90 J ournal of Natural Products, 2001, Vol. 64, No. 1
Notes
Ta ble 2. Cytotoxic Activities of 1, 1a , 2, 3, 3a , 3b, and
Controls against HL-60 Cells
mL) in pyridine (0.3 mL) at 40 °C for 12 h. The crude mixture
was passed through a Sep-Pak C18 cartridge with H2O-MeCN
(4:1; 1:1, each 10 mL) mixtures as solvents. The H2O-MeCN
(1:1) eluate was further passed through a Toyopak IC-SP M
cartridge (Tosoh, Tokyo, J apan) with EtOH (10 mL) to give a
mixture of the 1-[(S)-N-acetyl-R-methylbenzylamino]-1-deoxy-
alditol acetate derivatives of the monosaccharides,8 which was
then analyzed by HPLC under the following conditions:
solvent, MeCN-H2O (2:3); flow rate, 0.8 mL/min; detection,
UV 230 nm. The derivatives of L-arabinose, D-xylose, and
D-glucose were detected as follows: tR (min) 13.09 (derivative
of L-arabinose), 13.90 (derivative of D-xylose), 17.42 (derivative
of D-glucose).
compound
IC50 (µM)
1
1a
0.016
a
2
3
3a
0.014
0.00025
0.19
3b
8.3
etoposide
adriamycin
methotrexate
0.025
0.0072
0.012
a
IC50 > 10 µM.
Alk a lin e Hyd r olysis of 1. Compound 1 (6 mg) was treated
with 4% KOH in EtOH (2 mL) at room temperature for 30
min. The reaction mixture was neutralized by an Amberlite
IR-120B (Organo, Tokyo, J apan) column and passed through
a Sephadex LH-20 (Pharmacia, Uppsala, Sweden) column to
give a crude deacyl triglycoside and 3,4-dimethoxybenzoic acid
(0.8 mg). Purification of the glycoside was carried out by Si
gel column chromatography eluting with CHCl3-MeOH-H2O
(40:10:1) to afford 1a (4.7 mg) as a pure compound.
CCP PX-8010 controller, a Tosoh UV-8000, or a Tosoh RI-8010
detector, and a Rheodyne injection port with a 2-mL sample
loop for preparative HPLC and a 20-µL sample loop for
analytical HPLC. A Capcell Pak C18 column (10 mm i.d. × 250
mm, 5 µm, Shiseido, Tokyo, J apan) was used for preparative
HPLC, and a Capcell Pak C18 column (4.6 mm i.d. × 250 mm,
5 µm, Shiseido) was employed for analytical HPLC. The
following materials and reagents were used for bioassays:
Inter Med Immuno-Mini NJ -2300 microplate reader (Tokyo,
J apan); 96-well flat-bottom plate (Iwaki Glass, Chiba, J apan);
HL-60 cells (ICN Biomedicals, Costa Mesa, CA); RPMI 1640
medium (GIBCO BRL, Rockville, MD); MTT (Sigma, St. Louis,
MO).
Com p ou n d 1a : amorphous solid; [R]28 -18.0° (c 0.10,
D
MeOH); IR (film) νmax 3360 (OH), 2960, 2925, 2870, and 2850
1
(CH), 1740 (CdO), 1077, 1045 cm-1; H NMR (C5D5N) δ 5.39
(1H, br d, J ) 4.3 Hz, H-6), 5.12 (1H, d, J ) 7.7 Hz, H-1′′),
5.08 (1H, d, J ) 7.9 Hz, H-1′′′), 4.46 (1H, d, J ) 5.6 Hz, H-1′),
4.29 (1H, m, H-4′′), 4.23 (1H, m, H-16), 4.03 (1H, m, H-3′), 3.84
(1H, m, W1/2 ) 21.0 Hz, H-3), 3.41 (1H, q, J ) 7.4 Hz, H-20),
1.33 (3H, d, J ) 7.4 Hz, Me-21), 1.08 (3H, s, Me-19), 0.94 (3H,
s, Me-18), 0.93 (3H, d, J ) 6.5 Hz, Me-26), 0.88 (3H, d, J ) 6.4
Hz, Me-27); 13C NMR (C5D5N), see Table 1; FABMS (negative
mode) m/z 857 [M - H]-; FABMS (positive mode) m/z 881
[M + Na]+.
P la n t Ma ter ia l. The bulbs of O. saundersiae were pur-
chased from a nursery in Heiwaen, Nara, J apan, in October
1996. The bulbs were cultivated, and the flowered plant was
identified by Y. Sashida. A voucher of the plant is on file in
our laboratory (96/96-OS-2).
Extr a ction a n d Isola tion . The crude saponin fraction
prepared from O. saundersiae bulbs (16.2 kg) was subjected
to Si gel column chromatography, eluting with stepwise
gradients of CHCl3-MeOH (9:1; 4:1; 2:1) and finally with
MeOH alone, to give six fractions (I-VI).7 Fraction III was
further separated by an ODS Si gel column eluting with
MeOH-H2O (4:1) into four fractions (IIIa-IIId). Fraction IIId
was chromatographed on Si gel eluting with CHCl3-MeOH
(9:1) and ODS Si gel with MeCN-H2O (3:2) to give 1 and 2 in
impure form. Final purification was carried out by preparative
HPLC using MeCN-H2O (7:3) to yield 1 (11.2 mg) and 2 (18.5
mg) in pure form.
Com p ou n d 2: amorphous solid; [R]25 -54.4° (c 0.10,
D
MeOH); UV (MeOH) λmax 260 nm (log ꢀ 4.02); IR (KBr) νmax
3410 (OH), 2930 and 2880 (CH), 1720 (CdO), 1705 (CdO),
1690 (CdO), 1585 and 1500 (aromatic ring), 1055, 1030 cm-1
;
1H NMR (C5D5N) δ 7.69 (2H, s, H-2′′′′ and H-6′′′′), 5.68 (1H,
dd, J ) 8.9, 7.1 Hz, H-2′′), 5.56 (1H, dd, J ) 8.1, 6.1 Hz, H-2′),
5.39 (1H, br d, J ) 4.0 Hz, H-6), 5.15 (1H, d, J ) 7.1 Hz, H-1′′),
5.04 (1H, d, J ) 7.8 Hz, H-1′′′), 4.58 (1H, d, J ) 6.1 Hz, H-1′),
4.30 (1H, m, H-4′′), 4.17 (1H, m, H-16), 4.16 (1H, m, H-3′), 3.96
(3H, s, OMe), 3.82 (3H × 2, s, OMe × 2), 3.80 (1H, m, H-3),
3.23 (1H, q, J ) 7.4 Hz, H-20), 1.99 (3H, s, Ac), 1.33 (3H, d,
J ) 7.4 Hz, Me-21), 1.07 (3H, s, Me-19), 1.01 (3H, s, Me-18),
0.89 (3H, d, J ) 6.1 Hz, Me-26), 0.87 (3H, d, J ) 6.1 Hz,
Me-27); 13C NMR (C5D5N), see Table 1; FABMS (negative
mode) m/z 1093 [M - H]-; HRFABMS (positive mode) m/z
1117.5170 [M + Na]+ (calcd for C55H82O22Na, 1117.5196).
Com p ou n d 1: amorphous solid; [R]25 -56.0° (c 0.10,
D
MeOH); UV (MeOH) λmax 257 nm (log ꢀ 4.10); IR (KBr) νmax
3420 (OH), 2925 (CH), 1720 (CdO), 1705 (CdO), 1690 (CdO),
1
1595 and 1515 (aromatic ring), 1040 cm-1; H NMR (C5D5N)
δ 8.05 (1H, dd, J ) 8.5, 1.7 Hz, H-6′′′′), 7.91 (1H, d, J ) 1.7
Hz, H-2′′′′), 7.04 (1H, d, J ) 8.5 Hz, H-5′′′′), 5.68 (1H, dd, J )
8.9, 7.1 Hz, H-2′′), 5.56 (1H, dd, J ) 8.0, 6.0 Hz, H-2′), 5.39
(1H, br d, J ) 4.2 Hz, H-6), 5.13 (1H, d, J ) 7.1 Hz, H-1′′),
5.04 (1H, d, J ) 7.8 Hz, H-1′′′), 4.58 (1H, d, J ) 6.0 Hz, H-1′),
4.29 (1H, m, H-4′′), 4.18 (1H, m, H-16), 4.16 (1H, m, H-3′), 3.81
(3H, s, OMe), 3.80 (1H, m, H-3), 3.79 (3H, s, OMe), 3.23 (1H,
q, J ) 7.4 Hz, H-20), 1.99 (3H, s, Ac), 1.32 (3H, d, J ) 7.4 Hz,
Me-21), 1.08 (3H, s, Me-19), 1.01 (3H, s, Me-18), 0.89 (3H, d,
J ) 6.1 Hz, Me-26), 0.86 (3H, d, J ) 6.1 Hz, Me-27); 13C NMR
(C5D5N), see Table 1; FABMS (negative mode) m/z 1063 [M -
H]-; HRFABMS (positive mode) m/z 1087.5108 [M + Na]+
(calcd for C54H80O21Na, 1087.5090).
Acid Hyd r olysis of 1. A solution of 1 (3 mg) in 1 M HCl
(dioxane-H2O, 1:1, 2 mL) was heated at 95 °C for 2 h under
an Ar atmosphere. After cooling, the reaction mixture was
neutralized using an Amberlite IRA-93ZU (Organo, Tokyo,
J apan) column and passed through a Sep-Pak C18 cartridge
(Waters, Milford, MA), eluting with H2O-MeCN (4:1, 10 mL)
followed by MeOH (10 mL), to give a sugar fraction (1 mg).
The sugar fraction was dissolved in H2O (1 mL), to which (-)-
R-methylbenzylamine (5 mg) and Na[BH3CN] (8 mg) in EtOH
(1 mL) were added. After being set aside at 40 °C for 4 h
followed by addition of HOAc (0.2 mL) and evaporation to
dryness, the reaction mixture was acetylated with Ac2O (0.3
Alk a lin e Hyd r olysis of 2. Compound 2 (6.5 mg) was
subjected to alkaline hydrolysis as described for 1 to give 1a
(5.0 mg) and 3,4,5-trimethoxybenzoic acid (1.1 mg).
Red u ction of 3a . A mixture of 3a 1 (60 mg) and NaBH4 (90
mg) in MeOH (12 mL) was stirred for 30 min at room
temperature. The reaction mixture was chromatographed on
Si gel eluting with CHCl3-MeOH-H2O (60:10:1) and ODS Si
gel with MeCN-H2O (1:1) to yield 3b (12.1 mg).
Com p ou n d 3b: amorphous solid; [R]25 -6.7° (c 0.10,
D
MeOH); IR (film) νmax 3420 (OH), 2948 (CH), 1076 cm-1; H
1
NMR (C5D5N) δ 5.39 (1H, br d, J ) 4.1 Hz, H-6), 5.15 (1H, d,
J ) 7.6 Hz, H-1′′), 4.83 (1H, d, J ) 7.5 Hz, H-1′), 4.49 (1H, dd,
J ) 7.9, 4.5 Hz, H-16), 4.18 (1H, br d, J ) 9.1 Hz, H-22), 4.14
(1H, dd, J ) 9.2, 3.6 Hz, H-3′), 3.80 (1H, m, H-3), 1.32 (3H, d,
J ) 7.2 Hz, Me-21), 1.10 (3H, s, Me-19), 1.06 (3H, s, Me-18),
0.89 (3H, d, J ) 6.6 Hz, Me-26), 0.87 (3H, d, J ) 6.5 Hz, Me-
27); 13C NMR (C5D5N), see Table 1; FABMS (negative mode)
m/z 697 [M - H]-; FABMS (positive mode) m/z 721 [M + Na]+.
HL-60 Cell Cu ltu r e Assa y. HL-60 leukemia cells were
maintained in RPMI 1640 medium containing 10% fetal bovine
serum supplemented with L-glutamine, 100 units/mL penicil-
lin, and 100 µg/mL streptomycin. The cells (3 × 104 cells/mL)
were continuously treated with each compound for 72 h, and