In solid tumors, when O(2) partial pressure drops below 10 mmHg, ATP levels rapidly decrease due to the Warburg effect. It is known that certain macrocyclic polyamines catalyze the chemical hydrolysis of ATP with release of inorganic phosphate. Since tumor cells have diminished ATP levels as compared to normal cells, we attempted to deplete cellular ATP with macrocyclic polyamines in an effort to inhibit tumor cell proliferation. Five macrocyclic polyamines, related to the budmunchamine family of alkaloids, were prepared by total synthesis. They were the [17]-N(4) macrocycle 1, the [16]-N(4) macrocycle 20, the [18]-N(4) macrocycle 13, the [20]-N(5) macrocycle 8, and the [13]-N(3) macrocycle 17. Each one of them hydrolyzed ATP in vitro with release of P(i); the largest ring macrocycle 8 was the most efficient catalyst, while the smallest ring macrocycle 17 was the least efficient (P(i) released in these runs was on the order of 40-100 microM). The linear polyamine spermine had no hydrolytic effect on ATP. The macrocycles were found to be cytotoxic when assessed by means of a MTT assay against two human prostate cell lines, DuPro and PC-3, with resultant ID(50) values ranging between 0.5 and 1.8 microM. Colony forming efficiency (CFE) assays performed on DuPro cells, where the macrocycles were used in a concentration range of 1-8 microM, confirmed the cytotoxic effect of each macrocycle. Each killed 3-4 log of DuPro cells. The smallest ring 17 was the least cytotoxic after 24 h of incubation, although after 144 h of incubation it showed significant cytotoxicity at 8 microM. The macrocycles were equally efficient in depleting the intracellular ATP pools; after a 24 h incubation with each macrocycle other than 17 at 1-8 microM concentrations, cellular ATP concentrations were decreased by 3 orders of magnitude. The decrease in ATP levels was more pronounced after a 72 h incubation, when even 17 reduced ATP by 2 orders of magnitude. A linear pentamine of established cytotoxicity was without effect on the ATP pools. The macrocycles depleted almost entirely the intracellular pools of polyamines and were efficiently taken up by cells. A rough correlation could be established between the cytotoxic effect of the macrocyclic polyamines and their ATP-ase like activity in the DuPro cell line. As ATP is a scarce metabolite in cancer cells, where it can only be replenished through the very ATP-inefficient glycolytic pathway; macrocyclic polyamines appear to be promising new anticancer agents.

The application of high-performance liquid chromatography on-line coupled with atmospheric-pressure chemical-ionization mass spectrometry (HPLC-APCI-MS), electrospray-ionization mass spectrometry (ESI-MS), as well as with tandem mass spectrometry (MS/MS), for the analysis of natural products is discussed. The separation, detection and structure elucidation of polyamine-containing compounds contained in plant material and spider venoms was performed by means of these techniques. The use of the methods for biochemical investigations of diverse natural materials is proposed.

Dedicated to Prof. Dr. H. J. Veith on the occasion of his 60th birthdayThe lyophilized venom of the spider Agelenopsis aperta (Aranea: Agelenidae) has been re-analyzed by online coupled high-performance liquid chromatography and atmospheric-pressure chemical-ionization mass spectrometry (HPLC-UV(DAD)-APCI-MS and MS/MS). Thus, 33 acylpolyamines with 11 different molecular masses, present in various concentrations, were detected, and their structures were disclosed. Different types of possible fragmentation as well as principles of structure elucidation are discussed. The method can be used for rapid separation and analysis of spider-venom constituents.Last year, high-performance liquid chromatography (HPLC), coupled on-line with atmospheric-pressure chemical-ionization mass spectroscopy (APCI-MS), has been successfully used for the isolation and structure elucidation of polyamines from the venom of the spider Paracoelotes birulai (Aranea: Amaurobiidae) [22]. It gave an idea that this selective and sensitive method could be used for analysis and structure elucidation of new acylpolyamines present in Agelenopsis aperta venom in low concentrations.Since APCI-MS produces by collision-induced decomposition (CID) at low activation energies (`100 eV) quasi-molecular ions with low fragmentation, it was interesting to compare results obtained by on-line-coupled HPLC and APCI mass spectrometer (HPLC-APCI-MS and HPLC-APCI-MS/MS) with other analytical methods such as chemical-ionization (CI), matrix-assisted laser-desorption ionization (MALDI), fast-atom-bombardment (FAB), continuous-flow-frit-FAB, and electrospray-ionization (ESI) mass spectrometry.In this work, a number of novel polyamine-containing toxins from the venom of Agelenopsis aperta are reported.

Dedicated to Prof. Dr. Manfred Hesse on the occasion of his retirement A recently developed new and divergent approach for the solid-phase synthesis of polyamines and polyamine derivatives was extended to the preparation of linear pentamines, and it was applied to the synthesis of three quartets of isomeric polyamine spider toxins. The twelve synthetic acylpolyamines were investigated by HPLC-UV(DAD)-MS and HPLC-UV(DAD)-MS/MS and compared with the natural products in the complex mixture of the venom of Agelenopsis aperta. The comparative investigation supported the structures and assignments of seven previously found toxins and allowed the identification of an additional five polyamine derivatives in the natural sample. The MS/MS study of the isomerically pure polyamine derivatives revealed furthermore a characteristic pattern for the fragmentation of these compounds, which can possibly be used as evidence in the trace analysis of other polyamine derivatives.

The lyophilized venom of the spider Paracoelotes birulai (Araneidae: Amaurobiidae) has been analyzed. A number of acylpolyamines were found and separated. By on-line coupled high-performance liquid chromatography and atmospheric-pressure chemical-ionization mass spectrometry, the structures of the three most abundant compounds PB 490 (N-(16-guanidino-4-hydroxy-4,8,12-triazahexadecyl)-2-(4-hydroxyindol-3yl)acetamide; Fig. 3, b), PB 421 (N-(16-guanidino-4-hydroxy-4,8,12-triazahexadecyl)-2-(4-hydroxybenzamide; Fig. 4, a), and PB 448 (N-(16-amino-4-hydroxy-4,8,12-triazahexadecyl)-2-(4-hydroxyindol-3-yl)acetamide; Fig. 5,b) were elucidated. Two different types of polyamines were found in the a-palutoxins and compared with acylpolyamines from the Agelenidae spider family. The results of this investigation will initiate further chemotaxonomical studies on spiders.Introduction. ± Spider venoms attract the undivided attention of scientists interested in these unique natural mixtures of different classes of biologically active compounds. Chemists elucidate the structures of the constituents of spider venoms, biologists try to derive the mechanisms of the interactions of venoms with the spiders prey and create new specific insecticides, while pharmacists search for new drugs. The continuing discovery of new spider species also stimulates these investigations.Depending on the family and species, spider venoms contain, in diverse ratios, various natural compounds like amino acids, polyamines, purine bases, acylpolyamines, peptides, polypeptides or proteins; sometimes just some of these compounds, sometimes all of them together are present. In the last two decades, the assumption that only proteins are responsible for the toxicity of spider venoms was revised, and the activities of the compounds present in the venoms have been investigated. At the present time, several classes of natural products are believed to be the toxic constituents of spider venoms. Acylpolyamines, peptides, polypeptides, proteins, either by themselves or coacting with one another, are responsible for the toxic action of the venom.Being available in only milligram amounts, spider venoms are quite difficult to investigate by classical methods, and the successful application of NMR spectroscopy or elemental analysis for structure elucidation is more of an exception than a generally applied procedure.Paracoelotes birulai, known from several sources as Paracoelotes luctuosus