Driven from the excellent EPI activity of the 2-phenylquinoline derivatives,1,2 we aimed to improve their safety profile while maintaining inhibition activity. In doing so, we decided to separate EPI optimization for the two different microorganisms. For S. aureus we focused attention on 2-phenylquinoline class while for NTM we designed new 3-phenylquinolone derivatives developed starting from the isoflavone nucleus known to possess EPI activity.

3d rendering of a bacteria under microscope


All compounds were designed by using a traditional medicinal chemistry approach because of the lack of the crystal structures of both S. aureus NorA and M. avium MAV_1406. The synthesized compounds were initially tested for their capability to inhibit the ethidium bromide (EtBr) efflux, a known fluorescent substrate of many efflux pumps. Once excluded antimicrobial effect, active compounds were then assessed for their synergistic activity with different antibiotics against resistant S. aureus and M. avium strains. For the most interesting derivatives, cytotoxicity evaluation was performed on human cells and, for the best NorA inhibitors, preliminary PK properties were evaluated.


In the S. aureus field, seven series of mono- or di-methoxy 2-phenylquinoline derivatives (35 analogues – Figure 1) were synthesized and tested according to the procedure described in the methods. Overall, methoxy introduction led to an excellent increase in NorA inhibition by showing a complete restoration of ciprofloxacin activity against resistant S. aureus strains. In particular, the two best compounds resulted non-toxic at the used concentrations needed to gain antibiotic restoration by exhibiting a selectivity index ≥ 50. Moreover, in order to exclude an unspecific mechanism of efflux pump inhibition, membrane depolarization or disruption was assessed by fluorescent assays. In addition, preliminary PK studies were carried out for both compounds, which showed a good metabolic stability and a poor metabolic inhibition at the concentrations needed for NorA EPI activity.

Different routes to optimize S. aureus and M. avium EPI activities

Driven from the excellent results achieved by methoxy introduction, especially at C-6 position of the 2-phenylquinoline core, we synthesized a small set of derivatives (12 analogues – Figure 1) having different substituents on the oxygen at C-6. By biological data, we noticed that a bulky lipophilic moiety was preferred over polar portions, thereby leaving room for further chemical modifications.

In the M. avium field, given the known EPI activity of the natural compound biochanin A against nontuberculous mycobacteria, we merged its isoflavone nucleus with the 2-phenylquinoline scaffold of our hit compounds, thereby obtaining novel 3-phenylquinolone derivatives (8 analogues – Figure 1).4 All synthesized compounds were evaluated by ethidium bromide efflux assays and synergistic activity in combination with a panel of antibiotics against M. smegmatis and M. avium strains. In particular, three of them showed an excellent increase in EPI activity with respect to the 2-phenylquinoline hits, boosting several fold antibiotic activity. However, the three best compounds suffered from cytotoxicity issues against human cells at the needed concentrations to obtain EPI activity.4 Thus, we undertook a challenging chemical effort aimed to decorate with different substituents the C-6 and C-7 positions of the 3-phenylquinolone core in order to obtain derivatives having a reduced cytotoxicity while maintaining a good M. avium EPI activity. The new derivatives (27 analogues – Figure 1) were initially tested to evaluate their CC50 against human cells and most of them exhibited an increased value fulfilling our aims. Subsequently, some compounds retained the good M. smegmatis EPI activity of their parents both in ethidium bromide and synergistic assays. Interestingly, some derivatives unexpectedly disclosed a potent direct antimycobacterial activity comparable to the common used antibiotic clarithromycin against M. smegmatis. To date, biological tests against M. avium are ongoing.


Standing by for a full characterization of the two methoxy derivatives, necessary prior to move forward animal studies, we are planning some derivatives based on the recent results obtained by lipophilic substituents at C-6 of the quinoline core. However, findings achieved so far, in the S. aureus field, strength the approach to develop EPI to fight AMR. On the other hand, the promising compounds as NTM EPIs pave the way for further studies in this still not very explored field.


1) Sabatini, S.; Gosetto, F.; Manfroni, G.; Tabarrini, O.; Kaatz, G.W.; Patel, D.; Cecchetti, V. Evolution from a natural flavones nucleus to obtain 2-(4-propoxyphenyl)quinoline derivatives as potent inhibitors of the S. aureus NorA efflux pump. J. Med. Chem. 2011, 54, 5722-5736.

2) Sabatini, S.: Gosetto, F.; Iraci, N.; Barreca, M.L.; Massari, S.; Sancineto, L.; Manfroni, G.; Tabarrini, O.; Dimovska, M.; Kaatz, G.W.; Cecchetti, V. Re-evolution of the 2􀇦phenylquinolines: ligand-based design, synthesis, and biological evaluation of a potent new class of Staphylococcus aureus NorA efflux pump inhibitors to combat antimicrobial resistance. J. Med. Chem. 2013, 56, 4975-4989.

3) Machado, D.; Cannalire, C.; Costa, S.S.; Manfroni, G.; Tabarrini, O.; Cecchetti, V.; Couto, I.; Viveiros.; Sabatini, S. Boosting effect of 2􀇦phenylquinoline efflux inhibitors in combination with macrolides against Mycobacterium smegmatis and Mycobacterium avium. ACS Infect. Dis. 2015, 1, 593-603.

4) Cannalire, R.; Machado, D.; Felicetti, T.; Costa, S.S.; Massari, S.; Manfroni, G.; Barreca, M.L.; Tabarrini, O.; Couto, I.; Viveiros, M.; Sabatini, S.; Cecchetti, V. Natural isoflavone biochanin A as a template for the design of new and potent 3-phenylquinolone efflux inhibitors against Mycobacterium avium. 2017, 140, 321-330.

A part of this work has been funded by the Italian FFC – Del. Sondrio Valchiavenna (Grant FFC#17/2017)

Author affiliation

Doctorate School and Department of Pharmaceutical Sciences, University of Perugia


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