Pyridylpiperazine efflux pump inhibitor boosts in vivo antibiotic efficacy against K. pneumoniae

Antimicrobial resistance is a global problem, rendering conventional treatments less effective and requiring innovative strategies to combat this growing threat. The tripartite AcrAB-TolC efflux pump is the dominant constitutive system by which Enterobacterales like Escherichia coli and Klebsiella pneumoniae extrude antibiotics. Here, we describe the medicinal chemistry development and drug-like properties of BDM91288, a pyridylpiperazine-based AcrB efflux pump inhibitor. In vitro evaluation of BDM91288 confirmed it to potentiate the activity of a panel of antibiotics against K. pneumoniae as well as revert clinically relevant antibiotic resistance mediated by acrAB-tolC overexpression. Using cryo-EM, BDM91288 binding to the transmembrane region of K. pneumoniae AcrB was confirmed, further validating the mechanism of action of this inhibitor. Finally, proof of concept studies demonstrated that oral administration of BDM91288 significantly potentiated the in vivo efficacy of levofloxacin treatment in a murine model of K. pneumoniae lung infection.


Appendix Table
. PK profile of a single dose BDM91288 (5) (30 mg/kg, orally, formulated in 10% hydroxypropyl-β-cyclodextrin) (see Fig EV3A Appendix supplementary methods: synthesis of PyrPip compounds 1'-13 General Solvents for synthesis, analysis and purification were purchased as analytical grade from commercial suppliers and used directly without further purification.Chemical reagents were purchased from Fisher scientific, Fluorochem, Enamine or Sigma-Aldrich as reagent grade and used without further purification. LC-MS Waters system was equipped with a 2747 sample manager, a 2695 separation module, a 2996 photodiode array detector (200-400 nm) and a Micromass ZQ2000 detector (scan 100-800).XBridge C18 column (50 mm x 4.6 mm, 3.5µm, Waters) was used.The injection volume was 20 µL.A mixture of water and acetonitrile was used as mobile phase in gradient-elution.The pH of the mobile phase was adjusted with HCOOH and NH4OH to form a buffer solution at pH 3.8.The analysis time was 5 min (at a flow rate at 2 mL/min), 10 min (at a flow rate at 1 mL/min) or 30 min (at a flow rate at 1 mL/min).Purity (%) was determined by reversed phase HPLC, using UV detection (215 nm).All final compounds showed purity greater than 95%.
UPLC-MS Waters system was equipped with a UPLC I SMP MGR-FTN sample manager, an ACQUITY UPLC I-Class eK photodiode array detector (210-400 nm) and an ACQUITY QDa (Performance) detector (scan 30-1250).Acquity BEH C18 column (50 mm x 2.1 mm, 1.7 µm, Waters) was used.The injection volume was 0.5 µL.A mixture of water and acetonitrile was used as mobile phase in gradient-elution.The pH of the mobile phase was adjusted with HCOOH and NH4OH to form a buffer solution at pH 3.8.The analysis time was 5 min (at a flow rate at 600 µL/min), 10 min (at a flow rate at 600 µL/min) or 30 min (at a flow rate at 600 µL/min).Purity (%) was determined using UV detection (215 nm), and all isolated compounds showed purity greater than 95%.
HRMS analysis was performed on a LC-MS system equipped with a LCT Premier XE mass spectrometer (Waters), using a XBridge C18 column (50 mm x 4.6 mm, 3.5µm, Waters).A gradient starting from 98% H2O 5 mM Ammonium Formate pH 3.8 and reaching 100% MeCN 5 mM Ammonium Formate pH 3.8 within 3 min at a flow rate of 1 mL/min was used.
NMR spectra were recorded on a Bruker DRX-300 spectrometer.The results were calibrated to signals from the solvent as an internal reference [e.g.2.50 (residual DMSO-d6) and 39.52 (DMSO-d6) ppm for ¹H and ¹³C NMR spectra respectively].Chemical shifts (δ) are in parts per million (ppm) downfield from tetramethylsilane (TMS).The assignments were made using one-dimensional (1D) 1 H and 13 C spectra and two-dimensional (2D) HSQC-DEPT, COSY and HMBC spectra.NMR coupling constants (J) are reported in Hertz (Hz), and splitting patterns are indicated as follows: s for singlet, brs for broad singlet, d for doublet, t for triplet, q for quartet, dd for doublet of doublet, ddd for doublet of doublet of doublet, m for multiplet, δ for chemical shift, J for coupling constant.
HRMS and NMR spectra for compound BDM91288 (5) are provided as Source data file.

Procedure B: Boc-cleavage with HCl
In a round-bottomed flask containing the corresponding Boc-protected compound (0.05-4.02 mmol, 1.0 eq.) in 1,4-dioxane (0.4-16 mL) was added HCl 4M in 1,4-dioxane (10-27 eq.).The mixture was stirred at room temperature during 1-2 days.The solvent was then evaporated under reduced pressure, petroleum ether was added and the mixture was filtered to give the desired compounds.
Procedure C: Introduction of piperazine in position 3 of the quinoline by nucleophilic aromatic substitution The chlorinated derivative (0.70-10.7 mmol, 1.0 eq.), Boc-piperazine (1.5-2.6 eq.) and triethylamine (1.3-2.6 eq.) were dissolved in acetonitrile (2.0-39.0mL) under argon.The mixture was heated at 80 °C for 3 h to 2 days, cooled to room temperature.The reaction was washed with a 1M HCl aqueous solution, extracted twice with EtOAc.The organic layers were combined, washed with a saturated solution of NaCl, dried over MgSO4, evaporated under vacuum and purified by flash chromatography.