Yokosuka, A.; Sashida, Y. J . Nat. Prod. 2001,
64, 1069-1072.
(7) Yamori, T.; Matsunaga, A.; Sato, S.; Yamazaki, K.; Komi, A.; Ishizu,
K.; Mita, I.; Edatsugi, H.; Matsuba, Y.; Takezawa, K.; Nakanishi, O.;
Kohno, H.; Nakajima, Y.; Komatsu, H.; Andoh, T.; Tsuruo, T. Cancer
Res. 1999, 59, 4042-4049.
(8) Sargent, J . M.; Taylor, C. G. Br. J . Cancer 1989, 60, 206-210.
NP020114J