Structures And Biological Activity Of Alkaloids And Other Natural Products

两栖动物皮肤提供了广泛的生物活性生物碱，其中许多具有独特的药理活性和治疗潜力。这些生物碱包括蝙蝠毒素，它是钠通道的有效激活剂，组氨酸毒素，它是烟碱受体通道的非竞争性阻滞剂，矮米毒素/异矮米毒素/同矮米毒素和相关同系物，其中一些由于对钠通道的影响而具有肌强直和心脏强直活性，以及依比替丁，一种非常有效和选择性的烟碱激动剂，具有有效的抗伤害活性。其他生物碱包括十氢喹啉类、吡咯利嗪类、吲哚利嗪类、喹诺利嗪类、左咪嗪类和各种三环生物碱，包括螺吡咯利嗪肟类、藻毒素、假茶碱类、环五喹啉利嗪类和尾藻碱类。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。我们的天然产品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，其次是紫外（UV）、红外（IR）、质谱（MS）和核磁共振（NMR）数据的在线分析。这些技术，连同微化学反应的发展，包括氢化、酰化、顺式二醇的丁基硼化和甲醛和甲酸的n -甲基化GC分析，已经负责800多种生物碱的详细表征，代表了蛙皮提取物的26个结构类别。HPLC-MS可以研究所有的生物碱，即使是那些高分子量或极性的生物碱，也不能用GC，但由于缺乏广泛的大气压力化学电离（APCI）或电喷雾电离（ESI），因此只能提供有限的结构见解。使用电子冲击电离（EIMS）的气相色谱-质谱分析提供了丰富的、可诊断的碎片模式，而化学电离（CIMS）提供了分子量，在氘化氨中，还提供了可交换OH和NH基团的数量。这种开创性的光谱研究已经扩展到在碰撞激活CIMS模式下开发和应用串联质谱，展示和阐明与传统EIMS不同和互补的片段。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（识别官能团，如OH，羰基，双键和三键等）扩展到提供有价值的立体化学见解（顺式或反式环连接，Bohlmann波段分析，以指示氢在碳上的方向邻近氮等）。在FTIR中，我们小组在去年应用了一种新的方法，叫做甲基计数，我们还在开发中，它可以在红外光谱中详细地集成CH，大大提高了我们在结构解析过程中的能力。手性气相色谱分析已经用合成样品确定了许多生物碱的绝对立体化学性质。GC-MS和GC-FTIR，在某些情况下结合详细的核磁共振分析甚至合成进行结构验证，已经描绘了400多种生物碱的结构。目前，微探针核磁共振分析已应用于少量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生物碱已经鉴定出大约100种新的生物碱，其中一些是新的结构类，包括n -甲基十氢喹啉类、二烷基胺类和脱氢肼类。某些混合甲虫被发现含有蝙蝠毒素，似乎是在毒镖蛙和某些鸟类中发现的蝙蝠毒素的饮食来源。螨虫、蚂蚁、甲虫和千足虫是许多类两栖动物皮肤生物碱的食物来源，特别是以低碳毒素和许多具有支链碳骨架的嘧啶为食的甲螨，以其他具有线性碳骨架的嘧啶为食的金蚁，以三环瓢虫碱生物碱为食的甲虫和以螺吡咯利齐啶为食的siphonotid千足虫。从蚂蚁中进一步发现了新的生物碱。已经发现蜘蛛蛾（Eudulophasia）的幼虫从植物（Spigelia）中吸收ryanodine。利用高效液相色谱-紫外-质谱法对一种古老的黑眼蟾蜍皮肤提取物进行了重新检测，发现其中至少含有5种蟾二烯内酯和3种核桃烯内酯。这一结果与最初的生物测定一致，即瓦阿拜碱提取物对心肌功能所必需的Na/ k - atp酶的抑制作用。在这里，负离子质谱和在高效液相色谱溶剂中使用氘水有助于确定部分结构。其中一种矮毛菌毒素PTX 251D对蚊子和火蚁具有对映选择性接触毒性。两栖动物生物碱的主要生物学靶点似乎是电压敏感离子通道和配体门控离子通道，特别是钠、钙和烟碱通道。某些彪马毒素被发现激活伤害感觉通路，可能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.