Scaffold Simplification Yields Potent Antibacterial Agents That Target Bacterial Topoisomerases

Abstract

This work describes the lead optimization of a promising class of antibacterial compounds, derived from a previously reported N-[4-(4-fluorophenoxy)phenyl]-6-(methylsulfonyl)-2,6-diazaspiro [3.4]octane-8-carboxamide (LK1819), through systematic scaffold simplification. A novel series of amide derivatives were designed and synthesized, exploring key structural variations, including the replacement of the diphenyl ether core with a biphenyl system. All compounds were evaluated for in vitro antibacterial activity against the ESKAPE panel of pathogens. The most potent simplified analogs demonstrated exceptional, broad-spectrum activity, with minimum inhibitory concentrations (MICs) that were 10 to 100 times lower than the control antibiotic ciprofloxacin against many strains. Mechanistic studies using a reporter system and enzymatic assays revealed that the compounds do not inhibit protein synthesis but disrupt DNA replication, exhibiting a dose-dependent inhibitory effect on bacterial topoisomerase I and DNA gyrase. The compounds showed moderate toxicity against human cell lines, consistent with their DNA-targeting mechanism, but cytotoxicity assays indicated a sufficient selectivity window. We conclude that scaffold simplification successfully yielded highly potent antibacterial agents with a defined mechanism of action, presenting a promising foundation for further development as antibiotics and potentially as anticancer agents.

Keywords: DNA gyrase; DNA replication inhibitors; ESKAPE pathogens; amino biphenyls; amino diphenyl ethers; antibacterial agents; scaffold simplification; topoisomerase I.

Figure 1

Parent compounds structures of and design of target substances. The scaffold moietys subjected to modification are marked with colored letters. Red (A) is the A ring of the bisaryl fragment, blue (B) is the B ring of the bisaryl fragment, and green (C) is the carboxamide moiety.

Scheme 1

Synthesis of series compounds 1a–g. Reagents and conditions: (i) 1-fluoro-4-nitrobenzene, K₂CO₃/DMF, 130 °C, 18 h; (ii) ammonium formate, 10%Pd/C, EtOH, reflux, 2 h; (iii) N-Boc-proline (or N-Boc-isoproline), HBTU, triethylamine, DMF, rt, 12 h.

Scheme 2

Synthesis of series compounds 2a–i. Reagents and conditions: (i) N-Boc-proline (or N-Boc-isoproline), HBTU, triethylamine, DMF, rt, 12 h; (ii) corresponding arylboronic acid, Cs₂CO₃, [bis (diphenylphosphino)ferrocene] dichloro palladium (II), dioxane–water (10:1), 105 °C, 6 h.

Figure 2

Screening of antibacterial activity mechanism of the test compounds 1e, 2c, and 2b using the pDualrep2 reporter system. Shown is an agar plate containing a lawn of E. coli transformed with the pDualrep2 plasmid and 2 µL spots of compounds tested at a concentration of 20 µg/mL. Erythromycin (5 μg/mL) and ciprofloxacin (2.5 μg/mL) 2 μL spots were used as controls. Red fluorescence is induced by translation-stalling compounds, and green fluorescence is induced by substances that damage DNA. (a) ΔtolC strain; (b) lptD strain; (c) K12 strain.

Figure 3

The influence of the studied substances on the enzymes functioning: (a) Klenow fragment polymerization; (b) DNA gyrase relaxation; (c) topoisomerase I relaxation; and (d) topoisomerase I relaxation with different concentrations of 2c.

Figure 4

MTT test results of the compounds 2b and 2c against six human cell lines.