In vitro antifungal activity of organic compounds derived from amino alcohols against onychomycosis

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Medical

Microbiology

In

vitro

antifungal

activity

of

organic

compounds

derived

from

amino

alcohols

against

onychomycosis

César

Augusto

Caneschi

_,

_Angelina

_Maria

_de

_Almeida

_,

_Francislene

_Juliana

_Martins

_,

Mireille

Le

Hyaric

_,

_Manoel

_Marques

_Evangelista

_Oliveira

_,

_Gilson

_Costa

_Macedo

_,

Mauro

Vieira

de

Almeida

_,

_Nádia

_Rezende

_Barbosa

_Raposo

a_{UniversidadeFederaldeJuizdeFora,FaculdadedeFarmácia,NúcleodePesquisaeInovac¸ãoemCiênciasdaSaúde(NUPICS),Juizde} Fora,MG,Brazil

b_{UniversidadeFederaldeJuizdeFora,InstitutodeCiênciasExatas,DepartamentodeQuímica,JuizdeFora,MG,Brazil} c_{Fundac¸ãoOswaldoCruz,InstitutoNacionaldeInfectologiaEvandroChagas,LaboratóriodeMicologia,RiodeJaneiro,RJ,Brazil} d_{UniversidadeFederaldeJuizdeFora,InstitutodeCiênciasBiológicas,DepartamentodeParasitologia,MicrobiologiaeImunologia,Juiz} deFora,MG,Brazil

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Received18August2016 Accepted7December2016 Availableonline9February2017 AssociateEditor:LuisHenrique SouzaGuimarães Keywords: Aminoalcohols Amides Lipophilicity Antifungalactivity Onychomycosis

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Onychomycosisisafungalinfectionofthenailcausedbyhighdensitiesoffilamentous fungiandyeasts.Treatmentforthisillnessislong-term,andrecurrencesarefrequently detected.Thisstudyevaluatedinvitroantifungalactivitiesof12organiccompoundsderived fromamino alcoholsagainststandard fungal strains,suchas Trichophytonrubrum CCT 5507URM1666,TrichophytonmentagrophytesATCC11481,andCandidaalbicansATCC10231. Theantifungalcompoundsweresynthesizedfromp-hydroxybenzaldehyde(4a–4f)and

p-hydroxybenzoicacid(9a–9f).Minimuminhibitoryconcentrationsandminimumfungicidal concentrationsweredeterminedaccordingtoClinicalandLaboratoryStandardsInstitute protocolsM38-A2,M27-A3,andM27-S4.Theamineseries4b–4e,mainly4cand4e com-pounds,wereeffectiveagainstfilamentousfungiandyeast(MICfrom7.8to312␮g/mL).On theotherhand,theamideseries(9a–9f)didnotpresentinhibitoryeffectagainstfungi,except amide9c,whichdemonstratedactivityonlyagainstC.albicans.Thisallowedustoinferthat thepresenceofaminegroupandintermediatecarbonnumber(8C–11C)initsaliphaticside chainseemstobeimportantforantifungalactivity.Althoughthesecompoundspresent cytotoxicactivityonmacrophagesJ774,ourresultssuggestthatthesearomaticcompounds mightconstitutepotentialasleadermoleculesinthedevelopmentofmoreeffectiveand lesstoxicanalogsthatcouldhaveconsiderableimplicationsforfuturetherapiesof ony-chomycosis.

©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

∗ _{Correspondingauthor.}

E-mail:[email protected](N.R.Raposo).

http://dx.doi.org/10.1016/j.bjm.2016.12.008

1517-8382/©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Onychomycosis is a fungal infection of the nails caused bydermatophyticfungiand yeasts.1–5 _{Themainetiological}

agentsareTrichophyton,Epidermophyton,andCandidafungi.6,7

Onychomycosisisanemergingglobalhealthproblem,asit represents50%ofnaildisorders1,8,9 _{andaffects2–9%ofthe}

worldpopulation.2,10_{Thispathologicalconditionaffects}

qual-ityoflife,withlossofthepatientself-esteemandmayresult inphysical,occupational,andsociallimitations.2,11,12

Notableamongthemainfactorsrelatedto onychomyco-sisarehumidity,wearingclosedshoes,repetitivenailtrauma, genetic predisposition, and chronic diseases, such as dia-betes, HIV, and immunosenescence.2,10,13–16 _{Moreover, the}

mainetiological agentsofonychomycosisininfectedareas are Trichophyton rubrum (60%), Trichophyton mentagrophytes

(20%), Epidermophyton floccosum (10%), and Candida albicans

(5–10%).1–3,9_{TheT.mentagrophytescomplexisparticularly}

dif-ficulttoidentifybecauseofthemorphologicalfeaturesand theinter-specificrelationshipswithinthisgroupareunclear, requiringmoleculartoolstoidentifyspeciesinthiscomplex.17

Despite recent advances in medicine, treating ony-chomycosis remains challenging due to the anatomical characteristicsofnailsandthepoorefficacyofcurrently avail-abletreatments.1,18–20 _{Existingtherapiesforonychomycosis}

areonlypartiallyeffective, andrecurrenceand consequent fungal resistance are common when combined with poor medication compliance.21–25 _{Furthermore, oral antifungal}

drugs are associated with undesirable side effects, such as hepatotoxicity.8,10,19 _{Therefore, new alternatives to}

pre-vent, diagnose, and treat onychomycosis are of utmost importance.26_{Thus,the discoveryofnovelantifungal}

com-pounds is urgently necessary to develop more effective, economical therapies with fewer side effects. Amine and amidegroupsaresignificantfunctionalgroupsinmedications due totheir reactive and chemical properties,27 _{and these}

groupscomprise peptides found invarious proteins.28 _Our

previousdatashowthataminoalcoholshaveantimicrobial

and immunologicalactivitiesagainstTrypanosomacruzi.29–32

Thus, the present study evaluated the antifungalactivities ofamphiphilicaromaticaminoalcoholsandamidesagainst differentstrainsoffungiassociatedwithonychomycosis.

Materials

and

methods

Chemistry

Amino alcohols 4a–4f were synthesized from

p-hydroxybenzaldehyde1,asdescribedpreviouslybyAlmeida et al. (2013) and amides 9a–9f were synthesized from

p-hydroxybenzoic acid 5.29 _{Briefly, compound 1 was}

previously O-alkylated and then submitted to a direct reductiveaminationreactioninthepresenceof 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris),resulting intargeted aminoalcohols4a–4fwith32–87%yield(Fig.1).Amphiphilic aromaticamides9a–9fwasobtainedfromO-alkylatedesters

7a–7fpreparedfromcarboxylicacid5.Afterhydrolysisofthe ethylester,thecarboxylgroupwasconvertedtoacylchloride andtreatedwithTris,resultinginthedesiredamides(Fig.2).

Fungalstrains

ExperimentswereconductedusingT.rubrumCCT-5507URM 1666obtainedfromtheCollectionofTropicalCrops(CCT) pro-videdbytheAndréToselloFoundation(Campinas,SãoPaulo, SP,Brazil),T.mentagrophytesATCC11481andC.albicansATCC 10231fromtheAmericanTypeCultureCollection(ATCC)were providedbytheNationalInstituteofQualityControlin Health-OswaldoCruzFoundation(RiodeJaneiro,RJ,Brazil).

Molecularidentification

Genomic DNA was extracted from T. mentagrophytes ATCC 11481toidentifythefungalstraincomplex.Partial sequenc-ing of the internal transcribed spacer (ITS) region was evaluated using ITS1(TCCGTAGGTGAACCTGCGG) and ITS4

O O 2 a-f 4 a-f O O H H N H 1) K2CO3 1) 1 eq 2) 0.5 eq KI 2) 2 eq NaBH4 3) HCI 4M refluxo 5h MeOHanid n=3,5,7,8,9,11,13 X=Cl, Br 4a: n=3 n=7 n=8 n=9 n=11 n=13 4b: 4c: 4d: 4e: 4f: DMF, 70ºC CH3(CH2)nX H3C(H2C)n H2N H3C(H2C)n HO OH OH OH OH OH .HCI OH 1 3

HO HO _{DMF, 70ºC} O O O O O O O N H 8 a-f 9 a-f 7 a-f O 1. K2CO3

1– Oxalyl chloride, toluene, 0ºC-r.t. 2– Tris, K2CO3, DMA, 60ºC 1. KOH,EtOH,reflux 2. HCI 4M 2. CH3(CH2)nCI,KI CH3(CH2)n CH3(CH2)n CH3(CH2)n OH OH OH OH OH OC2H5 OC2H5 H2SO4 EtOH, reflux 5 6 n=3,5,7,8,9,11 9a: n=3 n=7 n=8 n=9 n=11 n=5 9b: 9c: 9d: 9e: 9f:

Fig.2–SynthesisoftheamideseriesadaptedfromAlmeidaetal.(2013).29

(TCCTCCGCTTATTGATATGC).33 _{Briefly, the conditions were}

100ng DNA, 10pmol of each primer, and an annealing temperatureof58◦C.Automatedsequencingwasevaluated usingthe SequencingPlatformatFundac¸ão Oswaldo Cruz-PDTIS/FIOCRUZ, Brazil. The sequences were edited using Sequencher4.9softwareandcomparedwithBLAST.TheITS sequence isolated from T. mentagrophytes ATCC 11481 was depositedinGenbankunderaccessionnumberNCBI/GenBank KX132909.Thisprocedurewasnotnecessaryforotherstrains usedhereinbecausethesestandardstrainshavebeen inves-tigated recently by different authors,34,35 confirming the characterizationofthestrains.

Antifungalactivity

Minimuminhibitoryconcentration(MIC)assay

The experiment was conducted using broth dilution. The MICassaywasperformedaccordingtoClinicaland Labora-toryStandardsInstitute(CLSI)protocolsM38-A2,36_M27-A3,37

andM27-S4,38_{asdescribedpreviously.Allanalyseswere}

per-formed intriplicate. A filamentous fungal suspensionwas preparedusing7-dayculturesmaintainedintubes contain-ingSabourauddextroseagar(SDA)at28±2◦C,foryeastthe microdilutiontestisincubatedat35±2◦C.Then,threewashes were performedbyadding 2mLof0.85% sterilesalineand 20␮LTween-80.Thesuspensionswereanalyzedusinga spec-trophotometer (Libra S12; Biochrom, Cambourne, UK) and 89–90%transmittanceatawavelengthof530nm.39_The

sus-pensionswerediluted1:50(v/v)inRPMI-1640culturemedium (Sigma,St.Louis,MO,USA)buffered with3(N-morpholino) propanesulfonicacid(MOPS;JTBaker,Griesheim,Germany), resulting in a suspension containing 0.4–5.0×104_CFU/mL.

TheC.albicansATCC10231cultureswereusedafter48hof growth in tubes with SDA at 37±2◦C. The cultures were dilutedandanalyzedwithaspectrophotometerasdescribed above.Finally,thesuspensionwasdilutedinRPMI-1640 cul-turemediumandbufferedwithMOPStoprovideasuspension of5–25×102_CFU/mL.37,40

The amphiphilic aromatic amino alcohol and amide compoundsweresolubilizedinRPMI-1640culturemedium,

bufferedwithMOPS,andtestedforfilamentousfungiatfinal concentrations of 7.8–1000␮g/mLand 39.06–5000␮g/mLfor yeasts.Fungalgrowthwasassessedbyadding100␮L RPMI-1640 culture medium buffered with MOPS containing the fungalinoculum.Thefungiwerehomogenizedfor2minand incubatedinasterile96-wellplateat28±2◦Cforsevendays (dermatophytes)or37±2◦Cfor48h(yeasts).Thefungiwere analyzedvisuallyusingaSMZ800microscope(Nikon,Melville, NY).Terbinafine,ketoconazole,andamphotericinBwereused asreferencedrugsandassessedinaccordancewiththe M38-A236_andM27-A337_protocols.

Minimumfungicidalconcentration(MFC)analysis

The MFC of filamentous fungi was determined by plating 10␮L from wells without fungal growth to a new sterile 96-wellmicroplatecontaining200␮LofSabourauddextrose broth (SDB),as describedpreviously.41 _{TheMFC was}

deter-minedasthelowestconcentrationofthearomaticcompound that reduced the initialfungal count>99.9%. The MFCfor

C.albicanswasevaluatedusing5␮Lfromwellswithout fun-gal growth, which was transferredtocryotubes containing 1000␮LSDB.Theanalysiswasconductedasdescribedabove. Allexperimentswereperformedintriplicate,andtheresults arepresentedasgeometricmeansofthereplicates.

Cellviabilityassay

Cytotoxic effects in J774 cells were assessed by culturing macrophages (5×105_{) with different concentrations of the}

amine compounds (4b–4e) (0.1–100␮g/mL) in 96-well tis-sue cultureplatesat37◦Cin5%CO2 for48h.Cell viability

was determined by the colorimetric MTT assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide].42

Thecytotoxicityresultsweredisplayedaspercentageofcell viability comparing withuntreatedcells (100%ofviability). Absorbance of the solubilized MTT formazan product was spectrophotometricallymeasuredat540nmusinga Spectra-Max 190 microplate reader (Molecular Devices, Sunnyvale, CA,USA).Allsampleswererunintriplicateandanalyzedon thesamedaytominimizeday-to-dayvariation.

Table1–Invitroinhibitoryactivityofamphiphilicaromaticaminoalcoholsandamides.

Compounds Fungalstrains

TrichophytonrubrumCCT 5507URM1666 Trichophytonmentagrophytes ATCC11481 Candidaalbicans ATCC10231

MIC MFC MIC MFC MIC MFC

4a 62.5 500 125 125 >5000 ND 4b 62.5 125 15.62 15.62 78 312.5 4c 7.8 15.62 15.62 15.62 31.25 31.25 4d 31.25 31.25 31.25 125 62.5 125 4e 15.62 62.5 7.8 62.5 15.65 15.65 4f >1000 ND 62.5 125 500 >5000 9a >1000 ND >1000 ND >5000 ND 9b >1000 ND >1000 ND >5000 ND 9c >1000 ND >1000 ND 625 >5000 9d >1000 ND >1000 ND >5000 ND 9e >1000 ND >1000 ND >5000 ND 9f >1000 ND >1000 ND >5000 ND Terbinafine 0.19 0.19 0.03 0.03 NE NE Ketoconazole 1 4 0.25 0.25 NE NE AmphotericinB NE NE NE NE 0.125 0.5

ND,notdetermined(MIC>1000␮g/mLorMIC>5000␮g/mL);NE,notevaluated;MIC,minimuminhibitoryconcentration;MFC,minimum fungicidalconcentration.Resultsareexpressedas␮g/mL.

Results

ThesequenceobtainedbypartialsequencingoftheITSregion (NCBI/GenBankKX132909)wasisolatedfromATCC11481and was100%concordantwiththatofT.mentagrophytesJX122271 depositedinGenbank.TheMICandMFCvaluesofthe com-pounds, as well as those ofterbinafine, ketoconazole, and amphotericinB,wereassessedinthefungalstrainsT.rubrum, T.mentagrophytes,andC.albicans(Table1).

AsdescribedinTable1,theamineseries(4b–4e)was effec-tiveagainstT.rubrumCCT5507URM1666(MIC≤62.5␮g/mL and MFC≤500␮g/mL), T. mentagrophytes ATCC 11481 (MIC≤31.25␮g/mL and MFC≤125␮g/mL), and C. albicans

ATCC 10231 (MIC≤62.5␮g/mL and MFC≤312.5␮g/mL). Compound 4c almost abolished growth of T. rubrum CCT 5507 URM 1666 (MIC=7.8␮g/mL and MFC=15.62␮g/mL) and T. mentagrophytes ATCC 11481 (MIC=15.62␮g/mL and MFC=15.62␮g/mL).Notably,this effectwasquitesimilar to thatcaused byterbinafine(0.03–1␮g/mL) and ketoconazole (0.25–16␮g/mL)(Table1),whichwerethe clinicalantifungal reference drugs (Table 1). Moreover, compounds 4a and 4f

moderately inhibited growth of T. rubrum and T. mentagro-phytes, suggesting that antifungal activity may be related to the number of carbon atoms (8C–12C) in the aliphatic chain.Nevertheless,amides9a–9ffailedtoinhibitanyofthe filamentousfungaloryeaststrains(Table1),exceptamide9c,

whichdemonstratedactivityagainstC.albicansATCC10231 (fungistatic at 625␮g/mL). Amphotericin B, which is used clinically,inhibited growth ofC. albicans (MIC=0.125␮g/mL andMFC=0.5␮g/mL)(Table1).

Aimingtoruleout thepossibilityofcytotoxicactivity of compounds4b–4e,weaccessedinanothersetofexperiments theaminemoleculesonmacrophagesJ774.Resultsdepicted inFig. 3show that compounds 4b–4d at0.1–10␮g/mL dis-playedlower cytotoxic effect on macrophage (cell viability

100 90 80 70 60 50 40 30 20 10 0 100 Concentration (µg/mL) 50 10 1 0.1 Cell viability (%) 4b 4c 4d 4e Control

Fig.3–Invitrocellviabilityassayofcompounds4b–4eon J774macrophages.

morethan80%).Thecompound4edecreasedtheviabilityof macrophagesby44.1%,44.4%and49.9%attheconcentration 0.1,1and10␮g/mL,respectively(Fig.3).Allcompounds4b–4e

reducedsignificantlythecellviabilityonlyatthe concentra-tionof50and100␮g/mL.

Discussion

WeidentifiedanewpartialsequenceintheITSregionfromthe

T.mentagrophytesATCC11481-NCBI/GenBankKX132909 com-plex due tothe difficulties of identifying different species using only phenotypic characteristics, as reported previ-ouslybyPackeuetal.(2013).43_{Moreover,thisprocedurewas}

not necessary forother strains used hereinbecause these standard strains have been investigatedrecently by differ-ent authors,18,34,44,45 _{confirmingthe characterizationofthe}

strains.

Importantly, the amphiphilic character of the organic compoundsreflectstheirabilitytointeractandpenetrate bio-logicalmembranesinducingdifferenteffects,29,32,46 _suchas

increasedantimicrobialactivity.47_{Lipophilicityisan}

impor-tantcharacteristicofantifungalagents,suchasterbinafine andketoconazole,andisalsoevidentindrugslike ampho-tericinB,whichhaveproveneffectivenesstotreatsystemic fungal infections.46 The relevance of the lipophilic chain inamino alcoholswas reportedby Almeida et al.(2013),29

whoassessedantibacterialactivityonsomebacterialstrains, suchas Pseudomonas aeruginosa, Escherichia coli, Staphylococ-cusaureus,Staphylococcusepidermidis,andmethicillin-resistant

S. aureus, demonstrating that lipophilic compounds show potentantimicrobialactivities.Inthisstudy,weextendedand addedtothesefindingsbydemonstratingthatamino alco-hols4b–4e showed significant antifungalactivity, although lipophiliccompound4aandthemostlipophilicaminoalcohol

4fonlydisplayedantifungalactivityagainsttwoofthefungal strainstested.

Theevaluationof the structure–activityrelationships of aminesshowedthatincreasingthenumberofcarbonsinthe aliphaticchaintoeightfavoredantifungalaction, corroborat-ingandextendingpreviouslypublisheddata.48

Thisreductioninantifungalpotential canbeassociated withsizeofthecarbonicsidechains3Cand13Cfor4aand

4fcompounds,respectively.Theresultsobtainedwith com-pound4econtradictthathigherlipophilicityofthecompound permits penetration into the target cell and consequently itspharmacologicalaction, asreportedbyGushchinaet al. (2015).49_{However,thishypothesiswasconfirmedbyAlmeida}

et al. (2013),29 _{who demonstrated the same profile with}

amino alcohol compounds, which show less antibacterial actionasthecarbonchainiselongated.Dolezaletal.(2002) reportedmarkedactivityagainstClorellavulgarisafter associ-atinglipophilicitywithitsalkoxysubstituent.50_Smolarzetal.

(2005)discoveredthattheantifungalpotentialof 2-carboxy-3,5-dimethoxy-E-stilbeneagainstTrichophytonspp.strainsis relatedtothesamephysicochemicalcharacteristicsreported above.51_{Althoughamidesarefoundindifferent}

pharmaceu-ticalcompounds,52,53 _{compounds9a–9fdidnotsignificantly}

inhibitgrowthoffilamentousfungioryeast,exceptcompound

9c,whichhasanalkylchainbearingeightcarbonatoms. Thediscrepancybetweentheantifungalactivitiesofamino alcohols 4a–4f and those of amides 9a–9f suggests that aminegroupscouldcontributetoantifungalaction,asonly aminecompoundsareinahydrochlorideform,whichhelps withtheirwatersolubilityandabsorption.Shahetal.(2015) observedsimilarresultswhentheyassessedamineandamide compoundstoxictoC.albicans.54_{Thus,antifungalpotentialis}

relatedtothesizeofthelateralaliphaticchainandcandirectly improveefficacy.55Surprisingly,ourdatasuggestthat inter-mediatesizealiphaticaminechainsshowedanotableeffect, justifyingadditionalexperiments.

Previousstudieshavereportedadirectcorrelationbetween

invitrocytotoxicityandinvivoacutetoxicityinanimalsand humans56_{;therefore,investigatingthetoxicologicaleffectsof}

new antifungal compounds in normalcells are important. Herein, the cytotoxicity of compounds 4b–4e was evalu-atedusinganMTTassaywithmacrophages.Compound4e

decreasedviabilityofmacrophagesby44.1%,44.4%,and49.9% atconcentrations of0.1, 1,and 10␮g/mL,respectively. Our dataalsoshowthatcompounds4b–4ereducedcellviability onlyat50and100␮g/mL,indicatingthathigher concentra-tionsreducedmacrophageviabilityandfungalgrowth.These resultssuggestthatlesstoxicitywasassociatedwiththe pres-enceofanintermediatenumberofcarbonsinthesidechain, ascompoundswitheightcarbonsinthealiphaticchainwere more cytotoxic than those with 7, 9, or 11 carbons. Thus, additional in vitrocytotoxic assays should beconductedto determinethesafetyprofileofanypotentialdrugcandidate fortherapeuticapplicationsinanimalsandhumans.

AccordingtotheWorldHealthOrganization(WHO), resis-tantmicroorganisms(includingbacteria,fungi,viruses,and parasites) can withstand attack by antimicrobialdrugs, so standard treatmentscan becomeineffectiveand infections persist, increasing the risk of spread to other microbes, resulting in resistance.57 _{The 2014 WHO report on global}

surveillanceofantimicrobialresistancerevealedthat antimi-crobialresistance,includingantifungalresistance,isnolonger apredictionforthefuture,asitisoccurringworldwidenow and isincreasingtherisk ofbeing unabletoproperlytreat commoninfectionsinthecommunityandhospitals.Forthis reason, fostering innovative research and developing new vaccines,diagnostics,infectiontreatmentoptions,andother toolsisurgentlyneeded.22,58–60

In summary, the data presentedhere demonstrate that amphiphilic aromatic amino alcohols (4b–4e) markedly inhibitedstandardstrainsoffungi,suchasT.rubrum,T. men-tagrophytesandC.albicans.Althoughthesecompoundshave presentedcytotoxiceffectonmacrophages;theymight con-stitutepotentialasleadermoleculesinthedevelopmentof innovativeantifungal(aimingeffectivenessandsafety)forthe treatmentoffungalinfection,includingonychomycosis.

Conflicts

of

interest

Noconflictofinterestwasdeclared.

Acknowledgements

ThisresearchwassupportedbyCAPES,CNPq,FAPEMIG,and PROPESQ/UFJF.

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