Class Of Conjugates Research Articles

Polymer–Ferrocene Conjugates Containing an Ester Function in the Connecting Links

Ferrocene, the parent of the metallocene family of organotransition metal compounds, has come to occupy a significant niche in cancer research. Developmental work in the authors' laboratory has been focused on the synthesis of antiproliferative ferrocene conjugates in which the bioactive ferrocene unit is covalently, yet bioreversibly bound (anchored) to water-soluble carrier polymers designed in accordance with requisite biomedical specifications. The anchoring link in most of these conjugates has been an aliphatic spacer containing the biofissionable amide group. In this communication the synthesis of a class of ferrocene conjugates is reported in which the ferrocene group is carrier-anchored through spacers containing an ester link, of interest here because of potentially different drug release behavior. The carriers are polyamides equipped with variously spaced hydroxyl side groups, to which the ferrocenylation agent, 4-ferrocenylbutanoic acid, is connected through esterification. The coupling reactions, mediated by carbodiimide agent and catalyzed by 4-(dimethylamino)pyridine, are carried out in DMF at temperatures not exceeding 65°C, and the water-soluble product polymers are isolated in yields of typically 70–85% by precipitation, aqueous dialysis, and freeze-drying. With the molar feed ratios chosen in these coupling experiments, the incorporation of ferrocene, assessed by 1H NMR spectroscopy, corresponds to iron contents of roughly 2.5–5.5%, by mass. The conjugates will be included in a forthcoming bioactivity screening program.

Modulation of Quinol/Quinone-Thioether Toxicity by Intramolecular Detoxication

A variety of cytotoxic, mutagenic, and carcinogenic conjugates of GSH require processing by enzymes of the mercapturic acid pathway to produce toxicity. However, metabolism of quinone-thioethers by gamma-GT can result in either activation or detoxication. For example, inhibition of gamma-GT completely protects against the nephrotoxicity caused by 2-bromo-bis-(glutathion-S-yl)hydroquinone and 2,3,5-tris-(glutathion-S-ly)hydroquinone, whereas the same protocol potentiates the nephrotoxicity of 2,5-dichloro-3-(glutathion-S-yl)hydroquinone and 2,5,6-trichloro-3-(glutathion-S-yl)hydroquinone. Which of these two scenarios occur as a consequence of metabolism by gamma-GT appears to be determined by the relative rate at which the product is transported into cells and/or interacts with cellular constituents, and the rate which the product undergoes intramolecular detoxication (cyclization) to a 1,4-benzothiazine. The same reaction may also explain why the mercapturic acid metabolite of menadione is nephrotoxic following systemic administration, whereas the GSH conjugate is without activity. Species differences exist in susceptibility to both 2-bromo-bis-(glutathion-S-ly)hydroquinone and 2,3,5-tris(glutathion-S-ly)hydroquinone induced nephrotoxicity. In this case, however, susceptibility does not correlate with renal gamma-GT activity, but rather to differences in the rate at which the corresponding cysteine and N-acetylcysteine conjugates undergo N-acetylation/N-deacetylation cycling. Thus the guinea pig--which is the only other rodent species (in addition to the rat), that is susceptible to 2-bromo-bis-(glutathion-S-ly)hydroquinone and 2,3,5-tris-(glutathion-S-ly)hydroquinone mediated nephrotoxicity--expresses the lowest activity of renal gamma-GT but exhibits the highest N-deacetylation:N-acetylation ratio. Differences in kinetics of these two reactions therefore contribute to species susceptibility. The toxicity of quinol/quinone thioethers is dependent upon a number of physiological, biochemical, and electrochemical factors. The rates at which quinol-thioethers undergo oxidation, with the concomitant generation of reactive oxygen species (IV, Fig. 1), macromolecular arylation (V, Fig. 1), intramolecular cyclization (VI, Fig. 1), and acetylation-deacetylation cycling (III, Fig. 1) is dependent upon the substrate in question. All these factors will contribute to the cell, tissue, and species susceptibility of this interesting class of GSH conjugates.

Multivalent melanotropic peptide and fluorescent macromolecular conjugates: new reagents for characterization of melanotropin receptors.

Radioreceptor binding studies have documented the presence of melanotropin receptors on some but not all of the various human melanoma cell lines that have been studied. Using a newly developed class of multivalent fluorescent melanotropin-macromolecular conjugates, we have demonstrated for the first time the presence of specific melanotropin receptors on all of the melanoma cell lines, both mouse and human, melanotic as well as amelanotic, that were investigated. The conjugates developed by us consisted of multiple copies of both a potent melanotropin analogue and a fluorophore, both arranged in a pendent fashion on a biologically inert macromolecule. While the multivalency of these conjugates may have established stronger binding with the melanotropin receptors on the cell surface (perhaps by establishing simultaneous multiple interactions), the presence of multiple copies of the fluorophore also greatly increased the level of detection in fluorescence labeling experiments. Membrane receptor-hormone-associated phenomena, such as capping and internalization of the receptor-ligand complex, also were observed. The details of these methods are described using B-16 mouse melanoma cells as a model system. The demonstration of MSH receptors as a common marker for melanoma suggests that this methodology might be employed for early clinical detection and anatomical localization of melanoma. These results also offer the possibility that substitution of the fluorophore in these conjugates by a chemical agent of (chemo-)therapeutic relevance may provide a powerful tool for site specific (tumor) targeting and cytotoxicity.

Quinol diglucuronides are predominant conjugated metabolites found in bile of rats following intratracheal instillation of benzo[a]pyrene.

[3H]Benzo[a]pyrene (B[a]P) was administered to male Sprague-Dawley rats via intratracheal instillation, and bile was collected over a period of 6 h. Conjugated metabolites of B[a]P in bile were separated by paper chromatography or reversed-phase ion-pair HPLC and quantified by liquid scintillation spectrometry. In paper chromatographic analysis, a class of conjugates more polar than thioether conjugates was recognized. These conjugates were identified as quinol diglucuronides by hydrolyzing with beta-glucuronidase and analyzing products of the hydrolysis with HPLC, and by migration on paper relative to a standard of 3,6-quinol diglucuronide. From this analysis, relative amounts of conjugated metabolites of B[a]P in bile were 37.3% quinol diglucuronides, 19.9% thioether conjugates, 33.3% monoglucuronide and sulfate conjugates, and 9.4% unconjugated metabolites. Analysis by reversed-phase ion-pair HPLC provided improved resolution among the conjugates in bile. In particular, the 3,6-quinol diglucuronide was resolved from the 1,6- and 6,12-quinol diglucuronides, with identification of peaks being based on sensitivity to hydrolysis with beta-glucuronidase and elution of standards of these diglucuronides. The elution position of thioether conjugates was identified by their insensitivity to hydrolysis with beta-glucuronidase and arylsulfatase and by synthesis of thioether conjugates in V79 (XEM-2) cells, which express cytochrome P450IA1 and have relatively high levels of glutathione S-transferases but low levels of UDP-glucuronyltransferases and sulfotransferases. From the reversed-phase ion-pair HPLC analysis, relative amounts of conjugates in bile were 10.4% 1,6- and 6,12-quinol diglucuronides, 20.8% 3,6-quinol diglucuronide, 30.4% thioether conjugates, 17.8% monoglucuronides, 6.2% sulfate conjugates, and 14.4% unconjugated metabolites. These studies provide the first report of the biosynthesis of quinol diglucuronide conjugates of B[a]P in vivo and demonstrate that they are excreted into bile in significant quantities.

The design, synthesis and study of siderophore-antibiotic conjugates Siderophore mediated drug transport

The use of conjugates of microbial iron chelators (siderophores) and antibiotics for illicit transport of antibiotics into cells is a potentially powerful method for the rational design of therapeutic agents. The structural complexity of most natural siderophores has impeded progress in this area. Described here are the design, syntheses and preliminary biological studies of several siderophore-beta-lactam antibiotic conjugates. Both hydroxamic-acid-based and catechol-based conjugates with and without amino acid spacers to carbacephalosporins were synthesized and demonstrated to be effective inhibitors of Escherichia coli X580. Mutant selection was noted for each class of conjugates. Mutants selected from exposure of the E. coli to the hydroxamate conjugates were susceptible to the catechol conjugates and vice versa. Combinations of hydroxamate- and catechol-carbacephalosporin conjugates were most effective inhibitors of E. coli X580.

Ecdysone metabolism in adult crickets, Gryllus bimaculatus

The fate of injected [ 3H]ecdysone has been investigated in female and male adults of the Mediterranean field cricket, Gryllus binaculatus (de Geer). The metabolism is similar in both sexes and at various stages of adult life. Several classes of apolar metabolites (A1–A5) represent the major compounds. The amount of polar conjugates is low in all tissues, as are the concentrations of 20-hydroxyecdysone. Ovaries are the only organs capable of storing considerable amounts of ecdysteroids. The amount of radiolabelled ecdysteroid activity (mostly [ 3H]ecdysone) excreted during the first 24 hr after injection is high. The chemical identity of the apolar metabolites is not yet known. A2, which is the major apolar compound, has recently been identified as a complex of ecdysone conjugates with abundant long-chain fatty acids (Hoffman et al., 1985 Life Sci. 37, 185–192). Incubations with tissue homogenates in vitro have shown that several organs are capable of converting ecdysone into apolar compounds. Apolar ecdysteroid acyl esters represent a newly identified class of ecdysone conjugates from insects. Their role in regulation of free ecdysteroid titres during the reproductive period in female crickets is discussed.

Apolar ecdysteroid esters in adult female crickets, [formula omitted][formula omitted

Six hours after injection of 0.5 uCi 3H-ecdysone into the hemocoele of adult female crickets, several labelled compounds could be separated from hemolymph and tissues by silicic acid column chromatography, TLC, and HPLC. The amount of conjugated, polar ecdysteroids was low in all tissues. whereas apolar metabolites were predominant in all tissues. The apolar compound A2, which is the most abundant in quantity, could be hydrolyzed by porcine liver esterase, yielding ecdysone and various long chain fatty acids. This represents a new class of apolar ecdysteroid conjugates not yet found in other insects.

A new class of apolar ecdysteroid conjugates: esters of 20-hydroxy-ecdysone with long-chain fatty acids in ticks

Summary Nymphs of the argasid tick Ornithodoros moubata rapidly transform most of the ingested molting hormone 20-hydroxy-ecdysone (20-hydroxy-E) into a family of 4 major apolar conjugates which are hydrolyzable by carboxylic-ester hydrolase. These conjugates—new among zoo- or phytoecdysteroids—are composed of 20-hydroxy-E esterified at C22 with the common long-chain fatty acids C16:0, C18:0, C18:1, or C18:2. The chemical structure of the purified metabolites was identified by fatty acid analysis, by CI/D mass spectrometry and by comparison with a chemically prepared 20-hydroxy-E-22-palmitate. The significance of the apolar metabolic pathway is discussed. It may serve to inactivate molting hormones which could be ingested with blood from hosts that feed on plants containing phytoecdysteroids. In addition, the conjugates may act as a storage form of hormones for the developing embryo.