Structures And Biological Activity Of Alkaloids And Other Natural Products

两栖动物的皮肤提供了多种具有生物活性的生物碱，其中许多具有独特的药理活性和治疗潜力。这些生物碱包括蛙毒素（batrachotoxins），其是钠通道的有效激活剂;组蛋白毒素（histrionicotoxins），其是烟碱受体通道的非竞争性阻断剂; pumiliotoxins/allopumiliotoxins/homopumiliotoxins和相关同系物，其中一些由于对钠通道的作用而具有肌强直和心强直活性;以及地棘蛙素（epibatidine），其是一种极其有效和选择性的烟碱激动剂，具有有效的抗伤害感受活性。其他生物碱包括十氢喹啉、吡咯里嗪、吲嗪、喹嗪、来米嗪和各种三环生物碱，包括螺吡咯里嗪肟、gephyrotoxins、假菲林胺、环戊喹嗪和coccinellines。有机化合物的结构解析现在几乎完全基于光谱分析，使用紫外（UV）、红外（气相IR）、质谱（MS）和核磁共振（NMR）光谱技术。我们的天然产物项目依赖于强大的分离和光谱技术的发展，用于分析从两栖动物皮肤，节肢动物和其他来源的提取物中获得的复杂混合物中微量的生物碱和其他化合物。关键技术是气相色谱（GC）或高效液相色谱（HPLC）分离，然后在线分析UV、IR、MS，希望还有NMR数据。这些技术，沿着微化学反应的发展，包括氢化，酰化，顺式二醇的丁基硼化和甲醛和甲酸的GC分析上的N-甲基化，已经负责详细表征超过800种生物碱，代表青蛙皮肤提取物中的约26个结构类别。HPLC-MS允许研究所有生物碱，即使是那些不GC的高分子量或极性的生物碱，但由于缺乏大气压化学电离（APCI）或电喷雾电离（ESI）的广泛碎片化，因此只能提供有限的结构见解。使用电子碰撞电离（EIMS）的GC-MS分析提供了丰富的、诊断性的碎片模式，而化学电离（CIMS）提供了分子量，并且使用氘代氨提供了可交换的OH和NH基团的数量。这种开创性的光谱研究已扩展到开发和应用串联质谱的碰撞激活CIMS模式，展示和阐明不同于传统的EIMS和互补的碎片。气相GC-FTIR（傅里叶变换红外）的分析潜力已经允许从红外的传统用途（鉴定官能团，如OH、羰基、双键和三键等）扩展，提供有价值的立体化学见解（顺式或反式环连接，Bohlmann带分析以指示氢在邻近氮的碳上的取向等）。一种新的FTIR方法，在过去的一年中，由我们的小组应用，我们仍在开发，命名为甲基计数，允许详细的CH积分在IR光谱，大大提高了我们的能力，在结构解析的过程中。手性气相色谱分析已经用合成样品建立了许多生物碱的绝对立体化学。GC-MS和GC-FTIR，在某些情况下结合详细的NMR分析，甚至合成结构验证，已经描绘了400多种生物碱的结构。用微探针进行核磁共振分析，现已应用于一些只有10微克的生物碱样品。目前从中南美洲和马达加斯加的两栖动物和节肢动物中提取的提取物已鉴定出约100种新生物碱，其中一些代表新的结构类别，包括N-甲基十氢喹啉、二烷基胺和脱氢叠氮烷。某些苜蓿甲虫被发现含有毒箭蛙毒素，似乎是毒箭蛙和某些鸟类中发现的毒箭蛙毒素的饮食来源。螨、蚂蚁、甲虫和千足虫是许多种类两栖动物皮肤生物碱的食物来源，特别是甲螨的pumiliotoxins和许多具有支链碳骨架的叠氮烷，蚁蚁的其他具有线性碳骨架的叠氮烷，甲虫的三环胭脂碱生物碱和siphonotid千足虫的螺吡咯烷。从蚂蚁中提取的其他新生物碱已在结构上得到确定。已经发现蜘蛛蛾（Eudulophasia）的幼虫可以从植物（Spigelia）中螯合ryanodine。用HPLC-UV-MS法重新检测了黑蟾皮的老提取物，发现至少有5种蟾蜍二烯内酯和3种强心内酯。该结果与原始生物测定一致，即该提取物抑制哇巴因与心肌功能必需的Na/K-ATP酶结合。在这里，负离子MS和在HPLC溶剂中使用氘代水可用于分配部分结构。其中一种短尾虫毒素PTX 251D对蚊和火蚁具有对映体选择性触杀作用。两栖类生物碱的主要生物靶点似乎是电压敏感性和配体门控离子通道，特别是钠、钙和烟碱通道。发现某些短小毒素可以激活伤害性感觉通路，推测是通过与钠通道的相互作用。

A wide range of biologically active alkaloids, many of which have unique profiles of pharmacological activity and therapeutic potential, has been provided by amphibian skin. 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 and epibatidine, an extremely potent and selective nicotinic agonist with potent antinociceptive activity. Further alkaloids include decahydroquinolines, pyrrolizidines, indolizidines, quinolizidines, lehmizidines, and a variety of tricyclic alkaloids, including spiropyrrolizidine oximes, gephyrotoxins, pseudophrynamines, cyclopentaquinolizidines and coccinellines. Structure elucidation of organic compounds is now based almost exclusively on spectroscopic analysis, using ultraviolet (UV), infrared (vapor-phase IR), mass (MS), and nuclear magnetic resonance (NMR) spectral techniques. Our natural products program has relied on the development of powerful separation and spectral techniques for the analysis of alkaloids and other compounds present in minute amounts in complex mixtures obtained in extracts from amphibian skin, arthropods, and other sources. The key techniques are gas chromatographic (GC) or high performance liquid chromatographic (HPLC) separation, followed by analysis online of UV, IR, MS and hopefully, NMR data. These techniques, along with development of microchemical reactions including hydrogenation, acylation, butylboronation of cis-diols and N-methylation on GC analysis with formaldehyde and formic acid, 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.). A new method in FTIR, applied during the last year by our group and still being developed by us, named methyl counting, allows a detailed CH integration in the IR spectrum, greatly enhancing our ability in the process of structure elucidation. 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 a few alkaloid samples of only 10 ug. Current extracts from amphibians and arthropods of Central and South America and Madagascar have led to identification of about 100 new alkaloids, some representing new structural classes, including N-methyldecahydroquinolines, dialkylamines and dehydroizidines. Certain melyrid beetles were found to contain batrachotoxins and appear likely to be the dietary source of batrachotoxins found in poison dart frogs and certain birds. The mites, ants, beetles and millipedes that are dietary sources of many classes of amphibian skin alkaloids have been identified, notably oribatid mites for pumiliotoxins and many izidines with branched carbon skeletons, myrmicine ants for other izidines with linear carbon skeletons, beetles for the tricyclic coccinelline alkaloids 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. The use of HPLC-UV-MS to re-examine an old extract of a Melanophryniscus toad skin has revealed at least 5 bufadienolides and 3 cardenolides. This result is consistent with the original bioassay, the inhibition by that extract of ouabain-binding to a Na/K-ATP-ase enzyme, essential in heart muscle function. Here, negative-ion MS and use of deuterated water in the HPLC solvent were useful in assigning partial structures. One of the pumiliotoxins, namely PTX 251D, had enantioselective contact toxicity for mosquitoes and fire ants. The major biological targets for the amphibian 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.