3-Aryl-4-methyl-2-quinolones Targeting Multiresistant Staphylococcus aureus Bacteria

Article abstract of DOI:10.1002/cmdc.201200551

The NorA efflux pump lowers intracellular fluoroquinolone concentrations by expelling antibiotics through the membrane of Staphylococcus aureus. We identified 3-aryl-4-methyl-2-quinolin-2-ones as compounds able to restore the activity of the NorA substrate, ciprofloxacin, against resistant S.aureus strains, and acting as efflux pump inhibitors (EPI). In particular, 5-hydroxy-7-methoxy-4-methyl-3-phenylquinolin-2-one (6c) presents both an EPI and an antimicrobial effect. Its efficacy and safety make it a potential candidate for further investigations.

Full text of DOI:10.1002/cmdc.201200551

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DOI: 10.1002/cmdc.201200551
3-Aryl-4-methyl-2-quinolones Targeting Multiresistant
Staphylococcus aureus Bacteria
Anne Dolꢀans-Jordheim,[a, b, c] Jean-Baptiste Veron,[d] Olivier Fendrich,[a, b]
Emmanuelle Bergeron,[a, b] Adrien Montagut-Romans,[d] Yung-Sing Wong,[d] Bianca Furdui,[d, e]
Jean Freney,[a, b, c] Charles Dumontet,[a, c, f, g] and Ahcꢁne Boumendjel*[d]
The NorA efflux pump lowers intracellular fluoroquinolone con-
centrations by expelling antibiotics through the membrane of
Staphylococcus aureus. We identified 3-aryl-4-methyl-2-quino-
lin-2-ones as compounds able to restore the activity of the
NorA substrate, ciprofloxacin, against resistant S. aureus strains,
and acting as efflux pump inhibitors (EPI). In particular, 5-hy-
droxy-7-methoxy-4-methyl-3-phenylquinolin-2-one (6c) pres-
ents both an EPI and an antimicrobial effect. Its efficacy and
safety make it a potential candidate for further investigations.
The membrane-based bacterial efflux pumps contribute to the
emergence of resistance against antibiotics, inducing failures
during chemotherapies. This phenomenon, which was first dis-
covered in 1980 with the transport of tetracyclines out of the
enterobacteria, is due to numerous efflux systems described
both in Gram-negative and Gram-positive bacteria.[1–6] In the
Staphylococcus aureus genome (2.8 Mb), approximately 253
open reading frames (ORFs) have been described as encoding
putative transport pumps. One of these pumps (NorA) is
a drug/proton antiporter, belonging to the major facilitator su-
perfamily (MFS) that effluxes a broad spectrum of anti-infec-
tious molecules, such as fluoroquinolones, ethidium bromide,
rhodamine and acridines, conferring a multidrug resistance
(MDR) phenotype to the bacteria.[7–9] Therefore, the search for
new molecules to reinforce the antibiotic arsenal against more
and more virulent bacteria is needed. The circumvention or re-
version of MDR to antibiotics could be achieved through two
strategies. The first is based on the search for novel antibiotics
that are not substrates of the efflux pumps, and the second
strategy relies on the inhibition of the efflux system by using
efflux pump inhibitors (EPIs) in association with antibiotics.[9]
The latter strategy could lead to an extension of the clinical
utility of existing antibiotics and a decrease of the adminis-
tered antibiotic doses.[10]
In the last decade, many EPIs were reported, but so far none
was brought to the clinic, mainly because of high toxicity and/
or low in vivo efficacy.[11–19] In this regard, the naturally occur-
ring alkaloid, reserpine, is frequently used as a positive control
of EPI;[20] unfortunately, it is too toxic at clinically relevant
doses.
In this study, we targeted novel molecules that have intrinsic
antibacterial activity and act as EPIs with the aim to get syner-
gy gains, thus, allowing us to investigate phenyl quinolones.
While phenyl quinolones share structural similarities with fluo-
roquinolones, conferring them potential antibiotic activity, they
are also structurally close to 3-phenyl-1,4-benzothiazines
(Figure 1), known as inhibitors of S. aureus NorA multidrug
efflux pump.[16] After screening different subclasses of aryl qui-
nolones, we focused our investigations on 3-aryl-4-methyl-2-
quinolones, shown in Figure 1. The emphasized structural
modifications mainly concerned the methoxylation and hy-
droxylation at C-5 and C-7 positions. This substitution pattern
was adopted because it showed its usefulness among flavo-
[a] A. Dolꢀans-Jordheim, O. Fendrich, E. Bergeron, J. Freney, C. Dumontet+
Universitꢀ Claude Bernard Lyon 1, 69008 Lyon (France)
[b] A. Dolꢀans-Jordheim, O. Fendrich, E. Bergeron, J. Freney
Research Group on Bacterial Opportunistic Pathogens and Environment
UMR 5557-Ecologie Microbienne Lyon, Universitꢀ Claude Bernard Lyon 1
CNRS, VetAgro Sup, 69600 Villeurbanne (France)
[c] A. Dolꢀans-Jordheim, J. Freney, C. Dumontet+
Hospices Civils de Lyon, 69000 Lyon (France)
[d] J.-B. Veron, A. Montagut-Romans, Y.-S. Wong, B. Furdui, A. Boumendjel+
Universitꢀ Joseph Fourier, Grenoble I/CNRS, UMR 5063
Dꢀpartement de Pharmacochimie Molꢀculaire, BP 53
38401 Grenoble Cedex 9 (France)
E-mail: Ahcene.Boumendjel@ujf-grenoble.fr
[e] B. Furdui
Dunarea de Jos University, Department of Chemistry
Domneasca 47, 800008 Galati (Romania)
[f] C. Dumontet+
INSERM U1052, Centre de Recherche en Cancꢀrologie de Lyon
69000 Lyon (France)
[g] C. Dumontet+
CNRS UMR 5286, Centre de Recherche en Cancꢀrologie de Lyon
69000 Lyon (France)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201200551.
Figure 1. The general structures of studied 3-aryl-2-quinolones and the
structure of 3-phenyl-1,4-benzothiazines.[16]
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noids, structurally close compounds that were found as prom-
ising inhibitors of several efflux pumps.[21]
rivatives 6c and 6d. In this case, a chromatographic separation
was necessary to isolate the three pure compounds.
Chemistry
Results and Discussion
Access to the investigated compounds shown in Figure 1 was
accomplished starting from the key blocks, 2-aminoacetophe-
nones 1–3 (Scheme 1). Compound 1 is commercially available,
and derivatives 2 and 3 were prepared according to a previous-
ly reported method from our laboratory.[22] The next step was
the conversion of key acetophenones 1–3 to 3-aryl-4-methyl-
quinolin-2-ones (5). Thus, treatment of 1–3 with arylacetic
acids in the presence of bis(2-oxo-3-oxazolidinyl)phosphinic
chloride (BOP-Cl) as a coupling agent and triethylamine afford-
ed the corresponding amides 4. The cyclization of obtained
amides 4 to quinolones 5 was conducted with tBuOK in
tBuOH. The synthesis of A-ring hydroxylated analogs 6 was
achieved by treatment of the methoxylated derivatives 5 with
boron tribromide (BBr3) in dichloromethane. During the O-de-
methylation step, we found that treatment of 5,7-dimethoxy-
quinolone (5c) with BBr3 provided the fully O-demethylated
compound 6b as well as the two partially O-demethylated de-
The ability of the synthesized compounds to restore the activi-
ty of ciprofloxacin was assessed by the microdilution method,
through the determination of the minimum inhibitory concen-
trations (MICs) of ciprofloxacin on S. aureus ATCC 29213.[23,24]
The results are shown in Table 1. To determine the structural
features of 2-quinolones responsible for the activity, we used
the simplest compound (5a) as the starting hit. Derivative 5a
has a 2-fold reduced MIC compared with ciprofloxacin at
98.7 mm. Using compound 5a, we evaluated the impact of me-
thoxylation at the C-5 and C-7 position. The introduction of
a methoxy group at C-5 (5b) led to a sensitive increase of ci-
profloxacin activity. The methoxylation at both C-5 and C-7 po-
sitions (5c) induced a positive impact on the activity. Next, we
decided to investigate the role of the 3-aryl ring by varying its
structure. In order to do this, we used derivative 5c as a model
by keeping all positions unmodified and introduced different
aryl rings chosen among substituted phenyl rings and hetero-
cycles. As shown in Table 1, only
substitution with
thoxyphenyl (5d)
higher-activity
a
para-me-
provided
derivative,
a
whereas substitution with het-
erocycles (5e and 5 f) was not
fruitful. It should be highlighted
that in the case of a halogenated
phenyl ring, the activity was not
measured due to poor solubility.
Next, we examined the A-ring
hydroxylation effect. The intro-
duction of a hydroxyl group at
C-5 provided derivative 6a,
which was less active than the
methoxy analogue 5b. The O-
demethylation at one of the two
methoxy groups of compound
5c led to compounds 6c and
6d. As shown in Table 1, com-
pound 6c showed the highest
activity in the series. The hy-
droxylation at both C-5 and C-7
positions (6b) had
a similar
effect as the dimethoxylated an-
alogue. Finally, the evaluation of
analogue 6e having
a CH2-
group as a spacer between the
3-phenyl group and C-3, led to
a decrease of activity compared
with compound 6c.
Having identified the most
active compound (6c), we
searched for a short, straightfor-
ward and a scale-up compatible
Scheme 1. A) Synthesis and B) O-demethylation of 3-aryl-4-methyl-2-quinolones.
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curve of ciprofloxacin alone and the curve of ciprofloxacin in
association with compound 6c (Figure 2).
Table 1. Effect of compounds 5a–f and 6a–e on reduction of ciprofloxa-
cin in S. aureus.
Interestingly, we also observed a bactericidal activity on En-
terococcus strains (E. faecalis and E. faecium), and even some
vancomycin-resistant strains (VRE), suggesting that other
Gram-positive cocci could be targeted by this association.
In order to investigate the role of NorA efflux pump in the
reversal effect of compound 6c on ciprofloxacin, we performed
checkerboard assays using S. aureus 1199B, which overexpress
NorA derived from a MSSA bloodstream isolate and S. aureus
K1712, a norA knock-out strain derived from the S. aureus
8325.4 strain.[18] First, the lack of NorA did not influence on the
intrinsic bactericidal activity of 6c, since the MIC value of 6c
alone on S. aureus K1712 was not different from the MIC of the
mother strain 8325.4 (62.5–125 mm or 17.6–35.1 mgmLÀ1).
Second, we observed a synergy between ciprofloxacin and
compound 6c on strains 1199, 1199B and 8325.4 with an in-
creased effect on strain 1199B, and no synergy between these
two molecules on strain K1712. Furthermore, compound 6c
did not show any potentiation effect on ciprofloxacin on
S. aureus K1712 being deleted in NorA, suggesting that NorA is
involved in this activity. In addition, no potentiation by 6c was
observed on the bactericidal activity of several non-NorA sub-
Compd
R1
R2
Ar
Concentration MIC
[mm]
reduction[a]
5a
5b
5c
5d
5e
5 f
6a
6b
6c[b]
H
OMe
H
H
Ph
Ph
98.7
36
12.8
13.2
15.5
2
2
1
2
2
2
1
2
4
2
4
2
4
OMe OMe Ph
OMe OMe 4-OMe-Ph
OMe OMe 3-indolyl
OMe OMe 2-thiophenyl 22
OH
OH
OH
H
OH
OMe Ph
Ph
Ph
75
27.7
31.25
3.9
46
31
20
6d
6e
Reserpine
OMe OH
OH OMe Benzyl
Ph
[a] Fold reduction of ciprofloxacin at the given concentration. [b] For
comparison purposes, the most active compound 6c was evaluated at
two different concentrations.
method for its preparation
(Scheme 2). Hence, we devel-
oped a two-step method start-
ing from the commercially avail-
able 3,5-dimethoxyaniline (7).
This compound was partially O-
demethylated according to
a known procedure,[25] providing
Scheme 2. Rapid, alternative two-step synthesis of compound 6c.
aminophenol 8. The latter was condensed with the
commercially available ethyl 3-oxo-2-phenylbuta-
noate by using microwave irradiation to provide the
desired compound (6c) in one step with high yield
(90%) and purity. This method allows the quick prep-
aration of multigram quantities of 6c in a short
period of time.
Because of its potent effect in restoring the anti-
bacterial activity of ciprofloxacin, we focused our fur-
ther investigation on derivative 6c. First, we investi-
gated the intrinsic antibacterial activity, and we
found that 6c presented
a bactericidal activity
(MIC=62.5–125 mm or 17.6–35.1 mgmLÀ1). The latter
activity was effective against a variety of bacteria, in-
cluding methicillin-sensitive S. aureus (MSSA) strains,
methicillin-resistant S. aureus (MRSA) strains and gly-
copeptide-intermediate S. aureus (GISA) strains. A
checkerboard assay was conducted to measure the
synergy between 6c and some classic antibiotics on
three types of S. aureus strains (MSSA, MRSA and
GISA). On all of them, we observed a synergy (FICꢀ
0.5) between compound 6c and ciprofloxacin. This
synergy was confirmed using a time–kill curve, in
which 2 log10 reduction was observed between the
Figure 2. Time–kill curves of S. aureus obtained by association of ciprofloxacin and 6c:
À1
À1
&
^
*
À1
alone ( ); 0.5 mgmL ciprofloxacin ( ); 0.125 mgmL ciprofloxacin ( ); 31.25 mm 6c
alone (ꢃ); 0.125 mgmLÀ1 ciprofloxacin and 31.25 mm 6c ( ); 0.5 mgmL ciprofloxacin and
*
~
31.25 mm 6c ( ).
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(2.1 equiv) was added. The solution was stirred overnight at RT.
tBuOH was evaporated in vacuo and a saturated solution of NH4Cl
(20 mL) was added. After extraction in AcOEt (2ꢃ20 mL), the or-
ganic layer was washed with water (10 mL), brine (10 mL), dried
over MgSO4 and concentrated. Finally, the crude material was puri-
fied by column chromatography (silica gel, CH2Cl2).
strates (oxacillin, erythromycin, tetracycline and vancomycin),
strengthening the idea that 6c is a NorA inhibitor. We further
performed an efflux inhibition assay using S. aureus 1199B
strain previously loaded with ethidium bromide (EBr), which is
a substrate of NorA and whose efflux can be measured by loss
of fluorescence. EtBr efflux was inhibited by 6c with a potency
that is comparable to reserpine, the reference used as NorA in-
hibitor, confirming that this pump was targeted by 6c for its
EPI activity (6c: IC50 =18 mm versus reserpine: IC50 =14 mm). Be-
cause NorA substrates and inhibitors could induce similar ef-
fects on P-glycoprotein (P-gp), in our hand, no effect on P-gp
was observed even at high concentrations (results not shown).
Toxicity evaluation of compound 6c showed that no toxicity
was observed with concentrations up to 100 mm
(20.07 mgmLÀ1) on HME-1 cells in vitro. For in vivo experiments,
no weight loss or death was observed with BALB/cByJ mice
treated with 6c, indicating that the compound was not toxic
at the tested doses. However, additional studies with various
administration schemes would be necessary before concluding
on this point.
4-Methyl-3-phenylquinolin-2-one (5a): White powder (167 mg
starting from 1 mmol of the corresponding amide 4, 71%); Rf =
0.42 (CH2Cl2/MeOH, 99:1); 1H NMR (400 MHz, CDCl3): d=11.03 (s,
1H); 7.59 (dd, J1 =7.0 Hz, J2 =2.9 Hz, 1H), 7.43 (dd, J1 =7.2 Hz, J2 =
3.2 Hz, 2H), 7.38 (dd, J1 =7.3 Hz, J2 =3.0 Hz, 1H), 7.22 (dd, J1 =
7.2 Hz, J2 =7.0 Hz, 2H), 7.10–7.00 (m, 3H), 2.05 ppm (s, 3H); MS
(ESI+): m/z: 236 [M+H]+; Anal. calcd for C16H13NO: C 81.68, H 5.57,
N 5.95, found: C 81.64, H 5.53, N 5.94.
5-Methoxy-4-methyl-3-phenylquinolin-2-one (5b): White powder
(344 mg starting from 2 mmol of the corresponding amide 4,
65%); Rf =0.23 (CH2Cl2/MeOH, 99:1); mp: 3208C (decomposition);
1H NMR (400 MHz, CD3OD): d=7.77 (d, J=8 Hz, 1H), 7.43–7.26 (m,
3H), 7.26 (d, J=8 Hz, 2H), 6.92–6.86 (m, 2H), 3.89 (s, 3H), 2.29 ppm
(s, 3H); MS (ESI+): m/z: 266 [M+H]+; Anal. calcd for C17H15NO2: C
76.96, H 5.70, N 5.28, found: C 76.93, H 5.66, N 5.26.
In conclusion, by using a simple design strategy and very
simple chemistry, we accessed a novel class of antibacterial
agents that target resistant S. aureus strains through an EPI ac-
tivity. The most promising derivative (6c) can be prepared in
two steps with high yield. The lack of toxicity as evidenced by
cellular and in vivo investigations make the lead compound
(6c) a serious candidate for preclinical trials as a novel antibac-
terial agent.
5,7-Dimethoxy-4-methyl-3-phenylquinolin-2-one (5c): White
powder (841 mg starting from 3 mmol of the corresponding amide
4, 98%); Rf =0.2 (CH2Cl2/MeOH, 99:1); mp: 266–2688C; 1H NMR
(400 MHz, [D6]DMSO): d=11.57 (s, 1H), 7.37–7.29 (m, 2H), 7.29–
7.26 (m, 1H), 7.14–7.12 (m, 2H), 6.44 (ls, 1H), 6.30 (ls, 1H), 3.78 (s,
3H), 3.76 (s, 3H), 2.28 ppm (s, 3H); MS (ESI+): m/z: 296 [M+H]+,
318 [M+Na]+; Anal. calcd for C18H17NO3: C 73.20, H 5.80, N 4.74,
found: C 73.15, H 5.79, N 4.72.
5,7-Dimethoxy-3-(4-methoxyphenyl)-4-methylquinolin-2-one
(5d): White powder (390 mg starting from 1.5 mmol of the corre-
sponding amide 4, 80%); Rf =0.19 (CH2Cl2/MeOH, 99:1); mp: 282–
Experimental Section
Chemistry
1
2848C; H NMR (400 MHz, [D6]DMSO): d=12.12 (s, 1H), 7.24 (d, J=
General: NMR spectra were recorded on a Bruker AC-400 instru-
ment (400 MHz). Electrospray ionization (ESI) mass spectra were ac-
quired by the Analytical Department of Grenoble University on an
Esquire 300 Plus Bruker Daltonis instrument with a nanospray inlet.
Combustion analyses were performed at the Analytical Department
of Grenoble University. IR spectra were collected on a Bruker
Vector 22 spectrometer. Thin-layer chromatography (TLC) used
Merck silica gel F-254 plates (thickness 0.25 mm). Flash chromatog-
raphy used Merck silica gel 60, 200–400 mesh. Unless otherwise
stated, reagents were obtained from commercial sources (Sigma–
Aldrich and Acros Organics) and were used without further purifi-
cation. Derivative 3 was prepared according to a previously report-
ed method from our laboratory.[22]
8.8 Hz, 2H), 6.95 (d, J=8.8 Hz, 2H), 6.37 (d, J=2.4 Hz, 1H), 6.21 (d,
J=2.4 Hz, 1H), 3.85 (s, 3H), 3.83 (s, 3H), 3.78 (s, 3H), 2.46 ppm (s,
3H); MS (ESI+): m/z: 326 [M+H]+, 348 [M+Na]+; Anal. calcd for
C19H19NO4: C 70.14, H 5.89, N 4.31, found: C 70.08, H 5.84, N 4.28.
5,7-Dimethoxy-3-(indol-3-yl)-4-methylquinolin-2-one (5e): White
powder (300 mg starting from 2 mmol of the corresponding amide
4, 45%); Rf =0.27 (CH2Cl2/MeOH, 95:5); mp: 3358C (decomposi-
1
tion); H NMR (400 MHz, [D6]DMSO): d=11.52 (s, 1H), 11.19 (s, 1H),
7.41 (m, 1H), 7.30 (s, 1H), 7.14 (m, 1H), 7.08 (m, 1H), 6.97 (m, 1H),
6.49 (ls, 1H), 6.34 (ls, 1H), 3.84 (s, 3H), 3,81 (s, 3H), 2.41 ppm (s,
3H); MS (ESI+): m/z: 335 [M+H]+, 357 [M+Na]+; Anal. calcd for
C20H18N2O3: C 71.84, H 5.43, N 8.38, found: C 71.81, H 5.40, N 8.34.
N-(2-Acetyl-5-methoxy-3methoxyethoxymethoxyphenyl)aryla-
mide (4): Acetophenone derivative 1, 2 or 3 was dissolved in dime-
thylformamide (DMF; 7 mLmmolÀ1) under argon atmosphere and
treated with Et3N (5 equiv). Bis(2-oxo-3-oxazolidinyl)phosphinic
chloride (BOP-Cl; 2 equiv) and arylacetic acid (2 equiv) were added.
The reaction was stirred at RT for 48 h, then a solution of NaHCO3
(5%, 10 mL) was added. The solution was partially evaporated in
vacuo, and the crude was extracted with AcOEt (2ꢃ20 mL),
washed with brine (2ꢃ10 mL), dried over MgSO4 and concentrated.
Purification over column chromatography (silica gel, CH2Cl2) afford-
ed the pure product.
5,7-Dimethoxy-4-methyl-3-(thiophen-2-yl)quinolin-2-one
White powder (284 mg starting from 1.5 mmol of the correspond-
(5 f):
ing amide 4, 63%); Rf =0.24 (CH2Cl2/MeOH, 99:1); mp: 3108C (de-
1
composition); H NMR (400 MHz, CDCl3): d=12.57 (s, 1H), 7.42 (m,
1H), 7.11 (m, 1H), 7.02 (m, 1H), 6.48 (ls, 1H), 6.23 (ls, 1H), 3.84 (s,
6H), 2.62 ppm (s, 3H); MS (ESI+): m/z: 302 [M+H]+; Anal. calcd for
C16H15NO3S: C 63.77, H 5.02, N 4.65, found: C 63.74, H 4.98, N 4.61.
Synthesis of derivatives 6a–d: Compound 5b or 5c (450 mg,
1.52 mmol) was dissolved in CH2Cl2 (15 mLmmolÀ1). BBr3 (1 equiv)
was slowly added, and after 3 h, H2O (5 mL) was added. The solu-
tion was filtered and washed with H2O (10 mL). Column chroma-
tography (silica gel) using a gradient of CH2Cl2/MeOH (100:0–95:5)
3-Aryl-4-methylquinolin-2-one (5): Amides 4 obtained from the
previous step were dissolved in tBuOH (6 mLmmolÀ1), and tBuOK
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allowed to purify 6a and separate the different hydroxylated iso-
mers 6b–d.
Susceptibility studies and MIC evaluation: Bacterial susceptibilities to
different molecules were assessed by the determination of antimi-
crobial minimal inhibitory concentration (MIC) values using the
broth microdilution method according to the Clinical and Labora-
tory Standards Institute (CLSI; Wayne, PA, USA) methodology and
the Comitꢀ de l’Antibiogramme de la Sociꢀtꢀ FranÅaise de Micro-
biologie (CASFM; Paris, France), using Mueller–Hinton II (MH II).[23,24]
The effect of combining test compound on the MICs of ciprofloxa-
cin was also investigated.
5-Hydroxy-4-methyl-3-phenylquinolin-2-one (6a): White powder
(188 mg, 50%); Rf =0.30 (CH2Cl2/MeOH, 97:3); mp: 3188C (decom-
position); 1H NMR (400 MHz, CD3OD): d=7.70 (d, J=8 Hz, 1H),
7.44–7.35 (m, 3H), 7.25 (d, J=8 Hz, 2H), 6.81–6.75 (m, 2H),
2.27 ppm (s, 3H); MS (ESI+): m/z: 252 [M+H]+; Anal. calcd for
C16H13NO2: C 76.48, H 5.21, N 5.57, found: C 76.45, H 5.18, N 5.55.
Toxicity: In vitro toxicity was evaluated by cell-survival experiments
using the MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide) and HME-1 cells as previously described.[26] In vivo
toxicity was evaluated on female BALB/cByJ mice (Charles River,
L’Arbresle, France) with intraperitoneal injections of compound 6c
(15 mgkgÀ1 per day) for 5 days and daily weighing of animals.
5,7-Dihydroxy-4-methyl-3-phenylquinolin-2-one
(6b):
Beige
powder (344 mg starting from 1.5 mmol of 5b and using 5 equiv
of BBr3, 86%); Rf =0.14 (CH2Cl2/MeOH, 95:5); mp: 3208C (decompo-
1
sition); H NMR (400 MHz, [D6]DMSO): d=11.34 (s, 1H), 9.82 (s, 1H),
7.38–7.30 (m, 3H), 7.15 (m, 2H), 6.20 (ls, 1H), 6.12 (ls, 1H), 3.34 (s,
3H), 2.33 ppm (s, 3H); MS (ESI+): m/z: 268 [M+H]+, 290 [M+Na]+;
Anal. calcd for C16H13NO3: C 71.90, H 4.90, N, 5.24, found: C 71.87,
H 4.86, N, 5.22.
5-Hydroxy-7-methoxy-4-methyl-3-phenylquinolin-2-one
(6c):
White powder (375 mg, 89%); Rf =0.33 (CH2Cl2/MeOH, 95:5); mp:
1
159–1608C; H NMR (400 MHz, [D6]DMSO): d=11.46 (s, 1H), 10.26
Evaluation of synergy
(s, 1H), 7.39–7.37 (m, 2H), 7.30 (m, 1H), 7.16 (m, 2H), 6.34 (ls, 1H),
6.22 (ls, 1H), 3.74 (s, 3H), 2.36 ppm (s, 3H); MS (ESI+): m/z: 282
[M+H]+; Anal. calcd for C17H15NO3: C 72.58, H 5.37, N 4.98, found:
C 72.54, H 5.33, N 4.95.
Checkerboard combination studies: The MICs of ciprofloxacin in
combination with compound 6c were determined on S. aureus
strains by the broth microdilution technique using a two-dimen-
sional checkerboard with twofold dilutions of each drug. Growth
control wells containing medium were included in each plate. Mi-
croplates were incubated for 24 h at 378C. The fractional inhibitory
concentration index (FICi) was the sum of the fractional inhibitory
concentration (FIC) of ciprofloxacin (FICcip) and the FIC of com-
pound 6c (FIC6c) calculated as follows: FICcip =(MIC of ciprofloxacin
in combination)/(MIC of ciprofloxacin alone) and FIC6c =(MIC of 6c
in combination)/(MIC of compound 6c alone). The synergy was de-
termined as follows using the lowest FICi value of the checker-
board: FICiꢀ0.5: synergy, FICi=0.5–1.0: additive activity, FICi>
1.0–2.0: no interaction, and FICi>2.0: antagonism.[27]
7-Hydroxy-5-methoxy-4-methyl-3-phenylquinolin-2-one
(6d):
Beige powder (17 mg starting from 1.5 mmol of 5b and using
1 equiv of BBr3, 4%); Rf =0.18 (CH2Cl2/MeOH, 95:5); mp: 290–
1
2928C (decomposition); H NMR (400 MHz, [D6]DMSO): d=11.50 (s,
1H), 10.08 (s, 1H), 7.40–7.36 (m, 2H), 7.31 (m, 1H), 7.15 (m, 2H),
6.35 (ls, 1H), 6.20 (ls, 1H), 3.79 (s, 3H), 2.30 ppm (s, 3H); MS (ESI+):
m/z: 282 [M+H]+; Anal. calcd for C17H15NO3: C 72.58, H 5.37, N
4.98, found: C 72.51, H 5.31, N 4.96.
3-Benzyl-5-hydroxy-7-methoxy-4-methylquinolin-2(1H)-one (6e):
Compound 6e was prepared according to two different methods:
a) the same method applied for the synthesis of 6a–d starting
from crude 3-benzyl-5,7-dimethoxy-4-methylquinolin-2-one, b) ac-
cording to scheme 2. Compound 6e was obtained as white
powder to give 6e as a white powder (259 mg according to
scheme 2 and starting from 1 mmol of 8), 88%): Rf =0.36 (CH2Cl2/
MeOH, 95:5); mp: 2788C (decomposition); 1H NMR (400 MHz,
[D6]DMSO): d=11.45 (s, 1H), 10.23 (s, 1H), 7.12–7.23 (m, 5H), 6.34
(d, J=2.45 Hz, 1H), 6.2 (d, J=2.45 Hz, 1H), 3.96 (s, 2H), 3.72 (s,
3H), 2.53 ppm (s, 3H); MS (ESI+): m/z: 296 [M+H]+, 318 [M+Na]+;
Anal. calcd for C18H17NO3: C 73.20, H 5.80, N 4.74, found: C 73.14, H
5.77, N 4.72.
Ethidium bromide efflux: The loss of ethidium bromide (EtBr) cation
from S. aureus strain 1199B loaded with EtBr was determined fluo-
rometrically.[7,28] Cells were grown overnight on MH agar and bac-
terial suspensions were realized (OD660 of 0.8 to 1) in buffer
(110 mm NaCl, 7 mm KCl, 50 mm NH4Cl, 0.4 mm Na2HPO4, 52 mm
Tris base and 0.2% glucose, adjusted pH 7.5 with HCl). Ethidium
loading of cells was accomplished by the addition of ethidium and
CCCP (final concentrations, 10 mgmLÀ1 and 100 mm, respectively).
After 20 min of incubation at 378C, cells were pelleted and then re-
suspended in fresh buffer with or without various concentrations
of compound 6c or reserpine, and the fluorescence of the suspen-
sion was monitored continuously (lex =530 nm, lem =600 nm).
Time–kill studies: Colony-forming units (CFU) of S. aureus strain
ATCC 29213 were determined in six-well plates using MH broth in-
oculated with exponentially growing bacteria to a final concentra-
tion of 106 CFUmLÀ1. Each assay included a growth control well
with no antibiotic, two wells with ciprofloxacin alone
(0.125 mgmLÀ1 or to 0.5 mgmLÀ1 final concentration), one well with
6c alone (31.25 mm=7.87 mgmLÀ1) and two wells with a combina-
tion of ciprofloxacin (0.125 or 0.5 mgmLÀ1) and 6c (31.25 mm). Cul-
tures were incubated at 378C, and samples were recovered hourly
between 1–5 h and after 22 h. Samples were serially diluted and
plated onto MH II agar media for colony counts.[29] Synergy and an-
tagonism were defined respectively as ꢁ2log10 CFUmLÀ1 fold de-
crease and ꢁ2log10 CFUmLÀ1 fold increase by the combination
compared with the most active single molecule.[30]
Biology
Bacterial strains: The strains of S. aureus used in this study were
ATCC 29213, the two mutated strains SA-1199B (over-expressing
norA and also possesses an A116E Grl A substitution) and SA-K1712
(norA-deleted) and their isogenic parents, respectively SA-1199 and
SA 8325-4[7] kindly provided by Dr. G. W. Kaatz (Detroit, USA). To
evaluate the spectrum of activity of 6c, ten methicillin-resistant
S. aureus (MRSA) and five vancomycin-intermediate S. aureus (VISA)
isolates were also employed as well as 16 Enterococcus isolates
(seven E. faecalis and nine E. faecium) kindly provided by Dr. S.
Boisset and Dr. O. Dumitrescu (Lyon, France).
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Received: November 28, 2012
Published online on && &&, 0000
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FULL PAPERS
Stop the resistance! 3-aryl-4-methyl-2-
quinolones are NorA efflux pump inhibi-
tors that are able to restore the sensitiv-
ity of resistant Staphylococcus aureus
bacteria toward fluoroquinolones. The
most active compound is nontoxic, can
be easily prepared in two steps, and
shows intrinsic antibacterial activity.
A. Dolꢀans-Jordheim, J.-B. Veron,
O. Fendrich, E. Bergeron,
A. Montagut-Romans, Y.-S. Wong,
B. Furdui, J. Freney, C. Dumontet,
A. Boumendjel*
&& – &&
3-Aryl-4-methyl-2-quinolones
Targeting Multiresistant
Staphylococcus aureus Bacteria
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ChemMedChem 0000, 00, 1 – 7
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