Alkaloids in bufonid toads (melanophryniscus): temporal and geographic determinants for two argentinian species.

Bufonid toads of the genus Melanophryniscus represent one of several lineages of anurans with the ability to sequester alkaloids from dietary arthropods for chemical defense. The alkaloid profile for Melanophryniscus stelzneri from a location in the province of Córdoba, Argentina, changed significantly over a 10-year period, probably indicating changes in availability of alkaloid-containing arthropods. A total of 29 alkaloids were identified in two collections of this population. Eight alkaloids were identified in M. stelzneri from another location in the province of Córdoba. The alkaloid profiles of Melanophryniscus rubriventris collected from four locations in the provinces of Salta and Jujuy, Argentina, contained 44 compounds and differed considerably between locations. Furthermore, alkaloid profiles of M. stelzneri and M. rubriventris strongly differed, probably reflecting differences in the ecosystem and hence in availability of alkaloid-containing arthropods.

A siphonotid millipede (Rhinotus) as the source of spiropyrrolizidine oximes of dendrobatid frogs.

Poison frogs of the neotropical family Dendrobatidae contain a wide variety of lipophilic alkaloids, which are accumulated from alkaloid-containing arthropods. A small millipede, Rhinotus purpureus (Siphonotidae), occurs microsympatrically with the dendrobatid frog Dendrobates pumilio on Isla Bastimentos, Bocas del Toro Province, Panamá. Methanol extracts of this millipede contain the spiropyrrolizidine O-methyloxime 236, an alkaloid previously known only from skin extracts of poison frogs, including populations of D. pumilio. Thus, R. purpureus represents a likely dietary source of such alkaloids in dendrobatid frogs.

Evidence for biosynthesis of pseudophrynamine alkaloids by an Australian myobatrachid frog (pseudophryne) and for sequestration of dietary pumiliotoxins.

Australian myobatrachid frogs of the genus Pseudophryne have only two classes of alkaloids in skin extracts, pseudophrynamines (PSs) and pumiliotoxins (PTXs). The former are unique to such Australian frogs, while the PTXs occur worldwide in all other genera of frogs/toads that contain lipophilic alkaloids. The major alkaloid of wild-caught frogs from one population of Pseudophryne semimarmorata was PTX 267C, while PSs were only minor or trace alkaloids. Captive-raised frogs from the same parental stock had no PTXs, but had larger amounts of PSs. A PTX fed to captive-raised frogs accumulated into skin along with dihydro and hydroxy metabolites. Thus, Pseudophryne frogs appear to biosynthesize PSs, but to sequester into skin dietary PTXs. In addition, biosynthesis of PSs appears reduced when high levels of dietary PTXs have accumulated into skin. This is the first evidence indicating that certain frogs are capable of synthesizing rather than merely sequestering alkaloids. A wide range of PSs, including many with molecular weights >500, were detected using both GC-mass spectral and LC-mass spectral analysis.

Ammonia chemical ionization tandem mass spectrometry in structural determination of alkaloids. II. Tetraponerines from pseudomyrmecine ants.

Chemical ionization tandem mass spectrometry (CI-MS/MS) of alkaloids with ammonia reagent gas and collision-activated dissociation as well as EI-MS/MS were applied to the tetraponerine alkaloids in extracts from six pseudomyrmecine ants of the genus Tetraponera. The MS/MS techniques along with gas chromatography Fourier transform infrared (GC/FTIR) spectra allowed identification in two extracts of seven of the eight known tetraponerines. The EI-MS/MS fragmentations proved diagnostic for the ring system and the CI-MS/MS patterns for the C-8 or C-9 substitution, while the Bohlmann bands in FTIR spectra were diagnostic for the C-8 or C-9 configurations. An Indian ant (T. allaborans) had T-2, T-4 and T-8, while a Chinese ant (T. binghami) had T-5, T-6, T-7 and T-8. Four other ants, T. rufonigra (India), T. penzigi (Africa), T. clypeata (Africa) and T. sp. cf. emeryi (Africa), had no tetraponerines.

Structure of alkaloid 275A, a novel 1-azabicyclo[5.3.0]decane from a dendrobatid frog, Dendrobates lehmanni: synthesis of the tetrahydrodiastereomers.

The principal alkaloid 275A in skins of the Colombian poison frog Dendrobates lehmanni has been identified as the pyrrolo[1,2-a]azepane (1), the first occurrence in nature of this "izidine" system. Tetrahydro-1 proved identical to one of the four synthetic diastereomers, 2a--2d, thereby establishing that 1 has the 5Z,10E relative stereochemistry. Alkaloid 1 is often accompanied by other congeners, in particular a 5Z,10Z diastereomer 15, a dihydro analogue 16, and a ketone 17. Such izidines in frogs may arise from dietary ants, as do other classes of izidines.

3-methyl-4-phenylpyrrole from the ants Anochetus kempfi and Anochetus mayri.

The cephalic extracts of the ant Anochetus kempfi were found to contain 2,5-dimethyl-3-isoamylpyrazine (1) and 3-methyl-4-phenylpyrrole (2). The structures of these compounds were established from their spectral data and by comparison with synthetic samples. This is the first report of a phenylpyrrole found in an insect and only the third report of a pyrrole from ants.

Specificity of ascorbate analogs for ascorbate transport. Synthesis and detection of [(125)I]6-deoxy-6-iodo-L-ascorbic acid and characterization of its ascorbate-specific transport properties.

Cellular ascorbic acid accumulation occurs in vitro by two distinct mechanisms: transport of ascorbate itself or transport and subsequent intracellular reduction of its oxidized product, dehydroascorbic acid. It is unclear which mechanism predominates in vivo. An easily detectable compound resembling ascorbate but not dehydroascorbic acid could be a powerful tool to distinguish the two transport activities. To identify compounds, 21 ascorbate analogs were tested for inhibition of ascorbate or dehydroascorbic acid transport in human fibroblasts. The most effective analogs, competitive inhibitors of ascorbate transport with K(i) values of 3 microM, were 6-deoxy-6-bromo-, 6-deoxy-6-chloro-, and 6-deoxy-6-iodo-L-ascorbate. No analog inhibited dehydroascorbic acid transport. Using substitution chemistry, [(125)I]6-deoxy-6-iodo-L-ascorbate (1.4 x 10(4) mCi/mmol) was synthesized. HPLC detection methods were developed for radiolabeled and nonradiolabeled compounds, and transport kinetics of both compounds were characterized. Transport was sodium-dependent, inhibited by excess ascorbate, and similar to that of ascorbate. Transport of oxidized ascorbate and oxidized 6-deoxy-6-iodo-L-ascorbate was investigated using Xenopus laevis oocytes expressing glucose transporter isoform GLUT1 or GLUT3. Oxidation of ascorbate or its analog in media increased uptake of ascorbate in oocytes by 6-13-fold compared with control but not that of 6-deoxy-6-iodo-L-ascorbate. Therefore, 6-deoxy-6-iodo-L-ascorbate, although an effective inhibitor of ascorbate transport, either in its reduced or oxidized form was not a substrate for dehydroascorbic acid transport. Thus, radiolabeled and nonradiolabeled 6-deoxy-6-iodo-L-ascorbate provide a new means for discriminating dehydroascorbic acid and ascorbate transport in ascorbate recycling.

Ammonia chemical ionization tandem mass spectrometry in structure determination of alkaloids. I. Pyrrolidines, piperidines, decahydroquinolines, pyrrolizidines, indolizidines, quinolizidines and an azabicyclo[5.3.0]decane.

CI-MS/MS of alkaloids with ammonia reagent gas and collision-induced dissociation provides unique structural information for certain mono- and bi-cyclic alkaloids. This technique was applied to solve the structures of 195C, a 4,6-disubstituted quinolizidine alkaloid found in frog skin and an ant, and 275A, a novel 3,5-disubstituted azabicyclo[5.3.0]decane found in frog skin.

Absence of skin alkaloids in captive-raised Madagascan mantelline frogs (Mantella) and sequestration of dietary alkaloids.

Mantelline frogs of the genus Mantella contain a variety of pumiliotoxin, allopumiliotoxin and homopumiliotoxin alkaloids in their skin. Pyrrolizidines, indolizidines and quinolizidines are also present. In contrast, captive-raised frogs (Mantella aurantiaca) have no alkaloids detectable in skin extracts. Frogs fed alkaloid-dusted fruit flies accumulate alkaloids into their skin. Thus, these mantelline frogs, like the neotropical dendrobatid frogs, appear dependent on dietary sources for their skin alkaloids and have the requisite alkaloid-sequestering system(s).

Pseudophrynaminol: a potent noncompetitive blocker of nicotinic receptor-channels.

(+/-)-Pseudophrynaminol inhibited carbamylcholine-elicited sodium-22 influx with an IC50 value of about 0.3 microM in both rat pheochromocytoma PC12 cells (ganglionic-type nicotinic receptor) and human medulloblastoma TE671 cells (neuromuscular-type nicotinic receptor). The inhibition in both cell lines appeared to be noncompetitive in nature. In rat cerebral cortical membranes, pseudophrynaminol had only low affinity (Ki 35 microM) for the agonist site on central nicotinic receptors at which [3H]nicotine binds. Pseudophrynaminol, at 10 microM, had marginal effects on a variety of other central receptors, and even at 100 microM inhibited batrachotoxin-elicited sodium-22 influx in a synaptoneurosomal preparation by only 40%. It had no effect at 30 microM on acetylcholinesterase and was a weak inhibitor of butyrylcholinesterase. Thus, pseudophrynaminol appears to be a potent, rather specific, noncompetitive inhibitor of ganglionic and neuromuscular nicotinic receptor-channels.

Synthesis and nicotinic activity of epiboxidine: an isoxazole analogue of epibatidine.

Synthetic (+/-)-epiboxidine (exo-2-(3-methyl-5-isoxazolyl)-7-azabicyclo[2.2.1]heptane) is a methylisoxazole analog of the alkaloid epibatidine, itself a potent nicotinic receptor agonist with antinociceptive activity. Epiboxidine contains a methylisoxazolyl ring replacing the chloropyridinyl ring of epibatidine. Thus, it is also an analog of another nicotinic receptor agonist, ABT 418 ((S)-3-methyl-5-(1-methyl-2-pyrrolidinyl)isoxazole), in which the pyridinyl ring of nicotine has been replaced by the methylisoxazolyl ring. Epiboxidine was about 10-fold less potent than epibatidine and about 17-fold more potent than ABT 418 in inhibiting [3H]nicotine binding to alpha 4 beta 2 nicotinic receptors in rat cerebral cortical membranes. In cultured cells with functional ion flux assays, epiboxidine was nearly equipotent to epibatidine and 200-fold more potent than ABT 418 at alpha 3 beta 4(5) nicotinic receptors in PC12 cells. Epiboxidine was about 5-fold less potent than epibatidine and about 30-fold more potent than ABT 418 in TE671 cells with alpha 1 beta 1 gamma delta nicotinic receptors. In a hot-plate antinociceptive assay with mice, epiboxidine was about 10-fold less potent than epibatidine. However, epiboxidine was also much less toxic than epibatidine in mice.

Alkaloid 223A: the first trisubstituted indolizidine from dendrobatid frogs.

The structure of alkaloid 223A (1), the first member of a new class of amphibian alkaloids, purified by HPLC from a skin extract of a Panamanian population of the frog Dendrobates pumilio Schmidt (Dendrobatidae) has been established as (5R,6S,8R,9S)- or (5S,6R,8S,9R)-6,8-diethyl-5-propylindolizidine, based on GC-MS, GC-FTIR, and 1H-NMR spectral studies. Three higher homologs of 223A, namely alkaloids 237L (2), 251M (3), and 267J (4), have been detected in other extracts, and tentative structures are proposed.

A 1,4-disubstituted quinolizidine from a Madagascan mantelline frog (Mantella).

A major alkaloid, 217A, in skin extracts of a mantelline frog (Mantella baroni) was isolated by HPLC and the structure defined by 1H-NMR spectral analysis as (1R,4S,10S)- or (1R,4R,10R)- 1-methyl-4-(Z)-(1-pent-2-en-4-ynyl) quinolizidine (1). Four other alkaloids-207I (2), 231A (3), 233A (4), and 235E' (5)-detected in frog skin extracts are proposed to be 1,4-disubstituted quinolizidines based on diagnostic mass and FTIR spectra.

A new secotrinervitane diterpene isolated from soldiers of the Madagascan termite species, Nasutitermes canaliculatus.

Reported herein is the X-ray crystallographic structure of a novel 10-oxygenated secotrinervitane diterpene, 3 alpha, 10 alpha-diacetoxy-7,16-secotrinervita-7,11,15(17)-triene (4), from soldiers of the endemic Madagascan termite Nasutitermes canaliculatus, which was compared with an energy-minimized structure obtained by computer molecular modeling. We also report 1H- and 13C-NMR and MS data for this new diterpene.

Chemistry of venom alkaloids in someSolenopsis (Diplorhoptrum) species from Puerto Rico.

A number of 15-carbon alkaloids have been identified in venom extracts of four Puerto Rican species of ants in the genusSolenopsis (Diplorhoptrum). Workers of a species from El Verde produced thecis andtrans isomers of 2-methyl-6-nonylpiperidine with the latter isomer predominating. The same compounds were identified in queens of a species from Río Grande, but in this species no alkaloids were detected in worker extracts. Workers of aDiplorhoptrum species collected on Mona Island produced primarily atrans-2-methyl-6-(Z-4-nonenyl)piperidine,3, with smaller amounts of thecis isomer, whereas the major compound found in the queens of the same species on Mona Island was (5Z,9Z)-3-hexyl-5-methylindolizidine, identical with the alkaloid produced by queens of a species collected on Cabo Rojo. Surprisingly, workers of the Cabo Rojo species produced (5Z,9Z)- and (5E,9E)-3-butyl-5-propylindolizidine (4 and5, respectively) reported earlier as the 223AB indolizidines from skins of dendrobatid frogs. The possible significance of the qualitative and quantitative differences in the venom alkaloids synthesized by queens and workers is discussed as is the possibility that ants containing such alkaloids may serve as a dietary source for the skin alkaloids used by certain frogs in chemical defense.

A new subclass of alkaloids from a dendrobatid poison frog: a structure for deoxypumiliotoxin 251H.

Deoxypumiliotoxin 251H [4], representing a new subclass of the pumiliotoxin-A class of alkaloids, has been isolated from a dendrobatid frog, Epipedobates tricolor. The structure, elucidated by nmr, gc-Ftir, and mass spectral analyses, is proposed to be an 8-methyl-6-(5-hydroxy-2-methylhexylidene)-1-azabicyclo[4.3.0]nonan e. The absolute stereochemistry and the relative configuration of the hydroxyl group are unknown.

An uptake system for dietary alkaloids in poison frogs (Dendrobatidae).

The skin of poison frogs (Dendrobatidae) contains a wide variety of alkaloids that presumably serve a defensive role. These alkaloids persist for years in captivity, but are not present in captive-raised frogs. Alkaloids fed to poison frogs (Dendrobates, Phyllobates, Epipedobates) are readily accumulated into skin, where they remain for months. The process can be selective; an ant indolizidine is accumulated, while an ant pyrrolidine is not. Frogs (Colostethus) of the same family, which do not normally contain alkaloids, do not accumulate alkaloids. Such an alkaloid uptake system provides a means of maintaining skin alkaloids and suggests that some if not all such 'dendrobatid alkaloids' may have a dietary origin.

Alkaloids in Madagascan frogs (Mantella): pumiliotoxins, indolizidines, quinolizidines, and pyrrolizidines.

Brightly colored ranid frogs of the genus Mantella are found only in rain forests of Madagascar. Gc-ms and gc-Ft-ir analyses of skin alkaloids of seven different species, including four populations of Mantella madagascariensis, are reported. All contain one or more representatives of the pumiliotoxin A (PTX-A) class with the 13,14-dihydro derivatives 309A and 325A found in major amounts in the four populations of M. madagascariensis, while 307A (PTX-A) is found in two populations of M. madagascariensis and in three additional species, Mantella aurantiaca, Mantella viridis, and Mantella crocea. The latter three species also contain appreciable quantities of 323A (PTX-B). The four populations of M. madagascariensis show major amounts of two 1,4-disubstituted quinolizidines, 217A and 231A, and a 5,8-disubstituted indolizidine, 217B, in addition to many minor or trace quinolizidines and indolizidines. Such disubstituted quinolizidines and indolizidines are present as trace alkaloids in the six other species of Mantella, along with 3,5-disubstituted indolizidines, 3,5-disubstituted pyrrolizidines, the decahydroquinoline cis-195A, tricyclic alkaloids, and homopumiliotoxins. A new alkaloid class, which appears to contain a quinolizidine moiety, is seen in M. aurantiaca and M. crocea and is represented by 235C and several congeners.

Alkaloids from bufonid toads (Melanophryniscus): decahydroquinolines, pumiliotoxins and homopumiliotoxins, indolizidines, pyrrolizidines, and quinolizidines.

Skins of bufonid toads of the genus Melanophryniscus contain several classes of alkaloids: decahydroquinolines, pumiliotoxins, allopumiliotoxins, homopumiliotoxins, both 3,5- and 5,8-disubstituted indolizidines, 3,5-disubstituted pyrrolizidines, and a 1,4-disubstituted quinolizidine. Tricyclic alkaloids, including precoccinelline [193A] and alkaloid 236, an oxime methyl ether, are present in one population of Melanophryniscus stelzneri.