An Efficient Buchwald–Hartwig/Reductive Cyclization for the Scaffold Diversification of Halogenated Phenazines: Potent Antibacterial Targeting, Biofilm Eradication, and Prodrug Exploration

Abstract

Bacterial biofilms are surface-attached communities comprised of nonreplicating persister cells housed within a protective extracellular matrix. Biofilms display tolerance toward conventional antibiotics, occur in ∼80% of infections, and lead to >500000 deaths annually. We recently identified halogenated phenazine (HP) analogues which demonstrate biofilm-eradicating activities against priority pathogens; however, the synthesis of phenazines presents limitations. Herein, we report a refined HP synthesis which expedited the identification of improved biofilm-eradicating agents. 1-Methoxyphenazine scaffolds were generated through a Buchwald-Hartwig cross-coupling (70% average yield) and subsequent reductive cyclization (68% average yield), expediting the discovery of potent biofilm-eradicating HPs (e.g., 61: MRSA BAA-1707 MBEC = 4.69 μM). We also developed bacterial-selective prodrugs (reductively activated quinone-alkyloxycarbonyloxymethyl moiety) to afford HP 87, which demonstrated excellent antibacterial and biofilm eradication activities against MRSA BAA-1707 (MIC = 0.15 μM, MBEC = 12.5 μM). Furthermore, active HPs herein exhibit negligible cytotoxic or hemolytic effects, highlighting their potential to target biofilms.

Phenazine Antibiotic‐Inspired Discovery of Bacterial Biofilm‐Eradicating Agents

“…In addition, 2,4‐diBr‐1‐OHPhz demonstrates no hemolysis activity at 200 μ m (highest test concentration), confirming that this HP was not eradicating biofilms through a membrane‐lysis mechanism, similar to QAC compounds . Positive biofilm eradication without hemolytic, or membrane‐lysing, activities provides some initial mechanistic insights and is ideal for the development of new therapeutic 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 …”

“…From this series of HPs, we identified several highly potent biofilm‐eradicating HPs that are substituted at the 7‐position of the HP scaffold, including 7‐phenoxy HP (MIC=0.1 μ m ; MBEC=2.35 μ m against MRSA‐1707) and 7‐chloro HP (MIC=0.1 μ m , MBEC=4.69 μ m against MRSA‐1707; Figure ) . Interestingly, both 9‐substituted HPs synthesized during these investigations were completely inactive against MRSA‐1707 in MIC assays (>1000‐fold less potent than the 7‐substituted HPs, Figure ).…”

“…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 third‐generation, modular HP synthesis was developed utilizing a 1) palladium‐catalyzed Buchwald–Hartwig cross coupling between anilines and 2‐bromo‐3‐nitroanisole, followed by a 2) base‐promoted reductive cyclization to afford 1‐methoxyphenazine analogues (Figure ) . The initial Buchwald–Hartwig step utilized 6 mol % bis(dibenzylideneacetone)palladium(0) and enabled the coupling of 21 anilines with 2‐bromo‐3‐nitroanisole to afford the desired nitroarene intermediates in 21–94 % yield (70 % average yield).…”