Ueoka et al.
plates which contained 100 µL of Lm/egfp suspension (1 × 106
cells/mL) was added 100 µL of test solution (sample dissolved in
MeOH) then the plates were incubated in a low temperature
incubator at 25 °C for 72 h. To determine the growth inhibitory
activity of 2 at 10 µg/mL, the fluorescent signals were measured
after 72 h of incubation.
TABLE 2. IC50 Values of Compounds 1, 2, and 3 (µg/mL)
1
2
3
Plasmodium falciparum
B16F10
P388
HeLa
EA-hy926
10
0.5
0.1
0.05
0.5
0.2
10
1.4
0.7
3
1.2
5.2
Extraction and Isolation. The sponge was collected by
dredging at a depth of 150 m on a seamount named Oshima-
Shinsone (28°52′40′′N, 129°33′19′′E) near Amami-oshima Is-
land, southern Japan and identified as Agelas gracilis (voucher
specimen ZMAPOR19857 was deposited at the Zoological
Museum, University of Amsterdam). The frozen sample (900
g) was extracted with MeOH (3 × 3 L) and concentrated in
vacuo. The extract was suspended in H2O (1 L) and extracted
with CHCl3 (2 × 1 L) and n-BuOH (2 × 1 L). The CHCl3 extract
was partitioned between 90% MeOH and n-hexane. The 90%
MeOH layer was adjusted to 60% MeOH by addition of H2O
and extracted with CHCl3. The CHCl3 layer was concentrated
and separated by ODS flash chromatography with aq MeOH to
give six fractions (A-F). Fraction B (70% MeOH) was gel-
filtered with MeOH to give 11 fractions. The first fraction was
separated by silica gel open column chromatography with CHCl3/
MeOH/CH3COOH (95:5:1) and the active fraction was further
purified by reversed-phase HPLC (C18-stationary phase, 10 ×
250 mm) with 70% MeOH to yield 0.6 mg of gracilioether B
(2). The n-BuOH and 60% MeOH layers were combined and
similarly separated by ODS flash chromatography. The 70%
MeOH and 70% MeCN eluates and fraction C (70% MeCN)
from the CHCl3 layer were combined and gel-filtered with
MeOH. The fast eluting fractions were collected and separated
by silica gel open column chromatography with CHCl3/MeOH/
CH3COOH (95:5:1), followed by reversed-phase HPLC (C18-
stationary phase, 20 × 250 mm) with 70% MeOH to afford
fractions A′ and B′. The fraction A′ was purified by reversed-
phase HPLC (C18-stationary phase, 10 × 250 mm) with 70%
MeOH to give 0.3 mg of gracilioether C (3). The fraction B′
was purified by reversed-phase HPLC (C18-stationary phase, 10
× 250 mm) with 50% MeCN giving 0.5 mg of gracilioether
A (1).
1.4
C exhibited moderate cytotoxicity, while A showed less
cytotoxicity.
Conclusion
Gracilioether A (1), a new member of the plakortin family,
was isolated from the deep-sea sponge Agelas gracilis along
with two analogues, gracilioethers B (2) and C (3). Gracilio-
ethers A-C (1-3) showed antimalarial activity; 2 was the most
active. Gracilioether B (2) was also antiprotozoan against
Leishmania major. Gracilioether A (1) seems to derive from a
common biosynthetic precursor of 1-3. From this hypothesis,
2 and 3 are likely to retain the same absolute stereochemistry
at C-6 and C-8.
Experimental Section
Assay for the Cytotoxicity against P388 Cells. P388 murine
leukemia cells were cultured in RPMI-1640 medium containing
10% fetal bovine serum, 100 µg/mL of kanamycin, and 10 µg/mL
of 2-hydroxyethyl disulfide at 37 °C under an atmosphere of 5%
CO2. To each well of the 96-well microplate containing 100 µL of
tumor cell suspension (1 × 104 cells/mL) was added 100 µL of
test solution dissolved in RPMI-1640 medium then the plate was
incubated in a CO2 incubator at 37 °C for 96 h. After addition of
50 µL of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide (MTT) saline solution (1 mg/mL) to each well, the plate
was incubated for 3 h under the same condition to stain live cells.
After the incubation, the plate was centrifuged and the supernatants
were removed and cells were dissolved in 150 µL of DMSO to
determine the IC50 values.
Assay for the Cytotoxicity against HeLa Cells. HeLa cells
were cultured in Dulbecco’s Modified Eagle’s Medium contain-
ing 10% fetal bovine serum, 2 µg/mL of gentamycin, 2 µg/mL
of antibiotic-antimicotic, and 0.3 M NaHCO3 (adjusted to pH
7.0-7.4 with 2 M HCl) at 37 °C under an atmosphere of 5%
CO2. To each well of the 96-well microplate containing 200 µL
of tumor cell suspension (1 × 104 cells/mL) was added test
solution after the 24 h preincubation and the plate was incubated
for 72 h. To determine the IC50 values, the plate was processed
as described for P388 cells.
Antimalarial Assay. Plasmodium falciparum ItG strain was
cultured in a suspension of 5% (v/v) type 0 (+) human red blood
cells. The culture medium consisted of RPMI 1640 supplemented
with 25 mM Hepes, 0.3 mM hypoxanthine, 16 mM NaHCO3, and
10% type 0 (+) human plasma. Compounds were dissolved in
MeOH before use. Each solution was diluted to the desired
concentration with culture medium. Control medium contained
MeOH in quantities equal to those used for experimental cultures.
To each well of the 96-well plates containing 38 µL of P. falciparum
ItG suspension (parasitemia: 5%) was added 2 µL of test solution,
then the plates were incubated at 37 °C for 48 h. To determine the
IC50 value, Giemsa-stained smears were made after 48 h of
incubation and parasite numbers were counted.
Gracilioether A (1): colorless solid; [R]34.6 +20 (c 0.03,
D
MeOH); UV (MeOH) 248.5 nm (ꢀ 9200); IR (film) 1697, 1540
cm-1; HRESIMS m/z377.1942 (M + Na)+, C19H30O6Na, (∆ +
0.2 mmu); 1H NMR (CD3OD) and 13C NMR (CD3OD), see
Table 1.
Gracilioether B (2): colorless solid; [R]34.6 -120 (c 0.03,
D
MeOH); UV (MeOH) 287.5 nm (ꢀ 2900); IR (film) 1624 cm-1
;
HRESIMS m/z 343.1887 (M + Na)+, C19H28O4Na, (∆ + 0.2 mmu);
1H NMR (CD3OD) and 13C NMR (CD3OD), see Table 1.
Gracilioether C (3): colorless solid; [R]31.7 -24 (c 0.02,
D
MeOH); UV (MeOH) 287.0 nm (ꢀ 7000); IR (film) 1697, 1539
cm-1; HRESIMS m/z345.2057 (M + Na)+, C19H30O4Na, (∆ +
1.5 mmu); 1H NMR (CD3OD) and 13C NMR (CD3OD), see
Table 1.
Preparation of Acetonide 1a. Gracilioether A (1; 0.2 mg) was
reacted with Zn powder (4.0 mg) in Et2O (100 µL) containing
AcOH (7 µL) and stirred overnight at rt. The mixture was filtered
and separated by reversed-phase HPLC (C18-stationary phase,
10 × 250 mm; with 50% MeCN) to give triol 1. Triol 1 was
dissolved in 160 µL of CH2Cl2 and 40 µL of 2,2-dimethoxypro-
pane containing catalytic amounts of PPTS, then stirred overnight
at rt, followed by reversed-phase HPLC separation (C18-station-
ary phase, 10 × 250 mm; with 70-100% MeOH) to yield
acetonide 1a.
Preparation of MTPA Esters 1b and 1c. Gracilioether A (1;
100 µg and 50 µg, respectively) was reacted with R-(-)- or S-(+)-
MTPACl (5 µL) in 100 µL of CH2Cl2 containing 1 mg of DMAP
for 5 min, respectively. The mixtures were partitioned between 0.1
M NaHCO3 and CHCl3. The CHCl3 layers were washed with 0.1
M HCl and H2O, then the organic layers were concentrated and
Antileishmanial Assay. Fluorescence signals of Lm/egfp pro-
mastigotes cultured in 199 medium supplemented with 10% fetal
bovine serum and 25 mM HEPES buffer in 96-well plates at 25
°C were measured by fluorescence microplate reader with excitation
at 485 nm and emission at 538 nm. To each well of the 96-well
4206 J. Org. Chem. Vol. 74, No. 11, 2009