Halichonadins K and L, new dimeric sesquiterpenoids from a sponge Halichondria sp.

Article abstract of DOI:10.1021/ol3014705

Two new structurally unique dimeric sesquiterpenoids, halichonadins K (1) and L (2), were isolated from an Okinawan marine sponge Halichondria sp. The structures of 1 and 2 were elucidated on the basis of spectroscopic analysis including a single crystal X-ray diffraction analysis and chemical conversion. Halichonadins K (1) and L (2) are homodimers of the eudesmane sesquiterpene linked with a piperidine ring through amide bonds. Halichonadin K (1) showed moderate cytotoxicity against KB cells.

Full text of DOI:10.1021/ol3014705

ORGANIC
LETTERS
2012
Vol. 14, No. 13
3498–3501
Halichonadins K and L, New Dimeric
Sesquiterpenoids from a Sponge
Halichondria sp.
Naonobu Tanaka,† Shohei Suto,† Haruaki Ishiyama,† Takaaki Kubota,†
Akihito Yamano,‡ Motoo Shiro,‡ Jane Fromont,§ and Jun’ichi Kobayashi*,†
Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812,
Japan, Rigaku Corporation, Akishima 196-8666, Japan, and Western Australian
Museum, Locked Bag 49, Weishpool DC, WA 6986, Australia
jkobay@pharm.hokudai.ac.jp
Received May 29, 2012
ABSTRACT
Two new structurally unique dimeric sesquiterpenoids, halichonadins K (1) and L (2), were isolated from an Okinawan marine sponge Halichondria
sp. The structures of 1 and 2 were elucidated on the basis of spectroscopic analysis including a single crystal X-ray diffraction analysis and
chemical conversion. Halichonadins K (1) and L (2) are homodimers of the eudesmane sesquiterpene linked with a piperidine ring through amide
bonds. Halichonadin K (1) showed moderate cytotoxicity against KB cells.
Marine sponges belonging to the genus Halichondria are
known to be a source of sesquiterpene isothiocyanates,
isonitriles, and formamides and dimeric sesquiterpenoids
with a urea linkage.1,2 During our search for structurally
unique metabolites from Okinawan marine sponges, we
reported the isolation of a series of sesquiterpenoids,
halichonadins AꢀF, from Halichondria spp.3 Recently,
we have also reported dimeric sesquiterpenoids, halicho-
nadins GꢀI, and a sesquiterpenoid, halichonadin J, from
the extracts of an Okinawan marine sponge Halichondria
sp. (NSS-2).4 Further investigation of the extracts resulted
in the isolation of two new dimeric sesquiterpenoids,
halichonadins K (1) and L (2). In this Letter, we describe
the isolation and structure elucidation of 1 and 2.
The sponge Halichondria sp. (NSS-2, 1.0 kg, wet weight)
collected at Unten Port, Okinawa, was extracted with
MeOH, and the extracts were partitioned between CHCl3
and water. The CHCl3-soluble materials were subjected to
silica gel columns and then purified using C18 HPLC to
afford halichonadins K (1, 0.00094%, wet weight) and L
(2, 0.00093%) together with a known eudesmane-type
sesquiterpenoid, halichonadin C.3a
Halichonadin K (1)5 was obtained as an optically active
colorless amorphous solid {[R]21D ꢀ25.2 (c 1.07, MeOH)}.
IR absorption bands at 1738 and 1650 cmꢀ1 implied the
presence of ester and amide carbonyl functionalities, re-
spectively. The molecular formula, C40H65N3O5, was es-
tablished by the HRAPCIMS (m/z 668.49987 [MþH]þ,
† Hokkaido University.
‡ Rigaku Corporation.
§ Western Australian Museum.
€
(1) Wright, A. D.; Koning, G. M. J. Nat. Prod. 1996, 59, 710–716.
(2) Zhan, Z.-J.; Ying, Y.-M.; Ma, L.-F.; Shan, W.-G. Nat. Prod. Rep.
1
Δþ0.17 mmu). The H and 13C NMR spectra (Table 1)
2011, 28, 594–629.
suggested that 1 has two sesquiterpene moieties {units A
(3) (a) Ishiyama, H.; Hashimoto, A.; Fromont, J.; Hoshino, Y.;
Mikami, Y.; Kobayashi, J. Tetrahedron 2005, 61, 1101–1105. (b) Kozawa,
S.; Ishiyama, H.; Fromont, J.; Kobayashi, J. J. Nat. Prod. 2008, 71,
445–447. (c) Ishiyama, H.; Kozawa, S.; Aoyama, K.; Mikami, Y.;
Fromont, J.; Kobayashi, J. J. Nat. Prod. 2008, 71, 1301–1303.
(4) Suto, S.; Tanaka, N.; Fromont, J.; Kobayashi, J. Tetrahedron
Lett. 2011, 52, 3470–3473.
(5) Halichonadin K (1): colorless amorphous solid; ꢀ[R1]21 ꢀ25.2 (c
D
;
1H and 13C
1.07, MeOH); IR (film) vmax 3284, 1738, and 1650 cm
NMR (Table 1); HRAPCIMS: m/z 668.49987 [MþH]þ (calcd for
C40H66N3O5, 668.49970).
r
10.1021/ol3014705
Published on Web 06/21/2012
2012 American Chemical Society

(C-1ꢀC-15) and B (C-10ꢀC-150)}. They were identical to
those of halichonadin H,4 a homodimer of sesquiterpene
with the eudesmane skeleton. The gross structure of units
A and B were confirmed by detailed analysis of the 2D
NMR spectra (Figure 1).
Figure 2. Selected NOESY correlations and relative configura-
tions for units A (C-1ꢀC-15) and C (C-100ꢀC-900) of halichona-
din K (1) (protons of methyl groups in unit A were not shown).
dichloromethane/diisopropyl ether gave a cocrystal of 1
and hydroquinone which is an additive of diisopropyl
ether.6 The single crystal X-ray diffraction analysis of the
crystal revealed the relative stereochemistry of 1.7 The
ORTEP drawing of 1 without hydroquinone was shown
in Figure 3. In addition, the analysis disclosed the absolute
stereochemistry of 1 {Flack parameter, 0.26(20), calcu-
lated using 3893 Friedel pairs}.8 Therefore, the absolute
configurations at 11 chiral centers of halichonadin K (1)
were assigned as 5S, 6S, 7S, 10R, 50S, 60S, 70S, 100R, 200R,
400S, and 600S.
Figure 1. Selected 2D NMR correlations for halichonadin K (1).
Furthermore, the 1H and 13C NMR spectra showed the
signals of the linker moiety {unit C (C-100ꢀC-900)}, namely
one ester and two amide carbonyl groups, one methoxy
group, three sp3 methines, and three sp3 methylenes
(Table 1). Among them, two sp3 methines {C-200 (δC
60.3) and C-600 (δC 60.5)} and one sp3 methylene {C-800
(δC 53.4)} were ascribed to those bearing a nitrogen atom.
The gross structure of unit C was assigned as follows. The
1
1Hꢀ H COSY spectrum demonstrated the connectivities
of C-200 to C-600 (Figure 1). HMBC correlations for protons
of nitrogen bearing sp3 methylene (H2-800) to C-200, C-600,
and C-900 and 900-OMe to C-900 revealed the presence of a
piperidine ring (C-200ꢀC-600 and 200-N) and the connectivity
of 200-N to a methyl acetate moiety (C-800 and C-900). The
existence of a hydroxy group at C-400 was implied by the
chemical shift of C-400 (δC 62.3). This was supported by the
downfield shift for H-400 (Δδ 1.12 ppm) of the 400-p-
bromobenzoate (1a) of halichonadin K (1) (Supporting
Information). The connectivities among units AꢀC
through amide bonds were disclosed by HMBC correla-
tions for H-200/C-100 and H-600/C-700 and NOESY cross-
peaks of H-200/60-NH and H-600/6-NH. Therefore, the gross
structure of 1 was assigned as shown in Figure 1.
The relative configuration of unit A was deduced to be
the same as that of halichonadin H4 based on the NOESY
analysis (Figure 2). Resemblance of the 13C chemical shifts
for units A and B of 1 implied that both units havethe same
relative configuration (Table 1). In unit C, NOESY corre-
lations for 400-OH/H-200 and 400-OH/H-600 were observed,
suggesting that the piperidine ring adopts the chair con-
formation and the axial orientations for H-200, H-600, and
400-OH (Figure 2). Thus, the relative configurations for
each unit of 1 were elucidated. However, their relative
relationship could not been assigned by the NOESY
analysis.
Figure 3. ORTEP drawing of halichonadin K (1).
Halichonadin L (2)9 was obtained as an optically active
colorless amorphous solid {[R]21D ꢀ14.3 (c 0.73, MeOH)}.
An IR absorption band at 1647 cmꢀ1 implied the
presence of amide carbonyl functionality. The molec-
ular formula, C47H72N4O4, was established by the
(7) Crystallographic data for halichonadin K (1) have been deposited
at the Cambridge Crystallographic Data Center (deposition number
CCDC 882486).
Although a crystal of the 400-p-bromobenzoate (1a) was
not obtained, crystallization of halichonadin K (1) from
(8) (a) Flack, H. D. Acta Crystallogr., Sect. A 1983, A39, 876–881. (b)
Flack, H. D.; Bernardinelli, G. J. Appl. Crystallogr. 2000, 33, 1143–1148.
(c) Flack, H. D.; Bernardinelli, G. J. Chirality 2008, 20, 681–690.
(9) Halichonadin L (2): colorless amorphousꢀs1olid; [R]21 ꢀ14.3 (c
D
1H and 13C NMR
0.73, MeOH); IR (film) vmax 3274 and 1647 cm
;
(6) 0.01% of hydroquinone is added as a stabilizer to diisopropyl
ether used for the crystallization.
(Table 1); HRAPCIMS: m/z 757.56233 [MþH]þ (calcd for C47H73N4O4,
757.56263).
Org. Lett., Vol. 14, No. 13, 2012
3499

Table 1. 1H (600 MHz) and 13C (150 MHz) NMR Data for Halichonadins K (1) and L (2) in C5D5N
1
2
position
13C
1H
13C
1H
1
42.2
24.5
38.5
147.3
56.6
46.7
50.1
18.7
40.6b
37.7
27.0
21.8
16.6
107.3
17.3
42.2
24.5
37.5
147.5
56.2
46.7
50.2
18.7
40.5b
37.6
26.9
21.7
16.5
107.5
17.3
173.6
60.3
37.5
62.3
37.8
60.5
173.5
53.4
172.0
ꢀ
1.31, 1.14 (1H each, m)
42.3
24.6
38.6
147.4b
56.6
46.9
50.3
18.8b
40.6
37.7
27.0
21.9
16.6
107.4b
17.4
42.3
24.6
38.6
147.6b
56.6
46.9
50.3
18.9b
40.6
37.7
27.1
22.0
16.7
107.6b
17.4
174.3
61.8b
36.4b
61.9
36.6b
61.6b
174.3
58.9
171.2
41.6
36.3
140.1
129.1
128.9
126.6
ꢀ
1.32, 1.19 (1H each, m)
1.52 (2H, m)
2
1.49 (2H, m)
3
2.25, 1.86 (1H each, m)
2.29, 1.95 (1H each, m)
ꢀ
4
ꢀ
5
1.96 (1H, m)
2.09 (1H, m)
6
4.24 (1H, m)
4.28 (1H, m)
7
1.26 (1H, m)
1.42 (1H, m)
8
1.38, 1.27 (1H each, m)
1.43, 1.32 (1H each, m)
1.42, 1.16 (1H each, m)
ꢀ
9
1.39, 1.10 (1H each, m)
10
11
ꢀ
2.12 (1H, m)
2.15 (1H, m)
12
13
14
15
10
20
30
40
50
60
70
0.90 (3H, d, J = 7.0 Hz)
0.98 (3H, d, J = 6.8 Hz)
1.09 (3H, d, J = 6.8 Hz)
5.03, 4.91 (1H each, brs)
0.73 (3H, s)
1.05 (3H, d, J = 7.0 Hz)
5.08 (1H, brs), 4.91a (1H, m)
0.73 (3H, s)
1.31, 1.14 (1H each, m)
1.32, 1.19 (1H each, m)
1.52 (2H, m)
1.49 (2H, m)
2.25, 1.86 (1H each, m)
2.29, 1.95 (1H each, m)
ꢀ
ꢀ
1.96 (1H, m)
2.09 (1H, m)
4.24 (1H, m)
4.28 (1H, m)
1.33 (1H, m)
1.42 (1H, m)
80
90
1.38, 1.27 (1H each, m)
1.43, 1.32 (1H each, m)
1.42, 1.16 (1H each, m)
ꢀ
1.39, 1.10 (1H each, m)
100
110
120
130
140
1050 0
100
ꢀ
2.18 (1H, m)
2.24 (1H, m)
0.95 (3H, d, J = 7.0 Hz)
1.03 (3H, d, J = 6.7 Hz)
1.15 (3H, d, J = 6.7 Hz)
5.15, 5.02 (1H each, brs)
0.73 (3H, s)
1.07 (3H, d, J = 7.0 Hz)
5.05 (1H, brs), 4.91a (1H, m)
0.73 (3H, s)
ꢀ
ꢀ
200
4.48 (1H, dd, J = 10.9, 3.2 Hz)
4.04 (1H, m)
300
2.25 (2H, m)
2.26 (2H, m)
4
4.35 (1H, brs)
4.38 (1H, brs)
2.38 (2H, m)
500
2.35 (2H, m)
600
4.49 (1H, dd, J = 9.2, 4.1 Hz)
4.04 (1H, m)
700
800
900
ꢀ
ꢀ
3.98, 3.85 (1H each, d, J = 18.1 Hz)
3.59 (2H, m)
ꢀ
ꢀ
1000
1100
1200
1300,1700
1400,1600
1500
NH-60
NH-6
NH-900
OMe
OH
ꢀ
3.71, 3.68 (1H each, m)
2.99 (2H, t, J = 7.8)
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
7.29 (2H, m)
ꢀ
ꢀ
7.29 (2H, m)
ꢀ
ꢀ
7.21 (1H, m)
ꢀ
8.06 (1H, brd, J = 8.9 Hz)
8.12 (1H, brd, J = 9.0 Hz)
ꢀ
8.50 (1H, brd, J = 7.0 Hz)
8.32 (1H, brd, J = 9.3 Hz)
8.84 (1H, brt, J = 5.1 Hz)
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
51.2
ꢀ
3.58 (3H, s)
ꢀ
6.28 (1H, brs)
ꢀ
6.68 (1H, brs)
a Signals were overlapped with that of HOD. b Signals may be interchangeable.
HRAPCIMS (m/z 757.56233 [MþH]þ, Δꢀ0.30 mmu).
The 1H and 13C NMR spectra of 2 were similar to those
of 1, and the signals for a phenethylamine moiety in
2 were discerned in place of the resonances of a methoxy
group in 1 (Table 1). These data implied that 2 is a
dimer of the eudesmane-type sesequiterpene having a
piperidine ring and a phenethylamine moiety. Analysis
1
of the 1Hꢀ H COSY and HMBC spectra suggested the
gross structrue of 2 as shown in Figure 4.
(10) Kunishima, M.; Kawachi, C.; Hioki, K.; Terao, K.; Tani, S.
Tetrahedron 2001, 57, 1551–1558.
3500
Org. Lett., Vol. 14, No. 13, 2012

Scheme 2. Possible Biogenetic Path of Halichonadins K (1)
and L (2)
Figure 4. Selected 2D NMR correlations for halichonadin L (2).
Scheme 1. Derivatization of Halichonadin K (1) to Halichona-
din L (2a)
N-phenethylacetamide moieties, respectively, con-
nected with a nitrogen atom of the piperidine ring.
A possible biogenetic path of halichonadins K (1) and L
(2) is proposed as shown in Scheme 2. A plausible bioge-
netic intermediate (X) seems to be derived by condensation
of glyoxylic acid and lysine. Halichonadins K (1) and L (2)
might be generated from X and two molecules of halicho-
nadin C.3a
To elucidate the stereochemistry of halichonadin L (2),
derivatization of halichonadin K (1) to 2a was carried out
(Scheme 1). Halichonadin K (1) was treated withK2CO3 in
MeOH/water (1:1) to give the demethyl derivative (1b).
Condensation of 1b and phenethylamine by DMT-MM10
in MeOH furnished halichonadin L (2a). Since 1H NMR
data (Supporting Information) and optical rotation
Halichonadin K (1) showed cytotoxicity against human
epidermoid carcinoma KB cells (IC50 10.6 μg/mL) in vitro.
{[R]18 ꢀ10.3 (c 0.20, MeOH)} of derived halichonadin
D
L (2a) were in agreement with those of natural halichona-
din L (2), the absolute stereochemistry of 2 was concluded
as shown in Scheme 1.
Acknowledgment. We thank S. Oka and A. Tokumitsu,
Equipment Management Center, Hokkaido University,
for measurements of MS spectra and Z. Nagahama and
K. Uehara for their help with the sponge collection. This
work was partly supported by a Grant-in-Aid for Scientific
Research from the Ministry of Education, Culture, Sports,
Science and Technology of Japan.
Halichonadins K (1) and L (2) are structurally unique
homodimers of the eudesmane sesquiterpene. To the best
ofour knowledge, theyare the first example of the isolation
of dimeric sesquiterpenoids linked with a piperidine ring
from a natural source, while some homo- and hetero
sesquiterpene dimers such as halichonadins A,3a E,3b
and GꢀI,4 and N,N0-bis{(1Z,4Z)-7RH-germacra-1(10),
4-dienyl}urea11 were reported from sponges of the gen-
era Halichondria and Axinyssa. Furthermore, hali
chonadins K (1) and L (2) have a methyl acetate and
Supporting Information Available. Experimental sec-
tion, 1D and 2D NMR spectra, and X-ray crystallo-
graphic data (CIF) for halichonadins K and L and their
derivatives. This material is available free of charge via
the Internet at http://pubs.acs.org.
(11) Satitpatipan, V.; Suwanborirux, K. J. Nat. Prod. 2004, 67, 503–
505.
The authors declare no competing financial interest.
Org. Lett., Vol. 14, No. 13, 2012
3501