Nematocidal thiocyanatins from a southern Australian marine sponge Oceanapia sp.

Article abstract of DOI:10.1021/np049977y

Investigations of a southern Australian marine sponge, Oceanapia sp., have yielded two new β methyl branched bisthiocyanates, thiocyanatins D ₁ (3a) and D₂ (3b), along with two new thiocarbamate thiocyanates, thiocyanatins E₁ (4a) and E₂ (4b). The new thiocyanatins belong to a rare class of bioactive marine metabolite previously only represented by thiocyanatins A-C (1, 2a/b). Structures were assigned on the basis of detailed spectroscopic analysis, with comparisons to the known bisthiocyanate thiocyanatin A (1) and synthetic model compounds (5-7). The thiocyanatins exhibit potent nematocidal activity, and preliminary structure-activity relationship investigations have confirmed key characteristics of the thiocyanatin pharmacophore.

Full text of DOI:10.1021/np049977y

J . Nat. Prod. 2004, 67, 1277-1282
1277
Nem a tocid a l Th iocya n a tin s fr om a Sou th er n Au str a lia n Ma r in e Sp on ge
Ocea n a pia sp .
Robert J . Capon,*,† Colin Skene,† Edward Hsiang-Te Liu,‡ Ernest Lacey,§ J ennifer H. Gill,§ Kirstin Heiland,# and
Thomas Friedel#
Centre for Molecular Biodiversity, Institute for Molecular Biosciences, University of Queensland, St. Lucia,
Queensland 4072, Australia, School of Chemistry, University of Melbourne, Parkville, Victoria 3010, Australia,
Microbial Screening Technologies Pty. Ltd., Kemps Creek, New South Wales 2171, Australia, and Novartis Animal Health
Pty. Ltd., Kemps Creek, New South Wales 2171, Australia
Received J anuary 6, 2004
Investigations of a southern Australian marine sponge, Oceanapia sp., have yielded two new â methyl
branched bisthiocyanates, thiocyanatins D1 (3a ) and D2 (3b), along with two new thiocarbamate
thiocyanates, thiocyanatins E1 (4a ) and E2 (4b). The new thiocyanatins belong to a rare class of bioactive
marine metabolite previously only represented by thiocyanatins A-C (1, 2a /b). Structures were assigned
on the basis of detailed spectroscopic analysis, with comparisons to the known bisthiocyanate thiocyanatin
A (1) and synthetic model compounds (5-7). The thiocyanatins exhibit potent nematocidal activity, and
preliminary structure-activity relationship investigations have confirmed key characteristics of the
thiocyanatin pharmacophore.
Recently we reported1 the isolation, structure elucida-
tion, synthesis, and biological activity of the first examples
of a family of novel bisthiocyanate nematocides, thiocyan-
atins A-C (1-2a ,b), isolated from a southern Australian
marine sponge, Oceanapia sp. Marine natural products
possessing a thiocyanate functionality are rare, with the
earliest reports (1989 and 1992) by Faulkner et al. describ-
ing sesquiterpene monothiocyanates from a Palauan sponge,
Axinyssa () Trachyopsis) aplysinoides.2,3 By comparison,
the thiocyanatins are acyclic lipids with twin terminal
thiocyanate moieties. The closest known natural analogues
to the thiocyanatins are also marine metabolites, being a
selection of bisisothiocyanates reported (1987) by Scheuer
et al. from a Fijian sponge, Pseudaxinyssa sp.4 Our ongoing
interest in the thiocyanatins stems from the observation
that thiocyanatin A (1) exhibits potent nematocidal activity
against the commercial livestock parasite Haemonchus
contortus (LD99 ) 1.3 µg/mL). Furthermore, the thiocyan-
atins feature a structure motif readily accessible by syn-
thesis. In light of these properties we were keen to further
explore our Oceanapia sp. extract as a possible source of
new bioactive thiocyanatins, to extend the selection of
available natural thiocyanatins and further define the
pharmacophore. In this report we describe four new nema-
tocidal thiocyanatins as pairs of inseparable isomers; the
isomeric â methyl branched bisthiocyanates, thiocyanatins
D1 (3a ) and D2 (3b), and the isomeric thiocyanate-thiocar-
bamates, thiocyanatins E1 (4a ) and E2 (4b).
Haemonchus contortus (LD99 ) 135 µg/mL). The extract
was decanted, concentrated in vacuo, and triturated with
CH2Cl2 to afford soluble material that displayed enhanced
nematocidal activity. Further solvent triturations using
hexane/diethyl ether mixtures followed by C18 HPLC
afforded the previously isolated metabolites thiocyanatins
A-C (1-2a /b), as well as a series of new closely related
analogues (3a /b and 4a /b). The nematocidal activity was
attributed to thiocyanatin A (LD99 ) 1.3 µg/mL), as well
as inseparable mixtures of thiocyanatins D1 (3a ) and D2
(3b) (LD99 ) 3.1 µg/mL) and thiocyanatins E1 (4a ) and E2
(4b) (LD99 ) 8.3 µg/mL).
High-resolution (+)ESIMS analysis of the inseparable
mixture 3a /b yielded a pseudomolecular ion (M + Na, m/z
393.1995) consistent with a molecular formula (C19H34S2N2O,
∆ mmu ) -3.9) requiring 4 DBE. A strong and character-
istic IR absorbance (2156 cm-1) was consistent with
incorporation of all 4 DBE and the N and S heteroatoms
into two thiocyanate functionalities, an observation further
supported by the appearance of two nonequivalent thiocy-
anate 13C NMR resonances (13C, 112.3 (C) and 112.8 (C)
ppm). The NMR data for 3a /b (see Table 1) also revealed
a 2°-OH methine (1H, δ 3.58 (m); 13C, 71.9 (CH)), compa-
rable to that observed in thiocyanatin A, as well as a 2°-
Me (1H, δ 1.05 (CH); 13C, 18.6 (CH3)) â to a deshielded
diastereotopic terminal methylene (1H, δ 2.79 (J ) 12.6,
7.5 Hz) and 3.00 (J ) 12.6, 5.6 Hz); 13C, 41.4 (CH2) ppm).
Analysis of the 2D NMR COSY and gHMBC spectra for
3a /b clearly supported the latter spin system (with cor-
1
Resu lts a n d Discu ssion
relations across the subunit -CH(CH3)CH2SCN), as did H
As reported earlier,1 the crude ethanolic extract from an
Oceanapia sp. collected off southern Australia during
scientific trawling operations exhibited significant nema-
tocidal activity against the commercial livestock parasite
NMR comparisons with the synthetic model compound (S)-
2-methylbutyl thiocyanate (5)6 (see Table 1). As with
thiocyanatin A (1), NMR analysis of 3a /b was unable to
unambiguously position the 2°-OH on the carbon chain.
Further to this, the 13C NMR spectrum of 3a /b revealed a
doubling of selected resonances that was interpreted as
being due to 2°-OH regioisomers brought about by the
unsymmetrical nature of the termini (branched vs un-
branched).
Dedicated to the late Dr. D. J ohn Faulkner (Scripps) and the late
Dr. Paul J . Scheuer (Hawaii) for their pioneering work on bioactive marine
natural products.
* 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.
As was the case with thiocyanatin A (1), placement of
the 2°-OH along the alkyl chain in 3a /b was achieved by
interpretation of key fragmentations in the EI mass
‡ University of Melbourne.
§ Microbial Screening Technologies Pty. Ltd.
#
Novartis Animal Health Pty. Ltd.
10.1021/np049977y CCC: $27.50
© 2004 American Chemical Society and American Society of Pharmacognosy
Published on Web 05/19/2004

1278 J ournal of Natural Products, 2004, Vol. 67, No. 8
Capon et al.
Ta ble 1. 1H NMR (400 MHz, CDCl3) Data for Thiocyanatins A (1), D1/2 (3a /b), and E1/2 (4a /b) and the Models 5 and 7
position
H2-1
1
3a /b
4a /ba
5b
7a
2.95, t (7.4)l
2.79, dd (7.5, 12.6)c
3.00, dd (5.6, 12.6)c
1.9, md
2.81, t (7.2)h
2.80, dd (7.6, 12.9)
3.01, dd (5.7, 12.9)
1.82, m
2.81, t (7.2)l
H-2/H2-2
2-Me
CHOH
H2-15
H2-16
CH2’s
1.85, m (7.4)m
1.58, quin (7.1)i
1.58, quin (7.2)m
1.05, d (6.8)e
3.58, m
1.05, d (6.6)
3.60, m
3.50, m
3.50, m
n
o
1.83, quin (7.4)f
2.94, t (7.4)g
1.2-1.7, m
1.81, quin (7.3)j
3.02, t (7.2)k
1.2-1.5, m
p
q
1.2-1.5, m
1.2-1.6, m (H2-3)
1.2-1.7, m
a
b
d
g
h
i
j
k
400 MHz, d4-MeOH. 300 MHz, CDCl3. c 3b H2-16. 3b H-15. e 3b 15-Me. f 3b H2-2. 3b H2-1. 4b H2-16. 4b H2-15. 4b H2-2. 4b
H2-1. Also H2-16. Also H2-15. As for H2-2. o As for H2-1.
l
m
n
thiocyanatin A (1), much the same as thiocyanatins B (2a )
and C (2b) are alternate dehydration products of thiocy-
anatin A (1).
The inclusion of a â methyl branch in 3a /b compared to
thiocyanatin A (1) introduces a second asymmetric center.
Whereas thiocyanatin A (1) possessed a single chiral center
about the 2°-OH, the pseudo-symmetric nature of the
molecule made it impossible to determine the enantio
purity of 1; thiocyanatin A (1) did not exhibit an optical
rotation. It has been shown for a large number of labdane
diterpenes in which the asymmetric ring system is sepa-
rated by two methylene groups from an asymmetric center
in the side chain that the molar rotation contributions for
the two chiral subunits are additive.7 We have successfully
used this technique to assign absolute stereochemistry to
a number of marine natural products.8-14 Insofar as
thiocyanatins D1 (3a ) and D2 (3b) possess a “chiral” 2°-OH
comparable to thiocyanatin A (1), it is reasonable to assume
that this functionality does not contribute significantly to
the optical properties of the 3a /b mixture, requiring that
the observed [Φ]D ) -13.7° be largely if not solely due to
the â methyl branched chiral center. Comparison of this
measurement with that reported for the known (S)-2-
methylbutyl thiocyanate (5)6 ([Φ]D ) +23.2°) supports
assignment of an R stereochemistry about the â methyl
branch in both thiocyanatins D1 (3a ) and D2 (3b).
High-resolution (+)ESIMS analysis of the inseparable
mixture 4a /b yielded a pseudomolecular ion (M + Na, m/z
397.1949) consistent with a molecular formula (C18H34S2-
N2O2, ∆ mmu ) -2.7) requiring 3 DBE and leading to the
initial impression that 4a /b were monohydrolysis ana-
logues of thiocyanatin A (1). The NMR data for 4a /b (see
Table 1) revealed discrete resonances for the two terminal
methylene groups, one substituted by a thiocyanate (1H, δ
3.02 (m); 13C, 34.9 (CH2); gHMBC correlation CH2-SCN
(113.7 ppm)) and the other substituted by a thiocarbamate
(1H, δ 2.81 (m); 13C, 30.4 (t) ppm; gHMBC correlation CH2-
SCONH2 (172.0 ppm)). These observations, together with
doubling of selected resonances in the 13C NMR spectrum,
suggested that 4a /b was a mixture of 2°-OH regioisomers
of a mono-thiocarbamate analogue of thiocyanatin A (1).
EI mass spectrometric analysis of 4a /b revealed intense
ions at m/z 186 and 200 of the general formula [NCS(CH2)n-
HCdOH]•+, consistent with the regioisomeric structures as
shown, thiocyanatins E1 (4a ) and E2 (4b). To confirm this
assignment, the model bisthiocarbamate 7 was prepared
(63%) from methanolic HCl hydrolysis of synthetic thiocy-
anatin A (1). It is noteworthy that the NMR data for the
thiocarbamate terminus in the model compound 7 were in
excellent agreement with that recorded for 4a /b (see Table
1), and the EI mass spectrum for 7 did not reveal diagnostic
C-C cleavages R to the 2°-OH. This latter observation is
consistent with the interpretation of the diagnostic ions in
the EI mass spectrum of 4a /b and may reflect the suscep-
spectrum. Diagnostic C-C cleavage R to the 2°-OH in 3a /b
yielded ions of the general formula [NCS(CH2)nHCdOH]•+
at m/z 186, 200, and 214. The proposed structure for
thiocyanatin D1 (3a ) is as indicated since this is the only
2°-OH regioisomer capable of giving rise to an ion at m/z
200 (as shown in the structure diagram). Similarly, the
favored structure for 3b as shown represents an alternate
biosynthetic alkylation of thiocyanatin A (1). In this way
thiocyanatins D1 (3a ) and D2 (3b) can be viewed as two
alternate biosynthetic â methyl branched analogues of

Nematocidal Thiocyanatins from Oceanapia sp.
J ournal of Natural Products, 2004, Vol. 67, No. 8 1279
C18 HPLC, mixture A was resolved by GC into six compo-
nents, four of which eluted early with similar retention
times (42.3 (3%), 43.2 (18%), 43.6 (15%), and 43.8 (11%)
min), while the remaining two eluted several minutes later,
also with similar retention times (51.4 (28%) and 51.6 (24%)
min). This GC analysis suggests that mixture A features
most if not all the proposed structures shown in Figure 1.
Analysis of the 1H NMR and (+)ESI-HRMS data for
mixture B suggested dehydration analogues along the lines
of those indicated in Figure 1. The presence of only a single
1H NMR (CDCl3) 2°-Me resonance (δ 0.95) was consistent
with â methyl branching adjacent to an SCONH2 termini,
while the remaining 1H NMR resonances compared well
with thiocyanatins B and C (2a /b). On the basis of the
hypothesis that the alkene thiocyanatins are biosynthetic
dehydration products of the corresponding natural alco-
hols,1 and given that the SCONH2 functionality most likely
arises from nonregiospecific hydrolysis of SCN moieties in
suitable natural precursors, SCN and SCONH2 asymmetri-
cally terminated thiocyanatins would be expected to give
rise to multiple alkene regioisomers (such as shown in
Figure 1). GC analysis of mixture B revealed at least one
major (95%) and one minor (5%) component (eluting at 50.7
and 51.0 min, respectively).
1H NMR and (+)ESI-HRMS analyses of mixture C were
consistent with â methyl branched analogues of thiocyan-
atins B and C (2a /b), which based on arguments compa-
rable to those detailed above indicated that mixture C may
be comprised of a selection of alkene regioisomers (see
Figure 1). GC analysis of mixture C revealed major
components eluting at 43.2 (38%) and 51.2 (45%) min, with
additional minor components eluting at 42.2 (8%) and 50.8
(9%) min, not inconsistent with the proposed series of regio-
and stereoisomeric alkenes shown in Figure 1.
While we acknowledge that the complex status of mix-
tures A-C precludes unambiguous structure assignments,
the observations summarized above and in Figure 1 are
disclosed at this time as an illustration of the breadth of
metabolic variety that Oceanapia assembles around the
thiocyanatin molecular motif.
F igu r e 1. Minor thiocyanatins detected and tentatively identified in
mixtures A-C.
In summary, the Oceanapia sp. has yielded a remarkable
new class of natural products, several examples of which
display potent nematocidal activity. In addition to isolating
and identifying thiocyanatins A (1), B (2a ), C (2b), D1 (3a ),
D2 (3b), E1 (4a ), and E2 (4b) and drawing attention to a
series of minor metabolites (Figure 1), we have prepared
the synthetic â methyl branched thiocyanate 5 and thio-
carbamate 6 and the bisthiocarbamate 7. We have also
prepared monothiocyanate (8) and trithiocyanate (9) ana-
logues of thiocyanatin A (1), as well as the deoxy analogue
10. Furthermore, as outlined in Scheme 1, we have
prepared a chain elongated C18 homologue 17 of thiocy-
anatin A (1). Together with natural thiocyanatins, these
synthetic analogues provide some preliminary SAR obser-
vations, based around the most potent natural example of
this structure class, thiocyanatin A (1). Some noteworthy
analogues and their nematocidal activity (LD99) are listed
in Figure 2. Even this limited selection of analogues
highlights the importance of both the 2°-alcohol and SCN
functionalities and the influence of chain length on nema-
tocidal activity.
tibility of the thiocarbamate functionality to EI fragmenta-
tion in advance of C-C cleavage R to the 2°-OH.
Our analysis of the Oceanapia extract also yielded three
minor thiocyanatin fractions (mixture A (<0.5 mg), mixture
B (<0.7 mg), and mixture C (<4 mg)) eluting as single
peaks by C18 HPLC.
1H NMR and (+)ESI-HRMS analysis of mixture A
suggested a selection of â methyl branched and further
hydrolyzed (SCN to SCONH2) analogues of thiocyanatins
1
E1/E2 (3a /b). More specifically, resonances in the H NMR
(CDCl3) spectrum for mixture A attributed to 2°-methyls
(δ 1.05 and 0.95) and deshielded diastereotopic methylene
protons (δ 2.68 and 2.78) were consistent with â methyl
branching adjacent to both SCN and SCONH2 termini. This
analysis was confirmed by direct spectroscopic comparison
with authentic samples of both the S thiocyanate 6 and
its thiocarbamate analogue 7, which revealed a character-
istic 1H NMR chemical shift difference between a 2°-Me â
to a SCN (∼δ 1.05) versus a SCONH2 (∼δ 0.95). The
remaining 1H NMR (CDCl3) spectral data for mixture A
were consistent with key resonances in thiocyanatins E1/
E2 (3a /b), while the 13C NMR (CDCl3) data revealed a
resonance (20 ppm) consistent with the new â branched
methyl termini. A list of plausible mixture A components
is shown in Figure 1. Although an inseparable mixture by
While the nematocidal mode of action of the thiocyan-
atins has yet to be determined, it is interesting to note that
synthetic thiocyanates attracted early attention as poten-
tial insecticides. Researchers in the mid to late 1900s
observed that alkyl thiocyanates could under appropriate
enzymatic conditions release HCN, and they went on to

1280 J ournal of Natural Products, 2004, Vol. 67, No. 8
Capon et al.
Sch em e 1a
subjected to C18 HPLC (2.0 mL/min 85-100% MeOH/water
through a Zorbax Eclipse XDB 5 µm 250 × 9.4 mm C18 column)
to give in order of elution thiocyanatin A (1) (11.0 mg, 0.03%),
an inseparable mixture of thiocyanatins D1 and D2 (3a /b) (4.6
mg, 0.013%), and an inseparable mixture of thiocyanatins B
and C (2a ,b) (23.2 mg, 0.066%). The 1:1 hexane/Et2O-soluble
material was treated in a similar fashion to give in order of
elution an inseparable mixture of thiocyanatins E1 and E2 (4a /
b) (5.4 mg, 0.015%), thiocyanatin A (1) (15.3 mg, 0.043%), an
inseparable mixture of thiocyanatins D1 and D2 (3a /b) (3.8 mg,
0.011%), and an inseparable mixture of thiocyanatins B and
C (2a ,b) (1.6 mg, 0.004%). Also recovered were the HPLC
inseparable mixtures A (<0.5 mg, <0.001%), B (<0.7 mg,
<0.001%), and C (<4 mg, <0.01%). All reported yields are
measured against the dry weight of the sponge sample. The
known thiocyanatins A-C were identical in all respects to
those previously reported.1
Th iocya n a tin s D1 a n d D2 (3a /b): clear viscous oil; [R]21
D
-3.7° (c 0.41, CHCl3); IR (CHCl3) νmax 3500 (br), 2156 cm-1
;
1H NMR (see Table 1); 13C NMR (100 MHz, CDCl3) δ 18.6 (2-
Me), 25.5, 25.6, 26.6, 27.86/27.91 (C-14), 28.78/28.82 (C-13),
29.3, 29.4, 29.5, 29.6, 29.9 (C-15), 33.8 (C-2), 34.1 (C-16), 35.2
(C-3), 37.4/37.5 (C-7/9 [3a ], C-8/10 [3b]), 41.4 (C-1), 71.9 (C-8
[3a ], C-9 [3b]), 112.3 (16-SCN), 112.8 (1-SCN); (+)ESIMS m/z
393 [(M + Na)+], 353 [(M + H)+ - H2O]; (+)ESI-HRMS m/z
393.1995 [(M + Na)+, calcd for C19H34S2N2ONa, 393.2010].
Th iocya n a tin s E1 a n d E2 (4a /b): clear viscous oil; 1H
NMR (see Table 1); 13C NMR (100 MHz, d4-MeOH) δ 26.6, 26.7,
28.9 (C-14), 29.7 (C-3), 29.9, 30.0, 30.2, 30.3, 30.4 (C-1), 30.5,
30.6, 30.69, 30.74, 31.1 (C-15), 31.6 (C-2), 34.9 (C-16), 38.4 (C-
7/9 [4a ], C-8/10 [4b]), 72.4 (C-8 [4a ], C-9 [4b]), 113.7 (SCN),
172.0 (SCONH2); (+)ESIMS m/z 397 [(M + Na)+]; (+)ESI-
HRMS m/z 397.1949 [(M + Na)+, calcd for C18H34S2N2O2Na,
397.1960].
(S)-2-Meth ylbu tyl Th iocya n a te (5).6 A mixture of LiAlH4
(0.35 g, 9.18 mmol) and dry ether (15 mL) was added dropwise
to a solution of (+)-(S)-2-methylbutyric acid (0.47 g, 4.59 mmol)
in dry ether (10 mL) under gentle reflux. Stirring was
continued at 40 °C for 16 h, after which the excess LiAlH4 was
filtered and the filtrate treated with EtOAc (20 mL) followed
by 10% HClaq (20 mL). The aqueous phase was extracted with
ether (10 mL × 2) and the combined organic phase washed
sequentially with 10% HClaq (10 mL), H2O (10 mL), and brine
(10 mL), then dried (anhydrous MgSO4). The ether was
removed under reduced pressure to give (S)-2-m eth ylbu ta n ol
(0.17 g, 42%) as a pale yellow oil. The crude alcohol (0.17 g,
1.93 mmol) in dry CH2Cl2 (20 mL) was treated with Et3N (0.4
mL, 2.90 mmol) and the mixture chilled to -10 °C, at which
point methansulfonyl chloride (0.15 mL, 2.04 mmol) was added
dropwise and the reaction stirred at -10 °C for 1 h. The
reaction mixture was poured onto ice and extracted with
additional CH2Cl2 (10 mL × 2), after which the combined
organic extract was washed sequentially with ice-cold water
(10 mL × 2), 4% w/v aqueous oxalic acid (10 mL × 2), 2%
NaHCO3 (10 mL × 2), and brine (10 mL), then dried (anhy-
drous Na2SO4). The CH2Cl2 was removed under reduced
pressure to give the (S)-2-m eth ylbu tyl m eth a n esu lfon a te
(0.16 g, 51%). A mixture of crude mesylate (0.16 mg, 0.96
mmol) and KSCN (93 mg, 0.96 mmol) in dry THF (20 mL) was
stirred at reflux for 16 h. The reaction was cooled to RT and
the solvent removed under reduced pressure. The residue was
partitioned between H2O (10 mL) and ether (10 mL) and
further extracted with ether (10 mL × 2), after which the
combined organic phase was sequentially washed with H2O
(10 mL) and brine (10 mL) and then dried (anhydrous MgSO4).
Evaporation of the ether followed by elution through a silica
SPE cartridge (eluent 8% EtOAc/hexane) yielded the desired
(S)-2-m eth ylbu tyl th iocya n a te (5) (20 mg, 16%) as a pale
yellow oil, along with unreacted mesylate (57 mg): 1H NMR
a
a, PPh3; b, NaHMDS, O2; c, mCPBA; d, LAH, e, H+; f, TsCl; g, KSCN.
explore the agrochemical potential of this effect. For
example, C12 and C13 n-alkyl and sec-alkyl thiocyanates
were noted to be outstanding insecticides against adult
female fruit-tree red spider mites,15 while various other
alkyl thiocyanates were described as lethal to mice and
houseflies16 and selected beetles17 through the action of
HCN. Although these early investigations focused on
synthetic alkyl thiocyanates, it has long been known that
certain plants and insects employ HCN as a defensive
agent through the intermediary of cyanogenic glycosides.
At this stage the chemical ecology of the thiocyanatins
remains unknown, although it is tempting to speculate that
naturally occurring alkyl thiocyanates such as the thiocy-
anatins may function as chemical defense agents, counter-
ing predatory attack through localized enzyme-mediated
release of HCN.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. See ref 1.
An im a l Ma ter ia l. See ref 1.
Extr a ction a n d Isola tion . The EtOH extract of the
Oceanapia sp. was decanted and concentrated in vacuo (800
mg), and the nematocidal components were extracted by
trituration with CH2Cl2 (329 mg), as detailed in an earlier
report.1 The CH2Cl2-soluble material was then subjected to a
further series of triturations to give hexane (92.6 mg), 1:1
hexane/Et2O (103.1 mg), and Et2O soluble (73.5 mg) along with
an insoluble (54.3 mg) fraction. The hexane-soluble material
was treated with MeOH (0.5 mL), the precipitated fats and
steroids (12.4 mg) were removed, and the supernatant was
(see Table 1); [R]14 +18.8° (c 1.0, CHCl3); IR (film) νmax 2156
D
cm-1; (+)ESIMS m/z 152 [(M + Na)+].
(S)-2-Meth ylbu tyl Th ioca r ba m a te (6). (S)-2-Methylbutyl
thiocyanate (5) (8.4 mg, 0.065 mmol) was dissolved in MeOH
(40 mL) and dry HCl gas passed through the solution for 2 h
at 0 °C. The solution was then stirred at RT for 16 h before

Nematocidal Thiocyanatins from Oceanapia sp.
J ournal of Natural Products, 2004, Vol. 67, No. 8 1281
F igu r e 2. Selected SAR data on thiocyanatins.
removal of the solvent under reduced pressure to afford (S)-
mL) and Et2O (5 mL). The aqueous phase was extracted with
Et2O (3 × 5 mL) and the combined organic phase washed with
brine (5 mL), dried (anhydrous MgSO4), filtered, and concen-
trated under reduced pressure. Purification by silica column
chromatography using 20% EtOAc/hexane as eluent yielded
8 (59 mg, 73%): IR νmax (film) 3354, 2154 cm-1; 1H NMR (300
MHz, CDCl3) δ 1.27-1.39 (m, methylene envelope), 1.49-1.54
(m, 2-H2, 15-H2), 2.91 (t, J ) 7.2 Hz, 16-H2 (1-H2)), 3.53 (m,
8-H), 3.58 (t, J ) 6.6 Hz, 1-H2 (16-H2)); 13C NMR (100 MHz,
CDCl3) 25.4, 25.5, 25.6, 27.8, 28.7, 29.2, 29.3, 29.4, 29.5, 29.7,
32.6, 33.9, 37.2, 37.3, 62.7, 71.7, 122.4; (+)ESIMS m/z 338
(M + Na, 100) 316 (M + H, 80); (+)ESI-HRMS m/ z 338.2126
(M + Na) (calcd 338.2130).
2-methylbutyl thiocarbamate (6) (6.8 mg, 71%) as a pale yellow
1
oil: [R]18 +35° (c 0.5, CHCl3); H NMR (400 MHz, CDCl3) δ
D
0.90 (t, J ) 7.2 Hz, 4-H3), 0.94 (d, J ) 6.8 Hz, 2-Me), 1.22 (m,
3-H), 1.46 (m, 3-H), 1.60 (m, 2-H), 2.78 (dd, J ) 13.2, 7.2 Hz,
1-H), 2.94 (dd, J ) 13.2, 5.6 Hz, 1-H); 13C NMR (100 MHz,
CD3OD) 11.3, 18.6, 28.5, 35.3, 36.8, 169.5; (+)ESIMS m/z 149
(M + H2, 60); (+)ESI-HRMS m/z 170.0614 (M + Na) (calcd for
C6H13NOSNa 170.0616).
Syn th esis of 1,16-Dith ioca r ba m yl-8-h yd r oxyh exa d ec-
a n e (7). Synthetic thiocyanatin A (1)1 (43 mg, 0.12 mmol) was
dissolved in MeOH (50 mL) and dry HCl gas passed through
the solution for 2 h at 0 °C. The solution was then stirred at
RT for 16 h before removal of the solvent under reduced
pressure to afford 1,16-dithiocarbamyl-8-hydroxyhexadecane
1,8,16-Tr ith iocya n a toh exa d eca n e (9). A mixture of the
tritosylate described above (0.70 g, 0.95 mmol) and KSCN (0.20
g, 2.09 mmol) in dry THF (25 mL) was stirred at reflux for 16
h, after which the solvent was removed and the residue
partitioned between H2O (10 mL) and Et2O (10 mL). The
aqueous phase was extracted with Et2O (3 × 10 mL), and the
combined organic phase was washed with brine (20 mL), dried
(anhydrous MgSO4), filtered, and concentrated under reduced
pressure. Purification by silica column chromatography using
10% EtOAc/hexane as eluent yielded 9 (0.31, 81%): IR νmax
(film) 2152 cm-1; 1H NMR (400 MHz, CDCl3) δ 1.30-1.50 (m,
methylene envelope), 1.72 (m, 3-H2, 14-H2), 1.80 (m, 2-H2, 15-
H2), 2.91 (t, J ) 7.2 Hz, 1-H2, 16-H2), 3.02 (m, 8-H); 13C NMR
(100 MHz, CDCl3) 26.7, 26.8, 27.7, 27.8, 28.6, 28.7, 28.8, 28.9,
29.0, 29.7, 29.8, 33.9, 34.0, 35.2, 35.3, 51.6, 111.4, 112.4; (+)-
ESIMS m/z 339 (M - SCN, 100), 420 (M + Na, 60); (+)ESI-
(7) (30.7 mg, 65%): IR (KBr) νmax 3385 (br), 3182, 1651 cm-1
;
1H NMR (see Table 1); 13C NMR (100 MHz, CD3OD) 26.6, 26.7
(C6, C10), 29.8 (C3, C14), 30.2, 30.3 (C1, C16), 30.4 (C4, C13),
30.6, 30.7, 30.8 (C5, C11, C12), 31.7 (C2, C15), 38.4 (C7, C9);
(+)ESIMS m/z 415 (M + Na, 100); (+)ESI-HRMS m/z 415.2067
(M + Na) (calcd for C18H36N2O3S2Na 415.2065).
1,8-Dih yd r oxy-16-t h iocya n a t oh exa d eca n e/8,16-Dih y-
d r oxy-1-th iocya n a toh exa d eca n e (8). To a stirred suspen-
sion of 1,8,16-trihydroxyhexadecane1 (0.28 g, 0.74 mmol),
DMAP (6.5 mg, 0.053 mmol), and TsCl (0.28 g, 1.47 mmol) in
dry CH2Cl2 (5.5 mL) was added Et3N (0.21 mL, 1.48 mmol) at
0 °C. After the mixture was stirred at RT for 16 h, additional
TsCl (0.28 g, 1.47 mmol) and Et3N (0.21 mL, 1.48 mmol) were
added and stirring continued for a further 16 h. The reaction
mixture was concentrated under reduced pressure and the
residue triturated with EtOAc. The EtOAc extract was con-
centrated under reduced pressured and subjected to silica
column chromatography using 20-40% EtOAc/hexane as
eluent to give a mixture containing the C-1 and C-16 mono-
tosylates, the C-1 and C-16 ditosylates, and the tritosylate
derivative. The mono-, di-, and tritosylates were separated by
silica chromatography. As described in an earlier report,1 the
ditosylate was used to prepare synthetic thiocyanatin A (1).
The mixed monotosylates (0.11 g, 0.26 mmol) were combined
with KSCN (0.05 g, 0.52 mmol) in dry THF (5 mL) and stirred
at reflux under N2 for 16 h. The solvent was removed under
reduced pressure and the residue partitioned between H2O (5
HRMS m/z 420.1567 (M + Na) (calcd for
420.1578).
C19H31N3S3Na
1,16-Dith iocya n a toh exa d eca n e (10). A mixture of com-
mercially available 1,16-hexadecanediol (0.25 g, 0.97 mmol),
HBr (45%) (0.31 mL, 2.42 mmol), and concentrated H2SO4 (0.11
mL) was stirred at 40 °C for 5 h, then diluted with H2O (5
mL) and Et2O (5 mL). The aqueous phase was extracted with
Et2O (2 × 5 mL), and the combined organic phase washed with
H2O (2 × 5 mL), saturated NaHCO3aq (3 × 5 mL), and brine
(5 mL), then dried (anhydrous MgSO4). Filtration followed by
concentration under reduced pressure gave 1,16-dibromohexa-
decane (0.18 g, 48%) as a pale yellow oil, which was then
dissolved in dry THF (20 mL) and KSCN (0.11 g, 1.15 mmol)

1282 J ournal of Natural Products, 2004, Vol. 67, No. 8
Capon et al.
was added. The mixture was stirred at reflux for 16 h, after
which the solvent was removed under reduced pressure. The
residue was partitioned between H2O (10 mL) and Et2O (10
mL), and the aqueous phase extracted with Et2O (3 × 10 mL).
The combined organic phase was washed with brine (10 mL),
dried (anhydrous MgSO4), filtered, and concentrated under
reduced pressure. Purification by silica SPE cartridge using
5% EtOAc/hexane as eluent afforded the dithiocyanate 10 (0.13
g, 83%): IR νmax (film) 2152 cm-1; 1H NMR (300 MHz, CDCl3)
δ 1.23-1.29 (m, methylene envelope), 1.43 (m, 3-H2, 14-H2),
1.82 (quin, J ) 7.5 Hz, 2-H2, 15-H2), 2.94 (t, J ) 7.2 Hz, 1-H2,
16-H2); 13C NMR (100 MHz, CDCl3) 27.9, 28.8, 29.3, 29.4, 29.5,
29.6, 29.8, 34.0, 112.4; (+)ESIMS m/z 363 (M + Na, 100), 341
(M + H, 60), 282 (M - SCN, 70); (+)ESI-HRMS m/z 363.1902
(M + Na) (calcd for C18H32N2S2Na 363.1905).
saturated NaHCO3aq (25 mL), H2O (25 mL), and brine (25 mL),
then dried (anhydrous Na2SO4). The crude material was
recrystallized from hexane/EtOAc to yield 1,9,18-trihydroxy-
octadecane (15) (61 mg, 32%) as a white solid: mp 58-62 °C;
1
IR νmax (KBr) 3310 cm-1; H NMR (400 MHz, CDCl3) δ 1.12-
1.31 (m, methylene envelope), 1.36-1.45 (m, 2-H2, 17-H2), 3.42
(m, 9-H2), 3.45 (t, J ) 6.6 Hz, 1-H2, 18-H2); 13C NMR (100 MHz,
CDCl3) 26.6, 26.7, 26.8, 26.9, 30.3, 30.4, 30.5, 30.6, 30.7, 30.8,
38.5, 64.1, 73.1; (+)ESIMS m/z 325 (M + Na, 100), 341 (M +
K, 10); (+)ESI-HRMS m/z 325.2710 (M + Na) (calcd for
C
18H38O3Na 325.2719).
9-Hyd r oxy-1,18-d ith iocya n a to-octa d eca n e (17). To a
stirred suspension of 15 (61 mg, 0.20 mmol), DMAP (2.5 mg,
0.02 mmol), and p-TsCl (80.9 mg, 0.42 mmol) in dry CH2Cl2
(2 mL) was added Et3N (59 µL, 0.42 mmol) at 0 °C, and the
mixture stirred at RT for 16 h, after which DMAP (1 mg, 8
µmol), p-TsCl (40 mg, 0.21 mmol), and Et3N (30 µL, 0.21 mmol)
was added at 0 °C. The reaction was stirred at RT for a further
16 h, then concentrated under reduced pressure. The residue
was purified by silica chromatography using 30% EtOAc/
hexane as eluent to yield 1,16-ditosylate 16 (52 mg, 42%). A
mixture of 16 (52 mg, 0.085 mmol) and KSCN (21 mg, 0.21
mmol) in dry THF (10 mL) was stirred at reflux for 20 h, after
which solvent was removed under reduced pressure and the
residue partitioned between H2O (10 mL) and Et2O (10 mL).
The aqueous phase was extracted with Et2O (3 × 10 mL) and
the combined organic phase washed with brine (10 mL), dried
(anhydrous MgSO4), filtered, and concentrated under reduced
pressure. Purification by silica column chromatography using
20% EtOAc/hexane yielded 9-hydroxy-1,18-dithiocyanato-oc-
1,9,18-Tr ih yd r oxyocta d eca n e (15). A mixture of com-
mercially available 9-bromo-1-nonanol (5 g, 22.4 mmol), dihy-
dropyran (2.1 mL, 23.3 mmol), and concentrated HCl (22 µL)
was stirred at RT for 18 h. The reaction mixture was diluted
with Et2O (20 mL) and washed with H2O (20 mL), saturated
NaHCO3aq (2 × 20 mL), and brine (20 mL), then dried
(anhydrous MgSO4), filtered, and concentrated under reduced
pressure. Purification by silica column chromatography using
10% EtOAc/hexane as eluent yielded the THP-protected
alcohol 11 (5.74 g, 84%). A mixture of 11 (5.74 g, 18.9 mmol)
and PPh3 (4.46 g, 18.9 mmol) in MeCN (45 mL) was stirred at
reflux for 16 h, then concentrated under reduced pressure to
give the corresponding Wittig salt 12 (10.6 g, 99%) as a
colorless oil. To a stirred solution of 12 (4.89 g, 8.66 mmol) in
dry THF (35 mL) and DMPU (11 mL) under N2 at RT was
added dropwise NaHMDS (8.66 mL, 8.66 mmol, 1 M solution
in THF). Stirring was continued for 30 min, after which O2
was passed into the reaction, and stirring continued at 55 °C
for 16 h. Saturated NH4Claq was added and the mixture poured
into H2O (150 mL). The aqueous phase was extracted with
EtOAc (3 × 50 mL), and the combined organic phase washed
with H2O (2 × 100 mL), brine (100 mL), then dried (anhydrous
MgSO4), filtered, and concentrated under reduced pressure.
Purification by silica column chromatography using 5% EtOAc/
hexane as eluent yielded the dimer alkene 13 (1.11 g, 61%) as
a clear oil: 1H NMR (300 MHz, CDCl3) δ 1.29 (m, methylene
envelope), 1.49-1.63 (m, 3′-H2, 4′-H2, 8′-H2, 9′-H2), 1.72 (m,
2′-H2, 7′H2), 1.82 (m, 7-H2, 12-H2), 2.01 (8-H2, 11-H2), 3.39 (dt,
J ) 9.6, 6.6 Hz, 1-H2, 16-H2), 3.50 (m, 5′-H2, 10′-H2), 3.72 (dt,
J ) 9.6, 6.9 Hz, 1-H2, 16-H2), 3.87 (m, 5′-H2, 10′-H2), 4.57 (t, J
) 4.5 Hz, 1′-H2, 6′-H2), 5.34 (m, 9-H2, 10-H2); 13C NMR (75
MHz, CDCl3) 19.6, 25.5, 26.2, 27.2, 29.2, 29.4, 29.5, 29.7, 30.7,
62.3, 67.6, 98.8, 129.8; (+)ESIMS m/z 285 (M - (THP)2 + H,
100), 475 (M + Na, 50); (+)ESI-HRMS m/z 475.3746 (M + Na)
(calcd for C28H52O4Na 475.3763). A mixture of 13 (0.41 g, 0.91
mmol) and mCPBA (1.00 g, 5.3 mmol) in dry CH2Cl2 (80 mL)
was stirred at RT under N2 for 16 h. The reaction mixture
was washed with saturated NaHCO3aq (3 × 80 mL), H2O (80
mL), and brine (80 mL) and dried (anhydrous MgSO4), then
filtered and concentrated under reduced pressure. Purification
by silica column chromatography using 15% EtOAc/hexane as
eluent afforded the corresponding epoxide 14 (0.31 g, 73%).
To a stirred suspension of LiAlH4 (0.13 g, 3.32 mmol) in dry
Et2O (20 mL) under N2 at RT was added dropwise a solution
of 14 (0.31 g, 0.66 mmol) in dry Et2O (20 mL) maintaining a
gentle reflux. Refluxing was continued for 18 h, and then the
reaction was quenched with EtOAc followed by addition of 1
M HClaq (40 mL) and H2O (40 mL). The aqueous phase was
extracted with Et2O (3 × 40 mL) and the combined organic
extract washed with 1 M HClaq (3 × 40 mL), H2O (3 × 40 mL),
and brine (40 mL), then dried (anhydrous MgSO4) and filtered.
Removal of the solvent under reduced pressure yielded a
product (0.30 g, 0.63 mmol), which was taken up in MeOH (2
mL) and treated with added concentrated H2SO4 (0.1 mL) at
RT for 16 h, then concentrated under reduced pressure. The
residue was diluted with Et2O (25 mL) and washed with
tadecane (17) (20 mg, 62%): IR νmax (KBr) 3503, 2152 cm-1
;
1H NMR (300 MHz, CDCl3) δ 1.29-1.52 (m, methylene
envelope), 1.82 (quin, J ) 7.8 Hz, 2-H2, 15-H2), 2.94 (t, J )
6.9 Hz, 1-H2, 16-H2), 3.58 (m, 9-H); 13C NMR (100 MHz, CDCl3)
25.5, 25.6, 27.8, 27.9, 28.7, 28.8, 29.2, 29.3, 29.4, 29.5, 29.6,
29.8, 29.9, 34.0, 34.1, 37.4, 37.5, 71.9, 112.4; (+)ESIMS m/z
407 (M + Na, 100), 423 (M + K, 80); (+)ESI-HRMS m/z
407.2167 (M + Na) (calcd for C20H36N2OS2Na 407.2167).
Ack n ow led gm en t. We acknowledge the CSIRO Division
of Oceanography and the crew and scientific personnel aboard
the O. R. V. Franklin for collection of the Oceanapia specimen.
We also acknowledge technical support by A. Loveless, taxo-
nomic classification by L. Goudie, and high-resolution mass
measurements by S. Duck of Monash University. This research
was supported by the Australian Research Council and No-
vartis Animal Health Australasia Pty Ltd.
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