Synthesis of omeprazole analogues and evaluation of these as potential inhibitors of the multidrug efflux pump NorA of Staphylococcus aureus

Article abstract of DOI:10.1128/AAC.01306-05

A series of 11 pyrrolo[1,2-α]quinoxaline derivatives, 1a to 1k, sharing structural analogies with omeprazole, a eukaryotic efflux pump inhibitor (EPI) used as an antiulcer agent, was synthesized. Their inhibitory effect was evaluated using Staphylacoccus aureus strain SA-1199B overexpressing NorA. By determinations of the MIC of norfloxacin in the presence of these EPIs devoid of intrinsic antibacterial activity and used at 128 μg/ml, and by the checkerboard method, compound 1e (MIC decrease, 16-fold; fractional inhibitory concentration index [ΣFIC], 0.18) appeared to be more active than compounds 1b to 1d, reserpine, and omeprazole (MIC decrease, eightfold; ΣFIC, 0.31), followed by compounds 1a and 1f (MIC decrease, fourfold; ΣFIC, 0.37) and 1g to 1k (MIC decrease, twofold; ΣFIC, 0.50 to 0.56). By time-kill curves combining norfloxacin (1/4 MIC) and the most efficient EPIs (128 μg/ml), compound 1e persistently restored the bactericidal activity of norfloxacin (inoculum reduction, 3 log₁₀ CFU/ml at 8 and 24 h), compound If led to a delayed but progressive decrease in the number of viable cells, and compounds 1b to 1d and omeprazole acted synergistically (inoculum reduction, 3 log₁₀ CFU/ml at 8 h but further regrowth), while compound 1a and reserpine slightly enhanced norfloxacin activity. The bacterial uptake of norfloxacin monitored by high-performance liquid chromatography confirmed that compounds 1a to 1f increased antibiotic accumulation, as did reserpine and omeprazole. Since these EPIs did not disturb the Δψ and ΔpH, they might directly interact with the pump. A structure-activity relationships study identified the benzimidazole nucleus of omeprazole as the main structural element involved in efflux pump inhibition and highlighted the critical role of the chlorine substituents in the stability and efficiency of compounds 1e to 1f. However, further pharmacomodulation is required to obtain therapeutically applicable derivatives. Copyright

Full text of DOI:10.1128/AAC.01306-05

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 2007, p. 831–838
0066-4804/07/$08.00ϩ0 doi:10.1128/AAC.01306-05
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 51, No. 3
Synthesis of Omeprazole Analogues and Evaluation of These
as Potential Inhibitors of the Multidrug Efflux Pump NorA
of Staphylococcus aureusᰔ†
C´eline Vidaillac,1,2 Jean Guillon,1 Corinne Arpin,2 Isabelle Forfar-Bares,1 Boubakar B. Ba,3
Jean Grellet,3 St´ephane Moreau,1 Daniel-Henri Caignard,4
Christian Jarry,1 and Claudine Quentin2*
Equipe d’Accueil 2962, Laboratoire de Chimie-Physique et Min´erale,1 Equipe d’Accueil 525, Laboratoire de Microbiologie,2
and Equipe d’Accueil 525, Laboratoire de Pharmacocin´etique et Pharmacie Clinique, Facult´e de Pharmacie,3
Universit´e Victor Segalen Bordeaux 2, 146 rue L´eo Saignat, 33076 Bordeaux Cedex, France, and
Institut de Recherche Servier, 125 Chemin de Ronde, 78290 Croissy-sur-Seine, France4
Received 6 October 2005/Returned for modification 8 November 2005/Accepted 2 November 2006
A series of 11 pyrrolo[1,2-a]quinoxaline derivatives, 1a to 1k, sharing structural analogies with omeprazole, a
eukaryotic efflux pump inhibitor (EPI) used as an antiulcer agent, was synthesized. Their inhibitory effect was
evaluated using Staphylococcus aureus strain SA-1199B overexpressing NorA. By determinations of the MIC of
norfloxacin in the presence of these EPIs devoid of intrinsic antibacterial activity and used at 128 ␮g/ml, and by the
checkerboard method, compound 1e (MIC decrease, 16-fold; fractional inhibitory concentration index [⌺FIC], 0.18)
appeared to be more active than compounds 1b to 1d, reserpine, and omeprazole (MIC decrease, eightfold; ⌺FIC,
0.31), followed by compounds 1a and 1f (MIC decrease, fourfold; ⌺FIC, 0.37) and 1g to 1k (MIC decrease, twofold;
⌺FIC, 0.50 to 0.56). By time-kill curves combining norfloxacin (1/4 MIC) and the most efficient EPIs (128 ␮g/ml),
compound 1e persistently restored the bactericidal activity of norfloxacin (inoculum reduction, 3 log10 CFU/ml at
8 and 24 h), compound 1f led to a delayed but progressive decrease in the number of viable cells, and compounds
1b to 1d and omeprazole acted synergistically (inoculum reduction, 3 log10 CFU/ml at 8 h but further regrowth),
while compound 1a and reserpine slightly enhanced norfloxacin activity. The bacterial uptake of norfloxacin
monitored by high-performance liquid chromatography confirmed that compounds 1a to 1f increased antibiotic
accumulation, as did reserpine and omeprazole. Since these EPIs did not disturb the ⌬␺ and ⌬pH, they might
directly interact with the pump. A structure-activity relationships study identified the benzimidazole nucleus of
omeprazole as the main structural element involved in efflux pump inhibition and highlighted the critical role of the
chlorine substituents in the stability and efficiency of compounds 1e to 1f. However, further pharmacomodulation
is required to obtain therapeutically applicable derivatives.
In recent years, active efflux has become recognized as a major
component of microbial resistance to most classes of antibiotics.
This mechanism is mediated by efflux pumps, which are mem-
brane-associated transporters promoting antibiotic extrusion
from the cell via an energy-dependent process. Some efflux
pumps selectively expel specific antibiotics, while others, referred
to as multidrug resistance (MDR) pumps, export a broad array of
structurally unrelated compounds (26, 30, 33, 36). MDR is con-
ferred mainly by the efflux systems of the major facilitator super-
family (MFS) of gram-positive bacteria, with the most studied
pump being NorA of Staphylococcus aureus, while the resistance/
nodulation/division efflux systems are the major contributors in
gram-negative bacteria (26, 30, 33, 36).
tractive since a single EPI that is active against MDR pumps and
can be used as adjunct therapy should (i) decrease intrinsic resis-
tance, (ii) reverse acquired resistance, and (iii) reduce the emer-
gence of mutants that are highly resistant to a wide range of
existing antibiotics (22, 25, 27, 29, 39, 43). Among the candidate
EPIs, drugs that are in clinical use for other indications are of
interest, since a large amount of data with respect to their phar-
macokinetics and toxicity are already available (29). Several of
these nonantibiotics have been demonstrated to act as EPIs on
NorA and other MDR pumps of gram-positive organisms but at
concentrations that are too high to be achieved at therapeutic
dosages and/or too toxic. These drugs include reserpine (antihy-
pertensive agent) (1, 9, 16–18, 20, 21, 28, 47), verapamil (antiar-
rhythmic) (1, 47), omeprazole (antiulcer) (1), paroxetine (antide-
pressant) (16, 44), and chlorpromazine (neuroleptic) (17). A
pharmacomodulation of paroxetine has been conducted to iden-
tify the structural features involved in efflux pump inhibition and
design new, more potent EPIs (44). Chlorpromazine derivatives
have been compared to elucidate the way that they inhibit bac-
terial efflux pumps (16). However, no such investigations have
been performed with omeprazole, although it was the most active
bacterial EPI in a previous comparative study (1).
Strategies used to combat efflux-mediated resistance are based
on the search for either new antibiotics bypassing the efflux sys-
tems or efflux pump inhibitors (EPIs). The latter solution is at-
* Corresponding author. Mailing address: Laboratoire de Microbi-
ologie, UFR des Sciences Pharmaceutiques, Universit´e Victor Segalen
Bordeaux 2, 146 rue L´eo Saignat, 33076 Bordeaux Cedex, France.
Phone: (33) 5 57 57 10 75. Fax: (33) 5 56 90 90 72. E-mail: claudine
.quentin@bacterio.u-bordeaux2.fr.
† Supplemental material for this article may be found at http://aac
.asm.org/.
We previously described a novel approach to design pyr-
rolo[1,2-a]quinoxaline derivatives as interesting bioactive ana-
ᰔ Published ahead of print on 13 November 2006.
831

832
VIDAILLAC ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
of tubes containing each drug alone at the same concentrations were also included.
Tubes were assessed visually for growth after an 18-h incubation period at 37°C. The
effect of drug combinations was estimated at the point of maximal effectiveness by
the fractional inhibitory concentration index (⌺FIC), i.e., the sum of the fractional
inhibitory concentration of each drug, which in turn is defined as the MIC of each
drug when used in combination divided by the MIC of the drug when used alone (7).
The ⌺FIC data were interpreted according to the following criteria: synergy was
defined as an ⌺FIC of Յ0.5, addition was defined as 0.5 Ͼ ⌺FIC Յ 1, indifference
was defined as Ͼ1 ⌺FIC Ͻ2, and antagonism was defined as an ⌺FIC of Ն2. All
experiments were carried out at least three times, and results are expressed as the
mode values.
Time-kill curves. Interactions between norfloxacin and the most efficient EPIs or
the reference EPIs on SA-1199B were also evaluated by the time-kill curve method
(7). An initial bacterial inoculum of 106 CFU/ml in the logarithmic phase was
prepared as described above for MIC determinations. The final concentrations were
16 ␮g/ml of norfloxacin and 128 ␮g/ml of the EPIs. Surviving cells were enumerated
by dilution plating in triplicate onto MH agar at 0 (inoculum control), 2, 4, 8, and
24 h of incubation at 37°C. Results are expressed as the log percentage of survival
and represent the mean values of three different experiments. The limit of sensitivity
was set at 1 log10 CFU/ml to avoid any carryover effect.
Norfloxacin and omeprazole assays. For the uptake experiments, bacterial
cultures of SA-1199 and SA-1199B were grown to mid-exponential phase in 300
ml of MH broth in a shaken water bath for 4 to 5 h at 37°C. Cells were
centrifuged, pelleted, washed, and resuspended in sodium phosphate buffer (50
mM) at pH 7.0. The suspension was then divided into 0.2-ml aliquots, and
bacteria were enumerated by dilution plating as indicated above. Each suspen-
sion was incubated with norfloxacin at one-quarter the MIC alone or in combi-
nation with each EPI at 128 ␮g/ml at time zero (T0). After 5, 10, 15, and 20 min,
cells were separated from the extracellular medium by differential centrifugation
through a water-impermeable silicone-paraffin oil barrier (1.029 g/cm3) and
directly lysed by the orthophosphoric acid present in the bottom of the tube. The
amounts of norfloxacin in the lysate were determined by high-performance liquid
chromatography (HPLC) with a Supercosil C18 column (Waters, Guyancourt,
France). The mobile phase consisted of 80% phosphate buffer (H3PO4-KH2PO4
[10 mM] at pH 2.5) and 20% acetonitrile. The excitation and emission wave-
lengths were 240 and 280 nm, respectively. Protein concentrations were deter-
mined with a commercially available kit based on the use of bicinchoninic acid
and bovine serum albumin as standards (Pierce, IL). Results were expressed as
ng drug accumulated/mg cell protein. Experiments were done in triplicate, and
results given are the means of the three experiments. The norfloxacin and
omeprazole concentrations were also determined during the time-kill curve
experiments in the supernatants at time zero T0 and at 24 h (T24) by HPLC using
the conditions indicated above for the antibiotic. For omeprazole and com-
pounds 1a to 1f, the mobile phase consisted of 65% phosphate buffer (50 mM
Na2HPO4, pH 6.5) and 35% acetonitrile. The wavelength of detection was 302
nm for omeprazole (48), 280 nm for compounds 1a to 1d, and 300 nm for
compounds 1e to 1f.
⌬␺ and ⌬pH determinations. Accumulation of the membrane-permeant cat-
ion tetraphenylphosphonium and salicylic acid was used to measure the trans-
membrane electrical potential, ⌬␺, and the ⌬pH, respectively, in the absence or
presence of reserpine (33 ␮M), carbonyl cyanide m-chlorophenylhydrazone
(CCCP) (100 ␮M), omeprazole, or the new compounds 1a to 1f (128 ␮g/ml).
After incubation in a shaken bath for 4 h at 37°C, MH broth cultures were
divided into aliquots that were placed in ice. The protein content of one aliquot
was determined as indicated above. These samples were then warmed for 5 min
to room temperature. To measure the ⌬␺, cells were labeled with 1 ␮M [3H]tet-
raphenylphosphonium (29 Ci/mmol; Amersham Biosciences Corp., Bucking-
hamshire, United Kingdom) for 10 min in the presence or absence of the EPIs.
One aliquot was treated with 4% DMSO (vol/vol) for 10 min prior to labeling in
order to obtain a background value. For the ⌬pH, EPIs were added to samples
for 20 min. Cells were then labeled with 20 ␮M [14C]salicylic acid (47 mCi/mmol;
Perkin-Elmer, Wellesley, MA). For both experiments, samples were centrifuged
for 10 min at 4°C and 13,000 ϫ g, and the radioactivity present in both phases
(cells and supernatant) was measured using a scintillation counter. The ⌬␺ and
⌬pH were calculated by using a cell volume of 4.2 ␮l/mg of cell protein as
previously described (17, 38). The results of these experiments, repeated three
times, were expressed as the mean values Ϯ standard deviations.
logues of pyridine, quinoxaline, or quinoline derivatives (11, 12).
The aim of this study was to design a series of new pyrrolo[1,2-
a]quinoxaline compounds mimicking the omeprazole structure
and to evaluate their efficiency as EPIs in a model targeting NorA
by rationally selected tests. A preliminary structure-activity rela-
tionship study allowed us to identify the structural elements of
these new omeprazole analogues implicated in EPI activity.
MATERIALS AND METHODS
Chemical syntheses: general procedures for preparation. (i) 2-[{(Pyrrolo[1,
2-a]quinoxalin-4-yl)methyl}thio]benzimidazoles 6a to 6g, 2-[{(pyrrolo[1,2-a]qui-
noxalin-4-yl)methyl}thio]imidazoles 6h to 6i, 2-[{(pyrrolo[1,2-a]quinoxalin-4-
yl)methyl}thio]pyridine 6j, and 2-[{(pyrrolo[1,2-a]quinoxalin-4-yl)methyl}thio]-
7-methoxypyrrolo[1,2-a]quinoxaline 6k.
Sodium hydroxide (9.23 mmol) was slowly added over 5 min to a stirred
solution of a 2-mercapto derivative (4.62 mmol) in ethanol (30 ml). A solution of
4-chloromethylpyrrolo[1,2-a]quinoxaline 5a to 5d (4.62 mmol) in 40 ml of etha-
nol was slowly added to the 2-mercapto derivative solution at 0°C and stirred for
16 h at room temperature. After the solvent was removed under reduced pres-
sure, the residue was poured into a 2.5% NaOH solution and extracted with
chloroform. The organic layer was dried over Na2SO4, and concentrated to give
compounds 6a to 6k.
(ii) 2-[{(Pyrrolo[1,2-a]quinoxalin-4-yl)methyl}sulfinyl]benzimidazoles 1a to 1g,
2-[{(pyrrolo[1,2-a]quinoxalin-4-yl)methyl}sulfinyl]imidazoles 1h and 1i, 2-[{(pyr-
rolo[1,2-a]quinoxalin-4-yl)methyl}sulfinyl]pyridine 1j, and 2-[{(pyrrolo[1,2-a]qui-
noxalin-4-yl)methyl}sulfinyl]-7-methoxypyrrolo[1,2-a]quinoxaline 1k.
A solution of m-chloroperbenzoic acid (2 mmol) in chloroform (20 ml) was
added dropwise to ice-cooled solutions of compounds 6a to 6k (2 mmol) in
chloroform (35 ml). The reaction mixture was stirred at 0°C for 1 h and then
washed with a saturated NaHCO3 solution and dried (Na2SO4). After removal of
the solvent, the residue was triturated in ethyl acetate, filtered, washed with ethyl
acetate, dried, and recrystallized from ethanol to give compounds 1a to 1k (see
the supplemental material).
Bacterial strains and media. The wild-type clinical isolate S. aureus SA-1199
and its overproducing NorA mutant strain SA-1199B (18, 19, 21) were gener-
ously provided as gifts from G. W. Kaatz (University of Michigan). These strains
were routinely cultured on Mueller-Hinton (MH) agar (Bio-Rad, Marnes-la-
Coquette, France) and broth adjusted to contain 20 ␮g/ml of Ca2ϩ and 10 ␮g/ml
of Mg2ϩ (AES, Bruz, France) at 37°C. They were stored in 30% glycerol–brain
heart broth at Ϫ80°C.
Antibiotic and chemicals. Norfloxacin, reserpine, and omeprazole were pur-
chased from Sigma-Aldrich (Saint Quentin Fallavier, France). Solutions of re-
serpine, omeprazole, and the candidate inhibitors 1a to 1k were extemporane-
ously prepared in 100% dimethyl sulfoxide (DMSO), but the highest final
concentration of DMSO present in all assays (4%, vol/vol) caused no inhibition
of bacterial growth (data not shown).
Antibiotic susceptibility tests. Antibiotic susceptibilities of SA-1199, SA-1199B,
and the survivors of the time-kill curve experiments were determined by the agar
diffusion method (31) using MH medium alone or supplemented with EPIs at 128
␮g/ml. Six antibiotics were tested, including all available fluoroquinolone disks, i.e.,
norfloxacin (5 ␮g), ciprofloxacin (5 ␮g), ofloxacin (5 ␮g), levofloxacin (5 ␮g), spar-
floxacin (5 ␮g), moxifloxacin (5 ␮g), and chloramphenicol (30 ␮g). After overnight
incubation at 37°C, the inhibition zone diameters were measured. Results are ex-
pressed as the mean values of multiple (at least three) independent experiments.
MICs of norfloxacin and EPIs were determined for SA-1199, SA-1199B, and
the survivors of the time-kill experiments by a broth dilution method in tubes
under standard conditions (31). Bacterial suspensions were prepared from an
MH broth culture obtained after incubation at 37°C in a stirred water bath for 4
to 5 h and further diluted 1/10 in MH broth (Ϸ106 CFU/ml). Norfloxacin
concentrations ranged between 128 and 0.1 ␮g/ml, and the EPI concentrations
ranged between 512 and 1 ␮g/ml. In a second series of experiments, antibiotic
solutions were combined with each EPI at a final concentration of 128 ␮g/ml.
After an 18-h incubation period at 37°C, the MIC was defined as the lowest
concentration that inhibited any visible growth. All tests were done at least in
triplicate, and the mode values were retained.
Checkerboard assay. Interactions between norfloxacin and the EPIs on SA-
1199B and the survivors of the time-kill experiments were evaluated by a checker-
board titration assay in tubes (7). The bacterial inoculum was prepared as described
above for MIC determinations. Norfloxacin was tested at seven concentrations (64
to 1 ␮g/ml), and each EPI was tested at six concentrations (512 to 16 ␮g/ml). Rows
RESULTS
Chemistry. A series of new pyrrolo[1,2-a]quinoxaline deriv-
atives, compounds 1a to 1k (Fig. 1), has been synthesized by

VOL. 51, 2007
OMEPRAZOLE ANALOGUES AS POTENTIAL NorA INHIBITORS
833
FIG. 1. Synthesis of pyrrolo[1,2-a]quinoxaline derivatives 1a to 1k, new structural analogues of omeprazole.
the pharmacomodulation of a basic heterocyclic synthon re-
lated to the structure of omeprazole. This synthesis has been
achieved by starting from various substituted 2-nitro-anilines in
six steps. The Clauson-Kaas reaction (8) of anilines with 2,5-
dimethoxytetrahydrofuran in acetic acid gave the pyrrolic de-
rivatives 2a to 2d (12), which were reduced using BiCl3-NaBH4
treatment to provide the expected 1-(2-aminophenyl)pyrroles
3a to 3d (37). The reaction of chloroacetyl chloride with 3a to
3d (12) led to chloracetamides 4a to 4d (10). The 4-chloro-
methylpyrrolo[1,2-a]quinoxalines 5a to 5d were prepared by

834
VIDAILLAC ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 1. In vitro activities of reserpine, omeprazole, and the
pyrrolo͓1,2-a͔quinoxaline derivatives 1a to 1k as EPIs by
the disk diffusion method for SA-1199Ba
tives 1a to 1k were found to lack any intrinsic antibacterial
activity (MIC Ͼ 512 ␮g/ml).
In a first step of screening, the high-throughput disk diffu-
sion method was used to characterize the profiles of the test
strains, to identify the preferential substrate and the optimal
concentration of the reference EPIs, and to detect any EPI
activity of the tested compounds. SA-1199B exhibited reduced
inhibition zones towards fluoroquinolones, particularly nor-
floxacin and ciprofloxacin, and a slightly decreased inhibition
zone towards chloramphenicol compared to SA-1199 (Table
1). At concentrations less than 32 ␮g/ml, omeprazole had no
effect on norfloxacin and ciprofloxacin activity, while at 128
␮g/ml, it was evident. Thus, the latter concentration was sub-
sequently used for all tested EPIs in order to maximize the
chance of observing an effect. Actually, at 128 ␮g/ml, all new
synthesized compounds increased the inhibition zones of the
fluoroquinolones, particularly those of the most affected drugs,
norfloxacin and ciprofloxacin, and thus acted as EPIs. In a
second step of screening, these data were confirmed by MIC
determinations. S. aureus strain SA-1199B exhibited norfloxa-
cin MICs that were 32 times higher than those for SA-1199,
and the presence of each EPI at 128 ␮g/ml resulted in a
reduction in the norfloxacin MIC (2- to 16-fold) for SA-1199B
and, to a much lesser degree, for SA-1199 (Table 2). Com-
pound 1e appeared to be the most active, as determined by the
disk diffusion method.
EPI
concn
(␮g/ml)
Inhibition zone diameter (mm)
Strain
EPI
Nor Cip Ofx Lvx Spx Mxf
C
SA-1199
27
26
27
30
30
31
27
SA-1199B
6
13
12
12
9
13
22
20
18
16
22
19
15
13
19
22
24
24
25
19
15
15
14
17
17
18
21
20
19
19
23
20
18
18
23
25
25
25
26
21
20
19
19
20
19
21
24
24
24
22
25
23
21
21
24
24
24
24
27
23
23
22
22
22
22
27
28
27
27
27
28
27
27
26
28
28
27
29
28
27
27
27
27
27
27
27
28
28
28
28
30
29
27
27
28
28
28
28
28
27
27
27
27
27
27
24
25
25
24
24
25
24
24
24
25
26
26
27
27
25
25
25
25
25
25
Res
Om
128
64
32
16
128
64
32
16
128
128
128
128
128
128
128
128
128
128
128
14
12
6
6
1a
1b
1c
1d
1e
1f
1g
1h
1i
10
13
15
16
18
10
6
6
6
1j
1k
6
6
a Abbreviations: Res, reserpine; Om, omeprazole; Nor, norfloxacin; Cip, cip-
rofloxacin; Ofx, ofloxacin; Lvx, levofloxacin; Spx, sparfloxacin; Mxf, moxifloxacin;
C, chloramphenicol.
Effect of norfloxacin and EPI combinations by the checker-
board method. In a third step of screening, the combinations
between the preferential substrate and the EPIs were investi-
gated by both reference methods. The checkerboard method
aims at evaluating the bacteriostatic effect of antibiotic com-
binations according to their concentrations at a fixed time of
18 h (7). This method was applied to the combination of
norfloxacin with reserpine, omeprazole, and each new com-
pound, compounds 1a to 1k, on SA-1199B, and the interpre-
tation was adapted to antibiotic-EPI combinations by calculat-
ing the ⌺FIC with the MIC of these atoxic EPIs taken as 1,024
␮g/ml, i.e., the value that was twice the maximal concentration
tested. By this mean, reserpine, omeprazole, and the five can-
didate inhibitors 1a to 1f appeared to be synergistic on SA-
1199B, while the other new compounds, compounds 1g to 1k,
were only additive. The rank order of decreasing activity of the
EPIs was identical to that found by MIC determinations, i.e.,
compound 1e was more active than reserpine, omeprazole, and
the cyclization of these amides, 4a to 4d, in refluxing phospho-
rus oxychloride according to the Bischler-Napieralski reaction
procedure (3, 45). The 4-chloromethylpyrrolo[1,2-a]quinoxa-
lines 5a to 5d (10) were then condensed with various substi-
tuted 2-mercapto derivatives in ethanol in the presence of
sodium hydroxide to give the corresponding sulfides 6a to 6k
(6, 15). The sulfoxides 1a to 1k were obtained by low-temper-
ature oxidation of sulfides 6a to 6k with m-chloroperbenzoic
acid in chloroform (4, 23).
Antibiotic and EPI susceptibilities of the S. aureus strains. A
strategy including four steps has been designed to investigate
the efficiency of new EPIs in a model targeting NorA and using
SA-1199 and SA-1199B. In preliminary experiments, the MICs
of the reference and putative EPIs were determined in order to
subsequently use them at concentrations devoid of bacterial
toxicity and hence to evaluate their efficiency only as EPIs.
Reserpine, omeprazole, and pyrrolo[1,2-a]quinoxaline deriva-
TABLE 2. In vitro activities of reserpine, omeprazole, and the pyrrolo͓1,2-a͔quinoxaline derivatives 1a to 1k as EPIs by
norfloxacin MIC determinations for SA-1199 and SA-1199B and by the checkerboard method for SA-1199Ba
MIC (␮g/ml) of Nor ϩ EPI:
Nor MIC EPI concn
Strain
(␮g/ml)
(␮g/ml)
Res
Om
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
2
SA-1199
SA-1199B
2
64
128
128
1
8
1
8
1
1
8
1
8
1
8
1
4
1
16
2
2
32
2
32
2
32
16
16/32 16/64 16/128 16/64 16/64 16/64 8/64 16/128 32/64 32/64 32/64 32/64 16/256
0.31 0.31 0.37 0.31 0.31 0.31 0.18 0.37 0.56 0.56 0.56 0.56 0.5
32
32
Optimal concn (␮g/ml)b
⌺FIC
a Abbreviations: Nor, norfloxacin; Res, reserpine; Om, omeprazole.
b Optimal norfloxacin/EPI concentrations by the checkerboard method, at which the ⌺FIC was calculated.

VOL. 51, 2007
OMEPRAZOLE ANALOGUES AS POTENTIAL NorA INHIBITORS
835
FIG. 2. Time-kill curves of SA-1199B for norfloxacin alone or combined with the EPIs. Norfloxacin was used at one-quarter the MIC (16 ␮g/ml)
alone (N) or combined with omeprazole (NϩOm) and (a) reserpine (NϩRes) or new derivatives 1a to 1d (Nϩ1a-d) and (b) 1e and 1f (Nϩ1e-f)
at 128 ␮g/ml.
compounds 1b to 1d, followed by compounds 1a, 1f, and 1g to
1k (Table 2).
(54 Ϯ 8 ␮g/ml), and compound 1a (72 Ϯ 9 ␮g/ml), while
compounds 1e and 1f appeared to be more stable (98 Ϯ 9
␮g/ml at T24). Consequently, the inhibitory effect of the EPIs in
time-kill experiments partially paralleled their stability.
Effect of EPIs on norfloxacin bacterial uptake. Finally, a
fourth step ensured that the previously demonstrated synergis-
tic effect was actually related to the increase in the antibiotic
substrate accumulation by the putative EPIs. The intrabac-
terial accumulation of norfloxacin at 16 ␮g/ml alone or com-
bined with the reference and the most efficient EPIs at 128
␮g/ml was determined by HPLC. In SA-1199 (Fig. 3a), the
accumulation of norfloxacin reached near equilibrium at 5 min
and thereafter remained at the same level up to 20 min, at
around 400 to 450 ng/mg cell protein. The addition of reser-
pine, omeprazole, and compounds 1a to 1f barely increased the
final level of norfloxacin accumulation (10%). In SA-1199B
(Fig. 3b), the accumulation of norfloxacin was significantly
lower than that observed in SA-1199, reaching 235 ng/mg cell
protein at 5 min and progressively up to 350 ng/mg cell protein
at 20 min. The addition of all EPIs substantially increased the
norfloxacin accumulation in SA-1199B, up to 270 to 315 ng/mg
cell protein at 5 min and around 400 to 450 ng/mg cell protein
at 20 min, increasing by 40% of the final level of norfloxacin
accumulation. Thus, the synergistic effect demonstrated by the
EPIs was correlated with the partial restoration of norfloxacin
uptake.
Effect of the inhibitors on the ⌬␺ and ⌬pH. In order to
investigate the basis of the EPI effect of omeprazole and re-
lated compounds on NorA, the ⌬␺ and ⌬pH of the cells were
determined for SA-1199B, alone and in the presence of these
drugs, compared to reserpine and CCCP, used as controls. The
⌬␺ of SA-1199B cells in MH broth was Ϫ126 mV Ϯ 6 mV. In
the presence of reserpine or omeprazole, this value was slightly
lowered, to Ϫ140 Ϯ 10 mV and Ϫ140 Ϯ 6 mV, respectively,
while in the presence of the uncoupler agent CCCP, it became
null. Compounds 1a to 1f had activity similar to that of omepra-
zole, leading to a ⌬␺ of Ϫ130 Ϯ 10 mV. The ⌬pH of SA-1199B
in MH was ϩ0.55 Ϯ 0.05 over the entire 20-min time course of
the experiment. The addition of reserpine, omeprazole, or com-
pounds 1a to 1f had no effect (ϩ0.55 Ϯ 0.05). In contrast, the
⌬pH was abolished by CCCP (⌬pH ϭ 0 Ϯ 0.1). Thus, the putative
Effect of norfloxacin and EPI combinations by the time-kill
curve method. The objective of the time-kill curve method is to
evaluate the bactericidal effect of antibiotic combinations ac-
cording to the time at some selected concentrations (7). This
method was applied to the combination of norfloxacin (one-
quarter the MIC, i.e., 16 ␮g/ml) with the reference EPIs,
omeprazole and reserpine, and the most active compounds,
compounds 1a to 1f, at 128 ␮g/ml, on SA-1199B. Norfloxacin
(Fig. 2) or the EPIs alone (data not shown) at the concentra-
tions used did not significantly inhibit the growth of the test
organism (difference Յ1 log10 CFU/ml). According to the
usual definition, all tested combinations appeared to be syner-
gistic by this method since they decreased the bacterial growth
significantly more than either norfloxacin or each EPI alone.
Indeed, the combination of norfloxacin with the reference
EPIs showed that reserpine, after a short initial decrease in
viable cells, hardly enhanced the norfloxacin activity. In con-
trast, omeprazole produced a 4-log10 decrease of the starting
inoculum at 8 h, followed by a regrowth, leading to a bacterial
count similar to that of the inoculum at 24 h. Compound 1a
behaved similarly to reserpine. Compounds 1b to 1d gave
curves similar to those for omeprazole but were less active in
reversing norfloxacin activity, particularly at 24 h. Finally, com-
pound 1f led to a delayed but progressive decrease in viable
cells, while compound 1e demonstrated the strongest activity,
yielding to a deeper decrease in the initial inoculum at 8 h and
persistently restoring the bactericidal activity of norfloxacin
(Fig. 2b). Surviving cells recovered in each experiment were
tested for their antibiotic and/or EPI susceptibilities. They
exhibited the same disk diffusion pattern as SA-1199B and
gave the same norfloxacin MIC (64 ␮g/ml) and the same ⌺FIC
(0.18) by the checkerboard method as SA-1199B, simulta-
neously used as control. Thus, survivors were not mutants with
increased resistance to norfloxacin or EPI inhibition. The
HPLC assay showed that during the time kill curve experi-
ments, norfloxacin concentrations were identical at T0 (16 Ϯ
0.8 ␮g/ml) and T24 (16 Ϯ 1.5 ␮g/ml). In contrast, the EPI
concentrations considerably decreased from T0 (128 Ϯ 10 ␮g/
ml) to T24 for omeprazole (40 Ϯ 8 ␮g/ml), compounds 1b to 1d

836
VIDAILLAC ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 3. Intracellular accumulation of norfloxacin alone or combined with EPIs. Norfloxacin was used at one-quarter the MIC (16 ␮g/ml) alone
(N) or combined with omeprazole (NϩOm) and (a) reserpine (NϩRes) or new derivatives 1a to 1d (Nϩ1a-d) and (b) 1e and 1f (N ϩ 1e-f) at 128
␮g/ml. Intracellular accumulation of norfloxacin in (a) SA-1199 and (b) SA-1199B is shown.
EPIs did not significantly influence the electrical potential and the
trans-membrane pH gradient of the bacterial cells, at least at the
concentrations used.
aureus (1, 20–22, 32). However, SA-1199B exhibited high-level
fluoroquinolone resistance that was not completely reversed by
the EPIs, as particularly apparent in norfloxacin accumulation
experiments. In fact, SA-1199B is a double-target (A116E in
GrlA)/efflux mutant (21). A lesser affinity of the drug for its
modified target might lead to a decreased accumulation into
the bacterial cell that cannot be amended by EPIs. Accord-
ingly, we observed a total restoration of the norfloxacin sus-
ceptibility by reserpine, using MIC determinations, for a single
NorA mutant (SA-1) selected in our laboratory (2). Although
SA-1199B has been used mostly to investigate the EPI activity
on NorA, allowing comparisons to data reported in the liter-
ature, a single NorA mutant such as SA-1 might be more
convenient for further studies.
By this strategy, all novel derivatives appeared to have EPI
properties, and one compound (compound 1e) was consis-
tently more active than omeprazole. Indeed, compound 1e at
128 ␮g/ml reduced the norfloxacin MIC for SA-1199B by 16-
fold. The “minimum potentiating concentration” required to
enhance the activity of the preferential substrate against a test
strain by at least eightfold (22), which was also the “minimal
effective concentration” of EPI that produced the maximal
reduction in the substrate MIC (32), was 64 ␮g/ml, and the
lowest active concentration (i.e., capable of reducing the nor-
floxacin MIC by twofold) was 32 ␮g/ml. At concentrations
Ն256 ␮g/ml, compound 1e shifted the norfloxacin MIC to 4
␮g/ml. Other commercially available drugs such as verapamil
paroxetine, chlorpromazine, and their derivatives (1, 47), the
novel P-gp inhibitors currently being developed for the treat-
ment of drug resistance tumors (9, 22, 32), and EPIs selected
by the screening of plant extracts (34) exhibited similar poten-
tiating effects on NorA. The screening of a chemical library
and the further synthesis of indole derivatives have yielded
inhibitors of NorA with a much higher potency (28, 41), but
their clinical safety has not been established. However, al-
though proton pump inhibitors are well-tolerated, maximal
serum concentrations following usual doses are well below the
concentrations at which compound 1e acted as an EPI (5, 14).
This limited series of 11 pyrrolo[1,2-a]quinoxaline deriva-
tives, designed as omeprazole analogues, has been synthesized
in order to initiate a preliminary structure-activity relationship
DISCUSSION
In this study, a series of 11 new pyrrolo[1,2-a]quinoxaline
derivatives, compounds 1a to 1k, was synthesized from com-
mercially available nitroanilines and developed as a template
for the design of new bioisosters of omeprazole by various
pharmacomodulations in order to obtain new compounds en-
dowed with a higher activity as bacterial EPIs. These deriva-
tives were then evaluated in a model targeting the NorA efflux
pump of S. aureus (18–21, 28, 42, 47). Indeed, although NorA
modestly contributes to wild-type fluoroquinolone resistance,
first-step mutants, by diminishing the intracellular drug con-
centrations, tend to subsequently accumulate additional target
mutations under treatment, leading to the commonly encoun-
tered high-level fluoroquinolone-resistant clinical strains (2).
Moreover, NorA is the prototype of other MFS pumps with 12
transmembrane segments, such as PmrA in Streptococcus pneu-
moniae (35), and has generally served as the model for study-
ing EPIs of MDR pumps in gram-positive organisms (22). EPIs
that are active on NorA are hence expected to inhibit at least
some of the related pumps.
The model used to assess the potential EPI activity of the 11
new pyrrolo[1,2-a]quinoxaline derivatives, compounds 1a to
1k, was based on the isogenic strains SA-1199 and SA-1199B,
classically used to investigate NorA inhibition. Our results re-
garding the susceptibilities of these strains to norfloxacin and
the reference EPIs by MIC determinations (1, 9, 16, 17, 19–21,
32, 42), time-kill curves (1), and accumulation assays (16, 42,
46) were in agreement with the literature. The antibiotic re-
sistance and inhibition profile of SA-1199B reflected the over-
expression of NorA, a pump that preferentially exports the
hydrophilic fluoroquinolones pefloxacin and ciprofloxacin
rather than the more hydrophobic drugs of this class, sparfloxa-
cin and moxifloxacin, and has a limited effect on the neutral
chloramphenicol (18–21, 28, 42, 47). The slight potentiation of
norfloxacin by the EPIs on wild-type strain SA-1199 is related
to the low-level expression of NorA in wild-type strains of S.

VOL. 51, 2007
OMEPRAZOLE ANALOGUES AS POTENTIAL NorA INHIBITORS
837
study, allowing further pharmacomodulations. Biological re-
sults highlighted the importance of the benzimidazole moiety
(compounds 1a to 1f), since its replacement by an imidazole
(compounds 1h and 1i), a pyridine (compound 1j), or a pyr-
rolo[1,2-a]quinoxaline ring (compound 1k) resulted in a signif-
icant loss of potency. Moreover, methoxy-substituted com-
pounds 1b to 1f, whatever the substitution position, on either
the benzimidazole or the pyrrolo[1,2-a]quinoxaline nucleus,
were more potent that the unsubstituted compound 1a. The
introduction of a second methoxy substituent (compound 1d)
was not beneficial compared to the monosubstituted deriva-
tives 1b and 1c. Otherwise, the introduction of one chlorine
atom at position 7 or 8 of the pyrroloquinoxaline heterocycle
provided an efficient restoration of the bactericidal activity of
norfloxacin (compounds 1e and 1f compared to compound 1c).
Nevertheless, the 7-chloro-substituted compound 1e was more
active than its 8-cholor-substituted homologue, compound 1f.
By reference to the omeprazole chemical structure, the re-
placement of the pyridine ring by a pyrrolo[1,2-a]quinoxaline
nucleus (compounds 1a to 1f) did not substantially change the
EPI activity. However, whatever the other structural modifica-
tions were, omeprazole and pyrrolo[1,2-a]quinoxaline ana-
logues 1a to 1f acted as EPIs on the NorA efflux pump only at
high concentrations (64 to 128 ␮g/ml). Finally, the N-methyl-
ated compound 1g, compared to compound 1a, was devoid of
EPI activity, suggesting the importance of the amino benzimid-
azolyl proton in the EPI activity.
This structure-activity relationship study might also contrib-
ute to analyses of the mode of action of omeprazole and its
analogues as bacterial EPIs. Indeed, the inhibition of MFS
pumps in gram-positive organisms, such as the single-compo-
nent system NorA, can be due either to the disturbance of the
source of energy or to a direct interaction with the pump by
steric obstruction or substrate competition. Compounds 1a to
1f, as omeprazole and reserpine (17), did not significantly
modify the electrical potential and the trans-membrane pH
gradient, in contrast to CCCP (17), and thus did not seem to
alter the source of energy of this proton-dependent pump.
Hence, these EPIs might interact directly with NorA. How-
ever, a mode of action analogous to that of omeprazole with
the eukaryotic Hϩ/Kϩ pump is unlikely, since the pH is not
favorable for the activation of our compounds into a reactive
sulfenamide (13, 14). Cloning of the norA gene and random
mutagenesis followed by the selection of isolates resistant to
the combination of norfloxacin and omeprazole, as previously
performed for Bmr and reserpine in Bacillus subtilis and re-
serpine (24), would be helpful to elucidate the mode of action
of these EPIs.
bility of the nonchlorinated compounds than the chlorinated
ones. It also might be due to the reversible versus irreversible
binding of these EPIs to the lipophilic binding pocket of NorA:
EPI removal should allow the pump to function de novo,
conferring norfloxacin resistance, thus permitting the bacterial
multiplication to start again. Indeed, such reversible binding
has been observed for omeprazole and the Hϩ/Kϩ-ATPase
(40). Finally, one cannot exclude that compounds 1a to 1d
transiently induced efflux pumps other than NorA, in contrast
with compounds 1e and 1f.
In conclusion, a series of new omeprazole analogues has
been synthesized and evaluated by a cost-effective and time-
saving general strategy. Concordant results showed that all
novel derivatives had EPI properties, and one compound
(compound 1e) was more active than omeprazole, particularly
in restoring the bactericidal activity of norfloxacin over a pro-
longed period. A structure-activity relationship study identified
the benzimidazole nucleus as the key element of efflux pump
inhibition and the chlorine at position 7 of the pyrroloquinoxa-
line heterocycle as a critical substitution. However, further
pharmacomodulation is required to obtain therapeutically ap-
propriate EPIs.
ACKNOWLEDGMENTS
This work was supported by the Institut de Recherche SERVIER
(fellowship attributed to C.V.).
We are grateful to J. Lestage for assistance in the HPLC studies and
M. Hugues for helpful discussion on ⌬␺ and ⌬pH experiments. We
thank an anonymous reviewer for critical review of the manuscript and
pertinent suggestions.
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