Amphibian skin has provided a wide range of biologically active alkaloids, many of which have unique profiles of pharmacological activity and therapeutic potential. These alkaloids include batrachotoxins, which are potent activators of sodium channels, histrionicotoxins, which are noncompetitive blockers of nicotinic receptor-channels, pumiliotoxins/allopumiliotoxins/homopumiliotoxins and related congeners, some of which have myotonic and cardiotonic activity due to effects on sodium channels, epibatidine, an extremely potent and selective nicotinic agonist with potent antinociceptive activity and epiquinamide, a nicotinic agonist selective for certain receptor subtypes. Further alkaloids include decahydroquinolines, pyrrolizidines, indolizidines, quinolizidines, lehmizidines, and a variety of tricyclic alkaloids, including spiropyrrolizidine oximes, gephyrotoxins, pseudophrynamines, cyclopentaquinolizidines, coccinellines and coccinelline analogs. The batrachotoxins were also found to occur in certain toxic birds and dietary beetles. Structure elucidation of organic compounds is now based almost exclusively on spectroscopic analysis, using ultraviolet (UV), infrared (IR), mass (MS), and nuclear magnetic resonance (NMR) spectral techniques. Our natural products program has relied on the development of powerful spectral techniques for the analysis of alkaloids and other compounds present in minute amounts in complex mixtures obtained in extracts from amphibian skin and other sources. The key techniques are gas chromatographic (GC) or high performance liquid chromatographic (HPLC) separation, followed by analysis online of UV, IR and MS data. These techniques, along with development of microchemical reactions including hydrogenation, acylation, butylboronation of cis-diols and reductive N-methylation on GC analysis with formaldehyde, have been responsible for the detailed characterization of over 800 alkaloids, representing some 26 structural classes in frog skin extracts. HPLC-MS allows study of all alkaloids, even those of high molecular weight or polarity that do not GC, but gives only limited structural insights because of lack of extensive fragmentation with either atmospheric pressure chemical ionization (APCI) or electrospray ionization (ESI). GC-MS analysis using electron impact ionization (EIMS) provides rich, diagnostic patterns of fragmentation, while chemical ionization (CIMS) provides molecular weight and, with deuterated ammonia, the number of exchangeable OH and NH groups. Such pioneering spectroscopic research has been extended to developing and applying tandem mass spectrometry in the collision-activated CIMS mode, demonstrating and elucidating fragmentations different from and complementary to conventional EIMS. The analytical potential of vapor-phase GC-FTIR (Fourier transform IR) has allowed extension from traditional uses of IR (identification of functional groups like OH, carbonyl, double and triple bonds, etc.), to providing valuable stereochemical insights (cis- or trans-ring junctions, Bohlmann band analysis to indicate orientation of hydrogens on carbons adjacent to nitrogen, etc.). Chiral GC analysis has established with synthetic samples the absolute stereochemistry of many alkaloids. GC-MS and GC-FTIR, in conjunction in some cases with detailed NMR analysis and even synthesis for structural verification, have delineated structures of over 400 alkaloids. NMR analysis with microprobe has now been applied to alkaloid samples of only 10 ug. Current extracts from amphibians of Central and South America and Madagascar have led to identification of about 100 new alkaloids, some representing new structural classes, including N-methyldecahydroquinolines and dehydroizidines. Mites, ants, beetles and millipedes that are dietary sources of certain classes of amphibian skin alkaloids have been identified, notably mites for pumiliotoxins and certain izidines, ants for other izidines, melyrid beetles for the batrachotoxins and siphonotid millipedes for the spiropyrrolizidines. Further novel alkaloids from ants have been structurally defined. The sequestration of ryanodine from plants (Spigelia) by larvae of the spider moth (Eudulophasia) has been discovered. One of the pumiliotoxins, namely PTX 251D had enantioselective contact toxicity for mosquitoes. The major biological targets for the alkaloids appear to be both voltage-sensitive and ligand-gated ion channels, in particular sodium, calcium and nicotinic channels. Certain pumiliotoxins were found to activate nociceptive sensory pathways, presumably through interaction with sodium channels. Analysis of the activity of alkaloids at nicotinic channels has continued, including studies with high density ganglionic-type nicotinic channels discovered in an insulinoma cell line. In vivo (mice) effects of nicotinic agonists, antagonists, and positive modulators, such as galanthamine, are being studied in search of potential agents for treatment of Alzheimer's disease.
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