Further Alkaloids Common to Ants and Frogs: Decahydroquinolines and a Quinolizidine

15 Pages·2002·0.68 MB·English

Journal of Chemical Ecology, Vol. 25, No. 5, 1999
FURTHER ALKALOIDS COMMON TO ANTS AND
FROGS: DECAHYDROQUINOLINES AND A
QUINOLIZIDINE
TAPPEY H. JONES,*,1 JEFFREY S. T. GORMAN,1 ROY R. SNELLING,2
JACQUES H. C. DELABIE,3 MURRAY S. BLUM,4
H. MARTIN GARRAFFO,5 POONAM JAIN,5 JOHN W. DALY,5 and
THOMAS F. SPANDE5
lDepartment of Chemistry, Virginia Military Institute
Lexington, Virginia 24450-0304
2Los Angeles County Museum of Natural History
900 Exposition Blvd., Los Angeles, California 90007
3Divisao de Zoologia, Centro de Pesquesa do Cacau, CEPLAC
Caixa Postal 7, 15600 Itabuna, Bahia, Brazil
4Department of Entomology, University of Georgia
Athens, Georgia 30602
5Laboratory of Bioorganic Chemistry
National Institute of Diabetes and Digestive and Kidney Diseases
National Institutes of Health
Bethesda, Maryland 20892
(Received May 11, 1998; accepted January 15, 1999)
Abstract—Three alkaloids—two minor decahydroquinolines (DHQs) and a
major quinolizidine—were detected in an extract of a Brazilian myrmicine ant
(Solenopsis (Diplorhoptrum) sp. picea group). One DHQ (3) was identical to
a known frog-skin alkaloid, cis-195A (cis-5-methyl-2-propyldecahydroquino-
line), while the second DHQ, an isomer of 3, designated 195J, was assigned
a tentative cis-2-methyl-5-propyldecahydroquinoline structure (2) based on
mass and infrared spectra. The third alkaloid proved identical to the frog-skin
alkaloid 195C, for which a structure had not been previously proposed.
Mass and infrared spectral analysis, including chemical ionization tandem
mass spectrometry, indicated a 4-methyl-6-propylquinolizidine structure (1)
for 195C. The four possible diastereomers were synthesized and the
(6Z,10E)-4-methyl-6-propylquinolizidine diastereomer (1b) was identical to
the natural alkaloid. Skin extracts of a population of a Madagascan mantelline
frog contained, among other alkaloids, minor amounts of the same alkaloid
*To whom correspondence should be addressed.
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0098-0331/99/0500-1179$16.00/0 © 1999 Plenum Publishing Corporation
1180 JONES ET AL.
triad 1-3 with 1 again predominating. The common occurrence of alkaloids
1-3 in both ant and frog supports the hypothesis that ants are a likely dietary
source for sequestered frog-skin alkaloids and brings to six, the alkaloid
classes common to ant and frog.
Key Words—Alkaloids, mass spectrometry, infrared spectroscopy, amphibians,
ants, decahydroquinolines, quinolizidines.
INTRODUCTION
A variety of saturated nitrogen heterocycles have been found as venom com-
ponents in species of the myrmicine genus Solenopsis (Diplorhoptrum), com-
monly known as thief ants. These include 2,5-dialkylpyrrolidines, 2,6-di-
alkylpiperidines, 3,5-dialkylpyrrolizidines, and 3,5-dialkylindolizidines (Jones
et al., 1996 and references therein). A pair of 2,5-disubstituted decahydro-
quinolines were recently reported in extracts of virgin queens of a Solenop-
sis (Diplorhoptrum) ant from Puerto Rico (Spande et al., 1998). In this
report, we describe the first isolation from an animal source of a 4,6-dia-
lkylquinolizidine, namely (6Z,10E)-4-methyl-6-propylquinolizidine (Figure 1,
1b), as a major alkaloid in workers of a Solenopsis (Diplorhoptrum) species
obtained near Itabuna, Brazil. In addition, smaller amounts of a cis-fused
2-methyl-5-propyldecahydroquinoline (2) and the isomeric 5-methyl-2-propyl-
decahydroquinoline (3) were detected.
The rich array of alkaloids, detected over the past three decades in skin
extracts from certain dendrobatid, mantelline, myobatrachid, and bufonid anu-
rans (Daly et al., 1993), is now thought to originate from dietary sources (Daly
et al., 1992, 1994a,b, 1997), which for such frogs/toads consist of small, often
tiny, arthropods. Some of the alkaloid-containing dendrobatid frogs have been
proposed to be "ant specialists" (Toft, 1980, 1995; Donnelly, 1991; Caldwell,
1996), and, including the present work, representatives of six of the approxi-
mately 20 major structural classes of alkaloids detected in extracts of frog skins
have now been identified in myrmicine ants, especially Solenopsis (Diplorhop-
trum) species (Jones and Blum, 1983; Daly, 1995; Spande et al., 1998).
The identification of quinolizidine 1b has made it possible to assign this
structure to alkaloid 195C, previously detected in many frog skin extracts but
whose structure had not been established. In addition, the decahydroquinolines 2
and 3 were shown to correspond to alkaloids present in frog skin extracts. These
results reinforce the dietary hypothesis for amphibian alkaloids since the same
mixture of 1b, 2, and 3 has been found in the Brazilian Solenopsis (Diplorhop-
trum) species and in frogs of the species Mantella betsileo.
ANT AND FROG SKIN ALKALOIDS 1181
FIG. 1. Structures of 1a-1d, 2, and 3.
METHODS AND MATERIALS
Chemical Analyses. Gas chromatographic-mass spectral analyses were per-
formed with Hewlett-Packard model 5890 gas chromatograph equipped with a 30-
m x 0.32-mm Rtx-5 column. Vapor-phase FTIR spectra were obtained from a
Hewlett-Packard model 5965B detector interfaced with a Hewlett-Packard 5890
gas chromatograph fitted with a 30-m x 0.25-mm Rtx-5 Amine column. IR spectra
of liquids were obtained with a Perkin-Elmer 1600 series FTIR instrument. Mass
spectra were obtained in the EI mode from a Shimadzu QP-5000 GC-MS equipped
with a 30-m x 0.32-mm Rtx-5 column. High-resolution mass spectrometry (HR-
MS) was performed with a JEOL SX102 instrument equipped with a 15-m x 0.20-
mm HP-5 column. EI-MS, EI-MS/MS and CI-MS/MS (NH3) data were obtained
with a Finnigan GCQ instrument equipped with a 25-m x 0.25-mm Rtx-5 column.
Ants. Two collections of Solenopsis (Diplorhoptrum) sp. picea group were
made near Itabuna, Bahia, Brazil, and immediately placed in small vials contain-
ing methylene chloride. Voucher specimens were deposited in the collection of
the Los Angeles County Museum of Natural History, Los Angeles, California.
Frogs. Collections of mantelline frogs, including three populations of Man-
tella betsileo Grandidier 1872, were made in Madagascar (Garruffo et al., 1993a;
Daly et al., 1996). The Mantella betsileo populations were from near Antanam-
baobe (11 skins, December 1990), Ambavala (8 skins, January 1994), and
Farakaraina (12 skins, December 1993). Skins were extracted with methanol,
1182 JONES ET AL.
and alkaloid fractions were obtained as described (Garraffo et al., 1993a; Daly
et al., 1994a). Quinolizidine 195C, a major alkaloid in skin extracts of Mantella
betsileo from Antanambaobe and Ambavala, was isolated from the combined
alkaloid fractions from these two populations on a reverse-phase column with
a linear gradient of CH CN-H O (10:90 to 90:10; cf. Garraffo et al., 1997).
3 2
Voucher specimens are in the collection of the American Museum of Natural
History (New York City).
Synthesis (see Scheme 1)
2-[3-(6-Propyl-2-pyridyl)-propyl]-1,3-dioxolane (5). A solution of the
anion derived from 1.0 g (5 mmol) of 2-[3-(6-methyl-2-pyridyl)-propyl]l,3-diox-
olane (4) (Jones et al., 1996) in 25 ml of diethyl ether and 4.0 ml of 1.3 M
2-butyllithium in hexanes, was added to 0.7 ml (7 mmol) of iodoethane in 15
ml of diethyl ether. The mixture was stirred 1 hr, then worked up in the usual
manner to provide 1.1 g of 5 that was >90% pure by gas chromatographic
analysis. GC-FTIR: 3066, 2959, 2882, 1580, 1454, 1401, 1249, 1140, 1052,
940 cm-1. MS, m/z (rel %): 235(M+, 2), 234(2), 207(10), 190(13), 164(10),
162(28), 136(10), 135(100), 107(10), 73(29), HR-MS m/z: calcd for C H NO ,
14 21 2
235.1572; observed, 235.1589.
cis-2-[3-(6-Propyl-2-piperidyl)-propyl]-1,3-dioxolane (8). A solution of
1.018 g of dioxolane 5 and 0.5 g of Rh-on-alumina catalyst (5%) in 50 ml of
ethanol was hydrogenated at 3 atm for 4 hr. After filtration, the solvent was
removed in vacuo. GC-MS analysis of the residue showed it to be homogeneous.
GC-FTIR: 2934, 2881, 2800, 1453, 1400, 1331, 1137, 1055, 941 cm-1. MS,
m/z (rel %): 240(M+-1, 2), 198(18), 138(15), 136(14), 127(10), 126(100), 96(9),
84(10), 73(24). HR-MS m/z: calcd for M+, C H NO , 241.2042; observed,
14 27 2
241.2036.
cis- and trans 2-[3-(6-Methyl-2-piperidyl)-propyl]-1,3-dioxolane (6 and 7).
For the sodium-in-ethanol reduction, a solution containing 2.0 g (10 mmol) of
pyridine acetal 4 in 80 ml of absolute ethanol was stirred and heated to reflux
under a drying tube and 16 g of sodium metal were added in small pieces over 45
min. The mixture was stirred at reflux for 3 hr, then hydrolyzed by slowly adding
several volumes of water. The solvent was removed in vacuo, and the organic
residue was extracted with 3 x 25 ml of diethyl ether. The total ether extract was
dried over anhydrous K CO , filtered, and the solvent removed in vacuo. The
2 3
resulting crude liquid was Kugelrohr-distilled at reduced pressure to give 1.5 g
of a colorless oil. GC-MS analysis showed the presence of two components (6
and 7) with identical mass spectra in a 1:4 ratio. The isomers were separated
by column chromatography by using 200 g of alumina and a solvent gradient
as previously described (MacConnell et al., 1971) to give 0.10 g of the first
eluting isomer (6) and 0.34 g of the second eluting isomer (7). GC-FTIR of
ANT AND FROG SKIN ALKALOIDS 1183
cis-piperidine 6: 2934, 2881, 2802, 2718, 2605, 1438, 1400, 1325, 1137, 1056,
940, 819 cm-1. GC-FTIR of frans-piperidine 7: 2935, 2882, 2654, 1456, 1400,
1378, 1263, 1138, 1057, 940, 813 cm-1. Typical MS, m/z (rel %): 212(M+-1, 1),
99(8), 98(100), 82(4), 81(4), 73(19). HR-MS m/z: calcd for MM, C H NO ,
12 22 2
212.1651; observed, 212.1663; calcd for C H N, 98.0970; observed, 98.0973.
6 12
For catalytic hydrogenation, a solution of 1.90 g (9 mmol) of dioxolane
4, 15 drops of triethylamine, and 0.7 g of Rh-on-alumina catalyst in 50 ml of
ethanol was hydrogenated at 3 atm for 3 hr. After filtration, the solvent was
removed in vacuo. Diethyl ether was added to the residue, and the solution was
washed with water and dilute NaOH. The ether layer was dried over anhydrous
K CO , filtered, and the solvent was removed in vacuo to give 1.85 g of a color-
2 3
less oil. GC-MS analysis showed that this oil was homogeneous and had a mass
spectrum and retention time identical to that of cis-piperidine 6.
4-Methyl-6-propyl-quinolizidines (1a-1d). For derivation from a mixture of
piperidines 6 and 7, a solution containing the mixture of piperidines 6 and 7 from
a sodium-in-ethanol reduction of 5 g (25 mmol) of 4 in 70 ml of freshly distilled
CH C1 was treated with an aqueous solution of KCN. The pH was adjusted to
2 2
6 with dilute HC1, and the mixture stirred overnight. The organic layer was sepa-
rated, dried over anhydrous K CO , filtered, and the solvent was removed in vacuo
2 3
to give 3.6 g of a liquid. A solution of 0.19 g (1 mmol) of this liquid was taken
up in 5 ml of diethyl ether under argon, cooled to 0°C with an ice bath, and 2.2
ml of 2 M n-propylmagnesium chloride were added. The mixture was stirred at
room temperature for 2 hr then quenched with water and NH Cl. The aqueous
4
layer was extracted with 20 ml of diethyl ether. The ether extract was dried over
anhydrous K CO , filtered, and the solvent was removed in vacuo. GC-MS anal-
2 3
ysis of the residue showed the presence of four components (la-Id) with virtu-
ally identical mass spectra in a 1:1:6:2 ratio. The GC-FTIR spectra of these
compounds are shown in Figures 2 and 3. Typical MS, m/z (rel %): 195(M+, 1),
194(1), 180(5), 153(11), 152(100), 124(5), 82(5), 69(8), 67(8), 55(15). HR-MS,
calcd for C H N, 195.1987; observed for the four GC peaks in order of elution:
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la, 195.1972; 1b, 195.1987; 1c, 195.1973; 1d, 195.1982.
For derivation from cis-piperidine 6, a sample of cis-piperidine 6 purified
from the sodium-in-ethanol reduction of 4 was treated in a similar fashion except
THF was used in place of diethyl ether as the solvent in the Grignard alkylation.
GC-MS analysis of the residue showed the presence of la and 1c in a 1:5 ratio.
For derivation from trans-piperidine 7, a similar treatment of an isolated
sample of trans-piperidine 7 from the sodium-in-ethanol reduction of 4 provided
1b and 1d in a 1:2 ratio.
For derivation from cis-piperidine 8, a sample of cis-piperidine 8 from the
hydrogenation of 5 was treated in a similar fashion except that an excess of 2.0
M methylmagnesium bromide was used in place of n-propylmagnesium bromide
to give la and 1d in a 1:2 ratio.
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FIG. 2. Vapor-phase FTIR spectra of two synthetic diastereomers of quinolizidine
195C.
RESULTS AND DISCUSSION
A broad survey of ant extracts is in progress in an effort to identify ant alka-
loids that may be dietary sources for some of the over 400 alkaloids that have
been characterized in skin extracts from four genera of diurnal, mainly terrestrial
dendrobatid frogs of tropical Central and South America; one genus of diurnal,
ANT AND FROG SKIN ALKALOIDS 1185
FIG. 3. Vapor phase FTIR spectra of two synthetic diastereomors of quinolizidine
195C. (see Fig. 2.)
mainly terrestrial mantelline frogs of Madagascar; one genus of nocturnal, ter-
restrial myobatrachid frogs of Australia; and one genus of diurnal, terrestrial
bufonid toads of southeastern South America (Daly et al., 1993; Daly, 1995).
Previously, several ant alkaloids of four structural classes had been detected in
skin extracts of frogs/toads (Table 1). Representatives of two further structural
1186 JONES ET AL.
ANT AND FROG SKIN ALKALOIDS 1187
1188 JONES ET AL.
classes of alkaloids, now discovered in ants and shared with frogs, are also pre-
sented in Table 1. These alkaloids are relatively simple and all can potentially
originate from a linear carbon-chain precursor.
Three isomeric alkaloids with molecular weight 195 (C H N) were
13 25
detected by GC-MS in an extract of a Brazilian myrmicine ant, Solenopsis
(Diplorhoptrum) sp. picea group. They occurred in a ratio of about 3:1:1. The
last-eluting alkaloid on GC was identified by its mass spectral fragmentation pat-
tern, FTIR spectrum, and cochromatography with an authentic sample as a cis-
fused 5-methyl-2-propyldecahydroquinoline (3), designated cis-195A. This was
previously detected in skin extracts from dendrobatid frogs (Daly et al., 1993)
and skin extracts from four species of mantelline frogs (Daly et al., 1996). The
second-eluting alkaloid was also a decahydroquinoline, based on the mass spec-
tral fragmentation, FTIR spectrum, and the presence of an exchangeable hydro-
gen. This decahydroquinoline had the same Bohlmann band pattern in its FTIR
spectrum as cis-195A, indicating that the H(2) and H(8a) hydrogens were cis
(Garraffo et al., 1994) and had absorption peaks in the fingerprint region that
are diagnostic for cis-fused decahydroquinolines, i.e., H(4a) and H(8a) were cis
(Tokuyama et al., 1991; Garraffo et al., 1994). Thus, the relative stereochemistry
at C(2), C(4a), and C(8a) are considered to be the same as in cis-195A. The rel-
ative stereochemistry at C(5) is undetermined. [A 5-epi-cis-19SA structure was
excluded based on direct comparison with synthetic material provided by Grieco
and Parker (1988).] The mass spectral fragmentation shows a substantially larger
loss of methyl (32 vs. 5%) in this decahydroquinoline that in cis-195A, and, thus,
it appears to have the methyl and propyl side-chains interchanged so that the
favored A-cleavage results in loss of methyl. Cleavage of C F from such deca-
3 7
hydroquinolines occurs to a significant extent due to loss of carbons 6-8 and
can be major, depending on the nature of the C(2) substituent (Yu et al., 1970;
Spande et al., 1998). The proposed structure of this decahydroquinoline is cis-2-
methyl-5-propyldecahydroquinoline (2). It has not been previously reported from
frog skin and is given the code designation cis-195J. The predominant alkaloid
of the Brazilian ant and the first to elute on GC had no exchangeable hydro-
gen and showed, on mass spectral fragmentation, a major loss of propyl and a
minor loss of methyl, consonant with A-cleavage of such substituents. One weak
fragment ion, m/z 124, was perplexing and misleading as it often indicates 3,5-
disubstituted indolizidines, which produce this ion from A-cleavage followed
by a McLafferty rearrangement (Garraffo et al., 1993b). The EI-MS/MS data
clarified the origin of this fragment. The FTIR spectrum had Bohlmann bands
weaker than those of disubstituted indolizidines and hence a 4-methyl-6-pro-
pylquinolizidine seemed the only reasonable structure. Alkylation of the inter-
mediate cyanopiperidines obtained from 6 and 7 (Grierson et al., 1986) afforded
all four possible quinolizidine diastereomers for comparison (Scheme 1). The
second of the four eluting diastereomers on GC was identical, with respect to GC