Synthesis of Omeprazole Analogues and Evaluation of These as Potential Inhibitors of the Multidrug Efflux Pump NorA of Staphylococcus aureus

A series of 11 pyrrolo[1,2-a]quinoxaline derivatives, 1a to1k, sharing structural analogies with omeprazole, a eukaryoticefflux pump inhibitor (EPI) used as an antiulcer agent, wassynthesized. Their inhibitory effect was evaluated using Staphylococcusaureus strain SA-1199B overexpressing NorA. By determinationsof the MIC of norfloxacin in the presence of these EPIs devoidof intrinsic antibacterial activity and used at 128 µg/ml,and by the checkerboard method, compound 1e (MIC decrease, 16-fold;fractional inhibitory concentration index [ ${Sigma}$ FIC], 0.18) appearedto be more active than compounds 1b to 1d, reserpine, and omeprazole(MIC decrease, eightfold; ${Sigma}$ FIC, 0.31), followed by compounds1a and 1f (MIC decrease, fourfold; ${Sigma}$ FIC, 0.37) and 1g to 1k (MICdecrease, twofold; ${Sigma}$ FIC, 0.50 to 0.56). By time-kill curves combiningnorfloxacin (1/4 MIC) and the most efficient EPIs (128 µg/ml),compound 1e persistently restored the bactericidal activityof norfloxacin (inoculum reduction, 3 log₁₀ CFU/ml at 8 and24 h), compound 1f led to a delayed but progressive decreasein the number of viable cells, and compounds 1b to 1d and omeprazoleacted synergistically (inoculum reduction, 3 log₁₀ CFU/ml at8 h but further regrowth), while compound 1a and reserpine slightlyenhanced norfloxacin activity. The bacterial uptake of norfloxacinmonitored by high-performance liquid chromatography confirmedthat compounds 1a to 1f increased antibiotic accumulation, asdid reserpine and omeprazole. Since these EPIs did not disturbthe ${Delta}$ ${psi}$ and ${Delta}$ pH, they might directly interact with the pump. A structure-activityrelationships study identified the benzimidazole nucleus ofomeprazole as the main structural element involved in effluxpump inhibition and highlighted the critical role of the chlorinesubstituents in the stability and efficiency of compounds 1eto 1f. However, further pharmacomodulation is required to obtaintherapeutically applicable derivatives.

INTRODUCTION

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Introduction

In recent years, active efflux has become recognized as a majorcomponent of microbial resistance to most classes of antibiotics.This mechanism is mediated by efflux pumps, which are membrane-associatedtransporters promoting antibiotic extrusion from the cell viaan energy-dependent process. Some efflux pumps selectively expelspecific antibiotics, while others, referred to as multidrugresistance (MDR) pumps, export a broad array of structurallyunrelated compounds (26, 30, 33, 36). MDR is conferred mainlyby the efflux systems of the major facilitator superfamily (MFS)of gram-positive bacteria, with the most studied pump beingNorA of Staphylococcus aureus, while the resistance/nodulation/divisionefflux systems are the major contributors in gram-negative bacteria(26, 30, 33, 36).

Strategies used to combat efflux-mediated resistance are basedon the search for either new antibiotics bypassing the effluxsystems or efflux pump inhibitors (EPIs). The latter solutionis attractive since a single EPI that is active against MDRpumps and can be used as adjunct therapy should (i) decreaseintrinsic resistance, (ii) reverse acquired resistance, and(iii) reduce the emergence of mutants that are highly resistantto a wide range of existing antibiotics (22, 25, 27, 29, 39,43). Among the candidate EPIs, drugs that are in clinical usefor other indications are of interest, since a large amountof data with respect to their pharmacokinetics and toxicityare already available (29). Several of these nonantibioticshave been demonstrated to act as EPIs on NorA and other MDRpumps of gram-positive organisms but at concentrations thatare too high to be achieved at therapeutic dosages and/or tootoxic. These drugs include reserpine (antihypertensive agent)(1, 9, 16-18, 20, 21, 28, 47), verapamil (antiarrhythmic) (1,47), omeprazole (antiulcer) (1), paroxetine (antidepressant)(16, 44), and chlorpromazine (neuroleptic) (17). A pharmacomodulationof paroxetine has been conducted to identify the structuralfeatures involved in efflux pump inhibition and design new,more potent EPIs (44). Chlorpromazine derivatives have beencompared to elucidate the way that they inhibit bacterial effluxpumps (16). However, no such investigations have been performedwith omeprazole, although it was the most active bacterial EPIin a previous comparative study (1).

We previously described a novel approach to design pyrrolo[1,2-a]quinoxalinederivatives as interesting bioactive analogues of pyridine,quinoxaline, or quinoline derivatives (11, 12). The aim of thisstudy was to design a series of new pyrrolo[1,2-a]quinoxalinecompounds mimicking the omeprazole structure and to evaluatetheir efficiency as EPIs in a model targeting NorA by rationallyselected tests. A preliminary structure-activity relationshipstudy allowed us to identify the structural elements of thesenew omeprazole analogues implicated in EPI activity.

MATERIALS AND METHODS

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Materials and Methods

Chemical syntheses: general procedures for preparation. (i) 2-[{(Pyrrolo[1, 2-a]quinoxalin-4-yl)methyl}thio]benzimidazoles6a to 6g, 2-[{(pyrrolo[1,2-a]quinoxalin-4-yl)methyl}thio]imidazoles6h to 6i, 2-[{(pyrrolo[1,2-a]quinoxalin-4-yl)methyl}thio]pyridine6j, and 2-[{(pyrrolo[1,2-a]quinoxalin-4-yl)methyl}thio]-7-methoxypyrrolo[1,2-a]quinoxaline6k.

Sodium hydroxide (9.23 mmol) was slowly added over 5 min toa stirred solution of a 2-mercapto derivative (4.62 mmol) inethanol (30 ml). A solution of 4-chloromethylpyrrolo[1,2-a]quinoxaline5a to 5d (4.62 mmol) in 40 ml of ethanol was slowly added tothe 2-mercapto derivative solution at 0°C and stirred for16 h at room temperature. After the solvent was removed underreduced pressure, the residue was poured into a 2.5% NaOH solutionand extracted with chloroform. The organic layer was dried overNa₂SO₄, and concentrated to give compounds 6a to 6k.

(ii) 2-[{(Pyrrolo[1,2-a]quinoxalin-4-yl)methyl}sulfinyl]benzimidazoles1a to 1g, 2-[{(pyrrolo[1,2-a]quinoxalin-4-yl)methyl}sulfinyl]imidazoles1h and 1i, 2-[{(pyrrolo[1,2-a]quinoxalin-4-yl)methyl}sulfinyl]pyridine1j, and 2-[{(pyrrolo[1,2-a]quinoxalin-4-yl)methyl}sulfinyl]-7-methoxypyrrolo[1,2-a]quinoxaline1k.

A solution of m-chloroperbenzoic acid (2 mmol) in chloroform(20 ml) was added dropwise to ice-cooled solutions of compounds6a to 6k (2 mmol) in chloroform (35 ml). The reaction mixturewas stirred at 0°C for 1 h and then washed with a saturatedNaHCO₃ solution and dried (Na₂SO₄). After removal of the solvent,the residue was triturated in ethyl acetate, filtered, washedwith ethyl acetate, dried, and recrystallized from ethanol togive compounds 1a to 1k (see the supplemental material).

Bacterial strains and media. The wild-type clinical isolate S. aureus SA-1199 and its overproducingNorA mutant strain SA-1199B (18, 19, 21) were generously providedas gifts from G. W. Kaatz (University of Michigan). These strainswere routinely cultured on Mueller-Hinton (MH) agar (Bio-Rad,Marnes-la-Coquette, France) and broth adjusted to contain 20µg/ml of Ca²⁺ and 10 µg/ml of Mg²⁺ (AES, Bruz, France)at 37°C. They were stored in 30% glycerol-brain heart brothat –80°C.

Antibiotic and chemicals. Norfloxacin, reserpine, and omeprazole were purchased from Sigma-Aldrich(Saint Quentin Fallavier, France). Solutions of reserpine, omeprazole,and the candidate inhibitors 1a to 1k were extemporaneouslyprepared in 100% dimethyl sulfoxide (DMSO), but the highestfinal 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 survivorsof the time-kill curve experiments were determined by the agardiffusion method (31) using MH medium alone or supplementedwith EPIs at 128 µg/ml. Six antibiotics were tested, includingall available fluoroquinolone disks, i.e., norfloxacin (5 µg),ciprofloxacin (5 µg), ofloxacin (5 µg), levofloxacin(5 µg), sparfloxacin (5 µg), moxifloxacin (5 µg),and chloramphenicol (30 µg). After overnight incubationat 37°C, the inhibition zone diameters were measured. Resultsare expressed 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 dilutionmethod in tubes under standard conditions (31). Bacterial suspensionswere prepared from an MH broth culture obtained after incubationat 37°C in a stirred water bath for 4 to 5 h and furtherdiluted 1/10 in MH broth ( ${approx}$ 10⁶ CFU/ml). Norfloxacin concentrationsranged between 128 and 0.1 µg/ml, and the EPI concentrationsranged between 512 and 1 µg/ml. In a second series ofexperiments, antibiotic solutions were combined with each EPIat a final concentration of 128 µg/ml. After an 18-h incubationperiod at 37°C, the MIC was defined as the lowest concentrationthat inhibited any visible growth. All tests were done at leastin triplicate, and the mode values were retained.

Checkerboard assay. Interactions between norfloxacin and the EPIs on SA-1199B andthe survivors of the time-kill experiments were evaluated bya checkerboard titration assay in tubes (7). The bacterial inoculumwas prepared as described above for MIC determinations. Norfloxacinwas tested at seven concentrations (64 to 1 µg/ml), andeach EPI was tested at six concentrations (512 to 16 µg/ml).Rows of tubes containing each drug alone at the same concentrationswere also included. Tubes were assessed visually for growthafter an 18-h incubation period at 37°C. The effect of drugcombinations was estimated at the point of maximal effectivenessby the fractional inhibitory concentration index ( ${Sigma}$ 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 incombination divided by the MIC of the drug when used alone (7).The ${Sigma}$ FIC data were interpreted according to the following criteria:synergy was defined as an ${Sigma}$ FIC of $≤$ 0.5, addition was defined as0.5 > ${Sigma}$ FIC $≤$ 1, indifference was defined as >1 ${Sigma}$ FIC <2,and antagonism was defined as an ${Sigma}$ FIC of $≥$ 2. All experiments werecarried out at least three times, and results are expressedas the mode values.

Time-kill curves. Interactions between norfloxacin and the most efficient EPIsor the reference EPIs on SA-1199B were also evaluated by thetime-kill curve method (7). An initial bacterial inoculum of10⁶ CFU/ml in the logarithmic phase was prepared as describedabove for MIC determinations. The final concentrations were16 µg/ml of norfloxacin and 128 µg/ml of the EPIs.Surviving cells were enumerated by dilution plating in triplicateonto MH agar at 0 (inoculum control), 2, 4, 8, and 24 h of incubationat 37°C. Results are expressed as the log percentage ofsurvival and represent the mean values of three different experiments.The limit of sensitivity was set at 1 log₁₀ CFU/ml to avoidany carryover effect.

Norfloxacin and omeprazole assays. For the uptake experiments, bacterial cultures of SA-1199 andSA-1199B were grown to mid-exponential phase in 300 ml of MHbroth in a shaken water bath for 4 to 5 h at 37°C. Cellswere centrifuged, pelleted, washed, and resuspended in sodiumphosphate buffer (50 mM) at pH 7.0. The suspension was thendivided into 0.2-ml aliquots, and bacteria were enumerated bydilution plating as indicated above. Each suspension was incubatedwith norfloxacin at one-quarter the MIC alone or in combinationwith each EPI at 128 µg/ml at time zero (T₀). After 5,10, 15, and 20 min, cells were separated from the extracellularmedium by differential centrifugation through a water-impermeablesilicone-paraffin oil barrier (1.029 g/cm³) and directly lysedby the orthophosphoric acid present in the bottom of the tube.The amounts of norfloxacin in the lysate were determined byhigh-performance liquid chromatography (HPLC) with a SupercosilC₁₈ column (Waters, Guyancourt, France). The mobile phase consistedof 80% phosphate buffer (H₃PO₄-KH₂PO₄ [10 mM] at pH 2.5) and20% acetonitrile. The excitation and emission wavelengths were240 and 280 nm, respectively. Protein concentrations were determinedwith a commercially available kit based on the use of bicinchoninicacid and bovine serum albumin as standards (Pierce, IL). Resultswere expressed as ng drug accumulated/mg cell protein. Experimentswere done in triplicate, and results given are the means ofthe three experiments. The norfloxacin and omeprazole concentrationswere also determined during the time-kill curve experimentsin the supernatants at time zero T₀ and at 24 h (T₂₄) by HPLCusing the conditions indicated above for the antibiotic. Foromeprazole and compounds 1a to 1f, the mobile phase consistedof 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 to1f.

${Delta}$ ${psi}$ and ${Delta}$ pH determinations. Accumulation of the membrane-permeant cation tetraphenylphosphoniumand salicylic acid was used to measure the transmembrane electricalpotential, ${Delta}$ ${psi}$ , and the ${Delta}$ pH, respectively, in the absence or presenceof reserpine (33 µM), carbonyl cyanide m-chlorophenylhydrazone(CCCP) (100 µM), omeprazole, or the new compounds 1a to1f (128 µg/ml). After incubation in a shaken bath for4 h at 37°C, MH broth cultures were divided into aliquotsthat were placed in ice. The protein content of one aliquotwas determined as indicated above. These samples were then warmedfor 5 min to room temperature. To measure the ${Delta}$ ${psi}$ , cells were labeledwith 1 µM [³H]tetraphenylphosphonium (29 Ci/mmol; AmershamBiosciences Corp., Buckinghamshire, United Kingdom) for 10 minin the presence or absence of the EPIs. One aliquot was treatedwith 4% DMSO (vol/vol) for 10 min prior to labeling in orderto obtain a background value. For the ${Delta}$ pH, EPIs were added tosamples for 20 min. Cells were then labeled with 20 µM[¹⁴C]salicylic acid (47 mCi/mmol; Perkin-Elmer, Wellesley, MA).For both experiments, samples were centrifuged for 10 min at4°C and 13,000 x g, and the radioactivity present in bothphases (cells and supernatant) was measured using a scintillationcounter. The ${Delta}$ ${psi}$ and ${Delta}$ pH were calculated by using a cell volumeof 4.2 µl/mg of cell protein as previously described (17,38). The results of these experiments, repeated three times,were expressed as the mean values ± standard deviations.

RESULTS

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Results

Chemistry. A series of new pyrrolo[1,2-a]quinoxaline derivatives, compounds1a to 1k (Fig. 1), has been synthesized by the pharmacomodulationof a basic heterocyclic synthon related to the structure ofomeprazole. This synthesis has been achieved by starting fromvarious substituted 2-nitro-anilines in six steps. The Clauson-Kaasreaction (8) of anilines with 2,5-dimethoxytetrahydrofuran inacetic acid gave the pyrrolic derivatives 2a to 2d (12), whichwere reduced using BiCl₃-NaBH₄ treatment to provide the expected1-(2-aminophenyl)pyrroles 3a to 3d (37). The reaction of chloroacetylchloride with 3a to 3d (12) led to chloracetamides 4a to 4d(10). The 4-chloromethylpyrrolo[1,2-a]quinoxalines 5a to 5dwere prepared by the cyclization of these amides, 4a to 4d,in refluxing phosphorus oxychloride according to the Bischler-Napieralskireaction procedure (3, 45). The 4-chloromethylpyrrolo[1,2-a]quinoxalines5a to 5d (10) were then condensed with various substituted 2-mercaptoderivatives in ethanol in the presence of sodium hydroxide togive the corresponding sulfides 6a to 6k (6, 15). The sulfoxides1a to 1k were obtained by low-temperature oxidation of sulfides6a to 6k with m-chloroperbenzoic acid in chloroform (4, 23).

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FIG. 1. Synthesis of pyrrolo[1,2-a]quinoxaline derivatives 1a to 1k, new structural analogues of omeprazole.

Antibiotic and EPI susceptibilities of the S. aureus strains. A strategy including four steps has been designed to investigatethe efficiency of new EPIs in a model targeting NorA and usingSA-1199 and SA-1199B. In preliminary experiments, the MICs ofthe reference and putative EPIs were determined in order tosubsequently use them at concentrations devoid of bacterialtoxicity and hence to evaluate their efficiency only as EPIs.Reserpine, omeprazole, and pyrrolo[1,2-a]quinoxaline derivatives1a to 1k were found to lack any intrinsic antibacterial activity(MIC > 512 µg/ml).

In a first step of screening, the high-throughput disk diffusionmethod was used to characterize the profiles of the test strains,to identify the preferential substrate and the optimal concentrationof the reference EPIs, and to detect any EPI activity of thetested compounds. SA-1199B exhibited reduced inhibition zonestowards fluoroquinolones, particularly norfloxacin and ciprofloxacin,and a slightly decreased inhibition zone towards chloramphenicolcompared to SA-1199 (Table 1). At concentrations less than 32µg/ml, omeprazole had no effect on norfloxacin and ciprofloxacinactivity, while at 128 µg/ml, it was evident. Thus, thelatter concentration was subsequently used for all tested EPIsin order to maximize the chance of observing an effect. Actually,at 128 µg/ml, all new synthesized compounds increasedthe inhibition zones of the fluoroquinolones, particularly thoseof the most affected drugs, norfloxacin and ciprofloxacin, andthus acted as EPIs. In a second step of screening, these datawere confirmed by MIC determinations. S. aureus strain SA-1199Bexhibited norfloxacin MICs that were 32 times higher than thosefor SA-1199, and the presence of each EPI at 128 µg/mlresulted in a reduction in the norfloxacin MIC (2- to 16-fold)for SA-1199B and, to a much lesser degree, for SA-1199 (Table2). Compound 1e appeared to be the most active, as determinedby the disk diffusion method.

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TABLE 1. In vitro activities of reserpine, omeprazole, and the pyrrolo[1,2-a]quinoxaline derivatives 1a to 1k as EPIs by the disk diffusion method for SA-1199B^a

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TABLE 2. In vitro activities of reserpine, omeprazole, and the pyrrolo[1,2-a]quinoxaline derivatives 1a to 1k as EPIs by norfloxacin MIC determinations for SA-1199 and SA-1199B and by the checkerboard method for SA-1199B^a

Effect of norfloxacin and EPI combinations by the checkerboard method. In a third step of screening, the combinations between the preferentialsubstrate and the EPIs were investigated by both reference methods.The checkerboard method aims at evaluating the bacteriostaticeffect of antibiotic combinations according to their concentrationsat a fixed time of 18 h (7). This method was applied to thecombination of norfloxacin with reserpine, omeprazole, and eachnew compound, compounds 1a to 1k, on SA-1199B, and the interpretationwas adapted to antibiotic-EPI combinations by calculating the ${Sigma}$ FIC with the MIC of these atoxic EPIs taken as 1,024 µg/ml,i.e., the value that was twice the maximal concentration tested.By this mean, reserpine, omeprazole, and the five candidateinhibitors 1a to 1f appeared to be synergistic on SA-1199B,while the other new compounds, compounds 1g to 1k, were onlyadditive. The rank order of decreasing activity of the EPIswas identical to that found by MIC determinations, i.e., compound1e was more active than reserpine, omeprazole, and compounds1b to 1d, followed by compounds 1a, 1f, and 1g to 1k (Table2).

Effect of norfloxacin and EPI combinations by the time-kill curve method. The objective of the time-kill curve method is to evaluate thebactericidal effect of antibiotic combinations according tothe time at some selected concentrations (7). This method wasapplied to the combination of norfloxacin (one-quarter the MIC,i.e., 16 µg/ml) with the reference EPIs, omeprazole andreserpine, and the most active compounds, compounds 1a to 1f,at 128 µg/ml, on SA-1199B. Norfloxacin (Fig. 2) or theEPIs alone (data not shown) at the concentrations used did notsignificantly inhibit the growth of the test organism (difference $≤$ 1 log₁₀ CFU/ml). According to the usual definition, all testedcombinations appeared to be synergistic by this method sincethey decreased the bacterial growth significantly more thaneither norfloxacin or each EPI alone. Indeed, the combinationof norfloxacin with the reference EPIs showed that reserpine,after a short initial decrease in viable cells, hardly enhancedthe norfloxacin activity. In contrast, omeprazole produced a4-log₁₀ decrease of the starting inoculum at 8 h, followed bya regrowth, leading to a bacterial count similar to that ofthe inoculum at 24 h. Compound 1a behaved similarly to reserpine.Compounds 1b to 1d gave curves similar to those for omeprazolebut were less active in reversing norfloxacin activity, particularlyat 24 h. Finally, compound 1f led to a delayed but progressivedecrease in viable cells, while compound 1e demonstrated thestrongest activity, yielding to a deeper decrease in the initialinoculum at 8 h and persistently restoring the bactericidalactivity of norfloxacin (Fig. 2b). Surviving cells recoveredin each experiment were tested for their antibiotic and/or EPIsusceptibilities. They exhibited the same disk diffusion patternas SA-1199B and gave the same norfloxacin MIC (64 µg/ml)and the same ${Sigma}$ FIC (0.18) by the checkerboard method as SA-1199B,simultaneously used as control. Thus, survivors were not mutantswith increased resistance to norfloxacin or EPI inhibition.The HPLC assay showed that during the time kill curve experiments,norfloxacin concentrations were identical at T₀ (16 ±0.8 µg/ml) and T₂₄ (16 ± 1.5 µg/ml). In contrast,the EPI concentrations considerably decreased from T₀ (128 ±10 µg/ml) to T₂₄ for omeprazole (40 ± 8 µg/ml),compounds 1b to 1d (54 ± 8 µg/ml), and compound1a (72 ± 9 µg/ml), while compounds 1e and 1f appearedto be more stable (98 ± 9 µg/ml at T₂₄). Consequently,the inhibitory effect of the EPIs in time-kill experiments partiallyparalleled their stability.

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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 µg/ml) alone (N) or combined with omeprazole (N+Om) and (a) reserpine (N+Res) or new derivatives 1a to 1d (N+1a-d) and (b) 1e and 1f (N+1e-f) at 128 µg/ml.

Effect of EPIs on norfloxacin bacterial uptake. Finally, a fourth step ensured that the previously demonstratedsynergistic effect was actually related to the increase in theantibiotic substrate accumulation by the putative EPIs. Theintrabacterial accumulation of norfloxacin at 16 µg/mlalone or combined with the reference and the most efficientEPIs at 128 µg/ml was determined by HPLC. In SA-1199 (Fig.3a), the accumulation of norfloxacin reached near equilibriumat 5 min and thereafter remained at the same level up to 20min, at around 400 to 450 ng/mg cell protein. The addition ofreserpine, omeprazole, and compounds 1a to 1f barely increasedthe final level of norfloxacin accumulation (10%). In SA-1199B(Fig. 3b), the accumulation of norfloxacin was significantlylower than that observed in SA-1199, reaching 235 ng/mg cellprotein at 5 min and progressively up to 350 ng/mg cell proteinat 20 min. The addition of all EPIs substantially increasedthe norfloxacin accumulation in SA-1199B, up to 270 to 315 ng/mgcell protein at 5 min and around 400 to 450 ng/mg cell proteinat 20 min, increasing by 40% of the final level of norfloxacinaccumulation. Thus, the synergistic effect demonstrated by theEPIs was correlated with the partial restoration of norfloxacinuptake.

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FIG. 3. Intracellular accumulation of norfloxacin alone or combined with EPIs. Norfloxacin was used at one-quarter the MIC (16 µg/ml) alone (N) or combined with omeprazole (N+Om) and (a) reserpine (N+Res) or new derivatives 1a to 1d (N+1a-d) and (b) 1e and 1f (N + 1e-f) at 128 µg/ml. Intracellular accumulation of norfloxacin in (a) SA-1199 and (b) SA-1199B is shown.

Effect of the inhibitors on the ${Delta}$ ${psi}$ and ${Delta}$ pH. In order to investigate the basis of the EPI effect of omeprazoleand related compounds on NorA, the ${Delta}$ ${psi}$ and ${Delta}$ pH of the cells weredetermined for SA-1199B, alone and in the presence of thesedrugs, compared to reserpine and CCCP, used as controls. The ${Delta}$ ${psi}$ of SA-1199B cells in MH broth was –126 mV ± 6mV. In the presence of reserpine or omeprazole, this value wasslightly lowered, to –140 ± 10 mV and –140± 6 mV, respectively, while in the presence of the uncoupleragent CCCP, it became null. Compounds 1a to 1f had activitysimilar to that of omeprazole, leading to a ${Delta}$ ${psi}$ of –130 ±10 mV. The ${Delta}$ pH of SA-1199B in MH was +0.55 ± 0.05 overthe entire 20-min time course of the experiment. The additionof reserpine, omeprazole, or compounds 1a to 1f had no effect(+0.55 ± 0.05). In contrast, the ${Delta}$ pH was abolished byCCCP ( ${Delta}$ pH = 0 ± 0.1). Thus, the putative EPIs did notsignificantly influence the electrical potential and the trans-membranepH gradient of the bacterial cells, at least at the concentrationsused.

DISCUSSION

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Discussion

In this study, a series of 11 new pyrrolo[1,2-a]quinoxalinederivatives, compounds 1a to 1k, was synthesized from commerciallyavailable nitroanilines and developed as a template for thedesign of new bioisosters of omeprazole by various pharmacomodulationsin order to obtain new compounds endowed with a higher activityas bacterial EPIs. These derivatives were then evaluated ina model targeting the NorA efflux pump of S. aureus (18-21,28, 42, 47). Indeed, although NorA modestly contributes to wild-typefluoroquinolone resistance, first-step mutants, by diminishingthe intracellular drug concentrations, tend to subsequentlyaccumulate additional target mutations under treatment, leadingto the commonly encountered high-level fluoroquinolone-resistantclinical strains (2). Moreover, NorA is the prototype of otherMFS pumps with 12 transmembrane segments, such as PmrA in Streptococcuspneumoniae (35), and has generally served as the model for studyingEPIs of MDR pumps in gram-positive organisms (22). EPIs thatare active on NorA are hence expected to inhibit at least someof the related pumps.

The model used to assess the potential EPI activity of the 11new pyrrolo[1,2-a]quinoxaline derivatives, compounds 1a to 1k,was based on the isogenic strains SA-1199 and SA-1199B, classicallyused to investigate NorA inhibition. Our results regarding thesusceptibilities of these strains to norfloxacin and the referenceEPIs by MIC determinations (1, 9, 16, 17, 19-21, 32, 42), time-killcurves (1), and accumulation assays (16, 42, 46) were in agreementwith the literature. The antibiotic resistance and inhibitionprofile of SA-1199B reflected the overexpression of NorA, apump that preferentially exports the hydrophilic fluoroquinolonespefloxacin and ciprofloxacin rather than the more hydrophobicdrugs of this class, sparfloxacin and moxifloxacin, and hasa limited effect on the neutral chloramphenicol (18-21, 28,42, 47). The slight potentiation of norfloxacin by the EPIson wild-type strain SA-1199 is related to the low-level expressionof NorA in wild-type strains of S. aureus (1, 20-22, 32). However,SA-1199B exhibited high-level fluoroquinolone resistance thatwas not completely reversed by the EPIs, as particularly apparentin norfloxacin accumulation experiments. In fact, SA-1199B isa double-target (A116E in GrlA)/efflux mutant (21). A lesseraffinity of the drug for its modified target might lead to adecreased accumulation into the bacterial cell that cannot beamended by EPIs. Accordingly, we observed a total restorationof the norfloxacin susceptibility 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 EPIactivity on NorA, allowing comparisons to data reported in theliterature, a single NorA mutant such as SA-1 might be moreconvenient for further studies.

By this strategy, all novel derivatives appeared to have EPIproperties, and one compound (compound 1e) was consistentlymore active than omeprazole. Indeed, compound 1e at 128 µg/mlreduced the norfloxacin MIC for SA-1199B by 16-fold. The "minimumpotentiating concentration" required to enhance the activityof the preferential substrate against a test strain by at leasteightfold (22), which was also the "minimal effective concentration"of EPI that produced the maximal reduction in the substrateMIC (32), was 64 µg/ml, and the lowest active concentration(i.e., capable of reducing the norfloxacin MIC by twofold) was32 µg/ml. At concentrations $≥$ 256 µg/ml, compound1e shifted the norfloxacin MIC to 4 µg/ml. Other commerciallyavailable drugs such as verapamil paroxetine, chlorpromazine,and their derivatives (1, 47), the novel P-gp inhibitors currentlybeing developed for the treatment of drug resistance tumors(9, 22, 32), and EPIs selected by the screening of plant extracts(34) exhibited similar potentiating effects on NorA. The screeningof a chemical library and the further synthesis of indole derivativeshave yielded inhibitors of NorA with a much higher potency (28,41), but their clinical safety has not been established. However,although proton pump inhibitors are well-tolerated, maximalserum concentrations following usual doses are well below theconcentrations at which compound 1e acted as an EPI (5, 14).

This limited series of 11 pyrrolo[1,2-a]quinoxaline derivatives,designed as omeprazole analogues, has been synthesized in orderto initiate a preliminary structure-activity relationship study,allowing further pharmacomodulations. Biological results highlightedthe importance of the benzimidazole moiety (compounds 1a to1f), since its replacement by an imidazole (compounds 1h and1i), a pyridine (compound 1j), or a pyrrolo[1,2-a]quinoxalinering (compound 1k) resulted in a significant loss of potency.Moreover, methoxy-substituted compounds 1b to 1f, whatever thesubstitution position, on either the benzimidazole or the pyrrolo[1,2-a]quinoxalinenucleus, were more potent that the unsubstituted compound 1a.The introduction of a second methoxy substituent (compound 1d)was not beneficial compared to the monosubstituted derivatives1b and 1c. Otherwise, the introduction of one chlorine atomat position 7 or 8 of the pyrroloquinoxaline heterocycle providedan 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 its8-cholor-substituted homologue, compound 1f. By reference tothe omeprazole chemical structure, the replacement of the pyridinering by a pyrrolo[1,2-a]quinoxaline nucleus (compounds 1a to1f) did not substantially change the EPI activity. However,whatever the other structural modifications were, omeprazoleand pyrrolo[1,2-a]quinoxaline analogues 1a to 1f acted as EPIson the NorA efflux pump only at high concentrations (64 to 128µg/ml). Finally, the N-methylated compound 1g, comparedto compound 1a, was devoid of EPI activity, suggesting the importanceof the amino benzimidazolyl proton in the EPI activity.

This structure-activity relationship study might also contributeto analyses of the mode of action of omeprazole and its analoguesas bacterial EPIs. Indeed, the inhibition of MFS pumps in gram-positiveorganisms, such as the single-component system NorA, can bedue either to the disturbance of the source of energy or toa direct interaction with the pump by steric obstruction orsubstrate competition. Compounds 1a to 1f, as omeprazole andreserpine (17), did not significantly modify the electricalpotential and the trans-membrane pH gradient, in contrast toCCCP (17), and thus did not seem to alter the source of energyof this proton-dependent pump. Hence, these EPIs might interactdirectly with NorA. However, a mode of action analogous to thatof omeprazole with the eukaryotic H⁺/K⁺ pump is unlikely, sincethe pH is not favorable for the activation of our compoundsinto a reactive sulfenamide (13, 14). Cloning of the norA geneand random mutagenesis followed by the selection of isolatesresistant to the combination of norfloxacin and omeprazole,as previously performed for Bmr and reserpine in Bacillus subtilisand reserpine (24), would be helpful to elucidate the mode ofaction of these EPIs.

Furthermore, time-kill data showed a late regrowth in the presenceof subinhibitory concentrations of norfloxacin with omeprazoleor its analogues 1a to 1d. This phenomenon has already beenreported for reserpine and omeprazole without a definite explanation(1). In this study, we have demonstrated that these survivorswere not mutants with enhanced norfloxacin resistance or reducedsusceptibility to EPI inhibition and that the antibiotic wasstable over the time-kill experiment period. In contrast, omeprazoleand the related compounds underwent a substantial albeit variabledegradation. The regrowth observed after 8 h with compounds1b to 1d, in contrast to compounds 1e and 1f, might be relatedto a greater instability of the nonchlorinated compounds thanthe chlorinated ones. It also might be due to the reversibleversus irreversible binding of these EPIs to the lipophilicbinding pocket of NorA: EPI removal should allow the pump tofunction de novo, conferring norfloxacin resistance, thus permittingthe bacterial multiplication to start again. Indeed, such reversiblebinding has been observed for omeprazole and the H⁺/K⁺-ATPase(40). Finally, one cannot exclude that compounds 1a to 1d transientlyinduced efflux pumps other than NorA, in contrast with compounds1e and 1f.

In conclusion, a series of new omeprazole analogues has beensynthesized and evaluated by a cost-effective and time-savinggeneral strategy. Concordant results showed that all novel derivativeshad EPI properties, and one compound (compound 1e) was moreactive than omeprazole, particularly in restoring the bactericidalactivity of norfloxacin over a prolonged period. A structure-activityrelationship study identified the benzimidazole nucleus as thekey element of efflux pump inhibition and the chlorine at position7 of the pyrroloquinoxaline heterocycle as a critical substitution.However, further pharmacomodulation is required to obtain therapeuticallyappropriate 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 studiesand M. Hugues for helpful discussion on ${Delta}$ ${psi}$ and ${Delta}$ pH experiments.We thank an anonymous reviewer for critical review of the manuscriptand pertinent suggestions.

FOOTNOTES

* Corresponding author. Mailing address: Laboratoire de Microbiologie, UFR des Sciences Pharmaceutiques, Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France. Phone: (33) 5 57 57 10 75. Fax: (33) 5 56 90 90 72. E-mail: claudine.quentin{at}bacterio.u-bordeaux2.fr.

Published ahead of print on 13 November 2006.

Supplemental material for this article may be found at http://aac.asm.org/.

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