2-Dehydrococcinelline, a new defensive alkaloid from the ladybird beetle Anatis ocellata (Coccinellidae)

Article abstract of DOI:10.1021/np9702695

2-Dehydrococcinelline (6), a novel coccinellid defensive alkaloid, has been isolated from the European ladybird Anatis ocellata. Its structure was established by spectroscopic methods and confirmed by chemical correlation with precoccinelline (1).

Full text of DOI:10.1021/np9702695

1148
J . Nat. Prod. 1997, 60, 1148-1149
Notes
2-Deh yd r ococcin ellin e, a New Defen sive Alk a loid fr om th e La d ybir d Beetle
An a tis ocella ta (Coccin ellid a e)
B. Lebrun,† J . C. Braekman,*,† D. Daloze,† and J . M. Pasteels‡
Laboratory of Bioorganic Chemistry and Laboratory of Animal and Cellular Biology, Faculty of Sciences, University of
Brussels, 50 Av. F. Roosevelt, 1050 Brussels, Belgium
Received J une 2, 1997X
2-Dehydrococcinelline (6), a novel coccinellid defensive alkaloid, has been isolated from the
European ladybird Anatis ocellata. Its structure was established by spectroscopic methods
and confirmed by chemical correlation with precoccinelline (1).
Ladybird beetles (Coleoptera; Coccinellidae) are well
protected against predation and a variety of alkaloids,
which contribute to this protection, have been charac-
terized from these beetles.1,2 Several of these alkaloids
belong structurally to the 2-methylperhydro-9b-azaphe-
nalene ring system [e.g. precoccinelline (1), coccinelline
(2), hippodamine (3), and convergine (4)] which appears
to be specific to these insects. In a preliminary chemical
survey of a variety of coccinellid beetles,3 we reported
the presence in the European species Anatis ocellata (L.)
of two alkaloids [AO1 (M+, 191) and AO2 (M+, 207)], the
mass spectra of which suggested that they are the
N-oxide/free base pair of an alkaloid belonging to the
2-methyldecahydro-9b-azaphenalene ring system. At
that time, because of lack of biological material, the
structure elucidation of AO1 and AO2 could not be
achieved. In the present study we report the isolation
and structure determination of the major alkaloid AO2
obtained by extraction of whole bodies of A. ocellata.
tions of the extract (352 mg) on alumina and silica gel
led to the isolation of the main Dragendorff positive
compound AO2, 1.4 mg. HREIMS measurements coupled
to the presence of 13 signals in the proton decoupled
13C NMR spectrum showed the molecular formula to be
C13H21NO. Characteristic fragment ions were observed
at m/ z 191.1667 (46; C13H21N), 190.1599 (93; C13H20N),
188.1448 (27; C13H18N), 176.1438 (100; C12H18N), 162
(20) and 148 (16) reminiscent of the mass spectrum of
hippocasine N-oxide (5),4 a defensive alkaloid isolated
20 years ago by Ayer et al.4 from Hippodamia caseyi, a
ladybird indigenous to Western Canada. Loss of O and
OH from the molecular ion is distinctive of a N-oxide
1
group. The H and 13C NMR spectra of AO2 contained
signals for one trisubstituted double bond, one vinylic
methyl group, three methines R to the nitrogen atom,
and seven methylenes. All of these data were sugges-
tive of formula 6, the connectivities of which were
further corroborated by a 2D NMR study (1H-1H COSY,
1
HMQC, HMBC). The complete assignments of the H
and 13C NMR signals are reported in the Experimental
Section. Most noteworthy in the 1H-1H COSY spectrum
were the correlations between H-3 (δ 5.19) and H-3a (δ
3.97) and H2-1 (δ 2.17 and 2.56), and between the latter
and H-9a (δ 3.51).
AO2 thus has the same connectivity as hippocasine
N-oxide (5), the structure of which has been determined
by X-ray diffraction analysis.4 At the time, neither 13C
1
nor high-field H NMR spectra of 5 had been reported
so that we could not make an accurate comparison
between 5 and AO2, but the reported chemical shift for
the vinylic proton of 5 (δ 5.42, instead of 5.19 for AO2)
suggested that the two compounds were not identical
and could thus be stereoisomeric. The establishment
of the relative configuration of AO2 was based on the
following NMR arguments. The 13C chemical shifts of
the carbon atoms adjacent to the nitrogen atom for
coccinelline (2), convergine (4), and AO2 are reported
in Table 1. It appears from the examination of this table
that C-3a is shielded in convergine while C-6a is
shielded in coccinelline. These shieldings can be at-
tributed to gauche interactions between H-3a/H7ax and
H-3a/H-9ax in convergine and between H-6a/H-3ax and
H-6a/H-1ax in coccinelline. In this respect, AO2 behaves
as coccinelline, suggesting that they have the same
The insects (35 adults) were exhaustively extracted
with CH3OH, and successive chromatographic separa-
† Laboratory of Bioorganic Chemistry.
‡ Laboratory of Animal and Cellular Biology.
X Abstract published in Advance ACS Abstracts, September 15, 1997.
S0163-3864(97)00269-3 CCC: $14.00
© 1997 American Chemical Society and American Society of Pharmacognosy

Notes
J ournal of Natural Products, 1997, Vol. 60, No. 11 1149
Ta ble 1. Comparison of the 13C NMR Chemical Shifts (δ) of
the Carbon Atoms Adjacent to the Nitrogen Atom in 2, 4, and 6
affording 352 mg of an orange oil which was chromato-
graphed over alumina (eluent: CH2Cl2/MeOH, gradient
from 100:0 to 80:20 and then MeOH 1% NH4OH). The
fractions showing Dragendorff positive spots by TLC
were combined and flash chromatographed on silica gel
(eluent: CH2Cl2/MeOH 0.1% NH4OH, gradient from
98:2 to 90:10). This afforded 1.4 mg of compound 6,
homogeneous by TLC, exhibiting the following proper-
ties: oil; [R] + 8 at 579 nm and + 19 at 407 nm (CH2-
Cl2, c ) 0.16); IR (film) νmax 2926, 1660, 1442, 1382, 956
cm-1; 1H NMR (CDCl3, 600 MHz) δ 5.19 (1H, br s, H-3),
3.97 (1H, br s, H-3a), 3.51 (1H, br s, H-9a), 3.14 (1H, t,
J ) 12 Hz, H-6a), 2.90 (1H, tt, J ) 13.5 and 5 Hz,
H-9ax), 2.70 (1H, tt, J ) 13.5 and 4.5 Hz, H-4ax), 2.56
(1H, br t, J ) 14 Hz, H-1ax), 2.17 and 2.20 (2H, m,
H-1eq and H-7ax), 1.98 (1H, dq, J ) 12 and 4 Hz,
H-6ax), 1.74 (3H, s, H3-10), 1.64 (1H, dt, J ) 13 and 5
Hz, H-8ax), 1.58 (1H, m, H-8eq), 1.52 and 1.43 (3H, m,
H2-5 and H-4eq), 1.26 (2H, m, H-6eq and H-7eq); 13C
NMR (CDCl3, 150.87 MHz) δ 133.4 (C-2), 121.7 (C-3),
71.6 (C-3a), 69.2 (C-9a), 59.4 (C-6a), 33.2 (C-1), 27.2 (C-
6), 26.6 (C-7), 26.3 (C-4), 23.6 (C-9), 22.2 (C-10), 18.7
(C-5), 17.2 (C-8); HREIMS M+ at m/ z 207.1615 (calcd
for C13H21NO, 207.1623; 8). Characteristic fragment
ions were observed at m/ z 191.1667 (46; C13H21N),
190.1599 (93; C13H20N), 188.1448 (27; C13H18N), 176.1438
(100; C12H18N), 162 (20) and 148 (16).
carbon
atom
coccinelline
2-dehydrococcinelline
convergine
(2)
(6)
(4)
C-9a
C-6a
C-3a
72.7
59.0
72.7
69.2
59.4
71.6
74.2
73.5
58.0
relative configuration. Catalytic hydrogenation of AO2
led to precoccinelline (1), thus confirming the configu-
ration attributions. The hydrogenation proceeds selec-
tively from the less hindered face (si-si face) of the
double bond to give the compound with an equatorial
methyl group. From all these results it can be deduced
that AO2 is 2-dehydrococcinelline (6).
A minor Dragendorff positive derivative was also
isolated from the CH3OH extract during the separation
procedures. Its mass spectrum (EIMS) presented a
molecular ion at m/ z 191, indicating that traces of the
corresponding free base (AO1) may be present in the
beetle.
A. ocellata belongs to the tribe Coccinellini. Until now
members of this tribe were found to produce alkaloids
with either 2-methylperhydro-9b-azaphenalene, ho-
motropane or long-chain skeletons, and these alkaloids
were never found in members of other subtribes.1 The
identification of 2-dehydrococcinelline in A. ocellata thus
confirms this pattern.
Ca ta lytic Hyd r ogen a tion of 2-Deh yd r ococcin el-
lin e. A solution of 2-dehydrococcinelline (1.4 mg) in
CH3OH containing Pd/C was hydrogenated under 3 atm
of hydrogen overnight. The solution was filtered over
silica gel and flash chromatographed (silica gel; CH2-
Cl2/MeOH 0.1% NH4OH, 98:2). This afforded 0.5 mg
of a compound showing chromatographic and spectro-
Exp er im en ta l Section
Gen er al Exper im en tal P r ocedu r es. HREIMS were
performed on a Fisons Autospec instrument. The 1H
and 13C NMR spectra were recorded in CDCl3 at 600
and 150.87 MHz, respectively, using a Varian Unity 600
instrument. The IR spectrum was obtained on a Bruker
IFS 48 FT instrument as a film on a NaCl disk. The
optical rotations were measured on a Perkin-Elmer 141
polarimeter (Hg vapor lamp) in a 10 cm cell at 20 °C.
Thin layer chromatography analyses (TLC) were per-
formed on 0.25 mm Polygram silica gel SILG/UV254
precoated plates (Macherey Nagel) or on 0.2 mm neutral
alumina 60 F254 precoated plates (Merck, type E).
Column chromatographies were performed over silica
gel (MN Kieselgel 0.04-0.063 mm), using the flash
technique or over MN neutral alumina. GC analyses
were performed on a Varian 3700 apparatus equipped
with an OV-1701 capillary column (Rescom, 25 m, 0.32
mm i.d.).
1
scopic properties (GC, TLC, MS and H NMR) identical
to those of precoccinelline (1).
Ack n ow led gm en t. This work was supported by
grants from the Belgian Fund for Basic Research (Grant
2.4513.90-96) and the French Community of Belgium
(ARC 93/98-137). We thank Mr. C. Maerschalk for the
NMR spectra and Mr. C. Moulard for the mass spectra.
Refer en ces a n d Notes
(1) Daloze, D.; Braekman, J . C.; Pasteels, J . M. Chemoecology 1995,
5/ 6, 173-183.
(2) King, A. G.; Meinwald, J . Chem. Rev. 1996, 96, 1105-1122.
(3) Pasteels, J . M.; Deroe, C.; Tursch, B.; Braekman, J . C.; Daloze,
D.; Hootele, C. J . Insect Physiol. 1973, 19, 1771-1784.
(4) Ayer, W. A.; Bennett, M. J .; Browne, L. M.; Purdham, J . T. Can.
J . Chem. 1976, 54, 1807-1813.
Extr a ction a n d Isola tion . A total of 35 adult
specimens of Anatis ocellata collected near Brussels
were ground and exhaustively extracted with MeOH
NP9702695