Gracilioethers A-C, antimalarial metabolites from the marine sponge Agelas gracilis

Article abstract of DOI:10.1021/jo900380f

(Chemical Equation Presented) Three new antiprotozoan compounds, gracilioethers A-C (1-3), have been isolated from the marine sponge Agelas gracilis. Their structures were elucidated on the basis of spectroscopic and chemical methods. Gracilioethers A-C s

Full text of DOI:10.1021/jo900380f

Gracilioethers A-C, Antimalarial Metabolites from the Marine
Sponge Agelas gracilis
Reiko Ueoka,† Yoichi Nakao,*,¶,† Shizuka Kawatsu,† Junko Yaegashi,†
Yoshitsugu Matsumoto,† Shigeki Matsunaga,† Kazuo Furihata,† Rob W. M. van Soest,‡ and
Nobuhiro Fusetani*,§,†
Graduate School of Agricultural and Life Sciences, The UniVersity of Tokyo, Bunkyo-ku, Tokyo 113-8657,
Japan, and Zoological Museum, UniVersity of Amsterdam, 1090GT Amsterdam, The Netherlands
ayocha@waseda.jp; anobu@fish.hokudai.ac.jp
ReceiVed February 20, 2009
Three new antiprotozoan compounds, gracilioethers A-C (1-3), have been isolated from the marine
sponge Agelas gracilis. Their structures were elucidated on the basis of spectroscopic and chemical
methods. Gracilioethers A-C showed antimalarial activity against Plasmodium falciparum with IC50
values of 0.5-10 µg/mL, whereas gracilioether B (2) also showed antileishmanial activity.
Introduction
for drug leads from Japanese marine invertebrates, we found
that the deep-sea sponge Agelas gracilis collected in southern
Japan showed considerable antimalarial activity in the lipophilic
extract. Bioassay-guided fractionation of the extract afforded
three new compounds of the plakortin family named gracilio-
ethers A-C.
Protozoan infection is increasingly becoming a threat to
human beings; for example 300-500 million people are infected
by malaria worldwide each year and one to three million die.1
Numberous attempts including use of the malaria vaccine have
been made to control this disease, but the administration of
antimalarial drugs is still most effective at this moment. Because
of increasing resistance to existing antimalarial drugs, there is
an urgent need for the development of antimalarial drugs with
new structures and modes of action. Marine natural products
have been explored for this purpose, which resulted in the
discovery of several drug candidates, including manzamines2
and cyclic peroxides.3 In the course of our continuing search
Results and Discussion
* To whom correspondence should be addressed. Y.N.: phone/fax +81-3-
5286-2568. N.F.: phone/fax +81-138-40-8884.
The CHCl3 soluble materials of the MeOH extract of the
sponge were fractionated by the modified Kupchan procedure4
to yield hexane, CHCl3, and 60% MeOH layers, the last of which
was combined with the n-BuOH extract of the water-soluble
portion of the MeOH extract and sequentially separated by ODS
flash chromatography, gel-filtration, and silica gel open column
chromatography. The active fraction was finally purified by
† The University of Tokyo.
¶ Present address: School of Advanced Science and Engineering, Waseda
University, Tokyo 169-8555, Japan.
‡ University of Amsterdam.
§ Present address: Graduate School of Fisheries Sciences, Hokkaido Univer-
sity, Hakodate 041-8611, Japan.
(1) Sachs, J.; Malaney, P. Nature (London) 2002, 415, 680–685.
(2) (a) Sakai, R.; Higa, T. J. Am. Chem. Soc. 1986, 108, 6404–6405. (b)
Ang, K. K. H.; Holmes, M. J.; Higa, T.; Hamann, M. T.; Kara, U. A. K.
Antimicrob. Agents Chemother. 2000, 44, 1645–1649.
(3) Kawanishi, M.; Kotoku, N.; Itagaki, S.; Horii, T.; Kobayashi, M. Bioorg.
Med. Chem. 2004, 12, 5297–5307.
(4) Kupchan, S. M.; Britton, R. W.; Ziegler, M. F.; Sigel, C. W. J. Org.
Chem. 1973, 38, 178–179.
10.1021/jo900380f CCC: $40.75
Published on Web 04/29/2009
2009 American Chemical Society
J. Org. Chem. 2009, 74, 4203–4207 4203

Ueoka et al.
13C (ppm)
TABLE 1. 1H and 13C NMR Data for Gracilioethers A (1), B (2), and C (3) in CD3OD
1
2
3
no.
1H (ppm)
13C (ppm)
1H (ppm)
4.85 s
13C (ppm)
1H (ppm)
4.82 s
1
2
3
4
5
6
7
169.0
86.8
174.6
89.4
43.6
103.0
43.1
168.8
84.3
174.1
142.1
142.1
99.0
169.0
83.8
174.3
140.7
142.8
99.6
4.86 s
2.60 (d, 10.7)
6.40 s
6.44 s
1.38 (dd, 13.4, 12.4)
2.34 (dd, 13.4, 5.5)
1.82 m
2.13 (dd, 11.0, 10.7)
3.37 (d, 8.0)
3.92 (dq, 8.0, 6.5)
1.29 (d, 6.5)
1.74 (2H, q, 7.6)
1.94 m
2.08 m
2.08 m
6.62 (dd, 15.8, 8.9)
5.82 (d, 15.8)
43.6
1.78 m
1.94 (d, 10.3)
1.78 m
5.29 m
5.29 m
44.3
8
9
10
11
12
13
43.6
42.0
84.5
68.1
20.3
34.0
41.4
155.3
131.7
204.2
26.8
40.7
135.7
135.9
69.3
23.7
19.5
4.16 (quint, 5.5, 6.2)
1.19 (d, 6.2)
2.18 m
2.23 s
2.05 m
19.4
2.13 m
14
15
0.93 (t, 7.6)
8.0
32.7
1.10 (t, 7.6)
1.78 (quint 7.6)
1.83 (quint 7.6)
0.75 (t, 7.6)
1.32 m
1.50 m
0.82 (t, 7.6)
3.65 s
12.0
33.0
1.17 (t, 7.2)
1.78 m
1.83 (m, 7.2)
0.75 (t, 7.2)
1.21 m
1.39 m
0.78 (t, 7.6)
3.65 s
12.4
32.8
1.72 (dq, 13.7, 7.3)
1.91 (dq, 13.7, 7.3)
1.05 (t, 7.3)
1.12 (qdd, 7.6, 13.5, 8.8)
1.64 (qdd, 7.6, 13.5, 3.3)
0.93 (t, 7.6)
16
17
9.5
26.2
8.1
29.5
8.1
30.2
18
19
12.7
51.3
11.8
51.1
11.9
51.1
3.65 s
repetitive reversed phase HPLC to yield gracilioethers A (1)
and C (3). The CHCl3 layer was processed to afford gracilioether
B (2).
H-19/C-1 and H-2/C-3), and correlations for H-7/C-6 and
15 indicated that b was connected to c via C-6; and (3) cross
peaks observed for H-5/C-3 suggested that the R,ꢀ-unsatur-
ated ester was connected to c via C-4 (Figure 1). The
remaining two deshielded carbons [δ 89.4 (C-4) and δ 84.5
(C-10)] could be assigned to those of a 1,2-dioxetane ring.
To fulfill the unsaturation and the molecular formula, C-3
and C-6 should be connected via an ether linkage to form a
five-membered ring, which was the common structural feature
to that of cladocrocin A. With all this information, the planar
structure of 1 was determined as shown. The geometry of
the ∆2-double bonds was determined as Z by comparison of
the chemical shift of H-2 (δΗ 4.86) with literature data
(δ for Z 4.85, δ for E 5.23).9
Gracilioether A (1) had a molecular formula of C19H30O6,
which was established by HR-ESIMS analysis [m/z 377.1942,
(M + Na)+, ∆ + 0.2 mmu]. Analysis of the 1H NMR data in
conjunction with the HSQC5 spectrum revealed the presence
of three ethyls, one methoxyl, one methyl, five methines
(including two oxymethines), one methylene, and one pro-
tonated sp2 carbon (Table 1). The 13C NMR spectrum further
showed the presence of one carbonyl, one oxygenated
nonprotonated sp2 carbon, and two oxygenated quaternary
carbons (Table 1). Although gracilioether A (1) was suggested
1
to be related to cladocrocin A by comparing the H and 13C
NMR data,6 the gross structure of 1 was quite different on
the basis of a detailed analysis of 2D NMR data including
COSY, HOHAHA,7 HSQC, and HMBC8 spectra. Four partial
structures a-d were deduced from COSY and HOHAHA
analysis (Figure 1). Assignment of C-5 and C-8 was difficult
due to overlapped carbon signals at δ 43.6. However, mutual
HMBC correlations between CH-5 and CH-10 as well as
correlations from H-9 to C-10 and C-11 could connect two
partial structural units c and d. The connection of the partial
structures a to d was unambiguously established by HMBC
data: (1) correlations H-14/C-4 and H-16/C-6 connected two
ethyl units at C-4 and C-6; (2) cross peaks observed for H-5/
C-13 could connect a to c via C-4, the terminal methoxy
group was a part of R,ꢀ-unsaturated ester (correlations for
The relative stereochemistry of 1 was elucidated on the
basis of NOESY data (Figure 2). NOESY correlations (H-
5/H-9, -11, -14, -15, and -16, H-9/H-17 and -18, H-10/H-8,
and H-11/H-17 and -18) suggested that three ethyl groups
(H-5, H-9, and a 2-oxygenated ethyl group) were on the same
face of the ring. Since the relative stereochemistry of C-10
and C-11 could not be determined by NOESY analysis, NOE
analysis was carried out for the acetonide 1a that was
prepared according to Scheme 1. One of the acetonide
methyls at δ 1.31 showed NOE’s to H-10, while the other
acetonide methyl at δ 1.41 showed NOE’s to H-12, indicating
that 1a was a 1,2-syn-acetonide, revealing the relative
stereochemistry of 1 as shown in Figure 2.
FIGURE 1. Key correlations and partial structures of 1.
4204 J. Org. Chem. Vol. 74, No. 11, 2009
FIGURE 2. Selected NOE’s for 1 and 1a.

Antimalarial Metabolite Gracilioethers
SCHEME 1
The absolute stereochemistry of 1 was determined by
application of the modified Mosher’s method to the secondary
hydroxy group at C-11. Treatment of 1 with R-(-)- or S-(+)-
MTPACl yielded S-(-)- and R-(+)-MTPA esters 1b and 1c,
respectively. The ∆δ value distribution pattern clearly indicated
11R configuration (Figure 3).10
ester 3a indicated the 9E-geometry (Figure 5). The absolute
stereochemistry at C-11 was determined as S based on Mosher
analysis.1
Gracilioethers A-C showed promising antimalarial activi-
ties (IC50 0.5-10 µg/mL) against Plasmodium falciparum as
shown in Table 2, while gracilioether B inhibited growth of
Leishmania major (68% at 10 µg/mL). Gracilioethers B and
Gracilioether B (2) had a molecular formula of C19H28O4 as
established by HR-ESIMS [m/z 343.1887, (M + Na)+, ∆ +0.2
mmu]. Three partial structures a-c deduced from COSY and
HOHAHA data were connected by key HMBC correlations as
follows: (1) correlations for H-12 and 10/C-11 indicated that
the terminal methyl ketone was conjugated with the double bond
between C-9 and 10; (2) the terminal methoxy group at the other
side of the molecule was a part of the R,ꢀ,γ,δ-unsaturated ester
(correlations for H-19/C-1 and H-2 and H-5/C-3); (3) crosspeaks
observed for H-14/C-4 and H-16/C-6 connected two ethyl units
at C-4 and C-6, respectively; and (4) the H-8 and H-5/C-6
correlations connected C-6 to C-5, -7, and -15 (Figure 4). To
fulfill the molecular formula, C-3 was connected to C-6 via an
ether linkage. Thus, the planar structure of 2 was assigned as
FIGURE 3. ∆δS-R values (ppm) of MTPA esters 1b and 1c.
1
shown. The 2Z-geometry was deduced from the H chemical
shift values,9 whereas the 9E-geometry was indicated on the
basis of a large coupling constant of 15.8 Hz.
The molecular formula of gracilioether C (3) was determined
as C19H30O4 on the basis of HR-ESIMS [m/z 345.2057, (M +
Na)+, ∆ + 1.5 mmu], suggesting that 3 was a dihydro-derivative
of 2. Reduction of the C-11 conjugated ketone was straight-
forward from the NMR data (δH 4.16; δC 69.3) assigned as 11-
dihydrogracilioether B. The geometry of ∆2 was determined as
Z on the basis of 1H chemical shift values9 as well. Though the
coupling constant between H-9 and H-10 could not be deter-
mined due to the overlapped signals, the large coupling constant
(J ) 15.1 Hz) observed for the corresponding protons in MTPA
FIGURE 4. Key correlations and partial structures of 2 and 3.
(5) Kay, L. E.; Keifer, P.; Saarinen, T. J. Am. Chem. Soc. 1992, 114, 10663–
10665.
(6) D’Auria, M. V.; Paloma, L. G.; Minale, L.; Riccio, R.; Zampella, A. J.
Nat. Prod. 1993, 56, 418–423.
(7) Bax, A.; Summers, M. F. J. Am. Chem. Soc. 1986, 108, 2093–2094.
(8) Bax, A.; Azolos, A.; Dinya, Z.; Sudo, K. J. Am. Chem. Soc. 1986, 108,
8056–8063.
(9) Bellur, E.; Bo¨ttcher, D.; Bornscheuer, U.; Langer, P. Tetrahedron:
Asymmetry 2006, 17, 892–899.
(10) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem. Soc.
1991, 113, 4092–4096.
FIGURE 5. ∆δS-R values (ppm) of MTPA esters 3a and 3b.
J. Org. Chem. Vol. 74, No. 11, 2009 4205

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

Antimalarial Metabolite Gracilioethers
separated by reversed-phase HPLC (C18-stationary phase, 10 × 50
mm; with 70-100% MeOH) to afford S-(-)- and R-(+)-MTPA
esters 1b and 1c, respectively.
Preparation of MTPA Esters 3a and 3b. Gracilioether C (3;
100 µg each) was similarly processed to give S-(-)- and R-(+)-
MTPA esters 3a and 3b, respectively.
tion for the Promotion of Science and Technology, and a Grant-
in-aids from JSPS (Area B 19310138, Area B 16380142) and
MEXT (Priority Area 16073207).
Supporting Information Available: NMR spectra for com-
pounds 1, 2, 3, 1a-c. This material is available free of charge
via the Internet at http://pubs.acs.org.
Acknowledgment. This work was partly supported by a
Waseda University Grant for Special Research Projects (2008A-
059), the Nissui Research Foundation, the Tokyo Ohka Founda-
JO900380F
J. Org. Chem. Vol. 74, No. 11, 2009 4207