DOI: 10.1002/cmdc.201000006

Efflux Pump Inhibitors: A Strategy to Combat P-Glycoprotein and the NorA Multidrug Resistance PumpLing Zhang and Shutao Ma*[a]

Introduction

The problem of drug resistance is becoming increasingly signif-icant in the treatment of cancers and bacterial infections. Thewell-known resistance mechanisms include target modification,enzymatic drug inactivation, minimizing drug accumulationwithin the cell, and efflux pumps. In particular, multidrug resist-ance (MDR) mediated by efflux pumps is becoming even moreimportant. MDR often appears after the long-term use of asingle drug, and it is usually characterized by resistance to aseries of unrelated compounds.[1] The most important form ofMDR is mediated by membrane efflux proteins such as P-gly-coprotein (P-gp) in mammalian cells[2] and the NorA pump inStaphylococcus aureus.[3] Because the activity of such efflux pro-teins can be blocked by efflux pump inhibitors (EPIs), theseefflux proteins are potentially effective targets for the treat-ment of cancer and bacterial infections. An alternative ap-proach is the development of new non-substrate agents thatcan bypass the action of efflux pumps.[4]

Efflux-mediated drug resistance was first reported in mam-malian cancer cells and was then discovered in bacteria withtetracyclines in early 1980s. Afterward, efflux pumps were rec-ognized as a primary cause of bacterial resistance to a wide va-riety of antimicrobials including b-lactams, tetracyclines, fluoro-quinolones, and macrolides.[5] These efflux pumps protect thecell by transporting cancer-fighting agents and antibiotics outof the cytoplasm, thus limiting the steady-state accumulationof drugs.

Efflux pumps are transmembrane transport proteins, and aregrouped into two mechanistically distinct categories. The firstgroup are primary adenosine triphosphate (ATP) binding cas-settes (ABC pumps), which are fueled by ATP hydrolysis. Thesecond group are fueled by electrochemical transmembraneproton or sodium ion gradients, and members include themajor facilitator superfamily (MFS), small-multidrug resistance(SMR), resistance–modulation–division (RND), and multipledrug and toxin extrusion (MATE) pumps.[6]

EPIs are of interest for the treatment of MDR bacterial infec-tions and tumors. In particular, the combination of EPIs withantibiotics or anticancer drugs is a promising approach to revi-talizing the clinical use of “old” drugs that are extruded by

efflux pumps. To qualify as EPIs, candidate compounds mustmeet the following criteria: 1) They must be nontoxic ; a favora-ble toxicity profile is a prerequisite for any novel EPI. 2) Theymust be selective toward specific target efflux pumps only.3) They must be appropriate for the given application route;inhibitors must have different properties depending on the ap-plication route in question, such as improving bioavailability.[7]

Over the past few years, natural and synthetic EPIs havebeen described and investigated.[8–19] However, only the studiesof those EPIs that target P-gp in resistant tumor cells and theNorA pump in resistant S. aureus strains are comparativelymature. In this minireview, we summarize the recent advancesin the development of EPIs of P-gp and NorA according totheir various chemical structures.

Inhibitors of P-gp

P-gp is a high-molecular-weight head-to-tail dimeric trans-membrane protein originally isolated from mutant Chinesehamster ovary cells in 1976.[20] P-gp is present in many normaltissues as well as in various drug-resistant cancer cells ; this cancause failure in chemotherapy, as it is able to transport a varie-ty of anticancer drugs out of the cell.

P-gp expression is mediated by the mdr1 gene, which is lo-cated on chromosome 7. It belongs to a superfamily of ABCtransporters, of which there are more than 40 members.[21]

Each P-gp monomer consists of multiple transmembrane do-mains and an ATP binding site.[2, 22] Importantly, the overexpres-sion of P-gp is often associated with MDR, as it can efflux awide range of large hydrophobic and amphiprotic sub-strates.[17] P-gp inhibitors, which are similar to P-gp substrates,have a large chemical and structural diversity.

Multidrug resistance (MDR) is the cause of an ever-increasingnumber of problems in the treatment of cancers and bacterialinfections. The active efflux of drugs contributes significantlyto this phenomenon. This minireview summarizes recent ad-

vances in combating MDR, with particular emphasis on naturaland synthetic efflux pump inhibitors of P-glycoprotein in resist-ant tumor cells and of the NorA MDR pump in Staphylococcusaureus.

[a] L. Zhang, Prof. S. MaDepartment of Medicinal ChemistrySchool of Pharmaceutical SciencesShandong University, 44 West Culture Road, Jinan 250012 (PR China)Fax: (+ 86) 531-88911612E-mail : mashutao@sdu.edu.cn

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The main mechanisms of P-gp inhibitors involve the regula-tion of P-gp expression by binding to drug binding sites[18] ormodulator binding sites.[19] Recently, many candidate com-pounds have been identified that inhibit MDR, including calci-um channel blockers, calmodulin inhibitors, protein kinase Cinhibitors, oligonucleotides, immunosuppressive agents, quino-lines, indole alkaloids, detergents, steroids, and anti-estrogens.

Over the last decade, the development of MDR modulatorshas been fraught with difficulty, and no agent has yet dis-played any significant clinical efficacy.[21, 23] This is largely due tothe inherent toxicity of these compounds or to the heightenedtoxicity of the anti-neoplastic agents when co-administeredwith modulators. Nevertheless, there are three generations ofEPIs. First-generation EPIs are therapeutic drugs such as theimmunosuppressant cyclosporin A and the calcium channelblocker verapamil. They are known to inhibit the efflux pump.However, these agents cannot be used clinically, as their vari-ous pharmacological drawbacks impede the delivery of anti-cancer drugs. Chemical modifications of first-generation inhibi-tors gave rise to second-generation EPIs such as the verapamilderivative KR30031,[24] and PSC833 (commonly known as val-spodar)[25] based on cyclosporin D. Clinical trials for third-gener-ation EPIs, including elacridar (GF120918), tariquidar (XR9576),zosuquidar (LY335979), laniquidar (R101933), and ontogen(OC144-093) have already been performed.[7] Most third-gener-ation EPIs bind P-gp in a noncompetitive manner. Nearly alldata from the clinical trials are somewhat unsatisfactory, butthey do reveal a promising future for P-gp inhibitors.[21]

Imidazoles

OC144-093 (2-[4-(3-ethoxy-1-propenyl)phenyl]-4,5-bis-[4-(2-pro-pylamino)phenyl]-1H-imidazole) was proven to be a novel andnontoxic modulator of P-gp (Figure 1). The structure–activityrelationships (SARs) were explored by using solid-phase combi-natorial chemistry.[26] Efforts to decrease the molecular weightresulted in 2,5-disubstituted imidazoles (such as compound 1),but these new modulators have low in vitro potency. Consider-ing its excellent potency and half-life, OC144-093 was chosenas a candidate for clinical trials.[27]

Pharmacokinetic studies in healthy male volunteers (400 mgp.o. administration) revealed the presence of OC144-093 at 3–

5 mm in plasma after 3–4 h, which is much higher than the1 mm concentration required for full reversal of P-gp activity inpreclinical models. No adverse effects or toxicity toward thecentral nervous system were observed. The oral bioavailabilityof OC144-093 in humans was estimated to be >60 % withfood intake, and its terminal half-life ~22 h, suggesting thatonce- or twice-a-day oral dosing would be sufficient to main-tain therapeutic levels.[27]

Dantzig et al.[28] reported that OC144-093 is probably not aP-gp transport substrate according to their experimental re-sults. OC144-093 was found to restore [3H]vinblastine accumu-lation in CEM/VLB1000 cells, showing 25-fold greater potencythan cyclosporin A in this model system. Furthermore, it wasalso found to be a potent inhibitor of P-gp-mediated ATPase,with an IC50 value of 0.16 mm.

Co-administration of OC144-093 with docetaxel enhancedthe oral bioavailability of docetaxel to a limited extent (8–26 %)in humans, and was shown to have a good safety profile aswell.[29] Combination of ONT-093 with paclitaxel was evaluatedin phase I trials. The combination was well tolerated, but anapparent pharmacokinetic interaction was found between pa-clitaxel and higher doses of ONT-093.[30]

Acridone carboxamides

The dihydropyridine azidopine is described as a photoaffinityligand for calcium channels and P-gp. GF120918 (9,10-dihydro-5-methoxy-9-oxo-N-{4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]phenyl}-4-acridine carboxamide hydrochlo-ride; Figure 2), also known as elacridar/GG918/GW0918, can

compete with azidopine (IC50 = 0.16 mm). Similarly, the calciumchannel blocker verapamil and the cytostatic agent vinblastinecan inhibit azidopine labeling with higher IC50 values as well(45 and 4 mm, respectively).[31] Traunecker et al.[32] found thatGF120918 can completely reverse resistance for drugs such asetoposide, vinblastine, doxorubicin, taxotere, and paclitaxel,the anticancer activities of which were decreased in the doxor-ubicin-selected P-gp-expressing human sarcoma cell line MES-Dx5, with IC50 values in the nanomolar range. GF120918 wasconfirmed to have minimal toxicity in phase I trials and to in-hibit P-gp at doses readily attainable in patients.[33]

GF120918 was found to be a good and safe P-gp inhibitoralternative for cyclosporin A in enhancing the oral bioavailabil-Figure 1. Imidazoles as EPIs of P-gp.

Figure 2. Structure of the third-generation EPI elacridar (GF120918).

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ity of paclitaxel.[34] The apparent bioavailability for orally ad-ministered paclitaxel is 6 % as a single agent,[35] and 50 % incombination with GF120918.[34] Importantly, GF120918 has noimmunosuppressive activity. Therefore, it may be a better can-didate, especially for repeated administration, than cyclospo-rin A as an EPI.

Studies carried out by Kruijtzer et al.[36] in 16 patients withsolid tumors revealed that co-administration of GF120918 withtopotecan resulted in a significant increase in the efficacy oftopotecan. The apparent oral bioavailability increased from40.0 to 97.1 % in the presence of GF120918.

In preclinical studies, GF120918 showed a high affinity to P-gp, producing P-gp inhibitory activity at a readily attainableconcentration. Experimental data from phase I trials revealedonly a minor influence of GF120918 on the pharmacokineticsof doxorubicin, whereas greater toxicity might be expected atthe tissue level.[37]

GF120918 was also found to be equivalent to reserpine inenhancing the in vitro activity of norfloxacin and ciprofloxacinagainst S. aureus expressing MDR pumps. Further studiesshowed four- to eightfold decreases in MIC values of these flu-oroquinolones (FQs) toward SA-1199B, an S. aureus strain over-expressing the NorA pump.[38]

Ellipticine analogues

It has been determined that some ellipticine analogues are in-hibitors of the efflux pump in drug-resistant mouse lymphomaT-cells. Among these tested ellipticines, compound 2 (Figure 3)showed significant activity, with 20.95-fold efficacy at2 mg mL�1 compared with the control (verapamil, 5 mg mL�1).Encouragingly, the activity ratio of compound 2 to the controldrug was too high to be measured at a concentration of20 mg mL�1. At the same concentration, the values for com-pounds 3 and 4 were 212.12 and 90.60, respectively.[39]

Verapamil and its derivatives

Verapamil is the first reported P-gp inhibitor.[40] However, itsunexpected side effects in the cardiovascular system make it

unsuitable for clinical use. To improve its MDR-reversing activi-ty and to decrease its cardiovascular toxicity, several new vera-pamil derivatives were designed and synthesized. Amongthese compounds, MM36, which bears a 9-anthranylmethylside chain, and CTS41, with a 9H-thioxanthenylethyl side chain,presented distinct and significant dose-dependent activitieswithout cardiovascular side effects (Figure 4).[41] In particular,CTS41 was 41-fold more potent than verapamil in K-562/doxRcells at 4 mm.[42]

The SARs of verapamils showed the contribution of a basiclinker between two aromatic residues. Thus, new flexible mole-cules were designed to bind with high affinity to P-gp, with abasic nitrogen atom at a defined distance from the aromaticgroups.[43]

Tetrahydroisoquinolines

Tariquidar (XR9576) is one of the most potent third-generationP-gp modulators, and contains a highly hydrophobic tetrahy-droisoquinoline–ethylphenylamine substructure (Figure 5).[44] Ithas been used as a basis for the rational design of new MDRmodulators. QSAR and molecular modeling studies have identi-fied the important structural features and pharmacophores ofthe XR compound series (of which tariquidar is a member),which will be used for the design of new P-gp inhibitors.[45]

Colabufo et al.[46] designed and synthesized a series of 6,7-di-methoxytetrahydroisoquinoline derivatives based on tariquidar.These modifications not only had no effect on the pharmacoki-netic profiles of these chemotherapeutic drugs, but the deriva-tives also showed high potency and specificity for P-gp. Thebest results were obtained for compounds 5 and 6 (EC50

values: 1.64 and 4.86 mm, respectively). SARs indicated that sat-uration of the double bond of the linkage or shifting it intothe tetraline ring decreases the P-gp inhibitory activity of thederivatives. Furthermore, it was discovered that in the pres-ence of compound 5, the antiproliferative effect of bicaluta-mide at 72 h was ameliorated in LnCap cells (androgen-depen-dent, EC50 from 51.9�6.1 to 17.8�2.6 mm) and was restored inFigure 3. Analogues of ellipticine as EPIs of P-gp.

Figure 4. Verapamil and its derivatives.

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PC-3 cells (androgen-independent, EC50 from 150�2.4 to 60�3.5 mm).[47]

Another new P-gp modulator, 6,7-dimethoxy-1-(3,4-dime-thoxy)benzyl-2-(N-n-octyl-N’-cyano)guanyl-1,2,3,4 tetrahydroiso-quinoline (7), was explored and evaluated for its MDR-revers-ing activity and cardiovascular effects. The results of in vitroassays for MDR reversal activity in several cell lines, such asMCF-7, MCF-7/ADR, and K562/A02, showed that compound 7exhibits a well-defined trend in MDR reversal. Moreover, theantitumor activity of adriamycin was significantly enhanced bythe co-administration of compound 7 in SCID nude mice.[48]

Several new derivatives with structure similar to that of tari-quidar (i.e. , containing the tetrahydroisoquinoline–ethylphenyl-amine substructure) were recently synthesized. In contrast toXR9576, nearly all these derivatives containing a smaller hydro-phobic bicyclic portion at a specific orientation were identifiedas the most potent amide derivatives. Among them, com-pound 8 is a good example.[49]

Analogues with the hydrophobic group attached at variouspositions of the tetrahydroisoquinoline skeleton were also pre-pared and evaluated as P-gp inhibitors. Some of them, such ascompound 9, were shown to enhance the anticancer activityof doxorubicin in the K562/DOX cell line.[50]

d-a-Tocopheryl-poly(ethylene glycol) succinate (TPGS)

Nonionic surfactants are commonly used in oral preparationsto enhance the bioavailability of water-insoluble drugs. Suchsurfactants are less toxic toward biological membranes and arebetter able to dissolve drugs of poor aqueous solubility. Inrecent years, a number of studies have focused on P-gp effluxactivity for several commonly used pharmaceutical excipientssuch as nonionic surfactants.

TPGS 1000 (d-a-tocopheryl-poly(ethylene glycol 1000) succi-nate) was developed in the1950s as a water-soluble form ofvitamin E (Figure 6). It consists ofa hydrophilic head group and alipophilic alkyl tail. Due to itssurface-active properties,TPGS 1000 can be used as a solu-bilizer, an emulsifier, and a vehi-cle for lipid-based drug delivery.It is in current clinical use to en-hance the bioavailability of am-prenavir. TPGS 1000 was recentlydescribed as an active oral ab-sorption enhancer for improvingthe bioavailability of poorly ab-sorbed drugs. In addition, TPGSincreases the sensitivity of P-gp-expressing cells toward severalcytotoxic P-gp substrates and ef-fectively blocks the polarized

transport of rhodamine 123 and paclitaxel, as determined intransport assays.[51]

Among the TPGS analogues with various chain lengths(TPGS 200, 238, 400, 600, 1000, 2000, 3400, 3500, 4000, and6000), TPGS 1000 has the greatest potency in inhibiting P-gpactivity.[52] SAR studies indicate that the introduction of a hy-drophobic moiety such as cholesterol and substituting toco-pherol into the basic TPGS structure, could impart the resultingderivatives with greater P-gp inhibitory activity thanTPGS 1000.

With polypropylene glycol succinate (PPG) derivatives per-forming well, hydrophilic group replacement was proven to beeven more beneficial. TPPG 1000, which was prepared by re-placing the poly(ethylene glycol) side chain of TPGS 1000 witha PPG side chain, was found to be the most promising. It notonly approached cyclosporin A in in vitro inhibitory activity,but also afforded a statistically significant enhancement in thebioavailability of raloxifene (a P-gp substrate) in rats, or rather,

Figure 5. Compounds sharing a chemical structure similar to that of tariquidar.

Figure 6. Example of nonionic surfactants as EPIs of P-gp.

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TPPG 1000 effected 93�2 % of rhodamine 123 efflux inhibitionin a Caco-2 cell monolayer model.[53] There had been strongevidence in support of the proposal : incorporating a large sur-factant molecule into the cell membrane afforded a decreasein ATPase activity, because the substrate inducing ATPasecannot bind to the allosteric binding site.[54]

Cage dimeric 1,4-dihydropyridines

Compound H17 was found to be highly active in humancancer cells and shows no potential to induce P-gp activity(Figure 7). Compound JW41, prepared by Coburger et al. ,[38]

differs from H17 by just one aromatic substitution, but bothcompounds at 1 mm (the lowest inhibitory concentration) showbetter activity than verapamil. However, at the highest concen-trations, H17 led to an almost threefold increase in cellularuptake of daunorubicin, whereas JW41 exhibited only half theactivity of H17.

Macrocyclic bisbibenzyls

Dihydroxychantol A (DHA), a macrocyclic bisbibenzyl com-pound extracted from the liverwort Asterella angusta, has beenidentified as an antifungal agent (Figure 8). DHA was recentlyfound to give the highest potency, increasing the cytotoxicityof adriamycin toward K562/A02 cells with 3.84-fold MDR rever-sal at 5 mm and 8.18-fold MDR reversal at 20 mm, whereas com-pound 9 effected only 1.78-fold and 3.25-fold reversal, respec-tively, at the same respective concentrations. Furthermore,DHA can block the efflux of drugs by inhibiting P-gp functionand its expression pathway.[55]

Inhibitors of the NorA MDR Pump

Bacterial resistance to antibiotics has become an increasinglyserious problem in recent years. Among the various mecha-nisms of bacterial resistance to drugs, efflux pumps have beengiven particular attention, since they have been recognized asone of the most significant aspects in MDR bacterial infec-tions.[56]

Drug-resistant pathogens have evolved the MRSA (methicil-lin-resistant S. aureus) phenotype and the VRSA (vancomycin-resistant S. aureus) phenotype. Vancomycin was previously thedrug of last resort for the treatment of MRSA infections.[57] Un-fortunately, bacterial resistance has also emerged againstnewer agents such as linezolid and daptomycin shortly aftertheir use in the clinic.[58]

The NorA protein encoded by norA gene in S. aureus is adrug/protein antiporter that belongs to the major facilitator su-perfamily (MFS) and is responsible for the efflux of a widerange of substances such as norfloxacin, ethidium bromide,berberine, and acriflavine.[59]

MDR S. aureus has posed a significant obstacle in the treat-ment of bacterial infections. Many approaches have beenmade to explore new EPIs to facilitate the activity of existingantimicrobials, for use in antibiotic–EPI combination therapies.

Flavones and flavonolignans

Chrysosplenol D (3’,4’,5-trihydroxy-3,6,7-trimethoxyflavone;Figure 9) was initially isolated from the Chinese medicinalplant Artemisia annua L and is able to potentiate the activity ofnorfloxacin. This activity is presumably due to its inhibition ofMDR efflux pumps in S. aureus.[60]

Recently, the flavonolignan 5’-methoxyhydnocarpin-D8 (11)was identified as a potent inhibitor of the NorA pump(Figure 10). Based on the structure of the natural product 11, anumber of hydnocarpin-type flavonolignans were designedand synthesized for activity test purposes. Among the testedflavonolignan derivatives, compounds 12 and 13, in which thehydroxy group is removed from ring A, or other modificationsare made on rings B and C, showed activity similar to, or evenbetter than, that of compound 11.

SARs indicated that flavonolignans with or without free phe-nolic hydroxy groups at the C5 and C7 positions are all similar-ly active analogues. The most potent compound 14, whichlacks the hydroxy groups on ring A, contains a 4’-propoxy

Figure 7. Cage dimeric 1,4-dihydropyridines.

Figure 8. Macrocyclic bisbibenzyl analogues as inhibitors of P-gp.

Figure 9. Structure of chrysosplenol D.

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group, indicating that alkylation at the 4’-position with smalllipophilic side chains might be important for the improvementof antibacterial activity.[61]

Indoles

It has been reported[62] that the 2-phenyl-1H-indole INF55 sig-nificantly decreases the IC50 value of ciprofloxacin againstS. aureus SA-1199, to eightfold lower than that of reserpine

(Figure 11). SAR studies suggestedthat 5-nitro-2-(3-methoxycarbonyl)-phenyl-1H-indole was slightly morepotent than INF55. In contrast,compounds lacking 5-nitro groups,as exemplified by compound 15(Figure 12), were much less potentthan INF55. Among them, the 3’-methoxycarbonyl derivative 16was found to be the most potentderivative, displaying slightly

higher potency than INF55. Another important finding wasthat the analogue 17, which bears a 4’-hydroxymethyl group,was nearly equipotent with INF55 against wild-type and NorA-pump-overexpressing S. aureus strains. Therefore, compound17 is not only the best compound to progress into furtherstudies of mutual prodrugs employing labile ester linkages,but also a promising candidate for incorporation into dual-action mutual prodrugs that target the NorA pump.[62] In addi-tion, the substitution at C5 of INF55 was very important for in-hibitory activity. Compounds with the C5 substituent removedor replaced with electron-withdrawing groups such as carbon-yl, sulfonic, and nitrile groups show loss of inhibitory activity,whereas compound 18, with a nitrile group, showed activitysimilar to that of INF55.

It is well known that the plant antimicrobial berberine canbe readily extruded by bacterial MDR efflux pumps. Thus,

efflux pump inhibition increases the antimicrobial activity ofberberine. In another related study, a berberine–INF55 conju-gate linked through a methylene group resulted in a highly ef-fective antimicrobial agent SS14 (Figure 13), which can eludeefflux pump action and accumulate to high concentrations inbacteria. SS14 was 100-fold more active against S. aureus thanberberine, and even more effective (200–400-fold) againstS. aureus mutants overexpressing the NorA pump.[63]

In further studies, almost all newly synthesized 2-aryl-5-nitro-1H-indoles were found to be promising NorA pump inhibitors.In particular, compound 19 (Figure 14) showed the highest ac-tivity against strains such as K2361 overexpressing the NorApump, potentiating berberine activity by >15-fold at a con-centration of 0.8 mg mL�1.[64]

A recent QSAR model derived from genetic algorithm (GA)and partial least-squares (PLS) analyses can be used in the pre-diction of activity of new 2-aryl-5-nitro-1H-indole derivatives.With this model, 2-(2-azidomethyl-5-phenoxyphenyl)-5-nitro-1H-indole was predicted to be able to decrease the MIC of ber-berine to 0.091 mg mL�1 against the S. aureus K2361 strain over-expressing the NorA pump.[65]

Figure 10. Flavones and flavonolignans.

Figure 11. Structure of INF55.

Figure 12. 5-nitro-2-(3-methoxycarbonyl)phenyl-1H-indoles as EPIs of NorA.

Figure 13. Berberine and the berberine–INF55 conjugate SS14 as inhibitorsof NorA.

Figure 14. Compound 19 derived from 2-aryl-5-nitro-1H-indole as an inhibi-tor of NorA.

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Piperines

Piperine, which consists of a 1,3-benzodioxolane group, penta-dienoic acid, and piperidine, was shown to effect a twofold de-crease in the MIC of ciprofloxacin when used in combinationagainst S. aureus SA-1199 (Figure 15). Sangwan et al.[66] carriedout some modifications to compartment C of piperine by re-placing the piperidine moiety with aliphatic and aromaticamines. The obtained products (compounds 20–24) showed adecrease in the MIC of ciprofloxacin by fourfold at a concentra-tion of 25 mg mL�1 (Figure 16).

SAR studies showed that the nature of the basic moiety isimportant for its response toward drug potentiation. Relativeto piperine, with its piperidinyl group as the basic moiety, re-placement by small branched alkyl substituents such as isopro-pyl, isobutyl, or diisopropyl groups exhibited slightly better po-tentiation than straight chains such as n-propyl or n-butylgroups. Among them, the anilinyl moiety was found to be thebest substituent. On the other hand, introduction of electron-withdrawing groups (e.g. , nitrile) in the anilinyl moiety, as incompound 20, showed potentiation of ciprofloxacin. In partic-ular, the substitution by ortho-, meta-, and para-phenylaceta-mide moieties for the piperidinyl moiety exhibited the best po-tentiating effect for the drug.

After screening a library of 200 piperine analogues, a fewcompounds were identified to be two- or fourfold morepotent than the parent molecule in potentiating the activity ofciprofloxacin at a significantly lower minimal effective concen-tration. The two most active EPIs, namely SK-20 and SK-56,were more potent than known EPIs such as reserpine and vera-

pamil (Figure 17). These inhibitors acted in a synergisticmanner with ciprofloxacin by substantially increasing its activi-ty against both NorA-pump-overexpressing and wild-typeS. aureus isolates.[67]

The structural modification of piperine can result in EPIswith significantly enhanced activity; this is summarized as fol-lows: 1) The introduction of an alkyl group at C4 is the majorcontributor to potentiation, and ethyl and n-propyl groupshave the maximal effect. 2) Replacement of the piperidinylmoiety with an aromatic amine such as anisidine or toluidine(with an ethyl or n-propyl group at C4) shows maximum po-tentiation. 3) Unsaturation is an important contributor to po-tentiation; however, di- and tetrahydro derivatives show muchless activity than the corresponding unsaturated analogues.4) Reduction of the amide carbonyl group leads to a decreasein potentiation. 5) Piperine derivatives containing a 3,4-methyl-enedioxyphenyl or a 4-methoxyphenyl group in compartmen-t A (Figure 15) show enhanced potentiation as well.[67]

Pyridines

A pentasubstituted pyridine was isolated from the extract ofJatropha elliptica (Pohl) Muell Arg. , namely 2,6-dimethyl-4-phe-nylpyridine-3,5-dicarboxylic acid di-ethyl ester 25 (Figure 18). Com-pound 25 is an EPI against theNorA pump of S. aureus, enhancingthe activity of ciprofloxacin, nor-floxacin, acriflavine, and ethidiumbromide, but it does not potenti-ate the activity of pefloxacin, asthe latter is not a substrate of theNorA pump. The MIC of ciprofloxa-cin against S. aureus SA-1199Breaches 3 mg mL�1 in the presenceof compound 25 at 100 mg mL�1,whereas it is only 16 mg mL�1 inthe absence of 25.[14]

Phenylpiperidine-selective serotonin reuptake inhibitors(PSSRIs) can block the NorA pump in S. aureus. Preliminary op-timization of the aryloxymethyl substituent at C3 of paroxetineindicated that arylalkene and arylthioether moieties retain orenhance their activity as NorA EPIs ; the pharmacophore piperi-dine amine is necessary for their activity, and replacement ofthis group with N-methyl or N-acetyl significantly diminishesinhibition of the NorA pump. Moreover, the piperidine deriva-tives lacking a 4-fluorophenyl substituent were generally less

Figure 15. Structure of piperine with compartments A–C indicated.

Figure 17. Examples of EPIs of NorA obtained by screening 200 piperine an-alogues.

Figure 18. 2,6-Dimethyl-4-phenylpyridine-3,5-dicarboxyl-ic acid diethyl ester.

Figure 16. Compounds with modifications in compartment C of piperine.

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active than their precursors. 4-Fluorophenyl and 4-unsubstitut-ed piperidines bearing a 5-bromo-2-chloroaryl group at C3were the most potent NorA EPIs in each series. Ether-linkedand alkenyl-linked 5-bromo-2-chloro derivatives 26 and 27showed 50 % inhibition of the NorA pump in S. aureus strainSA-K2361 at 40–50 mm (Figure 19). In particular, aryloxy-linkedand alkene-linked 4-fluorophenyl analogues 28 and 29 werethe most potent NorA EPIs.[68]

The PSSRIs were found to possess low to moderate intrinsicantimicrobial activity against strains of S. aureus, P. aeruginosa,and E. coli that have efflux-related MDR phenotypes. Their ac-tivity, however, was unaffected by the efflux-related pheno-type, indicating that these PSSRIs are not substrates for the in-volved pumps.[68]

Fluoroquinolones

FQs are used extensively as antibiotics. To overcome efflux-mediated resistance to FQs, the co-administration of EPI withFQ is being pursued to overcome efflux-mediated resistance toantibiotics. FQ–EPI conjugates were designed and synthesizedbased on the strategy of introducing the dipeptide moiety intothe C7 aminoquinolone structures. Among the synthesizedFQ–EPIs, compound 30, which was prepared by attaching anaryl urea to the C7 amine group of ofloxacin, is one of themost potent NorA pump inhibitors reported to date(Figure 20). It effects >84 % inhibition of efflux at 10 mm, simi-lar to carbonyl cyanide m-chlorophenyl hydrazone (CCCP).[69]

Phenothiazines and thioxanthenes

Phenothiazines and two geometric stereoisomers of the thio-xanthene flupentixol were investigated against S. aureus byKaatz et al.[70] Experimental data indicated that they possess in-trinsic antistaphylococcal activity. Among them, thioridazineand prochlorperazine were the most potent compounds withrespect to inhibition of NorA-mediated ethidium bromideefflux. In addition, trans-(E)-flupentixol was twofold morepotent than its cis isomer against S. aureus SA-8325-4

(Figure 21). However, the mechanism by which phenothiazinesand thioxanthenes exert their antimicrobial effect and/or po-tentiate the activity of a vast array of antibacterial agents isnot completely understood. It may involve an interaction withthe pump to a certain extent.[70]

Benzothiophenes

Chabert et al.[71] demonstrated that the sulfur analogues ofINF55 and benzothiophene derivatives 31–34 showed high in-hibitory activity toward the NorA efflux pump (Figure 22).

N-Caffeoylphenalkylamides

N-trans-feruloyl-4’-O-methyldopamine, a methanolic extractfrom Mirabilis jalapa Linn. (Nyctaginaceae), showed moderateactivity as an EPI against S. aureus overexpressing the NorApump, causing an eightfold decrease in the MIC value of nor-floxacin at 292 mm (MPC8 = 292 mm). Among several synthe-sized tryptamine derivatives, N-trans-3,4-O-dimethylcaffeoyltryptamine (35) was chosen as a lead compound for its compa-rable potentiation of norfloxacin in MDR S. aureus to that ofthe standard reserpine (Figure 23).

Figure 19. Some phenylpiperidine-selective serotonin reuptake inhibitors(PSSRIs) as inhibitors of NorA.

Figure 20. Analogues of fluoroquinolone as inhibitors of NorA.

Figure 21. Examples of phenothiazines and thioxanthenes as EPIs of NorA.

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SARs indicated that a hydroxy group on the aromatic ring isbetter than a methoxy group or no substitution for the cin-namic portion, while the methoxy-substituted compound 36(MPC8 = 162 mm) gave better results than the hydroxy substitu-tion for the amine part. In particular, compound 37 (MPC8 =

57 mm) have the best results.[72]

Macrolides

Lipophilic tetrasaccharide murucoidins, extracted from Ipo-moea murucoides, have been identified as inhibitors of MDRpumps in S. aureus. These microbiologically inactive tetrasac-charides 38–41 (MIC>512 mg mL�1) were shown to increasenorfloxacin potency against the SA-1199B strain overexpressingthe NorA pump by fourfold at 25 mg mL�1, while compound 42exerted the same effect at the much lower concentration of5 mg mL�1 (Figure 24).[73]

Similarly, two microbiologically inactive orizabins 43 and 44displayed a strong synergistic effect in combination with nor-floxacin (Figure 25). In particular, compound 44 at 25 mg mL�1

was shown to increase the activity of norfloxacin by fourfold (8versus 32 mg mL�1) against the MDR strain SA-1199B.[74]

Pyrrolo[1,2-a]quinoxalines

A series of pyrrolo[1,2-a]quinoxalines having a structure similarto that of omeprazole were synthesized and evaluated fortheir activity against S. aureus SA-1199B. In the presence of theomeprazole analogue 45 at 128 mg mL�1, the MIC of norfloxa-cin was decreased 16-fold (Figure 26).[75]

Others

Grapefruit oil (GFO) can modulate bacterial sensitivity to anti-bacterial agents. Preliminary data suggested that 4-{[(E)-5-(3,3-dimethyl-2-oxiranyl)-3-methyl-2-pentenyl]oxy}-7H-furo[3,2-g]chromen-7-one (46), one of the components of GFO, couldenhance the susceptibility of MRSA to norfloxacin. Comparedwith the NorA EPI reserpine (10 mg L�1), compounds 46 and47, isolated from grapefruit, decreased the MIC of norfloxacinby 20-fold (Figure 27).[76]

5,9-Dimethyldeca-2,4,8-trienoic amides and 9-formyl-5-meth-yldeca-2,4,8-trienoic amides were prepared for biological evalu-ation. Among them, compounds 48–58 were found to be themost effective inhibitors against S. aureus SA-1199B, effecting a

Figure 23. N-Caffeoylphenalkylamides as inhibitors of NorA.

Figure 24. Some of the lipophilic tetrasaccharide murucoidins as inhibitorsof NorA.

Figure 22. Benzothiophenes as inhibitors of NorA.

Figure 25. Orizabins as inhibitors of NorA.

Figure 26. Pyrrolo[1,2-a]quinoxaline as an inhibitor of NorA.

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Efflux Pump Inhibitors

decrease in the MIC value of ciprofloxacin by fourfold at25 mg mL�1 (Figure 28).[77]

Epicatechin gallate and epigallocatechin gallate exhibited afourfold potentiation of the activity of norfloxacin against anorfloxacin-resistant S. aureus strain overexpressing the NorA

pump, and inhibited the extrusion of ethidium bromide tosome extent (Figure 29). However, they also stimulated effluxparadoxically when their concentrations were increased to�20 mm.[25]

Outlook

MDR to anticancer drugs and antibiotics is a crucial clinicalproblem that needs to be solved. MDR appears after long-termuse of a single drug and is often characterized by resistance toa series of structurally unrelated compounds. It is well knownthat efflux pump systems contribute strongly to MDR. EPIs in

combination with anticancer drugs or antibiotics can not onlyincrease the intracellular concentration of drugs that are expel-led by efflux pumps, but can also restore the efficacy of drugsagainst resistant strains and thus decrease the capacity for fur-ther acquired resistance.[16] Unfortunately, the research and de-velopment of EPIs has been fraught with many disappoint-ments. None of the inhibitors in clinical trials has been success-ful in circumventing chemoresistance so far. One of the mainreasons for this is the multifaceted nature of drug resistanceinherent to cancer tissues; there is much more to the chemo-resistance of tumors than P-gps.[78] On the other hand, EPI/efflux pump–substrate combinations have some drawbackssuch as low stability, low selectivity, and low bioavailability.

Nonetheless, research into identifying potential EPIs is ongo-ing both in academic institutions and in the pharmaceutical in-dustry.[79] In particular, the co-administration of an EPI and ananticancer drug has already been successful to some degree.[80]

To date, many EPIs with diverse chemical structures have beendescribed and identified; these can be extracted from theirnatural source, derived from such extracts, or they can be ob-tained by chemical modification of known efflux pump sub-strates.

This minireview has highlighted a number of EPIs of P-gpand the NorA pump according to their structural features aswell as their mechanisms of action and pharmacological prop-erties. In conclusion, candidate EPIs must be nontoxic, highlyselective, and appropriate for the particular application routein question. Reserpine is an example of a natural product thathas not undergone further development owing to its neuro-toxicity at concentrations required to inhibit the NorApump.[81] Therefore, there is an urgent need to identify a newgeneration of EPIs to overcome such problems.

Acknowledgements

This research was supported by the “Major R&D Program of NewDrugs”—National S&T Key Special Subject of China(2009ZX09103-115), National Natural Science Foundation ofChina (20872081).

Keywords: cancer · inhibitors · multidrug resistance · NorApump · P-glycoprotein

Figure 29. Structures of epicatechin gallate and epigallocatechin gallate.

Figure 27. Two components of grapefruit oil as inhibitors of NorA.

Figure 28. Examples of 5,9-dimethyldeca-2,4,8-trienoic amides and 9-formyl-5-methyldeca-2,4,8-trienoic amides as inhibitors of NorA.

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Received: January 6, 2010Revised: February 24, 2010Published online on April 1, 2010

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