C-2 phenyl replacements to obtain potent quinoline-based Staphylococcus aureus NorA inhibitors

Article abstract of DOI:10.1080/14756366.2020.1719083

NorA is the most studied efflux pump of Staphylococcus aureus and is responsible for high level resistance towards fluoroquinolone drugs. Although along the years many NorA efflux pump inhibitors (EPIs) have been reported, poor information is available ab

Full text of DOI:10.1080/14756366.2020.1719083

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
C-2 phenyl replacements to obtain potent
quinoline-based Staphylococcus aureus NorA
inhibitors
Tommaso Felicetti, Gianmarco Mangiaterra, Rolando Cannalire, Nicholas
Cedraro, Donatella Pietrella, Andrea Astolfi, Serena Massari, Oriana
Tabarrini, Giuseppe Manfroni, Maria Letizia Barreca, Violetta Cecchetti,
Francesca Biavasco & Stefano Sabatini
To cite this article: Tommaso Felicetti, Gianmarco Mangiaterra, Rolando Cannalire, Nicholas
Cedraro, Donatella Pietrella, Andrea Astolfi, Serena Massari, Oriana Tabarrini, Giuseppe
Manfroni, Maria Letizia Barreca, Violetta Cecchetti, Francesca Biavasco & Stefano Sabatini
(2020) C-2 phenyl replacements to obtain potent quinoline-based Staphylococcusꢀaureus
NorA inhibitors, Journal of Enzyme Inhibition and Medicinal Chemistry, 35:1, 584-597, DOI:
10.1080/14756366.2020.1719083
To link to this article: https://doi.org/10.1080/14756366.2020.1719083
© 2020 The Author(s). Published by Informa
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2020, VOL. 35, NO. 1, 584–597
https://doi.org/10.1080/14756366.2020.1719083
SHORT COMMUNICATION
C-2 phenyl replacements to obtain potent quinoline-based Staphylococcus aureus
NorA inhibitors
a
b
a
ꢀ
ꢀ
Tommaso Felicetti , Gianmarco Mangiaterra , Rolando Cannalire
, Nicholas Cedrarob, Donatella Pietrellac
,
Andrea Astolfia, Serena Massaria , Oriana Tabarrinia , Giuseppe Manfronia , Maria Letizia Barrecaa
,
Violetta Cecchettia , Francesca Biavascob
and Stefano Sabatinia
a
ꢀ
Department of Pharmaceutical Sciences, Chemistry and Technology of the Drug Section, Universita degli Studi di Perugia, Perugia, Italy;
b
c
ꢀ
Department of Life and Environmental Sciences, Universita Politecnica delle Marche, Ancona, Italy; Department of Pharmaceutical Sciences,
ꢀ
Biochemical Sciences and Health Section, Universita degli Studi di Perugia, Perugia, Italy
ABSTRACT
ARTICLE HISTORY
Received 5 December 2019
Revised 10 January 2020
Accepted 12 January 2020
NorA is the most studied efflux pump of Staphylococcus aureus and is responsible for high level resistance
towards fluoroquinolone drugs. Although along the years many NorA efflux pump inhibitors (EPIs) have
been reported, poor information is available about structure-activity relationship (SAR) around their nuclei
and reliability of data supported by robust assays proving NorA inhibition. In this regard, we focussed
efforts on the 2-phenylquinoline as a promising chemotype to develop potent NorA EPIs. Herein, we
report SAR studies about the introduction of different aryl moieties on the quinoline C-2 position. The
new derivative 37a showed an improved EPI activity (16-fold) with respect to the starting hit 1. Moreover,
compound 37a exhibited a high potential in time-kill curves when combined with ciprofloxacin against
SA-1199B (norAþ). Also, 37a exhibited poor non-specific effect on bacterial membrane polarisation and
showed an improvement in terms of “selectivity index” in comparison to 1.
KEYWORDS
Antimicrobial resistance
breakers; efflux pump
inhibitors; NorA;
Staphylococcus aureus;
antimicrobial resistance
Introduction
non-specific role in the early stages of antibiotic exposure, thereby
allowing microorganisms to develop more specific and effective
mechanisms of resistance4,8,9. Therefore, the use of efflux pump
inhibitors (EPIs) in combination with extruded drugs may be a
major strategy in the development of effective antimicrobial treat-
ments. To date, little has been done in terms of EPI development
and no inhibitors have ever reached the clinical use10.
Among the six multi-drug resistant bacterial species termed
ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus,
Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas
aeruginosa and Enterobacter species), S. aureus and its methicillin-
resistant strain (MRSA) represent a serious problem worldwide,
due to its acquired resistance to several classes of antibiotics,
encoded by the SCC-mec cassettes11. Moreover, the trans-
membrane protein NorA, belonging to the Major Facilitator
Superfamily, is commonly overexpressed in S. aureus resistant
strains and strongly upregulated in response to fluoroquinolones
treatment. NorA can extrude different toxic compounds, including
the fluoroquinolone ciprofloxacin (CPX) and the dye ethidium
bromide (EtBr), by an antiporter mechanism exploiting the proton
motive force12.
Antimicrobial resistance (AMR) is a complex threat for human
health and represents a hot topic in drug discovery1. The use of
large amounts of antibiotics to control human and animal infec-
tions and in animal breeding has created unprecedented condi-
tions for the rising and spread of antibiotic resistance among
bacterial populations. The “right drug for the right bug” approach
remains a distant perspective, currently replaced by an empiric-
ally-guided consumption. Recently, the World Health Organisation
(WHO) has included AMR in the “ten threats to global health in
2019”, forecasting an imminent return to a time when we were
unable to easily treat common infections2. Considering the micro-
bial promptness in achieving successful machinery escaping anti-
biotic activity also towards new drugs3,4, the use of non-antibiotic
adjuvant molecules targeting resistance mechanisms, in co-admin-
istration with antibacterials, is a valid approach to recover drug
sensitivity in resistant strains5,6. The fascinating idea to “freeze”
resistance would allow antibiotics, for which resistance occurred,
to recover their activity thereby renewing our armamentarium to
fight microbial infections. Amongst the wide range of resistance
mechanisms developed by bacteria, one of the most common is
the drug extrusion from the cell, which can reduce intracellular
drugs to sub-inhibitory concentrations allowing bacteria to grow
in the presence of routinely adopted therapeutic doses7. Indeed,
for some drugs, microorganisms can only acquire resistance in the
Along the years many NorA EPIs have been discovered by
three different approaches: i) screening libraries of natural or syn-
thetic molecules; ii) repurposing molecules with known biological
activity and iii) designing and synthesising new com-
presence of efflux pump activity. Most likely, efflux pumps play a pounds10,13–16
. The lack of NorA structural information has
ꢀ
Department of Pharmaceutical Sciences, Universita degli Studi di Perugia, Via del Liceo 1,
CONTACT Stefano Sabatini
Perugia, 06123, Italy; Francesca Biavasco
stefano.sabatini@unipg.it
ꢀ
Department of Life and Environmental Sciences, Universita Politecnica delle Marche, via Brecce
f.biavasco@univpm.it
Bianche, Ancona, 60131, Italy
ꢀ
T.F. and G.M. contributed equally to the work.
ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
585
4
6
R4
Et
1
Starting hit R = a; R = -H
Best NorA EPI 2 R4 = b; R6 = -OMe
N
a =
Et
R4 = alkylamino chain needed
to retain NorA EPI acitivy
N
b =
C-6 represents the
best position to
introduce -OMe group
OPr
Shift of aryl at C-3 position
> NTM EPI activity
< NorA EPI activity
OR4
N
N
N
R6
R6 NorA EPI activity
-OMe > -OBz > -H = -OH >> -O-alkylamino chains
N
N
present work
S
OPr
N
OPr
S
S
N-alkylation reduces NorA EPI activity
Cl
N
OPr
Figure 1. Known SAR around the 2-phenylquinoline scaffold and new designed compounds.
strongly hampered the identification of potent NorA EPIs. a methoxy group on the C-6 position of the 2-phenylquinoline
core strongly improved NorA EPI activity while lowering host cell
toxicity19; iii) the replacement of the methoxy group at C-6 pos-
ition with a benzyloxy moiety retained NorA inhibition activity
(although increasing human cell toxicity), while a free hydroxy
group or different O-alkylamino chains at C-6 yielded less potent
analogues20; iv) the shift of the aryl portion from C-2 to C-3 pos-
ition of the quinoline core increased EPI activity towards nontu-
berculous mycobacteria (NTM) while decreasing that against S.
No examples of structure-based drug design have been so far
reported for EPI identification, which, therefore, relies on ligand-
based drug design approaches or classical medicinal chemistry
strategies. In addition, the risk for a strategy aimed at identifying
an EPI relies on the poor availability of quick and smart biological
screenings able to early identify active molecules. On the contrary,
due to the poor knowledge of the NorA efflux mechanisms and
the lack of biophysical experiments validating a true NorA inhib-
ition, too frequently molecules have been described in literature
as NorA EPIs when they are not. The ability of a compound to act
in synergy with CPX against S. aureus resistant strains seems not
to be sufficient to consider that molecule as NorA EPI. Additional
experiments are needed to rule out non-specific effects of com-
pounds boosting antibiotic activity. Since many efflux pumps
work through the proton motive force, its disruption by depolaris-
ing the bacterial membrane is the most common example of a
non-specific effect still resulting in efflux pump inhibition.
However, selective membrane depolarisation in microorganisms
appears challenging and often compounds result very toxic by
eliciting the same effect on eukaryotic cells. Conversely, an actual
NorA EPI must exert a strong synergistic effect with CPX (reducing
its MIC at least 4-fold) against overexpressing norA S. aureus
strains while resulting poorly active or ineffective against both
wild-type and norA knock-out strains. In addition, NorA efflux
inhibition activity should be demonstrated by phenotypic assays
(i.e. EtBr efflux assays) in norA overexpressing S. aureus strains and
non-specific EPI effect needs to be excluded by performing bac-
terial membrane polarisation experiments. Moreover, the identified
EPIs should not possess any intrinsic antibacterial effect at the
concentrations needed to reach synergism with antibiotics in
order to prevent a potential interference throughout the syner-
gism with antibacterials.
aureus (Figure 1)21,22
.
In this work, we focus efforts on the exploration of the C-2
position by replacing the 4’-proproxyphenyl substituent with dif-
ferently substituted pyridine or thiophene moieties in order to
identify potential isosteric replacements (Figure 1). Indeed, previ-
ous SAR studies did not cover such a portion on the quinoline
scaffold. Although more potent 2-phenylquinoline-based NorA
EPIs have been identified by us, we selected the previously
reported compound 1 as starting hit23, because the lack of sub-
stituents on the quinoline benzene ring allows for a better com-
parison with the new C-2 modified analogues (Figure 1).
Considering the role of O-alkylamino chains on the quinoline C-4
position to retain NorA EPI activity, we introduced two different
chains (a, O-ethyl-N,N-diethylamino and b, O-ethylpiperidine) for
each new C-2 aryl quinoline scaffold. In particular, chain “a” was
selected because present in the starting hit 1 and chain “b” since
it resulted the best chain in the last work, where the most potent
methoxy 2-phenylquinoline derivative
2
was identified
(Figure 1)19.
Materials and methods
Chemistry
All starting materials, reagents and solvents were purchased from
common commercial suppliers and were used as such, unless
otherwise indicated. Organic solutions were dried over anhydrous
Na2SO4 and concentrated with a rotary evaporator at low pres-
sure. All reactions were routinely checked by thin-layer chroma-
tography (TLC) on silica gel 60F254 (Merck) and visualised by using
UV or iodine. Flash chromatography separations were carried out
on Merck silica gel 60 (mesh 230–400). Melting points were deter-
In this direction, we are currently working on 2-phenylquino-
line nucleus giving rise to a really promising class of NorA EPIs.
2-Phenylquinoline derivatives have shown an excellent NorA EPI
activity, widely restoring CPX antibacterial activity against resistant
S. aureus strains. Efforts aimed at delineating a robust structure-
activity relationship (SAR) investigation around this scaffold high-
lighted some significant findings: i) an alkylamino chain on the
oxygen at quinoline C-4 position is needed to retain NorA EPI
mined in capillary tubes (Stuart SMP30) and are uncorrected.
1
activity and preferred over N-1 position17,18; ii) the introduction of Yields were of purified products and were not optimised. H NMR

586
T. FELICETTI ET AL.
spectra were recorded at 200 or 400 MHz (Bruker Avance DRX-200 brine, dried over Na2SO4 and evaporated under vacuum to a solid
or 400, respectively) while 13C NMR spectra were recorded at that was purified by flash column chromatography.
101 MHz (Bruker Avance DRX-400). Chemical shifts are given in
ppm (d) relative to TMS. Spectra were acquired at 298 K. Data
N-(2-acetylphenyl)pyridine-2-carboxamide (16)
processing was performed with standard Bruker software
XwinNMR and the spectral data are consistent with the assigned
structures. The purity of the tested compounds was evaluated by
combustion analysis using a Fisons elemental analyser, model
EA1108CHN, and data for C, H, and N are within 0.4% of the the-
oretical values (ꢁ95% sample purity).
General procedure A: time, 90 min; used chloride, pyridine-2-car-
bonyl chloride 7 (0.57 g, 4.06 mmol); purification, (EP/EtOAc 60/40).
Derivative 1624 was obtained as a grey solid (0.71 g, 73% yield),
1
mp 107.5–108.0 ꢂC. H NMR (400 MHz, CDCl3): d ¼ 2.69 (s, 3H, CH3),
7.27 (t, J ¼ 7.3 Hz, 1H, H-4’), 7.67–7.71 (m, 2H, H-5’ and H-6’),
8.06–8.20 (m, 3H, H-3, H-5 and H-3’), 8.76–8.87 (m, 2H, H-4 and H-
6), 13.31 ppm (s, 1H, NH).
N-(2-acetylphenyl)-5-propoxypyridine-2-carboxamide (14)
SOCl2 (1.2 mL, 16.44 mmol) was slowly added at 0 ꢂC to 5-propoxy-
pyridine-2-carboxylic acid 3 (0.39 g, 2.20 mmol), then the reaction
mixture was stirred at 60 ꢂC for 30 min. The excess of SOCl2 was
removed under reduced pressure to give 5-propoxypyridine-2-car-
bonyl chloride (5) as a yellow oil that immediately was dissolved
in dry THF (6 mL) and added to a mixture of aminoacetophenone
13 (0.29 g, 2.20 mmol) and Et3N (0.9 mL, 6.60 mmol) in dry THF
(14 mL). The reaction was stirred at rt for 2 h, then it was poured
N-(2-acetylphenyl)nicotinamide (17)
General procedure A: time, 72 h; used chloride, nicotinoyl chloride
8 (0.57 g, 4.06 mmol); purification, (EP/EtOAc 60/40). Derivative
1724 was obtained as a white solid (0.96 g, 99% yield), mp
114.0–115.0 ꢂC. 1H NMR (200 MHz, CDCl3): d ¼ 2.69 (s, 3H, CH3),
7.12 (m, 1H, H-4’), 7.37–7.42 (m, 1H, H-4), 7.61 (dt, J ¼ 1.5 and
8.7 Hz, 1H, H-5’), 7.95 (dd, J ¼ 1.4 and 8.3 Hz, 1H, H-3’), 8.27–8.33
in ice/water and the pH was adjusted to ’8 with 2 N HCl. The (m, 1H, H-5), 8.75 (dd, J ¼ 1.8 and 8.5 Hz, 1H, H-6’), 8.90 (d,
mixture was extracted by EtOAc and the organic layers were J ¼ 8.4 Hz, 1H, H-6), 9.27 (d, J ¼ 1.9 Hz, 1H, H-2), 12.73 ppm (s,
washed by brine, dried over Na2SO4 and evaporated to dryness.
After purification by flash chromatography column (CH2Cl2/acet-
one 98/2), derivative 14 was obtained as a yellow solid (0.41 g,
63% yield), m.p. 132.0–134.0 ꢂC. 1H NMR (200 MHz, DMSO-d6):
d ¼ 1.02 (t, J ¼ 7.5 Hz, 3H, OCH2CH2CH3), 1.73–1.87 (m, 2H,
OCH2CH2CH3), 2.59 (s, 3H, CH3), 4.00 (t, J ¼ 6.6 Hz, 2H,
OCH2CH2CH3), 7.11 (t, J ¼ 7.6 Hz, 1H, H-4’), 7.23–7.28 (m, 1H, H-4),
7.56 (t, J ¼ 8.4 Hz, 1H, H-5’), 7.90 (d, J ¼ 7.9 Hz, 1H, H-6’), 8.15 (d,
J ¼ 8.5 Hz, 1H, H-3’), 8.39 (d, J ¼ 2.1 Hz, 1H, H-6), 8.96 (d, J ¼ 8.7 Hz,
1H, H-3), 13.37 ppm (s, 1H, NH).
1H, NH).
N-(2-acetylphenyl)isonicotinamide (18)
General procedure A: time, 20 h; used chloride, isonicotinoyl chlor-
ide
9
(3.45 g, 24.40 mmol); purification, (EP/EtOAc 50/50).
Derivative 1824 was obtained as a white solid (3.52 g, 60% yield),
1
mp 111.0–112.0 ꢂC. H NMR (200 MHz, CDCl3): d ¼ 2.66 (s, 3H, CH3),
7.16 (dt, J ¼ 1.2 and 7.9 Hz, 1H, H-4’), 7.61 (dt, J ¼ 1.6 and 7.4 Hz,
1H, H-5’), 7.83–7.86 (m, 2H, H-3 and H-5), 7.94 (dd, J ¼ 1.5 and
8.0 Hz, 1H, H-6’), 8.73–8.80 (m, 2H, H-2 and H-6), 8.89 (dd,J ¼ 1.1
and 8.5 Hz, 1H, H-3’), 12.85 ppm (s, 1H, NH).
N-(2-acetylphenyl)-6-propoxynicotinamide (15)
SOCl2 (0.6 mL, 8.22 mmol) was slowly added at 0 ꢂC to 6-propoxy-
nicotinic acid 4 (0.20 g, 1.10 mmol), then the reaction mixture was
stirred at 60 ꢂC for 30 min. The excess of SOCl2 was removed
under reduced pressure to give 6-propoxynicotinoyl chloride (6)
as a yellow oil that immediately was dissolved in dry THF (4 mL)
N-(2-acetylphenyl)thiophene-2-carboxamide (19)
General procedure A: time, 20 h; used chloride, thiophene-2-car-
bonyl chloride 10 (0.34 g, 2.34 mmol); purification, (EP/EtOAc
50/50). Derivative 1924 was obtained as a yellow solid (0.33 g, 58%
1
and added to a mixture of aminoacetophenone 13 (0.15 g, yield), mp129.0–131.0 ꢂC. H NMR (200 MHz, CDCl3): d ¼ 2.68 (s, 3H,
1.10 mmol) and Et3N (0.7 mL, 5.50 mmol) in dry THF (10 mL). The CH3), 7.04–7.15 (m, 2H, H-4 and H-4’), 7.51–7.61 (m, 2H, H-3 and
reaction was stirred at rt for 2 h, then it was poured in ice/water
H-5’), 7.79 (dd, J ¼ 1.1 and 5.0 Hz, 1H, H-5), 7.91 (dd, J ¼ 1.6 and
and the pH was adjusted to ’8 with 2 N HCl. The mixture was
extracted by EtOAc and the organic layers were washed by brine,
dried over Na2SO4 and evaporated to dryness. After purification
by flash chromatography column (CH2Cl2/acetone 98/2), derivative
15 was obtained as a brown oil (0.18 g, 56% yield). 1H NMR
(200 MHz, DMSO-d6): d ¼ 0.81 (t, J ¼ 7.3 Hz, 3H, OCH2CH2CH3),
1.57–1.72 (m, 2H, OCH2CH2CH3), 2.60 (s, 3H, CH3), 3.89 (t,
J ¼ 7.3 Hz, 2H, OCH2CH2CH3), 6.48 (d, J ¼ 8.7 Hz, 1H, H-6’), 7.18 (t,
J ¼ 7.5 Hz, 1H, H-4’), 7.59 (t, J ¼ 7.7 Hz, 1H, H-5’), 7.78–7.84 (m, 1H,
H-5), 8.00 (d, J ¼ 8.0 Hz, 1H, H-3’), 8.38–8.41 (m, 2H, H-2 and H-4),
11.91 ppm (s, 1H, NH).
8.0, 1H, H-6’), 8.83 (dd, J ¼ 1.2 and 8.5 Hz, 1H, H-3’), 12.70 ppm (s,
1H, NH).
N-(2-acetylphenyl)thiophene-3-carboxamide (20)
General procedure A: time, 12 h; used chloride, thiophene-3-car-
bonyl chloride 11 (0.41 g, 2.80 mmol); purification (EP/EtOAc
50/50). Derivative 2024 was obtained as a white solid (0.34 g, 50%
yield), mp 83.0–84.0 ꢂC. 1H NMR (200 MHz, CDCl3): d ¼ 2.67 (s, 3H,
CH3), 7.10 (dt, J ¼ 1.1 and 7.2 Hz, 1H, H-4’), 7.35 (dd, J ¼ 3.0 and
5.5 Hz, 1H, H-4), 7.57 (dt, J ¼ 1.6 and 8.6 Hz, 1H, H-5’), 7.63 (dd,
J ¼ 1.1 and 5.3 Hz, 1H, H-2), 7.91 (dd, J ¼ 1.6 and 8.0 Hz, 1H, H-6’),
8.11 (dd, J ¼ 1.1 and 3.0 Hz, 1H, H-5), 8.88 (dd, J ¼ 1.1 and 8.4 Hz,
1H, H-3’), 12.57 (s, 1H, NH).
General procedure A for the synthesis of compounds 16–21
A solution of acyl chlorides 7–12 (1 equiv.) in dry THF (4 ml per
mmol) was added to a solution of aminoacetophenone 13 (1
equiv) and Et3N (5 equiv.) in dry THF (5 ml per mmol of 13). The
reaction was stirred at rt for 90 min–72 h, then it was poured in
N-(2-acetylphenyl)-5-chlorothiophene-2-carboxamide (21)
General procedure A: time, 12 h; used chloride, 5-chlorothiophene-
ice/water. The pH was adjusted up to ’8 with 2 N HCl, the mix- 2-carbonyl chloride 12 (0.56 g, 3.08 mmol); purification, (EP/EtOAc
ture was extracted by EtOAc and the organic layer was washed by 50/50). Derivative 21 was obtained as a white solid (0.73 g, 85%

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
587
yield), mp 136.0–137.0 ꢂC. 1H NMR (200 MHz, CDCl3): d ¼ 2.67 (s, obtained as a white solid (0.53 g, 57% yield), mp > 300 ꢂC. 1H
1H, CH3), 6.93 (d, J ¼ 6.1 Hz, 1H, H-3), 7.11 (t, J ¼ 7.6 Hz, 1H, H-4’), NMR (400 MHz, DMSO-d6): d ¼ 6.64 (s, 1H, H-3), 7.35 (t, J ¼ 7.3 Hz,
1H, H-6), 7.68 (dt, J ¼ 1.4 and 8.1 Hz, 1H, H-7), 7.76 (d, J ¼ 8.2 Hz,
1H, H-8), 7.85 (d, J ¼ 6.2 Hz, 2H, H-3’ and H-5’), 8.08 (dd, J ¼ 1.3
and 8.1 Hz, 1H, H-5), 8.76 (d, J ¼ 5.1 Hz, 2H, H-2’ and H-6’),
11.94 ppm (s, 1H, OH).
7.52–7.62 (m, 2H, H-4 and H-5’), 7.91 (d, J ¼ 8.0 Hz, 1H, H-6’), 8.77
(d, J ¼ 8.4 Hz, H-3’), 12.66 ppm (s, 1H, NH).
General procedure B for the synthesis of compounds 22–29
Under N2 atmosphere in a pressure tube, NaOH powder (3 equiv)
was added to a solution of derivatives 14–21 (1 equiv) in dry
dioxane (2 ml per mmol). The reaction mixture was stirred at
110 ꢂC for 2–8 h, then it was poured in ice/water. The pH was
acidified up to 6 with 2 N HCl and the obtained precipitate was fil-
tered to give the desired compounds as solids. After crystallization
by Et2O/EtOH, compounds were used for the next reactions.
2–(2-Thienyl)quinolin-4-ol (27)
General procedure B: time, 8 h; starting material, N-(2-acetylphenyl)th-
iophene-2-carboxamide 1927 (0.20 g, 0.82 mmol). Compound 2724
was obtained as a pink solid (0.11 g, 62% yield), mp > 300 ꢂC. 1H
NMR (400 MHz, DMSO-d6): d ¼ 6.43 (s, 1H, H-3), 7.23–7.25 (m, 1H, H-
5’), 7.30 (t, J¼ 7.5 Hz, 1H, H-6), 7.63 (t, J¼ 7.1 Hz, 1H, H-7), 7.77–7.91
(m, 3H, H-8, H-3’ and H-4’), 8.03 (d, J¼ 8.0 Hz, 1H, H-5), 11.74 ppm (s,
1H, OH).
2–(5-Propoxypyridin-2-yl)quinolin-4-ol (22)
General procedure B: time, 6 h; starting material, N-(2-acetyl-
phenyl)-5-propoxypyridine-2-carboxamide 14 (0.23 g, 0.73 mmol).
Compound 22 was obtained as a white solid (0.20 g, 100% yield),
mp 190.0–191.0 ꢂC. 1H NMR (400 MHz, DMSO-d6): d ¼ 1.95 (t,
J ¼ 7.4 Hz, 3H, OCH2CH2CH3), 3.42–3.60 (m, 2H, OCH2CH2CH3), 4.09
(t, J ¼ 6.6 Hz, 2H, OCH2CH2CH3), 6.80 (s, 1H, H-3), 7.29 (t, J ¼ 7.4 Hz,
1H, H-6), 7.56–7.64 (m, 2H, H-7 and H4’), 8.00 (d, J ¼ 8.4 Hz, 1H, H-
3’), 8.05 (d, J ¼ 7.4 Hz, 1H, H-8), 8.20 (d, J ¼ 8.8 Hz, 1H, H-5), 8.45 (d,
J ¼ 2.7 Hz, 1H, H-6’), 11.81 ppm (s, 1H, OH).
2–(3-Thienyl)quinolin-4-ol (28)
General procedure B: time, 8 h; starting material, N-(2-acetylphe-
nyl)thiophene-3-carboxamide 20 (0.20 g, 0.82 mmol). Compound
2824 was obtained as a white solid (0.13 g, 72% yield), mp >
300 ꢂC. 1H NMR (400 MHz, DMSO-d6): d ¼ 6.47 (s, 1H, H-3), 7.30 (t,
J ¼ 7.1 Hz, 1H, H-6), 7.63 (dd, J ¼ 1.2 and 7.0 Hz, 1H, H-7), 7.68–7.77
(m, 3H, H-8, H-4’ and H-5’), 8.01 (d, J ¼ 8.1 Hz, 1H, H-5), 8.24–8.31
(m, 1H, H-4’), 11.64 ppm (s, 1H, OH).
2–(5-Chloro-2-thienyl)quinolin-4-ol (29)
2–(6-Propoxypyridin-3-yl)quinolin-4-ol (23)
General procedure B: time, 8 h; starting material, N-(2-acetyl-
phenyl)-5-chlorothiophene-2-carboxamide 21 (0.50 g, 1.79 mmol).
Compound 29 was obtained as a yellow solid (0.32 g, 73% yield),
mp > 300 ꢂC. 1H NMR (200 MHz, DMSO-d6): d ¼ 6.74 (s, 1H, H-3),
7.28 (d, J ¼ 4.1 Hz, 1H, H-3’), 7.40 (t, J ¼ 7.1 Hz, 1H, H-6), 7.70 (dt,
J ¼ 2.0 and 7.1 Hz, 1H, H-7), 7.81–7.92 (m, 2H, H-8 and H-4’), 8.06
(d, J ¼ 8.2 Hz, 1H, H-5), 11.64 ppm (s, 1H, OH).
General procedure B: time, 6 h; starting material, N-(2-acetyl-
phenyl)-6-propoxynicotinamide 15 (0.75 g, 2.51 mmol). Compound
23 was obtained as a white solid (0.40 g, 57% yield), mp
212.5–215.0 ꢂC.1H NMR (400 MHz, DMSO-d6): d ¼ 0.86 (t, J ¼ 7.5 Hz,
3H, OCH2CH2CH3), 1.65–1.74 (m, 2H, OCH2CH2CH3), 3.90
(t, J ¼ 7.4 Hz, 2H, OCH2CH2CH3), 6.28 (s, 1H, H-3), 6.53 (d, J ¼ 8.8 Hz,
1H, H-5’), 7.27–7.30 (m, 1H, H-6), 7.60–7.66 (m, 2H, H-7 and H-8),
7.87 (dd, J ¼ 1.5 and 9.4 Hz, 1H, H-4’), 8.03 (d, J ¼ 7.9 Hz, 1H, H-5),
8.38 (d, J ¼ 2.5 Hz, 1H, H-2’), 11.45 ppm (s, 1H, OH).
General procedure C for the synthesis of compounds 30a,b–37a,b
To a solution of derivatives 22–29 (1 equiv) in dry DMF (10 per
mmol), K2CO3 (4 equiv) and 2-chloro-N,N-dimethylethylamine
hydrochloride or 1–(2-chloroethyl)piperidine hydrochloride (3
equiv) were added. The reaction mixture was stirred at 80 ꢂC for
2–12 h, then it was poured in ice/water and extracted with EtOAc.
The organic layers were washed with brine, dried over Na2SO4
and evaporated under vacuum to give crude oils. After purifica-
tion by flash chromatography column, compounds (30b, 32b,
33b, 34a, 35a, 36a, 36b and 37b) were obtained as solids.
Differently, to a solution of the compounds (30a, 31a, 31b, 32a,
33a, 34b, 35b and 37a) in Et2O, HClgas was bubbled and, after fil-
tration, compounds were collected as hydrochloride solids.
2-Pyridin-2-ylquinolin-4-ol (24)
General procedure B: time, 2 h; starting material, N-(2-acetylphe-
nyl)pyridine-2-carboxamide 16 (0.50 g, 2.08 mmol). Compound
2424 was obtained as a white solid (0.28 g, 61% yield), mp
236.5–237.0 ꢂC. 1H NMR (200 MHz, DMSO-d6): d ¼ 6.83 (s, 1H, H-3),
7.27 (t, J ¼ 6.8 Hz, 1H, H-6), 7.51–7.65 (m, 2H, H-7 and H-5’),
7.90–8.05 (m, 3H, H-8, H-3’ and H-4’), 8.22 (d, J ¼ 7.5 Hz, 1H, H-5),
8.77 (d, J ¼ 4.9 Hz, 1H, H-6’), 11.90 ppm (s, 1H, OH).
2-Pyridin-3-ylquinolin-4-ol (25)
General procedure B: time, 7 h; starting material, N-(2-acetylphe-
nyl)nicotinamide 1725 (0.20 g, 0.83 mmol). Compound 2524 was
obtained as a yellow solid (0.13 g, 70% yield), mp 243.0–244.0 ꢂC.
1H NMR (400 MHz, DMSO-d6): d ¼ 6.37 (s, 1H, H-3), 7.32 (t,
J ¼ 7.0 Hz, 1H, H-6), 7.58 (dd, J ¼ 4.8 and 7.9 Hz, 1H, H-4’),
7.63–7.71 (m, 2H, H-7 and H-5’), 8.07 (d, J ¼ 7.9 Hz, 1H, H-8), 8.20
(d, J ¼ 7.9 Hz, 1H, H-5), 8.72 (d, J ¼ 4.5 Hz, 1H, H-6’), 8.99 (d,
J ¼ 1.8 Hz, 1H, H-2’), 11.81 ppm (s, 1H, OH).
N,N-diethyl-2-f[2–(5-propoxypyridin-2-yl)quinolin-4-yl]oxygethan-
amine hydrochloride (30a)
General procedure C: time, 4 h; starting materials, 2–(5-propoxy-
pyridin-2-yl)quinolin-4-ol 22 (0.20 g, 0.71 mmol) and (2-
chloroethyl)diethylamine hydrochloride. After purification (CH2Cl2/
MeOH 97/3) and hydrochlorination, compound 30a was obtained
as a white solid (0.04 g, 17% yield), mp 212.0–213.0 ꢂC. 1H NMR
(400 MHz, DMSO-d6): d ¼ 0.99 (t, J ¼ 7.4 Hz, 3H, OCH2CH2CH3), 1.29
(t, J ¼ 7.4 Hz, 6H, NCH2CH3 x 2), 1.73–1.82 (m, 2H, OCH2CH2CH3),
2-Pyridin-4-ylquinolin-4-ol (26)
General procedure B: time, 8 h; starting material, N-(2-acetylpheny- 3.21–3.28 (m, 4H, NCH2CH3 x 2), 3.71–3.75 (m, 2H, OCH2CH2N),
l)isonicotinamide1826 (1.00 g, 4.16 mmol). Compound 2624 was 4.14 (t, J ¼ 6.6 Hz, 2H, OCH2CH2CH3), 4.93–4.97 (m, 2H, OCH2CH2N),

588
T. FELICETTI ET AL.
7.70–7.75 (m, 2H, H-6 and H-4’), 7.97 (t, J ¼ 7.7 Hz, 1H, H-7), 8.11 (s, 3.99 (t, J ¼ 8.1 Hz, 2H, OCH2CH2CH3), 4.95–5.00 (m, 2H, OCH2CH2N),
1H, H-3), 8.39–8.42 (m, 2H, H-8 and H-3’), 8.53 (d, J ¼ 2.6 Hz, 1H, H- 6.60 (d, J ¼ 9.2 Hz, 1H, H-5’), 7.71 (t, J ¼ 7.4 Hz, 1H, H-6), 7.75 (s, 1H,
6’), 8.80 (d, J ¼ 8.6 Hz, 1H, H-5), 11.32 ppm (s, 1H, HCl). 13C NMR H-3), 7.97 (t, J ¼ 7.5 Hz, 1H, H-7), 8.40–8.43 (m, 2H, H-8 and H-4’),
(101 MHz, DMSO-d6): d ¼ 8.93, 10.74, 22.32, 47.32, 49.64, 66.26, 8.45–8.51 (m, 1H, H-5), 9.22 (s, 1H, H-2’), 11.19 ppm (s, 1H, HCl).
70.51, 99.93, 120.29, 122.01, 123.27, 125.30, 125.43, 125.57, 127.96, 13C NMR (101 MHz, DMSO-d6): d ¼ 11.25, 21.63, 22.50, 22.76, 51.30,
127.99, 133.39, 133.72, 138.98, 154.37, 157.76 ppm. Anal calcd for 52.89, 54.49, 64.87, 99.72, 119.64, 123.10, 124.06, 124.26, 127.26,
C23H30ClN3O2: C, 66.41; H, 7.27; N, 10.10; found: C, 66.38; H, 7.28; 132.97, 133.15, 138.69, 142.37, 142.59, 154.07, 161.47, 164.12. Anal
N, 10.13.
calcd for C24H30ClN3O2: C, 67.36; H, 7.07; N, 9.82;found: C, 67.42; H,
7.06; N, 9.80.
4–(2-Piperidin-1-ylethoxy)-2–(5-propoxypyridin-2-yl)quinoline (30b)
General procedure C: time, 4 h; starting materials, 2–(5-propoxy- N,N-diethyl-2-[(2-pyridin-2-ylquinolin-4-yl)oxy]ethanamine hydro-
pyridin-2-yl)quinolin-4-ol 22 (0.23 g, 0.81 mmol) and 1–(2-chloroe-
thyl)piperidine hydrochloride. After purification (CH2Cl2/MeOH
95/5), compound 30b was obtained as a white solid (0.11 g, 33%
yield), mp 90.0–91.0 ꢂC. 1H NMR (400 MHz, CDCl3): d ¼ 1.05 (t,
J ¼ 7.4 Hz, 3H, OCH2CH2CH3), 1.39–1.45 (m, 2H, piperidine CH2),
chloride (32a)
General procedure C: time, 12 h; starting materials, 2-pyridin-2-
ylquinolin-4-ol
24
(0.30 g,
1.35 mmol)
and
(2-
chloroethyl)diethylamine hydrochloride. After purification (CH2Cl2/
MeOH 95/5) and hydrochlorination, compound 32a was obtained
as a white solid (0.10 g, 21% yield), mp 173.0–174.0 ꢂC. 1H NMR
(400 MHz, DMSO-d6): d ¼ 1.28 (t, J ¼ 7.2 Hz, 6H, NCH2CH3 x 2),
3.21–3.29 (m, 4H, NCH2CH3 x 2), 3.67–3.71 (m, 2H, OCH2CH2N),
4.76 (t, J ¼ 4.9 Hz, 2H, OCH2CH2N), 7.52 (ddd, J ¼ 1.1, 4.8 and
9.1 Hz, 1H, H-3’), 7.60 (dt, J ¼ 1.1 and 7.0 Hz, 1H, H-6), 7.79 (dt,
J ¼ 1.4 and 7.0, 1H, H-7), 7.99 (dt, J ¼ 1.8 and 8.0 Hz, 1H, H-5’),
7.81–8.06 (m, 2H, H-3 and H-4’), 8.25 (dd, J ¼ 1.0 and 8.2 Hz, 1H, H-
8), 8.59 (d, J ¼ 7.9 Hz, 1H, H-5), 8.73 (dd, J ¼ 1.5 and 5.0 Hz, 1H, H-
6’), 10.70 ppm (s, 1H, HCl). 13C NMR (101 MHz, DMSO-d6): d ¼ 9.06,
47.52, 49.92, 63.66, 120.90, 121.72, 122.35, 125.41, 126.91, 129.29,
130.99, 137.94, 148.52, 149.58, 155.25, 156.97, 161.51 ppm. Anal
calcd for C20H24ClN3O: C, 67.12; H, 6.76; N, 11.74;found: C, 67.21;
H, 6.76; N, 11.70.
1.52–1.63 (m, 4H, piperidine CH2
x 2), 1.78–1.97 (m, 2H,
OCH2CH2CH3), 2–49-2.61 (m, 4H, piperidine NCH2 ꢃ 2), 2.97 (t,
J ¼ 5.9 Hz, 2H, OCH2CH2N), 4.02 (t, J ¼ 6.5 Hz, 2H, OCH2CH2CH3),
4.46 (t, J ¼ 5.9 Hz, 2H, OCH2CH2N), 7.31 (dd, J ¼ 2.8 and 8.7 Hz, 1H,
H-4’), 7.45 (t, J ¼ 7.3 Hz, 1H, H-6), 7.66 (t, J ¼ 7.1 Hz, 1H, H-7), 7.89
(s, 1H, H-3), 8.03 (d, J ¼ 8.2 Hz, 1H, H-8), 8.17 (d, J ¼ 8.1 Hz, 1H, H-
5), 8.36 (d, J ¼ 2.5 Hz, 1H, H-6’), 8.56 ppm (d, J ¼ 8.8 Hz, 1H, H-3’).
13C NMR (101 MHz, CDCl3): d ¼ 10.47, 22.51, 24.17, 26.07, 54.99,
57.63, 66.75, 69.96, 97.85, 121.09, 121.55, 121.91, 122.49, 125.34,
129.00, 129.72, 136.91, 148.95, 155.98, 157.37, 161.99 ppm. Anal
calcd for C24H29N3O2: C, 73.63; H, 7.47; N, 10.73;found: C, 73.70; H,
7.46; N, 10.71.
N,N-diethyl-2-f[2–(6-propoxypyridin-3-yl)quinolin-4-yl]oxygethan-
amine hydrochloride (31a)
4–(2-Piperidin-1-ylethoxy)-2-pyridin-2-ylquinoline (32b)
General procedure C: time, 2 h; starting materials, 2–(6-propoxy- General procedure C: time, 4 h; starting materials, 2-pyridin-2-
pyridin-3-yl)quinolin-4-ol 23 (0.20 g, 0.71 mmol) and (2- ylquinolin-4-ol 24 (0.21 g, 0.93 mmol) and 1–(2-chloroethyl)piperi-
chloroethyl)diethylamine hydrochloride. After purification (CH2Cl2/ dine hydrochloride. After purification (CHCl3/MeOH 97/3), com-
MeOH 95/5) and hydrochlorination, compound 31a was obtained pound 32 b was obtained as a white solid (0.21 g, 69% yield), mp
as a white solid (0.19 g, 65% yield), mp 188.0–190.0 ꢂC. 1H NMR 77.0–79.5 ꢂC. 1H NMR (400 MHz, DMSO-d6): d ¼ 1.33–1.35 (m, 2H,
(400 MHz, DMSO-d6): d ¼ 0.89 (t, J ¼ 7.6 Hz, 3H, OCH2CH2CH3), 1.29 piperidine CH2), 1.44–1.50 (m, 4H, piperidine CH2 x 2), 2.46–2.49
(t, J ¼ 7.1 Hz, 6H, NCH2CH3 x 2), 1.72–1.82 (m, 2H, OCH2CH2CH3), (m, 4H, piperidine NCH2 ꢃ 2), 2.84 (t, J ¼ 6.4 Hz, 2H, OCH2CH2N),
3.21–3.31 (m, 4H, NCH2CH3 x 2), 3.61–3.68 (m, 2H, OCH2CH2N), 4.41 (t, J ¼ 6.8 Hz, 2H, OCH2CH2N), 7.48 (ddd, J ¼ 1.2, 5.1 and
4.00 (t, J ¼ 7.6 Hz, 2H, OCH2CH2CH3), 4.87–5.01 (m, 2H, OCH2CH2N), 8.9 Hz, 1H, H-3’), 7.55 (dt, J ¼ 1.9 and 7.6 Hz, 1H, H-6), 7.75 (dt,
6.60 (d, J ¼ 9.7 Hz, 1H, H-5’), 7.72 (t, J ¼ 7.5 Hz, 1H, H-6), 7.77 (s, 1H, J ¼ 1.8 and 7.3 Hz, 1H, H-7), 7.97 (dt, J ¼ 2.5 and 8.4 Hz, 1H, H-5’),
H-3), 7.98 (t, J ¼ 7.8 Hz, 1H, H-7), 8.37 (d, J ¼ 8.3 Hz, 1H, H-5), 8.41 7.99–8.01 (m, 2H, H-3 and H-4’), 8.11 (d, J ¼ 7.9 Hz, 1H, H-8), 8.56
(dd, J ¼ 2.4 and 9.6 Hz, 1H, H-4’), 8.52–8.54 (m, 1H, H-8), 9.26 (s, (dd, J ¼ 2.0 and 7.9 Hz, 1H, H-5), 8.70 ppm (dd, J ¼ 1.2 and 5.4 Hz,
1H, H-2’), 11.16 ppm (s, 1H, HCl). 13C NMR (101 MHz, DMSO-d6): d 1H, H-6’). 13C NMR (101 MHz, DMSO-d6): d ¼ 24.30, 26.07, 54.79,
¼ 9.01, 11.25, 22.49, 47.58, 49.82, 51.31, 65.19, 100.03, 112.58, 57.41, 67.17, 98.46, 121.19, 121.60, 122.01, 125.19, 126.67, 129.44,
119.61, 123.10, 123.56, 123.72, 127.45, 133.25, 138.72, 142.69, 130.69, 137.77, 148.71, 149.57, 155.66, 157.19, 162.04 ppm. Anal
143.02, 153.97, 161.44, 164.62 ppm. Anal calcd for C23H30ClN3O2: C, calcd for C21H23N3O: C, 75.65; H, 6.95; N, 12.60;found: C, 75.74; H,
66.41; H, 7.27; N, 10.10;found: C, 66.37; H, 7.30; N, 10.12.
6.93; N, 12.54.
4–(2-Piperidin-1-ylethoxy)-2–(6-propoxypyridin-3-yl)quinoline
N,N-diethyl-2-[(2-pyridin-3-ylquinolin-4-yl)oxy]ethanamine hydro-
hydrochloride (31b)
chloride (33a)
General procedure C: time, 5 h; starting materials, 2–(6-propoxy- General procedure C: time, 5 h; starting materials, 2-pyridin-3-
pyridin-3-yl)quinolin-4-ol 23 (0.30 g, 1.09 mmol) and 1–(2-chloroe- ylquinolin-4-ol 25 (0.30 g, 1.35 mmol) and (2-
thyl)piperidine hydrochloride. After purification (CH2Cl2/MeOH 99/ chloroethyl)diethylamine hydrochloride. After purification (CH2Cl2/
1) and hydrochlorination, compound 31 b was obtained as a white MeOH 90/10) and hydrochlorination, compound 33a was obtained
solid (0.25 g, 54% yield), mp 223.0–224.0 ꢂC. 1H NMR (400 MHz, as a white solid (0.17 g, 35% yield), mp 181.0–182.0 ꢂC. 1H NMR
DMSO-d6): d ¼ 0.89 (t, J ¼ 7.4 Hz, 3H, OCH2CH2CH3), 1.32–1.42 (m, (400 MHz, DMSO-d6): d ¼ 1.29 (t, J ¼ 7.1 Hz, 6H, NCH2CH3 x 2),
1H, piperidine CH), 1.67–1.90 (m, 7H, piperidine CH, piperidine 3.12–3.25 (m, 4H, NCH2CH3 ꢃ 2), 3.70–3.71 (m, 2H, OCH2CH2N),
CH2 x 2 and OCH2CH2CH3), 3.03–3.10 (m, 2H, piperidine NCH2), 4.90–4.93 (m, 2H, OCH2CH2N), 7.71 (t, J ¼ 7.5 Hz, 1H, H-6), 7.91 (t,
3.37–3.40 (m, 2H, piperidine NCH2), 3.67–3.68 (m, 2H, OCH2CH2N), J ¼ 7.7 Hz, 1H, H-7), 7.98 (s, 1H, H-3), 8.09 (t, J ¼ 6.1 Hz, 1H, H-5’),

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
589
8.25 (d, J ¼ 8.3 Hz, 1H, H-8), 8.35 (d, J ¼ 8.2 Hz, 1H, H-5), 8.98 (d, N,N-diethyl-2-f[2–(2-thienyl)quinolin-4-yl]oxygethanamine (35a)
General procedure C: time, 4 h; starting materials, 2–(2-thienyl)qui-
J ¼ 7.9 Hz, 1H, H-6’), 9.26 (d, J ¼ 8.1 Hz, 1H, H-4’), 9.68 (s, 1H, H-2’),
11.31 ppm (s, 1H, HCl). 13C NMR (101 MHz, DMSO-d6): d ¼ 24.30,
26.07, 54.79, 57.41, 67.17, 98.46, 121.19, 121.60, 122.01, 125.19,
126.67, 129.44, 130.69, 137.77, 148.71, 149.57, 155.66, 157.19,
162.04 ppm. Anal calcd for C20H24ClN3O: C, 67.12; H, 6.76; N,
11.74;found: C, 67.31; H, 6.75; N, 11.70.
nolin-4-ol
27
(0.30 g,
1.32 mmol)
and
(2-
chloroethyl)diethylamine hydrochloride. After purification (CH2Cl2/
MeOH 98/2), compound 35a was obtained as a white solid
(0.26 g, 60% yield), mp 84.0–86.0 ꢂC. 1H NMR (400 MHz, CDCl3):
d ¼ 0.99 (t, J ¼ 7.0 Hz, 6H, NCH2CH3 ꢃ 2), 2.58 (q, J ¼ 7.0 Hz, 4H,
NCH2CH3 x 2), 2.93 (t, J ¼ 5.5 Hz, 2H, OCH2CH2N), 4.37 (t, J ¼ 5.4 Hz,
2H, OCH2CH2N), 7.17–7.19 (m, 1H, H-4’), 7.47 (t, J ¼ 7.3 Hz, 1H, H-6),
7.54 (s, 1H, H-3), 7.65–7.69 (m, 2H, H-7 and H-5’), 7.84 (d,
J ¼ 8.4 Hz, 1H, H-8), 8.02–8.04 ppm (m, 2H, H-5 and H-3’). 13C NMR
(101 MHz, CDCl3): d ¼ 12.50, 47.53, 51.37, 68.08, 98.05, 120.52,
121.94, 125.77, 127.52, 128.61, 128.68, 129.89, 130.70, 145.74,
148.71, 153.64, 161.91 ppm. Anal calcd for C19H22N2OS: C, 69.90; H,
6.79; N, 8.58;found: C, 69.99; H, 6.77; N, 8.58.
4–(2-Piperidin-1-ylethoxy)-2-pyridin-3-ylquinoline (33b)
General procedure C: time, 4 h; starting materials, 2-pyridin-3-
ylquinolin-4-ol 25 (0.30 g, 1.35 mmol) and 1–(2-chloroethyl)piperi-
dine hydrochloride. After purification (CH2Cl2/MeOH 95/5), com-
pound 33 b was obtained as a white solid (0.26 g, 58% yield), mp
1
101.5–102.5 ꢂC. H NMR (400 MHz, DMSO-d6): d ¼ 1.33–1.36 (m, 2H,
piperidine CH2), 1.47–1.52 (m, 4H, piperidine CH2 x 2), 2.47–2.51
(m, 4H, piperidine NCH2 x 2), 2.81–2.85 (m, 2H, OCH2CH2N), 4.47 (t,
J ¼ 5.8 Hz, 1H, OCH2CH2N), 7.52–7.58 (m, 2H, H-6 and H-5’), 7.64 (s,
1H, H-3), 7.74 (t, J ¼ 7.4 Hz, 1H, H-7), 7.99 (d, J ¼ 8.4 Hz, 1H, H-8),
8.10 (d, J ¼ 8.2 Hz, 1H, H-5), 8.61 (d, J ¼ 7.9 Hz, 1H, H-6’), 8.65 (d,
J ¼ 4.7 Hz, 1H, H-4’), 9.43 ppm (s, 1H, H-2’). 13C NMR (101 MHz,
DMSO-d6): d ¼ 24.29, 26.05, 54.76, 57.50, 67.37, 99.59, 120.49,
121.97, 124.13, 126.39, 129.36, 130.73, 134.96, 135.17, 148.94,
149.06, 150.73, 155.87, 162.26 ppm. Anal calcd for C21H23N3O: C,
75.65; H, 6.95; N, 12.60;found: C, 75.71; H, 6.93; N, 12.57.
4–(2-piperidin-1-ylethoxy)-2–(2-thienyl)quinoline hydrochloride (35b)
General procedure C: time, 5 h; 2–(2-thienyl)quinolin-4-ol 27
(0.30 g, 1.32 mmol) and 1–(2-chloroethyl)piperidine hydrochloride.
After purification (CH2Cl2/MeOH 95/5) and hydrochlorination, com-
pound 35 b was obtained as a white solid (0.29 g, 58% yield), mp
1
208.0–209.5 ꢂC. H NMR (400 MHz, DMSO-d6): d ¼ 1.66–1.69 (m, 2H,
piperidine CH2), 1.76–1.89 (m, 4H, piperidine CH2 ꢃ 2), 3.01–3.14
(m, 2H, piperidine NCH2), 3.42–3.55 (m, 2H, piperidine NCH2),
3.62–3.71 (m, 2H, OCH2CH2N), 4.87–4.89 (m, 2H, OCH2CH2N), 7.28
(t, J ¼ 4.1 Hz, 1H, H-4’), 7.59 (t, J ¼ 7.4 Hz, 1H, H-6), 7.62 (s, 1H, H-3),
7.83 (t, J ¼ 7.6 Hz, 1H, H-7), 7.88 (d, J ¼ 4.5 Hz, 1H, H-5’), 8.16 (d,
J ¼ 6.9 Hz, 1H, H-8), 8.30 (d, J ¼ 8.2 Hz, 1H, H-5), 8.34 (bs, 1H, H-3’),
11.33 ppm (s, 1H, HCl). 13C NMR (101 MHz, DMSO-d6): d ¼ 21.61,
22.81, 52.82, 54.64, 64.21, 99.15, 120.07, 122.80, 126.68, 129.13,
129.63, 130.14, 131.68, 132.03, 144.46, 146.35, 152.64, 162.50 ppm.
Anal calcd for C20H23ClN2OS: C, 64.07; H, 6.18; N, 7.47;found: C,
64.11; H, 6.17; N, 7.45.
N,N-diethyl-2-[(2-pyridin-4-ylquinolin-4-yl)oxy]ethanamine (34a)
General procedure C: time, 4 h; starting materials, 2-pyridin-4-
ylquinolin-4-ol
26
(0.30 g,
1.35 mmol)
and
(2-
chloroethyl)diethylamine hydrochloride. After purification (CH2Cl2/
MeOH 90/10), compound 34a was obtained as a white solid
(0.22 g, 51% yield), mp 73.5–74.0 ꢂC. 1H NMR (400 MHz, CDCl3):
d ¼ 1.09 (t, J ¼ 6.1 Hz, 6H, NCH2CH3 x 2), 2.70 (q, J ¼ 7.3 Hz, 4H,
NCH2CH3 x 2), 3.05 (t, J ¼ 6.0 Hz, 2H, OCH2CH2N), 4.34 (t, J ¼ 6.2 Hz,
2H, OCH2CH2N), 7.19 (s, 1H, H-3), 7.50 (t, J ¼ 7.2 Hz, 1H, H-6), 7.71
(t, J ¼ 6.8 Hz, 1H, H-7), 7.97–7.99 (m, 2H, H-3’ and H-5’), 8.08 (d,
J ¼ 8.9 Hz, 1H, H-8), 8.18 (d, J ¼ 8.3 Hz, 1H, H-5), 8.71–8.77 ppm (m,
2H, H-2’ and H-6’). 13C NMR (101 MHz, CDCl3): d ¼ 12.08, 48.06,
51.40, 67.68, 98.19, 120.92, 121.66, 121.80, 126.15, 129.43, 130.29,
147.29, 149.20, 150.41, 155.83, 162.52 ppm. Anal calcd for
C20H23N3O: C, 74.74; H, 7.21; N, 13.07;found: C, 74.81; H, 7.19;
N, 13.01.
N,N-diethyl-2-f[2–(3-thienyl)quinolin-4-yl]oxygethanamine (36a)
General procedure C: time, 4 h; starting materials, 2–(3-thienyl)qui-
nolin-4-ol
28
(0.30 g,
1.32 mmol)
and
(2-
chloroethyl)diethylamine hydrochloride. After purification (CH2Cl2/
MeOH 95/5), compound 36a was obtained as a white solid
(0.13 g, 30% yield), mp 66.0–67.0 ꢂC. 1H NMR (400 MHz, CDCl3):
d ¼ 1.12 (t, J ¼ 7.3 Hz, 6H, NCH2CH3 ꢃ 2), 2.71 (q, J ¼ 7.0 Hz, 4H,
NCH2CH3
ꢃ 2), 3.07 (t, J ¼ 6.1 Hz, 2H, OCH2CH2N), 4.34 (t,
J ¼ 6.1 Hz, 2H, OCH2CH2N), 7.09 (s, 1H, H-3), 7.39–7.47 (m, 2H, H-6
and H-8), 7.66 (dt, J ¼ 1.4 and 8.4 Hz, 1H, H-7), 7.80 (dd, J ¼ 1.3
and 5.0 Hz, 1H, H-5’), 7.98 (dd, J ¼ 1.1 and 4.4 Hz, 1H, H-4’), 8.02 (d,
J ¼ 8.5 Hz, 1H, H-5), 8.12 ppm (dd, J ¼ 1.1 and 8.3 Hz, 1H, H-2’). 13C
NMR (101 MHz, CDCl3): d ¼ 11.92, 47.48, 51.29, 67.16, 98.55, 120.39,
121.64, 124.44, 125.18, 126.27, 126.85, 128.95, 129.96, 143.12,
149.19, 154.51, 161.85 ppm. Anal calcd for C19H22N2OS: C, 69.90; H,
6.79; N, 8.58;found: C, 69.94; H, 6.78; N, 8.56.
4–(2-Piperidin-1-ylethoxy)-2-pyridin-4-ylquinoline hydrochloride (34b)
General procedure C: time, 4 h; 2-pyridin-4-ylquinolin-4-ol 26
(0.30 g, 1.35 mmol) and 1–(2-chloroethyl)piperidine hydrochloride.
After purification (CH2Cl2/MeOH 99/1) and hydrochlorination, com-
pound 34 b was obtained as a white solid (0.30 g, 61% yield), mp
1
183.5–185.0 ꢂC. H NMR (400 MHz, DMSO-d6): d ¼ 1.33–1.42 (m, 2H,
piperidine CH2), 1.66–1.93 (m, 4H, piperidine CH2 ꢃ 2), 3.04–3.09
(m, 2H, piperidine NCH2), 3.51–3.54 (m, 2H, piperidine NCH2),
3.63–3.67 (m, 2H, OCH2CH2N), 4.93 (t, J ¼ 5.0 Hz, 2H, OCH2CH2N),
7.69 (t, J ¼ 7.3 Hz, 1H, H-6), 7.87 (t, J ¼ 7.0 Hz, 1H, H-7), 8.02 (s, 1H,
H-3), 8.15 (d, J ¼ 8.4 Hz, 1H, H-8), 8.35 (d, J ¼ 8.1 Hz, 1H, H-5), 8.92
(d, J ¼ 6.7 Hz, 2H, H-3’ and H-5’), 9.08 (d, J ¼ 6.7 Hz, 2H, H-2’ and
H-6’), 11.42 ppm (s, 1H, HCl). 13C NMR (101 MHz, DMSO-d6):
d ¼ 21.66, 22.72, 52.74, 54.52, 64.34, 101.11, 121.09, 122.73, 124.99,
128.38, 129.29, 131.92, 143.00, 148.19, 152.38, 153.36, 162.55 ppm.
Anal calcd for C21H24ClN3O: C, 68.19; H, 6.54; N, 11.36;found: C,
68.01; H, 6.56; N, 11.40.
4–(2-Piperidin-1-ylethoxy)-2–(3-thienyl)quinoline (36b)
General procedure C: time, 3 h; starting materials, 2–(3-thienyl)qui-
nolin-4-ol 28 (0.30 g, 1.32 mmol) and 1–(2-chloroethyl)piperidine
hydrochloride. After purification (CH2Cl2/MeOH 98/2), compound
36b was obtained as a white solid (0.27 g, 61% yield), mp
109.5–111.0 ꢂC. 1H NMR (400 MHz, CDCl3): d ¼ 1.44–1.48 (m, 2H,
piperidine CH2), 1.60–1.66 (m, 4H, piperidine CH2 ꢃ 2), 2.55–2.61
(m, 4H, piperidine CH2 ꢃ 2), 2.98 (t, J ¼ 6.8 Hz, 2H, OCH2CH2N),
4.40 (t, J ¼ 6.0 Hz, 2H, OCH2CH2N), 7.08 (s, 1H, H-3), 7.40–7.48 (m,
2H, H-6 and H-8), 7.66 (dt, J ¼ 1.5 and 8.3 Hz, 1H, H-7), 7.80 (dd,

590
T. FELICETTI ET AL.
J ¼ 1.4 and 5.0 Hz, 1H, H-5’), 7.97 (dd, J ¼ 1.1 and 4.2 Hz, 1H, H-4’),
8.01 (d, J ¼ 8.2 Hz, 1H, H-5), 8.13 ppm (dd, J ¼ 1.1 and 8.3 Hz, 1H,
H-2’). 13C NMR (101 MHz, CDCl3): d ¼ 24.01, 25.94, 55.13, 57.52,
66.67, 98.55, 120.41, 121.68, 124.43, 125.18, 126.27, 126.85, 128.94,
129.97, 143.12, 149.18, 154.52, 161.78 ppm. Anal calcd for
C20H22N2OS: C, 70.97; H, 6.55; N, 8.28;found: C, 70.85; H, 6.56;
N, 8.30.
FIC values for 37a and CPX were calculated according to the
following equation: MIC in combination/MIC alone. FICI values
were calculated by the sum of FIC values of 37a and CPX.
The loss of EtBr from S. aureus SA-1199B was determined
fluorometrically as previously described30. The effect of various
concentrations of tested compounds on the EtBr efflux of SA-
1199B was compared to that in their absence, allowing the calcu-
lation of the percentage reduction in efflux.
Haemolysis assays were performed as previously described31.
Cell viability assays for compounds 1 and 37a were performed
on human leukemic monocyte cell line (THP-1). THP-1 cells were
grown in RPMI 1640 supplemented with 10% heat-inactivated foe-
tal calf serum, 10,000 units of penicillin, and 10 mg of strepto-
mycin/mL overnight to confluence. Monolayers were treated for
24 h at 37 ꢂC with scalar concentration of tested compounds
(0 ꢄ 250 mg/mL). Cell viability was then evaluated using an ATP
bioluminescence kit (Via Light kit; Cambrex). Results are expressed
as 50% cytotoxic concentration (CC50). The CC50 was defined as
the concentration required to reduce the cell number by 50%
compared to that for the untreated controls. Each concentration
was tested in triplicate.
Membrane potential assays were carried out by measuring the
effect of 1 and 37a on the membrane potential using the
BacLight Bacterial Membrane Potential Kit (Molecular Probes, Life
Technologies) according to the manufacturer’s instructions. Briefly,
SA-1199B was grown in MHB at 37 ꢂC until reaching an OD600 of
0.6. Bacterial cells were then washed in PBS and diluted to 1 ꢃ 106
CFU/mL with filtered PBS (filter 0.22 mm) in flow cytometry tubes.
Ten microliters of 3 mM 3,3-diethyloxacarbocyanine iodide
(DiOC2(3) in DMSO) was added to each tube (final concentration
30 mM) and mixed. Then, 1 or 37a from a stock solution in DMSO
(10 mg/mL) was added to reach final concentrations of 1, 5, and
10 mg/mL. As positive control, 10 mL of 500 mM carbonyl cyanide
m-chlorophenylhydrazone (CCCP, final concentration 5 mM) was
used to eradicate the proton gradient by eliminating the mem-
brane potential. The samples were analysed after 30 min by meas-
(2-f[2–(5-Chloro-2-thienyl)quinolin-4-yl]oxygethyl)diethylamine
hydrochloride (37a)
General procedure C: time, 4 h; starting materials, 2–(5-chloro-2-
thienyl)quinolin-4-ol
29
(1.00 g,
4.14 mmol)
and
(2-
chloroethyl)diethylamine hydrochloride. After purification (CH2Cl2/
MeOH 95/5) and hydrochlorination, compound 37a was obtained
as a white solid (0.50 g, 32% yield), mp 189.0–191.0 ꢂC. 1H NMR
(400 MHz, DMSO-d6): d ¼ 1.28 (t, J ¼ 7.2 Hz, 6H, NCH2CH3 ꢃ 2),
3.19–3.26 (m, 4H, NCH2CH3 ꢃ 2), 3.65–3.83 (m, 2H, OCH2CH2N),
4.76–4.78 (m, 2H, OCH2CH2N), 9.76 (d, J ¼ 4.1 Hz, 1H, H-3’), 7.54 (t,
J ¼ 7.0 Hz, 1H, H-6), 7.63 (s, 1H, H-3), 7.74 (dt, J ¼ 1.1 and 7.0 Hz,
1H, H-7), 7.92 (d, J ¼ 8.3 Hz, 1H, H-8), 8.05 (d, J ¼ 4.0 Hz, 1H, H-4’),
8.18 (d, J ¼ 8.1 Hz, 1H, H-5), 11.07 ppm (s, 1H, HCl). 13C NMR
(101 MHz, DMSO-d6): d ¼ 9.01, 47.40, 49.68, 64.01, 98.10, 120.29,
122.47, 126.48, 127.91, 128.10, 128.81, 131.44, 132.64, 141.11,
145.34, 152.34, 161.72 ppm. Anal calcd for C19H22Cl2N2OS: C, 57.43;
H, 5.58; N, 7.05;found: C, 57.50; H, 5.56; N, 7.04.
2–(5-Chloro-2-thienyl)-4–(2-piperidin-1-ylethoxy)quinoline (37b)
General procedure C: time, 3 h; starting materials, 2–(5-chloro-2-
thienyl)quinolin-4-ol 29 (0.29 g, 1.20 mmol) and 1–(2-chloroethyl)-
piperidine hydrochloride. After purification (CH2Cl2/MeOH 95/5),
compound 37b was obtained as a white solid (0.14 g, 35% yield),
1
mp 99.0–101.0 ꢂC. H NMR (400 MHz, CDCl3): d ¼ 1.47–1.51 (m, 2H,
piperidine CH2), 1.66–1.71 (m, 4H, piperidine CH2 ꢃ 2), 2.58–2.65
(m, 4H, piperidine NCH2 ꢃ 2), 3.03 (t, J ¼ 5.7 Hz, 2H, OCH2CH2N),
4.44 (t, J ¼ 5.7 Hz, 2H, OCH2CH2N), 6.92 (d, J ¼ 4.0 Hz, 1H, H-3’),
7.01 (s, 1H, H-3), 7.39–7.43 (m, 2H, H-6 and H-4’), 7.64 (dt, J ¼ 1.5
and 8.6 Hz, 1H, H-7), 7.94 (d, J ¼ 8.2 Hz, 1H, H-8), 8.07 ppm (d,
J ¼ 7.3 Hz, 1H, H-5). 13C NMR (101 MHz, CDCl3): d ¼ 23.72, 25.52,
55.00, 57.27, 66.34, 93.14, 120.55, 121.62, 124.45, 125.40, 127.07,
128.72, 130.22, 133.31, 144.40, 148.91, 152.48, 161.60 ppm. Anal
calcd for C20H21ClN2OS: C, 64.42; H, 5.68; N, 7.51;found: C, 64.50;
H, 5.66; N, 7.50.
uring the fluorescence using
a
cytometer Attune NxT
(ThermoFisher Scientific) with a laser emitting at 488 nm and col-
lecting in the green and red channels. The red to green fluores-
cence ratio was determined and normalised against the emission
from the DiOC2(3) blank tube having 1 ml of the bacterial suspen-
sion and a final concentration of DiOC2(3) of 30 mM. The results
are presented as the percentage of depolarised membranes com-
pared with the drug-free control.
In silico ADME
Microbiological procedures
Quinoline derivatives 1,
2 and 37a were built using the
Schrodinger Maestro Interface32 and then imported in MetaSite33.
The protonation state of the compounds was normalised at a pH
of 7.5. The MetaSite analysis was performed using the liver
enzymes model. Compond 37a was built in SeeSAR34 which auto-
matically assigns the proper geometry, protonation state and
tautomeric form of the ligands using ProToss method35. The
Optibrium models for PK properties predictions were downloaded
from Optibrium webpage36.
The strains of S. aureus used in this study included SA-1199B,
which overexpresses norA and possesses an A116E GrlA substitu-
tion, and its isogenic parent SA-1199 (norA wt). MICs assays were
performed using broth microdilution method in 96 wells-micro-
titer plates, following the CLSI guidelines28.
Chequerboard assays and time-kill curves were performed as
previously described29. The formers were performed using 2-fold
increasing concentrations of both antibiotic (from 0.001 to 20 mg/
mL) and compounds (from 0.39 to 25 mg/mL), while time-kill
curves were performed testing CPX concentrations ranging from
Results and discussion
1= x to 1x MIC alone and in combination with the compound 37a
4
Chemistry
at 3.13 and 6.25 mg/mL. The dynamic of the bactericidal activity of
the combination CPX-Compound was evaluated by CFU counts
after 2, 4, 6, 8 and 24 h incubation at 37 ꢂC.
All designed compounds 30a,b–37a,b were synthesised according
to the procedure depicted in Scheme 1. Chlorination of acids 3

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
591
Scheme 1. (i) SOCl2, 60 ꢂC, 30 min; (ii) Et3N, dry THF, rt, 90 min-6h, 50–99%; (iii) NaOH, dry dioxane, 110ꢂC, 2–8 h, 57–100%; (iv) chloroalkylamine hydrochloride,
K2CO3, dry DMF, 80 ꢂC, 2–12 h, 17–69%.
and 4 with SOCl2 afforded acyl chlorides 5 and 6 that were imme- Chequerboard assays
diately reacted with the commercially available aminoacetophe-
The five derivatives showing the best synergistic activity were
none 13 in presence of Et3N to give the amide derivatives 14 and
tested by chequerboard assays in combination with CPX against S.
15. Similarly, coupling reaction of commercially available acyl
aureus SA-1199B (Figure 2), also including reference compounds 1
chlorides 7–12 with 13 afforded amides 16–2024 and 21.
and 2. The two quinoline analogues (35b and 36b) with the C-2
Following the same procedure reported by Brouwer et al.37, ring
unsubstituted thiophene portion did not retain any synergistic
closure of 14–21 through NaOH in dioxane at 110 ꢂC in a pressure
effect with CPX when their concentration did fall below 25 mg/mL.
tube gave the C-2 aryl quinoline derivatives 22, 23, 24–2824 and
On the other hand, pyridine derivatives 30a and 30b at concen-
29. O-alkylation of 4-hydroxyquinolines 22–29 with 2-chloro-N,N-
trations of 6.25 and 12.5 mg/mL, respectively, reduced the CPX MIC
dimethylethylamine hydrochloride or 1–(2-chloroethyl)piperidine
by 4-fold, thereby retaining an EPI activity comparable to 1.
hydrochloride afforded desired quinoline analogues 30a,b–37a,b
(Scheme 1).
Interestingly, the chlorothiophene derivative 37a showed the best
results being able, up to 0.39 mg/mL, to decrease by 4-fold the
CPX MIC against SA-1199B, resulting about 16-fold more active
than the starting hit 1. Actually, compound 37a exhibited a syner-
gistic activity with CPX greater than that shown by compound 2,
Synergistic assays
In order to quickly identify derivatives having significant synergis-
tic activity, the 16 compounds were firstly assayed at 25 mg/mL in
combination with scalar concentrations of CPX against the norA
overexpressing S. aureus strain SA-1199B (norAþ/GrlA mutation)38
(data not shown). Four compounds (30a, 30b, 35b and 36b)
showed a significant synergistic effect producing a 4-fold reduc-
tion of the antibiotic MIC while derivative 37a exhibited an
impressive CPX MIC reduction (32-fold). Worthy of note, among
the five active compounds, three were thiophene derivatives (35b,
36b and 37a) displaying their activity regardless of the position of
the sulphur atom on the thiophene ring. On the contrary, only
pyridine-2-yl-5-OPr derivatives (30a and 30b) exhibited a signifi-
cant synergistic effect whereas all the remaining pyridine ana-
logues (31a, 31b, 32a, 32b, 33a, 33b, 34a and 34b) lost EPI
which produced a weaker and less significant effect (2-fold MIC
reduction) at the same concentration (0.39 mg/mL).
Thus, when rationalising SAR information, it appears clear as
the replacement of the phenyl portion at quinoline C-2 with a thi-
ophene ring was well tolerated when both O-ethylamino chains
were present at the quinoline C-4 position. Interestingly, in the
case of derivative 37a, the substitution of the H-bond acceptor
–OPr group with a chlorine atom yielded very good results, paving
the way for future modifications. On the contrary, the replacement
of the phenyl portion at the quinoline C-2 position with pyridine
moieties, regardless the presence of –OPr group, afforded less
potent EPI derivatives. 30a and 30b compounds, when tested at
the concentration of 25 mg/mL (synergistic assays) showed a
potent synergistic effect with CPX while at lower concentrations
activity regardless of the nitrogen atom position within the pyri- (chequerboard assays), they displayed a poor ability to decrease
dine moiety. the CPX MIC.

592
T. FELICETTI ET AL.
Figure 2. Chequerboard assays of compounds 30a, 30b, 35b, 36b, 37a and reference compounds 1 and 2 in combination with CPX against SA-1199B.
Focussing on the best identified compound 37a, to indirectly fast method to indirectly evaluate NorA inhibition. Thus, to dem-
rule out that its synergism with CPX against SA-1199B was due to onstrate that the synergistic effect of 37a with CPX was due to
non-specific effects, this derivative was tested at 25 mg/mL (a con- the NorA efflux inhibition, we performed EtBr efflux assays against
centration 64-fold higher than that needed to reduce by 4-fold SA-1199B. Interestingly, at 50 mM compound 37a exhibited
CPX MIC) in combination with scalar concentrations of CPX 74.3 3.5% of EtBr efflux inhibition, thus confirming its ability to
against the S. aureus strain SA-1199 (norA wild-type). Interestingly, inhibit NorA efflux pump. Indeed, we commonly consider as active
compound 37a did not exhibit any significant synergistic effect NorA EPIs those compounds having an EtBr efflux inhibition
with CPX in presence of a norA basal expression (data not shown). ꢁ70%17,18
Whether the synergistic activity of 37a with CPX was due to a
mechanism different from that of the NorA inhibition, we would
.
Time-kill curves
have likely to observe a synergistic effect with CPX also against
SA-1199 wild-type strain. Consequently, the observed high differ-
ence in the synergistic activity of 37a with CPX against SA-1199B
and SA-1199 strongly supports a 37a-mediated NorA inhibition.
Subsequently, we decided to test the synergistic effect of com-
pound 37a (at 3.13 and 6.25 mg/mL) when combined with CPX in
time-kill curve analysis against SA-1199B (Figure 3). Interestingly,
at both concentrations, derivative 37a exhibited a strong effect
on the bactericidal activity of CPX, thereby proving its efficacy
MIC evaluation
when combined with CPX over 24 h. Indeed, the combination of
1
Considering the very promising results of derivative 37a, it was compound 37a (at both the used concentrations) and CPX at =
4
tested alone against SA-1199B in order to prove that the synergis- MIC yielded the same bactericidal effect as CPX tested at its MIC.
tic activity with CPX was not influenced by a direct antibacterial Moreover, the same concentrations of 37a combined with CPX at
effect. Worthy of note, up to 25 mg/mL, this compound did not its MIC showed a strongly significant and definitely higher bacteri-
show any antibacterial activity. This result is essential for a poten- cidal activity than that shown by CPX alone over the all 24 h.
tial EPI compound; indeed, a poor or absent antibacterial effect is These findings highlight the high potential of the chlorothiophene
a key requirement to avoid the evolutionary pressure on microor- derivative 37a in potentiating CPX activity against resistant
ganisms that can evolve resistance only towards compounds strains.
endowed with an antimicrobial activity39. In this case, compound
37a reduces CPX MIC (from 10 mg/mL to 2.5 mg/mL) at a concen-
tration ꢁ64-fold lower than its MIC. As a confirmation of the syn-
Cytotoxicity assays
ergistic effect of 37a with CPX, we calculated the FIC values for
both compounds: FIC37a < 0.0156 and FICCPX ¼ 0.25. The sum of
both FIC values led to a FIC index (FICI) < 0.27 showing a signifi-
cant synergistic effect as widely recognised for FICI values <0.540.
Therefore, although FICI evaluation is mainly used to determine
the synergism between two antimicrobial agents, the EPI 37a,
devoid of any antibacterial effect, exhibited a clear synergistic
activity with the fluoroquinolone CPX.
In order to evaluate the toxic effects of the derivative 37a, haem-
olysis assays were performed at scalar compound concentrations
in comparison with the starting hit 1 (Figure 4). Although deriva-
tive 37a showed a slightly higher haemolytic effect than the start-
ing hit 1, when considering the activity-toxicity relationship, the
chlorothiophene derivative 37a exhibited an improved safety pro-
file. In particular, 37a at 20 mg/mL exhibited about 10% of haemo-
lytic effect, a value 3-fold higher than the starting hit 1, when
tested at the same concentration. However, at concentrations ꢅ
5 mg/mL (about 12-fold higher than that needed to reach syner-
gism with CPX), compound 37a reduced its haemolytic activity
below 2%.
EtBr efflux assays
Since EtBr is a known NorA substrate resulting fluorescent only
when inside bacterial cells, monitoring bacterial fluorescence of
In addition, to further evaluate the toxic profile of compound
SA-1199B, overexpressing norA gene, through a fluorimeter is a 37a, we determined its CC50 against human monocytic cells

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
593
Figure 3. Time-kill curves of CPX and combination of compound 37a with different concentrations of CPX against SA-1199B.
Figure 4. Haemolysis assays of compounds 37a and starting hit 1.

594
T. FELICETTI ET AL.
(THP-1); compound 1 was also tested for comparison (Table 1). energy source (protons) that efflux pumps use to extrude their sub-
To note, on THP-1 cells derivative 37a exhibited a CC50 of strates by an antiport mechanism. A typical example of a com-
pound inhibiting efflux pumps activity by this non-specific
mechanism is represented by carbonyl cyanide m-chlorophenyl
hydrazone (CCCP). Therefore, in order to know whether compound
37a might counteract the NorA-mediated CPX efflux by disrupting
proton motive force, we performed membrane polarisation assays
against SA-1199B treated with 37a at three different concentrations
(1, 5, and 10 mg/mL); starting hit 1 (at the same concentrations) for
comparison and CCCP at 1 mg/mL as positive control were included.
Membrane polarisation was assessed by cytofluorimeter analysis
using the fluorescent probe 3,3-diethyloxacarbocyanine iodide
(DiOC2(3)) as its distribution is proton gradient-sensitive. DiOC2(3)
in the presence of a bacterial membrane potential exhibits red
fluorescence that shifts to green emission as the membrane poten-
tial is lost, thereby allowing calculation of the percentage of mem-
brane polarisation by a red/green fluorescence ratio41.
Overall, the effect of the concentrations of 1 and 37a on S.
aureus membrane polarisation was dose-dependent. Indeed, both
1 and 37a at 10 mg/mL extensively (>50%) depolarised S. aureus
membrane. On the contrary, when 1 and 37a were tested at 5
and 1 mg/mL, starting 1 retained a significant depolarising effect
on the bacterial membrane while 37a reduced its influence on
the proton motive force. Indeed, at 5 and 1 mg/mL, compound
37a was able to depolarise only about 20% of the bacterial mem-
brane similarly to 2, as previously reported by us19. Since 37a
retained a significant synergistic effect with CPX at a concentra-
tion as low as 0.39 mg/mL, we can observe that most of its activity
is due to the NorA inhibition and not to a non-specific effect on
the S. aureus membrane.
6.33 mg/mL, a concentration slightly lower than the CC50 of start-
ing hit 1. However, when compared with compound 2, previously
tested by us19, the toxicity of the chlorothiophene derivative 37a
resulted significantly increased. Therefore, it appeared evident that
the replacement of the C-2 pOPr phenyl moiety (compound 1)
with a 5-chloro-2-thiophene portion (compound 37a) led to a sig-
nificant increase in the synergistic activity with CPX against SA-
1199B while retaining the same toxic profile. Therefore, comparing
both the concentrations needed to reduce by 4-fold CPX MIC and
the respective CC50 values on THP-1 of 1 and 37a, it was clear
that 37a exhibited
a significant improvement in terms of
“selectivity index” (16.2 for 37a and 1.6 for 1—Table 1). On the
other hand, the presence of a –OMe substituent at the C-6 and an
O-ethylpiperidine chain at C-4 of the quinoline core resulted
essential to reduce the cytotoxicity against THP-1 cells as well as
to confer a potent NorA EPI activity. Thus, thinking in terms of EPI
activity, the introduction of the chlorothiophene moiety at the
quinoline C-2 position is strongly recommended to boost the syn-
ergistic effect with CPX against SA-1199B; yet the increase in tox-
icity towards human cells should be considered. Therefore, these
results could suggest further chemical modifications leading to
new potent and safe quinoline-based NorA EPIs.
Membrane polarisation assays
Since efflux pumps need a proton gradient along the bacterial
membrane, its modification/disruption can lead to an efflux inhib-
ition. However, this effect cannot be related to a specific EPI activ-
ity; on the contrary, it relies on a non-specific interruption of the
Thus, the replacement of the pOPr-phenyl moiety of com-
pound 1 with the chlorothiophene portion (compound 37a),
could reduce the depolarising effect on the bacterial membrane
while increasing the synergistic effect with CPX; interestingly, this
improvement in EPI activity can be related to a significant NorA
inhibition thereby excluding an extensive disruption of the proton
motive force (Figure 5).
Table 1. Cytotoxicity assays against THP-1 cells of compounds
1, 2 and 37a and their calculated “selectivity index.”
Compound
CC50 (mg/mL)
SIa
1
2
37a
10.0
>100b
6.33
1.6
>128
16.2
aSI, selectivity index calculated by the ratio between CC50 val-
ues and minimum EPI concentration able to reduce by 4-fold
CPX MIC.
In silico ADME studies
We have previously demonstrated that our potent quinoline-based
NorA EPI 2 exhibited a good metabolic stability in both in silico
bData from Ref. 19.
Figure 5. Membrane polarisation assays of compounds 1 and 37a against SA-1199B at three different concentrations (1, 5 and 10mg/mL) using the BacLight Bacterial
Membrane Potential Kit. CCCP was used as positive control at 1 mg/mL (5 mM). % of membrane polarisation was calculated from the red/green fluorescence ratio by
comparing bacterial cells in the presence of compounds with untreated cells.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
595
Figure 6. Predicted reactivity for compounds 1 (A), 2 (B) and 37a (C). The atoms are colour-coded based on their predicted reactivity (red: high reactivity; blue: low
reactivity). The blue sphere highlights the most probable site of metabolism.
Table 2. Predicted physicochemical and ADME descriptors for derivative 37a.
reactivity problems encountered with the introduction of the C-6
methoxy group as in derivative 2.
Desidered value
37a
361
3.62
1.41
2
MWa
<500
<5
>1
In addition, it is noteworthy that 37a obeyed the Lipinski’s rule
of five, showing a lower molecular weight when compared to
derivatives 1 and, in particular, 2 (361, 379 and 431 Da, respect-
ively) and had computed physicochemical and pharmacokinetic
descriptors in line with the recommended guidelines for orally
dosed compounds (Table 2)42.
LogDb
LogSc
HBAd
<5
HBDe
<10
<180
>–5
þ
1
26.6
tPSA (Å2)f
Caco-2g
HIAh
ꢄ4.48
þ
2C9 pKii
<6
5.02
Stabilityj
Stable
Stable
Conclusions
aMolecular weight.
bLogarithm of the octanol/water partition coefficient at pH ¼ 7.4.
In order to identify new potent EPIs to counteract the rapid
insurgence of bacterial resistance towards common antibiotics,
we made a further effort to build a robust SAR delineation
around the quinoline-based NorA inhibitors. Herein, we have
described the design, synthesis and biological evaluation of new
2-arylquinoline derivatives. In particular, a new chlorothiophene
analogue (37a) endowed with high synergistic effect with CPX
against SA-1199B, was identified. To balance the lack of biophys-
ical experiments proving compound binding to NorA pump and
in an attempt to exclude potential non-specific effects, we car-
ried out further experiments supporting that a NorA inhibition
could produce the observed synergistic activity of 37a. Indeed,
the demonstration that our compound did inhibit EtBr efflux in
a phenotypic assay and did not extensively depolarise S. aureus
membrane strongly underpinned that 37a can specifically inhibit
NorA. In addition, time-kill curves of this EPI combined with CPX
displayed the high potential of an EPI in boosting CPX bacteri-
cidal effect.
cIntrinsic aqueous solubility: logS ꢁ 1 corresponds to intrinsic aqueous solubility
greater than 10 mM.
dNumber of hydrogen bond acceptors.
eNumber of hydrogen bond donors.
fTopological polar surface area.
gApparent permeability across monolayers of the Caco-2 line of human epithelial
colorectal adenocarcinoma cells.
hHuman intestinal adsorption: classification of “þ” for compounds which are
ꢁ30% adsorbed and “ꢄ” for compounds which are <30% adsorbed.
ipKi values for CYP2C9 affinity. The defined threshold is to avoid drug-drug inter-
actions due to inhibition of CYP2C9.
jHuman liver microsomal stability. All property values were calculated using
Optibrium in SeeSAR34,36
.
and in vitro experiments. However, demethylation of the C-6
methoxy group of 2 produced the most abundant metabolite
after 2 h of incubation with mouse liver microsomes19.
Therefore, given the comparable EPI activity between deriva-
tives 2 and 37a, a prediction of the metabolic stability for the lat-
ter compound was planned by means of MetaSite software33, with
the inclusion of the initial hit 1 and the potent derivative 2 for
comparison. In agreement with the previous results19, the in silico
protocol correctly predicted the methoxy moiety as the most
reactive group as well as the most probable site of metabolism of
quinoline 2 (Figure 6). By contrast, no highly reactive atoms were
foreseen for derivatives 1 and 37a (Figure 6). These results sug-
gested that the chlorothiophene portion of compound 37a repre-
sented a less reactive moiety than the pOPr chain present in
derivatives 1 and 2, underlining that the new potent EPI 37a
could couple the good biological activity of 2 with an enhanced
metabolic stability. Indeed, the comparison between the NorA EPI
activity and predicted metabolic stability of analogues 1, 2 and
Data collected on 2-arylquinoline derivatives will be useful to
definitely obtain a set of compounds having potent NorA EPI
activity, poor non-specific effects on bacterial membrane and an
acceptable cytotoxic profile in order to justify the use of these
EPIs in animal models of infections.
Disclosure statement
No potential conflict of interest was reported by the authors.
ORCID
Rolando Cannalire
https://orcid.org/0000-0003-0460-6731
https://orcid.org/0000-0003-1572-4290
https://orcid.org/0000-0002-9992-6318
37a showed that the use of the chlorothiophene moiety could Donatella Pietrella
lead to potent NorA quinoline-based EPIs missing the metabolic Serena Massari

596
T. FELICETTI ET AL.
Oriana Tabarrini
https://orcid.org/0000-0003-2693-5675
https://orcid.org/0000-0003-0207-3927
https://orcid.org/0000-0003-3530-5042
https://orcid.org/0000-0001-8558-7004
https://orcid.org/0000-0002-8582-2227
https://orcid.org/0000-0003-0971-3536
18. Astolfi A, Felicetti T, Iraci N, et al. Pharmacophore-based
repositioning of approved drugs as novel Staphylococcus
aureus NorA efflux pump inhibitors. J Med Chem 2017;60:
1598–604.
19. Felicetti T, Cannalire R, Pietrella D, et al. 2-Phenylquinoline S.
aureus NorA efflux pump inhibitors: evaluation of the
importance of methoxy group introduction. J Med Chem
2018;61:7827–48.
20. Felicetti T, Cannalire R, Nizi MG, et al. Studies on 2-phenyl-
quinoline Staphylococcus aureus NorA efflux pump inhibi-
tors: new insights on the C-6 position. Eur J Med Chem
2018;155:428–33.
21. Cannalire R, Machado D, Felicetti T, et al. Natural isofla-
vone biochanin A as a template for the design of new
and potent 3-phenylquinolone efflux inhibitors against
Giuseppe Manfroni
Maria Letizia Barreca
Violetta Cecchetti
Francesca Biavasco
Stefano Sabatini
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