Halichonadin F and the Cu(I) complex of halichonadin C from the sponge Halichondria sp.

Article abstract of DOI:10.1021/np800164s

A new sesquiterpenoid with an aromadendrane skeleton, halichonadin F (1), and the Cu(I) complex of haliconadin C (2) were isolated from a marine sponge Halichondria sp., and the structures and relative stereochemistries of 1 and the complex of 2 were elucidated on the basis of spectroscopic data and chemical methods.

Full text of DOI:10.1021/np800164s

J. Nat. Prod. 2008, 71, 1301–1303
1301
Halichonadin F and the Cu(I) Complex of Halichonadin C from the Sponge Halichondria sp.
Haruaki Ishiyama,† Shingo Kozawa,† Kazuki Aoyama,‡ Yuzuru Mikami,‡ Jane Fromont,§ and Jun’ichi Kobayashi*,†
Graduate School of Pharmaceutical Sciences, Hokkaido UniVersity, Sapporo 060-0812, Japan, Medical Mycology Research Center, Chiba
UniVersity, Chiba 260-0856, Japan, and Western Australian Museum, Locked Bay 49, Welshpool DC, WA 6986, Australia
ReceiVed March 13, 2008
A new sesquiterpenoid with an aromadendrane skeleton, halichonadin F (1), and the Cu(I) complex of haliconadin C
(2) were isolated from a marine sponge Halichondria sp., and the structures and relative stereochemistries of 1 and the
complex of 2 were elucidated on the basis of spectroscopic data and chemical methods.
A number of sesquiterpene isothiocyanates, isonitriles, and
formamides have been isolated from marine sponges of the genus
Halichondria,1 and these compounds are thought to have a role in
maintaining ecological systems, for example, an allomone in the
browser-prey relationship.2,3 During a search for structurally
unique metabolites from Okinawan marine sponges,4,5 a new
sesquiterpenoid with the aromadendrane skeleton, halichonadin F
(1), and the Cu(I) complex of halichonadin C (2) were isolated
from a sponge Halichondria sp. In this paper, we describe the
isolation and structure elucidation of 1 and 2.
Table 1. 1H and 13C NMR Data of Halichonadin F (1) in
CDCl3
a
a
position
δH
δC
H coupled with Cb
1
2.20 (m)
1.88 (m)
1.88 (m)
1.53 (m)
1.73 (m)
1.28 (m)
2.01 (m)
1.37 (m)
0.58 (t, 10.2)
0.65 (m)
1.84 (m)
2.09 (m)
1.82 (m)
54.2
27.1
6
2a
2b
3a
3b
4
5
6
7
8a
8b
9a
9b
10
11
12
13
14
15
34.6
36.1
39.2
28.5
26.4
19.4
10
7
40.2
60.1
20.3
28.5
15.8
16.0
16.9
1.00 (s)
0.98 (s)
0.92 (d, 5.4)
1.30 (s)
6, 13
11
3
9, 10
The sponge Halichondria sp. (SS-1163) collected off Unten Port,
Okinawa, was extracted with MeOH, and the MeOH extract was
partitioned between EtOAc and water. The EtOAc-soluble materials
were subjected to passage over a silica gel column (n-hexane/
EtOAc, 1:1 f MeOH). The fraction eluted with MeOH was
separated on a silica gel column (CHCl3/MeOH, 95:5 f 7:3), and
then the fraction eluted with CHCl3/MeOH (7:3) was separated on
a silica gel column (EtOAc/MeOH, 5:1 f MeOH) to yield
halichonadine F (1, 0.7 mg, 0.0001% wet wt) and the Cu(I) complex
of halichonadin C (2, 2.6 mg, 0.0004%) together with six known
related terpenoids, halichonadins A and C,5 acanthenes B, C,6 and
E,7 and epipolasin B.8a
a δ in ppm. b HMBC correlations.
The molecular formula, C15H27N, of 1 was established by
HREIMS [m/z 221.2144 (M+), ∆ 0.1 mmu]. IR (3285 cm-1) data
suggested the presence of an amino and/or hydroxyl functionality.
The gross structure of 1 was deduced from detailed analysis of the
1H and 13C NMR data (Table 1) aided with 2D NMR experiments
(1H-1H COSY, HOHAHA, HMQC, and HMBC). The 13C NMR
data indicated that 1 possessed two sp3 quaternary carbons, five
sp3 methines, four sp3 methylenes, and four methyl groups. Among
them, one sp3 quaternary carbon (δC 60.1) was ascribed to that
bearing a nitrogen atom. The 1H-1H COSY and HOHAHA spectra
revealed connectivities of C-1 to C-5 and C-9 and C-4 to C-14
(Figure 1). HMBC correlations of H-5 to C-10, H3-15 to C-9 and
C-10, H3-12 to C-6 and C-13, and H3-13 to C-1 suggested the
presence of an aromadendrane skeleton (Figure 1). Thus, the gross
Figure 1. Selected 2D NMR correlations for halichonadin F (1).
structure of halichonadin F was elucidated to be 1 with an
aromadendrane skeleton.
ROESY correlations of H-1 to H-6, H-2 to H-4 and H3-15, and
H-4 to H-5 in the aromadendrane skeleton indicated R-orientations
of H-1, H-6, H-7, Me-14, and Me-15 and ꢀ-orientations of H-4
and H-5 (Figure 2).
To confirm the relative stereochemistry of halichonadin F (1), a
diastereomer (3) of 1 was prepared from commercially available
(+)-aromadendrene (4).9 Treatment of 4 with thiocyanic acid
(HSCN) in CHCl3 provided the enantiomer (5) of epipolasin B,8b
which was hydrolyzed by NaOH in glycol to give compound 3
(Scheme 1). The 1H NMR resonances of 3 were not identical with
those of 1. Acetylation of 1 and 3 provided the acetoamides 6 and
* To whom correspondence should be addressed. Tel: +81-11-706-3239.
Fax: +81-11-706-4989. E-mail: jkobay@pharm.hokudai.ac.jp.
† Hokkaido University.
‡ Chiba University.
§ Western Australian Museum.
7, respectively (Scheme 2). The H NMR resonance for Me-15 in
1
10.1021/np800164s CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 06/14/2008

1302 Journal of Natural Products, 2008, Vol. 71, No. 7
Notes
Scheme 3. Generation of Halichonadin C from the Cu(I)
Complex of Halichonadin C (2)
Scheme 4. Formation of the Cu(I) Complex of Halichonadin C
(2) from Halichonadin C
Figure 2. Selected ROESY correlations and relative stereochemistry
for halichonadin F (1).
Scheme 1. Synthesis of the C-10 Epimer (3) of Halichonadin F
(1) from (+)-Aromadendrene (4)a
methods. Halichonadin F (1) is a new aromadendrane sesquit-
erpenoid having an amino group, although aromadendrane
sesquiterpenoids with a thioisocyanate group,8a an isonitrile
group,12 and an N-formylamide13 group, and a dimeric sesquit-
erpenoid with eudesmane and aromadendrane skeletons linked
through a urea fragment7 have been isolated from marine sponges
Epipolasis kushimotoensis, Axinella cannabina, or Halichondria
sp. Halichonadin F (1) and the Cu(I) complex of halichonadin
C (2) showed antimicrobial activity against Micrococcus luteus
(MIC, 4 µg/mL, both), Trichophyton mentagrophytes (MIC, 16
and 8 µg/mL, respectively), and Cryptococcus neoformans (MIC,
16 µg/mL, both).
a Reagents and conditions: (a) HSCN, CHCl3, rt, 2 days (74%); (b) NaOH,
glycol, 165 °C, 3 h (6%).
Scheme 2. Acetylation of Halichonadin F (1) and Compound 3
Experimental Section
General Experimental Procedures. Optical rotations were recorded
on a JASCO DIP-1000 polarimeter. IR spectra were taken on a JASCO
FT/IR-5300 IR spectrometer. 1H and 13C NMR spectra were recorded
on a Bruker AMX-600 NMR spectrometer and a JEOL ECA500 NMR
spectorometer. The 7.26 and 77.0 ppm resonances of residual CDCl3
were used as internal references for 1H and 13C NMR spectra,
respectively. EI and HREI mass spectra were obtained on a DX-303
mass spectrometer. ESI and HRESI mass spectra were obtained on a
JEOL JMS-SX102A spectrometer.
Animal Material. A voucher specimen (SS-1163) was deposited at
the Graduate School of Pharmaceutical Sciences, Hokkaido University.
Extraction and Isolation. The sponge (0.55 kg, wet wt) was
extracted with MeOH (1 L × 3). The methanolic extract (24.5 g) was
partitioned between H2O (300 mL) and EtOAc (300 mL × 3). The
EtOAc-soluble materials (2.9 g) were subjected to passage over a silica
gel column (n-hexane/EtOAc, 1:1 f MeOH) to yield the Cu(I) complex
of halichonadin C (2, 2.6 mg, 0.0004%, wet wt). The fraction eluted
with MeOH was separated on a silica gel column (CHCl3/MeOH, 95:5
f 7:3), and then the fraction eluted with CHCl3/MeOH (7:3) was
separated on a silica gel column (EtOAc/MeOH, 5:1 f MeOH) to yield
halichonadin F (1, 0.7 mg, 0.0001%).
6 was δ 1.20, while that in 7 was δ 1.41, indicating that the
stereochemistry of C-10 in 1 was opposite of that in 3.
The Cu(I) complex of halichonadin C (2) showed a molecular
ion peak at m/z 756 (M+) in the ESIMS. HRESIMS analysis of 2
revealed the molecular formula to be C48H75CuN3 [m/z 756. 5254
(M+), ∆ -0.3 mmu]. IR (2182 cm-1) and 13C NMR (δC 155.3 and
54.9) data suggested the presence of an isonitrile functionality. The
1H and 13C NMR data of 2 were similar to those of halichonadin
C.5 The molecular formula C48H75CuN3 and the 1H and 13C NMR
data of 2 suggested that the structure of 2 was the Cu(I)-coordinated
trimer of halichonadin C. To confirm the complex structure of 2,
compound 2 was treated with KCN to give a Cu(I)-free compound
Halichonadin F (1): colorless, amorphous solid; [R]24D +19 (c 0.10,
MeOH); IR (NaCl) νmax 3285 and 2925 cm-1; 1H and 13C NMR (Table
1); EIMS m/z 221 [M]+; HREIMS m/z 221.2144 (calcd for C15H27N,
221.2143).
1
(Scheme 3), whose H NMR data were identical with those of
halichonadin C. Furthermore, natural halichonadin C was treated
with CuCl in CH2Cl2 to yield a Cu(I)-coordinated complex (Scheme
4),10 whose 1H NMR data were identical with those of the isolated
Cu(I)-coordinated trimer of halichonadin C (2). Therefore, the
structure of compound 2 was concluded to be a complex of Cu(I)
coordinated to three molecules of halichonadin C, possessing a
eudesmane skeleton with an isonitrile group at C-6.
In conclusion, the structures and relative stereochemistries of
halichonadin F (1) and the Cu(I) complex of halichonadin C (2)
were elucidated on the basis of spectroscopic data and chemical
Cu(I) complex of halichonadin C (2): colorless solid; mp 170-172
°C; [R]25 -6 (c 1.0, CHCl3); IR (NaCl) νmax 3270, and 1740 cm-1
;
D
1H and 13C NMR (Tables 1 and 2); ESIMS m/z 756 [H]+; HRESIMS
m/z 756.5254 (calcd for C48H75CuN3, 756.5257).
Diastereomer (3) of Halichonadin F (1). To (+)-aromadendrene
4 (Wako Pure Chemical Co., Ltd., 182.4 mg, 0.893 mmol) was added
a HSCN chloroform solution8b (2 mL), and the reaction mixture was
stirred at room temperature for 2 days. The reaction mixture was filtered
and concentrated in vacuo. Purification of the residue by column
chromatography on silica gel (pentane) gave 5 (174.9 mg) as a colorless
oil. Isothiocyanate 5 (59.2 mg, 0.225 mmol) was treated with NaOH

Notes
Journal of Natural Products, 2008, Vol. 71, No. 7 1303
dd, J ) 14.1, 6.9 Hz, H-9); EIMS m/z 263 [M]+; HREIMS m/z 263.2266
(calcd for C17H29ON, 263.2249).
Table 2. 1H and 13C NMR Data of the Cu(I) Complex of
Halichonadin C (2) in CDCl3
Generation of Halichonadin C from the Cu(I) Complex of
Halichonadin C (2). The Cu(I) complex of halichonadin C (2) (0.1
mg, 1.3 µmol) was treated with KCN (1.0 mg, 154 µmol) in MeOH
(50 µL) at room temperature for 3 h, and the reaction mixture was
filtered and concentrated in vacuo. Purification of the residue by column
chromatography on silica gel (n-hexane/EtOAc, 9:1) gave halichonadin
C (0.05 mg, 50%): [R]24D -72 (c 0.025, CHCl3). The 1H NMR spectrum
of the generated halichonadin C was identical with that of natural
halichonadin C; ESIMS m/z 231 [M]+; HRESIMS m/z 231.1987 (calcd
for C16H25N, 231.1987).
a
a
position
δH
δC
H coupled with Cb
1a
1b
2a
2b
3a
3b
4
5
6
7
8
9a
9b
10
11
12
13
14a
14b
15
16
1.45 (m)
1.28 (m)
1.64 (m)
1.59 (m)
2.34 (m)
1.96 (m)
41.5
23.5
37.5
144.9
55
54.9
48.2
18.1
39.3
2.07 (m)
3.50 (t, 10.8)
1.56 (m)
Formation of the Cu(I) Complex of Halichonadin C (2) from
Halichonadin C. To a CH2Cl2 (1 mL) solution of natural halichonadin
C (36.8 mg, 0.159 mmol) was added CuCl (5.0 mg, 0.051 mmol), and
the reaction mixture was stirred at room temperature for 5 h. The
reaction mixture was filtered and concentrated in vacuo. Purification
of the residue by column chromatography on silica gel (EtOAc/MeOH,
1.54 (m)
1.54 (m)
1.22 (m)
37.2
27.4
20.9
15.7
107.8
2.08 (m)
0.98 (d, 7.2)
0.86 (d, 7.2)
4.95 (s)
5:1) gave compound 2 (23.4 mg, 0.031 mmol, 20%): [R]23 +60 (c
D
7, 11, 13
7, 11, 12
3, 5
1
0.64, MeOH). The H NMR spectrum was identical with that of the
natural Cu(I) complex of halichonadin C (2); ESIMS m/z 756 [M]+;
HRESIMS m/z 756.5248 (calcd for C48H75CuN3, 756.5257).
4.53 (s)
0.68 (s)
5
16.6
152.7
1, 5, 9, 10
Acknowledgment. We thank S. Oka and A. Tokumitsu, Center for
Instrumental Analysis, Hokkaido University, for EIMS and ESIMS
measurements, and Z. Nagahama and K. Uehara for their help with
the sponge collection. This work was partly supported by a Grant-in-
Aid from the Uehara Memorial Foundation and Grant-in-Aid for
Scientific Research from the Ministry of Education, Culture, Sports,
Science and Technology of Japan.
a δ in ppm. b HMBC correlations.
(16 mg) in glycol (0.6 mL) at 165 °C for 3 h. The reaction mixture
was diluted with saturated aqueous K2CO3 (1 mL), extracted with CHCl3
(3 × 0.5 mL), dried with K2CO3, and concentrated in vacuo. Purification
with silica gel column chromatography (CHCl3/MeOH, 4:1 f 0:1)
provided 3 (3.1 mg, 0.014 mmol) in 6% yield as a colorless, amorphous
References and Notes
solid: [R]20D -16 (c 0.10, MeOH); IR (NaCl) νmax 3340 and 2925 cm-1
;
(1) Wright, A. D.; Ko¨ning, G. M. J. Nat. Prod. 1996, 59, 710–716.
(2) (a) Faulkner, D. J. Nat. Prod. Rep. 1984, 1, 551–598. (b) Faulkner,
D. J. Nat. Prod. Rep. 1987, 4, 539–590. (c) Edenborough, M. S.;
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1975, 97, 4763–4764.
(4) (a) Tsuda, M.; Endo, T.; Perpelescu, M.; Yoshida, S.; Watanabe, K.;
Fromont, J.; Mikami, Y.; Kobayashi, J. Tetrahedron 2003, 59, 1137–
1141. (b) Endo, T.; Tsuda, M.; Okada, T.; Mitsuhashi, S.; Shima, H.;
Kikuchi, K.; Mikami, Y.; Fromont, J.; Kobayashi, J. J. Nat. Prod.
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(5) Ishiyama, H.; Hashimoto, A.; Fromont, J.; Hoshino, Y.; Mikami, Y.;
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(6) Burgoyne, D. L.; Dumdei, E. J.; Andersen, R. J. Tetrahedron 1993,
49, 4503–4510.
(7) Kozawa, S.; Ishiyama, H.; Fromont, J.; Kobayashi, J. J. Nat. Prod.
2008, 71, 445–447.
(8) (a) Tada, H.; Yasuda, F. Chem. Pharm. Bull. 1985, 33, 1941–1945.
(b) da Silva, C. C.; Almagro, V.; Zukerman-Schpector, J.; Castellano,
E. E.; Marsaioli, A. J. J. Org. Chem. 1994, 59, 2880–2881.
(9) Gijsen, H. J. M.; Kanai, K.; Stork, G. A.; Wijnberg, J. B. P. A.; Orru,
R. V. A.; Seelen, C. G. J. M.; van der Kerk, S. M.; de Groot, A.
Tetrahedron 1990, 46, 7237–7246, and references therein.
(10) Treatment of natural halichonadin C with CuI gave a trace of the Cu(I)-
coordinated halichonadin C (2), although CuI rather than CuCl has
been used for formation of a Cu(I)-coordinated trimer of p-tolyl
isocyanide.11
1H NMR (600 MHz, CDCl3) δ 0.51 (1H, dd, J ) 10.9, 9.2 Hz), 0.57
(1H, m), 0.91 (1H, br s), 0.99 (6H, s), 1.00 (3H, s), 1.21-1.32 (4H,
m), 1.44 (1H, m), 1.59-1.74 (5H, m), 1.99 (1H, m); EIMS m/z 221
[M]+; HREIMS m/z 221.2129 (calcd for C15H27N, 221.2143).
Acetylation of Halichonadin F (1). To a solution of halichonadin
F (1; 0.1 mg, 0.45 µmol) in pyridine (0.1 mL) was added Ac2O (0.1
mL), and the mixture was reacted at room temperature for 5 h. The
mixture was concentrated and purified with silica gel column chroma-
tography (n-hexane/EtOAc, 1:1 f 0:1) to provide 6 (0.09 mg, 0.34
µmol) in 76% yield.
6: colorless, amorphous solid; [R]23D -88 (c 0.04, CHCl3); IR (NaCl)
ν
max 3300, 2925 cm-1;1H NMR δ 0.54 (1H, t, J ) 10.8 Hz, H-6), 0.62
(1H, m, H-7), 0.92 (3H, d, J ) 7.2 Hz, H-14), 0.98 (3H, s, H-13),
1.00(3H, s, H-12), 1.01 (1H, m, H-8), 1.20 (3H, s, H-15), 1.26 (1H, m,
H-9), 1.32 (2H, m, H-3), 1.40 (1H, m, H-5), 1.66 (2H, m, H-2), 1.76
(1H, dt, J ) 14.4, 6.0 Hz, H-8), 1.90 (3H, s, NAc), 2.00 (1H, m, H-4),
2.06 (1H, dd, J ) 13.2, 6.0 Hz, H-9), 2.45 (1H, q, J ) 9.0 Hz, H-1);
EIMS m/z 263 [M]+; HREIMS m/z 263.2230 (calcd for C17H29ON,
263.2249).
Acetylation of Compound 3. To a solution of compound 3 (0.1
mg, 4.5 µmol) in pyridine (0.1 mL) was added Ac2O (0.1 mL), and
the mixture was reacted at room temperature for 5 h. The mixture was
concentrated and purified with silica gel column chromatography
(hexane/EtOAc, 1:1 f 0:1) to provide 7 (0.08 mg, 3.0 µmol) in 67%
(11) Toth, A.; Floriani, C.; Chiesi-Villa, A.; Guastini, C. J. Chem. Soc.,
Dalton Trans. 1988, 1599–1604.
(12) Fattorusso, E.; Magno, S.; Mayol, L.; Santacroce, C.; Sica, D.
Tetrahedron 1974, 30, 3911–3913.
(13) Fattorusso, E.; Magno, S.; Mayol, L.; Santacroce, C.; Sica, D.
Tetrahedron 1975, 31, 269–270.
yield. 7: colorless, amorphous solid; [R]21 +17 (c 0.04, CHCl3); H
1
D
NMR δ 0.50 (1H, t, J ) 10.2 Hz, H-6), 0.59 (1H, m, H-7), 0.94 (3H,
d, J ) 7.2 Hz, H-14), 1.00 (3H, s, H-13), 1.01 (3H, s, H-12), 1.05
(1H, m, H-8), 1.20 (1H, m, H-9), 1.26 (2H, m, H-2), 1.41 (3H, s, H-15),
1.51 (1H, q, J ) 10.0 Hz, H-5), 1.68 (2H, m, H-3), 1.69 (1H, m, H-8),
1.78 (1H, m, H-1), 1.96 (3H, s, NAc), 2.01 (1H, m, H-4), 2.80 (1H,
NP800164S