From Quinoline to Quinazoline-Based S. aureus NorA Efflux Pump Inhibitors by Coupling a Focused Scaffold Hopping Approach and a Pharmacophore Search

Abstract

Antibiotic resistance breakers, such as efflux pump inhibitors (EPIs), represent a powerful alternative to the development of new antimicrobials. Recently, by using previously described EPIs, we developed pharmacophore models able to identify inhibitors of NorA, the most studied efflux pump of Staphylococcus aureus. Herein we report the pharmacophore-based virtual screening of a library of new potential NorA EPIs generated by an in-silico scaffold hopping approach of the quinoline core. After chemical synthesis and biological evaluation of the best virtual hits, we found the quinazoline core as the best performing scaffold. Accordingly, we designed and synthesized a series of functionalized 2-arylquinazolines, which were further evaluated as NorA EPIs. Four of them exhibited a strong synergism with ciprofloxacin and a good inhibition of ethidium bromide efflux on resistant S. aureus strains coupled with low cytotoxicity against human cell lines, thus highlighting a promising safety profile.

Keywords: NorA efflux pump inhibitors; Staphylococcus aureus; antibiotics; medicinal chemistry; quinazoline derivatives.

Figure 1

Chemical structures of previously reported quinoline NorA EPIs 1–5.

Figure 2

Overview of the developed KNIME workflow for the collection of 6,6 bicyclic cores (protocol details in the Experimental Section). In the figure is represented as example the fragmentation process for the approved drug Trimetrexate (DrugBank ID: DB01157).

Figure 3

(A) Schematic overview of applied in‐silico workflow (B). Fitting of compounds 1, 8 a and 10 a on ModB and ModC pharmacophoric models. Red sphere and vectors, H‐bond acceptor; blue sphere, positive charge; orange ring, aromatic ring; green sphere, hydrophobic moiety.

Scheme 1

Reagents and condition: i) Et₃N, dry THF, acyl chloride, rt – 60 °C, 3 h, 36–71 % or (for 18) DMAP, dry dioxane, 4‐propoxy benzoyl chloride, 100 °C, MW, 10 min, 43 %; ii) NaOH, dry dioxane, 110 °C, MW, 10 min, 40 % or tBuOK, tBuOH, rt – 90 °C, 90 min – 3 h, 75–90 %; iii) K₂CO₃, chloroalkylamines, dry DMF, 80–90 °C, 1–5 h, 11–84 % or (for 8 c and 8 d) 90–100 °C, MW, 10–15 min, 14–84 %; iv) POCl₃, 100 °C, 3 h, 80 %; v) 60 % NaH, 1‐(2‐hydroxyethyl)piperidine, dry DMF, rt, 3 h, 41 %; vi) 10 % Pd/C, ammonium formate, MeOH, reflux, 2 h, 14 %.

Scheme 2

Reagents and condition: i) a) SOCl₂, reflux, 1 h; b) aq. 33 % NH₃, CH₃CN, rt, 1 h, 95 %; ii) CuBr, 1‐(4‐propoxyphenyl)ethan‐1‐one, Cs₂CO₃, dry DMSO, 110 °C, 8 h, 30 %; iii) POCl₃, reflux, 3 h; iv) 2‐(diethylamino)ethan‐1‐ol or 1‐piperidinethaanol, NaH, dry THF, reflux, 1–3 h, 50–53 %.

Scheme 3

Reagents and condition: i) 4‐propoxyphenylboronic acid, Pd(PPh₃)₄, DME, aq. 2 M Na₂CO₃, MW, 100 °C, 15 min, 72 %; ii) 2‐(diethylamino)ethan‐1‐ol or 1‐piperidinethanol, NaH, dry THF, reflux, 1 h, 50–55 %.

Figure 4

Checkerboard assays: A) CPX MIC reduction against SA‐1199B in the presence of increasing (from 0.19 to 6.25 μg/mL) concentrations of derivatives 8 a–c, 10 a, 12 b, 12 c, 13 a and 13 b and reference compound 1. B) CPX MIC reduction against SA‐K2378 in the presence of increasing (from 0.19 to 12.5 μg/mL) concentrations of derivatives 8 b, 8 c, 12 c and 13 a. Assays performed in duplicate.

Figure 5

Time‐kill curves of CPX at $1/4$ x, $1/2$ x and 1 x MIC alone or in combination with compound 8 c (A) and 12 c (B) at 0.78 μg/mL against S. aureus strain SA‐1199B. Assays were performed testing at each time points 4 dilutions in duplicate.