A Modular Synthetic Route Involving N-Aryl-2-nitrosoaniline Intermediates Leads to a New Series of 3-Substituted Halogenated Phenazine Antibacterial Agents

Abstract

Pathogenic bacteria demonstrate incredible abilities to evade conventional antibiotics through the development of resistance and formation of dormant, surface-attached biofilms. Therefore, agents that target and eradicate planktonic and biofilm bacteria are of significant interest. We explored a new series of halogenated phenazines (HP) through the use of N-aryl-2nitrosoaniline synthetic intermediates that enabled functionalization of the 3-position of this scaffold. Several HPs demonstrated potent antibacterial and biofilm-killing activities (e.g., HP 29, against methicillin-resistant Staphylococcus aureus: MIC = 0.075 μM; MBEC = 2.35 μM), and transcriptional analysis revealed that HPs 3, 28, and 29 induce rapid iron starvation in MRSA biofilms. Several HPs demonstrated excellent activities against Mycobacterium tuberculosis (HP 34, MIC = 0.80 μM against CDC1551). This work established new SAR insights, and HP 29 demonstrated efficacy in dorsal wound infection models in mice. Encouraged by these findings, we believe that HPs could lead to significant advances in the treatment of challenging infections.

Pyrazine and Phenazine Heterocycles: Platforms for Total Synthesis and Drug Discovery

“…Our group has discovered a series of synthetically tunable halogenated phenazine antibacterial agents that eradicate surface-attached biofilms with excellent potency [ 24 , 25 , 57 , 58 , 59 , 60 , 61 ]. Biofilms are densely packed bacterial communities that consist of enriched populations of metabolically-dormant (non-replicating) persister cells demonstrating high levels of tolerance to all classes of antibiotics.…”

“…After the identification of HP-1 , we wanted to explore structure–activity relationships (SARs) of the HP scaffold by functionalizing the other positions on the phenazine heterocycle with various substituents ( Scheme 15 ) [ 24 , 58 , 59 , 60 , 61 ]. To do this, we devised modular chemical syntheses that enabled a diversity of functionalization at the 3- and 6-9 positions of the HP scaffold from available aniline starting materials.…”

“…To do this, we devised modular chemical syntheses that enabled a diversity of functionalization at the 3- and 6-9 positions of the HP scaffold from available aniline starting materials. Precursor 1-methoxyphenazines (e.g., 161 , 169 ) were rapidly accessed through a number of efficient synthetic routes, including (1) Buchwald–Hartwig cross-coupling reaction between diverse anilines 158 and 2-bromo-3-nitroanisole 159 , followed by a sodium-borohydride-mediated reductive cyclization to form the phenazine nucleus of 161 ; [ 24 ] and (2) a potassium- tert -butoxide-promoted reaction between aniline starting materials ( 158 ) and 5-chloro-2-nitroanisole 167 to form N -aryl-2-nitrosoaniline intermediates 168 , which were then subjected to N , O -Bis(trimethylsilyl)acetamide (BSA) to form the desired phenazine heterocycle [ 61 ]. From these synthetic efforts, 1-methoxyphenazine products (e.g., 161 and 169 ) were then demethylated using boron tribromide (BBr 3 ), and brominated with two equivalents of N -bromosuccinimide 163 (NBS) to yield target halogenated phenazines 164 and 170 [ 24 , 61 ].…”

“…Overall, this collection of novel halogenated phenazines (e.g., 28 , 155 – 157 ) demonstrate potent antibacterial activities against several Gram-positive pathogens, including Staphylococcus aureus , Staphylococcus epidermidis , and Enterococcus faecalis (antibacterial activities reported as minimum inhibitory concentrations, or MIC values) [ 24 , 25 , 57 , 58 , 59 , 60 , 61 ]. In addition, halogenated phenazines potently eradicate surface-attached bacterial biofilms that are innately tolerant to all classes of conventional antibiotics (biofilm-killing activities reported as minimum biofilm eradication concentrations, or MBEC values).…”

“…HP-14 ( 28 ) is one of the potent analogues we discovered during the course of these studies, and our group used this compound as a probe in transcriptomic profiling experiments to demonstrate that HPs induce rapid iron starvation in MRSA-1707 biofilms [ 25 ]. We have shown that several other HP analogues—such as HP-29 ( 157 )—also induce rapid iron starvation in MRSA biofilms [ 61 ]. In addition, HP-29 has demonstrated good efficacy in treating S. aureus and Enterococcus faecalis wound infections in mice.…”