187
6.96
3.27
7.55
2.78
H
N
H
N
116.2
36.6
119.1
35.8
3
N
NH3
N
NH2
N
N
O
O
7.61
136.8
9.00
136.4
Me
1H NMR
4
5
13C NMR
25
Stellettazole D (1): yellow amorphous solid; ½¡ꢀD +6.1°
1
stellettazole C
(c 0.10, MeOH), UV (MeOH): -max (log ¾) 263 (4.03) nm; IR
(KBr): ¯max 3346, 2964, 2928, 2871, 1652, 1598, 1537, 1449,
1386, 1310, 1247, 1135, 973 cm¹1; HRESI-TOF-MS: [M]+
m/z 373.2957, calculated for C22H37N4O 373.2962.
S. Tsukamoto, H. Kato, H. Hirota, N. Fusetani, Tetrahedron
Figure 2. NMR chemical shifts of cationic moiety.
The 1H NMR spectrum exhibited a spin system correspond-
ing to disubstituted ethylamine (from H-1¤ to H2-3¤), monosub-
stituted propylamine (from H2-9¤ to H2-12¤), and mutually
coupled heteroaromatic protons (H-5¤ and H-7¤). HMBC cross
peaks (Table 1) revealed that the heteroaromatic protons are
incorporated into an imidazolium ring, which is substituted by
both ethyl and propyl groups [H-5¤ (¤ 6.96)/C-4¤ (¤ 131.0) and
C-7¤ (¤ 136.8); H-7¤ (¤ 7.61)/C-4¤ and C-5¤ (¤ 116.2); H2-3¤/C-4¤
and C-5¤; H2-9¤ (¤ 4.02)/C-5¤ and C-7¤]. Acetylation of
stellettazole D (1) with Ac2O yielded acetamide 4.11 A newly
generated amide proton at ¤ 7.99 (H-12¤) coupled to H-11¤ (¤
3.25) indicated that stellettazole D (1) has an aminopropyl group.
The remaining norsesquiterpene unit was identified by a
combination of 2D NMR techniques including 1H-1H COSY,
edited-gsHSQC,12 and gHMBC experiments, the same as for
stellettamide B (3) (Table 1). The geometry of the two asym-
metric double bonds at C-2 and C-4 was assigned as 2E,4E by
NOESY correlations [H-3 (¤ 6.84)/H-5 (¤ 5.93), H-4 (¤ 6.44)/2-
6
7
8
9
S. Matsunaga, T. Yamashita, S. Tsukamoto, N. Fusetani,
Preparative HPLC was performed using a JAI LC-9104
recycling preparative HPLC system.
10 HRESI-TOF-MS data were recorded on a Waters LCT-Premier
XE mass spectrometer using positive electronspray ionization
with a Waters ACQUITY Ultra Performance LC system. NMR
spectra were recorded on a Bruker Avance DPX400 spec-
1
trometer. The H and 13C chemical shifts were referenced to
the CD3OD solvent signals at ¤H 3.33 and ¤C 49.0 or the
DMSO-d6 solvent signals at ¤H 2.50 and ¤C 39.5. IR and UV
spectra were recorded on a JASCO FTIR VALOR-III spec-
trometer and a JASCO V-560 spectrometer, respectively.
11 Stellettazole D (1, 0.5 mg) was dissolved in a mixture of Ac2O
and pyridine (1:1, 1 mL) and stirred at room temperature for
1 h. The solvent was removed in vacuo to generate the
acetamide 4 in quantitative yield. 1H NMR signals for 4
(DMSO-d6): ¤ 7.99 (1H, br s, H-12¤), 7.95 (1H, br s, H-1¤),
7.67 (1H, s, H-7¤), 7.01 (1H, s, H-5¤), 6.84 (1H, d, J = 11.1 Hz,
H-3), 6.44 (1H, dd, J = 15.0, 11.1 Hz, H-4), 5.93 (1H, dd,
J = 15.0, 8.3 Hz, H-5), 5.12 (1H, J = 7.2 Hz, H-9), 4.01 (2H, t,
J = 6.7 Hz, H2-9¤), 3.25 (2H, br t, J = 6.7 Hz, H2-11¤), 2.88
(2H, m, H2-2¤), 2.69 (2H, t, J = 6.7 Hz, H2-3¤), 2.32 (1H, dtq,
J = 8.3, 7.1, 6.7 Hz, H-6), 2.28 (3H, s), 2.08 (2H, tt, J = 6.7,
6.7 Hz, H2-10¤), 2.04 (2H, dt, J = 7.2, 6.2 Hz, H2-8), 1.94 (3H,
s, 2-Me), 1.74 (3H, s, 10-Me), 1.68 (3H, s, H3-11), 1.39 (2H,
dt, J = 7.1, 6.2 Hz, H2-7), 1.07 (3H, d, J = 6.7 Hz, 6-Me).
12 W. Willker, D. Leibfritz, R. Kerssebaum, W. Bermel, Magn.
Me (¤ 1.93)] and the proton-proton coupling constant (J4,5
15.0 Hz). To elucidate the stereochemistry at C-6, stellettazole
=
13
D (1) was treated with NaIO4 in the presence of RuCl3 to
25
generate (S)-2-methylglutaric acid14 (½¡ꢀD +24°, c 0.042),
thereby determining 6S-stereochemistry. Finally, the norsesqui-
terpene and aminopropylimidazolium units were determined to
be connected via an amide linkage on the basis of HMBC cross
peaks [H-3 (¤ 6.84), 2-Me (¤ 1.93) and H-1¤ (¤ 7.91)/C1 (¤
171.0)].
Interestingly, the cationic position of stellettazole D (1) was
different from stellettazole C, on the basis of the chemical shifts
of the imidazole and the terminal amide showing a typical
change by cation influence (Figure 2).15
Stellettazole D (1) was moderately cytotoxic against the
murine leukemia cell line P388 and against HeLa cells (IC50:
29.1 and 83.6 ¯g mL¹1, respectively). To our knowledge, this is
the first report of a stellettazole-type alkaloid from a marine
sponge other than Stelletta spp.16 This may suggest that
symbiotic bacteria in the marine sponge are the true producers
of stelletta-type alkaloids, such as stellettazoles, stellettadines,
and stellettamides. Further studies on the biosynthetic pathway
and biological activity of stellettazole D (1) are in progress.
13 P. H. J. Carlsen, T. Katsuki, V. S. Martin, K. B. Sharpless,
25
1779. (S)-2-Methylglutaric acid from 1: ½¡ꢀD +24°, (c 0.042,
MeOH). 1H NMR (CD3OD): ¤ 1.23 (3H, d, J = 7.1 Hz, H3-2),
1.83 (1H, dq, J = 15.0, 7.5 Hz, H-3), 1.97 (1H, dq, J = 15.0,
7.5 Hz, H-3), 2.49 (2H, t, J = 7.5 Hz, H2-4), and 2.61 (1H,
sext, J = 7.1 Hz, H-2).
15 E. Pretsch, P. Buehlmann, M. Badertscher, Structure Determi-
nation of Organic Compounds: Tables of Spectral Data, 3rd
ed. Springer-Verlag, Berlin, 2000, pp. 184, 233.
We are grateful to Dr. K. Yamada (Keio University) for
biological testing. We thank Dr. P. R. Bergquist (University of
Auckland) for the identification of the sponge. We wish to thank
Ms. W. Seki (Nippon Suisan Kaisha, Ltd.) for her technical
support.
16 a) J. W. Blunt, B. R. Copp, W. Hu, M. H. G. Munro, P. T.
J. W. Blunt, B. R. Copp, W. Hu, M. H. G. Munro, P. T.
J. W. Blunt, B. R. Copp, W. Hu, M. H. G. Munro, P. T.
J. W. Blunt, B. R. Copp, M. H. G. Munro, P. T. Northcote,
References and Notes
1
J. W. Blunt, B. R. Copp, M. H. G. Munro, P. T. Northcote,
2
M. Kuramoto, N. Miyake, Y. Ishimaru, N. Ono, H. Uno, Org.
Chem. Lett. 2011, 40, 186-187
© 2011 The Chemical Society of Japan