Halichonines A, B, and C, novel sesquiterpene alkaloids from the marine sponge Halichondria okadai Kadota

Article abstract of DOI:10.1039/c1cc15557a

Novel sesquiterpene alkaloids, halichonines A (1), B (2), and C (3), were identified from the marine sponge Halichondria okadai Kadota. By spectroscopic analyses and synthesis, their structures were revealed to include a 6,6-bicyclic ring system and two prenylated amine moieties. In addition, 2 induced apoptosis in HL60 human leukemia cells.

Full text of DOI:10.1039/c1cc15557a

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COMMUNICATION
Halichonines A, B, and C, novel sesquiterpene alkaloids from the marine
sponge Halichondria okadai Kadotaw
Osamu Ohno,a Tatsuhiko Chiba,b Seiji Todoroki,a Hideaki Yoshimura,c Norihito Maru,d
Ken Maekawa,e Hiroshi Imagawa,e Kaoru Yamada,d Atsushi Wakamiya,f
Kiyotake Suenaga*a and Daisuke Uemura*d
Received 8th September 2011, Accepted 13th October 2011
DOI: 10.1039/c1cc15557a
Novel sesquiterpene alkaloids, halichonines A (1), B (2), and
C (3), were identified from the marine sponge Halichondria
okadai Kadota. By spectroscopic analyses and synthesis, their
structures were revealed to include a 6,6-bicyclic ring system and
two prenylated amine moieties. In addition, 2 induced apoptosis
in HL60 human leukemia cells.
The marine sponge H. okadai Kadota (80 kg), collected
from the mediolittoral zone in Mie Prefecture, Japan, was
immersed in MeOH for 1 week at room temperature. Con-
centrated extract was partitioned with EtOAc and water, and
the EtOAc layer was chromatographed on TSK G3000S
polystyrene gel (EtOH/water). The fraction eluted with 40%
aqueous EtOH was partitioned with 1.2 N HCl and EtOAc.
The water layer was subsequently adjusted to pH 10 with
NaOHaq, and then extracted with EtOAc. The extract was
then chromatographed on silica gel (CHCl3/MeOH) using
Dragendorff’s reagent test-guided fractionation. Final purification
was achieved by preparative thin layer chromatography
(CHCl3/MeOH: 9/1), which gave halichonines A (1) (17.1 mg),
B (2) (2.0 mg) and C (3) (1.8 mg), as pale yellow amorphous powders.
The molecular formula of 2 [[a]2D8 +13.8 (c 0.2, CHCl3)] was
determined to be C29H52N2 by HR-ESIMS data (m/z 429.4220
[M + H]+, calcd for C29H53N2, 429.4209). The NMR spectral
data of 2 in CD3OD are shown in Table 1. A detailed analysis
of the COSY and HMQC spectra of 2 allowed us to elucidate
the following six partial structures: C1–C3, C5–C7, C9–C11,
C12–C13, C15–C18, and C19–C20. The connectivity of these
fragments was elucidated based on the HMBC techniques.
HMBC crosspeaks of H3, H5, H22 and H23 to quaternary
carbon C4 (dC 32.5), H3, H22 and H23 to C5 (dC 50.4), and
H22 and H23 to C3 (dC 42.2) established the connection
among C3, C4, C5, C22, and C23. HMBC correlations from
H5 and H24 to quaternary carbon C10 (dC 36.1) and C9
(dC 51.7), and of H24 to C1 (dC 39.2) constructed the connection
of C1, C5, C9, and C24 to C10. HMBC correlations from H25 to
C7 (dC 121.7), C8 (dC 135.4) and C9, and of H11 to C8 indicated
the connection of C7, C9 and C25 to C8. These observations
Marine sponges take seawater into their bodies, gather food
by filtration, and coexist with symbiotic microorganisms.1
They are widely known to be a rich source of biologically
active and structurally unique secondary metabolites. For
example, okadaic acid,2 halichondrins,3 and halichlorine4 have
already been isolated from the marine sponge Halichondria
okadai Kadota, a species abundant in the tidal zone in the
Pacific Ocean. These isolated compounds have good potential
for clinical or biological use. In fact, a synthetic analogue of
halichondrin B, eribulin mesylate (Halavent), was recently
approved for the treatment of patients with metastatic breast
cancer in European Union, the United States, Japan, and
many other countries.5 In our continuing search for biologically
active substances from marine organisms,6 we isolated novel
sesquiterpene alkaloids, halichonines A (1), B (2), and C (3)
(Fig. 1), from H. okadai Kadota. We describe here the isolation,
structure determination, and biological activities of 1, 2, and 3.
a Department of Chemistry, Faculty of Science and Technology,
Keio University, 3-14-1 Hiyoshi, Yokohama 223-8522, Japan.
E-mail: suenaga@chem.keio.ac.jp; Fax: +81-45-566-1819;
Tel: +81-45-566-1819
b Chemical Research Laboratory, Faculty of Liberal Arts,
Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan
c Department of Chemistry, Graduate School of Science,
Nagoya University, Chikusa, Nagoya 464-8602, Japan
d Department of Chemistry, Faculty of Science, Kanagawa University,
2946 Tsuchiya, Hiratsuka 259-1293, Japan.
E-mail: uemurad@kanagawa-u.ac.jp
e Faculty of Pharmaceutical Sciences, Tokushima Bunri University,
Yamashiro-cho, Tokushima 770-8514, Japan
f Institute for Chemical Research, Kyoto University, Uji,
Kyoto 611-0011, Japan
w Electronic supplementary information (ESI) available: Experimental
procedures, crystallographic information, spectral data for all new
compounds, and results of cell growth analysis. CCDC 842822. For
ESI and crystallographic data in CIF or other electronic format see
DOI: 10.1039/c1cc15557a
Fig. 1 Structures of halichonines A (1), B (2), and C (3).
Chem. Commun., 2011, 47, 12453–12455 12453
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Table 1 NMR data for halichonine B (2) in CD3OD
dC (mult.) dH (mult., J in Hz) HMBCc (1H - 13C)
a
b
Position
1
2
3
a
b
a
b
a
b
39.2 (t)
18.5 (t)
42.2 (t)
1.01 (m)
2.03 (m)
1.39 (m)
1.55 (m)
1.16 (m)
1.38 (m)
C2
C1, 10
C4, 22
C5, 22, 23
4
5
6
7
8
9
10
11
12
32.5 (s)
50.4 (d)
23.5 (t)
121.7 (d)
135.4 (s)
51.7 (d)
36.1 (s)
53.6 (t)
51.0 (t)
Fig. 2 COSY and selected HMBC correlations of 2.
1.18 (m)
1.95 (m, 2H)
5.34 (s)
C4, 6, 9, 10, 24
C25
1.84 (m)
2.31 (m, 2H)
C8, 12, 15
a
2.86 (dd, 7.8, 13.9)
3.11 (dd, 5.7, 13.9)
5.22 (m)
C11, 13, 14, 15
C11, 13, 14, 15
C26, 27
b
13
14
15
121.5 (d)
134.0 (s)
53.0 (t)
a
b
2.22 (m)
2.52 (m)
C11, 12, 17
C11, 12, 16
C17
16
17
18
19
20
21
22
23
24
25
26
27
28
29
24.3 (t)
26.2 (t)
48.0 (t)
45.8 (t)
119.0 (d)
137.4 (s)
21.1 (q)
32.6 (q)
12.8 (q)
21.6 (q)
24.7 (q)
16.7 (q)
24.6 (q)
16.7 (q)
1.43 (m, 2H)
1.45 (m, 2H)
2.55 (m, 2H)
3.18 (d, 6.5, 2H)
5.22 (m)
C15
C17, 19
C18, 20, 21
C28, 29
Fig. 3 The crystal structure of HCl complex of 2. The counter anions
of ClÀ and the solvent water molecules are omitted for clarity.
0.88 (s, 3H)
0.85 (s, 3H)
0.74 (s, 3H)
1.74 (s, 3H)
1.70 (s, 3H)
1.63 (s, 3H)
1.74 (s, 3H)
1.68 (s, 3H)
C3, 4, 5, 23
C3, 4, 5, 22
C1, 5, 9, 10
C9, 7, 8
C13, 14, 27
C13, 14, 26
C20, 21, 29
C20, 21, 28
3 were almost identical to those of 2, except for the signals
around the C3 moiety. Therefore, the structures of 1 and 3 were
elucidated based on a comparison of their spectroscopic data to
those of 2 (ESI, Tables S1 and S3w). A hydroxyl group was
revealed to be present at C3 of 1 by COSY and HMQC analyses.
The connectivity around C3 was revealed by the HMBC correlations
of H3/C5, H3/C22, H2/C3, H22/C3, and H23/C3. Meanwhile, a
carbonyl group was revealed to be present at position C3 of 3
based on its 1H and 13C NMR spectra (dC 216.7). From an
analysis of 1H-1H/13C-1H COSY spectra, the planar structure of
3 could be determined. Thus, 1–3 were revealed to possess
different levels of oxidation at position C3. The NOESY spectrum
of 1 demonstrated that the relative stereostructure of the 6,6-
bicyclic rings was the same as that of 2. The NOE correlations at
a
Recorded at 100 MHz. b Recorded at 600 MHz. c Recorded at 400 MHz.
indicated that 2 possessed a 6,6-bicyclic ring system. Meanwhile,
the existence of two prenyl moieties was demonstrated based on
HMBC correlations at H26/C13, H26/C14, H27/C13, H27/C14,
H28/C20, H28/C21, H29/C20, and H29/C21. In addition,
HMBC correlations at H18/C19 and H19/C18 suggested that
C18 was attached through nitrogen to a C19–C29 prenyl group.
This elucidation was also supported by the analyses of 1H NMR
and COSY spectra of 2 in DMSO-d6 (NH:dH 1.59). Similarly,
HMBC correlations at H11/C12, H11/C15, H12/C11, H12/C15,
H15/C11, and H15/C12 indicated that C11, C12 and C15 were
connected by an amine bond. Thus, the planar structure of 2 was
determined to be as shown in Fig. 2. The relative configuration of
the 6,6-bicyclic rings was determined by the NOESY spectrum
(ESIw, Fig. S19B). The NOE correlations at H1a/H3b, H3b/H5,
H1b/H2b, H2a/H3a, and H1b/H24 revealed that ring A had a
chair conformation. Furthermore, the NOE correlations at H1a/
H9 and H5/H9 indicated that ring B had a half-chair conformation.
In addition, 2 was successfully obtained as a crystal in the HCl-
salt form. The relative stereostructure of 2 was confirmed by
X-ray crystallographic analysis (Fig. 3 and S20 in ESIw),7 and it
completely coincided with those determined by the spectroscopic
analysis described above.
H1a/H3, H3/H5 and H3/H23, and the coupling constants (J2a,3
=
4.4 Hz, J2b,3 = 11.0 Hz) showed that the hydroxyl group at the C3
position of 1 had an equatorial orientation (ESIw, Fig. S19A).
Furthermore, the CD curve of 3 exhibited a negative Cotton effect
(De = À0.7) at 294 nm. Application of the octant rule8 clearly
indicated that the axial-oriented methyl group at C10 is located in
the upper position (ESIw, Fig. S22). Thus, the absolute configu-
ration of C5, C9, and C10 in 3 was clarified to be 5R, 9R, and 10S.
From the viewpoint of the likely similar biosynthesis within the
same collected sample, we propose that the configuration of 1–3
may be superimposed on each other. Each halichonine congener
included a sesquiterpene skeleton and two prenylated amine
moieties as novel and unique structural features. Therefore, they
are likely to be synthesized from a farnesol and an ornithine via
unusual biosynthetic routes. To the best of our knowledge, this is
the first report of the isolation of such new type of sesquiterpene
alkaloids from marine organisms.
The molecular formula of 1 [[a]2D5 +35.5 (c 0.55, CHCl3)] was
determined to be C29H52ON2by HR-ESIMS data (m/z 445.4166
[M + H]+, calcd for C29H53ON2, 445.4158). The molecular
formula of 3 [[a]2D5 +8.42 (c 0.19, CHCl3)] was determined to be
C29H50ON2 by EIMS data (m/z 442). The NMR signals of 1 and
For the confirmation of the structure including the relative
configuration at C3, synthesis of 1 was carried out. The synthesis
began with N-(4-aminobutyl)-2-nitrobenzenesulfonamide (4),
which was transformed to amine 6 in 42% yield in three steps.9
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lines (L1210 and PC13) using the MTT assay. After 72 hours
of incubation, each compound showed moderate growth-
inhibitory activity toward these cells (ESIw, Table S4). 2 was
then subjected to the trypan blue dye exclusion using HL60
human leukemia cells, and showed cytotoxicity (IC50 value:
0.60 mg mlÀ1). In addition, 2-induced cell death in HL60 cells
was suppressed in the presence of Z-VAD-FMK, an irrever-
sible and cell-permeable inhibitor of caspases (Fig. 4A). DNA
ladder analysis revealed that 2 induced DNA fragmentation in
HL60 cells (Fig. 4B). These results indicated that 2 induced
apoptosis in HL60 cells.
In conclusion, we isolated halichonines A (1), B (2), and C
(3) from the marine sponge H. okadai Kadota. Based on the
results of spectroscopic analyses, 1–3 were determined to be
novel sesquiterpene alkaloids with a 6,6-bicyclic ring system
possessing different levels of oxidation at position C3. In
addition, the relative stereostructure of 1 was confirmed by
successful synthesis. All three halichonine congeners showed
growth-inhibitory activities against mammalian cancer cells,
and 2 was revealed to induce apoptosis in HL60 cells. Thus,
these halichonines may deserve further studies.
Scheme 1 Total synthesis of (Æ)-1. Reagents and conditions: (a)
Boc2O (1.2 equiv.), Et3N (1.2 equiv), CH2Cl2, rt. (b) 1-bromo-3-
methyl-2-butene (3.0 equiv.), K2CO3 (2.5 equiv.), DMF, rt, 77% yield
for 2 steps. (c) TFA, CH2Cl2, 0 1C, 54% yield. (d) BAIB (1.2 equiv.),
TEMPO (0.1 equiv.), 69% yield. (e) 6 (2.7 equiv.), NaBH(OAc)3 (5.0
equiv.), DCE, rt, 44% yield. (f) 3-methyl-2-butenal (2.5 equiv.),
NaBH(OAc)3 (5.0 equiv.), DCE, rt, 32% yield. (g) PhSH (1.3 equiv.),
K2CO3 (2.3 equiv.), CH3CN, rt, 14% yield.
We thank Drs T. Sasamori, Kyoto University, K. Naka-
mura, Kanagawa University, Y. Yamano, Osaka University,
and K. Miyamoto, Keio University, for their contributions to
our studies. This work was supported in part by Grants-in-Aid
from the Ministry of Education, Culture, Sports, Science and
Technology of Japan (21221009 to D.U. and 20611006
to O.O.).
Notes and references
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Fig. 4 Induction of apoptosis in HL60 cells by 2. (A) HL60 cells were
preincubated (solid column) or not (open column) with 50 mM
Z-VAD-FMK, and then treated with 0 or 1 mg mlÀ1 of 2. After
24 h, cell viability was determined. Values are the means Æ SD of
quadruplicate determinations. (B) HL60 cells were incubated with the
indicated concentrations of 2 for 24 h. Cellular DNA was then
extracted and electrophoresed on an agarose gel.
5 (a) M. J. Towle, K. A. Salvato, J. Budrow, B. F. Wels,
G. Kuznetsov, K. K. Aalfs, S. Welsh, W. Zheng, B. M. Seletsky,
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7 See the ESIw.
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(Æ)-3b-Hydroxydrimenol (7) was prepared in 5 steps from
farnesol as previously reported.10 Oxidation of 7 with BAIB
and a catalytic amount of TEMPO gave aldehyde 8 (78% yield)
as a colorless oil.11 The aldehyde 8 was then subjected to
reductive amination with 6, to give amine 9 (44% yield).12
Reductive amination of 9 with 3-methyl-2-butenal afforded 10
in 32% yield. The nosyl protecting group of 10 was removed with
PhSH and K2CO3 to give (Æ)-1 and unidentified byproducts.
Finally, purification of the mixture using alumina column chromato-
graphy (hexane/EtOAc, CHCl3, and CHCl3/MeOH) provided
(Æ)-1 in 14% yield (Scheme 1). Synthetic 1 was identical to the
natural 1 in all respects (1H and 13C NMR, and HR-ESIMS
spectra). Thus, the relative stereostructure of 1 was confirmed.
Next, compounds 1–3 were examined with regard to their
growth-inhibitory activities against mammalian cancer cell
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