Biselyngbyaside, a macrolide glycoside from the marine cyanobacterium lyngbya sp.

Article abstract of DOI:10.1021/ol900579k

Bioassay-guided fractionation of the cytotoxic constituents of the marine cyanobacterium Lyngbya sp. led to the isolation of biselyngbyaside (1), a new 18-membered macrolide glycoside. The structure of 1 was established by spectroscopic analysis including

Full text of DOI:10.1021/ol900579k

ORGANIC
LETTERS
2009
Vol. 11, No. 11
2421-2424
Biselyngbyaside, a Macrolide Glycoside
from the Marine Cyanobacterium
Lyngbya sp.
Toshiaki Teruya,† Hiroaki Sasaki,† Kazuhiro Kitamura,† Takeshi Nakayama,‡ and
Kiyotake Suenaga†,*
Department of Chemistry, Faculty of Science and Technology, Keio UniVersity,
Hiyoshi 3-14-1, Kohoku-ku, Yokohama 223-8522, Japan, and Institute of Biological
Sciences, UniVersity of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan
suenaga@chem.keio.ac.jp
Received March 20, 2009
ABSTRACT
Bioassay-guided fractionation of the cytotoxic constituents of the marine cyanobacterium Lyngbya sp. led to the isolation of biselyngbyaside
(1), a new 18-membered macrolide glycoside. The structure of 1 was established by spectroscopic analysis including 2D-NMR techniques and
by synthetic studies. Biselyngbyaside (1) exhibits broad-spectrum cytotoxicity in a human tumor cell line panel.
The oceans are a good source of new biologically active
substances. Over the past few decades, a considerable number
of marine natural products have been reported.1 Several
compounds that are now used at the clinical trial level,2 such
as halichondrin B,3 bryostatin 1,4 and dolastatin 10,5 were
originally found in marine animals. Therefore, marine natural
products continue to receive increasing attention. In particu-
lar, cyanobacteria are prolific producers of bioactive second-
ary metabolites.6
macrolides have been isolated from marine sponges. These
include 18-membered macrolides, tedanolide9 and 13-deox-
ytedanolide9 from Tedania ignis and Mycale adhaerens,
respectively, laulimalide,9 a 20-membered ring from Caco-
spongia mycofijiensis, Hyatella sp., Fasciospongia sp.,
Dactylospongia sp., and a chromodorid nudibranch, and
peloruside A,10 a 16-membered ring macrolide from Mycale
hentscheli. Biselyngbyaside (1) has the same macrolide ring-
size as tedanolide and 13-deoxytedanolide. We report here
the isolation, structure determination, and biological activity
of 1.
In our ongoing efforts to identify novel marine cyanobac-
terial metabolites with antitumor activity, we found bise-
lyngbyaside (1). Over the past few years, several classes of
The marine cyanobacterium Lyngbya sp. (a voucher
specimen was deposited at Keio University), collected in
Okinawa Prefecture, was extracted with methanol. The
concentrated extract was partitioned between ethyl acetate
and water, and the ethyl acetate layer was concentrated and
partitioned between n-hexane and 90% aqueous methanol.
The concentrated 90% aqueous methanol layer was subjected
to fractionation guided by cytotoxicity against HeLa S3 cells
† Keio University.
‡ University of Tsukuba.
(1) Blunt, J. W.; Copp, B. R.; Hu, W.-P.; Munro, H. H. G.; Northcote,
P. T.; Prinsep, M. R. Nat. Prod. Rep. 2009, 26, 170–244.
(2) Molinski, T. F.; Dalisay, D. S.; Lievens, S. L.; Saludes, J. P. Nat.
ReV. Drug DiscoVery 2009, 8, 69–85.
(3) (a) Uemura, D.; Takahashi, K.; Yamamoto, T.; Katayama, C.;
Tanaka, J.; Okumura, Y.; Hirata, Y. J. Am. Chem. Soc. 1985, 107, 4796–
4798. (b) Hirata, Y.; Uemura, D. Pure Appl. Chem. 1986, 58, 701–710.
10.1021/ol900579k CCC: $40.75
Published on Web 05/11/2009
 2009 American Chemical Society

Table 1. NMR Spectral Data of 1 in CD3OD
position
1H (ppm)a
13C (ppm)b
HMBCc
H-2, 3, 17
1
172.1
42.9
2a
2b
3
4
5
6a
6b
7
8
9
10
11a
11b
12
13
14
15
16a
16b
17
18
19
20a
20b
21
22
23
24
25
26
27
1′
2.32 m
2.57 dd (14.7, 7.8)
4.50 m
5.42 m
5.44 m
2.31 m
2.32 m
3.47 m
77.4
131.9
129.3
36.8
H-4, 1′, 2a, 2b
H-3, 5
H-4, 6a, 6b
H-7, 5
89.0
133.0
138.4
34.0
H-6a, 6b, 9, 24, 25
H-6, 7, 25
H-7, 25, 26
H-9, 11a, 26
H-9, 13, 26
5.10 d (8.8)
2.68 m
1.94 m
2.30 m
5.55 m
6.05 dd (17.6, 10.3)
6.00 dd (17.6, 10.3)
5.52 m
2.22 m
2.39 m
41.5
Figure 1. Gross structure of 1 determined by 2D-NMR spectroscopy
1
133.3
135.3
132.1
132.7
39.6
H-11a
(bold lines, H-1H COSY; arrows, HMBC correlations).
H-16a, 16b
H-14, 15
with column chromatography (ODS silica gel, methanol-
water) and reversed-phase HPLC to give biselyngbyaside (1)
as a colorless oil. Biselyngbyaside (1) exhibited cytotoxicity
against HeLa S3 cells (IC50 ) 0.1 µg/mL).
5.51 m
5.13 d (8.8)
72.0
124.9
140.1
36.6
H-1’
H-20a, 20b, 27
H-20a, 20b
H-27, 18, 21
2.72 dd (14.7, 5.9)
2.92 dd (14.7, 6.8)
5.40 m
5.50 m
1.64 d (5.9)
3.15 s
1.55 s
1.02 d (6.3)
1.67 s
4.25 d (7.8)
3.22 dd (9.3, 7.8)
3.04 dd (9.3, 9.3)
3.44 dd (9.3, 8.8)
3.18 m
3.73 dd (11.7, 4.4)
3.85 dd (11.7, 2.0)
3.62 s
127.9
127.4
18.1
55.6
10.2
22.5
23.6
100.9
74.5
87.7
70.7
77.6
62.4
H-20a, 20b, 23
H-20a, 20b, 23
H-21, 22
H-7
H-7, 9
H-18, 20a, 20b
H-2’
H-1′, 3′, 4’
H-1′, 2′, 4′, 7’
H-3′
2′
3′
4′
5′
6’a
6’b
7′
The molecular formula of 1 was determined to be C34H52O9
by ESIMS [(M + Na)+, m/z 627.3487, ∆-2.2 mmu]. The
IR spectrum contained absorption bands for hydroxyl and
ester groups [3589, 3502 (br), 1725 cm-1]. The NMR data
for 1 are summarized in Table 1. The 1H NMR spectrum of
1 showed the presence of six methyl groups (δ 3.62, 3.15,
1.67, 1.64, 1.55, and 1.02). In the 13C NMR spectrum, 34
carbon signals were observed, including one carbonyl carbon
(δ 172.1), 12 olefinic carbons (δ 140.1, 138.4, 135.3, 133.3,
133.0, 132.7, 132.1, 131.9, 129.3, 127.9, 127.4, and 124.9),
and six methyl carbons (δ 61.0, 55.6, 23.6, 22.5, 18.1, and
10.2). The remaining carbon signals were assigned to six
methylenes and nine methines, based on the results of an
HMQC experiment. The presence of a 3-O-methylglucoside
moiety in 1 was readily revealed based on the coupling
constants in the 1H NMR spectra (Figure 1) and correlations
in the 1H-1H COSY spectra, and their ꢀ-anomeric structures
were determined by correlations in the NOESY spectrum
H-4’
H-4’
61.0
H-3′
a Recorded at 400 MHz. Coupling constants (Hz) are in parentheses.
b Recorded at 100 MHz. c Protons correlated to carbon resonances in 13C
column. Parameters were optimized for JCH ) 6 Hz.
and the 3-O-methylglucoside moiety in 1 were clarified by
HMBC correlations: H1′/C2, H25/C7, H25/C8, H25/C9,
H27/C18, H27/C19, and H27/C20. HMBC correlations H2/
C1 suggested the connectivity of C1-C2. Consequently, the
entire carbon chain was assembled, and all protons and
carbons were assigned, as shown in Figure 1 and Table 1,
respectively. The HMBC correlations H24/C7, H7/C24, H7′/
C3′, and H3′/C7′ determined that the two methoxy groups
were located at C7 and C3′. On the basis of its molecular
formula and degree of unsaturation, 1 should contain a
lactone ring. Considering the chemical shift of H17 (δH 5.51)
and the HMBC correlation H17/C1, the lactone ring must
be formed between C1 and C17. The double bond geometries
in 1 were determined from proton-proton coupling constants
and the NOE correlation from NOESY data as measured in
1
(H1′/H3′ and H1′/H5′) and the JCH value at the anomeric
carbons (δC1′ 160 Hz).11 This result was also supported by
the 1H NMR spectrum, where the magnitude of 3JH1′-2′ was
7.8 Hz as measured in CD3OD. Regarding the macrolide
moiety, three sets of proton spin systems, C2-C7, C9-C18,
and C20-C23, were determined based on the 1H-1H COSY
spectrum. The connectivities between these partial structures
2422
Org. Lett., Vol. 11, No. 11, 2009

Scheme 1. Degradation Experiment of Biselyngbyaside (1)
C6D6 (see Supporting Information). The geometries of the
two trisubstituted olefins in 1 were clarified to be 8E and
18Z, respectively, based on the NOESY correlation of H25/
H10, and H27/H18. The 4E, 12E, and 14E geometries were
determined from the H4-H5, H12-H13, and H14-H15
trans-coupling constants (15.1, 16.6, and 16.6 Hz, respec-
tively). In addition to these results, NOESY correlations were
observed between H13 and H11, and between H14 and H16.
A careful analysis of NOESY correlation observed between
H21 and H23 revealed a 21E geometry. Thus, the gross
structure of biselyngbyaside (1) was determined to be as
shown in Figure 1.
assigned based on the 2D-NMR spectra, and the ∆δ values
(δS-δR, Hz) were then calculated. These results established
Figure 2. ∆δ values (∆δS-R) in ppm for bis-S- and bis-R-MTPA
esters (3 and 4, 7 and 8).
The absolute stereochemistries of C3, C17, and the 3-O-
methylglucoside moiety in 1 were determined by the modi-
fied Mosher’s method12 and synthetic means. Methanolysis
of 1 gave methyl ester 2 along with elimination of the sugar
(Scheme 1). Since 1 is unstable under acidic and basic
conditions, hydrogenation and subsequent acid hydrolysis
provided 3-O-methylglucoside 5 and ꢀ-hydroxy methyl ester
6 accompanied by hydrogenolysis at C7 and C17 (Scheme
1). Derived products 2 and 6 were transformed into the (R)-
and (S)-MTPA esters, 3 and 4, and 7 and 8, respectively
that the absolute stereochemistries of C3 and C17 in 1 were
3R and 17R, respectively (Figure 2). Furthermore, tribro-
mobenzoylation of 5 afforded methyl 2,4,6-tri-O-(4-bro-
mobenzoyl)-3-O-methyl-R-D-glucose 9, which was identified
by 1H NMR data and CD data (see Supporting Information).
An authentic sample of 9 was prepared from D-glucose.
The absolute stereochemistries of C7 and C10 in 1 were
determined by enantioselective synthesis of the degradation
product from natural 1. The oxidative degradation of 1 into
biscarbamates 10 was effected by a three-step sequence.
The synthesis of degradation product 10 started from
known aldehyde 11a.13 The (2R)-aldehyde 11a was con-
verted into aldehyde 12 in three steps. Treatment of 12 with
1
(Scheme 1). The H NMR signals of these esters were
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Org. Lett., Vol. 11, No. 11, 2009
2423

LiCH2COOtBu gave a mixture of diastereomeric alcohols
13a and 13b, which was separated by silica gel column
chromatography. The stereochemistries of the hydroxyl
groups in 13a and 13b were determined by the modified
Mosher’s method.12 Conversion of 13a and 13b into the
respective biscarbamates 14a and 14b was achieved by a
Biselyngbyaside (1) exhibited cytotoxicity against HeLa
S3 cells with an IC50 value of 0.1 µg/mL. Biselyngbyaside
(1) was evaluated against a disease-oriented panel composed
of 39 human cancer cell lines (HCC panel) at the Japanese
Foundation for Cancer Research (see Supporting Informa-
tion).14 The average GI50 value across all of the cell lines
tested was 0.60 µM, and 1 exhibited differential cytotoxici-
ties: the central nervous system cancer SNB-78 (GI50 0.036
µM) and lung cancer NCI H522 (GI50 0.067 µM) were
especially sensitive. Biselyngbyaside (1) was COMPARE-
negative,14 indicating that it likely inhibits cancer cell
proliferation through a novel mechanism.
1
four-step sequence (Scheme 2). The H NMR data of 14a
Scheme 2
.
Synthesis of Degradation Product of Natural
Biselyngbyaside (1)
In summary, biselyngbyaside (1), with a potentially new
mechanism of action, was isolated from the marine cyano-
bacterium Lyngbya sp. The structure of biselyngbyaside (1)
was determined based on 2D-NMR spectra and synthetic
procedures. Biselyngbyaside (1) exhibits broad-spectrum
cytotoxicity in a human tumor cell line panel.
Acknowledgment. We thank Professor Isao Inoue (Univ.
of Tsukuba) for identifying the cyanobacterium. We also
thank Professor Hideo Kigoshi (Univ. of Tsukuba) for
performing the ESIMS analysis. We thank Dr. T. Yamori
(Screening Committee of Anticancer Drugs supported by
Grant-in-Aid for Scientific Research on Priority Area “Can-
cer” from The Ministry of Education, Culture, Sports,
Science and Technology, Japan) for screening in the human
cancer cell line panel. This work was supported by a Grant-
in-Aid for Young Scientists (B) from the Ministry of
Education, Culture, Sports, Science and Technology, Jaoan,
Keio Leading-edge Laboratory of Science and Technology,
Keio Gijuku Academic Development Funds, and Suntory
Institute for Bioorganic Research.
fully agreed with those of degradation product 10 from
natural biselyngbyaside (1). To determine the absolute
stereochemistry of 10, the enantiomeric (3R, 6R)-biscarbam-
ate ent-14a was prepared from (2S)-aldehyde 11b in the same
manner as described above, and both enantiomers were
analyzed by chiral HPLC. Of the two synthetic enantiomers
14a and ent-14a, the retention time of ent-14a was identical
to that for 10 from the natural product, which established
the absolute stereochemistry of 10. Based on these findings,
the complete stereostructure of biselyngbyaside (1) was
determined as shown in 1.
Supporting Information Available: Detailed experimen-
tal procedures, spectroscopic data and HCC panel data for
1. This material is available free of charge via the Internet
at http://pubs.acs.org.
OL900579K
(14) Yamori, T.; Matsunaga, A.; Sato, S.; Yamazaki, K.; Nakanishi, O.;
Kohno, H.; Nakajima, Y.; Komatsu, H.; Andoh, T.; Tsuruo, T. Cancer Res.
1999, 59, 4042–4049.
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Org. Lett., Vol. 11, No. 11, 2009