Structure and synthesis of a unique isonitrile lipid isolated from the marine mollusk actinocyclus papillatus

Article abstract of DOI:10.1021/ol200377w

The first chemical study of an Actinocyclidae nudibranch, Actinocyclus papillatus, resulted in the isolation of (-)-actisonitrile (1), a lipid based on a 1,3-propanediol ether skeleton. The structure was established by spectroscopic methods, whereas the a

Full text of DOI:10.1021/ol200377w

ORGANIC
LETTERS
2011
Vol. 13, No. 8
1897–1899
Structure and Synthesis of a Unique
Isonitrile Lipid Isolated from the Marine
Mollusk Actinocyclus papillatus
Emiliano Manzo,† Marianna Carbone,† Ernesto Mollo,† Carlo Irace,‡
Antonio Di Pascale,‡ Yan Li,§ Maria Letizia Ciavatta,† Guido Cimino,† Yue-Wei Guo,*,§
and Margherita Gavagnin*,†
Consiglio Nazionale delle Ricerche (CNR), Istituto di Chimica Biomolecolare (ICB),
Via Campi Flegrei, 34, 80078 Pozzuoli (Na), Italy, Dipartimento di Farmacologia
ꢀ
ꢀ
Sperimentale, Facolta di Farmacia, Universita ‘Federico II’ di Napoli, Italy, and State
Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, Shanghai 201203, PR China
ywguo@mail.shcnc.ac.cn; margherita.gavagnin@icb.cnr.it
Received November 30, 2010
ABSTRACT
The first chemical study of an Actinocyclidae nudibranch, Actinocyclus papillatus, resulted in the isolation of (-)-actisonitrile (1), a lipid based on
a 1,3-propanediol ether skeleton. The structure was established by spectroscopic methods, whereas the absolute configuration of the chiral
center was determined by comparing the optical properties of natural actisonitrile with those of (þ)- and (-)-synthetic enantiomers, opportunely
prepared. Both (-)- and (þ)-actisonitrile were tested in preliminary in vitro cytotoxicity bioassays on tumor and nontumor mammalian cells.
Lipids are undoubtedly representatives of chemical bio-
diversity. Fatty acids, fatty alcohols, and lipids containing
them are ubiquitous in nature, and a number of structures
occur in both terrestrial and marine organisms, even though
the latter organisms have proven to be a major source of
unique structures with promising bioactivity.1 In particular,
ether lipids usually occurring as minor components2,3 have
attracted the attention of medicinal chemists due to their
antineoplastic4 and other pharmacological activities.3,5
Typical ether lipids are monoalkyl ethers of glycerin, also
called alkyl/alkenyl glyceryl ethers.
In the course of our screening program for cytotoxic
compounds from marine mollusks, we have isolated a
unique etherlipidfrom asinglespecimenofthe nudibranch
Actinocyclus papillatus, belonging to the never studied
Actinocyclidae family. The structure of this novel com-
pound, which we named actisonitrile (1), is characterized
by the presence of a 1,3-propanediol moiety bearing an
isonitrile group at the C-2 position.
The isolation, structure elucidation, and synthesis of
actisonitrile are herein reported together with the results
of a preliminary bioactivity evaluation.
† Istituto di Chimica Biomolecolare (ICB).
‡
ꢀ
Universita `Federico II' di Napoli.
§ Chinese Academy of Sciences.
(1) Blunt, J. W.; Copp, B. R.; Munro, M. H. G.; Northcote, P. T.;
Prinsep, M. R. Nat. Prod. Rep. 2010, 27, 165–237 and previous issues in
this series..
(2) Snyder, F., Ed., Ether Lipids: Chemistry and Biology; Academic
Press: New York, 1972.
(3) Urata, K.; Takaishi, N. J. Am. Oil Chem. Soc. 1996, 73, 819–830.
(4) Ando, K.; Kodama, K.; Kato, A.; Tamura, G.; Arima Cancer
Res. 1972, 32, 125–129.
(5) Berger, F. M.; Hubbard, C. V.; Ludwig, B. J. Appl. Environ.
Microbiol. 1953, 1, 146–149.
r
10.1021/ol200377w
2011 American Chemical Society
Published on Web 03/15/2011

The mantle and the inner organs from one individual of
A. papillatus, collected by scuba along the coast of Wei Zhou
Island (South China Sea) during May 2007, were separately
extracted with acetone. The TLC chromatographic analysis
of the diethyl ether soluble portion of both extracts showed
the presence in the mantle of a main metabolite (Rf 0.70,
light petroleum ether/diethyl ether 1:1) which was not
detected in the inner organs. The subsequent fractionation
of the mantle extract (172.1 mg) by Sephadex LH20 chro-
matography, followed by further silica gel purification,
afforded pure actisonitrile (1, 8.8 mg, 5.0%) as the major
component (for details, see the Supporting Information).
Actisonitrile (1)6 showed in the HRESIMS spectrum a
sodiated molecular peak at m/z 390.2973 according to the
molecular formula C22H41NO3. The co-occurrence in the
ESIMS spectrum of a base peak at m/z 363, corresponding
to the loss of HCN, suggested the presence of an isonitrile
group. This hypothesis was further confirmed by the char-
acteristic IR absorption at 2140 cm-1 as well as by the
diagnostic signal at δ 158.7 in the 13C NMR spectrum.
Scheme 1. Synthesis of 1
1
The H NMR spectrum of 1 showed signals at δ 0.88
(3H, t, J = 7 Hz, H3-160), 1.20-1.38 (26H, overlapped
multiplets, H2-30/ H2-150), 1.57 (2H, m, H2-20), 3.48 (2H,
app t, J = 7 Hz, H2-10) that were attributed to a saturated
C16 fatty alcohol. A typical set of signals at δ 3.63 (2H, m,
H2-3), 3.96 (1H, m, H-2), 4.20 (1H, dd, J = 11, 7 Hz, H-1a),
4.30 (1H, dd, J = 11, 4 Hz Hz, H-1b)] that could be attri-
buted to a glyceryl-like fragment was also observed. The
proton spectrum was completed by a 3H singlet at δ 2.12
assigned to an acetyl group. These data were consistent
with a structure containing an acetylated 2-isonitrile-1,3-
propanediol moiety connected to a fatty alcohol residue
aminoglycerol) derivative. In this case, the isonitrile group
should originate by dehydration of a corresponding N-
formylserinol precursor. Even though glycerolipids are very
common in marine organisms,1 the occurrence of a certain
number of serinolipids from tunicates,7-10 sponges,11 and
cyanobacteria12 implies that both biosynthetic pathways
could be plausible. However, further studies are necessary to
elucidate the true origin of this molecule.
In order to assign the absolute configuration of C-2,
which is the only chiral center of actisonitrile (1), the
stereospecific total synthesis of both (-)- and (þ)-enantio-
mers was carried out. The synthesis of each compound was
accomplished in eight steps, as outlined in Scheme 1. The
levorotatory enantiomer (-)-1 was prepared starting from
the commercially available S-(-)-glycidyl trityl ether (2).
The opening of the epoxide 2 under basic conditions
allowed the introduction of the fatty alcohol hexadecanol
to the less substituted position. The subsequent mesylation
of the intermediate 3, followed by reaction with sodium
azide, led to the formation of C-N bound at the C-2
position of the 1,3-propanediol moiety affording the
1
through an ether linkage. Analysis of the H-1H COSY
experiment confirmed the defined spin systems. The 13C
NMR spectrum of 1 was very indicative. Along with the
expected resonances due to the alkyl chain carbons of the
fatty alcohol, the spectrum contained two oxygenated me-
thylene signals at δ 69.1 (C-3) and 62.7 (C-1) and a methine
carbon at δ 53.1 (C-2) indicating that the secondary carbon
in the C3 fragment had to be linked to the isonitrile group.
According to this structural hypothesis, the HMBC
spectrum of 1 showed diagnostic correlations between
C-3 and H2-10 as well as between the carbonyl of the acetyl
residue and H2-1.
Therefore, the structure of actisonitrile (1) was deter-
mined to be 3-(hexadecyloxy)-2-isonitrile-propyl acetate.
This compound exhibits structural features without any
precedent as it incorporates an isonitrile function in the
1,3-propanediol fragment. Actisonitrile (1) could be con-
sidered either a glyceryl-like ether in which the 2-OH group
is replaced by an isonitrile function or a serinol (2-
ꢁ ꢁ
´
(7) Gonzalez, N.; Rodrıguez, J.; Jimenez, C. J. Org. Chem. 1999, 64,
5705–5707.
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Lett. 2000, 2, 1605–1607.
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Matsunaga, S.; Fusetani, N. J. Org. Chem. 2007, 72, 1218–1225.
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Nakao, Y.; Fusetani, N.; Matsunaga, S. Tetrahedron 2007, 63, 6748–6754.
(11) Ueoka, R.; Fujita, T.; Iwashita, T.; van Soest, R. W. M.;
Matsunaga, S. Biosci. Biotechnol. Biochem. 2008, 72, 3055–3058.
(12) Wan, F.; Erickson, K. L. J. Nat. Prod. 1999, 62, 1696–1699.
(6) Actisonitrile (1): [R]D -7.0 (c = 0.27, CHCl3); CD (n-hexane)
[θ]204 850; IR (liquid film) 2925, 2854, 2140, 1748, 1228 cm-1; 1H NMR
values (CDCl3) δ 0.88 (3H, t, J = 7 Hz, H3-160), 1.20-1.38 (26H,
overlapped multiplets, H2-30/ H2-150), 1.57 (2H, m, H2-20), 2.12 (3H, s,
OCOCH3), δ 3.48 (2H, app t, J = 7 Hz, H2-10), 3.63 (2H, m, H2-3), 3.96
(1H, m, H-2), 4.20 (1H, dd, J = 11, 7 Hz, H-1a), 4.30 (1H, dd, J = 11 and
13
4 Hz Hz, H-1b); selected C NMR (CDCl3) δ 14.1 (C-160), 20.6
(COCH3), 29.6 (C-20), 53.1 (C-2), 62.7 (C-1), 69.1 (C-3), 72.0 (C-10);
ESIMS m/z 390 (M þ Na), 363 (M - HCN þ Na)þ; HR ESIMS calcd for
C22H41NO3Na 390.2984, found 390.2973.
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Org. Lett., Vol. 13, No. 8, 2011

Figure 2. CD curves (θ) of natural actisonitrile (blue), synthetic
(-)-actisonitrile (green), and synthetic (þ)-actisonitrile (red).
Figure 1. Δδ (ppm) for (i) Mosher derivatives 6a and 6b and (ii)
the corresponding detritylated MTPA amides 6c and 6d.
following the same synthetic strategy (for details, see the
Supporting Information).
corresponding azide 5. Because introduction of the azide
group is operated predominantly through a SN2 mechan-
ism, we decided to verify the inversion of configuration at
C-2 by applying the modified Mosher method13,14 to the
amino derivative 6, which was obtained by reduction with
Lindlar’s catalyst of azide 5. Treatment of compound 6 with
(R)- and (S)-MTPA chlorides in dry CH2Cl2 and DMAP
afforded the corresponding (S)-MTPA (6a) and(R)-MTPA
(6b) amides, respectively. The two Mosher derivatives were
characterized by 2D-NMR experiments (see the Supporting
Information). The Δδ (δS - δR) values observed for the
signals of protons close to the amino group at C-2 indicated
the R configuration (Figure 1i). With the aim of excluding a
possible interaction of the trityl residue on the observed Δδ
values that could invalidate the applied Mosher method, the
evaluation of the proton chemical shift differences was also
made on the detritylated MTPA amide derivatives (6c and
6d) obtained by acetolysis of 6a and 6b (see the Supporting
Information). According to the different priority order in 6c
and 6d with respect to 6a and 6b, the S configuration was
observed at C-2 (Figure 1ii).
Having confirmed the inversion of configuration at C-2,
amine 6 was submitted to a formylation reaction. Accord-
ingtoReddyetal.,15 thelessexpensiveammonium formate
was used as N-formylating agent. The resulted formyl
derivative 7 was then submitted to acetolysis to provide
by removal of the trityl group the corresponding alcohol 8.
Compound 8 was acetylated affording the intermediate 9.
The subsequent dehydration of formamide 9 with p-tosyl
chloride in pyridine led to (-)-3-(hexadecyloxy)-2-(R)-
isonitrile-propyl acetate, (-)-actisonitrile 1.
The optical properties including the CD absorption of
both synthetic products were compared with those of
natural 1 (Figure 2). The negative optical rotation value
and the positive CD profile of natural 16 showed results
similar to those of synthetic (-)-enantiomer,16 thus assign-
ing the R absolute stereochemistry.
By considering interest in the biological properties of
ether lipids,3-5 the bioactivity of both (-)- and (þ)-
actisonitrile enantiomers were tested in a fluorimetric
culture cytotoxicity assay on tumor and nontumor mam-
malian cells (see the Supporting Information). Without
showing appreciable bioselectivity, both enantiomers ex-
hibited a parallel concentration-dependent toxic profile,
displaying IC50 values within the micromolar range. In
particular, (-)-1 and (þ)-1 show moderate cytotoxicity
against nontumor H9c2 rat cardiac myoblast cells (IC50 23
( 6 and 23 ( 6 μM, respectively).
The selective distribution of actisonitrile (1) in the ex-
ternal tissues of A. papillatus as well as its cytotoxicity could
suggest a possible involvement of this molecule in the
defensive mechanisms of the nudibranch. Specific ecological
assays will be conducted in order to confirm this hypothesis.
Acknowledgment. This research work was financially
supported by the National S & T Major Project
(2009ZX09301-001), National Marine 863 Project
(2007AA09Z447 and 2007AA09Z402), NSFC grants
(Grant Nos. 30730108, 20721003, and 20772136), CAS key
project (Grant No. KSX2-YW-R-18), STCSM project
(No. 10540702900), and PRIN-MIUR 2007 Project “Anti-
tumor natural products and synthetic analogues”.
The dextrorotatory enantiomer (þ)-3-(hexadecyloxy)-
2-(S)-isonitrile-propyl acetate, (þ)-actisonitrile, was
prepared starting from (R)-(þ)-glycidyl trityl ether and
Supporting Information Available. Full experimental
procedures, 1D and 2D NMR spectra of compound 1,
and spectral data of all synthetic intermediates including
Mosher derivatives. This material is available free of
charge via the Internet at http://pubs.acs.org.
(13) Sullivan, G. R.; Dale, J. A.; Mosher, H. S. J. Org. Chem. 1973,
38, 2143–2147.
(14) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am.
Chem. Soc. 1991, 113, 4092–4096.
(15) Reddy, P. G.; Kumar, G. D. K.; Baskaran, S. Tetrahedron Lett.
(16) Synthetic (-)-(R)-actisonitrile: [R]D -7.9 (c = 0.20, CHCl3); CD
(n-hexane) [θ]204 748.
2000, 41, 9149–9151.
Org. Lett., Vol. 13, No. 8, 2011
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