A Highly Potent Class of Halogenated Phenazine Antibacterial and Biofilm-Eradicating Agents Accessed Through a Modular Wohl-Aue Synthesis

Abstract

Unlike individual, free-floating planktonic bacteria, biofilms are surface-attached communities of slow- or non-replicating bacteria encased within a protective extracellular polymeric matrix enabling persistent bacterial populations to tolerate high concentrations of antimicrobials. Our current antibacterial arsenal is composed of growth-inhibiting agents that target rapidly-dividing planktonic bacteria but not metabolically dormant biofilm cells. We report the first modular synthesis of a library of 20 halogenated phenazines (HP), utilizing the Wohl-Aue reaction, that targets both planktonic and biofilm cells. New HPs, including 6-substituted analogues, demonstrate potent antibacterial activities against MRSA, MRSE and VRE (MIC = 0.003–0.78 µM). HPs bind metal(II) cations and demonstrate interesting activity profiles when co-treated in a panel of metal(II) cations in MIC assays. HP 1 inhibited RNA and protein biosynthesis while not inhibiting DNA biosynthesis using 3H-radiolabeled precursors in macromolecular synthesis inhibition assays against MRSA. New HPs reported here demonstrate potent eradication activities (MBEC = 0.59–9.38 µM) against MRSA, MRSE and VRE biofilms while showing minimal red blood cell lysis or cytotoxicity against HeLa cells. PEG-carbonate HPs 24 and 25 were found to have potent antibacterial activities with significantly improved water solubility. HP small molecules could have a dramatic impact on persistent, biofilm-associated bacterial infection treatments.

Phenazine Antibiotic‐Inspired Discovery of Bacterial Biofilm‐Eradicating Agents

“…All substitutions of the HP at the 9‐position (R 9 =Me, Et, Br) led to inactive analogues. As previously mentioned, the loss of antibacterial activity regarding 9‐substituted HPs is likely due to steric interactions disrupting a critical metal(II)‐chelation event required for antibacterial activity …”

“…CBD pegs can be removed from their specialized lid, sonicated and plated out to determine the colony forming units (CFU) of viable biofilm cells. From our investigations with methicillin‐resistant S. aureus and Staphylococcus epidermidis (MRSA, MRSE) biofilm‐eradicating agents, we have determined that there is a ≥3‐log reduction in biofilm cells (eradication of ≥99.9 % biofilm cells) at the corresponding MBEC values of active biofilm‐eradicating agents …”

“…A second‐generation synthesis utilized diverse aniline building blocks that were incorporated into the HP scaffold through a Wohl–Aue reaction to probe substituents in the 6–9 positions (Figure ) . The Wohl–Aue reaction is a base‐promoted condensation between an aniline and nitroarene to afford a phenazine .…”

“…Mono‐substitution at the 8‐position (R 8 ; Figure A) of the HP scaffold has led to analogues with both increased and decreased antibacterial activities . Mono‐chlorination or bromination at R 8 results in HPs with increased antibacterial activities; however, methyl and phenyl ether analogues at R 8 resulted in HPs with decreased antibacterial activities compared to the parent HP.…”

“…With extensive SAR information in hand, we employed a series of diverse prodrugs designed to inactivate the critical metal‐chelating moiety of the HP scaffold, to avoid off‐target metal chelation and potential toxicity, until removal under particular conditions to liberate an active HP . Functionalization of the phenolic hydroxy group of the HP scaffold leads to analogues that are unable to bind metal(II) cations; however, several analogues are active antibacterial agents due to the cleavage of prodrug moieties and liberation of active HP agent, which then elicits antibacterial activities.…”