synthesis and in-vitro antimicrobial evaluation of a high-affinity iron chelator in combination with...

Synthesis and in-vitro antimicrobial evaluation of ahigh-affinity iron chelator in combination withchloramphenicolChun-Feng Zhua, Di-Hong Qiuc, Xiao-Le Kongd, Robert C. Hiderd and Tao Zhoub

aCollege of Pharmaceutical Sciences, Zhejiang University of Technology, bSchool of Food Science and Biotechnology, Zhejiang GongshangUniversity, Hangzhou, cFaculty of Life Science and Biotechnology, Ningbo University, Ningbo, China and dDivision of Pharmaceutical Science, King’sCollege London, London, UK

Keywordsantimicrobial activity; chloramphenicol;hexadentate; 3-hydroxypyridin-4-one; ironchelator

CorrespondenceTao Zhou, School of Food Science andBiotechnology, Zhejiang GongshangUniversity, Hangzhou, Zhejiang, 310035,China.E-mail: [email protected]

Received September 4, 2012Accepted November 14, 2012

doi: 10.1111/jphp.12013

Abstract

Objectives The objectives of this study were first to design and synthesize a hex-adentate chelator with high iron(III) affinity and, second, to evaluate its antimi-crobial activity in the presence and absence of chloramphenicol.Methods A hexadentate ligand was synthesized by conjugating a protected biden-tate compound onto a tripodal structure. The pKa values and iron affinity of thechelator were determined by spectophotometric titration. Minimum inhibitoryconcentrations were determined by visual inspection of broth turbidity. The bac-tericidal rates were calculated by counting the colony numbers on a light boardafter incubation with and without an antimicrobial agent.Key findings A hexadentate 3-hydroxypyridin-4-one was found to possess a highaffinity for iron(III), with a pFe value of 31.2 (negative logarithm of concentrationof the free iron(III) in solution (when [Fe3+]Total = 10-6 m; [Ligand]Total = 10-5 m;pH = 7.4). We found that this chelator had an appreciable inhibitory effect in vitroagainst the two bacterial strains Providencia stuartii and Staphylococcus aureus,particularly in the presence of chloramphenicol.Conclusions A 3-hydroxypyridin-4-one hexadentate ligand has potential as anantimicrobial agent. Combination therapy with this iron chelator plus chloram-phenicol has potential for the treatment of extracellular infections.

Introduction

Iron is essential for almost all microorganisms by virtue ofits unique chemical properties, namely the ability to coordi-nate and activate oxygen and the possession of an idealredox chemistry between ferrous and ferric iron for involve-ment in electron transport and metabolic processes. Thus,limiting the amount of available iron should in principleinhibit microbial growth. Many microorganisms haveevolved strategies to scavenge and absorb iron from theenvironment by the production and secretion ofsiderophores, which are low-molecular-weight compounds(500–1500 Da) possessing a high affinity and selectivity foriron(III).[1] Most siderophores are structured around one ofthree types of bidentate chelator structure–hydroxamate,catecholate and a-hydroxycarboxylate. Thus, chelators con-taining these structures may accelerate the growth of micro-organisms. Indeed, many ferrophilic organisms, such asVibrio vulnificus, Yersinia enterocolitica and mucoralesspecies can use desferrioxamine B (DFO), a hydroxamate

siderophore produced by streptomyces species, for efficientiron uptake via the specific outer membrane receptorDesA.[2,3] There is no report relating to the siderophoresproduced by Providencia stuartii, whereas Staphylococ-cus aureus has been demonstrated to use twohydroxycarboxylate-type siderophores, staphyloferrin A andstaphyloferrin B, via the transporters Hts and Sir, respec-tively, to access the transferrin iron pool.[4,5] Such uptakecan be interrupted by the introduction of high-affinityiron-selective chelating agents.[6] The iron affinity of theseagents must be extraordinarily high, so that they can effi-ciently compete with siderophores.

Antimicrobial activity of chelators has been reportedpreviously.[7–11] In principle, the structure of potentialantimicrobial chelators should differ from those ofsiderophores, otherwise the iron–chelator complex will beused by the microorganism, leading to a failure of inhibi-tion of microorganism growth. The antimicrobial activity

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And PharmacologyJournal of Pharmacy

Research Paper

© 2012 The Authors. JPP © 2012Royal Pharmaceutical Society 2013 Journal of Pharmacy and Pharmacology, 65, pp. 512–520512

of the clinically useful iron chelators DFO, deferiprone andexjade, all of which have been used for the treatment ofiron-overload disorders, has been investigated.[12–14]

However, deferiprone and exjade are bidentate and triden-tate ligands, respectively, which have relatively low ironaffinity at low iron concentrations, and DFO is asiderophore and thus is capable of stimulating the growthof many bacterial strains. More recently, we have demon-strated that hexadentate 3-hydroxypyridin-4-ones caninhibit the growth of both Gram-positive and Gram-negative bacteria.[15,16] The hexadentate hydroxypyridinonedeveloped in this study has a completely different structurefrom all known siderophores so, unlike ferroxamine, theiron complex is predicted not to gain access to the bacteriaby receptor-mediated transport. The Gram-negative bacte-rium P. stuartii is a common cause of antibiotic-resistantinfections,[17] and the Gram-positive bacterium S. aureus isone of the most commonly encountered pathogenic organ-isms and is a frequent cause of bloodstream, respiratorytract, skin and soft tissue infection. The latter is themultidrug-resistant organism associated with MRSA.[18]

Herein we report the synthesis of a hexadentate3-hydroxypyridin-4-one which possesses a short PEG sidechain to enhance its water solubility.

Interestingly, the combination of iron chelators with anti-biotics has also been demonstrated to have clinical poten-tial.[13,14] In this study we report the in-vitro inhibitory effectof the hexadentate chelator when used alone and in combi-nation with chloramphenicol against P. stuartii andS. aureus.

Materials and Methods

Chemistry

All chemicals were of AR grade (Sigma) and used withoutany further purification. 1H NMR and 13C NMR spectrawere recorded on a Bruker Avance 400 spectrometer(Bruker Corporation, Germany) with tetramethylsilane asan internal standard. Electrospray ionization (ESI) massspectra were obtained by infusing samples into an LCQDeca XP ion-trap instrument (ThermoFinnigan, San Jose,CA, USA). High-resolution mass spectra (HRMS) wereobtained on a QTOFMicro (Waters, USA) by direct infusingsamples into the ESI source. The synthetic route for thehexadentate hydroxypyridinone 7 is shown in Figure 1.

Synthesis of di-tert-butyl 4-(3-tert-butoxy-3-oxopropyl)-4-(2-(2-(2-methoxyethoxy)ethoxy)acetamido)heptanedioate (3)

A solution of acid 1 (2.136 g, 12 mmol), amine 2 (4.15 g,10 mmol), 1-hydroxybenzotriazole (HOBt, 12 mmol) and1,3-dicyclohexylcarbodiimide (DCC, 12 mmol) in N,N-

dimethylformamide (DMF, 40 ml) was stirred at room tem-perature overnight. The reaction mixture was filtered toremove the white precipitate, 1,3-dicyclohexylurea, and thefiltrate was concentrated under high vacuum. The residuewas dissolved in dichloromethane (150 ml), washed with5% NaHCO3, saturated NaCL, 5% cold HCl and saturatedNaCL successively, and dried over anhydrous Na2SO4. Afterremoval of the solvent, the residue was purified by silica-gel column chromatography using ethyl acetate–hexane(1 : 1 to 2 : 1) as an eluent to provide pure product 3(4.72 g, 82%) as a colourless oil. 1H NMR (CDCl3): d 1.43(s, CH3, 27H), 1.98 (m, CH2, 6H), 2.19 (m, CH2, 6H), 3.38(s, CH3, 3H), 3.57 (m, CH2, 2H), 3.68 (m, CH2, 6H), 3.90 (s,CH2, 2H), 6.46 (s, NH, 1H). ESI-MS: 576 ([M + H]+).

Synthesis of 4-(2-carboxyethyl)-4-(2-(2-(2-methoxyethoxy)ethoxy)acetamido)heptanedioic acid (4)

A solution of 3 in formic acid was stirred at room tempera-ture for 24 h. After removal of formic acid, toluene wasadded and then removed to get rid of residential formicacid. The crude product 4 was obtained as a white solidin 100% yield. 1H NMR (DMSO-d6): d 1.87 (m, CH2,6H), 2.13 (m, CH2, 6H), 3.24 (s, CH3, 3H), 3.45 (m, CH2,2H), 3.55 (m, CH2, 6H), 3.81 (s, CH2, 2H), 6.84 (s, NH, 1H),12.11 (br, COOH, 3H). 13C NMR (DMSO-d6): d 27.9(CCH2CH2), 29.0 (CCH2CH2), 56.4 (CCH2CH2), 57.9(CH3O), 69.3 (CH2), 69.4 (CH2), 69.6 (CH2), 70.2 (CH2),71.1 (CH2), 168.6 (CONH), 174.2 (COOH). ESI-MS: 408([M + H]+).

Synthesis of N1,N7-bis([3-(benzyloxy)-1,6-dimethyl-4-oxo- 1,4-dihydropyridin-2-yl]methyl)-4-(3-((3-(benzyloxy)-1,6-dimethyl-4-oxo-1,4-dihydropyridin-2-yl)methylamino)-3-oxopropyl)-4-(2-(2-(2-methoxyethoxy)ethoxy)acetamido)heptanediamide (6)

A mixture of triacid 4 (0.407 g, 1 mmol), amine 5 (0.851 g,3.3 mmol), HOBt (0.51 g, 3.3 mmol) and DCC (0.68 g,3.3 mmol) in dry DMF (15 ml) was stirred at room tem-perature for 2 days. After filtration, the filtrate was concen-trated, the residue was purified by silica-gel columnchromatography using CH2Cl2/MeOH (20 : 1 to 5 : 1) as aneluent to give the desired product 6 as a pale yellow powder(1.03 g, 91%). 1H NMR (DMSO-d6): d 1.83 (m, CH2, 6H),2.03 (m, CH2, 6H), 2.26 (s, CH3, 9H), 3.16 (s, CH3, 3H), 3.39(s, CH3, 9H), 3.50 (m, CH2, 8H), 3.76 (s, CH2, 2H), 4.33 (d,J = 4.8 Hz, CH2, 6H), 5.06 (s, CH2, 6H), 6.19 (s, pyridinoneC-5H, 3H), 6.83 (s, NH, 1H), 7.28–7.37 (m, Ph, 9H), 7.42(m, Ph, 6H), 8.09 (t, J = 4.8 Hz, NH, 3H). 13C NMR(DMSO-d6): d 20.0 (CH3), 29.1 (CCH2CH2), 30.0(CCH2CH2), 34.3 (NHCH2-pyridinone), 35.6 (NCH3), 56.8

Chun-Feng Zhu et al. Iron chelator as an antimicrobial agent

© 2012 The Authors. JPP © 2012Royal Pharmaceutical Society 2013 Journal of Pharmacy and Pharmacology, 65, pp. 512–520 513

(CCH2CH2), 57.9 (CH3O), 69.3 (CH2), 69.4 (CH2), 69.6(CH2), 70.2 (CH2), 71.0 (CH2), 72.1 (CH2Ph), 117.3 (C-5Hin pyridinone), 127.7 (CH in Ph), 128.1 (CH in Ph), 128.3(CH in Ph), 137.6 (C in Ph), 140.2 (C-2 in pyridinone),145.5 (C-3 in pyridinone), 147.8 (C-6 in pyridinone), 168.4(CONH), 171.9 (C-4 in pyridinone), 172.0 (CONH). ESI-MS: 1128.5 ([M + H]+).

Procedure for synthesis of N1,N7-bis((3-hydroxy-1,6-dimethyl-4-oxo-1,4 -dihydropyridin-2-yl)methyl)-4-(3-((3-hydroxy-1,6-dimethyl-4-oxo-1,4-dihydropyridin-2-yl)methylamino)-3-oxopropyl)-4-(2-(2-(2-methoxyethoxy)ethoxy)acetamido)heptanediamide (7)

To a suspension of 6 (1.128 g, 1 mmol) and concentratedhydrochloric acid (1 ml) in MeOH (30 ml) was added 5%palladium/charcoal (0.15 g). Hydrogenation was carried outat 30 psi H2 for 5–6 h. After filtration to remove the cata-lyst, the filtrate was concentrated to dryness. The residuewas purified by crystallization from methanol–acetone.

Hydrochlorides of hexadentate ligand 7 were obtained as awhite solid (0.891 g, 92%). 1H NMR (DMSO-d6): d 1.85 (m,CH2, 6H), 2.12 (m, CH2, 6H), 2.57 (s, CH3, 9H), 3.21 (s,CH3, 3H), 3.40 (m, CH2, 2H), 3.52 (m, CH2, 6H), 3.79(s, CH2, 2H), 3.88 (s, CH3, 9H), 4.56 (d, J = 5.0 Hz, CH2,6H), 6.87 (s, NH, 1H), 7.28 (s, Pyridinone C-5H, 3H), 8.93(t, J = 5.0 Hz, NH, 3H); 13C NMR (DMSO-d6): d 20.5 (CH3),29.2 (CCH2CH2), 30.0 (CCH2CH2), 34.7 (NHCH2-pyridinone), 39.1 (NCH3), 56.8 (CCH2CH2), 57.9 (OCH3),69.4 (CH2), 69.5 (CH2), 69.7 (CH2), 70.3 (CH2), 71.1 (CH2),112.7 (C-5H in pyridinone), 139.9 (C-2 in pyridinone),142.6 (C-3 in pyridinone), 148.5 (C-6 in pyridinone), 159.5(C-4 in pyridinone), 168.6 (CONH), 173.2 (CONH). ESI-MS: 858.4 ([M + H]+), 429.8 ([M+2H]2+); HRMS: calcd. forC41H60N7O13 ([M + H]+), 858.4249; found, 858.4208.

Physico-chemical properties of iron chelator

Determination of pKa

The titration system used in this determination comprisedan autoburette (Metrohm Dosimat 765 l ml syringe) and an

OO

OH

ONH

ORO

OOR

OR

O

NH

NHO

ONH

NH

O

N

N

N

OR'O

O

OR'

O

R'O

H2N

OBu-tO

OOBu-t

OBu-t

O

1

2

3: R = Bu-t

4: R = H

a

b

c

6: R' = Bn

7: R' = H

d

2

OO

O

2

OO

O

2

N

O

OBn

NH25

Figure 1 Synthesis of the hexadentate hydroxypyridinone 7. Reagents and conditions: a, HOBt, DCC, DMF, rt, 24 h; b, HCOOH, rt, overnight; c,HOBt, DCC, DMF, rt, 48 h; d, 30 psi H2, 5% Pd/C, HCl, MeOH.

Chun-Feng Zhu et al.Iron chelator as an antimicrobial agent


HP 8453 UV-visible spectrophotometer. KCl electrolytesolution (0.1 m) was used to maintain the ionic strength.The temperature of the test solutions was maintained in athermostatic cuvette holder at 25 � 0.1°C using a Cary 1controller. An argon atmosphere was applied to the entiretitration equipment. The initial sample concentration wasapproximately 7 ¥ 10-5 m. pKa values were analysed fromthese data by pHab.[19]

Determination of iron(III) affinity

The stability constant of the 7–iron(III) complex wasdetermined by spectrophotometric titration against thehydroxyl anion. The automatic titration system used inthis study comprised of an autoburette (Metrohm Dosimat765 ml syringe) and Mettler Toledo MP230 pH meter withMetrohm pH electrode (6.0133.100) and a reference elec-trode (6.0733.100). KCl electrolyte solution (0.1 m) wasused to maintain the ionic strength. The temperature ofthe test solutions was maintained in a thermostatic-jacketed titration vessel at 25°C � 0.1°C by using a TechneTE-8J temperature controller. The solution under investi-gation was stirred vigorously during the experiment. AGilson Mini-plus No. 3 pump with speed capabilityof20 ml/min was used to circulate the test solutionthrough a Hellem quartz flow cuvette. For stability con-stant determinations, a 50-mm path length cuvette wasused, and for pKa determinations, a cuvette path length of10 mm was used. The flow cuvette was mounted on an HP8453 UV-visible spectrophotometer. All instruments wereinterfaced to a computer and controlled by a Visual Basicprogram. Automatic titration and spectral scans adoptedthe following strategy: the pH of a solution was increasedby 0.1 pH unit by the addition of KOH (0.097 m for pKadeterminations and 1.0 m for stability constant determina-tions) for from the autoburette; when pH readings variedby <0.001 pH units over a 3-s period, an incubation periodwas activated. For pKa determinations, a period of 1 minwas adopted; for stability constant determinations, aperiod of 5 min was adopted. At the end of the equilib-rium period, the spectrum of the solution was thenrecorded. The cycle was repeated automatically until thedefined endpoint pH value was achieved. All the titrationdata were analysed with the pHab program.[19] The speciesplot was calculated with the HYSS program.[20] Analytical-grade reagent materials were used in the preparation of allsolutions.

Antimicrobial assay

Reagents

The media used in this study were brain–heart infusion(BHI) agar and BHI broth, which were purchased from

Hangzhou Microbial Reagent Co., Ltd (Hangzhou, China).Antimicrobial agents were tested in triplicate at severalappropriate concentrations for their antimicrobial effects.The solution of compound 7 was prepared by dissolving inde-ionized water. Chloramphenicol solution was obtainedby dissolving in ethanol (0.01 g in 1 ml of 95% ethanol),followed by dilution with de-ionized water. The solutionswere stored at 4°C.

Bacterial strains

P. stuartii and S. aureus were purchased from ChinaGeneral Microbiological Culture Collection (CGMCC).These two bacteria were inoculated into a tube containingan inclined plane of BHI agar and cultured at 37°C for 24 h.This gel was then used to inoculate 5 ml of BHI broth andincubated at 37°C for 24 h before transferring 50 mlinto another tube of fresh BHI broth. This transfer wasincubated at 37°C to an optical density of P. stuartiiand S. aureus of approximately 106 colony-forming units(cfu/ml).

Measurement of minimum inhibitoryconcentration

All assays were cultured at 37°C for 24 h in 15 ¥ 75-mmtubes. The incubation medium was BHI broth. All tubescontained 80 ml of antimicrobial agent at different concen-trations (0–3000 mg/ml) and 20 ml of bacterial inoculum,with a total volume of 100 ml. After incubation at 37°C for2 h, 900 ml of sterilized water was added to each tube toreach a final volume of 1 ml, and the tubes were incubatedat 37°C for 24 h. Minimum inhibitory concentrations(MIC) were determined by visual inspection of the turbid-ity of broth in tubes.[21] All assays were carried out intriplicate.

Bactericidal rate

A range of concentrations of 7 and chloramphenicol weretested. To a sterile test tube were added 80 ml of drug solu-tion (except for controls, which contained 80 ml of sterilizedwater) and 20 ml of bacterial inoculum. The contents of thetube were fully mixed by shaking and the tube was stoppledand kept in an incubator at 37°C for 2 h. Then 900 ml BHImedium was added and the test tubes were cultured at 37°Cfor 24 h. The number of colonies on the agar was countedon a light board. All the experiments were performed intriplicate. The bactericidal rate was calculated as follows:

RX X

Xo t

o

= − ×100% where R is the bactericidal rate, Xo is

the number of bacteria in the absence of antimicrobialagent (control) and Xt is the number of bacteria in the pres-ence of antimicrobial agent.



Statistical analysis

The data concerning the various antimicrobial treatmentswere statistical analysed using analysis of variance test (SASV8 software). Significant differences between the treatmentswere examined by Duncan’s multiple range test. P � 0.05was considered as statistically significant.

Results

Chemistry

Triacid 4 was synthesized by coupling of 2-(2-(2-methoxyethoxy)ethoxy)acetic acid (1) to amine 2 in thepresence of 1-hydroxybenzotriazole (HOBt) and 1,3-dicyclohexylcarbodiimide (DCC), followed by treatmentwith formic acid. Then we synthesized 2-(aminomethyl)-3-(benzyloxy)-1,6-dimethylpyridin-4(1H)-one, a benzyl-protected bidentate hydroxypyridine (5) that contains a freeamino group, starting from kojic acid as previouslyreported, with slight modification.[22] Amine 5 was conju-gated to the triacid 4 via amide bonds in the presence ofHOBt and DCC in N,N-dimethylformamide (DMF) atroom temperature, providing the protected hexadentateligand 6 in 91% yield. Deprotection of benzyl groups on 6was achieved by hydrogenation in the presence ofpalladium/charcoal, generating the hexadentate chelator 7in excellent yield (92%). All the compounds were fully char-acterized by 1H NMR, 13C NMR, mass spectrometry andhigh-resolution mass spectrometry.

Physicochemical characterisation

To demonstrate the iron(III) affinity of hexadentate chela-tor 7, we determined its pKa values and stability constantfor the corresponding iron(III) complex using an auto-mated titration system.[23–25] The titration data were ana-lysed with the pHab.[19]

pKa values

The pH-dependent UV spectra of 7 (Figure 2) wererecorded between 250 and 350 nm over the pH range 1.8–11.4 for the free ligand. The speciation spectra demonstratea clear shift in lmax from 280 to 310 nm, which reflects thepH dependence of the ligand ionization equilibrium. Theligand 7 can be considered as a trimer of the correspondingbidentate ligand and it therefore possesses two sets ofintrinsic pKa values. Using the spectrophotometric titrationmethod, the pKa values of 7 obtained from nonlinear least-squares regression analysis were found to be 2.556, 3.284,4.029, 7.725, 8.579 and 9.672. Of the six pKa values, thethree lower values correspond to the 4-oxo functions andthe higher three correspond to the 3-hydroxyl functions.

Iron(III) affinity

The stability constant of an iron–ligand complex is one ofthe key parameters related to the chelation efficacy of aligand. As iron(III) forms a 1 : 1 complex with a hexaden-tate ligand, the stability constant of the 7–iron(III) complexK1 can be expressed as follows:

Fe LH FeL H KFeL H

Fe LH3

3 1

3

33

3+ ++

++ + =�[ ][ ]

[ ][ ](1)

where L represents ligand. Thus, the addition of basefavours the formation of complex FeL by consumingprotons. However, the addition of a large amount of basecould result in dissociation of FeL because of the followingequilibrium:

FeL OH Fe OH L+ +− − −4 43� ( ) (2)

Indeed, the stability constant of the 7-iron(III) complex canbe determined by the competition of iron binding betweenchelator 7 and hydroxyl anion. The UV spectra of 7 in thepresence of iron at different pH values are shown inFigure 3. With the increase of pH, the absorbance at 460 nmdecreased, suggesting the decrease of iron complex FeLconcentration. The logK1 value was determined to be34.21 � 0.035.

The pFe3+ value, defined as the negative logarithm of con-centration of the free iron(III) in solution (when[Fe3+]Total = 10-6 m; [Ligand]Total = 10-5 m; pH = 7.4), is a moresuitable parameter for comparison than the stability con-stant, since it takes into account the effect of ligand basicity,denticity and the degree of protonation. The pFe3+ value of7, calculated by using the measured stability constant logK1

290 310

0.00

0.20

0.40

0.60

0.80

1.00

250 270 330 350

pH 11.364

pH 1.809

Wavelength (nm)

Ab

sorb

ance

Figure 2 UV spectra of hydroxypyridinone 7. [7] = 20.3 mM (in12.018 ml of 0.1 M KCl), pH was changed from 1.809 to 11.364 bythe addition of KOH (0.097 M) at 25°C.



and pKa values, is extremely high, being 31.2 (the equilva-lent figure for desferrioxamine is 26.6).

Antimicrobial assay

Minimum inhibitory concentrations

The MICs for hexadentate 7 against P. stuartii and S. aureuswere determined to be 320 and 240 mg/ml, respectively. TheMICs for chloramphenicol against P. stuartii and S. aureuswere 2.4 and 3.2 mg/ml, respectively.

Bactericidal rate

To evaluate the influence of the two compounds in combi-nation, the inhibition of the Gram-negative bacteriumP. stuartii and the Gram-positive bacterium S. aureus byiron chelator 7 and chloramphenicol was investigated indi-vidually and in combination. We investigated the antibacte-rial effect by using several concentrations of chelator 7together with a fixed concentration of chloramphenicol.The results for P. stuartii are shown in Figure 4. When chlo-ramphenicol was used alone at concentrations of 0.4, 0.6and 0.8 mg/ml, the bactericidal rates against P. stuartii weredetermined to be 13.8% (Figure 4a), 26.9% (Figure 4b) and38.8% (Figure 4c), respectively. When chelator 7 was usedalone at a concentration of 20, 30, 40, 50 and 60 mg/ml, thebactericidal rates against P. stuartii were determined to be5.5%, 8.2%, 12.7%, 14.7% and 17.6%, respectively. It wasfound that the combination of chelator 7 with chloram-phenicol was more effective than either chelator 7 or chlo-ramphenicol used alone. For instance, in the case of the

combination of 60 mg/ml chelator 7 with 0.4 mg/ml chlo-ramphenicol, the bactericidal rate was measured as 50.1%,which was higher than the aggregate of the two compoundswhen presented individually (31.4%) (Figure 4a); in thecase of the combination of 60 mg/ml chelator 7 with0.8 mg/ml chloramphenicol, the bactericidal rate was meas-ured as 85.4%, which again was higher than the aggregate ofthe two when presented individually (56.4%) (Figure 4c).Statistical analysis of the results indicated that in all cases asignificant difference occurred between the combinationtreatment and treatment with chelator 7 or chlorampheni-col alone on P. stuartii (P � 0.001).

0.00

0.10

0.20

0.30

0.40

0.50

0.60

450 500 550 600 650

pH 10.630

pH 12.962

Wavelength (nm)

Ab

sorb

ance

Figure 3 UV spectra of hydroxypyridinone 7 in the presence ofiron(III). [7] = 32.6 mM, [Fe] = 29.7 mM (in 18.062 ml of 0.1 M KCl), pHwas changed from 10.630 to pH 12.962 by the addition of KOH(1.0 M) at 25°C.

0

20

40

60

80

A-I A-II A-III A-IV A-V

(a)

Bac

teric

idal

rat

e (%

)

7 alone

7+0.4 µg/mL chloramphenicol

0.4 µg/mL chloramphenicol

0

20

40

60

80

100

B-I B-II B-III B-IV B-V

(b)

Bac

teric

idal

rat

e (%

)

7 alone7+0.6 µg/mL chloramphenicol0.6 µg/mL chloramphenicol

0

20

40

60

80

100

C-I C-II C-III C-IV C-V

(c)

Bac

teric

idal

rat

e (%

)

7 alone



Figure 4 The inhibitory effect of hydroxypyridinone 7 in combinationwith chloramphenicol against P. stuartii. Concentration of 7 in I-V was20, 30, 40, 50 and 60 mg/ml, respectively; concentration of chloram-phenicol in (a), (b) and (c) was 0.4, 0.6 and 0.8 mg/ml, respectively.



The results of the antimicrobial assay against S. aureus ispresented in Figure 5. With chloramphenicol at a concen-tration of 0.4, 0.6 and 0.8 mg/ml, the bactericidal ratesagainst S. aureus were determined to be 18.6% (Figure 5a),24.6% (Figure 5b) and 32.8% (Figure 5c), respectively.When chelator 7 was used alone at concentrations of 20, 30,40, 50 and 60 mg/ml, the bactericidal rates against S. aureuswere determined to be 7.8%, 10.6%, 14.2%, 19.1% and21.6%, respectively. It was found that the combination ofchelator 7 with chloramphenicol was more effective thaneither chelator 7 or chloramphenicol used alone, againdemonstrating an enhanced effect. For instance, in the case

of the combination of 60 mg/ml chelator 7 with 0.8 mg/mlchloramphenicol, the bactericidal rate was measured as61%, which was higher than the aggregate of the two indi-vidual values (54.4%) (Figure 5c). Again, in all cases, a sta-tistically significant difference was found between thecombination treatment and treatment with chelator 7 orchloramphenicol alone on S. aureus (P � 0.001).

Discussion

The hexadentate hydroxypyridinone 7 was synthesized andits physico-chemical properties were characterised. The pKavalues of 7 are similar to those of two previously reportedhexadentate hydropyridinones.[26] The logK1 value of7–iron(III) complex was found to be between those of ironcomplexes of two hexadentate hydropyridinones (33.76 and35.07), but the pFe3+ value of 7 is slightly higher, namely31.2, as compared with, respectively, 30.4 and 30.0.[26]

Although the iron affinity of chelator 7 is weaker thanthat of the strongest-known siderophore enterobactin(logK1 = 49, pFe = 35.5),[27] it is stronger than that of manysiderophores, such as desferrioxamine (logK1 = 30.6,pFe = 26.6), desferrichrome (logK1 = 29.07, pFe = 25.2) anddesferricrocin (logK1 = 30.4, pFe = 26.5).[28] These proper-ties render chelator 7 to be a potent agent for controllingbacterial growth.

This study confirms that hexadentate hydroxypyridi-nones can inhibit the growth of S. aureus in vitro.[15,16] Fur-thermore, this study is the first to report the inhibitoryeffect of hexadentate hydroxypyridinone against P. stuartii.Thus, the hexadentate hydroxypyridinone 7 can inhibit thegrowth of both Gram-negative and Gram-positive bacteriain vitro.

Enhanced antimicrobial effect of two unrelated antimi-crobial agents has recently received widespread interest (e.g.the existence of an enhanced antimicrobial effect betweensilver nanoparticles and commonly used antibiotics).[29,30]

Significantly, the chelator ethylenediaminetetraacetic acid(EDTA) has been demonstrated to enhance the inhibitoryeffect of fatty acid sucrose esters against S. aureus.[31] In thisstudy, we investigated the use of hexadentate iron chelatorsin the presence of chloramphenicol. The antimicrobialeffect of the combination treatment against both P. stuartiiand S. aureus was found to be stronger than the effect ofchelator and chloramphenicol alone. Chloramphenicol hasbeen shown to inhibit microbial protein synthesis in a widevariety of bacteria,[32] and this activity will render themicrobe more sensitive to iron deprivation.

Conclusion

A hexadentate hydroxypyridinone with high iron(III) affin-ity has been synthesized. This iron chelator was found toexhibit inhibitory activity against P. stuartii and S. aureus in

0

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A-I A-II A-III A-IV A-V

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B-I B-II B-III B-IV B-V

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0.6 µg/mLchloramphenicol

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C-I C-II C-III C-IV C-V

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Figure 5 The inhibitory effect of hydroxypyridinone 7 in combinationwith chloramphenicol against S. aureus. Concentration of 7 in I-V was20, 30, 40, 50 and 60 mg/ml, respectively; concentration of chloram-phenicol in (a), (b) and (c) was 0.4, 0.6 and 0.8 mg/ml, respectively.



vitro. The combination treatment of the chelator with chlo-ramphenicol was found to show an enhanced inhibitoryeffect, suggesting that a combined formulation of iron che-lator with antibiotics may possess clinical potential, particu-larly in the treatment of external infections, such as thosefound in wounds and ulcers.

Declarations

Conflict of interest

The Author(s) declare(s) that they have no conflicts ofinterest to disclose.

Funding

This research work was financially supported by theNational Natural Science Foundation of China (No.20972138), Zhejiang Provincial National Natural ScienceFoundation of China (No. LY12B02014), Qianjiang ScholarsFund of Zhejiang Province (No. 2010R10051), and Scien-tific Research Foundation for the Returned OverseasChinese Scholars, State Education Ministry of China([2009]1590).

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