In Vivo and In Vitro Activities and ADME-Tox Profile of a Quinolizidine-Modified 4-Aminoquinoline

15 Pages·2017·1.49 MB·English

molecules
Article
In Vivo and In Vitro Activities and ADME-Tox Profile
of a Quinolizidine-Modified 4-Aminoquinoline:
A Potent Anti-P. falciparum and Anti-P. vivax
Blood-Stage Antimalarial
NicolettaBasilico1,†,SilviaParapini2,†,AnnaSparatore3 ID,SergioRomeo3 ID,PaolaMisiano2,
LiviaVivas4,VanessaYardley4,SimonL.Croft4,AnnetteHabluetzel5,LeonardoLucantoni5,‡,
LaurentRenia6,BruceRussell6,§ ID,RossarinSuwanarusk6,§,FrancoisNosten7,8,
GiulioDondio9,ChiaraBigogno9,DanielaJabes10andDonatellaTaramelli2,* ID
1 DepartmentofBiomedical,SurgicalandDentalSciences(DiSBIOC),UniversityofMilan,ViaPascal36,
20133Milan,Italy;[email protected]
2 DepartmentofPharmacological&BiomolecularSciences(DiSFeB),UniversityofMilan,ViaPascal36,
20133Milan,Italy;[email protected](S.P.);[email protected](P.M.)
3 DepartmentofPharmaceuticalsSciences(DISFARM),UniversityofMilan,ViaMangiagalli25,
20133Milan,Italy;[email protected](A.S.);[email protected](S.R.)
4 DepartmentofImmunologyandInfection,FacultyofInfectiousandTropicalDiseases,
LondonSchoolofHygieneandTropicalMedicine(LSHTM),KeppelStreet,LondonWC1E7HT,UK;
[email protected](L.V.);[email protected](V.Y.);[email protected](S.L.C.)
5 SchoolofPharmacy,UniversityofCamerino,Viad’Accorso16,63032Camerino,MC,Italy;
[email protected](A.H.);l.lucantoni@griffith.edu.au(L.L.)
6 SingaporeImmunologyNetwork(SIgN),AgencyforScienceTechnologyandResearch,Biopolis,
Singapore138648,Singapore;[email protected](L.R.);[email protected](B.R.);
[email protected](R.S.)
7 ShokloMalariaResearchUnit,Mahidol-OxfordTropicalMedicineResearchUnit,
FacultyofTropicalMedicine,MahidolUniversity,MaeSot63110,Thailand;[email protected]
8 CentreforTropicalMedicineandGlobalHealth,NuffieldDepartmentofMedicineResearchbuilding,
UniversityofOxfordOldRoadCampus,OxfordOX37FZ,UK
9 AphadSrl,ViadellaResistenza65,20090Buccinasco,Milan,Italy;[email protected](G.D.);
[email protected](C.B.)
10 NeEDPharmaceuticalsSrl,VialeOrtles22/4,20139Milan,Italy;[email protected]
* Correspondence:[email protected];Tel.:[phone];Fax:[phone]
† Theseauthorscontributedequallytothework.
‡ Currentaddress:GriffithInstituteforDrugDiscovery,GriffithUniversity,Brisbane,QLD4111,Australia.
Received:24October2017;Accepted:29November2017;Published:1December2017
Abstract: Natural products are a prolific source for the identification of new biologically active
compounds. In the present work, we studied the invitro and invivo antimalarial efficacy and
ADME-Tox profile of a molecular hybrid (AM1) between 4-aminoquinoline and a quinolizidine
moietyderivedfromlupinine(Lupinusluteus). Theaimwastofindacompoundendowedwiththe
targetproductprofile-1(TCP-1: moleculesthatclearasexualblood-stageparasitaemia),proposed
by the Medicine for Malaria Venture to accomplish the goal of malaria elimination/eradication.
AM1 displayed a very attractive profile in terms of both invitro and invivo activity. By using
standard invitro antimalarial assays, AM1 showed low nanomolar inhibitory activity against
chloroquine-sensitive and resistant P. falciparum strains (range IC 16–53 nM), matched with a
50
high potency against P. vivax field isolates (Mean IC 29 nM). Low toxicity and additivity with
50
artemisininderivativeswerealsodemonstratedinvitro. Highinvivooralefficacywasobserved
inbothP.bergheiandP.yoeliimousemodelswithIC valuescomparableorbetterthanthoseof
50
chloroquine. Themetabolicstabilityindifferentspeciesandthepharmacokineticprofileinthemouse
modelmakesAM1acompoundworthfurtherinvestigationasapotentialnovelschizonticidalagent.
Molecules2017,22,2102;doi:10.3390/molecules22122102 www.mdpi.com/journal/molecules
Molecules2017,22,2102 2of15
Keywords: malaria;4-aminoquinoline;drugresistance;P.falciparum;P.vivax
1. Introduction
MalariaisaninfectiousdiseasecausedbyPlasmodiumparasites;twospecies,P.falciparumand
P.vivax,areresponsibleformorethan400,000deaths/yearworldwide. Themajorityoffatalities(90%),
mostlychildrenbelowtheageoffiveandpregnantwomen,occurinAfrica,andinparticularinthe
sub-Saharanregions[1].
Increasedfundingandeffortstofindnew,effectivetreatments,togetherwithimprovementsin
theuseofcontroltools(insecticide-treatednetsandresidualindoorspraying),succeededinreducing
malariaincidenceby41%between2000and2015. Similarly,globalmalariamortalitydecreasedby
62%overthesameyears. However,theemergenceofparasiteresistancetothecurrentcombination
therapies based on artemisinin derivatives (ACTs) may frustrate all these efforts [2–5]. Moreover,
in some of the countries where P. falciparum has been eliminated (Central Asia, Argentina, Belize,
Mexico,andlargepartsofChina)thereisgrowingevidenceforanincreaseinmorbidityandmortality
duetoP.vivaxinfection[6–8]. Forthesereasons,thediscoveryanddevelopmentofnewdrugsactive
againstdifferentPlasmodiumspeciesremainsahighpriority.
The4-aminoquinolineclassofdrugs,representedbytheclassicalantimalarialagentchloroquine
(CQ),hasbeenhighlysuccessfulinthetreatmentofmalariafordecadesbeforeresistanceemergedand
spread. Anothermemberofthefamily,amodiaquine(AQ),isstillusedinACT[9]. Fortheirstability,
bioavailability,andefficacy,quinoline-typedrugsareextremelyeffectiveasblood-stageschizonticidal
agents.However,theirusageishamperedbythelargediffusionofresistanceofP.falciparumandP.vivax
strainstoCQ,andcross-resistancetoAQandmefloquine.IncountrieswhereCQusewasdiscontinued,
studieshaveshownthat,intheabsenceofdrugpressure,CQ-resistantstrainsofP.falciparumhavebeen
replacedbyCQ-sensitiveisolates[10]. Therefore,thedevelopmentofCQanaloguesasmulti-species
antimalarialagentsmaystillbevalidandhasbeenrecentlyhighlightedandreviewed[11,12].
Chemical modification of the side chain of 4-aminoquinolines, either in length or with the
introduction of bulky basic groups, has been shown to overcome CQ resistance. Over the years,
several compounds active also in CQ-resistant strains have been synthesized and some of them,
includingonefromourgroup,reachedpre-clinicaltesting[13–15]. FerroquineandAQ13,forexample,
areinadvancedclinicalphaseIIevaluation[16–20].
Inordertoeliminatemalaria,theantimalarialtargetproductprofileshavebeenrecentlyrevised
bytheMedicineforMalariaVenture(MMV),highlightingtheimportanceofdevelopingcompounds
thatwillbeabletotargetdifferentparasitespeciesandstages,maintainingagoodADME/PKprofile
whichcanberelevantforcombinationtherapies[21].
A few years ago, we synthesized and studied a new series of 7-chloro-4-aminoquinolines
characterizedbythepresenceofabulkyquinolizidinylorquinolizidinylalkylsidechain,thelatter
derivedfromthenaturalproduct(−)-lupinine,analkaloidextractedfromLupinusluteusseeds[22].
Among them, 7-chloro-4-(N-lupinyl)aminoquinoline, named (−)-AM1, was selected for further
physicochemicalandbiologicalcharacterization. Theresultsindicatedthattheantiplasmodialactivity
of (−)-AM1 was likely due to inhibition of hemozoin formation. This was directly tested in a
beta-hematin inhibitory assay invitro, and it was substantiated by measuring the lipophilicity of
(−)-AM1 [23]. In fact, (−)-AM1 is more lipophilic than chloroquine and thus able to accumulate
at the interface between lipids and water where hemozoin formation is reported to occur [24].
TheefficacyagainstCQ-resistantstrainsofP.falciparumcouldbeascribedtotheabilityof(−)-AM1
to block drug efflux by hydrophobic interactions with PfCRT, the transporter responsible for
CQ resistance [23]. Due to the limited market supply of (−)-lupinine, the total synthesis of
7-chloro-4-(N-lupinyl)aminoquinoline, the most active compound of the series, was developed
resultinginaracemicproductcalled(±)-AM1(Figure1)[25].
Molecules 2017, 22, 2102 3 of 15
aminoquinoline, the most active compound of the series, was developed resulting in a racemic product
Molecules2017,22,2102 3of15
called (±)-AM1 (Figure 1) [25].

FFiigguurree 11.. SSttrruuccttuurree ooff iinnvveessttiiggaatteedd ccoommppoouunnddss:: 77--cchhlloorroo--NN--((((((11SS,,99aaRR))--ooccttaahhyyddrroo--11HH--qquuiinnoolliizziinn--11--yyll))
mmeetthhyyll))qquuiinnoolliinn--44--aammiinnee (−(−))--AAMM11;; 77-c-chhlolorroo--NN--((((((11RR,,99aaSS))--ooccttaahhyyddrroo--11HH--qquuiinnoolliizziinn--11--yyll))mmeetthhyyll))
qquuiinnoolliinn--44--aammiinnee ((++))--AAMM11..
Both the racemate and its purified enantiomers showed potent antimalarial in vitro activity
Both the racemate and its purified enantiomers showed potent antimalarial invitro activity
against both CQ-S (D10) and CQ-R (W-2) strains of P. falciparum, and a good therapeutic index [25].
againstbothCQ-S(D10)andCQ-R(W-2)strainsofP.falciparum,andagoodtherapeuticindex[25].
In the present work, we extended the previous observations by completing in vivo efficacy
Inthepresentwork,weextendedthepreviousobservationsbycompletinginvivoefficacystudies
studies of racemic AM1 and its purified enantiomers in two rodent malaria models (P. berghei and
ofracemicAM1anditspurifiedenantiomersintworodentmalariamodels(P.bergheiandP.yoelii)with
P. yoelii) with different susceptibilities to CQ, and activity studies against P. vivax clinical isolates.
differentsusceptibilitiestoCQ,andactivitystudiesagainstP.vivaxclinicalisolates. Wealsoevaluated
We also evaluated the ADME-Tox profiles in vivo and in vitro.
theADME-Toxprofilesinvivoandinvitro.
2. Results
2. Results
22..11.. IInn VViittrroo AAnnttiimmaallaarriiaall AAccttiivviittyy aaggaaiinnsstt PP.. ffaallcciippaarruumm aanndd PP..v vivivaaxx
RRaacceemmiicc AAMM11 aanndd iittss ppuurriififieedd eennaannttiioommeerrss ((FFiigguurree 11)) sshhoowweedd aann aannttiimmaallaarriiaall aaccttiivviittyy iinn vviittrroo iinn
tthhee llooww nnaannoommoollaarr rraannggee ((IICC5500 == 161–65–35 3nMnM) w)whehne ntetsetsetde dagaagianisnt sbtobtoht ChQC-QS -(SD(1D01) 0a)nadn CdQC-QR- sRtrsatirnasi nosf
oPf. Pfa.lfcaiplcairpuamru m(F(CFRC1R 1anadn dFCFCRR3)3, ),ininclculuddining gaa mmuultltididrruugg-r-reessisistatannt tppaarraassiittee TTMM9911CC223355 ((iissoollaatteedd iinn
TThhaaiillaanndd)) ((TTaabbllee 11)).. AAllll tthhee ccoommppoouunnddss hhaadd vveerryy llooww ttooxxiicciittyy aanndd aann eexxcceelllleenntt sseelleeccttiivviittyy iinnddeexx
((SSII >> 11000000)) aaggaaininsstt hhuummaann eennddootthheelliiaall cceellllss aanndd fifibbrroobbllaassttss,, aanndd mmoouussee cceellll lliinneess,, aass pprreevviioouussllyy
rreeppoorrtteedd [[2255]].. FFuurrththeermrmoorer,e (,±()±-A)-MA1M s1hoshwoewd erdemreamrkaarbklea balnetiamntailmaraialal raicatlivaicttyi vaigtyainagsta 1in0s dti1ff0erdeinffte frieenldt
fiiseolldatiesso loaft ePs. ovfivPa.xv wiviatxh wpiotthenpcoyte anbcoyuatb 3o tuimt3esti mhiegshherig thhearnt thhaant tohf aCtQof (CTQab(lTe a1b, lreig1h,tr icgohltucmonlu).m n).
TTaabbllee 11.. IInn vviittrroo aannttiimmaallaarriiaall aaccttiivviittiieess ooff tthhee AAMM11 rraacceemmaattee aanndd iittss eennaannttiioommeerrss oonn PP.. ffaallcciippaarruumm
llaabboorraattoorryy ssttrraaiinnss aanndd PP.. vviivvaaxx fifieelldd iissoollaatteess..
CoCmompopuonunddss D10 c(CQ-S) W-2PP ..c ff(aaCllcQcipi-paRar)urmumITC IM5C059(0n1(MCnM2)5a)3a FCR1 FCR3 P.Pv.i vvFaiixveaIlCdx 5 II0Cs(on50lM a(tn)eMbs )b
D10c(CQ-S) W-2c(CQ-R) TM91C235 FCR1 FCR3 FieldIsolates
(±)-AM1 16.15 35.44 - - - 29.72
(−()±-A)-MAM1 1 2136..9185 3553..4647 1-6.74 3-2.87 2-3.28 29.72-
(−)-AM1 23.98 53.67 16.74 32.87 23.28 -
(+)-AM1 17.49 35.77 - - - -
(+)-AM1 17.49 35.77 - - - -
CQ 23.72 437.62 199.46 181.93 112.56 86.66
CQ 23.72 437.62 199.46 181.93 112.56 86.66
aa TThheea asssasyayw awsaevs aeluvaatleudaatfetder a7f2tehri n7c2u bha tiinoncu,ubsaintigonth, eupsairnagsi ttehlea cptaatreadseihtey dlarocgtaetnea sdee(hpLyDdHro)gmenetahsoed ,(pmLeaDnHo)f
3mdeitffheoredn,t mexepaenri mofe n3t sd.ibffIeCr5e0nrte seuxltpsefrroimme1n0tfis.e lbd IiCso50la rteessufrlotms ftrhoemT h1a0i– fBieulrdm iessoelBaoterds efrr.ocmDa tthaea lTrehaadiy–pBuubrlmisheesde
iBno[r2d5]e.r. c Data already published in [25].
WWhheenn tteesstteedd iinn ccoommbbiinnaattiioonn ssttuuddiieess wwiitthh ddiihhyyddrrooaarrtteemmiissiinniinn ((DDHHAA)) uussiinngg DD1100 ((CCQQ--SS)) aanndd WW--22
((CCQQ--RR)) ppaarraassiitteess,, tthhee iissoobboollooggrraammss sshhoowweedd tthhaatt ((−−))--AAMM11 hhaadd aaddddiittiivvee aaccttiivviittyy wwiitthh DDHHAA ((FFIICC << 22)),,
wwiitthh aan onno-ns-isginginfiicfiacnatnttr etnredntdo wtoarwdaarndt aagnotnaigsomni(sFmig u(rFeig2uAr,eB )2.ATh,Be).r eTsuhlet sraerseusltims ialraer tsoimthiolasre otob tathinoesde
wobitthaiCneQd (wFiigthu rCeQ2C (F,Dig)u. re 2C,D).
Molecules2017,22,2102 4of15
Molecules 2017, 22, 2102 4 of 15

FigFuigreure2 .2. IsIosobboolologgrraamm aannaallyyssiiss ooff tthhee aannttiimmaallaarriiaall aaccttiivviittyy ooff ((−−)-)A-AMM1 1ini ncocmombinbaintiaotnio nwiwthi th
dihdyihdyrdoraoratertmemisiisnininin( D(DHHAA))a aggaaininsstt DD1100 cchhlloorrooqquuiinnee--sseennssitiitvive e(C(CQQ-S-)S )(A(A) )oor rWW22 cchhlolororoqquiunien-ere-rseisstiasntat nt
(CQ(C-QR)-R()B ()BP). Pfa. lfcailpcaipraurmums tsrtarianins.s. CCoommbbiinnaattiioonnss ooff DDHHAA wwitihth cchhlolororqouqiunien e(C(QC)Q a)rae rsehsohwonw inn i(nC,(DC),.D ).
DHDAHAfr afcrtaicotnioanlaIlC I5C050v avlaulueessw weererec caalclcuullaatteedd bbyy ddiivviiddiinngg tthhee IICC5500 ooff DDHHAA ccoommbbinineded wwitiht h(−()−-A)-MA1M o1r or
CQC,Qre, srpesepceticvtievleyl,yb, ybyth teheIC IC5050o offD DHHAAa alolonnee,, aanndd ppllootttteedd oonn tthhee hhoorriizzoonnttaal laaxxisi.s .TTheh ecocrorrersepsopnodnindgin g
frafcrtaioctnioanla(−l ()−-)A-AMM11o orrC CQQI ICC5500 vvaalluueess wweerree ccaallccuullaatteedd bbyy ddivivididiningg eeaachch fifixxeded cocnocnecnetnrtartaiotino nbyb ythteh IeCI5C0 50
ofothf eth(e− ()−-A)-AMM11o orrC CQQa alolonnee,,a anndd ppllootttteedd oonn tthhee vveerrttiiccaall aaxxiiss. .AAnn isisoobboololgorgarmam clcolsoes teot othteh deidagiaognoanl al
indinicdaitceasteasn aadnd iatdivdeiteivffee cet.ffCecutr. veCsusrivgensi fisciagnntilfyica(>n2tl)ya b(o>v2)e oarbobveleo wort hbeedloiawg otnhael idnidaigcoanteala nitnadgiocanties tic
antagonistic or synergistic effects, respectively.
orsynergisticeffects,respectively.
2.2. In Vivo Oral Efficacy Studies
2.2. InVivoOralEfficacyStudies
Racemic AM1, (−)-AM1, and (+)-AM1 were tested for oral in vivo efficacy in the four-day mouse
RacemicAM1,(−)-AM1,and(+)-AM1weretestedfororalinvivoefficacyinthefour-daymouse
infection model against P. berghei ANKA [26] and P. yoelii. The data, expressed as ED50 and ED90
infection model against P. berghei ANKA [26] and P. yoelii. The data, expressed as ED and ED
values, are reported in Table 2. 50 90
values,arereportedinTable2.
Table 2. Therapeutic efficacy of (±)-AM1, (−)-AM1, (+)-AM1, and chloroquine against P. berghei
TabAlNeK2.AT ahnedra Pp.e yuoteilciie ifnfifceacctiyoonf in(± m)-iAceM u1s,in(−g t)h-Ae MSt1an,(d+a)r-dA Mfou1r,-adnady cThelsotr. oquineagainstP.bergheiANKA
andP.yoeliiinfectioninmiceusingtheStandardfour-dayTest.
P. berghei a P. yoelii a
Compounds ED50 (95%P C.Ib)e r gheiaED90(95% CI) ED50(95% CIP).yoelEiiDa90 (95% CI)
(mg/kg) (mg/kg) (mg/kg) (mg/kg)
Compounds ED (95%CI) ED (95%CI) ED (95%CI) ED (95%CI)
(±)-AM1 2.5707 (1.26–4.29) 12.5980 (7.69–17.46) 50 90
(mg/kg) (mg/kg) (mg/kg) (mg/kg)
(−)-AM1 2.05 (1.31–2.79) 8.65 (7.06–10.23) 1.55 (1.25–1.92) 4.95 (3.08–7.94)
(±()+-A)-AMM11 2.717.6(01 (.216.0–84–.22.91)1) 12.25.803(7 (.16.94–6–127..6406))
(C−h)-loArMoq1uine 2.015.1(21 (.301.9–82–.17.92)6) 8.625.9(37 .(026.6–41–03..2232)) 1.55(1<.23 5–1.92) 584.5.9 (55.(338.0–86–.472.)9 4)
a C(D+1) -mAiMce 1were ino1c.u6l0at(e1d.0 i8.p–.2 w.1i1th) 4 × 1026 .i0n3fe(c1te.4d6 e–r2y.6th0r)ocytes/mouse. Four drug doses of compounds,
Chloroquine 1.12(0.98–1.26) 2.93(2.64–3.22) <3 58.5(5.38–6.42)
dissolved in standard suspending formula (SSV), were administered orally, once a day for four days,
a CsDta1rtminigce twwoer heoiunorsc uploastet-dinif.pec.tiwonit.h P4er×ce1n0t6 pianrfaescitteademeriyat hwroacsy etvesa/lumaoteuds eb.yF mouicrrdosrucogpdico seexsamoficnoamtiopno uonf ds,
dissolvedinstandardsuspendingformula(SSV),wereadministeredorally,onceadayforfourdays,starting
Giemsa-stained smears taken on day 4. ED50 and ED90 values were calculated by GraphPad Prism6.
twohourspost-infection. PercentparasitaemiawasevaluatedbymicroscopicexaminationofGiemsa-stained
All animal work at The London School of Hygiene and Tropical Medicine (LSHTM) was conducted
smearstakenonday4.ED50andED90valueswerecalculatedbyGraphPadPrism6.AllanimalworkatTheLondon
Schuonodleorf Hlicyegniecne e(aPnPdL7T0ro/8p4i2ca7l) Macecdoicridnieng(L StoH TUMK) wHaosmceo nOduffcicteed ruengduelartliiocnens:c eT(hPeP LA7n0/im84a2ls7 )(aScccioerndtiinfigc to
UKPHroocmedeuOrfefisc)e Arecgt uolfa 1ti9o8n6s.: TheAnimals(ScientificProcedures)Actof1986.
AlAlcllo cmompopuonudnsdss hshoowweeddv veerryy ggoooodd oorraall eefffificcaaccyy,, ssiimmiillaarr ttoo tthhaat toof fCCQQ. .TThhe eEEDD50 5d0idd indont odtifdfeifrf er
sigsnigifincifaicnatnlytlyb ebtewtweeenent htheet wtwooe nenaanntitoiommeerrss,, wwhheerreeaass tthhee EEDD9900 oof f(+(+)-)A-AMM1 1wwasa slolwoewr etrhathna tnhatht aotf of
Molecules2017,22,2102 5of15
(−)-AM1andtheracemate, andidenticaltothatofCQ.IntheP.yoeliimodel, whichisconsidered
relativelyCQ-resistant,the(−)-AM1enantiomermaintaineditsantimalarialactivity,whileCQhad
lowerefficacy,asexpected. Themaximumtolerateddose,MTD,inCD1micetreatedwith(−)-AM1
was greater than 100 mg/kg, i.p. No toxic clinical signs or mortality were noted up to this dose.
Similarresultswereobtainedusingthecompoundsasfreebasesoraswatersolublesalts,confirming
atherapeuMtiocleciunlesd 2e01x7, g22r, e21a0t2e rthanthatobservedforCQ(MTD50mg/kgi.p.). 5 of 15
2.3. InVivo(−T)-rAaMns1m ainsds itohne- rBalcoecmkainteg, aanndd idPernotpichayl ltaoc tthicatE offf eCcQts. In the P. yoelii model, which is considered
relatively CQ-resistant, the (−)-AM1 enantiomer maintained its antimalarial activity, while CQ had
Havinlogwdere emffiocancsyt, raas teexdpeacthedi.g Thhein mvaixvimouomr atolleerfafitecda cdyosae,g MaiTnDs,t ina sCeDx1u malicbe ltoreoadte-ds twaigthe s(−)i-nAMth1e four-day
test,weinwveass tgigreaatteerd ththane 1a0c0t imvigt/ykgo, fi.tph. eN(o− to)-xAic Mcli1niecanla snigtnios more mroorntatlihtye wtrearne snmotiesds iuopn tost tahgise sdoosfe.t herodent
Similar results were obtained using the compounds as free bases or as water soluble salts,
malariaparasiteP.bergheiANKAandAnophelesstephensimosquitoes. BALB/cmice,fourdaysafter
confirming a therapeutic index greater than that observed for CQ (MTD 50 mg/kg i.p.).
infectionwithP.berghei,weretreatedorallywith50mg/kgof(−)-AM1andanesthetizedandsubmitted
to the bite2s.3o. Ifn AVi.vso tTerpahnesmnsisisimon-oBsloqcukiintgo aensd oPnroephhyloacutirc Elaffteectrs. After 10 days, a high proportion (86.4%) of
mosquitoesinHfeacvtinegd dwemitohnsgtraamtede tao hciygthe isn fvriovom or(a−l e)-ffAicaMcy1 a-tgraeinastte adsemxuiacl eblhooods-tsetadgeoso icny thstes foounr-dthaye irmidgut
(CI 81.4–te1s0t,3 w.6e% in)v.eTstihgiastewd tahse nacotitvdityif ofef rtheen (t−)f-rAoMm1 ecnoannttrioomlemr oons qthue ittroanpsmosisistiiovni tsytag(e9s0 o%f t,hCe Irode8n0t .6–99.4%).
95 95
malaria parasite P. berghei ANKA and Anopheles stephensi mosquitoes. BALB/c mice, four days after
Similarly, there was not a significant difference in infection intensity, i.e., the number of oocysts
infection with P. berghei, were treated orally with 50 mg/kg of (−)-AM1 and anesthetized and
permidgut,betweenmosquitoesinfectedwithgametocytesfrom(−)-AM1-treatedmiceorcontrols:
submitted to the bites of A. stephensi mosquitoes one hour later. After 10 days, a high proportion
anaverage(86o.4f%5)4 o.1f 1m(oCsqIu9i5to3e9s .i7n7fe–c6te8d. 4w5it)ha gnadme3t9oc.1yt7es( CfroI9m5 (1−8)-.A4M4–15-t9re.a8t9ed) omoiccey hsotsstewd eoroecyfsotsu onnd t,hreeirs pectively
(Student’smt-itdegsutt p(C=I950 8.21.44)–.103.6%). This was not different from control mosquito positivity (90%, CI95 80.6–
99.4%). Similarly, there was not a significant difference in infection intensity, i.e., the number of
When tested for prophylactic activity (see M&M for details), no significant differences in
oocysts per midgut, between mosquitoes infected with gametocytes from (−)-AM1-treated mice or
parasitaemiain(−)-AM1treatedmicecomparedtocontrolmicewereseenduringtheentireevaluation
controls: an average of 54.11 (CI95 39.77–68.45) and 39.17 (CI95 18.44–59.89) oocysts were found,
period(daryesspe5c–ti9veplyo (sStt-uidnefnetc’st ito-tnes)t. pI =t 0c.a24n). b econcludedthatinthepresentmodel,(−)-AM1doesnot
showtransmisWsihoenn -btelsotecdk ifnogr porropphryolpachtiyc laaccttiivcitya c(tsievei tMy.&M for details), no significant differences in
parasitaemia in (−)-AM1 treated mice compared to control mice were seen during the entire
evaluation period (days 5–9 post-infection). It can be concluded that in the present model, (−)-AM1
2.4. MetabolicStabilityandBiotransformationStudiesinRat,Mouse,andHumanMicrosomes
does not show transmission-blocking or prophylactic activity.
Preliminary experiments were then conducted to explore the ADME properties of these
2.4. Metabolic Stability and Biotransformation Studies in Rat, Mouse, and Human Microsomes
compounds. Whenevaluatedinhepaticmicrosomes(Figure3),racemicAM1anditsenantiomers
Preliminary experiments were then conducted to explore the ADME properties of these
showed high metabolic stability (greater than 50% remaining at 30 min) in both rat and human
compounds. When evaluated in hepatic microsomes (Figure 3), racemic AM1 and its enantiomers
species,comparabletothatofCQ.Onthecontrary,inthemousemicrosomes,allthecompoundswere
showed high metabolic stability (greater than 50% remaining at 30 min) in both rat and human
moderatelyspuecniesst,a cbolmep(afrraobmle 1to6 tthoat4 o7f% CQre. mOna itnhien cgonattra3r0y, mini nth)e, wmoituhser amciecrmosiocmAesM, a1ll atnhed c(o+m)-pAouMnd1s lessstable
than(−)-AwMer1e .mSotdaenrdataerlyd urnesftearbelen (cferocmo m16 ptoo u47n%d srepmraoinpirnagn aot l3o0l manind), 7w-iethth roacxeymcioc uAmMa1r ainndw (+e)r-AeMm1e tabolized
less stable than (−)-AM1. Standard reference compounds propranolol and 7-ethoxycoumarin were
asexpected[27].
metabolized as expected [27].

Figure 3. Metabolic stability studies in mouse, rat, and human microsomes. Hepatic metabolic
Figure3.Metabolicstabilitystudiesinmouse,rat,andhumanmicrosomes.Hepaticmetabolicstability
stability of the AM1 racemate and its enantiomers was determined in the microsomes of mouse, rat,
oftheAM1anrda cheummaatne aspnedcieitss aefntearn t3i0o mmeinr sinwcuabsadtieotne. rm7-EinCe danidn pthroepmranicorlools owmerees uosfemd oaus sree,ferraetn,caen dhuman
speciesaftceorm3p0oumndins. Rinescuultbs aatrieo enx.pr7e-sEseCd aasn Mdeapnr o± pS.rDa.n, no =lo 2l. Mweetraeboulisc esdtabailsityre rfaenrgeen vcaeluceso: m>5p0:o Huinghd; s. Results
areexpres1s0e–d50a: MseMdieuamn; <±10:S L.oDw.,. n=2. Metabolicstabilityrangevalues: >50: High;10–50: Medium;

<10:Low.
Molecules2017,22,2102 6of15
Biotransformationstudieswereperformedonlyon(−)-AM1inrat,mouse,andhumanhepatic
microsomesinordertoidentifymajormetabolitesandhighlightpossibledifferencesamongspecies.
Molecules 2017, 22, 2102 6 of 15
AMMreoollpeeccuurlleeesss 22e0011n77,t, 2a222t,, i22v1100e22 chromatogram in the mouse and the MS spectra are sho6w6 ooffn 1155 in Figure S1
(SupplemeBnitoatrraynsMforamteartiioanl ss)t,uwdiehsi wleetreh epeprfroormpoeds eodnlys tornu (c−t)u-AreMs1o inf mrate, tmaobuoslei,t aensdi nhuamllatnh heepsaptiecc ies,together
Biotransformation studies were performed only on (−)-AM1 in rat, mouse, and human hepatic
microBsioomtreasn sinfo ormrdaetri oton isdteundtiiefsy wmearjoe rp mereftoarbmoelidte os nalnyd o hni g(−h)l-iAghMt 1p oinss ribatle, mdioffuesree,n acneds ahmumonagn s hpeepciaetsic.
with thmeircerotseonmtieos nin toirmdeers t,o aidreenstihfyo mwanjori nmeTtaabbolleite3s. anInd htihgehlisgahmt peostsaibbllee d,iftfhereenmceest aamboonlgit sepsecfoieus. nd in mouse
micrAos ormeperse isne notradtievre t oc hidreonmtiaftyo mgraajmor mine ttahbeo mlitoeus saen da nhdig hthlieg hMt Sp osspseibclter ad iafrfeer esnhcoews na mino nFgig supreec iSes1.
plasma(aSuftpepAArle 5 mrr0eeeppmnrreetgassre/eynnk ttMaagttaiiovvtereera iccalhhltsrrr)ooe, mmawtaahmttioolegge rrntaahtmmea priirenno ipttnhhocees le ummdd oosteuurdssueec. taTaunnrheddes ttorhhefee tm eMMentSStai bossoppnleeitctceittsmrr aain eaa sarreleal ntsshhhdeoo w[wsMpnne c+iiinne sHF,F tii]gog+uguoerreetfh tSeShr11 e metabolites
(Supplementary Materials), while the proposed structures of metabolites in all the species, together
arethew(sSaiutmhp ptehleefm orreentaetnallrtiyto hnMr teaitmeeresisap,l esa)cr, eiwe sshh.iolew tnh ein p rToapboles e3d. sIntr uthcetu sraems oef tmabeltea, btohlei temse itna baolll itthees sfopuecnides i,n t omgeotuhseer
with the retention times, are shown in Table 3. In the same table, the metabolites found in mouse
pwlaitshm tah ea frteetre n5t0io nm tgim/kegs ,o arrael sthreoawtmn einnt Taarbel ei n3c. lIund tehde. sTahmee rteatbelnet,i othne tmimeetas baonlidte s[ Mfo u+n dH i]n+ mofo tuhsee
plasma after 50 mg/kg oral treatment are included. The retention times and [M + H]+ of the
mmpleeattsaTambbaoaobl liliatteeefsts3e a.arr rIee5n 0tthvh emeiv ssgoaa/mmkagnee dffooorriran ala llvllt ritttehhrarroteemeem essapnpieten ccaiimeerses.e. tinacbloulditeeds. oTfh(e− r)e-tAenMti1ona ntidmtehse iarnpd ro[Mpo +se Hd]s+t rouf ctthuer es.
metabolites are the same for all three species.
Table 3. In vivo and in vitro main metabolites of (−)-AM1 and their proposed structures.
TTaabbllee 33.. IInn vviivvoo aanndd iinn vviittrroo mmaaiinn mmeettaabboolliitteess ooff ((M−−))e--AAtaMMbo11l iaacnnPddr ttohhdeeuiirrc tppsrrDooppeootessecetdde dssttrrIunuccVttuuitrrreeoss..a ndInVivo
MMeettaabbIoonlliiVcc iPPtrrroooddMuuciccttsrso DDsoeemtteeecctsteedd IInn VViittrroo aanndIdn IInVn iVVvioivvPoolasmaLevelsafter
MH+(((rrr..ttMtMM,..,,m HmHmHi+in++i n n) )) PPPPrrrrooooppppoooosssseeeedddd SSSSttttrrrruuuuccccttttuuuurrrreeee HuHIIImnunnM am VVV(ne((−a−i−tiit)ant)t)r-r-r-bAoAoAooMMMMMMlRi(c1−i11iia c cc P 1tr11r)ro r-o o μμAoμsssMdMoMRooMmummMa 1c t e eeto1ssssu DµsMeetectIeIInMnnd 5 V55VVoI00n0uiii v m vvmsmVooeogg gi PtPP///rkklklloaMagagg sss a OmoOmOmnurdarraaasaa LleILLll5 n eGee0GG vvvVamaaeeevvivlllgvsassaa /og gagkaaefeeMffgt t t eeeOorrr u rasleGavage
(r.t., min) HHuummaannAt 30RR MaaAttint30MMMooiuunssee AtM M12oo0uu Msseei n At120Min
At 30 Min At 120 Min
At 30 Min At 120 Min
330
330(6.313)0 + + + + + + + +
(63.310) + + + +
(6.1) + + + +
(6.1)
344
344 + + + ND
344(5(.539.4)94) + + + + + + ND ND
(5.9) + + + ND
(5.9)
N
346(4(((3.34344744.4..)776766)) ) CCClll OOO---NNNHHH+++ HHH NN +++ + +++ + +++ + +++ +
N
N
N
r.t.: retention time; ND: Not detectable.
r.t.: retention time; ND: Not detectable.
r.tr.:t :rerteetnetniotino tnimtiem; Ne;DN: NDo:tN doettedcteatbelcet.a ble.
The metabolites of (−)-AM1 ([M + H]+ = 330 at 6.1 min) were detected in all species but with
The metabolites of (−)-AM1 ([M + H]+ = 330 at 6.1 min) were detected in all species but with
ThddeiifffmfeerrTeeehnntaett bamabobeuultinnatdbdeoaasnlnioctceeefs,, (ioin−nf (a)a−-gg)Ar-reAeMeeMmm11ee nn((t[t[ M Mww ii+tt+h hH ttH]hh+ ee]= + mm33=iic0crr 3oao3sts oo06mm.1aaa tmll 6ssitnt.aa1)bb wiimlliiettyiryne dd)daaewtttaae.e. c ArtAee d mmd ianaess tsase l[lc[M Mtsep +de+ c HiHien]s]++ ba==ul l3t3 44sw66p i tieihssc iesbutwith
odbifsfeerrveendt aatb 4u.n7d maninc ea,n idn 5a.5g rmeeinm (ennott wpritehs etnhte i nm hiucrmosaonms, adl asttaa nboilti tsyh odwatna). sAu gmgaesstsi n[Mg t h+e Hpr]+e s=e n3c4e6 oifs
differenaoo tbbhssayeedbrrvvruoeendxdy daaltata t44ne..d77c emmo,rii nnian a apnnarddog 5t5ro..e55n eammmteiinnde ((nNnntoo-ottw xppirrdieetessh eemnntteth tiiaennb hhmouulimmitecaa,r nnofusss,,r odtdhmaaettraaa cnnlooosnttt fssaihhrbmooiwweldinnt ))yb ssyuud ggtahggteeea ssft.triiannAgggm tthhmeeen atpp srmrees/sszee[ nnM=cc e3e 2 +oo9ff H]+ = 346 is
a hydroxylated or a protonated N-oxide metabolite, further confirmed by the fragment m/z = 329
observe(aMd h-1ya7dt)r. 4oA.x7lyl lmtahteei dnf roaagrn mad epn5rtoa.5ttioonmnast iednde t(Nenc-tooetxdi pdaerre em rseeeftenarbtreoidlni tteoh, utfhumer tohacentra shc,oyddnfraiortqmaueinndoo bltiyzsi nthheo ep wforrantgio)mns euonfgt tgmhee/ zsp t=ai rn3eng29t thepresence
(M-17). All the fragmentations detected are referred to the octahydroquinolizine portion of the parent
ofahydc(oMrmo-1px7oy)u. lnAadtlle (tdih.eeo. ,f rrmaag/zmp 1er3no8tt aoatinnodna st1 ed5d2et, eNscete-eod F xaiigrdeu erree mfeSr2er)et. daF brtoao gltmhiteee no,tcf tumahr/zyt hd1re8or2q ucision nroelfiliazrtienmde eptdoo rtbthioyen thohyfed trhfoerx aypglaarmteendet ntm/z=329
compound (i.e., m/z 138 and 152, see Figure S2). Fragment m/z 182 is related to the hydroxylated
occotmahpyodurnodq u(ii.ne.o, limzi/nz e1 3p8o ratniodn ,1 5w2h, iscehe iFnicgluurdee sS 2t)h. eF rmagemtheynlet nmam/z in18o2 girso ruepla. teTdh et of rtahgem heyndt romx/yz la1t9e4d
(M-17).oActlalhtyhderofqruaignomliezinntea tpioorntisond, ewtehcicthe dinaclruedrees fethrer emdetthoytlehneamocintao hgyrodurpo.q Tuhien ofrlaizgmineentp mor/zt io19n4 oftheparent
soecetmahsy drerloaqteudin tool izthinee cphloorrtoioqnu, inwohliinceh iinncclluuddiensg tthhee mmeetthhyylleennaammiinnoo gbrroidugpe. T(Fhieg ufrrea gSm3)e.n Tt omg/ezt h1e9r4,
compotussheeneeesmdme ss( d irr.aeeetlla.aa, tteemmdda /yttooz st1tuhh3gee8g ccehahstlln ootdrrhooaqq1tuu 5iit2nnhoo,ell siinnpeeeoe siisFnnicbcilglluueu ddmriinneeggtS att2bhho)ee.l immtFeee rttcahhogyyullmleednn eaabnmmet iinntmhooe /bb zrrNiidd-1ogg8xee2i d((FieFs iiggoruunerr leeta hSStee33 ))d4.. -TTatoomoggiteenhttohhe-ee7rrh-,, ydroxylated
these data may suggest that the possible metabolite could be the N-oxide on the 4-amino-7-
octahydcthhroleosqreou qdiunaitonalo ilmzinianey.e Fspruaoggrgmteiesont ntt ,mhwa/zt h2tih0c2eh. 7p,i onwschslioubsldee esmtsruetthcatebuormeli tieest nhcooytul eeldlnu acbimed aittnheedo , Ngcar-onox upidproe. bToabnhl yet hbfere a r4ge-famemrerienndot -tm7o- /z194seems
chloroquinoline. Fragment m/z 202.7, whose structure is not elucidated, can probably be referred to
relatedtchthoel octrhholeqourcoihnqouloliinnrooel.qi nFuera ipngomortleiinonnt e m(si/enze c2 Fl0ui2g.d7u,ir new gSh3ot)s.h eTe shtmer ucecottnuhcreyen litesr nantaoiotm ne liounfc oitdhbae trmeidde,t gacabeno( lpFitreiog abuta 5rbe.l5y S mb3ei)n r. e(Thfeyordrgeredotx htyoe r,thesedata
the chloroquinoline portion (see Figure S3). The concentration of the metabolite at 5.5 min (hydroxy
othr eN c-holxoirdoeq)u iidneonlitnifei epdo ritni orna t( saene dF imguorues Se3 )m. Tichroe scoomncee nptrroaftiiloesn wofa tsh eto mo estmabaollli tteo abt e5 .i5d meninti f(ihedyd bryox ay
may suogr gNe-sotxitdhea) tidtehnetifpieods isni brlaet amnde tmabouosleit emiccorousoldmeb eprtohfielesN w-oasx itodoe somnallt htoe b4e- aidmenintifoie-d7 -bcyh lao roquinoline.
MorS /NM-oSx aidnea)ly isdise.n tified in rat and mouse microsome profiles was too small to be identified by a
FragmeMMntSS//mMMT/hSSe z aa Mnn2aa0Sll2y/yMs.s7iisSs,.. wsphecotrsae osf tar umcitnuorr emiestanbootliteel u[Mci +d Hat]e+ d= ,3c44a nat p5.r9o mbainb ilsy shboewrne fienr Friegdurteo St4h aendc hitl oroquinoline
The MS/MS spectra of a minor metabolite [M + H]+ = 344 at 5.9 min is shown in Figure S4 and it
portionis( sceoemThFpeaig tMiubSlre/eM wSSi3 tsh)p. eaTc tfhruaer tohcfeo arn mocxienindoitrzr emadte itpoarnboodoluiftcett .[h MTeh +me Hae]dt+a d=bi t3io4ol4ni t aoet f5 a1.t94 5 ma.m5inu mis ( isanhtoo(mwhiync dimnr oaFsixgsy uuronei rSt)4N tao-n otdxh eiitd e)identified
is compatible with a further oxidized product. The addition of 14 amu (atomic mass unit) to the
inratapnisad rceomnmt opcauantsi bebleem awtitcritirhbo usat oefdmu rttoeh tephrer o ofxofiirdlmeizasetdiwo npa rosofdt aou kocet.ts oTm ghareo lauldptdo. i tbioeni dofe 1n4t iafimeud (batyomaiMc mSa/sMs uSniat)n taol ythsei s.
parent can be attributed to the formation of a keto group.
ThpeaMrenTTSthh /ecea Mppnaa rSbreeenns attp t [[tMeMricb t++ur HHtae]]d+o+ ==tfo 33a 3t3h00me aa fninonddr o mtthhraeemt immoeneett taoaabfbb ooaoll ikittleeeistts oe [[M Mg[rM +o+ u HHp+]].++ H= = 33]44+66 =aann3dd4 [[M4M a ++t HH5]].++9 == m33444i4n wwieesrrees aahllssooow ddenetteeiccntteeddF igureS3and
in theT mheo puasree nptl a[Msm +a Hsa]+m =p 3l3e0s acnodlle tchtee dm aeftatebro olirteasl [tMre a+t mHe]+n =t 3o4f 6C aDn1d m[Mic +e Hw]i+t h= 35404 m wge/rkeg a olsfo ( d−)e-tAecMte1d
itiscom(iiFnnip gtthhaueetr eimmb Sloo5euu).ss wee piptllhaassmmaaaf ussaarmmthppelleerss occoxollilldeeccittzeeeddd aaffptteerrro oodrraaull cttrrtee.aaTttmmheeenntta oodff dCCiDDti11o mmniiccoeef ww1ii4tthha 55m00 mmugg(//akktggo oomff ((i−−c))--mAAMMas11s unit)tothe
(Figure S5).
parent c(Faingubree Sa5t)t. ributedtothefor mationofaketogroup.
Th eparent[M+H]+ =330a ndthemetabolites[M+H]+ =346and[M+H]+ =344werealso

detectedinthemouseplasmasamplescollectedafteroraltreatmentofCD1micewith50mg/kgof
(−)-AM1(FigureS4).
Molecules2017,22,2102 7of15
2.5. P450andhERGInteraction
ThepotentialforinhibitionofthemajorhumanP450isoenzymeswasalsoinvestigated. Racemic
AM1andthetwoenantiomersallshowedasimilarprofile. At3µM,allcompoundscausedstrong
inhibition(>60%)oftheCYP2D6activity,withonlyaslightinhibitionoftheremainingfourisoform
activities (Table 4). It is worth mentioning that CYP2D6 is the only P450 isoenzyme which is also
inhibitedbyCQ(Table4)andconfirmedindrug–druginteractionstudiesinhumans[28].
The4-aminoquinolineclassofmoleculesisassociatedwithpotentialcardiovascularsideeffects.
PreliminaryexperimentsofhERGbindingindicatedthat(±)-AM1and(−)-AM1inhibithERGwith
a Ki in the range of 0.5–0.8 µM, whilst chloroquine in the same experiments had a Ki of 2.2 µM.
Electrophysiologicalfunctionalassayswillberequiredtofurtherexcludethisliability.
Table4.ComparativeP450inhibitionpotentialofracemicAM1,itsenantiomers,andchloroquine.
P450Interaction—GentestKit
CYP1A2 CYP2C9 CYP2C19 CYP2D6 CYP3A4
Compounds
CECa MFC CEC AMMC BFC
Mean%Inhibitionat3µMb
(±)-AM1 <5 5.7±0.2 6.0±1.6 61.4±2.0 11.6±0.4
(+)-AM1 8.3±0.8 11.1±0.5 9.0±1.1 62.2±1.0 14.3±1.9
(−)-AM1 <5 <5 5.0±1.3 74.4±3.5 8.4±0.8
CQ <5 <5 <5 20.4±1.1 6.8±1.7
a Substrates: CEC: 3-Cyano-7-ethoxycoumarin; MFC: 7-Methoxy-4-trifluoromethylcoumarin; AMMC:
3-[2(N,Ndiethyl-N-methylamino)ethyl]-7-methoxy-4-methylcoumarin; BFC: 7-Benzyloxy-4-(trifluoromethyl)-
coumarin.bResultsareexpressedasMean±S.D.,n=2;<5%inhibitionisconsiderednoeffect.
2.6. Pharmacokinetics
Table5reportsthekineticparametersevaluatedafteroraladministration10mg/kgoftheAM1
racemateanditsenantiomerstoCD1mice. Nosignificantdifferencesinmaximalplasmaconcentration
achieved among the racemate or its enantiomers were observed. Cmax was about 0.10 µM for all
compoundsandtheAUC20min·µMafteroraladministrationof10mg/kg.
The(−)-AM1enantiomerwasalsoorallyinjectedtothemiceatthreeincreasingdoses(10,25,
and50mg/kg)anddoseproportionalitywasdisplayed,togetherwithaparallelkineticofelimination
ofthethreetreatments,independentlyfromthegivendose(FigureS5).
Table5.PharmacokineticparametersoftheAM1racemateanditsenantiomers.
(±)-AM1 (+)-AM1 (−)-AM1
Parametersa
10mg/kg
Cmax(µM) 0.0996 0.108 0.084
Cmax(ng/mL) 32.78 35.48 27.60
Tmax(min) 30 120 15
MRTlast(min) 196 162 200
AUClast(min·µM) 19.5 22.9 18.8
AUClast(min·ng/mL) 6421 7551 6179
a Pharmacokinetic parameters were determined by the non-compartmental analysis WinNonlin 5.1, linear
trapezoidal,uniformweight.Thefollowingparameterswerecalculated:Cmax(maximumplasmaconcentration),
Tmax(timetaketoreachCmax),MRT(meanresidencetime),AUC(areaunderthecurve).InvivoPKexperiments
wereperformedwithmaleCD-1miceorally-treatedbyasingledose(10mg/kg)oftheAM1racemateandsingle
enantiomers,eachdissolvedinSSV.Bloodsampleswerecollectedateightselectedtime-points(15,30,and60min;
2,4,6,8,and24h)underethyletheranaesthesia,andplasmasamplesanalyzedbyLC/MS/MS.
Molecules2017,22,2102 8of15
3. Discussion
Historically, the 4-aminoquinolines are a very successful chemical class of antimalarials,
which includes chloroquine and amodiaquine, still clinically used against P. vivax infection or
in combination therapy against P. falciparum. In addition to their direct antiparasitic activity,
theadvantagesofmostofthe4-aminoquinolinesarestability,highbioavailability,favorablePK/PD
propertiesafteroraladministration, andlowcost. Forthesereasons, althoughtheemergenceand
spreadofresistantP.falciparumstrainssignificantlyaffectedtheuseofchloroquineasmonotherapy,
researchonanewgenerationof4-aminoquinolineseffectiveagainstmultiresistantP.falciparumstrains
neverstoppedandnovelcompoundsarepresentlyindevelopment.
This is the case for ferroquine and for AQ-13, in which a ferrocenyl moiety or a short
diethylaminopropyl group replaced the side chain of chloroquine, respectively [16,29,30]. Both
compoundssuccessfullycompletedphaseIstudies[19,20]inhumanvolunteersandinasymptomatic
patients, and are presently under phase II efficacy studies, either alone or in combination (https:
//clinicaltrials.gov/ct2/show/NCT01614964,https://clinicaltrials.gov/ct2/show/NCT02497612).
Inbothcases,theabilitytoovercomeCQresistancehasbeenattributedtothemodificationof
thelateralsidechainofthe4-aminoquinolinering,withtheintroductionoftheferrocenemoietyas
inferroquine,orbyreducingitslengthasinAQ13. Similarly,thecompoundAM1,describedinthis
paper,wasobtainedbytheintroductionofabulky,basic,andlipophilicquinolizidinering(N-lupinyl)
inthelateralchainofa7-chloro-4-aminoquinoline[22],leadingtoacompoundmorelipophilicthan
CQ (AM1 LogD = 2.26 vs. CQ LogD = 0.90) [23]. Differently from the semisynthetic product
7.4 7.4
whichused(−)-lupinineasstartingmaterial,thetotalsynthesisofAM1leadtotheformationofa
racemate,whichwasthentestedforantimalarialactivityandtoxicityinvitrotogetherwiththeisolated
enantiomers. Thetotalsynthesisof(±)-AM1ischeapandstraightforward,andcanbeaccomplished
infourstepsusingcommerciallyavailablematerial[25].
TheAM1enantiomersdisplayedinvitroactivitysimilartotheracemate,whichsupportsfurther
studiesontheinvivoefficacyofthesecompoundsandontheanalysisoftheirpharmacokineticand
ADME/toxproperties.
Here, we report the activity of (±)-AM1 and its enantiomers in the low nanomolar range
documentedagainstlaboratorychloroquine-resistantP.falciparumstrains,aswellasthemultidrug
resistantstrainTM91C235strainfromThailand.IncombinationstudieswithDHA,(−)-AM1displayed
anadditiveeffectsimilartothatobtainedwithCQandDHAincombination. Therefore,theprofileof
thecompoundappearsgoodintermsofpossibleadministrationagainstCQ-resistantstrainsandin
combinationwithartemisinin.
Moreover,(±)-AM1displayedaninhibitoryactivitythree-foldhigherthanchloroquineagainst
tenfieldisolatesofP.vivax,aninfectionthataccountsforapproximately4%ofmalariacasesworldwide,
41% of which occur outside Africa [1]. The WHO’s recommendation for the treatment of P. vivax
infections is chloroquine, in non-chloroquine resistant areas, or ACT. However, in areas where
chloroquine-resistant P. vivax infections have been identified, ACT should be the preferred choice.
AllACTs,excepttheartesunate+sulfadoxine-pyrimethamine(AS+SP)combination,aredemonstrated
tobeeffectiveagainsttheblood-stageinfectionsofP.vivax. Theinvitroandinvivodatareportedfor
(±)-AM1supportitspossibledevelopmentagainstP.vivax,aswell.
TheAM1racemateanditsenantiomerswereorallyactiveatsuitabledosesintheclassicalinvivo
four-day test in mice as schizonticidal agents, with no significant differences in potency among
(±)-AM1 and its enantiomers. They were inactive against the parasite transmission stages in the
P.berghei/A.stephensimodel. Theseresultsareconsistentwithpreviousdataonthisclassofdrugs[31].
However,forother4-aminoquinolinesrecentlysynthesized(TDR58445andTDR58446),areduction
ofactivityinbothearlyandlategametocytesstageshasbeendescribed[32],althoughatarelatively
higherconcentration(1µM),comparedtotheactivityonasexualstages.
IntermsofinvitrometabolicstabilityandinterferencewiththeP450enzymes,racemicAM1and
its enantiomers did not differ significantly from chloroquine, used as reference drug. Similarly,
Molecules2017,22,2102 9of15
the binding assay to hERG indicated that these molecules may share a potential liability for
cardiovascularsideeffectswiththequinolineantimalarials,includingchloroquineandpiperaquine,
currently recommended by WHO for malaria therapy [33]. In the case of future developments,
morestringentmethodsofanalysiswillbenecessary.
PKstudiesinanimalswereperformedbyoralgavageaccordingtothetargetproductprofilefor
anantimalarialdrug. Theneedforacomplexformulation(SSV)wasdictatedbythepoorsolubilityof
thefreebases,whichcouldalsohaveanimpactontotaldrugdisposition.
This information is of relevance in respect to the invivo efficacy data. In fact, all efficacy
experimentshavebeenperformedwiththecompoundspreparedasfreebases,suggestingthatthe
invivodataobtainedsofarcouldunderestimatethepotentialofthetestcompoundasantimalarial.
The introduction of a bulky bicyclic tertiary amine in place of the diethylaminoalkyl chain
typical of CQ and AQ13 changed the metabolic profile, thus N-dealkylation is not the preferred
metabolicpathway. Thisaspectcouldbeimportantbecauseithasbeendemonstratedthatthemajor
CQmetabolites,desethylchloroquine(DEC)and,overall,bisdesethylchloroquine(bisDEC),areless
activethantheparentcompoundonCQ-resistantstrainsofP.falciparum[34]. SimilarlytoCQ,invivo
studiesonAQ13metabolitesshowedthatitsN-deethylationledtospecificchangesinlipophilicity
andincreasedthecross-resistanceofthiscompoundevenmorethanthatofCQ[35]. ThemainAM1
metaboliteseemstobeanN-oxideontheaminomethylenebridge. Intermsofmetabolicstability,
thisdifferencedoesnotincreasethehalf-lifeofAM1comparedtoCQ,butcouldchangetheinvitro
antimalarial profile. It remains to be elucidated how the invivo antimalarial activity of AM1 is
influencedbythesemetabolites.
In conclusion, we have demonstrated that the 4-aminoquinoline compounds remain of high
interestasantimalarialagents,andAM1inparticulardisplaysaveryattractiveprofileintermsof
invitroandinvivoactivity.
4. MaterialsandMethods
4.1. Compounds
RacemicAM1anditsenantiomers(Figure1)weresynthesizedaspreviouslydescribed[25,36].
Chloroquineandalltheotherreagents,unlessindicated,werepurchasedfromSigma(SigmaItalia,
Milan,Italy).
4.2. CulturesofP.falciparumandInVitroChemosensitivityAssays
ThestrainsD10and3D7(CQ-Sensitive),andW-2,FCR1,FCR3(CQ-Resistant),andTM91C235
(multidrug-resistant) of P. falciparum were cultured invitro as described by Trager and Jensen
with minor modifications [37]. Briefly, parasites were maintained at 5% hematocrit (human type
A+redbloodcells)inRPMI1640(EuroClone,Celbio,Pero,Italy)mediumwiththeadditionof10%
heat-inactivatedhumanserum,20mMHepes,and2mMglutamine(EuroClone). Thecultureswere
maintained at 37 ◦C in a standard gas mixture consisting of 1–3% O , 5% CO , and 92–94% N .
2 2 2
Compounds were dissolved in either water or DMSO and then diluted with medium to achieve
the required concentrations (final DMSO concentration <1%, which is non-toxic to the parasite).
Asynchronous cultures of P. falciparum with parasitaemia of 1–1.5% and 1% final hematocrit were
aliquoted into 96-well flat-bottom microplates (COSTAR) with serial dilutions of test compounds,
andincubatedfor72hat37◦C.Parasitegrowthwasdeterminedspectrophotometrically(OD650)by
measuringtheactivityoftheparasitelactatedehydrogenase(pLDH)accordingtoamodifiedversionof
themethodofMakler,incontrolanddrug-treatedcultures[38]. Theantimalarialactivityisexpressed
as50%inhibitoryconcentrations(IC );eachIC valueisthemeanandstandarddeviationofatleast
50 50
threeseparateexperimentsperformedinduplicate[22].
The antimalarial activity of (−)-AM1 was assayed in combination with dihydroartemisinin
(DHA)againstW-2(CQ-R)orD10(CQ-S)strainsofP.falciparumbypotentiationtestsaspreviously
Molecules2017,22,2102 10of15
described[39].CQwasusedasacontrol.Isobologramswereconstructedbyplottingapairoffractional
IC valuesforeachcombinationof(−)-AM1orCQwithDHA[40].
50
Anisobologramclosetothediagonalindicatesanadditiveeffect. Curvessignificantly(>2)above
orbelowthediagonalindicateantagonisticorsynergisticeffects,respectively.
4.3. InVitroAssaysagainstP.vivaxFieldIsolates
TenP.vivaxisolateswerecollectedfromJunetoJuly2010frommalariapatientsattendingthe
ShokloMalariaResearchUnit(SMRU)clinics,MaeSotregionoftheTakProvinceinnorth-western
ThailandundertheethicalguidelinesintheapprovedprotocolOXTREC027-05(CentreforClinical
VaccinologyandTropicalMedicine,UniversityofOxford,Oxford,UK).Thesesampleswerecollected
from patients with no prior antimalarial therapy and with microscopically confirmed P. vivax.
After written consent, blood samples were collected by venepuncture in 5 mL volume lithium
heparinizedtubes,whichwerethentransportedtothelaboratoryatSMRUwithin5hofcollection.
Thetenisolateswerechosenfortestingbecausethemajority(>80%)oftheparasiteswereatthemid
trophozoite stage (around 20 h post invasion) and no schizonts were detected. After platelet and
leukocyteremoval[41],thesusceptibilityoftheseP.vivaxisolatestoAM1,chloroquinediphosphate
(MW 515.9, Sigma-Aldrich®, Milan, Italy), and artesunate (Base MW282.3, Holly Pharmaceuticals
CoLtd.,Tustin,CA,USA)wasassessedaspreviouslydescribed[42,43].
4.4. InVivoEfficacyTestsandTransmission-Blocking/ProphylacticActivities
InvivoantimalarialefficacytestswereperformedusingP.berghei(CQsensitive,CQ-S)ANKA
andP.yoelii(CQresistant,CQ-R)rodentspeciesinthestandardfour-daysuppressivePeters’test[26].
GroupsoffiveCD1micewereinoculatedi.v. with4×106infectederythrocytes/mice. Thecompound,
dissolvedinstandardsuspendingformula(SSV)(0.5%sodiumcarboxymethylcellulose,0.5%benzyl
alcohol,0.4%Tween80,0.9%NaCl),wasadministeredorally,onceadayforaperiodoffourdays,
starting two hours post-infection. Four drug doses were used, ranging from 1 to 30 mg/kg to
determine ED and ED values. Parasitaemia was determined by microscopic examination of
50 90
Giemsa-stainedbloodfilmstakenondayfour,processedusingMICROSOFT®EXCELspreadsheets
(MicrosoftCorp.,Redmond,WA,USA),andexpressedaspercentagesofinhibitionfromthearithmetic
meanparasitaemiasofeachgroupinrelationtotheuntreatedgroup. ED andED valueswere
50 90
calculatedbyGraphPadPrism6(GraphPadSoftwareInc,LaJolla,CA,USA).
(−)-AM1wasalsotestedintransmission-blockingandprophylacticstudiesusingtheP.berghei/
A.stephensimodelasdescribed,withafewmodifications[44].
Forthetransmission-blockingtest,fourBALB/cfemalemice(18–20g)wereinfectedi.p. with
107P.berghei-infectedredbloodcells.Fourdayslater,onceconfirmedforgametocytecarriage,twomice
weretreatedwith50mg/kg(−)-AM1,throughtheoraladministrationof500µLofamethylcellulose
suspension, obtained by dissolving a 20 mg/mL ethanol stock solution in 0.5% methylcellulose
to a final 9% ethanol concentration. Two mice received the vehicle only as controls. One hour
after the treatment, mice were anesthetized and exposed to the bites of 100 sugar-starved female
mosquitoes,at20◦C.Tendaysafterthebloodmeal,samplesofmosquitomidgutsfromeachtreatment
(n=40) were dissected and P. berghei oocysts were manually counted using light microscopy at
400×magnification. Oocystnumberswerelog-transformedandthegeometricmeanswerecompared
usingtheStudent’st-test.
For the prophylactic test, the experimental drugs were administered at 50 mg/kg per os for
4consecutivedays;90minafterthelastadministration,micewereinfectedby6–10infectivemosquito
bites. ThickandthinGiemsa-stainedsmearswerepreparedandexaminedondays5–10post-infection
toassessparasitedensityperµLofblood.
Rearing and handling of experimental animals was in full compliance with the Italian
LegislativeDecreeonthe“protectionofanimalsusedforexperimentalandotherscientificpurposes”
(D.Lgs. 26of03/04/2014)andinfulladherencewiththeEuropeanDirective2010/63/UE.