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.

Synthesis and evaluation of halogenated nitrophenoxazinones as nitroreductase substrates for the detection of pathogenic bacteria

The synthesis and microbiological evaluation of 7-, 8- and 9-nitro-1,2,4-trihalogenophenoxazin-3-one substrates with potential in the detection of nitroreductase-expressing pathogenic microorganisms are described. The 7- and 9-nitrotrihalogenophenoxazinone substrates were reduced by most Gram negative microorganisms and were inhibitory to the growth of certain Gram positive bacteria; however, the majority of Gram positive strains that were not inhibited by these agents, along with the two yeast strains evaluated, did not reduce the substrates. These observations suggest there are differences in the active site structures and substrate requirements of the nitroreductase enzymes from different strains; such differences may be exploited in the future for differentiation between pathogenic microorganisms. The absence of reduction of the 8-nitrotrihalogenophenoxazinone substrates is rationalized according to their electronic properties and correlates well with previous findings

Phosphonopeptides Revisited, in an Era of Increasing Antimicrobial Resistance

Given the increase in resistance to antibacterial agents, there is an urgent need for the development of new agents with novel modes of action. As an interim solution, it is also prudent to reinvestigate old or abandoned antibacterial compounds to assess their efficacy in the context of widespread resistance to conventional agents. In the 1970s, much work was performed on the development of peptide mimetics, exemplified by the phosphonopeptide, alafosfalin. We investigated the activity of alafosfalin, di-alanyl fosfalin and β-chloro-L-alanyl-β-chloro-L-alanine against 297 bacterial isolates, including carbapenemase-producing Enterobacterales (CPE) (n = 128), methicillin-resistant Staphylococcus aureus (MRSA) (n = 37) and glycopeptide-resistant enterococci (GRE) (n = 43). The interaction of alafosfalin with meropenem was also examined against 20 isolates of CPE. The MIC50 and MIC90 of alafosfalin for CPE were 1 mg/L and 4 mg/L, respectively and alafosfalin acted synergistically when combined with meropenem against 16 of 20 isolates of CPE. Di-alanyl fosfalin showed potent activity against glycopeptide-resistant isolates of Enterococcus faecalis (MIC90; 0.5 mg/L) and Enterococcus faecium (MIC90; 2 mg/L). Alafosfalin was only moderately active against MRSA (MIC90; 8 mg/L), whereas β-chloro-L-alanyl-β-chloro-L-alanine was slightly more active (MIC90; 4 mg/L). This study shows that phosphonopeptides, including alafosfalin, may have a therapeutic role to play in an era of increasing antibacterial resistance

Synthesis and evaluation of novel 7- and 8-aminophenoxazinones for the detection of β-alanine aminopeptidase activity and the rapid identification of Pseudomonas aeruginosa in clinical samples

A series of novel 8-aminophenoxazin-3-one and 7-aminophenoxazin-3-one chromogens and their corresponding β-alanine derivatives were synthesized and evaluated for their ability to detect β-alanyl aminopeptidase activity in bacteria known to hydrolyse β-alanine derivatized substrates. The results provided insight into the structural requirements for effective visualization of enzymatic activity and the mechanism of formation of phenoxazinon-3-ones. 8-Aminophenoxazin-3-one substrates 23c, 23d and 23e were prepared in good to high overall yield and were selective for β-alanyl aminopeptidase activity in bacteria, producing a lighter agar background coloration facilitating visualization of colored colonies, with variable localization to the colonies, but had lower sensitivities for the detection of Pseudomonas aeruginosa in comparison to the analogous 7-aminophenoxazin-3-one substrates. The synthetic methodology employed here allows the preparation of a range of substrates for evaluation and the establishment of structure-activity relationships. For example, the 2-pentyl substituted aminophenoxazin-3-one 22b performed with analogous sensitivity to the corresponding 1-pentyl-7-aminophenoxazin-3-one substrate 1 used commercially, highlighting that the position of the pentyl substituent can be varied while maintaining detection sensitivity

Discovery of sisunatovir (RV521), an inhibitor of respiratory syncytial virus fusion

RV521 is an orally bioavailable inhibitor of respiratory syncytial virus (RSV) fusion that was identified after a lead optimization process based upon hits that originated from a physical property directed hit profiling exercise at Reviral. This exercise encompassed collaborations with a number of contract organizations with collaborative medicinal chemistry and virology during the optimization phase in addition to those utilized as the compound proceeded through preclinical and clinical evaluation. RV521 exhibited a mean IC50 of 1.2 nM against a panel of RSV A and B laboratory strains and clinical isolates with antiviral efficacy in the Balb/C mouse model of RSV infection. Oral bioavailability in preclinical species ranged from 42 to >100% with evidence of highly efficient penetration into lung tissue. In healthy adult human volunteers experimentally infected with RSV, a potent antiviral effect was observed with a significant reduction in viral load and symptoms compared to placebo

Synthesis and testing of chromogenic phenoxazinone substrates for β-alanyl aminopeptidase

Novel 7-N-(β-alanyl)aminophenoxazin-3-one salts 27a-d have been synthesized and tested as chromogenic substrates for β-alanyl aminopeptidase, which is present in Pseudomonas aeruginosa, the most common respiratory pathogen in patients with cystic fibrosis. The biological results show that 7-N-(β-alanyl)amino-1-pentylphenoxazin-3-one trifluoroacetate salt 27a is a chromogenic substrate for this bacterium, with a low degree of diffusion in nutrient media for growing bacterial cultures and a bright red colour, making it easily distinguishable from the agar background

Synthesis and Evaluation of Novel 7- and 8‑Aminophenoxazinones for the Detection of β‑Alanine Aminopeptidase Activity and the Reliable Identification of Pseudomonas aeruginosa in Clinical Samples

A series of novel 8-aminophenoxazin-3-one and 7-aminophenoxazin-3-one chromogens and their corresponding β-alanine derivatives were synthesized and evaluated for their ability to detect β-alanyl aminopeptidase activity in bacteria known to hydrolyze β-alanine derivatized substrates. The results provided insight into the structural requirements for effective visualization of enzymatic activity and the mechanism of formation of phenoxazinon-3-ones. 8-Aminophenoxazin-3-one substrates <b>23c</b>, <b>23d</b>, and <b>23e</b> were prepared in good to high overall yield and were selective for β-alanyl aminopeptidase activity in bacteria, producing a lighter agar background coloration facilitating visualization of colored colonies, with variable localization to the colonies, but had lower sensitivities for the detection of Pseudomonas aeruginosa in comparison to the analogous 7-aminophenoxazin-3-one substrates. The synthetic methodology employed here allows the preparation of a range of substrates for evaluation and the establishment of structure–activity relationships. For example, the 2-pentyl substituted aminophenoxazin-3-one <b>22b</b> performed with analogous sensitivity to the corresponding 1-pentyl-7-aminophenoxazin-3-one substrate <b>1</b> used commercially, highlighting that the position of the pentyl substituent can be varied while maintaining detection sensitivity