Products of metabolic activation of the antitumor drug ledakrin (nitracrine) in vitro.

The aim of this work was to characterize the products of metabolic activation of the antitumor drug ledakrin (Nitracrine) in model metabolic systems, where formation of drug-DNA adducts was previously discovered. The metabolic products obtained in different biological systems were compared with those obtained in experiments where chemical reducing agents were applied. Therefore, activation products were obtained in the presence of the microsomal fraction of rat liver and in the experiments with the reducing agents dithiothreitol, hydrazine hydrate, and SnCl(2). Furthermore, transformations of the drug with oxidoreductase enzymes DT-diaphorase and xanthine oxidase were observed. The ledakrin transformation products were separated and analyzed by HPLC with diode array detection. Structural studies of the products were performed by means of ESI-MS and NMR. Proton, carbon, and nitrogen assignments were made based upon DQF-COSY, ROESY, TOCSY, HSQC, and HMBC experiments. It was demonstrated during the reduction of ledakrin that a key metabolite, a compound with an additional five-membered ring attached to positions 1 and 9 of the acridine core and with the retained 9-aminoalkyl side chain, was formed in all the systems that were studied. It was determined that the reactive nitrogen atoms of this additional ring underwent further transformations resulting in the formation of a six-membered ring produced by the addition of a carbon atom to the dihydropyrazoloacridine ring. Furthermore, it was observed that positions 2 and 4 of ledakrin's acridine ring are susceptible to nucleophilic substitution as revealed by the studies with dithiothreitol. Additionally, although most products from the reduction of ledakrin were extremely unstable, 1-aminoacridinone, produced enzymatically and with dithiothreitol, exhibited persistent stability under the studied conditions.

The relevance of enzymatic oxidation by horseradish peroxidase to antitumour potency of imidazoacridinone derivatives.

The presented study concentrated on the oxidative enzymatic transformation of six imidazoacridinone derivatives exhibiting different antitumour activity. Horseradish peroxidase was applied as an enzymatic model system. The investigations aimed to evaluate: (1) whether the studied compounds can undergo oxidative biotransformation; (2) whether the susceptibility to such biotransformation relates to the structure and antitumour activity of these compounds; and (3) which elements of imidazoacridinone structure are involved in this kind of transformation. The reaction courses were followed by three methods: UV-VIS spectroscopy, electron paramagnetic resonance and high-performance liquid chromatography. It was shown that all the imidazoacridinones studied underwent enzymatic oxidation resulting in the formation of several products, spectra of which revealed that imidazoacridinone chromophore as well as alkylamino side-chain were involved in these biotransformations. The susceptibility to enzymatic oxidation turned out to be well correlated with antitumour activity of these compounds. It was demonstrated that the highly active antitumour 8-hydroxy derivatives underwent oxidative transformation far more readily than the less active 8-methoxy derivatives and analogues without substituent in position 8. The results indicated that the oxidation pathway of 8-hydroxy compounds was different from those observed for the remaining imidazoacridinones studied. It also differed from the pathway proposed earlier for mitoxantrone. Moreover, it was find out that not only the rate but also the mechanism of horseradish oxidation of 8-hydroxy derivatives depended on the reaction conditions. In the presence of excess of hydrogen peroxide, the drugs were exceptionally reactive giving rise to the mixture of many unstable products, among which compounds with both changed and unchanged chromophore structure were formed. However, the equimolar ratio of drug and hydrogen peroxide led to stable products, which resulted from the oxidation in aminoalkyl side-chain. The possible structures of products of imidazoacridinone enzymatic oxidation are discussed. In conclusion, the results presented in this paper indicate that the oxidative metabolic activation of imidazoacridinones may represent the crucial step in their biological action.