Hyrtioseragamines A and B, new alkaloids from the sponge hyrtios species

Article abstract of DOI:10.1021/ol102867x

Two novel alkaloids with a furo[2,3-b]pyrazin-2(1H)-one moiety and a guanidino group, hyrtioseragamines A (1) and B (2), have been isolated from an Okinawan marine sponge Hyrtios species. The structures of 1 and 2 were elucidated on the basis of spectrosc

Full text of DOI:10.1021/ol102867x

ORGANIC
LETTERS
2011
Vol. 13, No. 4
628–631
Hyrtioseragamines A and B, New Alkaloids
from the Sponge Hyrtios Species
Yohei Takahashi,† Yoshiro Iinuma,† Takaaki Kubota,† Masashi Tsuda,†
Mitsuhiro Sekiguchi,‡ Yuzuru Mikami,§ Jane Fromont, and Jun’ichi Kobayashi*,†
Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812,
Japan, Astellas Pharm, Inc., Tsukuba 350-8585, Japan, Medical Mycology Research Center,
Chiba University, Chiba 260-0856, Japan, and Western Australian Museum, Perth,
WA 6000, Australia
jkobay@pharm.hokudai.ac.jp
Received November 27, 2010
ABSTRACT
Two novel alkaloids with a furo[2,3-b]pyrazin-2(1H)-one moiety and a guanidino group, hyrtioseragamines A (1) and B (2), have been isolated from
an Okinawan marine sponge Hyrtios species. The structures of 1 and 2 were elucidated on the basis of spectroscopic data and chemical
conversions. Compounds 1 and 2 are the first natural products possessing a furo[2,3-b]pyrazine-related moiety.
Marine sponges have been recognized as a rich source of
bioactive secondary metabolites with unprecedented skele-
tons.1 During our continuing search for bioactive substances
from marine sponges,2 we have investigated extracts of an
Okinawan marine sponge Hyrtios sp. (SS-985) and isolated
two novel alkaloids, hyrtioseragamines A (1) and B (2),
possessing a furo[2,3-b]pyrazin-2(1H)-one and a guanidino
group. Here we describe the isolation and structure elucida-
tion of 1 and 2.
The sponge Hyrtios sp. (SS-985, 2.85 kg) collected off
Seragaki, Okinawa was extracted with MeOH. The MeOH
extract was partitioned between n-hexane and H2O, and
the aqueous layer was successively extracted with CHCl3,
EtOAc, and n-BuOH. CHCl3- and n-BuOH-soluble mate-
rials of the extract were subjected to C18 column chroma-
tography followed by repeated C18 HPLC to afford hyrtio-
seragamines A (1, 12.6 mg, 4.4 ꢀ 10-4% wet weight)3 and
B (2, 2.2 mg, 7.7 ꢀ 10-5%)4 together with known β-carbo-
line alkaloids, gesashidine A5 and dragmacidonamines
A and B.6
(3) Hyrtioseragamine A (1): yellow amorphous solid; UV (MeOH)
max 214 (log ε 4.3), 238 (4.1 sh), 270 (3.9), and 389 nm (4.1); IR (film)
λ
νmax 3343, 3171, 2922, 2604, 1645, 1548, and 1470 cm-1
;
1Hþand 13C
NMR (CD3OD) see Table 1; ESIMS (pos.) m/z 327 [MþH] ; HRE-
SIMS (pos.) m/z 327.15700 ([MþH]þ, calcd for C16H19N6O2,
327.15640).
(4) Hyrtioseragamine B (2): yellow amorphous solid; UV (MeOH)
max 222 (log ε 4.4), 250 (4.1 sh), 280 (3.8 sh), and 373 nm (4.0); IR (film)
λ
ν
max 3353, 3152, 2936, 2722, 1680, 1631, 1554, and 1453 cm-1; 1H and
† Hokkaido University.
13C NMR (CD3OD) see Table 1; ESIMS (pos.) m/z 497 [MþH]þ;
HRESIMS (pos.) m/z 497.20467 ([MþH]þ, calcd for C26H25N8O3,
497.20441).
‡ Astellas Pharm, Inc.
§ Chiba University.
Western Australian Museum.
(5) Iinuma, Y.; Kozawa, S.; Ishiyama, H.; Tsuda, M.; Fukushi, E.;
Kawabata, J.; Fromont, J.; Kobayashi, J. J. Nat. Prod. 2005, 68, 1109–
1110.
(6) Pedpradab, S.; Edrada, R.; Ebel, R.; Wrey, V.; Proksch, P. J. Nat.
Prod. 2004, 67, 2113–2116.
(1) Blunt, J. W.; Copp, B. R.; Munro, M. H. G.; Northcote, P. T.;
Prinsep, M. R. Nat. Prod. Rep. 2010, 27, 165–237.
(2) Kura, K.; Kubota, T.; Fromont, J.; Kobayashi, J. Bioorg. Med.
Chem. Lett. 2011, 21, 267–270.
r
10.1021/ol102867x
Published on Web 01/19/2011
2011 American Chemical Society

Table 1. 1H and 13C NMR Data of Hyrtioseragamines A (1) and B (2) in CD3OD at 300 Ka
1
2
HMBC
position
δ
C (mult.)
δH (mult., J in Hz)
HMBC (H to C)
position
δ
C (mult.)
δ
H (mult., J in Hz)
(H to C)
1
144.6
120.6
132.6
121.8
129.9
117.4
158.5
100.3
127.8
159.1
148.7
148.2
31.3
C
1
135.4
131.2
133.3
131.6
130.9
129.1
154.8
103.2
127.3
158.9
150.9
148.6
31.3
C
2
CH
CH
CH
CH
C
7.03 (brd, 8.0)
4, 6
2
CH
CH
CH
CH
C
7.67 (m)
7.72 (m)
7.71 (m)
8.16 (m)
1, 4, 6
3
7.25 (ddd, 8.0, 7.8, 1.4)
6.91 (ddd, 7.8, 7.6, 1.0)
7.64 (dd, 7.8, 1.4)
1, 2, 5
2, 3, 5, 6
1, 3, 7
3
1
4
4
6
5
5
1, 3, 7
6
6
7
C
7
C
8
CH
C
6.94 (s)
7, 8a, 12a
8
CH
C
6.88 (s)
7, 12a
8a
10
11
12a
14
15
16
18
8a
10
11
12a
14
15
16
18
20
30
40
40a
50
C
C
C
C
C
C
CH2
CH2
CH2
C
2.91b (t, 7.2)
2.08b (quin, 7.2)
3.34b (t, 7.2)
10, 11, 15, 16
11, 14, 16
CH2
CH2
CH2
C
2.82b (t, 7.2)
1.97b (quin, 7.2)
3.25b (t, 7.2)
10, 11, 15, 16
11, 14, 16
27.8
27.6
42.7
14, 15, 18
42.6
14, 15, 18
159.6
159.6c
146.3
100.3
159.5c
119.6
124.8
130.4
136.9
123.1
140.9
164.0
C
CH
C
7.15 (s)
20, 40, 40a, 20-CO
C
CH
CH
CH
CH
C
8.71 (d, 8.2)
40, 40a, 70, 80a
40a, 50, 60, 80
50, 80a
60
70
80
80a
20-CO
7.91 (dd, (8.2, 7.8)
8.12 (dd, 7.8, 8.4)
8.25 (d, 8.4)
40,d 40a, 60, 70
C
a 1H and 13C NMR spectra were recorded at 600 and 150 MHz, respectively. b 2H. c Interchangeable. d The correlation observed through four bonds
due to a long-range coupling.
The molecular formula, C16H18N6O2, of hyrtioseragamine
A (1) was established by HRESIMS (m/z 327.15700
[MþH]þ, Δ þ0.60 mmu). ESIMS (m/z 335 [MþD]þ) using
CD3OD as a mobile phase revealed that 7 out of 18 hydrogen
atoms were H/D exchangeable. IR absorptions indicated
the existence of OH and/or NH (νmax 3343 and 3171 cm-1
)
groups and a carbonyl group (1645 cm-1), while UV absorp-
tions (λmax 270 and 389 nm) were attributed to a conjugated
aromatic functionality.
The 1H NMR (Table 1) spectrum of 1 measured in CD3-
OD contained eight proton signals, three of which were
sp3 methylene protons and five of which were sp2 methine
protons. In the 13C NMR (Table 1) spectrum of 1, three sp3
methylenes were observed in the aliphatic carbon region
from δC 20 to 50. On the other hand, five sp2 methines and
eight sp2 quaternary carbons were observed in aromatic and
carbonyl carbons region from δC 100 to 160. The sp2 qua-
ternary carbon observed in the low-field region (C-18, δC
159.6) and a positive coloration in the Sakaguchi test implied
the presence of a guanidino group.
The 1H-1H COSY and HMBC spectrum of 1 in CD3OD
at 300 K revealed the presence of a 1,2-disubstituted ben-
zene ring (C-1-C-6) (Figure 1a). The chemical shift at C-1
(δC 144.6) suggested that an amino group was attached to
C-1. The connectivity of C-6 to C-8 was elucidated from
HMBC cross-peaks of H-5/C-7 and H-8/C-7 and the
Figure 1. Selected 2D NMR correlations for two partial struc-
tures (a and b) of hyrtioseragamine A (1) in CD3OD at 300 K.
ROESY correlation of H-5/H-8. The chemical shift of C-7
(δC 158.5) indicated that C-7 was connected to an oxygen
atom (O-13). H-8 showed a large 1JCH value (180.0 Hz) and
HMBC cross-peaks to C-8a (δC 127.8) and C-12a (δC 148.2),
indicating the existence of a 2,3,5-trisubstituted furan ring.
1
On the other hand, H-1H COSY correlations and the
HMBC correlation of H-16/C-18 suggested the existence of
a 1-propylguanidino group (Figure 1b). The connection of
C-14 to C-10 via C-11 was disclosed by HMBC correlations
of H2-14/C-10, H2-14/C-11, and H2-15/C-11. The strong IR
absorption at 1645 cm-1 and the chemical shift of C-10 (δC
159.1) and C-11 (δC 148.7) indicated that C-10 and C-11 were
an amide carbonyl and an imino carbon, respectively.
Org. Lett., Vol. 13, No. 4, 2011
629

Furthermore, it was implied by the molecular formula of
hyrtioseragamine A (1) that C-10 and C-11 in the partial
structure b were connected to C-8a or C-12a in the partial
structure a via N-9 and N-12 (Figure 1).
Figure 3. Selected 2D NMR correlations for hyrtioseragamine B
(2) in CD3OD at 300 K.
H-30/C-40, and H-30/C-40a, the chemical shifts of C-20 (δC
146.3) and C-80a (δC 140.9), and the 1JCH value of CH-30
(169.8 Hz). Given the molecular formula of 2, chemical shifts
of C-20 (δC 146.3) and C-40 (δC 159.5), and the HMBC cross-
peak of H-30 to a carbonyl carbon (C-20-CO, δC 164.0), it was
indicated that an amino carbonyl group and an amino group
were attached to C-20 and C-40, respectively. Additionally, the
ROESY correlation of H-2/H-30 implied the connectivity of
C-1 and C-40 via a secondary amino group (Figure 3).
Figure 2. Methylation of hyrtioseragamine A (1) and key 2D
NMR correlations for septamethyl derivative (3c) of hyrtioser-
agamine A (1) in DMSO-d6 at 350 K.
To elucidate the structure of hyrtioseragamine A, 1 was
methylated by methyl iodide under basic conditions to
afford three methylated derivatives 3a-3c (Figure 2). The
HMBC spectrum of 3c recorded in DMSO-d6 at 350 K
showed cross-peaks of N-9-Me/C-8a, N-9-Me/C-10, H2-14/
C-10, H2-14/C-11, and H2-15/C-11 (Figure 2). In addition,
N-9-Me showed the NOESY correlation to H-8. These
correlations disclosed that C-11 was connected to C-8a
and C-12a via an amide bond (N-9 and C-10) and an imine
nitrogen atom (N-12), respectively. Thus, the structure of
hyrtioseragamine A was elucidated to be 1.
Figure 4. Chemical conversion of hyrtioseragamine B (2) into
pyrimidine derivative (4) and key 2D NMR correlations for 4 in
C5D5N at 300 K.
To confirm the structure of hyrtioseragamine B (2) by
2D NMR cross-peaks from exchangeable protons, 2 was
treated with 2,4-pentanedione under basic conditions to
give a pyrimidine derivative (4) of 2 (Figure 4). Detailed
analysis of 1D and 2D NMR spectra of 4 in C5D5N at
300 K supported the structure of 2. The NH proton (δH
9.85) in 4 exhibited HMBC correlations to C-2, C-6, C-30,
C-40, and C-40a, indicating the connection of C-1 and C-40
via a secondary amino group. This connectivity was sup-
ported by ROESY correlations from C-1-NH to H-8 and
H-50. Furthermore, the presence of two exchangeable
protons coupling to each other (δH 8.88 and 8.48, J =
3.8 Hz) and the HMBC correlation from one of these (δH
8.48) and H-30 to C-20 and a carbonyl carbon (C-20-CO, δC
168.0), respectively, suggested the existence of a primary
amide group at C-20. Thus, the structure of hyrtioseraga-
mine B was elucidated to be 2.
IR and UV spectra of hyrtioseragamine B (2) were
similar to those of 1, implying the existence of OH and/
or NH (νmax 3353 and 3152 cm-1), carbonyl group(s)
(νmax 1631 cm-1), and a conjugated aromatic system
(λmax 280 and 373 nm). The HRESIMS of 2 showed the
molecular formula to be C26H24N8O3 (m/z 497.20467
[MþH]þ, Δ þ0.26 mmu).
1
The H NMR (Table 1) spectrum of 2 included 10 sp2
methine and 3 sp3 methylene protons, while the 13C NMR
(Table 1) spectrum of 2 showed 13 sp2 quaternary carbons,
10 sp2 methines, and 3 sp3 methylenes. These data indicated
that 2 had another aromatic ring in addition to the same
partial structure as that of 1.
Inspection of the 1H-1H-COSY, HMBC, and ROESY
spectra of 2 in CD3OD at 300 K and comparison of 1H and
13C NMR data of 2 with those of 1 disclosed that 2 had the
same partial structure as that of 1 (C-1-C-18) (Figure 3).
Another 1,2-disubstituted benzene ring (C-40a-C-80a) in 2
was revealed by the 1H-1H COSY and HMBC spectra. The
presence of a 2,4-disubstituted quinoline ring (C-10-C-80)
was deduced by HMBC correlations of H-50/C-40, H-30/C-20,
To the best of our knowledge, hyrtioseragamines A (1)
and B (2) are the first natural products possessing a furo-
[2,3-b]pyrazine-related moiety. In addition, quinolinecarboxylic
(7) Yin, S.; Boyle, G. M.; Carroll, A. R.; Kotiw, M.; Dearnaley, J.;
Quinn, R. J.; Davis, R. A. J. Nat. Prod. 2010, 73, 1586–1589 and
references therein.
630
Org. Lett., Vol. 13, No. 4, 2011

A diketopyperazine ring might be formed by kynure-
nine, which is a metabolite of tryptophan, and arginine
and nucleophilic addition of O-13 to C-7 followed by
aromatization to produce 1. Hyrtioseragamine B (2)
might be generated by nucleophilic addition of a pri-
mary amino group attached to C-1 of 1 to the carbonyl
group of C-4 in a quinolone form of the kynurenic
acid derivative. Since the diketopyperazine moiety is
well-known to be a microbial metabolite, compounds 1
and 2 might be produced by symbiotic microbes of the
sponge.
Scheme 1. Possible Biogenetic Path for Hyrtioseragamines A (1)
and B (2)
Hyrtioseragamines A (1) and B (2) showed antimicro-
bial activities against Aspergillus niger (MIC, 8.33 and
16.6 μg/mL, respectively) and Cryptococcus neoformans
(MIC, 33.3 and 16.6 μg/mL, respectively). Compounds 1
and 2 did not show cytotoxicity (IC50 > 10 μg/mL) against
murine lymphoma L1210 and human epidermoid carcino-
ma KB cells in vitro.
Acknowledgment. The authors thank Ms. S. Oka, Ms.
A. Tokumitsu, and Ms. T. Komiya, Instrumental Analysis
Division, Equipment Management Center, Creative Re-
search Institution, Hokkaido University, for measurements
of ESIMS; Dr. Eri Fukushi, Graduate School of Agricul-
ture, Hokkaido University, for measurements of a part of
NMR spectra; and Mr. Z. Nagahama and Mr. S. Furugen
for their help with collection of the sponges. This work was
partly supported by a research fellowship for young scien-
tists from the Japan Society for the Promotion of Science (to
Y.T.) and a Grant-in-Aid for Science Research from the
Ministry of Education, Culture, Sports, Science, and Tech-
nology of Japan.
Supporting Information Available. Detailed experimen-
tal section and 1D and 2D NMR data for hyrtioseragamines
A and B and their derivatives. This material is available free
of charge via the Internet at http://pubs.acs.org.
acid derivatives from marine organisms are very rare.7
Biogenetically, 1 might be generated from tryptophan and
arginine (Scheme 1).
Org. Lett., Vol. 13, No. 4, 2011
631