Original Papers
2
5
Fraction 7 (F-7, 1.64 g) eluted with ether/EtOAc 20:1 was puri-
fied by Sephadex LH-20 CC using MeOH as eluent to give 2 sub-
fractions (F-7-1 and F-7-2). Compound 17 (119.2 mg;
t = 21.8 min) was obtained by RP-HPLC (60% MeOH in H O;
Hypoxyside (4): colorless oil; [α]
D
+ 77 (c 0.2 MeOH); UV
(MeOH) λmax (log ε) 220 (2.7); IR (neat) vmax: 3365, 2951, 2869,
1715, 1669, 1556, 1456, 1362, 1146, 1031, 631, 601, 594,
585 cm ; HRESI‑MS (positive ion mode) m/z 407.2417 [M +
Na] , (calcd for C21H36 NaO , 407.2410). H and 13C NMR data,
−
1
R
2
+
1
2
mL/min) from F-7-1. Compound 14 (73.2 mg, t = 21.2 min)
R
6
was purified by RP-HPLC (57% MeOH in H O; 2 mL/min) from F-
see ▶ Table 3.
2
2
5
7
-2. Fraction 8 (F-8, 1.87 g) eluted with CH Cl /MeOH 100:0 was
Hypoxyolide A (5): white powder; [α]
(MeOH) λmax (log ε) 216 (3.5); CD (c 1.5 × 10− M, MeOH) λmax
D
+ 16 (c 0.1 MeOH); UV
2
2
6
subjected to ODS CC with a gradient elution of MeOH‑H O (20,
2
3
0, 50, 70, and 100%, v/v, 1 L each) to yield 8 subtractions (F-8-1
(Δε) 229 (− 11.7), 252 (0.45); IR (neat) vmax: 3364, 2924, 1752,
−
1
to F-8-8). Compound 16 (64.9 mg, t = 17.2 min) was purified
from the subfraction F-8-3 by RP-HPLC (45% MeCN in H O
1713, 1678, 1346, 1204, 1138, 988, 722, 606, 568, 456 cm ;
R
+
HRESI‑MS (positive ion mode) m/z 359.2060 [M + H] , (calcd for
2
1
13
[
0.01% TFA]; 2 mL/min). Ten subfractions (F-9-1 to F-9–10) were
C18H31O , 359.2070). H and C NMR data, see ▶ Table 4.
7
2
5
obtained from fraction 9 (F-9, 1.56 g) by ODS CC with a gradient
elution of MeOH–H O (20, 30, 50, 70, and 100%, v/v, 1 L each). F-
9
Hypoxyolide B (6): white powder; [α]
D
+ 3 (c 0.1 MeOH); UV
(MeOH) λmax (log ε) 279 (2.6); CD (c 1.5 × 10− M, MeOH) λmax
(Δε) 226 (1.57), 247 (− 0.38),292 (0.25); IR (neat) vmax: 3390,
2955, 2926, 2871, 1746, 1702, 1455, 1287, 1105, 1030, 983,
6
2
-7 (67 mg) was further purified with RP-HPLC (30% MeCN in H O
2
[
0.01% TFA]; 2 mL/min) to yield compound 15 (17.3 mg;
−
1
t = 16.7 min). Fraction 10 (F-10, 1.861 g) eluted with CH Cl /
834, 716 cm ; HRESI‑MS (positive ion mode) m/z 363.1788 [M +
Na] (calcd for C18H28Na O , 363.1784). H and 13C NMR data, see
R
2
2
+
1
MeOH 20:1 was separated by Sephadex LH-20 CC to give 9 sub-
fractions F-10–1 to F-10–9. Compound 19 (7.4 mg) was obtained
from F-10–4 (51.3 mg) by preparative TLC using CH Cl -MeOH
6
▶ Table 4.
2
2
ECD calculations
(
10:1, v/v). F-10–5 (43.4 mg) was purified by RP-HPLC (28%
MeCN in H O [0.01% TFA]; 2 mL/min) to give compounds 3
Systematic conformation analysis of 5a and 6a were conducted
with CONFLEX (version 7 Rev. A; CONFLEX Corporation) using the
MMFF94 molecular mechanics force field. Optimization with DFT
calculation at the B3LYP/6-31G(d) level in MeOH by the
Gaussian09 program (Revision C.01, Gaussian Inc.) [30] afforded
the MMFF minima. At the B3LYP/6-31G(d) level, the exciting
states were calculated using time-dependent density-functional
theory (TDDFT) methodology for 5a and 6a. The overall ECD spec-
tra were then produced on the basis of Boltzmann weighting of
each conformer as described in literature [31].
2
(
(
7.2 mg; tR = 16.1 min),
7 (21.3 mg; tR = 22.6 min), and 8
35.2 mg; t = 32.2 min). F-10–6 (61.4 mg) was separated by RP-
R
HPLC (38% MeOH in H O [0.01% TFA]; 2 mL/min) to afford com-
2
pounds 12 (2.1 mg; t = 14.2 min) and 13 (2.0 mg; t = 13.9 min).
R
R
Compound 18 (4.2 mg; t = 9.5 min) was obtained from the frac-
R
tion F-10–7 (33.4 mg) by RP-HPLC (38% MeOH in H O [0.01%
2
TFA]; 2 mL/min). Compounds 2 (2.8 mg; t = 25.2 min) and 1
R
(
(
122.3 mg; t = 27.5 min) were isolated from the fraction F-10–8
R
189.9 mg) by RP-HPLC (70% MeOH in H O [0.01% TFA]; 2 mL/
2
min), respectively. Fraction 11 (F-11, 804.7 mg) eluted with
CH Cl /MeOH 10:1 was separated into 9 subfractions F-11-1 to
Acid hydrolysis and determination of absolute
configurations of monosaccharides by HPLC
2
2
F-11–9 by ODS CC (MeOH–H O, 20, 30, 50, 70, and 100%, v/v,
2
1
L each). Compounds 9 (13.1 mg; t = 17.5 min) and 5 (6.7 mg;
The procedure reported in [32] was followed. Each glycoside
(1.0 mg) was hydrolyzed with 2 M HCl (2 mL) for 3 h at 90°C. After
R
tR = 21.7 min) were obtained from subfraction F-11–6 (99.3 mg)
by RP-HPLC (42% MeCN in H O [0.01% TFA]; 2 mL/min). F-11–7
extraction 3 times with CHCl (3 × 5 mL), the aqueous layer was
2
3
(
163.11 mg) was separated with MeOH on Sephadex LH-20 CC
evaporated to dryness under vacuum. The dried residue was dis-
solved in pyridine (0.5 mL, Sigma) containing L-cysteine methyl
ester (1.0 mg, Sigma) and heated at 60°C for 1 h. A solution of
O-tolylisothiocyanate (5.0 µL, Sigma) was added to the mixture
and heated at 60°C for another 1 h. The reaction mixture was
analyzed by HPLC (C18 column [Phenomenex Gemini, 5 µm,
followed by purification with RP-HPLC (32% MeCN in H O [0.01%
TFA]; 2 mL/min) to afford compounds 10 (78.3 mg; t = 18.1 min)
and 6 (6.5 mg; t = 24 min). F-11–8 (123.63 mg) was purified by
RP-HPLC (33% MeCN in H O [0.01% TFA]; 2 mL/min) to give com-
2
R
R
2
pounds 4 (9.8 mg; t = 21.5 min) and 11 (10.2 mg; t = 24.7 min).
R
R
2
5
5
,6-Epoxyphomol (2): colorless oil; [α]
MeOH) λmax (log ε) 218 (3.5); CD (c 1.2 × 10− M, MeOH) λmax (Δε)
D
+ 62 (c 0.2 MeOH); UV
4.6 × 250 mm]; mobile phase: MeCN–H O–1‰ formic acid
2
6
(
[25:75, v/v]; detection wavelength: 254 nm; flow rate: 0.8 mL/
min; column temperature: 35°C). The monosaccharides were
identified by comparison of their retention time with those of au-
2
1
4
23 (− 1.13), 246 (0.32); IR (neat) vmax: 3417, 2958, 2929, 2872,
682, 1651, 1556, 1538, 1455, 1270, 1235, 1151, 1039, 748,
−
1
+
57 cm ; HRESI‑MS (positive ion mode) m/z 429.2484 [M + H]
thentic samples treated in the same way (D-glucose: t = 19.5 min;
R
calcd for C22H37O , 429.2488)· H and 13C NMR data, see
1
L-glucose: t = 17.9 min; L-rhamnose: t = 33.6 min).
(
8
R
R
▶
Table 1.
2
5
Cytotoxicity assay
Fuscuyne (3): white powder; [α]
D
− 131 (c 0.2 MeOH); UV
(
2
8
MeOH) λmax (log ε) 209 (4.0); IR (neat) vmax: 3415, 2934, 2892,
The cytotoxicities of compounds 1–19 were investigated with the
Cell Counting Kit 8 (CCK8) cell viability assay [4,33]. K562,
SW480, HEPG2 cells were purchased from National Infrastructure
of Cell Line Resource. Cells were maintained in RPMI 1640 me-
871, 2223, 1669, 1439, 1425, 1127, 1098, 1052, 979, 908,
−
1
38, 804 cm ; HRESI‑MS (positive ion mode) m/z 315.1211 [M +
Na] , (calcd for C16H20 NaO , 315.1208). H and 13C NMR data,
+
1
5
see ▶ Table 2.
dium (Sigma) with 10% FBS (Gemini Bio-Products) and grown
4
with 5% CO in an incubator at 37°C. Cells (1 × 10 cells/well) were
2
Basnet BB et al. Cytotoxic Secondary Metabolites… Planta Med