Comparative in vitro and in vivo antimalarial activity of the indole alkaloids ellipticine, olivacine, cryptolepine and a synthetic cryptolepine analog

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ContentslistsavailableatSciVerseScienceDirect

Phytomedicine

jo u r n al h om e p a g e :w w w . e l s e v i e r . d e / p h y m e d

Comparative

in

vitro

and

in

vivo

antimalarial

activity

of

the

indole

alkaloids

ellipticine,

olivacine,

cryptolepine

and

a

synthetic

cryptolepine

analog

L.F.

Rocha

e

Silva

a,b,c

_,

_A.

_Montoia

a,b

_,

_R.C.N.

_Amorim

a

_,

_M.R.

_Melo

a

_,

_M.C.

_Henrique

a,b

_,

_S.M.

_Nunomura

a

_,

M.R.F.

Costa

d

_,

_V.F.

_Andrade

_Neto

e

_,

_D.S.

_Costa

a,c

_,

_G.

_Dantas

e

_,

_J.

_Lavrado

f

_,

_R.

_Moreira

f

_,

_A.

_Paulo

f

_,

A.C.

Pinto

g

_,

_W.P.

_Tadei

a

_,

_R.S.

_Zacardi

h

_,

_M.N.

_Eberlin

h

_,

_A.M.

_Pohlit

a,∗

a_National_Institute_for_Amazonian_Research,_Av._André_Araújo,_2936,_Aleixo,_69060-001_Manaus,_Amazonas,_Brazil b_Federal_University_of_Amazonas,_Av._General_Rodrigo_Otávio,_3000,_Coroado,_69077-000_Manaus,_Amazonas,_Brazil c_University_Center_of_the_North,_R._Dez_de_Julho,_873,_Centro,_69010-060_Manaus,_Amazonas,_Brazil

d_Heitor_Vieira_Dourado_Tropical_Medicine_Foundation,_Av._Pedro_Teixeira,_25,_Dom_Pedro,_69040-000_Manaus,_Amazonas,_Brazil e_Federal_University_of_Rio_Grande_do_Norte,_C.P._1524,_Campus_{Universitário}_Lagoa_Nova,_59078-970_Natal,_Rio_Grande_do_Norte,_Brazil f_University_of_Lisbon,_Alameda_da_{Universidade,}_1649-004_Lisbon,_Portugal

g_Federal_University_of_Rio_de_Janeiro,_Av._Athos_da_Silveira_Ramos,_149,_Cidade_{Universitária,}_21941-909_Rio_de_Janeiro,_Brazil

h_State_University_of_Campinas,_Campus_{Universitário}_Zeferino_Vaz_SN,_Cidade_{Universitaria}_Campinas,_13083-970_Campinas,_São_Paulo,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Keywords: Plasmodiumfalciparum Plasmodiumberghei Murinemacrophages Antiplasmodialactivity Indolealkaloids

a

b

s

t

r

a

c

t

Indole alkaloids ellipticine (1), cryptolepine triﬂate (2a), rationally designed 11-(4-piperidinamino)cryptolepinehydrogendichloride(2b)andolivacine(3)(anisomerof1)wereevaluated invitroagainstPlasmodiumfalciparumandinvivoinPlasmodiumberghei-infectedmice.1–3inhibited P.falciparum(IC50≤1.4␮M,orderofactivity:2b>1>2a>3).Invitrotoxicitytomurinemacrophages wasevaluatedandrevealedselectivityindices(SI)of10–12for2aandSI>2.8×102_for_1,_2b_and_3.₁ administeredorallyat50mg/kg/daywashighlyactiveagainstP.berghei(invivoinhibitioncompared tountreatedcontrol(IVI)=100%,meansurvivaltime(MST)>40days,comparableactivityto chloro-quinecontrol).1administeredorallyandsubcutaneouslywasactiveat10mg/kg/day(IVI=70–77%; MST=27–29days).3exhibitedhighoralactivityat≥50mg/kg/day(IVI=90–97%,MST=23–27days). Cryptolepine(2a)administeredorallyandsubcutaneouslyexhibitedmoderateactivityat50mg/kg/day (IVI=43–63%, MST=24–25 days). At 50mg/kg/day, 2b administered subcutaneously was lethal to infectedmice(MST=3days)andmoderatelyactivewhenadministeredorally(IVI=45–55%,MST=25 days).1and3arepromisingcompoundsfordevelopmentofantimalarials.

Introduction

Despitegreateffortandresourcesfortheeradicationofmalaria this diseasestillremains a gravepublichealth problem involv-inghundredsofthousandsofdeathsannually(WHO2010).While researchonvaccinesisatanadvancedstage,drugtherapyisstill theprincipletoolforthecontrolanderadicationofthedisease.The emergenceofstrainsofPlasmodiumfalciparumandP.vivaxwhich areresistanttoﬁrstandsecondlineantimalarials(multidrug resis-tantorMDR)havemotivatedthesearchfornewdrugsrepresenting newanddistinctchemicalclassesandmechanismsofactionthan thoseoftheantimalarial drugscurrentlyinuse. Chemical com-poundsofnovelstructureandofnaturaloriginrepresentamajor

∗ Correspondingauthor.Tel.:+559236433177;fax:+559236433079. E-mailaddresses:[email protected],[email protected](A.M.Pohlit).

sourceforthediscoveryanddevelopmentofnewdrugsfordiseases, especiallymalaria(Kauretal.2009;Schmidtetal.2012a,b).

Historically,plantsusedintraditionalmedicineas antimalar-ialsandfebrifugeshaveprovidedsubstanceswhichhaveproved tobe usefulas antimalarialsor have servedchemists as struc-turalmodelsforthedevelopmentofsemi-syntheticdrugsorpurely syntheticanalogs.Thisistrueofthemostimportant antimalar-ialnaturalproductsrevealedtodate:quinine(isolatedfromthe barkof Cinchonaspp.) and artemisinin(isolated fromArtemisia annua leaves). Thetherapeutic efﬁcacy and complex molecular structureofquinineleadtothedevelopmentofpurelysynthetic analogschloroquine,primaquine,meﬂoquine,amongothersinthe lastcentury.Morerecently,semi-syntheticderivatives(e.g.sodium artesunate, artemether, arteether and dihydroartemisinin) pre-paredinoneormorestepsfromisolatedartemisininhavebecome keypharmaceuticalcomponentsinformulationsusedinwhatis commonlycalledartemisinincombinationtherapy(ACT)forthe treatmentofresistantandMDRPlasmodiumfalciparuminfections 0944-7113© 2012 Elsevier GmbH.

http://dx.doi.org/10.1016/j.phymed.2012.09.008

Open access under the Elsevier OA license.

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Fig.1. Structuresofantimalarialcompoundsellipticine(1),cryptolepinetriﬂate(2a triﬂate),11-(4-piperidinamino)cryptolepinehydrogendichloride(2b)andolivacine (3).

(Plowe2009;Willcox2011).Theextractsofalargenumberofplant speciesincludingmanythatareusedintraditionalmedicinehave beenevaluatedforinvitroantiplasmodialactivitiesandsomehave alsobeentestedininvivomodels,usuallyinmiceinfectedwith Plasmodiumberghei,P.yoeliiorP.chabaudi.Insomecases,the con-stituent(s)responsiblefortheiractivitieshavebeenisolatedbut relativelyfewhavebeenstudiedfurthertoassesstheirpotential asleadcompoundsforthedevelopmentofnewantimalarialdrugs (Wright2005).

Inrecentyears,themonoterpeneindolealkaloidellipticine(1, Fig.1)hasbeenthesubjectofanumberofpharmacologicalstudies anditsderivativeshavebeenstudiedinclinicaltrialsagainst differ-entformsofcancer.Ellipticinehasbeenisolatedfromthealkaline ethanolextractofthebarkoftheAmazoniantreeAspidosperma vargasii(Apocynaceae)(Andrade-Netoetal.2007;Henriqueetal. 2010) which is used in traditional medicine as anantimalarial (Oliveiraetal.2003).Invitroantiplasmodialactivityof1wasﬁrst reportedbyAndrade-Netoetal.(2007).Recently,theantimalarial activityof1wasindependentlyconﬁrmedandthecomparableor superioractivityoffourderivativesof1againstP.falciparuminvitro wasdescribed(Passemaretal.2011;Pohlitetal.2012).

TherootsoftheWestAfricanclimbingshrubCryptolepis san-guinolenta(Lindl.)Schltr.(Apocynaceae)are atraditionallyused herbalformalariatreatment.DryaqueousrootextractsofC. san-guinolentahaveprovenefﬁcacyaccordingtoclinicaltrials(Willcox 2011).Cryptolepine(2a,Fig.1)isthemajoralkaloidconstituent intherootsofthisplantand2asulfate exhibitsinvitroactivity (IC50=0.44␮M)againstmultidrug-resistantK1strainofP.

falcipa-rum(Wrightetal.2001).However,2asulfatefailedtocuremalaria inmicewhen givenorallyand istoxicatadoseof20mg/kgto P.berghei-infectedmicewhenadministeredintraperitoneally(i.p.) (Cimangaetal.1997;Wrightetal.2001).Also,2ahasinvivochronic effectscausingnecrosisofrodentlivercellsatadoseof30mg/kg andalsodamagestheDNAoflymphocytesinvitro.Theseandother experimentalresultsdonotsupporttheuseoftheindolealkaloid cryptolepine(2a)asanantimalarial(Gopalanetal.2011;Willcox 2011).

Derivativesofcryptolepine(2a)havebeenintroducedhaving greaterantiplasmodialactivityandbettertoxicityproﬁles.Oneof themostpromisingofthesederivativesis2,7-dibromocryptolepine whichhaspotentinvitroactivity(IC50=50nM)againstK1strain

ofP.falciparumandexhibitssigniﬁcantsuppression(89–91%)of P.bergheigrowthininfected miceatdosesof 20–25mg/kg/day over4days.While2,7-dibromocryptolepinedoesnotapparently intercalateDNA bases asdoes2a,both thesecompounds dam-agelymphocyteDNAbasedonresultsofthecometassay(Wright etal. 2001;Gopalan etal. 2011).Recently,cryptolepine triﬂate

(2atriﬂate)andtensyntheticanalogsof2acontainingaminoalkyl side chainsat C-11 weresynthesized and screened for invitro antiplasmodialactivityandcytotoxicity.Oneofthemost promis-ingofthesecompoundswas11-(4-piperidinamino)cryptolepine hydrogendichloride(2b)whichexhibitedpotentinhibitory activ-ity(IC50=44nM)againstP.falciparumW2strainandwastheleast

toxicofallthecompoundstested, including2a triﬂateand had thelargestcytotoxicitytoantiplasmodialinhibitionratio(46.4:1) (Lavradoetal.2008,2011).

Olivacine (3) is a rare alkaloid which is isolated from Aspi-dosperma olivaceum. The antitumor activity of 3 has been the subjectofstudiesfordecades.Compound3andanalogshavealso beensynthesized(Besselièvre&Husson1981;Chevallier-Multon etal.1990;Guillonneauetal.2005).

Mechanistic studies demonstrate that cryptolepine (and it analogs)andellipticine(1)mayhaveimportantinhibitoryeffects on theformation of hemozoin in P.falciparum. Hemozoin for-mationis a fundamental process related tothesurvival of this parasitewithintheredblood cell.Hemeis toxictoPlasmodium spp.andisaby-productofdigestionofhemoglobinbyproteolytic enzymesintheparasite’sdigestivevacuole.Crystallizationofheme tohemozoinisadetoxifyingprocessthatoccursnaturallywithin thePlasmodiumdigestivevacuoleandisnecessaryforthe prolif-erationoftheseparasiteswithintheredbloodcell.Inhibitionof hemozoinformationisassociatedwithdeathofparasiteswithin theredbloodcell duetoosmoticimbalancesand othereffects. Earlymechanisticstudiesinvolvingchemicalassaysshowedthat ellipticine(1)caninhibithemecrystalgrowth(thatis,hemozoin formation)inthelab(ChongandSullivan2003).Inotherwork,it waspresumedthatcryptolepine(2a)interactsdirectlywithheme moleculesintheprocessofinhibitinghemozoinformation(Kumar etal.2007;Lavradoetal.2011).

Previousstudiespointtothefactthatlargeorsmallstructural differencesamonganalogousindolealkaloids,suchascryptolepine analogs and _{␤-carbolines} (harmane analogs), can lead to large differencesininvitroandinvivoantimalarialactivityand cyto-toxicity of these compounds. Thus, small structural differences probablymodulateandultimatelydeﬁnetheprimarymechanisms of action of these compounds (DNA intercalation, inhibition of hemepolymerization,inhibitionofproteinsynthesis,amongother mechanismsyettoberevealed)(Arzeletal.2001;Ancolioetal. 2002;VanBaelenetal.2009).

Inthepresentstudy,theantiplasmodialactivityofstructurally relatedindolealkaloidsellipticine(1),cryptolepinederivative2b andolivacine (3) isinvestigated forthefirst timein P. berghei-infectedmiceandthedataarecomparedtothoseforcryptolepine triflate(2a triflate). The in vitroantiplasmodial activityagainst chloroquine-resistantandchloroquinesensitivestrainsofP. falcip-arumandcytotoxicityofthesecompoundswasevaluatedinvitro againstmurinemacrophagesasameanstocomparatively eval-uateselectivityoftheantimalarialeffect.Theoverallaimofthis workwastoprovidecomparativeinvitroandespeciallyinvivo anti-malarialdataforallfourcompoundswhichmightleadtoinsights intotherelativeimportanceof cryptolepineand ellipticinering systems/skeletonsforthefurtherdevelopmentofantimalarials.

Materialsandmethods Chemicals

Ellipticine (1) used in this work was obtained from two sources.Synthetic 1waspurchased fromSigma–Aldrich (Stein-heim,Germany).Also,1wasisolatedfromthebarkofAspidosperma vargasii from INPA’s Ducke Reserve in Amazonas State, Brazil through an alkaline extraction sequence followed by column

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chromatography as described previously (Andrade-Neto et al. 2007;Henriqueetal.2010).Cryptolepinetriﬂate(2atriﬂate)and cryptolepineanalog2bwereobtainedbysynthesisasdescribed previously (Lavrado et al. 2008). Olivacine (3) was isolated fromAspidospermaolivaceumfromMinasGeraisState,Brazil,by acid–baseextraction.Thepurityofthesecompoundswaschecked byTLC,UPLC–MSandNMRandwas>98%.

CultureandtestforinvitroinhibitionofP.falciparumparasites Strains of P. falciparum used in this study were the anti-malarialdrug-susceptible3D7cloneoftheNF54isolateandthe chloroquine-resistant,pyrimethamine-resistantand cycloguanil-resistantK1strain.Parasiteswereculturedaccordingtothemethod ofTragerandJensen(1976)asmodiﬁedbyAndrade-Netoetal. (2007).Theparasiteculturewascarriedoutat37◦Cwitha hema-tocrit of 3–5% and in an atmosphere of 5% CO2. The parasites

weremaintainedinvitroin A+humanredbloodcells. The cul-turemediumwasRPMI1640(Sigma–Aldrich)supplementedwith 10% human serum and containing 25mM HEPES and 2mM l-glutamine. The micro-testwas performedusing the methodof Rieckmannetal.(1978)withmodificationswhichweredescribed inAndrade-Netoetal.(2007).Stocksolutionsofindolealkaloids ellipticine(1),cryptolepinetriflate(2atriflate),olivacine(3)and 11-substitutedcryptolepinehydrogendichlorideanalog2cwere preparedin DMSOat aconcentrationof 5.0mg/ml.Seven dilu-tionswere performed in culture medium (RPMI1640) of each stocksamplesolutionresultinginfinaltestconcentrations(well concentrations)of50–3.2×10−3␮g/ml.Eachdilutedsamplewas testedinduplicatein96-welltestplatescontainingasuspension ofparasitizedredbloodcellsatahematocritof3%andinitial para-sitemiaof1%ofsynchronizedyoungtrophozoites(ringforms).The finalvolumeofeachwellwas200␮l.Referenceantimalarial com-poundschloroquineandquininewereusedaspositivecontrolsat concentrationsrecommendedbyWHO(2001).Thetestplatewas incubatedfor48hat37◦Cunderthesamelowoxygengasmixture usedforparasiteculture.Aftertheincubationperiod,thinsmears werepreparedfromthecontentsofeachwellandevaluatedusing amicroscope.Thehalfmaximalinhibitory(IC50)responses

com-paredwiththedrug-freecontrolswereestimatedbyinterpolation usingMicrocalOrigin®_software.

TestforinvivosuppressionofPlasmodiumberghei

InvivoantimalarialactivitywasevaluatedusingP.bergheiNK65 strain(drug-sensitive).Thisstrainwasmaintainedbysuccessive passagesof blood formsfrommouse tomouse.Thetest proto-colisbasedonthe4-daysuppressivetestasdescribedbyPeters (1965).FemaleWebsterSwissmiceweighing26±2gwereusedin thisstudy.Animalswereinfectedintraperitoneallywith0.2mlof infectedbloodsuspensioncontaining1×105_parasitized

erythro-cytesandrandomlydividedintogroupsofthreeindividuals.Test groupsweretreatedorallyandsubcutaneouslyatdosesranging from100to1mg/kg/day.Positivecontrolgroupsreceivedadose of10mgchloroquine/kg/day orallyor subcutaneouslyand neg-ativecontrol groups received0.2mlof 2%DMSO orsaline. The animalsweretreatedfor4daysstarting24hafterinoculationwith P.berghei.Ondays5and7afterinoculationwithparasites,blood smearswerepreparedfromallmice,ﬁxedwithmethanol,stained withGiemsadye,thenmicroscopicallyexamined(1000× magni-ﬁcation).Parasitemiawasdeterminedincodedbloodsmearsby randomlycounting2000–4000erythrocytesinthecaseoflow par-asitemias(≤10%);orupto1000erythrocytesinthecaseofhigher parasitemias.Overallmortalitywasmonitoreddailyinallgroups duringaperiodof4weeksfollowinginoculation.Thedifference betweentheaverageparasitemiaofcontrolgroups(100%)andtest

groupswascalculatedasapercentageofparasitegrowth suppres-sion(PGS)accordingtotheequation:PGS=100×(A−B)/A,where AistheaverageparasitemiaofthenegativecontrolgroupandB correspondstotheparasitemiaofthetestgroup.

Cytotoxicitytest

Forthistest,macrophagesfromSwissmicewereusedwhich were described in Mota et al. (2012), with modiﬁcations. The macrophageswereobtainedatthetimeofusebycollectionwith cold, sterilephosphate salinesolution (PSB) fromthe exudates oftheperitonealcavityofmice.Aftercentrifugingtheperitoneal exudatesolutions,thesupernatantwasdiscardedandpelletwas re-suspendedin5mlofRPMImediumwithoutFBSforcounting themacrophagesinaNeubauerchamber.1× 105_cells_were_added

toeachwell.TheplatewasincubatedinaCO2incubatorat37◦C

for24h.Thecytotoxicityofthesampleswasdeterminedusingthe methylthiazoletetrazolium (MTT) colorimetric assay (Mosmann 1983).For theassays, the cells weretrypsinized, washed, sus-pendedinDMEM,anddistributedinto72wellsperplate(5×103

cellsperwell)thenincubatedfor18hat37◦C.Thesampleswere separatelydilutedinDMSOandtestedintriplicateatthefollowing concentrations:1.5,3.1,6.3,12.5,25,50,and100␮g/ml.In paral-lel,weevaluatedacontrolgroupconsistingofRPMI1640without FBS,acontrolgroupconsistingof1%DMSO(vehicle)andapositive control(chloroquine,BSPharma,BeloHorizonte,MG,Brazil)atthe sameconcentrationsusedforsubstances1–3.After24and48hof incubationat37◦C,100␮lofMTT(5mg/mlinRPMI1640without FBSandwithoutphenolred)wasaddedtoeachwell.After3hina CO2 incubatorat37◦C,thesupernatantwasremovedandadded

to100_␮l DMSOineach well.Theabsorbanceofeach wellwas obtainedfromaspectrophotometricreadingat562nm.The mini-mumlethaldosesthatinhibited50%ofcellgrowthwereobtained fromthedrugconcentrationresponsecurves.Resultsareexpressed inmean±standarddeviation.

Selectivityindex

Therelativecytotoxicitytoantiplasmodialactivityforagiven compound was evaluated as a selectivity index (SI), where SI=IC50(murinemacrophages)/IC50(P.falciparum).

Animalsandethicalapproval

AdultWebsterSwissalbinomice(26±2gweight)wereused fortheantimalarialandtoxicitytestsandreceivedwaterandfood adlibitum.InvivotestswereperformedusingGuidelinesforEthical ConductinTheCareandUseofAnimalsofFederalUniversityofRio GrandedoNorte(CEUA043/2010).

Results

Indole alkaloids ellipticine (1), cryptolepine triﬂate (2a triﬂate) and olivacine (3) and synthetic analog 11-(4-piperidinamino)cryptolepine hydrogen dichloride (2b) were assayedforinvitroactivityagainstPlasmodiumfalciparumK1e3D7 strainsandcytotoxicactivityagainstmurinemacrophages.From theIC50 valuesfor eachsubstanceagainstmurinemacrophages

andmalariaparasitestrainsitwaspossibletodetermineselectivity indices. The invitro resultsare presented in Table1. Olivacine (3)isapreviouslyknownindolealkaloidforwhichantimalarial activityhasnotbeenpreviouslydescribed.Itwastheleastactive compoundinvitro,however,itdidsigniﬁcantlyinhibitP.falciparum growth(IC50=1.2␮MagainstK1strain).Thepotentinvitroactivity

ofellipticine(1)reportedpreviouslywasconﬁrmedherein(IC50

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Table1

Medianinhibitionconcentrations(IC50)andselectivityindices(SI)forindolealkaloids1–3invitroagainstPlasmodiumfalciparumK1and3D7strainsandmurinemacrophages.

Compound IC50(␮M) SIa

Name No. P.falciparum Murinemacrophages

K1 3D7 Ellipticine 1 0.81 0.35 >4.1×102 _>5.0_×₁₀2_/>1.2_×₁₀3 Cryptolepinetriﬂate 2a 0.80 0.91 9.1 11/10 Cryptolepineanalog 2b 0.10 0.087 34 3.3× 102_/3.9_×₁₀2 Olivacine 3 1.4 1.2 >4.1×102 _>2.9_×₁₀2_/>3.4_×₁₀2 Chloroquinediphosphate 0.13 0.058 1.4×102 _1.1_×₁₀3_/2.4_×₁₀3 Quininesulfate 0.16 0.11 n.t. – Artemisinin 0.0021 0.0011 n.t. –

Datavaluesareexpressedastwo(2)signiﬁcantﬁguresaspertheprecisionofthemethodsused.n.t.:nottested;–:notcalculable.

a_SI₌_IC

50(“macrophages₎/IC_50(P._falciparum)andisreportedforK1/3D7strains.

respectively).Ellipticine(1)anditsstructuralisomerolivacine(3) weretheleastcytotoxiccompoundsinthisstudy(IC50>0.41mM,

highestconcentrationtested).This lowcytotoxicity contributed greatlytothehighselectivityindicesobtainedfor1and3against P.falciparum 3D7 (>1.2×103 _and _>3.4_×₁₀2_, _{respectively).} _The

compoundwiththemostinvitroactivityagainstP.falciparumwas rationallydesigned11-(4-piperidinamino)cryptolepinehydrogen dichloride (2b) (IC50=0.10 and 0.087␮M, against P. falciparum

K1and3D7strains,respectively)whichhadbeenselectedfrom amonganumberofsyntheticcryptolepineanalogsreportedearlier basedonitsfavorableinvitroantimalarialandcytotoxicproperties reportedinthatearlierwork(Lavradoetal.2008).Inthepresent work, cryptolepine analog 2b exhibited an IC50 value against

murinemacrophagesof34␮Mthus makingitthesecondmost cytotoxiccompoundafter2atriﬂate.Relativelyhighcytotoxicity againstmurinemacrophagesleadto2atriﬂateand2bexhibiting thelowestSIvalues.

Theindolealkaloids1–3wereevaluatedinvivoinP. berghei-infected mice in the 4-day suppressive test and the result is presentedinTable2.Ellipticine(1)washighlyactiveatanoraldose of50mg/kg/day(100%inhibitionversuscontrolsondays5and7). Atthissamedose,themeansurvivaltime(MST)oftheanimalswas >40days(limitoftheobservationperiodandidenticaltotheMST ofthecontrolsubstancechloroquine).Also,1hadgoodoral activ-ityonday5andgoodactivityviasubcutaneousinjectiononday7 at10mg/kg/day(77and70%inhibition,respectively;MST=27–29 days)andmoderateoralactivityat1mg/kg/day(61–67%inhibition, MST=22–23days).3exhibitedhighoralactivityat≥50mg/kg/day (90–97%inhibition,MST=23–27days)andlowtomoderateoral andsubcutaneousactivityat1and10mg/kg/day(7–64%inhibition, MST=24–27days).Cryptolepinetriﬂate(2atriﬂate)exhibitedonly moderateoralandsubcutaneousactivityat50mg/kg/day(43–63% inhibition,MST=24–25days).Atadoseof50mg/kg/day, subcuta-neouslyinjectedcryptolepinederivative2bwaslethaltoinfected mice(MST=3days)andoralactivityatthisdosewasmoderate (45–55%inhibition,MST=25days).At10mg/kg/day,2b adminis-teredorallyandsubcutaneouslyexhibitedlowtomoderateactivity (25–60%inhibition,MST=24days).

Discussion

Indolealkaloids1–3wereactiveagainstchloroquine-resistant K1andchloroquine-sensitive3D7strainsofP.falciparum.Thus,1–3 donotexhibitcross-resistancetochloroquine,though,asdiscussed below,likechloroquine,theyinhibithemozoinformation.

Theinvitroactivityof1againstP.falciparumwasdescribedfor theﬁrsttimebyAndrade-Neto(2007)(IC50=73nM,K1strain)and

wasrecentlyindependentlyconﬁrmed againstthe chloroquine-resistantFcM29-CameroonstrainofP.falciparum(IC50=1.13␮M)

(Passemaretal.2011;seealsoPohlitetal.2012).Presentedherein

aretheﬁrstdataontheantimalarialactivityofolivacine(3)which exhibitedimportantinvitroantimalarialactivityandlow cytotox-icity.

During intraerythrocytic infection, P. falciparum parasites crystallize toxic heme released during hemoglobin catabolism resultinginhemozoinformation.Inmechanisticstudies,1(ICG, IC50=7.9␮M) exhibited greater in vitroinhibition of hemozoin

crystalgrowth(ICG)thanquinine(ICG,IC50=17.1␮M).ICGby1

mayoccurbysurfacebindingorasubstratesequestration mecha-nism(ChongandSullivan2003).

Alternatively,1mayinhibitPlasmodiumspp.byinteractionwith DNA.Highlyplanar,1intercalatesDNAbasesinvitrowithhigh affinity. Also, 1 strongly inhibits DNA topoisomerase II invitro (Moodyetal.2007).Recently,formationofcovalentDNAadducts mediatedbyellipticineoxidationwithcytochromeP450and per-oxidaseswasproposedasamodeofaction(Kotrbovaetal.2011). Cryptolepinetriflate(2atriflate)exhibitedIC50valuesof0.80

and0.91␮MagainsttheK1and3D7strainsofP.falciparum, respec-tively,inagreementwithearlierreportsfor2aagainstK1(Cimanga etal. 1997;Wrightet al.2001)and 3D7strains(Lavrado etal. 2011).Herein,2atriﬂatewasthemosttoxiccompoundtomurine macrophages(IC50=9.4␮M)anditsselectivityindices(SI)werethe

lowestofallcompoundsstudied(SI=10–12).

Lavrado et al. (2008) synthesized analogs of 2a containing diamino-alkanesidechainsatC-11.Thebasisforthisapproachwas theobservationthatabasicaminosidechainisarequirementfor chloroquineaccumulationintheacidicdigestivevacuoleofthe par-asite.Theseanalogsof2awerepotentinhibitors(IC50=20–455nM)

of P.falciparum strainshavingdifferent drugresistance pheno-types(Lavradoetal.2008,2011).Herein,therationallydesigned 11-(4-piperidinamino)cryptolepine2bwhichhasoptimalinvitro antimalarialandSI(Lavradoetal.2008)wasthemostactive com-poundagainstP.falciparuminvitro.

Cryptolepine analog 2b was less toxic (IC50=34␮M) to

macrophages than 2a and exhibited good SI. Similarly, 2a tri-ﬂate and 2b exhibited low toxicity to HUVEC cells (IC50=1.18

and2.04␮M,respectively)(Lavradoetal.2008,2011).In previ-ouswork,2aexhibitedhighcytotoxic,genotoxic,DNAintercalating andtopoisomeraseIIinhibitoryproperties(Bonjeanetal.1998).2a iodide,itsderivativesandanalogs(e.g.2,7-dibromocryptolepine) causeDNAdamageinlymphocytes,butdonotaffecthumansperm. DuetoinvitroDNAdamageby2a,2,7-dibromocryptolepineand relatedcompounds,thesesubstancesmaynotbesuitablefor pre-clinicaldevelopmentasantimalarials(Gopalanetal.2011).

Ellipticine(1), olivacine (3) and related ellipticine-like com-poundshavereceivedattentionduetotheirhightoxicitytotumor cellsandlownumberofsideeffects.Thus,derivativesofthese com-poundsareexcellenttargetcompoundsforclinicalstudies(Sizum etal.1988;Jasztold-Howorkoetal.2004).Intumorcells,thereis evidencethatthemechanismofactioninvolvesDNAintercalation

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Table2

InvivosuppressionofPlasmodiumbergheiininfectedmiceandaveragemousesurvivaltimeafteroralandsubcutaneoustreatmentswithcompounds1–3.

Dose(mg/kg/day) %Parasiteinhibition Averagesurvivaltime±SD(day)

Oral Subcutaneous Oral Subcutaneous

Day5 Day7 Day5 Day7

Ellipticine(1) 50 100 100 NT NT >40 NT 10 77 42 33 70 27±2 29±3 1 67 61 0 44 22±4 23±5 Cryptolepine(2a) 50 43 50 63 61 24±5 25±4 10 38 28 21 46 18±3 21±3 1 3 5 16 31 17±3 22±2 Cryptolepineanalog(2b) 50 55 44 D D 25±4 3±1 10 47 25 60 30 24±4 24±4 Olivacine(3) 100 97 90 14 55 26±6 27±2 50 91 90 NT NT 23±2 NT 10 48 64 40 44 27±2 26±2 1 25 42 7 45 24±3 25±1 Chloroquine 10 99 98 98 99 >40 >40 Control 0 0 0 0 20±5 22±3

SD:standarddeviation;NT:nottested;–:notcalculable.

andinterferencewiththeactivityoftopoisomeraseIIwith

conse-quentcytotoxiceffectswhicharerelatedtosize,shapeandﬂatness

of1 and3 (Carvalhoand Laks2001;Braga etal. 2004). Impor-tantly,weobservedlow cytotoxicityfor1and 3againstmouse macrophageswhereas2atriﬂateandcryptolepinederivative2b exhibitedrelativelyhightoxicitytomacrophages.

Asseenabove,compounds1–3inhibitP.falciparuminvitro.Only cryptolepinehasbeenstudiedpreviouslyusinginvivo antimalar-ialmodels.So,theeffectsof1–3inP.berghei-infectedmicewere explored.Ellipticine(1)wasthemostactivecompoundinvivo sup-pressingparasitemiaby100%andprovidingMSTof>40daysatan oraldoseof50mg/kg/day.Remarkably,thiswasthesameresult obtainedforcontrolcompoundchloroquineat10mg/kg/dayforall animals.Also,upto77%inhibitionofparasitemiawasobserved for1atdosesof10mg/kg/day.Fewcompounds(e.g.chloroquine) signiﬁcantlyreduceparasitemiainmiceinfectedwithP.berghei. Atthe highest doseof 1 (50mg/kg/day), nomortalityor other signsofintoxicationwereobserved.Theinvivoantimalarial activ-ityof3waslowerthanthatofitsstructuralisomer 1.Notoxic effectswereobservedinmicewhichwereadministered3atupto 100mg/kg/day.

Theinvivoantimalarialactivityof2atriﬂatewaslowto moder-ate.Thus,2atriﬂateadministeredsubcutaneously(50mg/kg/day) inhibited P. berghei by 63% (day 5). Similarly, Kirby et al. (1995) observed that parasitemia in P. berghei-infected mice was not reduced by subcutaneously administered cryptolepine (113mg/kg/day).

Cryptolepineiodide(2aiodide)wastoxictoP.berghei-infected micebyintraperitonealinjectionat12.5mg/kg/day(Wrightetal. 2001).Toxicityisrelated totheintraperitonealrouteof admin-istration. However, no deaths were reported when 2a iodide was administered subcutaneously (at 113mg/kg/day) or orally (50mg/kg/day)(Wrightetal.2001).Herein,2atriﬂateexhibited notoxiceffects.

Cryptolepineanalog2bexhibitedmoderateactivityinvivoand acutetoxicity.Orallyat50mg/kg/day,2binhibitedthegrowthofP. bergheiby55%.However,subcutaneouslyat50mg/kg/day,2bkilled allmiceaftertheseconddose.Subcutaneously,at10mg/kg/day,2b causedulcerationattheplaceofinjectionandinhibitedP.berghei onlymoderately(60%).

Cryptolepine triflate (2a triflate) and derivatives (e.g. 2,7-dibromocryptolepine and 2b) exhibit limited efficacy in P. berghei-infectedmice.Also,cytotoxicity,acutetoxicityand geno-toxicityarepotentialdrawbackstotheiruseasdrugs.Importantly, structuralisomers1and3exhibitedgoodinvitroSIand1provided P.berghei-infectedmicewithmeansurvivaltimesgreaterthan40 days.Thesedatarevealthepotentialofellipticine(1)andolivacine (3)asantimalarialleads.

Acknowledgements

ThisworkwassupportedbygrantsfromtheBrazilianMalaria Network (CNPq 555665/2009-7 and CNPq 555669/2009-2), Bionorte CNPq 554317/2010-9, MCT/INPA/INPA, PRONEX/FINEP 023/2009andTheScienceandTechnologyFoundationofPortugal (PTDC/SAU-FAR/114864/2009 and PEst-OE/SAU/UI4013/2011). LFRS,AMP,AM,RCNA,GDandDCSthankFAPEAMand/orCNPqfor individualfellowshipsreceived.

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