(-)-Echinobetaine A: Isolation, structure elucidation, synthesis, and SAR studies on a new nematocide from a Southern Australian marine sponge, Echinodictyum sp.

Article abstract of DOI:10.1021/np049687h

A nematocidal agent present in a southern Australian marine sponge of the genus Echinodictyum has been isolated and identified by detailed spectroscopic analysis and total synthesis as the novel betaine (-)-echinobetaine A (6). Preliminary SAR investigations have been undertaken.

Full text of DOI:10.1021/np049687h

J. Nat. Prod. 2005, 68, 179-182
179
(-)-Echinobetaine A: Isolation, Structure Elucidation, Synthesis, and SAR
Studies on a New Nematocide from a Southern Australian Marine Sponge,
Echinodictyum sp.
Robert J. Capon,*,† Dat Vuong,† Ernest Lacey,§ and Jennifer H. Gill§
Centre for Molecular Biodiversity, Institute for Molecular Biosciences, University of Queensland,
St. Lucia, Queensland 4072, Australia, and Microbial Screening Technologies Pty. Ltd.,
Kemps Creek, New South Wales 2171, Australia
Received September 23, 2004
A nematocidal agent present in a southern Australian marine sponge of the genus Echinodictyum has
been isolated and identified by detailed spectroscopic analysis and total synthesis as the novel betaine
(-)-echinobetaine A (6). Preliminary SAR investigations have been undertaken.
During our investigations into new agrochemical agents
from Australian marine organisms we examined a sponge
specimen, Echinodictyum sp., collected by beam trawl at
a depth of 85 m during a 1995 scientific expedition to the
Great Australian Bight, Australia. The EtOH extract of
this Echinodictyum sp. displayed growth inhibitory proper-
ties against the bacteria Serratia marcescens, Micrococcus
luteus, and Staphylococcus aureus, but more importantly
it displayed excellent in vitro antiparasitic activity against
the endo parasite Haemonchus contortus. The latter para-
site inflicts serious economic damage to the agricultural
sector worldwide through its impact on the health and
productivity of livestock such as sheep. While agrochemi-
cals exist to combat such parasites, the increasing incidence
of resistance requires that the search for new and improved
antiparasitics be both vigorous and ongoing. Experience
has shown us that new antiparasitic lead compounds can
be discovered through the bioassay-directed exploration of
marine biodiversity,1-9 with Echinodictyum being a prom-
ising target for just such an investigation.
structure elucidation, and synthesis of a nematocidal agent,
(-)-echinobetaine A (6), from this same Echinodictyum sp.
Results and Discussion
The EtOH extract of the Echinodictyum specimen was
decanted, concentrated in vacuo, and triturated with CH2-
Cl2, after which the residue was partitioned between
n-BuOH and H2O. Whereas the n-BuOH solubles displayed
antibacterial activity and ultimately led to the isolation and
identification of the echinosulfonic acids A-C (2-4) and
echinosulfone (5),7 the crude H2O solubles displayed sig-
nificant antiparasitic activity against H. contortus.
The H2O solubles were further fractionated by elution
through Sephadex G-10 (H2O) followed by C18 HPLC (0.1%
TFA in H2O) to yield the nematocide (-)-echinobetaine A
(6) (H. contortus, LD99 83 µg/mL).
Before embarking on this study we reviewed the existing
chemical literature on the genus Echinodictyum. The first
published account (in 1983)10 reported the bioassay-
directed isolation and subsequent identification of the
previously known synthetic compound 4-amino-5-bromopy-
rrolo[2,3-d]pyrimidine (1), as a potential bronchodilator. To
the best of our knowledge the only other report of novel
metabolites from an Echinodictyum sp. was an early
account by us in 1999.7 In that report we described a series
of novel antibacterial agents, echinosulfonic acids A-C (2-
4) and echinosulfone (5). At that time our primary interest
in Echinodictyum focused on the in vitro antiparasitic
activity displayed by the EtOH extracts of at least two
specimens in our collection, both from the Great Australian
Bight. Although the echinosulfonic acids 2-4 and echino-
sulfone (5) were identified as natural antibacterial agents,
these compounds were not responsible for the antiparasitic
properties displayed by the Echinodictyum extract. After
our earlier report, we persisted in our investigations to the
point where we now report the isolation, characterization,
High-resolution ESI(+)MS analysis of 6 revealed a
highest mass ion at m/z 176 measuring for C8H18NO3 (∆
-0.4 mmu). Examination of the NMR (D2O, 400 MHz) data
for 6 revealed resonances consistent with an OMe (1H: δ
3.19 (s); 13C: 58.6 ppm), an NMe3 (1H: δ 2.97 (s); 13C: 53.3
ppm), and two deshielded diastereotopic methylenes (1H:
δ 3.31 (dd, J ) 13.6, 1.6 Hz) and 3.78 (dd, J ) 13.6, 8.0
1
Hz); 13C: 65.1 ppm; as well as H: δ 3.56 (m); 13C: 71.8
ppm) flanking a deshielded methine (1H: δ 3.15 (m); 13C:
41.6 ppm), accounting for all but CO2H of the proposed
molecular formula. Furthermore, the appearance of a 13C
NMR resonance at 175.1 ppm was consistent with the
remaining functional unit in 6 being a carboxylic acid.
Given the deshielded character of the methylene carbons,
and the co-occurrence of quaternary ammonium and car-
boxylic acid functionalities, the most probable structure for
(-)-echinobetaine A (6) was that shown. Analysis of the
* To whom correspondence should be addressed. Tel: +61 7 334 62979.
Fax: +61 7 334 62101. E-mail: r.capon@imb.uq.edu.au.
† University of Queensland.
§ Microbial Screening Technologies Pty. Ltd.
10.1021/np049687h CCC: $30.25
© 2005 American Chemical Society and American Society of Pharmacognosy
Published on Web 01/21/2005

180 Journal of Natural Products, 2005, Vol. 68, No. 2
Capon et al.
Table 1. NMR (D2O, 400 MHz) Data for (-)-Echinobetaine A
(6)
Scheme 2. Synthesis of the (()-2-Methoxy-γ-aminobutyric
Acid Betaine Methyl Ether (17)a
gHMBC
13C δ
1H δ (m, J Hz)
COSY
(1H-13C)
1
175.1
2
41.6 3.15 (m)
3-Ha, 3-Hb, 4-H2
3-Ha
65.1 3.78 (dd. J ) 13.6, H-2, 3-Hb
8.0 Hz)
NMe3, C-1
NMe3, C-1
3-Hb
3.31 (dd, J ) 13.6, 2-H, 3-Ha
1.6 Hz)
NMe3
4
OMe
53.3 2.97 (s)
71.8 3.56 (m)
58.6 3.19 (s)
C-3, NMe3
C-3, OMe
C-4
2-H
a
(a) Ag2O, MeI, 99%; (b) NaOH, 100%; (c) PhCH2Br, DMF, 30%; (d) i
Scheme 1. Synthesis of (()-Echinobetaine A (16)a
PTsOH, ii NMe3, 56%.
echinobetaine A (6) by NMR, ESI(+)MS, and IR and by
coelution on HPLC.
Of particular note, the nematocidal properties for (()-
echinobetaine A (16) were an order of magnitude weaker
than those for the natural product (-)-echinobetaine A (6)
(LD99 83 µg/mL), highlighting the significance of stereo-
chemistry in the echinobetaine A pharmacophore. Ongoing
studies are directed at assignment of absolute stereochem-
istry to (-)-echinobetaine A (6), as well as chiral resolution
of (()-echinobetaine A (16), to yield authentic synthetic
samples of both enantiomers to support future SAR inves-
tigations.
In an attempt to further define the nematocidal phar-
macophore revealed by echinobetaine A (6) we prepared
the isomer 17 in four steps from (()-R-hydroxybutyrolac-
tone (18) (see Scheme 2). In this sequence commercially
available 18 was converted to the methyl ether 19, which
was subsequently ring opened to the hydroxycarboxylate
salt 20. The salt 20 was then converted to the benzyl ester
21, which was in turn transformed into the target product,
(()-2-methoxy-γ-aminobutyric acid betaine methyl ether
(17). The isomeric synthetic analogue 17 displayed only
very modest nematocidal activity (LD99 438 µg/mL), whereas
all intermediates prepared during the synthesis of 16 and
17 were inactive. From this bioassay data it would appear
that the (-)-echinobetaine A (6) nematocidal pharama-
cophore does not extend to isomeric methoxy betaines in
general and does appear to be stereo-dependent.
(-)-Echinobetaine A (6) represents the first example of
a new class of antiparasitic natural products and illustrates
the all too often unrealized and untapped potential of small
molecular weight water-soluble marine metabolites. Given
the promising antiparasitic activity displayed by the
original Echinodictyum extract, we are confident that as
yet unidentified water-soluble metabolites with even greater
antiparasitic activity await discovery. Our future efforts
will extend synthetic studies to further explore and define
the (-)-echinobetaine A (6) pharmacophore, as well as
support ongoing exploration of Echinodictyum extracts to
detect, isolate, and identify new and hopefully more potent
antiparasitics.
a (a) PhCHO, 100%; (b) i KOH, ii HCl, 100%; (c) piperidine, 100%; (d)
LiAlH4, 96%; (e) MeI, NaH, 91%; (f) NBS, BaCO3, 95%; (g) NMe3, benzene,
98%; (h) 1 M HCl; (i) AcOHaq, 25%.
2D NMR COSY and gHMBC data for 6 (see Table 1)
supported this assignment; however, to provide unambigu-
ous evidence, we undertook the total synthesis of (()-
echinobetaine A (16) as outlined in Scheme 1.
Preparation of the benzoate intermediate 13 proceeded
in six steps following the method of Bosies et al. (see
Scheme 1).11 Quantitative conversion of the readily avail-
able diester 7 to the acetal 8 was followed by quantitative
conversion to the ester 9. Decarboxylation of 9 yielded 10
as a mixture of cis and trans isomers (3:4), while subse-
quent reduction and methylation returned the alcohol 11
(96%) and methyl ether 12 (91%), respectively. Opening of
the acetal in 12 with NBS yielded the key intermediate 13
(95%), which was smoothly converted to the corresponding
trimethylammonium 14 (98%). Acid hydrolysis of the
benzoate ester moiety in 14 returned the alcohol 15, while
unoptimized oxidation of 15 returned (()-echinobetaine A
(16) in 25% yield after purification by HPLC. Synthetic (()-
echinobetaine A (16) was identical with natural (-)-
Experimental Section
General Experimental Procedures. As previously de-
scribed.12
Animal Material. An Echinodictyum sp. (Museum of
Victoria Registry Number MVF88741) of marine sponge was
collected during a scientific expedition to the Great Australian
Bight aboard the RV Franklin in July 1995, as previously
described.7
Extraction and Isolation. The EtOH extract of the
Echinodictyum specimen was decanted and concentrated in
vacuo to yield a bright yellow-orange solid (4.08 g), which was

(-)-Echinobetaine A from Echinodictyum sp.
Journal of Natural Products, 2005, Vol. 68, No. 2 181
triturated with CH2Cl2. The CH2Cl2-insoluble materials were
partitioned between n-BuOH and H2O. The H2O solubles (2.96
g) were concentrated in vacuo, and the residue was fraction-
ated by Sephadex G-10 (H2O) chromatography (2.5 × 30 cm
column), followed by isocratic C18 HPLC (2.0 mL/min H2O +
0.1% TFA through a Zorbax 10 µm 250 × 10 mm column) to
yield (-)-echinobetaine A (6) (25 mg, 0.044% specimen dry
weight) as the TFA salt. All fractionation studies were sup-
ported by bioassay.
4-H2a), 4.15-4.11 (m, 2- and 4-H2b), 4.06 (d, J ) 7.6 Hz, CH2-
OH), 1.69 (br s, 3-H); major isomer 1H NMR (400 MHz, CDCl3)
δ 7.50-7.32 (m, Ph), 5.43 (s, 6-H), 4.32 (dd, J ) 11.6, 4.8 Hz,
2- and 4-H2a), 3.74 (dd, J ) 11.6, 11.6 Hz, 2- and 4-H2b), 3.52
(d, J ) 6.0 Hz, CH2OH), 2.39 (m, 3-H); 13C NMR (100 MHz,
CDCl3) δ 138.3, 138.1, 128.9, 128.8, 128.2, 126.0, and 125.9
(Ph), 101.8 and 101.4 (2 × C-6), 69.6 and 67.7 (2 × C-4 and
C-2), 61.5 and 60.9 (2 × CH2OH), 36.8 and 36.4 (2 × C-3); ESI-
(+)MS m/z 217 (M + Na).
3-Methoxymethyl-6-phenyl-[1,5]-dioxane (12). A dry
THF solution of the alcohol 11 (2.48 g, 0.013 mol) and NaH
(1.53 g, 0.064 mol) was refluxed under a N2 atmosphere for 1
h, after which MeI (9 mL, 0.11 mol) was added dropwise and
the reaction refluxed for a further 2 h. On cooling the reaction
mixture was extracted with Et2O (3 × 50 mL), and the organic
phase washed with brine (3 × 20 mL), dried (anhydrous
MgSO4), and concentrated in vacuo to yield the methyl ether
12 (2.46 g, 91%) as minor (36%) and major (64%) isomers:
minor isomer 1H NMR (400 MHz, CDCl3) δ 7.50-7.31 (m, Ph),
5.50 (s, 6-H), 4.21 (d, J ) 10.8 Hz, 2- and 4-H2a), 4.11-4.08
(m, 2- and 4-H2b), 3.76 (d, J ) 7.2 Hz, CH2OMe), 3.41 (s,
CH2OMe), 1.74 (br m, 3-H); major isomer 1H NMR (400 MHz,
CDCl3) δ 7.50-7.32 (m, Ph), 5.42 (s, 6-H), 4.28 (dd, J ) 11.8,
4.4 Hz, 2- and 4-H2a), 3.73 (dd, J ) 11.8, 11.8 Hz, 2- and 4-H2b),
3.32 (s, CH2OMe), 3.24 (d, J ) 5.6 Hz, CH2OH), 2.47 (m, 3-H);
13C NMR (75 MHz, CDCl3) δ 138.4, 138.2,128.5, 127.9, 125.8,
and 125.7 (Ph), 101.5 and 101.1 (2 × C-6), 71.3 and 70.6 (2 ×
CH2OMe), 69.5 and 67.6 (2 × C-4 and C-2), 58.7 and 58.6 (2 ×
CH2OMe), 34.6 and 34.4 (2 × C-3); ESI(+)MS m/z 231 (M +
Na).
3-Bromo-2-(methoxymethyl)-1-propyl Benzoate (13). A
mixture of the methyl ether 12 (2.46 g, 0.012 mol), freshly
recrystallized N-bromosuccimide (2.2 g, 0.006 mol), and BaCO3
(0.359 g, 0.002 mol) was refluxed in dry CH2Cl2 (30 mL) for
24 h under a N2 atmosphere. After cooling, the reaction was
concentrated in vacuo and the residue triturated with hexane,
filtered, and dried in vacuo to yield the bromobenzoate 13 (3.3
g, 95%): IR (neat) νmax 1724 cm-1; 1H NMR (300 MHz, CDCl3)
δ 8.04 (d, J ) 7.4 Hz, 2′-H and 6′-H), 7.58 (t, J ) 7.4 Hz, 4′-
H), 7.46 (t, J ) 7.4 Hz, 3′-H and 5′-H), 4.45 (dd, J ) 11.2, 6.4
Hz, 1-H2a), 4.38 (dd, J ) 11.2, 6.4 Hz, 1-H2b), 3.61 (d, J ) 5.6
Hz, 4-H2), 3.56 (dd, J ) 7.8, 5.0 Hz, 3-H2a), 3.51 (dd, J ) 7.8,
5.0 Hz, 3-H2b), 3.37 (s, OMe), 2.47 (m, 2-H); 13C NMR (75 MHz,
CDCl3) δ 166.0 (CO2Ph), 132.9, 129.8, 129.4, and 128.3
(CO2Ph), 70.9 (C-4), 63.6 (C-1), 58.9 (OMe), 40.5 (C-2), 31.9
(C-3); ESI(+)MS m/z 309/311 (M + Na).
(-)-Echinobetaine A (6): [R]22 -49° (c 0.59, MeOH); IR
D
(neat) νmax 1683 cm-1; 1H NMR (400 MHz, D2O) and 13C NMR
(100 MHz, D2O), see Table 1; HRESI(+) MS m/z 176.1283
(calcd for C8H18NO3, 176.1287).
Diethyl 6-Phenyl-[1,5]-dioxane-3,3-dicarboxylic Acid
Diester (8). A solution of diethyl bis-hydroxymethylmalonate
(7) (22.0 g, 0.1 mol), PhCHO (10.6 g, 0.1 mol), and p-TsOH
(0.2 g) was refluxed in toluene (200 mL) for 24 h using a Dean
Stark trap, after which the toluene was removed in vacuo and
the residue purified by distillation to afford the acetal 8 (7.64
1
g, 25%): bp1mmHg 160-170 °C; IR (Nujol) νmax 1741 cm-1; H
NMR (300 MHz, CDCl3) δ 7.43-7.31 (m, Ph), 5.46 (s, 6-H),
4.83 (d, J ) 11.5 Hz, 2- and 4-H2a), 4.31 (q, J ) 7.1 Hz, CO2CH2-
CH3), 4.17 (q, J ) 7.1 Hz, CO2CH2CH3), 4.14 (d, J ) 11.5 Hz,
2- and 4-H2b), 1.29 (t, J ) 7.1 Hz, CO2CH2CH3), 1.23 (t, J )
7.1 Hz, CO2CH2CH3); 13C NMR (75 MHz, CDCl3) δ 167.6 and
166.7 (2 × CO2CH2CH3), 137.3, 129.0, 128.1, and 126.0 (Ph),
101.6 (C-6), 69.4 (C-4 and C-2), 61.9 (CO2CH2CH3), 53.1 (C-3),
13.9 (CO2CH2CH3); ESI(+)MS m/z 331 (M + Na).
Ethyl 6-Phenyl-[1,5]-dioxane-3,3-dicarboxylic Acid Mo-
noester (9). An EtOH solution of the acetal 8 (6.58 g, 0.021
mol) and KOH (1.71 g, 0.03 mol) was stirred at RT for 4 h,
after which the EtOH was evaporated in vacuo and the residue
treated with 1 M HCl (40 mL) to return the monoester 9 as a
white solid (5.50 g, 95%): IR (KBr) νmax 3280-2544 (br), 1741,
1
1716 cm-1; H NMR (400 MHz, CDCl3) δ 7.45-7.35 (m, Ph),
6.03 (s, CO2H), 5.50 (s, 6-H), 4.88 (d, J ) 11.6 Hz, 2- and 4-H2a),
4.35 (q, J ) 7.2 Hz, CO2CH2CH3), 4.18 (d, J ) 11.6 Hz, 2- and
4-H2b), 1.34 (t, J ) 7.2 Hz, CO2CH2CH3); 13C NMR (75 MHz,
CDCl3) δ 171.9 (CO2H), 167.5 (CO2CH2CH3), 137.1, 129.3,
128.3, and 126.1 (Ph),101.8 (C-6), 69.3 (C-4 and C-2), 62.5
(CO2CH2CH3), 53.2 (C-3), 14.0 (CO2CH2CH3); ESI(+)MS m/z
303 (M + Na).
Ethyl 6-Phenyl-[1,5]-dioxane-3-carboxylic Acid Ester
(10). An anhydrous pyridine solution (10 mL) of the ester 9
(5.0 g, 0.018 mol) and piperidine (30 µL) was refluxed for 5 h,
after which the solution was added dropwise to ice cold HClaq
(37%, 13 mL). The resulting solution was extracted with CH2-
Cl2 (3 × 50 mL) and the combined organic layer dried
(anhydrous MgSO4) and concentrated in vacuo to afford the
decarboxylated product 10 as a mixture of minor (43%) and
(()-3-Trimethylammonium-2-(methoxymethyl)-1-pro-
pyl) Benzoate (14). A solution of the bromobenzoate 13 (3.3
g, 0.013 mol) in 23% trimethylamine in dry benzene (200 mL)
was stirred for 48 h, after which the resulting precipitate was
filtered and washed with Et2O (50 mL) to yield the insoluble
trimethylammonium salt 14 (3.40 g, 98%). Recrystallization
from methanol/EtOAc returned a white solid with a melting
point of 125-127 °C: IR (Nujol) νmax 1720 cm-1; UV (MeOH)
major (57%) isomers (3.0 g, 72%): IR (Nujol) νmax 1736 cm-1
;
minor isomer 1H NMR (300 MHz, CDCl3) δ 7.49-7.35 (m, Ph),
5.52 (s, 6-H), 4.73 (d, J ) 11.0 Hz, 2- and 4-H2a), 4.28 (q, J )
7.0 Hz, CO2CH2CH3), 4.11 (dd, J ) 11.0, 3.3 Hz, 2- and 4-H2b),
2.41 (br m, 3-H), 1.32 (t, J ) 7.0 Hz, CO2CH2CH3); major
isomer 1H NMR δ7.49-7.35 (m, Ph), 5.44 (s, 6-H), 4.47 (dd, J
) 11.9, 4.8 Hz, 2- and 4-H2a), 4.16 (q, J ) 7.0 Hz, CO2CH2-
CH3), 4.00 (dd, J ) 11.9, 11.9 Hz, 2- and 4-H2b), 3.42 (m, 3-H)
1.23 (t, J ) 7.0 Hz, CO2CH2CH3); 13C NMR (75 MHz, CDCl3)
δ 171.1 and 169.9 (2 × CO2CH2CH3), 138.0, 137.7, 129.0, 128.9,
128.2, 128.1, 126.1, and 126.0 (Ph), 101.8 and 101.3 (2 × C-6),
68.0 and 67.1 (2 × C-4 and C-2), 61.1 and 60.8 (2 × CO2CH2-
CH3), 39.9 (C-3), 14.1 (2 × CO2CH2CH3); ESI(+)MS m/z 259
(M + Na).
1
λmax (ꢀ) 229 (7400); H NMR (400 MHz, CDCl3) δ 7.98 (d, J )
7.8 Hz, 2′-H and 6′-H), 7.56 (t, J ) 7.8 Hz, 4′-H), 7.43 (t, J )
7.8 Hz, 3′-H and 5′-H), 4.55 (dd, J ) 11.8, 5.2 Hz, 1-H2a), 4.34
(dd, J ) 11.8, 7.2 Hz, 1-H2b), 3.86 (dd, J ) 13.4, 4.4 Hz, 3-H2a),
3.70 (dd, J ) 13.4, 3.2 Hz, 3-H2b), 3.61 (dd, J ) 9.2, 4.4 Hz,
4-H2a), 3.51 (s, NMe3), 3.46 (dd, J ) 9.2, 7.2 Hz, 4-H2b), 3.34
(s, OMe), 2.71 (br m, 2-H); 13C NMR (100 MHz, CDCl3) δ 166.3
(CO2Ph), 133.3, 129.5, 129.1, and 128.4 (CO2Ph), 71.4 (C-4),
65.8 (C-3), 64.3 (C-1), 59.1 (OMe), 53.6 (NMe3), 34.7 (C-2);
HRESI(+)MS m/z 266.1764 (calcd for C15H24NO3, 266.1756).
(()-3-Trimethylammonium-2-(methoxymethyl)-1-pro-
panol (15). The trimethylammonium salt 14 (1.65 g, 0.006
mol) was refluxed for 24 h in 1 M HCl (80 mL), then washed
with EtOAc, and the aqueous layer was concentrated in vacuo
to yield the alcohol 15 (840 mg, 87%). Recrystallization from
MeOH/EtOAc returned flake-like crystals with a melting point
of 123-124 °C: IR (Nujol) νmax 3275 cm-1; 1H NMR (400 MHz,
D2O) δ 3.49 (dd, J ) 11.6, 5.7 Hz, 3-H2a), 3.42 (dd, J ) 11.6,
5.7 Hz, 3-H2b), 3.36 (dd, J ) 10.4, 5.2 Hz, 1-H2a), 3.30 (dd, J )
10.4, 6.4 Hz, 1-H2b), 3.19 (d, J ) 4.0, Hz, 4-H2), 3.23 (s, OMe),
3.00 (s, NMe3), 2.19 (m, 2-H); 13C NMR (100 MHz, D2O) δ 71.5
(6-Phenyl-[1,5]-dioxan-3-yl)methanol (11). The mo-
noester 10 (3.3 g, 0.014 mol) was treated with LiAlH4 (2.3 g,
0.061 mol) in refluxing dry Et2O (20 mL) under a N2 atmo-
sphere for 3 h. The reaction was then quenched with ice and
following the dropwise addition of EtOAc (18 mL), water (35
mL), and 15% NaOH (9 mL) was extracted with Et2O (3 × 50
mL). After drying (anhydrous MgSO4) the organic phase was
concentrated in vacuo to yield the alcohol 11 (2.5 g, 94%) as
minor (42%) and major (58%) isomers: IR (Nujol) νmax 3390-
3308 (br) cm-1; minor isomer 1H NMR (400 MHz, CDCl3) δ
7.50-7.32 (m, Ph), 5.52 (s, 6-H), 4.25 (d, J ) 11.2 Hz, 2- and

182 Journal of Natural Products, 2005, Vol. 68, No. 2
Capon et al.
(C-4), 65.5 (C-3), 60.9 (C-1), 58.4 (OMe), 53.2 (NMe3), 36.0 (C-
2); HRESI(+)MS m/z 162.1489 (calcd for C8H20NO2, 162.1494).
(()-Echinobetaine A (16). A 54% AcOHaq solution of the
alcohol 15 (0.142 g, 0.97 mmol) and KMnO4 (0.278 g, 1.8 mmol)
was stirred for 3 h at 50 °C, after which the reaction was
filtered and concentrated in vacuo. The resultant solid was
eluted through a C18 solid-phase extraction (SPE) cartridge
with H2O. The solvent was removed in vacuo and the residue
subjected to isocratic C18 HPLC (2.0 mL/min H2O + 0.1% TFA,
Zorbax 10 µm 250 × 10 mm column) to yield (()-echinobetaine
A (16) (43 mg, 25%): IR, 1H NMR (400 MHz, D2O), and13C
NMR (100 MHz, D2O) identical with (-)-echinobetaine A (6);
HRESI(+)MS m/z 176.1282 (calcd for C8H18NO3, 176.1287).
(()-2-Methoxy-γ-butyrolactone (19). A solution of the
(()-2-hydroxy-γ-butyrolactone (18) (1.87 g, 18.3 mmol), MeI
(11 mL, 176 mmol), and Ag2O (17.8 g, 77 mmol) was refluxed
under a N2 atmosphere in CHCl3 (50 mL) for 1 h. The reaction
was cooled and filtered, and the clear solution was concen-
trated in vacuo to yield the methoxy butyrolactone 19 (2.10 g,
99%): 1H NMR (CDCl3, 400 MHz) δ 4.40 (m, 4-H2a), 4.23 (m,
4-H2b), 4.02 (t, J ) 7.6 Hz, 2-H), 3.56 (s, OMe), 2.50 (m, 3-H2a),
2.23 (m, 3-H2b); 13C NMR (75 MHz, CDCl3) δ 174.6 (C-1), 74.6
(C-2), 65.0 (C-4), 57.6 (OMe), 28.9 (C-3).
Sodium (()-4-Hydroxy-2-methoxybutyrate (20). A solu-
tion of the methoxy butyrolactone 19 (2.10 g, 18.1 mmol) in 2
M NaOH (6 mL) and MeOH (6 mL) was stirred for 16 h at RT
and the reaction mixture concentrated in vacuo to return the
crude sodium salt 20 (2.80 g, 100%), which was used without
further purification: 1H NMR (D2O, 300 MHz) δ 3.56 (dd, J )
8.1, 4.8 Hz, 2-H), 3.48 (t, J ) 7.2 Hz, 4-H2), 3.12 (s, OMe),
1.70 (m, 3-H2); 13C NMR (D2O, 75 MHz) δ 180.3 (C-1), 79.9
(C-2), 58.2 (C-4), 57.0 (OMe), 35.0 (C-3).
Benzyl (()-4-Hydroxy-2-methoxybutanoate (21). The
sodium salt 20 (2.80 g, 18.00 mmol), benzyl bromide (2.2 mL,
18.7 mmol), and 18-crown-6-(1,4,7,10,13,16-hexaoxacycloocta-
decane) (1.60 g, 6.2 mmol) were stirred at 40 °C in DMF (30
mL) under a N2 atmosphere for 4 days, after which the reaction
mixture was quenched with H2O (30 mL), extracted with Et2O,
washed with brine, dried over anhydrous Mg2SO4, and con-
centrated in vacuo to yield the benzyl ester 21 (1.20 g, 30%),
which was purified by rapid silica filtration eluting with 30%
ethyl acetate/petroleum spirit: 1H NMR (CDCl3, 400 MHz) δ
7.37 (m, OCH2Ph), 5.23 (d, J ) 12.4 Hz, OCH2aPh), 5.18 (d, J
) 12.4 Hz, OCH2bPh), 4.03 (dd, J ) 7.6, 4.8 Hz, 2-H), 3.76 (t,
J ) 5.2 Hz, 4-H2), 3.43 (s, OMe), 2.01 (m, 3-H2); 13C NMR (D2O,
100 MHz) δ 172.2 (C-1), 128.6, 128.5, 128.4, and 128.3
(OCH2Ph), (79.1 (C-2), 66.7 (OCH2Ph), 59.7 (C-4), 58.4 (OCH3),
35.1 (C-3).
and the organic phase washed sequentially with water, 5%
copper sulfate, and water, after which it was dried with
anhydrous MgSO4 and concentrated in vacuo to yield a 4-tosyl
intermediate (1.39 g, 68%). The tosyl intermediate was heated
to 95 °C with a solution of trimethylamine in benzene in a
sealed tube for 16 h, after which the reaction mixture was
concentrated in vacuo to a clear, viscous oil (1.60 g, 91%), which
was dissolved in 4 M HCl (15 mL) and dioxane (5 mL) and
stirred at RT for 16 h. The reaction mixture was concentrated
in vacuo to yield a viscous oil, which was purified by C18 SPE
(10% stepwise elution from 100% H2O to 100% MeOH). The
100% H2O fractions were combined, concentrated in vacuo, and
further purified by anion exchange SPE (preconditioned and
sample loaded from 10 mL of 0.1 M NaClaq, elution with H2O)
to yield the trimethylammonium 17 (350 mg, 56%): IR (neat)
1
νmax1736 cm-1; H NMR (D2O, 400 MHz) δ 3.89 (dd, J ) 8.0,
3.6 Hz, 2-H), 3.30 (dt, J ) 12.4, 4.8 Hz, 4-H2), 3.23 (s, OMe),
2.96 (s, NMe3), 2.20 (m, 3-H2a), 2.00 (m, 3-H2b); 13C NMR (D2O,
75 MHz) δ 174.8 (C-1), 76.8 (C-2), 62.8 (C-4), 58.1 (OMe), 53.1
(NMe3), 25.5 (C-3); HRESI(+)MS m/z 176.1282 (calcd for C8H18-
NO3, 176.1287).
Acknowledgment. We acknowledge the assistance of L.
Goudie in identifying sponge material, D. Howse for database
support, and T. Friedel and K. Heiland for facilitating access
to agrochemical assays. This research was partially funded
by both the Australian Research Council and Novartis Animal
Health Australasia Pty Ltd.
References and Notes
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(()-2-Methoxy-γ-aminobutyric Acid Betaine Methyl
Ether (17). A solution of the benzyl ester 21 (1.20 g, 5.35
mmol) and p-toluenesulfonyl chloride (2.04 g, 10.7 mmol) was
stirred in dry pyridine at 5 °C for 3 h. Diethyl ether was added
NP049687H