Structures And Biological Activity Of Alkaloids And Othe

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batrachotoxins，它是钠通道的有效激活剂，组氨酸毒素，它是尼古丁受体通道的非竞争性阻滞剂，pumiliotoxin /allopumiliotoxins/homopumiliotoxins和相关的同系物，其中一些由于对钠通道的作用而具有肌强直和心脏强直活性，依比替丁，一种非常有效和选择性的尼古丁激动剂，具有有效的抗伤活性和epiquinamide，一种对某些受体亚型有选择性的烟碱激动剂。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、藻毒素、假卟啉类、环五喹啉利嗪类、瓢虫碱类和瓢虫碱类似物。在某些有毒鸟类和食用甲虫中也发现了这种细菌毒素。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（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光谱技术的发展，用于分析从两栖动物皮肤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）和质谱（MS）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GC分析的还原性n -甲基化，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）。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。微探针核磁共振分析现已应用于仅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中提取的提取物已经鉴定出大约100种新的生物碱，其中一些代表了新的结构类别，包括脱氢肼。蚂蚁、甲虫和千足虫是某些种类两栖动物皮肤生物碱的食物来源，已被确定，特别是福蚁类蚂蚁为pumiliotoxins， melyrid甲虫为batrachotoxins， siphonotid千足虫为spiropyrolizidines，马达加斯加甲虫为一种新的类似coccinelline的生物碱，这种生物碱也存在于同域的mantellid青蛙中。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。生物碱在烟碱通道活性的微孔分析仍在继续，包括在胰岛素瘤细胞系中发现的高密度神经节型烟碱通道的研究。为了寻找治疗阿尔茨海默病的潜在药物，正在研究尼古丁激动剂、拮抗剂和阳性调节剂在体内（小鼠）的作用。高效液相色谱-质谱法在泰国蟾蜍属（安索尼亚属、Leptophryne属和Pedostipes属）中发现了蟾二烯内酯。这种钠/钾- atp酶抑制剂以前只在蟾属蟾蜍中被发现，直到25年前我们在新世界的蟾属蟾蜍中发现蟾二烯内酯。

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 dehydroizidines. Ants, beetles and millipedes that are dietary sources of certain classes of amphibian skin alkaloids have been identified, notably formicine ants for the pumiliotoxins, melyrid beetles for the batrachotoxins, siphonotid millipedes for the spiropyrrolizidines, and a Madagascan beetle for a novel coccinelline-like alkaloid, also present in sympatric mantellid frogs. 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. Microwell 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 are being studied in search of potential agents for treatment of Alzheimer's disease. HPLC-MS allowed the discovery of bufadienolides in three genera (Ansonia, Leptophryne and Pedostipes) of bufonid toads of Thailand. Such sodium/potassium-ATPase inhibitors were previously known only in toads of the genus Bufo, until our discovery of bufadienolides 25 years ago in toads of the New World genus Atelopus.