Isolation, structure determination, and synthesis of neodysiherbaine A, a new excitatory amino acid from a marine sponge.

Article abstract of DOI:10.1021/ol015798l

[structure: see text] A new excitatory amino acid, neodysiherbaine A (2), was isolated as a minor constituent of the aqueous extract from the marine sponge Dysidea herbacea. The structure was deduced by spectroscopic methods and established unambiguously

Full text of DOI:10.1021/ol015798l

ORGANIC
LETTERS
2001
Vol. 3, No. 10
1479-1482
Isolation, Structure Determination, and
Synthesis of Neodysiherbaine A, a New
Excitatory Amino Acid from a Marine
Sponge
,†
,‡
Ryuichi Sakai,* Tatsuki Koike,‡ Makoto Sasaki,* Keiko Shimamoto,§
Chie Oiwa,† Atsuko Yano,‡ Katsuji Suzuki,† Kazuo Tachibana,‡ and
Hisao Kamiya†
School of Fisheries Science, Kitasato UniVersity, Sanriku-cho, Iwate 022-0100, Japan,
Department of Chemistry, Graduate School of Science, The UniVersity of Tokyo, and
CREST, Japan Science and Technology Corporation (JSP), Hongo, Bunkyo-ku, Tokyo
113-0033, Japan, and Suntory Institutes for Bioorganic Research, Mishima-gun,
Osaka 618-8503, Japan
msasaki@chem.s.u-tokyo.ac.jp
Received March 6, 2001
ABSTRACT
A new excitatory amino acid, neodysiherbaine A (2), was isolated as a minor constituent of the aqueous extract from the marine sponge
Dysidea herbacea. The structure was deduced by spectroscopic methods and established unambiguously by the total synthesis. The present
synthesis, including as a key step cross-coupling of the 6/5-bicyclic core with an amino acid residue, is useful in constructing its structural
analogues.
In the course of our study to find neurologically active
secondary metabolites from marine organisms, we have
isolated a novel glutamate receptor agonist, dysiherbaine (1),
as a potent epileptogenic amino acid from the marine sponge
Dysidea herbacea.1 Dysiherbaine induces characteristic
epilepsy-like seizures in mice and has been characterized to
be the most potent epileptogenic excitatory amino acid yet
identified. Dysiherbaine activates neuronal non-NMDA type
glutamate receptors (GluR), namely, AMPA and kainic acid
(KA) receptors with considerable preference over KA
receptors.2 Moreover, Swanson and co-workers have recently
demonstrated highly unusual actions of 1 toward recombinant
KA receptors; in that, 1 could differentially activate one of
the activation sites within the subunit of a heteromeric
GluR5/KA2 receptor complex.3 This discrete affinity of 1
has enabled characterization of the unexpectedly complex
behavior of the heteromeric KA receptors. Because of these
unusual pharmacological properties of 1 to KA receptors,
and its potent epileptogenic action, 1 is anticipated to be a
useful tool to investigate GluR in the central nervous system.
These biological properties of 1 as well as the unique
structure have attracted the keen attention of synthetic
† Kitasato University.
(2) Sakai, R.; Swanson, G. T.; Shimamoto, K.; Contractor, A.; Ghetti,
A.; Tamura-Horikawa, Y.; Oiwa , C.; Kamiya, H. J. Pharm. Exp. Ther.
2001, 296, 655-663.
(3) Swanson, G. T. Abstracts of Papers, the 30th Annual Meeting of the
Society for Neuroscience, 2000.
‡ The University of Tokyo and CREST.
§ Suntory Institute for Bioorganic Research.
(1) Sakai, R.; Kamiya, H.; Murata, M.; Shimamoto, K. J. Am. Chem.
Soc. 1997, 119, 4112-4116.
10.1021/ol015798l CCC: $20.00 © 2001 American Chemical Society
Published on Web 04/14/2001

chemists. Recently, several groups, including us,4 have
independently accomplished the total synthesis of 1.5 This
work not only confirmed its chemical structure but also
defined the absolute stereochemistry. However, further
chemical studies are required to uncover the structure-
activity relationships (SAR) of 1, which may lead to
development of more selective GluR ligands.
We therefore searched for minor neuroactive components
in the aqueous extract of D. herbacea using the mouse assay
as a guide and found a new dysiherbaine analogue, neo-
dysiherbaine A (2), as a minor constituent. We now report
the isolation, structure determination, and total synthesis of
2 (Figure 1).
overall spectral pattern of 2 was similar to that of 1,
indicating that the structure of 2 is closely related to that of
1, although a significant difference, lack of a methyl signal,
was evident. A COSY experiment assigned all the non-
exchangeable proton signals, whereby large differences in
chemical shifts were observed in some signals for 1 and 2;
H-8 (∆δ1-2 ) -0.2), H-7 (+0.2), and H-9 (+0.3). These
shifts along with the difference in molecular formulas
between 1 and 2 (less CH3N with an additional oxygen in 2
in comparison with 1) allowed us to assign a hydroxyl group
at C8. The relative stereochemistry of the perhydrofuro[b]-
pyran ring is assigned to be the same as that for 1, since the
coupling pattern of all the protons on this ring system is
nearly identical to that of 1. However, we could not
determine the stereochemistry at quaternary C4 and correlate
stereochemistry at C2 to the bicyclic portion because a
minute amount of 2 hampered further 2D NMR studies. We
thus carried out the total synthesis of 2 to resolve the
remaining stereochemical ambiguities and ultimately provide
access to additional material for biological evaluation.
We decided to synthesize 2a as the most likely candidate.
The present synthesis relied on a strategy developed for the
total synthesis of 1,5 which was designed to be applicable
to a variety of its analogues. We envisioned that 2a would
be constructed by cross-coupling of organozinc compound
3 and vinyl triflate 4, which would be accessed from a
carbohydrate precursor (Scheme 1).
Figure 1. Structures of dysiherbaine (1) and neodysiherbaine A
(2).
Dysidea herbacea collected in Yap State, Micronesia, in
July 1998 was homogenized with water. The aqueous extract
was separated by a series of gel filtration chromatography
(Sephadex LH20 and BioGel P2). Fractions were pooled
according to their TLC profile and bioactivity. Purification
of the most active fraction afforded 1 (70 ppm of the wet
sponge). Another bioactive fraction eluted before 1 on the
BioGel P2 column was separated by DE52 anion exchange
Scheme 1. Retrosynthesis of NeodysiherbaineA (2a)
chromatography (Whattman). The UV (342 nm), 1H and 13
C
NMR, and ESIMS (m/z 330) spectra of the bioactive fraction
indicated that it contained shinorine (or mytilin A), a
ubiquitous mycosporine,6 as a major component. However,
an authentic compound presented by Prof. H. Nakamura of
Nagoya University confirmed that shinorine was not the
bioactive principal of this fraction. We thus suspected the
presence of a minor active principal, and finally, purification
by HPLC (C18) of this fraction afforded a small amount of
the active component, neodysiherbaine A (2, 0.26 mg).7
FABMS of 2 showed a molecular ion at m/z 290. From high-
resolution FABMS data, m/z 290.0865 ([M - H]-), its
formula was deduced to be C11H17NO8. In the1H NMR, the
Synthesis of vinyl triflate 4 commenced with tri-O-acetyl-
D-glucal (5), which was converted into olefin 6 by a three-
step sequence of reactions in 84% overall yield (Scheme 2).
Dihydroxylation of 6 proceeded stereoselectively to give
R-diol 7 in 79% yield along with 17% yield of the
corresponding â-diol. Routine protective group manipulations
led to alcohol 8 (76% overall), which upon oxidation and
Wittig reaction provided terminal olefin 9 in 83% yield.
Hydroboration followed by oxidative workup afforded
primary alcohol 10 (95% yield), which was then converted
to methyl ketone 11 in 83% overall yield. Kinetic deproton-
ation (KHMDS, THF, -78 °C) followed by treatment of
the derived enolate with Tf2NPh provided the desired enol
triflate 4, which was immediately used in the crucial coupling
reaction.
(4) Sasaki, M.; Koike, T.; Sakai, R.; Tachibana, K. Tetrahedron Lett.
2000, 3923-3926.
(5) (a) Snider, B. B.; Hawryluk, N. A.Org. Lett. 2000, 2, 635-638. (b)
Masaki, H.; Maeyama, J.; Kamada, K.; Esumi, T.; Iwabuchi, Y.; Hatakeya-
ma, S. J. Am. Chem. Soc. 2000, 122, 5216-5217.
(6) Chioccara, F.; Misuraca, G.; Novellino, E.; Prota, G. Tetrahedron
lett. 1979, 3181-3182.
1
(7) CD (H2O) λext 204 nm, ∆ꢀ ) 1.7; H NMR (400 MHz, D2O at 20
°C, HOD at δ 4.65 as an internal reference) δ 4.07 (brs, 1H, H-7), 3.99
(brs, 1H, H-6), 3.76 (t, 1H, J ) 3.7 Hz, H-8), 3.71 (dd, 1H, J ) 1 2.6, 2.5
Hz, H-10a), 3.55 (brs, 1H, H-9), 3.42 (brd, 2H, J ) 12.7 Hz, H-10b, H-2),
2.50 (dd, 1H, J ) 14.9, 1.5 Hz, H-3a), 2.42 (d, 1H, J ) 14.2 Hz, H-5a),
2.01(dd, 1H, J ) 14.0, 3.4 Hz, H-5b), 1.81 (dd, 1H, J ) 15.1, 12.1 Hz,
H-3b).
Treatment of 4 with organozinc reagent 38 in the presence
of PdCl2(PPh3)2 in THF-DMA (1:1) according to the
reported method4 furnished the desired cross-coupled product
1480
Org. Lett., Vol. 3, No. 10, 2001

Elaboration of 14 to the fully protected neodysiherbaine
A (18) was carried out according to the previous synthesis
of 1.4 Thus, epoxidation of 14 with m-chloroperbenzoic acid
(mCPBA) in CH2Cl2-pH 7.0 phosphate buffer (1:1) pro-
duced epoxide 15 as a 1:1 mixture of diastereomers in 88%
yield (Scheme 4). Upon treatment of this mixture with
Scheme 2. Synthesis of Enol Triflate 4a
Scheme 4a
a Reagents and conditions: (a) Et3SiH, BF3‚OEt2, CH2Cl2, 0 °C;
(b) NaOMe, MeOH, rt; (c) PhCH(OMe) , CSA, CH2Cl2, rt, 84%
(three steps); (d) OsO4, NMO, acetone-2H2O, rt, 79% (+ â-diol,
17%); (e) NaH, BnBr, DMF 0 °C f rt; (f) CSA, CH2Cl2-MeOH,
0 °C, 99% (two steps); (g) TBSOTf, 2,6-lutidine, CH2Cl2, rt; (h)
CSA, CH2Cl2-MeOH, 0 °C, 85% (two steps); (i) SO3‚Pyr, Et3N,
DMSO, CH2Cl2, rt; (j) Ph3P+CH3Br-, NaHMDS, THF, 0 °C, 83%
(two steps); (k) 9-BBN, THF, rt, then NaHCO3, H2O2, rt, 95%; (l)
SO3‚Pyr, Et3N, DMSO, CH2Cl2, rt; (m) MeMgBr, THF, -78 f 0
°C; (n) TPAP, NMO, 4 Å molecular sieves, CH2Cl2, rt, 83% (three
steps); (o) KHMDS, THF, -78 °C, then Tf2NPh, -78 f 0 °C.
a Reagents and conditions: (a) mCPBA, CH2Cl2-pH 7.0 phos-
phate buffer, rt, 88%; (b) CSA, CH2Cl2, rt; (c) 1 N NaOH, THF,
rt, 46% (two steps); (d) TPAP, NMO, 4 Å molecular sieves,
CH3CN, rt; (e) TMSCHN2, MeOH, rt 18: 43% (two steps) 19:
24% (two steps).
camphorsulfonic acid (CSA), 5-exo-ring closure proceeded
to yield an inseparable mixture of 6/5-bicyclic alcohol 16
and its δ-lactone, which was then hydrolyzed with aqueous
NaOH to give hydroxy acid 17 as an approximately 2:1
mixture of diastereomers at C4 (46% yield from 15). The
mixture of isomers was subjected to oxidation with TPAP/
NMO10 followed by treatment with trimethylsilyldiaz-
omethane and chromatography on silica gel to give protected
neodysiherbaine A (18) and its C4 epimer 19 in 43% and
24% overall yields, respectively. The stereochemistry at C4
of 18 was unambiguously determined by NOEs between
H2-3/H-5â and H-5â/H-6.
12 in 52% yield from 11 (Scheme 3). Subsequent desilylation
provided secondary alcohol 13 in 83% yield. Oxidation with
Dess-Martin periodinane9 followed by NaBH4 reduction at
low temperature led to alcohol 14 as a single stereoisomer
in 84% yield for the two steps.
Scheme 3a
Finally, hydrogenolysis of the benzyl groups of 18
provided diol 20 (77% yield), which was hydrolyzed with 6
N HCl to obtain the targeted 2a as its hydrochloride salt in
99% yield (Scheme 5). The 1H NMR spectra and biological
activity of synthetic 2a were identical with those of the
natural neodysiherbaine A. In addition, the CD spectrum
measured for synthetic 2a showed close agreement with that
for the natural sample. Thus, the complete structure, includ-
(8) Jackson, R. F. W.; Wishart, N.; Wood, A.; James, K.; Wythes, M. J.
J. Org. Chem. 1992, 57, 3397.
(9) (a) Dess, D. B.; Martin, J. C. J. Am. Chem. Soc. 1991, 113, 7277-
7278. (b) Ireland, R. E.; Liu, L. J. Org. Chem. 1993, 58, 2899-2900.
(10) Ley, S. V.; Norman, J.; Griffith, W. P.; Marsden, S. P. Synthesis
1994, 639-666.
a Reagents and conditions: (a) 3, PdCl2(PPh3)2, THF-DMA, 60
°C, 52% from 11; (b) n-Bu4NF, THF, rt, 83%; (c) Dess-Martin
periodinane, NaHCO3, CH2Cl2, rt; (d) NaBH4, THF-MeOH, -78
f 0 °C, 84% (two steps).
Org. Lett., Vol. 3, No. 10, 2001
1481

and 230 ( 15 nM, respectively. It is of particular interest
whether 2 shows discrete affinity within the subtypes of the
recombinant hetereomeric KA receptors. Detailed neurologi-
cal properties of 2 will be published elsewhere.
Scheme 5a
In summary, we have isolated a new excitatory amino acid,
neodysiherbaine A (2). The structure as well as the absolute
configuration was determined by spectroscopic methods. The
synthesis described herein should allow for the preparation
of various analogues of this excitatory amino acid, including
radiolabeled compounds. Further studies along this line are
underway and will be reported in due course.
a Reagents and conditions: (a) H2, Pd/C, MeOH, rt, 77%; (b) 6
N HCl, 60 °C, 99%.
Acknowledgment. This work was financially supported
in part by a Grant-in-Aid for Scientific Research from the
Ministry of Education, Science, Sports and Culture, Japan,
and Suntory Institute for Bioorganic Research. We are
grateful to the late Professor H. Nakamura, Nagoya Univer-
sity, for a sample of shinorine and valuable discussion. We
also thank Mr. Andy Tafileichig, Department of Resources
and Development, of the Yap State government for assistance
in sample collection.
ing the absolute configuration, of neodysiherbaine A was
unambiguously determined as depicted in structure 2a.
4-Epineodysiherbaine A was also synthesized from 19 in
84% overall yield in the same manner as described for the
synthesis of 2a from 18.
Neodysiherbaine A (2) induced dose-dependent behavioral
changes in mice. The ED50 value of 2 (15 pmol/mouse) was
comparable to that of 1 (13 pmol/mouse), although some
behavioral patterns observed for 2 were distinguishable from
that for 1. Epileptogenic activity of 4-epineodysiherbaine A
(ED50 ) 11.4 nmol/mouse) was much less potent than that
of 2. In our radioligand binding study using rat synaptic
membrane preparation, 2 displaced [3H]KA and [3H]AMPA
at IC50 values of 66 ( 5.2 and 227 ( 40 nM, respectively,
but not [3H]CGP39653, an NMDA receptor ligand. In the
same assay, the corresponding values for 1 were 33 ( 5.1
Supporting Information Available: Experimental pro-
cedures for the isolation of 2; FABMS, 1H NMR, and COSY
spectra of 2; experimental procedures for the synthesis of
1
2a; comparison of H NMR and CD spectra for 2 and 2a.
This material is available free of charge via the Internet at
http://pubs.acs.org.
OL015798L
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Org. Lett., Vol. 3, No. 10, 2001