Hyrtimomines A-C, new heteroaromatic alkaloids from a sponge hyrtios sp.

Article abstract of DOI:10.1021/ol400687b

Three new alkaloids, hyrtimomines A-C (1-3), were isolated from an Okinawan marine sponge Hyrtios sp. The structures of 1-3 were elucidated on the basis of spectroscopic analysis and application of a phenylglycine methyl ester (PGME) method. Hyrtimomines A (1) and B (2) are heteroaromatic alkaloids possessing a fused hexacyclic 6/5/6/6/7/5 ring system, while hyrtimomine C (3) is an alkaloid consisting of hydroxyindole and azepino-hydroxyindole moieties. Hyrtimomine A (1) exhibited cytotoxicity against KB and L1210 cells.

Full text of DOI:10.1021/ol400687b

ORGANIC
LETTERS
2013
Vol. 15, No. 8
2010–2013
Hyrtimomines AꢀC, New Heteroaromatic
Alkaloids from a Sponge Hyrtios sp.
Rei Momose,† Naonobu Tanaka,† Jane Fromont,‡ and Jun’ichi Kobayashi*,†
Graduate School of Pharmaceutical Sciences, Hokkaido University,
Sapporo 060-0812, Japan, and Western Australian Museum, Locked Bag 49,
Weishpool DC, WA 6986, Australia
jkobay@pharm.hokudai.ac.jp
Received March 14, 2013
ABSTRACT
Three new alkaloids, hyrtimomines AꢀC (1ꢀ3), were isolated from an Okinawan marine sponge Hyrtios sp. The structures of 1ꢀ3 were elucidated
on the basis of spectroscopic analysis and application of a phenylglycine methyl ester (PGME) method. Hyrtimomines A (1) and B (2) are
heteroaromatic alkaloids possessing a fused hexacyclic 6/5/6/6/7/5 ring system, while hyrtimomine C (3) is an alkaloid consisting of hydroxyindole
and azepino-hydroxyindole moieties. Hyrtimomine A (1) exhibited cytotoxicity against KB and L1210 cells.
Marine sponges have been recognized as a rich source
of bioactive secondary metabolites with fascinating
chemical structures.1 Among them, sponges belong-
ing to the genus Hyrtios are known to be a source
of heteroaromatic alkaloids with various structures.2
Previously, we have reported the isolation of indole
alkaloids, hyrtiosins A and B,3 gesashidine A,4 and
hyrtinadine A5 from Hyrtios spp. We have also isolated
alkaloids having a furo[2,3-b]pyrazin-2(1H)-one moiety,
hyrtioseragamines A and B, from Hyrtios sp.6 In our
continuing search for structurally unique metabolites
from Okinawan marine sponges, we investigated the
extracts of Hyrtios sp. (SS-163) and isolated three new
alkaloids, hyrtimomines AꢀC (1ꢀ3). In this Letter, we
describe the isolation and structure elucidation of 1ꢀ3.
The sponge Hyrtios sp. (SS-163, 3.3 kg, wet weight)
collected off Kerama Islands, Okinawa, was extracted
with MeOH, and the extracts were partitioned between
EtOAc and water. The EtOAc-soluble materials were
partitioned between n-hexane and 10% MeOH aq., while
the water layer was extracted with n-BuOH. Combined
10% MeOH aq.-soluble materials and n-BuOH-soluble
materials were first fractionated by silica gel column
chromatography, followed by fractionation by C18 column
chromatography. Next, fractions were further purified
by MCI gel CHP-20P column chromatography, and
final purification was achieved by C18 HPLC or HILIC
† Hokkaido University.
‡ Western Australian Museum.
(1) Blunt, J. W.; Copp, B. R.; Keyzers, R. A.; Munro, M. H. G.;
Prinsep, M. R. Nat. Prod. Rep. 2013, 30, 237–323.
(2) (a) Yamanokuchi, R.; Imada, K.; Miyazaki, M.; Kato, H.;
Watanabe, T.; Fujimuro, M.; Saeki, Y.; Yoshinaga, S.; Terasawa,
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Namikoshi, M.; de Voogd, N. J.; Yokosawa, H.; Tsukamoto, S. Bioorg.
Med. Chem. 2012, 20, 4437–4442. (b) Inman, W. D.; Bray, W. M.;
Gassner, N. C.; Lokey, R. S.; Tenney, K.; Shen, Y. Y.; TenDyke, K.;
Suh, T.; Crews, P. J. Nat. Prod. 2010, 73, 255–257. (c) Sauleau, P.;
Martin, M.-T.; Dau, M.-E. T. H.; Youssef, D. T. A.; Bourguet-
Kondracki, M.-L. J. Nat. Prod. 2006, 69, 1676–1679. (d) Youssef,
D. T. A. J. Nat. Prod. 2005, 68, 1416–1419. (e) Aoki, S.; Ye, Y.; Higuchi,
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(3) Kobayashi, J.; Murayama, T.; Ishibashi, M.; Kosuge, S.; Takamatsu,
M.; Ohizumi, Y.; Kobayashi, H.; Ohta, T.; Nozoe, S.; Sasaki, T. Tetra-
hedron 1990, 46, 7699–7702.
(6) Takahashi, Y.; Iinuma, Y.; Kubota, T.; Tsuda, M.; Sekiguchi,
M.; Mikami, Y.; Fromont, J.; Kobayashi, J. Org. Lett. 2011, 13, 628–
631.
(4) Iinuma, Y.; Kozawa, S.; Ishiyama, H.; Tsuda, M.; Fukushi, E.;
Kawabata, J.; Fromont, J.; Kobayashi, J. J. Nat. Prod. 2005, 68, 1109–
1110.
(5) Endo, T.; Tsuda, M.; Fromont, J.; Kobayashi, J. J. Nat. Prod.
2007, 70, 423–424.
(7) Hyrtimomine A (1): dark-brown amorphous solid; UV (MeOH)
λ
max 214 (ε 19 610), 293 (9680), 352 (7230 sh), 369 (9810), and 386 (8740)
1
nm; IR (KBr) vmax 3420, 1740ꢀ1640 (br), and 1200 cmꢀ1; H and 13C
NMR (Table 1); HRESIMS: m/z 314.09221 [M þ H]þ (calcd for
C19H12N3O2, 314.09240).
r
10.1021/ol400687b
Published on Web 04/09/2013
2013 American Chemical Society

HPLC to afford hyrtimomines A (1, 0.00009%, wet weight),
B (2, 0.00023%), and C (3, 0.00012%).
Hyrtimomine A (1)7 was obtained as a dark-brown
amorphous solid. TheUVspectrumsuggested the presence
of a conjugated aromatic chromophore. The molecular
formula of 1, C19H11N3O2, was established by the HRE-
SIMS (m/z 314.09221 [M þ H]þ, Δꢀ0.19 mmu), corre-
1
sponding to 16 degrees of unsaturation. The H NMR
spectrum showed signals of three D2O-exchangeable, six
aromatic, and two olefinic protons, while the 13C NMR
spectrum displayed the resonances of 17 aromatic and two
olefinic carbons (Table 1). From these data, 1 was eluci-
dated to be a heteroaromatic alkaloid with a highly con-
densed structure.
Figure 2. Structure and key 2D NMR correlations of hyrtimo-
mine A (1).
The structures of two partial units (units A and B) in 1
were assigned as follows. In unit A (N-1, C-2ꢀC-9, and
N-10), the presence of a 3,4-disubstituted-5-hydroxyindole
Hyrtimomine B (2)8 was obtained as an optically active
yellow amorphous solid {[R]22D ꢀ276.8 (c 0.027, MeOH)}.
The HRESIMS revealed the molecular formula of 2 to be
C20H13N3O4 (m/z 360.09789 [M þ H]þ, Δ þ0.01 mmu).
The 1H and 13C NMR spectra of 2 were similar to those of
1, and the signals due to one nitrogen bearing an sp3
methine (CH-9), one sp3 methylene (CH2-8), and one
carboxy group (C-11) in 2 were discerned in place of the
resonances of the Z-olefine (CH-8 and CH-9) in 1
(Table 1). The connectivity of C-8 to N-10 via C-9 was
1
moiety was suggested by analysis of the 1Hꢀ H COSY and
1
HMBC spectra (Figure 1). 1Hꢀ H COSY cross-peaks of
H-8/H-9 and H-9/10-NH and HMBC correlations for
H-8/C-2, H-8/C-3a, and H-9/C-3 disclosed that an ethen-
amine moiety (C-8 and C-9) was connected to C-3. The
geometry of the olefin was assigned as Z based on the
coupling constant for H-8/H-9 (J = 9.5 Hz). Similarly, the
structure of a 2,3-disubstituted-5-hydroxyindole moiety
(unit B, N-10ꢀC-70a) was deduced. The presence of an
oxygen attached to C-20 was implied by the chemical shift
of C-20 (δC 157.0). Thus, the structures of units A and B
were assigned as shown in Figure 1.
1
confirmed by 1Hꢀ H COSY cross-peaks of H2-8/H-9 and
H-9/10-NH, implying that the carboxy group (C-10) was
attached to C-9 (Figure 3). Thus, the gross structure of
hyrtimomine B (2) was elucidated as shown in Figure 3.
Figure 3. Selected 2D NMR correlations for hyrtimomine B (2).
Figure 1. Selected 2D NMR correlations for units A and B in
hyrtimomine A (1).
To assign the absolute configuration at C-9, hyrtimo-
mine B (2) was converted into the (S)- and (R)-PGME
(PGME = phenylglycine methyl ester) amides (2a and 2b,
respectively). TheΔδ values(Δδ =δS ꢀ δR) obtainedfrom
In addition to the 2D NMR correlations shown in
Figure 1, an HMBC correlation for H-9 to an sp2 qua-
ternary carbon (δC 152.3, C-80) was observed, suggesting
the connectivity of C-9 to C-80 via N-10. Given the degree of
unsaturation of 1 and a ROESY cross-peak of 10-NH/H-40,
the structure of hyrtimomine A (1) was elucidated as shown
in Figure 2.
1
the H NMR data for 2a and 2b indicated the absolute
configuration of C-9 in 2 to be S (Figure 4).9
Hyrtimomine C (3)10 was isolated as a yellow amorphous
solid. The HRESIMS indicated the molecular formula of 3
to be C19H13N3O3 (m/z332.10288 [M þ H]þ,Δꢀ0.11 mmu).
(8) Hyrtimomine B (2): yellow amorphous solid; [R]22 ꢀ276.8
(9) Yabuuchi, T.; Kusumi, T. J. Org. Chem. 2000, 65, 397–404.
(10) Hyrtimomine C (3): yellow amorphous solid; UV (MeOH) λmax
215 (ε 24 980), 272 (7430), 295 (7360), 380 (6680), and 474 (3720) nm; IR
(KBr) vmax 3427, 2926, 1733ꢀ1604 (br), 1588, 1205, and 1138 cmꢀ1; 1H
and 13C NMR (Table 1); HRESIMS: m/z 332.10288 [M þ H]þ (calcd for
C19H14N3O3, 332.10297).
D
(c 0.027, MeOH); UV (MeOH) λmax 219 (ε 11 780), 243 (6480 sh), 290
(5490), 327 (2750 sh), 343 (2980 sh), and 389 (4930) nm; IR (KBr) vmax
3383, 1645, 1592, 1572, and 1364 cmꢀ1 1H and 13C NMR (Table 1);
;
HRESIMS: m/z 360.09789 [M
360.09788).
þ
H]þ (calcd for C20H14N3O4,
Org. Lett., Vol. 15, No. 8, 2013
2011

Table 1. 1H and 13C NMR Data for Hyrtimomines AꢀC (1ꢀ3) in DMSO-d6
1
2
3
position
13C
1H
13C
1H
13C
1H
1
ꢀ
11.40 (1H, brs)
6.97 (1H, brs)
ꢀ
ꢀ
12.00 (1H, brs)
7.67 (1H, brs)
ꢀ
ꢀ
13.15 (1H, brs)
2
125.2
115.0
125.7
108.7
146.1
113.1
126.3
114.2
122.6
106.2
148.7
110.9
134.3
118.0
127.1
107.1
161.4
113.9
8.38 (1H, s)
3
ꢀ
3a
4
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
5
ꢀ
ꢀ
ꢀ
6
7.10 (1H, d,
J = 8.8 Hz)
7.43 (1H, d,
J = 8.8 Hz)
ꢀ
7.57 (1H, d,
J = 8.6 Hz)
7.99 (1H, d,
J = 8.6 Hz)
ꢀ
7.03 (1H, d,
J = 8.6 Hz)
7.99 (1H, d,
J = 8.6 Hz)
ꢀ
7
119.7
119.5
123.7
7a
8
134.9
110.9
132.6
30.4
130.2
183.9
5.39 (1H, d,
J = 9.5 Hz)
5.55 (1H, brd,
J = 9.5 Hz)
8.27 (1H, brs)
3.63, 3.48
(1H each, brs)a
5.20 (1H, brs)
ꢀ
9
121.9
58.7
56.9
4.25 (2H, m)
10
11
10
20
30
30a
40
50
60
ꢀ
ꢀ
8.97 (1H, brs)
ꢀ
ꢀ
170.7
ꢀ
13.78 (1H, brs)
ꢀ
ꢀ
ꢀ
ꢀ
12.40 (1H, brs)
8.04 (1H, brs)
ꢀ
157.0
95.8
ꢀ
154.7b
94.3
ꢀ
135.0
110.2
126.5
103.4
153.4
113.4
ꢀ
ꢀ
119.7
108.1
154.0
114.8
ꢀ
120.4
106.2
153.7
114.2
ꢀ
ꢀ
7.87 (1H, s)
ꢀ
7.77 (1H, brs)
ꢀ
6.43 (1H, brs)
ꢀ
6.94 (1H, d,
J = 8.6 Hz)
7.32 (1H, d,
J = 8.6 Hz)
ꢀ
6.99 (1H, brd,
J = 7.9 Hz)
7.45 (1H, d,
J = 7.9 Hz)
ꢀ
6.72 (1H, dd,
J = 8.6, 1.9 Hz)
7.41 (1H, d,
J = 8.6 Hz)
ꢀ
70
113.5
113.7
113.4
70a
80
127.9
152.3
127.5
157.9b
131.3
166.1
ꢀ
ꢀ
ꢀ
5-OH
50-OH
11.18 (1H, brs)
9.12 (1H, brs)
9.67 (1H, brs)
9.66 (1H, brs)
a Signals were overlapped with that of HOD. b Signals may be interchangeable.
Figure 5. Selected 2D NMR correlations for hyrtimomine C (3).
Figure 4. Δδ values [Δδ (in ppm) = δS ꢀ δR] obtained for the
(S)- and (R)-PGME amides (2a and 2b) of hyrtimoimine B (2).
moieties in 3 were confirmed by analysis of the 2D NMR
spectra measured in DMSO-d6, whereas no correlation sug-
gesting the connectivity of CH2-9 to the other atoms was
observed because of its broadening proton signal. On the
The 1H and 13C NMR spectra revealed the presence of one
ketone carbonyl group, one sp2 quaternary carbon, and
one sp3 methylene adjacent to a nitrogen atom as well as
two 5-hydroxyindole moieties (Table 1).
1
other hand, the H NMR spectrum measured in CD3OD
gave the sharp resonance of H2-9 {δH 4.38 (2H, s)}. In
the HMBC spectrum in CD3OD, correlations for H2-9 to
C-3, C-8, and C-80 were observed (Figure 5), indicating the
connectivities of C-3 to the sp2 methylene (C-9) through a
The structures of 3,4-disubstituted-5-hydroxyindole (N-1ꢀ
C-7a) and 3-monosubstituted-5-hydroxyindole (C-10ꢀC-70a)
2012
Org. Lett., Vol. 15, No. 8, 2013

ring system. Hyrtimomine C (3) is an alkaloid consisting of a
hydroxyindole and azepino-hydroxyindole moieties. These
alkaloids have an azepino-indole moiety in common, while
some azepino-indole alkaloids, hyrtiazepine,2c hyrtioreticu-
lins C and D,2a and clavicipitic acid,11 have been reported
to date.
Scheme 1. Possible Biogenetic Path of Hyrtimomines AꢀC
(1ꢀ3)
A possible biogenetic path of hyrtimomines AꢀC (1ꢀ3)
from hyrtiazepine is proposed as shown in Scheme 1.
Hyrtiazepine seems to be derived from 5-hydroxy-L-trypto-
phan and 5-hydroxyindole-3-aldehyde.3 Decarboxylation
and oxidation of hyrtiazepine might give hyrtimomine C (3).
Hyrtimomine B (2) might be derived by intramolecular
cyclization of hyrtiazepine and followed by decarboxylation
to generate hyrtimoimine A (1). Although the absolute
stereochemistry of hyrtiazepine has not been reported,2c the
absolute configuration of C-9 in hyrtimomine B (2) was
coincident with that of 5-hydroxy-L-tryptophan.
Hyrtimomine A (1) showed cytotoxicity against human
epidermoid carcinoma KB cells (IC50 3.1 μg/mL) and
muline leukemia L1210 cells (IC50 4.2 μg/mL) in vitro,
while hyrtimomines B (2) and C (3) did not show such
cytotoxicity (IC50 >10 μg/mL).
Acknowledgment. We thank S. Oka and A. Tokumitsu,
Equipment Management Center, Hokkaido University,
for measurements of MS spectra, and Z. Nagahama for his
help with sponge collection. This work was partly sup-
ported by a Grant-in-Aid for Scientific Research from the
Ministry of Education, Culture, Sports, Science and Tech-
nology of Japan.
ketone carbonyl group (C-8) and of C-9 to an sp2 quaternary
carbon (C-80) through a nitrogen atom (N-10). In addi-
tion, the connectivities among N-10, C-30, and C-4 via C-80
were disclosed by an HMBC cross-peak of H-20/C-80 and a
4J HMBC correlation for H-6/C-80. Therefore, the structure
of hyrtimomine C was elucidated to be 3.
Supporting Information Available. Experimental sec-
tion, 1D and 2D NMR spectra of hyrtimomines AꢀC and
the derivatives of hyrtimomine B. This material is avail-
able free of charge via the Internet at http://pubs.acs.org.
Hyrtimomines A (1) and B (2) are structurally unique
heteroaromatic alkaloids with a fused hexacyclic 6/5/6/6/7/5
(11) Robbers, J. E.; Otsuka, H.; Floss, H. G.; Arnold, E. V.; Clardy,
J. J. Org. Chem. 1980, 45, 1117–1121.
The authors declare no competing financial interest.
Org. Lett., Vol. 15, No. 8, 2013
2013