Bioorganic & Medicinal Chemistry Letters 24 (2014) 3506–3511

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier .com/ locate/bmcl

Effect of ferrocene-substituted porphyrin RL-91 on Candida albicansbiofilm formation

http://dx.doi.org/10.1016/j.bmcl.2014.05.0610960-894X/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Tel.: +381 (11) 3976034; fax: +381 (11) 3975808.E-mail address: nadastankovic@imgge.bg.ac.rs (N. Stankovic).

Rainer Lippert a, Sandra Vojnovic b, Aleksandra Mitrovic c, Norbert Jux d, Ivana Ivanovic-Burmazovic a,Branka Vasiljevic b, Nada Stankovic b,⇑a Friedrich-Alexander University Erlangen-Nuremberg, Department of Chemistry & Pharmacy, Egerlandstr. 1, 91058 Erlangen, Germanyb Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, PO Box 23, 11000 Belgrade, Serbiac Faculty of Chemistry, University of Belgrade, Studentski Trg 12-16, 11000 Belgrade, Serbiad Friedrich-Alexander University Erlangen-Nuremberg, Dep. Chemistry & Pharmacy and Interdisciplinary Center for Molecular Materials, Henkestr. 42, 91054 Erlangen, Germany

a r t i c l e i n f o a b s t r a c t

Article history:Received 11 March 2014Revised 15 May 2014Accepted 16 May 2014Available online 27 May 2014

Keywords:AntifungalPorphyrinsCandida albicansBiofilmFerrocene

Ferrocene-substituted porphyrin RL-91 exhibits antifungal activity against opportune human pathogenCandida albicans. RL-91 efficiently inhibits growth of both planktonic C. albicans cells and cells withinbiofilms without photoactivation. The minimal inhibitory concentration for plankton form (PMIC) wasestablished to be 100 lg/mL and the same concentration killed 80% of sessile cells in the mature biofilm(SMIC80). Furthermore PMIC of RL-91 efficiently prevents C. albicans biofilm formation. RL-91 is cytotoxicfor human fibroblasts in vitro in concentration of 10 lg/mL, however it does not cause hemolysis in con-centrations of up to 50 lg/mL. These findings open possibility for application of RL-91 as an antifungalagent for external antibiofilm treatment of medical devices as well as a scaffold for further developmentof porphyrin based systemic antifungals.

� 2014 Elsevier Ltd. All rights reserved.

7–11

Candida albicans is regular component of the normal humanmicro flora usually found in the respiratory, intestinal, and urogen-ital tracts. It is also a major human fungal pathogen causing infec-tions that range from those involving skin and superficial mucosaltissue to invasive systemic candidiasis.1,2 In their natural habitatCandida cells form biofilms, structured communities attached tohost tissue surfaces or surfaces of implanted medical devices.There is an estimation that upwards of 60% of all nosocomial infec-tions are due to microbial biofilms whose development in immu-nocompromised patients or patients with indwelling medicaldevices has wide-ranging consequences from prolonged hospital-ization to high mortality rates.3 They are difficult to eradicate,can cause device malfunction, and present continual source ofrecurring infections. Fully mature biofilms are usually formed after48 h and consist of yeasts, hyphae, and pseudohyphae withinstructurally highly heterogeneous exopolysaccharide matrix.4,5

Candida cells inside biofilm represent mixed population with dif-ferent growth and metabolic rates and gene expression.6 All thosemultiple factors that include architectural, biophysical, biochemi-cal, and metabolic properties of biofilm constituents contributeto formation of environmental niche which gives Candida cells highprotection from biocides, antifungal agents, and host immune

response. Despite of apparent abundance of antimycotic treat-ments there is still no efficient answer to Candida infections ofmedical devices.12 Resilience of biofilms to different groups of anti-mycotic,8,13,14 emerging resistance, especially to triazoles,15,16 andlack of safe systemic antifungals,17 are driving force behind con-stant search for novel agents and structures that can lead to devel-opment of new antifungal drugs.

Porphyrins are tetrapyrollic compounds that are widespreadthroughout living world in some of the most important mole-cules—hemoglobin, chlorophyll, cytochrome P450. Porphyrins areknown for their antibacterial, antifungal and antitumor activity,and are utilized as sensitizers in photodynamic therapy (PDT).18–20

meso-Tetrakis(4-ferrocenylpheny1)porphyrin, indicated as RL-91(Fig. 1), was chosen from a small library of meso-tetraphenylsubstituted porphyrins tested for their antifungal activity onCandida cells. RL-91 bears four ferrocene moieties covalentlyattached in the para-position of each of the four meso-phenyl rings,resulting in a very rigid structure that was previously described forits electron transfer abilities.21

The aim of this study was to examine antifungal activity offerrocene-substituted porphyrin RL-91 against C. albicans with-out photoactivation. It’s ability to prevent C. albicans biofilmsformation and activity against mature biofilms was alsoassessed, as well as in vitro cytotoxic effect on human fibroblastcell line.

Figure 1. Different derivatives of TPP. Arrow indicates order of decreasing hydrophobicity with MSre being the least hydrophobic.

R. Lippert et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3506–3511 3507

Under applied Lindsey conditions,22 RL-91 was synthesizedfrom 4-ferrocenylbenzaldehyde and pyrrole,23 according to a pro-cedure described by Schmidt et al.21 In general the solubility ofRL-91 was poor, but sufficient in dimethyl sulfoxide (DMSO) thatwas used as a solvent for RL-91, as well as for nystatin andvoriconazole in all further experiments. Synthesis of 5,10,15,20-tetrakis-phenyl-porphyrin (TPP), 5,10,15,20-tetrakis-(40-tert-butyl-phenyl)-porphyrin (TTBPP),24 5,15-(20,60-bis-(methoxymethylene)-40-tert-butylphenyl)-10,20-(40-tert-butylphenyl)-porphyrin and5,10,15,20-tetrakis-(20,60-bis-(methoxymethylene)-40-tert-butyl-phenyl)-porphyrin (4OMe, 8OMe),25 10,15,20-tris-(40-tert-butyl-phenyl)-5-(20-carboxy-phenyl)-porphyrin (MSre),26 and 5,15-bis-(40-tert-butyl-20,60-bis-(200,200-bis-(ethoxycarbonyl)-ethyl)-phenyl)-10,20-bis-(40-tert-butylphenyl)-porphyrin (FB-4x),27 was describedpreviously. In a statistical porphyrin synthesis, with appliedLindsey conditions,22 RL-61 and RL-XYL were synthesized fromtert-butyl-2-(4-formylphenoxy)acetate and 4-(tert-butyl)-2,6-dimethylbenzaldehyde, respectively.

C. albicans ATCC10231 strain was maintained on Sabourauddextrose (SD) agar.28 The minimum inhibitory concentration ofRL-91 for planktonic cells (PMIC) was assessed in SD medium usinga microdilution method. Inocula were adjusted to density0.5 � 105 cell/mL according to standard method for the MIC deter-mination of fermentative yeasts.29 After 24 h treatment, 5 lL ofcultures from microtiter plate were transferred to 5 mL of freshmedium, grown for the next 24 h and OD600nm was measured(InfiniteM200pro, Tecan, Männedorf, Switzerland). PMIC wasdefined as the highest dilution of compound at which no evidenceof growth was observed.

Candida biofilm formation was assessed as described by Kaganand co-workers with some modifications.30 For inhibition of biofilmformation concentrations of RL-91 (12.5, 25, 50, and 100 lg/mL)and nystatin (nystatin powder, Hemofarm, Vrsac, Serbia) (1.25,2.5, 5, and 10 lg/mL) were tested on standardized high density cellsuspension (5 � 106 cells/mL) in SD medium at 37 �C for 72 h. Inexperiments of biofilm disruption concentrations of RL-91 (100,200, and 500 lg/mL) and nystatin (10, 20, and 50 lg/mL) weretested on mature biofilms (48 h) in SD medium at 37 �C for 24 h.Upon the treatments the viability assay with 3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was performed.31

Synergistic effect of RL-91 (25 lg/mL), nystatin (2.5 lg/mL), andvoriconazole (8 lg/mL) was monitored as change of OD600nm ofstandardized C. albicans suspension (0.5 � 105 cell/mL) in SDmedium in quadruplicate during 24 h. Effects of photoactivationof RL-91 and TPP on antifungal properties were measured aschange of OD600mn of standardized C. albicans suspension in SD

medium in quadruplicate during 24 h. Cells were illuminated withblue LED light (400–420 nm) for 30 min from 10 cm distance.

Viability staining of Candida cells with propidium iodide (PI)and 4,6-diamidino-2-phenylin (DAPI) was done as described previ-ously with some modifications.32 Standardized cell suspension(OD600nm 1) was treated with RL-91 (50 or 100 lg/mL) in darkfor 3 h. Final concentrations of PI and DAPI were 100 lg/mL and1 lg/mL, respectively. Upon treatment cells were studied underOlympus BX51 fluorescent microscope and analyzed with Cytovi-sion 3.1 software (Applied Imaging Corp., San Jose, USA).

Cytotoxicity assay was performed on MRC5 cell line (humanlung fibroblast, obtained from ATCC) in RPMI-1640 medium (Gibco,Carlsbad, USA) supplemented with 100 lg/mL streptomycin,100 U/mL penicillin, and 10% (v/v) fetal bovine serum (FBS).MRC5 cells were treated with increasing concentrations (1, 10, 50,100 or 200 lg/mL) of RL-91 for 24 h and cytotoxicity was deter-mined using MTT reduction assay.33 Hemolysis assay with sheepred blood cells (RBC) (1% v/v, Torlak, Belgrade, Serbia) was doneas previously described,34 with 2.5, 25, or 250 lg/mL of RL-91 orTPP. Hemoglobin absorbance was measured at 405 nm (platereader Labsystem Multiscan RC, MTX Labsystems Inc., Vienna, USA).

In this study we assessed 9 substituted porphyrin (Fig. 1) forantifungal properties. Therefore, derivatives of TPP were synthe-sized (Fig. 1) that provide different electron donor/acceptor groupsin the periphery of the meso-phenyl rings to explore the effects ofthese porphyrins on their biological activity. In order to graduallyincrease the hydrophobicity of the porphyrin derivatives in rela-tion to TPP, TTBPP, with tert-butyl groups in the para-positions ofthe meso-phenyl rings, and RL-XYL, with tert-butyl groups in thepara-positions and additional methyl groups in the ortho-positionsof the meso-phenyl rings were synthesized. As representatives ofrelatively hydrophilic compounds porphyrins equipped with fouror eight methoxymethylene groups (4OMe, 8OMe), four ferrocenylentities (RL-91), ester groups (RL-61), malonates (FB-4x), and car-boxylic acids (MSre) were synthetized. All compounds were readilysoluble in DMF or DMSO. From a library of compounds only RL-91,bearing covalently attached ferrocene moiety, showed antifungalactivity.

The growth inhibition of planktonic cells occurred at PMIC ofRL-91 of 100 lg/mL without photoactivation (Fig. 2). The presenceof the compound caused fungistatic effect during the first 6 h ofcultivation at concentrations lower than 12.5 lg/mL (Fig. 2A).However, upon 24 h cultivation, the presence of 12.5 lg/mL causedabout 65% inhibition, while 50 lg/mL and caused above 95% inhi-bition in comparison to no treatment control. As a positive controlwe included polyene nystatin, renowned antifungal now for

Figure 2. Effect of RL-91 on planktonic Candida cells. (A) Fungistatic effect of RL-91 on Candida albicans in SD medium h, non-treated control; �, 3.12 lg/mL RL-91; N,6.25 lg/mL RL-91; 4, 12.5 lg/mL; d, 25 lg/mL; s, 50 lg/mL RL-91; j, 100 lg/mL RL-91; (B) fluorescent microscopy of Candida albicans cells, (C) 3 h treatment with DMSO;(D) with sub PMIC concentration of RL-91 (50 lg/mL), (E) and PMIC concentration of RL-91 (100 lg/mL). Candida cells were stained with DAPI (blue) and PI (red).

3508 R. Lippert et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3506–3511

decades in clinical use in treatment of mucocutaneous Candidainfections.35 PMIC of clinically used nystatin preparation wasdetermined to be 10 lg/mL.34

Changes in Candida cells viability in SD medium with the RL-91doses below PMIC (50 lg/mL) and PMIC (100 lg/mL) were demon-strated with PI/DAPI staining (Fig. 2D and E). We observed due to PIpermeability and binding to nucleic acid that can occur only in thedead cells yielding fluorescence in the red wavelength region thattreatment with doses of RL-91 higher than PMIC induced the mem-brane damage and cell death (Fig. 2E). Candida cells treated with50 lg/mL of RL-91 had mostly intact membranes although the celldeath was observable according to blue staining by DAPI that eas-ily passes the membrane of both living and dead cells stronglybinding to DNA and brightly staining condensed nuclei indicatingapoptosis (Fig. 2D). The presence of DMSO on Candida cells inapplied concentrations (up to 2.5%) did not cause any adverseeffects (Fig. 2C).

Formation of biofilm both on host tissues or medical devicessurfaces has negative clinical consequences as cells within the bio-films are protected from host immune responses and from theaction of antifungal agents. Therefore we investigated ability ofporphyrin RL-91 to prevent biofilm formation in concentrationslower and equal to minimal inhibitory concentrations for plank-tonic Candida cells. The biofilm inhibitory effect of RL-91 wasassessed in SD medium by the metabolic activity of cells and theirability to reduce MTT again not applying any photoactivation.Effect of RL-91 was compared to effects of polyene macrolide nys-tatin. RL-91 showed inhibitory effects on biofilm formation in subPMIC concentrations which was dose dependent (Fig. 3A). Increas-ing sub PMICs of 12.5, 25, and 50 lg/mL of RL-91 survived about65%, 50%, and 40% of the cells respectively, while the PMIC of100 lg/mL was fungicidal to 95% of cells in high density cell sus-pension used initially for biofilm cultivation. Sub PMIC concentra-tions of nystatin did not prevent biofilm formation, while atconcentration of 10 lg/mL about 20% of the cells survived (Fig. 3B).

Mature biofilms are structured formations in which embeddedcells are protected in multiple ways. Experiments with cell survivalin pre-formed biofilms showed that concentration of RL-91 of100 lg/mL (PMIC) represents minimal inhibitory concentrationfor sessile cells effectively diminishing survival of cells in biofilmby 80% (SMIC80). Further increase of concentration to 200 lg/mL

and 500 lg/mL did not add significantly to fungicidal effect onthe cells embedded in biofilm since 15% to 17% cells remained met-abolically active in the presence of high porphyrin concentrations(Fig. 3C). Nystatin in concentration of PMIC and two times PMICfor planktonic cells (10 and 20 lg/mL respectively) had no impacton preformed mature biofilms. Only concentration as high as50 lg/mL caused fungicidal effect giving 10% of cell survival(Fig. 3D).

In order to establish cytotoxicity of RL-91 to human cells itsantiproliferative activity against MRC5 human lung fibroblasts aswell as disruptive effect on cell membrane of sheep red blood cells(RBC) were investigated (Fig. S1). The concentration of 10 lg/mLwhich showed fungistatic effect on planktonic Candida cells caused80% inhibition of fibroblasts proliferation, while at PMIC concen-tration this inhibition was more than 90% (Fig. S1A). However, thistoxicity does not initially include membrane disruption step sinceRL-91 exhibited no hemolytic effect at low and medium doses (10and 25 lg/mL), while concentration of 250 lg/mL showed mediumhemolytic effect of 37% (Fig. S1B).

Candida albicans is human commensal fungus and opportunisticpathogen associated with 78% of fungal nosocomial infections andover 10% of all nosocomial infections.36 The major source of infec-tion are Candida biofilms formed on surfaces of host tissues, med-ical devices and implants.37 Biofilms show high resilience toconventional antifungal treatments therefore new molecules andtreatment strategies that would lead to biofilm growth inhibitionor biofilm eradication are constantly developed. Porphyrins areknown as photosensitizers (PS) in photodynamic therapy (PDT)of cancer cells,38 some of them as Photofrin�, Photosan III�, andLaserphyrin� being approved for clinical use. Photofrin� was testedagainst Candida biofilms showing significant reduction in biofilmformation upon irradiation of 18 J/cm2,39 and only recently por-phyrins XF-73 and TMPyP were shown to be efficient as photosen-sitizers in photodynamic therapy of Candida biofilms after 4 hincubation and 48.2 J/cm2 irradiation.40

The therapeutic effect of XF-73 and TMPyP is based on the elec-trostatic interaction of these positively charged photosensitizerswith the negatively charged outer cell wall of the Candida spe-cies.39 Upon irradiation of the attached photosensitizers with light,reactive oxygen species are generated that effectively cause irre-versible damage to the Candida cells, with first changes affecting

Figure 3. Inhibition of the Candida biofilm formation and fungicidal effect on Candida biofilms. Effect of increasing concentrations of RL-91 (A) and nystatin (B) on biofilmformation. Interaction of PMIC and supra PMIC concentrations of RL-91 (C) and nystatin (D) with 48 h mature Candida biofilms. Biofilm viability was estimated using MTTreduction assay. Columns represent % of the growth control (100%).

R. Lippert et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3506–3511 3509

the plasma membrane.41 RL-91 was developed as a model mole-cule to investigate light-initiated electron transfer in photosynthe-sis.21 We compared activity of 2, 4, 8, and 16 lM RL-91 in the darkand upon photoactivation by blue light (400–420 nm) with activityof equimolar concentrations of renowned photosensitizer TPPunder the same conditions (Fig. S2). TPP did not show any anti-fungal activity in the dark (Fig. S2A). However, hemolytic activityof 250 mg/mL of TPP on 1% sheep RBC was 87.5%, 2.4 times higherthan that of RL-91 indicating substantial potential of TPP for dis-turbing membrane integrity. Upon photoactivation TPP did notshow antifungal activity in concentration tested (Fig. S2B) probablydue to high peptone content of SD medium.42 Antifungal activity ofRL-91 did not change significantly after the photoactivation(Fig. S2D and E), suggesting that the presence of ferrocene moietyalone was sufficient for its pronounced biological activity. Theanomalous metabolism of the ferrocene material often showsunexpected biological activity but the detailed underlying mecha-nism is still inconclusive.43,44 The incorporation of ferrocene to aporphyrin increased the lipophilic character of the porphyrin andresulted in a higher membrane permeation ability.45 We testedsynergistic effect of two antifungals in clinical use which affectfungal cell membrane through two different mechanisms of actionwith sub PMIC concentrations of RL-91. Polyene nystatin with highaffinity to ergosterol makes structured barrel-like pores in plasmamembrane and can disrupt vacuolar membrane,46,47 while azoledrug voriconazole enters cell by facilitated diffusion and inhibitssynthesis of membrane sterols.48 RL-91 (25 lg/mL) and nystatin(2.5 lg/mL) in concentrations four-fold lower than their individualPMICs had completely inhibited growth of Candida cells (Fig. S3A).Although most of the C. albicans strains are very sensitive to voric-onazole,49 we estimated PMIC of ATCC10231 strain to be P16 lg/mL.Two-fold lower concentration of voriconazole (8 lg/mL) togetherwith 25 lg/mL of RL-91 had abolished growth of Candida cells insuspension (Fig. S3B). This findings suggests that RL-91 in subPMIC concentrations could be combined with other antifungalsknown for their high toxicity in order to reduce therapeuticconcentrations.

In this study antifungal activity of RL-91 without photoactiva-tion and its ability to inhibit both growth and biofilm formationin C. albicans was reported for the first time. This is importantknowing that the biofilm exopolymer matrix represents a physicalbarrier for the photosensitizers as well as light.14 MIC for plank-tonic Candida cells (PMIC) of RL-91 was 100 lg/mL with pro-nounced fungistatic effect in concentration as low as 10 lg/mL(Fig. 2A). The lowest fungistatic concentration of about 10 lg/mLof RL-91 for cells in suspension was equal to IC80 for human fibro-blasts (Fig. S1A) therefore leaving very narrow window for thedevelopment of RL-91 as systemic antifungal. PMICs of antifungalsagainst a range of C. albicans strains and clinical isolates varygreatly from 0.5 lg/mL to 1 mg/mL for amphotericin B,50 16 ng/mL to 512 lg/mL for fluconazole,51 0.54 lg/mL,52,53 5.29 lg/mL,54

and 10 lg/mL for nystatin,34 4 ng/mL to 0.125 lg/mL for micafun-gin.55 Because of protective effects of biofilm lifestyle SMIC forsessile Candida cells can vary in considerable degree ranging from4-fold for nystatin, 8-fold to 16-fold for chlorhexidine, 16-fold foramphotericin B, to 1000-fold and more in case of fluconazole andmiconazole.8,56 Besides biofilm effects Candida cells are developing‘classic’ resistance mechanisms to antifungals consisting of chang-ing of composition of drug targets, acquiring of genes coding forefflux pumps, etc.57,58 Unlike for all major classes of antifungals,excluding polyenes, resistance to porphyrins is not commonlyreported. In early stages of biofilm development RL-91 was effi-cient in prevention of biofilm formation in dose dependent man-ner. Results for nystatin showed that only PMIC concentration of10 lg/mL had significant effect on biofilm formation, giving about20% metabolically active cells upon 72 h incubation. RL-91 PMIC of100 lg/mL completely inhibited growth of planktonic Candidacells, while in biofilm prevention experiments same concentrationgave about 5% survivals. This discrepancy is probably due to differ-ent cell density of inocula which were standardized to 0.5 � 105 fordetermination of PMIC and 5 � 106 for biofilm formation, respec-tively. When examined under fluorescent microscope upon DAPI/PI staining, cells exposed to RL-91 PMIC appeared bright red dueto PI penetrating disrupted cell membrane or blue with condensed

3510 R. Lippert et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3506–3511

nuclei stained with DAPI (Fig. 2C) indicating apoptosis. Cells trea-ted sub PMIC of 50 lg/mL represented a mixture of viable cellswith diffuse chromatin, cells that stained mostly PI negative withcondensed nuclei, and a small portion of PI positive cells(Fig. 2D). When treated with 250 lg/mL of RL-91 sheep RBCshowed 37% hemolysis. This data suggest that porphyrin can dis-rupt cell membrane in dose dependent manner. Membrane disrup-tion is probably not a primary mode of action of RL-91 since PInegative cells show signs of apoptotic changes. This is in accor-dance with fact that porphyrins exert their activity by generatingreactive oxygen species (ROS) oxidizing and damaging multiplecellular targets—proteins, lipids, and RNA, with DNA being lessaccessible because of protective effect of nuclear envelope.18,19

Lack of preference towards specific lipid composition in cell mem-brane could be one of the reasons for good activity of RL-91 onmature Candida biofilms. Interactions between sterols and phos-pholipids in the membrane lipid rafts affect fluidity, asymmetry,and transport across the membranes.59 Alterations in the sterol/phospholipid composition of the fungal cell membrane duringbiofilm maturation may diminish drug uptake rendering cellsinsensitive to most membrane active antifungals.57,60 Our resultson nystatin influence on mature biofilm (Fig. 3D) where concentra-tion of 5� PMIC (50 lg/ml) gave 9.54% of metabolically active cellsare in accordance with literature data on diminished sensitivity ofcells in biofilm to ergosterol binding polyenes.61 Exopolysaccharidebiofilm matrix represents physical barrier to antifungals. Resultson susceptibility of mature biofilm to RL-91 (Fig. 3C) with SMIC80

equal to PMIC (100 lg/ml) imply good biofilm penetrability. Thirdpossible mechanism of RL-91 action could be via porphyrininterfering with the heme uptake system of the cell. Unlike Saccha-romyces cerevisiae, Candida, an opportunistic pathogen, can adaptits strategy for iron uptake to the physiological context,62 anduse heme as a sole iron source.63 This pathway can be used foruptake of other porphyrins mimicking heme. In our preliminarysusceptibility testing of various microorganisms to RL-91 S. cerevi-siae was completely insensitive to RL-91 (data not shown).

RL-91, a ferrocenyl substituted porphyrin is efficient in treat-ment of mature biofilms with good penetrability and SMIC in thesame order of magnitude to PMIC. It is promising agent in treat-ment of medical devices prone to development of Candida biofilmswhich are source of recurring fungal infections. In commonly usedbiocides such as ethanol (EtOH) and hydrogen peroxyde (H2O2)effective concentrations reducing 80% of Candida growth are about3 times higher for biofilms compared to planktonic cells.11 H2O2

has been used for disinfection of oral hygiene devices and contactlenses,64,65 while the utility of ethanol was investigated as centralvenous catheter lock therapy.11 Our results indicate that RL-91may have therapeutic potential in the treatment and disinfectionof contaminated medical devices and equipment and enhancementof the activity of other antifungals and biocides in treatment ofbiofilms associated with medical devices and equipment. There isalso strong possibility of further development of antifungal proper-ties of RL-91 since meso-tetraphenylporphyrins can be usefulscaffolds for numerous substitution and metal addition, whichcould substantially modify its bioactivity.

Acknowledgments

This work was supported by Ministry of Science andTechnological Development of the Republic of Serbia (Grantnumber: 173048, MSTD, 2011-2014). R. L. and I. I.-B. acknowledgethe support by the intramural grant from University Erlangen-Nürnberg (Emerging Field Initiative: Medicinal Redox InorganicChemistry). Authors are grateful to Dr. Jasmina Nikodinovic-Runicfor helpful suggestions during manuscript preparation.

Supplementary data

Supplementary data associated with this article can be found,in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.05.061.

References and notes

1. Cheng, M. F.; Yang, Y. L.; Yao, T. J.; Lin, C. Y.; Liu, J. S.; Tang, R. B.; Yu, K. W.; Fan,Y. H.; Hsieh, K. S.; Ho, M.; Lo, H. J. BMC Infect. Dis. 2005, 5, 22.

2. Andes, D. R.; Safdar, N.; Baddley, J. W.; Playford, G.; Reboli, A. C.; Rex, J. H.;Sobel, J. D.; Pappas, P. G.; Kullberg, B. J. Clin. Infect. Dis. 2012, 54, 1110.

3. Davey, M. E.; O’Toole, G. A. Microbiol. Mol. Biol. Rev. 2000, 64, 847.4. Chandra, J.; Kuhn, D. M.; Mukherjee, P. K.; Hoyer, L. L.; McCormick, T.;

Ghannoum, M. A. J. Bacteriol. 2001, 183, 5385.5. Hawser, S. P.; Douglas, L. J. Infect. Immun. 1994, 62, 915.6. Ramage, G.; Saville, S. P.; Thomas, D. P.; Lopez-Ribot, J. L. Eukaryot. Cell 2005, 4,

633.7. Tobudic, S.; Kratzer, C.; Lassnigg, A.; Presterl, E. Mycoses 2012, 55, 199.8. Lamfon, H.; Porter, S. R.; McCullough, M.; Pratten, J. J. Antimicrob. Chemother.

2004, 53, 383.9. Chandra, J.; Mukherjee, P. K.; Leidich, S. D.; Faddoul, F. F.; Hoyer, L. L.; Douglas,

L. J.; Ghannoum, M. A. J. Dent. Res. 2001, 80, 903.10. Watamoto, T.; Samaranayake, L. P.; Egusa, H.; Yatani, H.; Seneviratne, C. J. J.

Med. Microbiol. 2011, 60, 1241.11. Nett, J. E.; Guite, K. M.; Ringeisen, A.; Holoyda, K. A.; Andes, D. R. Antimicrob.

Agents Chemother. 2008, 52, 3411.12. Meis, J. F.; Verweij, P. E. Drugs 2001, 61, 13.13. Hawser, S. P.; Douglas, L. J. Antimicrob. Agents Chemother. 1995, 39, 2128.14. Taraszkiewicz, A.; Fila, G.; Grinholc, M.; Nakonieczna, J. Biomed. Res. Int. 2013,

2013, 150653.15. Sanglard, D.; Odds, F. C. Lancet Infect Dis 2002, 2, 73.16. Pfaller, M. A. Am. J. Med. 2012, 125, S3.17. Hamill, R. J. Drugs 2013, 73, 919.18. Stojiljkovic, I.; Evavold, B. D.; Kumar, V. Expert Opin. Invest. Drugs 2001, 10, 309.19. Calzavara-Pinton, P.; Rossi, M. T.; Sala, R.; Venturini, M. Photochem. Photobiol.

2012, 88, 512.20. Burda, W. N.; Fields, K. B.; Gill, J. B.; Burt, R.; Shepherd, M.; Zhang, X. P.; Shaw, L.

N. Eur. J. Clin. Microbiol. Infect. Dis. 2012, 31, 327.21. Schmidt, E.; Calderwood, T.; Bruice, T. Inorg. Chem. 1986, 25, 3718.22. Lee, C.-H.; Lindsey, J. S. Tetrahedron 1994, 50, 11427.23. Huang, G.; Li, B.; Liu, W.; Sh, i. L.; Ma, Y. J. Chem. Res. 2000, 10, 491.24. Drain, C. M.; Gong, X. Chem. Commun. 1997, 2117.25. Jux, N. Org. Lett. 2000, 2, 2129.26. Jasinski, S.; Ermilov, E. A.; Jux, N.; Röder, B. Eur. J. Org. Chem. 2007, 7, 1075.27. Jee, J. E.; Eigler, S.; Hampel, F.; Jux, N.; Wolak, M.; Zahl, A.; Stochel, G.; van

Eldik, R. Inorg. Chem. 2005, 44, 7717.28. Sigma-Aldrich, Sabouraud Dextrose Broth.29. Eucast Clin. Microbiol. Infect. 2003, 1. European Society of Clinical Microbiology

and Infectious Diseases.30. Kagan, S.; Jabbour, A.; Sionov, E.; Alquntar, A. A.; Steinberg, D.; Srebnik, M.; Nir-

Paz, R.; Weiss, A.; Polacheck, I. J. Antimicrob. Chemother. 2013.31. Hansen, M. B.; Nielsen, S. E.; Berg, K. J. Immunol. Methods 1989, 119, 203.32. Farrell, H.; Hayes, J.; Laffey, J.; Rowan, N. J. Microbiol. Methods 2011, 84, 317.33. Hansen, M. B.; Nielsen, S. E.; Berg, K. J Immunol Meth 1989, 119, 203.34. Stankovic, N.; Senerovic, L.; Bojic-Trbojevic, Z.; Vuckovic, I.; Vicovac, L.;

Vasiljevic, B.; Nikodinovic-Runic, J. J. Appl. Microbiol. 2013, 115, 1297.35. Zotchev, S. B. Curr. Med. Chem. 2003, 10, 211.36. Dumitru, R.; Hornby, J. M.; Nickerson, K. W. Antimicrob. Agents Chemother.

2004, 48, 2350.37. Douglas, L. J. Trends Microbiol. 2003, 11, 30.38. Dougherty, T. J.; Gomer, C. J.; Henderson, B. W.; Jori, G.; Kessel, D.; Korbelik, M.;

Moan, J.; Peng, Q. J. Natl Cancer Inst. 1998, 90, 889.39. Chabrier-Rosello, Y.; Foster, T. H.; Perez-Nazario, N.; Mitra, S.; Haidaris, C. G.

Antimicrob. Agents Chemother. 2005, 49, 4288.40. Gonzales, F. P.; Felgentrager, A.; Baumler, W.; Maisch, T. Future Microbiol 2013,

8, 785.41. Souza, R. C.; Junqueira, J. C.; Rossoni, R. D.; Pereira, C. A.; Munin, E.; Jorge, A. O.

Lasers Med. Sci. 2010, 25, 385.42. Oriel, S.; Nitzan, Y. Photochem. Photobiol. 2012, 88, 604.43. Cable, E. E.; Isom, H. C. Drug Metab. Dispos. 1999, 27, 255.44. Hanzlik, R. P.; Soine, P.; Soine, W. H. J. Med. Chem. 1979, 22, 424.45. Hegazy, W. H. Int. Res. J. Pure Appl. Chem. 2012, 2, 170.46. Neumann, A.; Baginski, M.; Czub, J. Journal of American Chemical Society 2010,

132, 18266.47. Recamier, K. S.; Hernandez-Gomez, A.; Gonzalez-Damian, J.; Ortega-Blake, I. J.

Membr. Biol. 2010, 237, 31.48. Mansfield, B. E.; Oltean, H. N.; Oliver, B. G.; Hoot, S. J.; Leyde, S. E.; Hedstrom, L.;

White, T. C. PLoS Pathog. 2010, 6, e1001126.49. Eucast, Voriconazole Rationale for the EUCAST-AFST clinical breakpoints, version

2.0; European Society of Clinical Microbiology and Infectious Diseases, 2010. p1.

50. Eucast, Amphotericin B Rationale for the EUCAST clinical breakpoints, version 1.0;European Society of Clinical Microbiology and Infectious Diseases, 2010. p 7.

R. Lippert et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3506–3511 3511

51. Eucast Fluconazole rationale for the EUCAST clinical breakpoints, version 1.0;European Society of Clinical Microbiology and Infectious Diseases, 2007.

52. Carrillo-Munoz, A. J.; Quindos, G.; Tur, C.; Ruesga, M. T.; Miranda, Y.; del Valle,O.; Cossum, P. A.; Wallace, T. L. J. Antimicrob. Chemother. 1999, 44, 397.

53. Arikan, S.; Ostrosky-Zeichner, L.; Lozano-Chiu, M.; Paetznick, V.; Gordon, D.;Wallace, T.; Rex, J. H. J. Clin. Microbiol. 2002, 40, 1406.

54. Sousa, A. S.; Gómez-Criado, C.; Baquero, F. Chemotherapy 1985, 31, 211.55. Eucast Micafungin and Candida spp.: Rationale for the EUCAST clinical

breakpoints, version 1.0; European Society of Clinical Microbiology andInfectious Diseases, 2013.

56. Vandenbosch, D.; Braeckmans, K.; Nelis, H. J.; Coenye, T. J. Antimicrob.Chemother. 2010, 65, 694.

57. Ghannoum, M. A.; Rice, L. B. Clin. Microbiol. Rev. 1999, 12, 501.58. Sanglard, D. Enferm. Infecc. Microbiol. Clin. 2002, 20, 462.59. Alvarez, F. J.; Douglas, L. M.; Konopka, J. B. Eukaryot. Cell 2007, 6, 755.60. Hitchcock, C. A.; Dickinson, K.; Brown, S. B.; Evans, E. G.; Adams, D. J. Biochem J

1990, 266, 475.61. Vediyappan, G.; Rossignol, T.; d’Enfert, C. Antimicrob. Agents Chemother. 2010,

54, 2096.62. Lesuisse, E.; Knight, S. A.; Camadro, J. M.; Dancis, A. Yeast 2002, 19, 329.63. Santos, R.; Buisson, N.; Knight, S.; Dancis, A.; Camadro, J. M.; Lesuisse, E.

Microbiology 2003, 149, 579.64. Kaspar, H.H.; Sibley, M.J.; Yung, G.H., U.S. Patent 4,568,517 1986.65. Curtis, J.; Nathoo, S.; Prencipe, M., U.S. Patent 5,639,445 1997.