Synthesis and Biological Evaluation of Novel Chromogenic Substrates for the Enhanced Detection of Pathogenic Bacteria

The present work investigated the preparation of phenoxazinone derivatives and evaluated their performances for the detection of pathogenic bacteria. The first method investigated the condensation of nitroaminophenol with tetrahalogenobenzoquinones; the corresponding nitrohalogenophenoxazinones were all characterised and evaluated for the detection of nitroreductase activity in a range of clinically important microorganisms. The detection of nitroreductase activity has been previously suggested for the monitoring of bacterial growth; however, nitrohalogenophenoxazinones were proven to be less suitable for this purpose than other, previously reported, nitroreductase substrates. The second route investigated the synthesis of phenoxazinone derivatives via the oxidative cyclisation of diamino-dihydroxydiphenylethers and of diaminobenzoquinones. The reactive intermediates were trapped and characterised in order to rationalize the mechanism of formation of aminophenoxazinones via this route. 7- and 8-Aminophenoxazinones derivatives were prepared and further derivatised with b-alanine. Similarly, some nitrohalogenophenoxazinones were reduced to their corresponding aminophenoxazinones and derivatised with b-alanine. Thirteen new chromogenic substrates were prepared, characterised and evaluated for their sensitivity to detect b-alanine aminopeptidase on agar medium; this enzyme is expressed by only three types of bacteria, the most important being Pseudomonas aeruginosa, a pathogen commonly known to affect cystic fibrosis sufferers. Their performance for the detection of Pseudomonas aeruginosa were compared to the lead compound (7-N-(b-alanyl)amino-1-pentylphenoxazin-3-one), the substrate contained in a commercially available medium, chromIDTM ID Ps. aeruginosa. The substrates, if hydrolysed, resulted in a low colouration of the colonies when compared to the lead compound; however, 2-pentyl substituted aminophenoxazinones were found to be less toxic and had an excellent affinity for the bacterial colonies.