Exploring Anti-Prion Glyco-Based and Aromatic Scaffolds

46 Pages·2017·9.96 MB·English

molecules
Review
Exploring Anti-Prion Glyco-Based and Aromatic
Scaffolds: A Chemical Strategy for the Quality of Life
MaríaTeresaBlázquez-Sánchez†,AnaM.deMatos† andAméliaP.Rauter*
CentrodeQuímicaeBioquímica,FaculdadedeCiências,UniversidadedeLisboa,EdC8,Piso5,CampoGrande,
1749-016Lisboa,Portugal;[email protected](M.T.B.-S.);[email protected](A.M.d.M.)
* Correspondence:[email protected];Tel.:[phone]or+351-964-408-824
† Bothauthorscontributedequallytothisreview.
AcademicEditor:MargaretA.Brimble
Received:24April2017;Accepted:17May2017;Published:24May2017
Abstract: Priondiseasesarefatalneurodegenerativedisorderscausedbyproteinmisfoldingand
aggregation,affectingthebrainprogressivelyandconsequentlythequalityoflife. Alzheimer’sis
alsoaproteinmisfoldingdisease,causingdementiainover40millionpeopleworldwide. Thereare
notherapeuticsabletocurethesediseases. Cellularprionproteinisahigh-affinitybindingpartnerof
amyloidβ(Aβ)oligomers,themosttoxicspeciesinAlzheimer’spathology. Thesefindingsmotivate
thedevelopmentofnewchemicalsforabetterunderstandingoftheeventsinvolved. Diseasecontrol
is far from being reached by the presently known therapeutics. In this review we describe the
synthesisandmodeofactionofmolecularentitieswithinterventioninpriondiseases’biological
processesand,ifknown,theirroleinAlzheimer’s. Adiversityofstructuresiscovered,basedon
glycans,steroidsandterpenes,heterocycles,polyphenols,mostofthemembodyingaromaticsanda
structuralcomplexity. Thesemoleculesmayberegardedaschemicaltoolstofostertheunderstanding
ofthecomplexmechanismsinvolved,andtoencouragethescientificcommunitytowardsfurther
developmentsforthecureofthesedevastatingdiseases.
Keywords: prion;proteinaggregation;amyloid;anti-prioncompounds;structureoptimization
1. Introduction
Priondiseases,alsocalledtransmissiblespongiformencephalopathies(TSEs),areinfectiousand
fatalneurodegenerativediseases. Theyaffectthebrainandthenervoussystem,causingdeterioration
ofmentalandphysicalabilities,amyloidplaqueformation,neuronaldeath,andproductionofholesin
thecortexthatappears,whenexaminedunderamicroscope,asasponge. Theclinicalsignsinhumans
mayincludememoryproblemsandpersonalitychanges,depression,lackofcoordination,involuntary
movementsorinsomnia.Inthelaterstagesofthedisease,dementiaandlossofabilitytomoveorspeak
arecommon. Priondiseasesarethesingleconformationaldiseasestransmissible,eitherexperimentally
orbynaturalroutes,mainlybyingestion. Prionshaveanextraordinaryresistancetoconventional
sterilizationproceduresandarecapabletobindtometalandplasticsurfaceswithoutlosinginfectivity.
Theyarecausedbyanabnormalconformationalisoform(PrPSc)ofthehost-encodedcellularprion
protein(PrPC)[1]. Full-lengthPrPCisa253aminoacid-longglycoproteinubiquitouslyexpressedin
allmammalsandanchoredtotheexternalcellmembranebyaglycosylphosphatidylinositol(GPI)
moiety [2]. Although there is evidence on its relevant physiological role as a cellular signalling
molecule[3–5],onceinthepresenceofinfectiousPrPScseeds,PrPCunitsareremodelledtoreproduce
theconformationofthemisfoldedisoform. NewlyformedPrPSc moleculesarethenincorporated
intoprionamyloidaggregatesthattendtoaccumulateinneurons,astrogliaandmicroglia,eventually
leading to synaptic damage and vacuolar neuropathology characteristic of TSEs [6]. The exact
mechanismbywhichPrPScaggregatesleadtoneurotoxicityis,nevertheless,poorlyunderstood.
Molecules2017,22,864;doi:10.3390/molecules22060864 www.mdpi.com/journal/molecules
Molecules2017,22,864 2of46
Human TSEs are rare and include familial Creutzfeld-Jacob disease (CJD) caused by
polymorphisms in the PRNP gene giving rise to PrPSc, sporadic CJD, as well as the variant CJD
triggeredbytheconsumptionofbovinePrPSc-contaminatedfood[1,7]. Attemptstodevelopsmall
Molecules 2017, 22, 864 2 of 45
organiccompoundstargetingeitherPrPC,PrPSc,ortheconversionfromoneconformertoanother
havebeenHcaurmriaend ToSuEts ianret hraerep aansdt itnwclouddee fcaamdielisa,l bCuretuetvzfeenldt-hJaocuobg dhisaeafesew (CoJrDa)l lcyauasveadi blayb ploelycommorppohuisnmdss have
beensihno twhen PtoRNsiPg ngiefincea gnitvlyingen rhisaen tcoe PthrPeScs, usrpvoirvaadlico fCmJDi,c easi nwfeelclt easd twhei tvhamriaonut sCeJPDr PtrSigc,gtehreedt rbayn sthpeo sition
ofsuchcoenfsfuemctspttioonh oufm boavninPer PPrSPcSch-caosnbtaemenincahteadll feonogdi n[1g,7a].n Adttleemdpttos troa tdheevreldoips asmppalol ionrtgianngicr ceosmulptsousondfsa r. The
targeting either PrPC, PrPSc, or the conversion from one conformer to another have been carried out
reasonsforthisincludeundesiredtoxicityandmetabolicprofileor,insomecases,reducedcompound
in the past two decades, but even though a few orally available compounds have been shown to
bloodbrainbarrierpermeability[8]. Yet,theexistenceofvariousphenotypicTSEvariantscausedby
significantly enhance the survival of mice infected with mouse PrPSc, the transposition of such effects
severatlod hiuffmeraenn PtrPPrSPc hScass tbreaeinn schisalpleonsgsiinbgl yanthd elemd atojo rrafthacerto driscaopnptoriinbtuintign rgestuolttsh seos efacro. nTsheec ruetaisvoensfa fiolru resin
theseathrcish ifnocrluedfefe uctnidveesitrheedr atopxeiucittiyc aanpdp rmoeatcahbeosli.c EparcohfilPe roPrS, cins tsroaminel ecaasdess,t oreadudciesdti ncoctmdpiosuenasde bilnocoudb ation
periodb,raanind baerarciehr opneremiesaabsilsiotyc i[a8]t.e Ydewt, tihthe eaxipstaerntciceu olfa vrapriaotutse prnheonfoptyrpioicn TdSEis vtrairbiaunttiso cnauinsetdh beyb sreavienra[l1 ,9,10]
and,inddifefeerde,nitt PhraPsScb setreaninssh iosw pnostshibaltyo tnhee pmaarjtoirc ufalcatrorm coolnetcruibluetminga ytoe xtheersted ciostnisnecctuetifvfee cftasilaugreasi ninst tdhief ferent
search for effective therapeutic approaches. Each PrPSc strain leads to a distinct disease incubation
prionstrains[11].
period, and each one is associated with a particular pattern of prion distribution in the brain [1,9,10]
Despitesomanydifficulties,theurgentneedforefficacioustreatmentsagainstpriondiseases
and, indeed, it has been shown that one particular molecule may exert distinct effects against different
keepsthescientificcommunitymotivatedtopursuingthediscoveryofnewanti-prionmoleculesup
prion strains [11].
untiltodayD.eIsnpitthe issop merasnpye dcitfifviceu,lwtiees,h tehree uinrgpenrot vneidede afonr oefvfiecravciioeuws torfeathtmeelnattse satgarienpsot rptrsiofno cduisseiansgeso nthe
develokpeempse nthteo sfcimenotilfeicc ucolemsmwuintihtya mnotit-ivpartieodn toa cptuivrsiutyinag nthde pdoistceonvteirayl oafg naeiwns atnTtiS-pErsio.nF mroomlecusulegs aurp- based
compouunntidl tsodtoaya.r Ionm thaitsi cpearnsdpehcteivteer, owaer ohmereaitnic pcroovmidpeo aunn odvse,rtvhieiswr eovf itehwe lapterests erenptosratsl sfooctuhseinsgy onnth these isand
modedoefvaeclotipomneonft tohfe mmoolesctuplerso mwiitshin agntmi-porlieocnu laacrtivenityti taiensd dpeostcernitbiaeld aignaitnhset lTiSteErsa. tFurroem,u slutigmara-tbealsyeda iming
compounds to aromatic and heteroaromatic compounds, this review presents also the synthesis and
tostimulatethedevelopmentofinnovativetherapiesagainstprion-mediateddiseases.
mode of action of the most promising molecular entities described in the literature, ultimately aiming
to stimulate the development of innovative therapies against prion-mediated diseases.
2. SynthesisandModeofActionofGlyco-BasedandAromaticScaffoldswithanti-PrionEffects
2. Synthesis and Mode of Action of Glyco-Based and Aromatic Scaffolds with anti-Prion Effects
2.1. Glyco-BasedMolecularEntities
Su2.l1f.a Gtelydcop-Boalysesda Mccohlaecruidlaer sE,natsititehse familyofheparanmimetics(HMs)andpentosepolysulfate(PPS)
areamongStuhlfeatmedo sptoalycstaivccehdarriudgess, taess ttehde ifnamexilpy eorfi mheepnatraalnm moidmeeltsicosf (pHrMiosn) danisde apseent(oFsieg uporely1su,slftartue ctures
A–C).(HPPepS)a raaren asmulofnatge t(hHe Sm)wosat sacfotiuven ddroungst hteesatemd yilno iedxppelraiqmueenstailn mToSdEesls[ 1o2f] parniodnr edpisoeratseed (Faisgaunree 1s,s ential
partosfttrhucetucreelsl uAl–aCr)r. eHceeppatroarn ususelfdatef o(HrSp)r wioans fuopuntadk oena tnhed aamsyalocidru pclaiaqlufeas cinto TrSfEosr [1c2e]l lanindf reecptoiortned[ 1a3s ]. Itis
knownant hesastenstuiallf aptairot nof dtheeg creelelualanr drecpeaptttoerr unssedo ffotrh persioenp uopltyaskae cacnhda arsi da ecrsutcriaulc ftauctroers fogro cvelel rinnfetchteionH [S1-3p]. rotein
It is known that sulfation degree and patterns of these polysaccharide structures govern the HS-protein
interactions [14] and are related to their anti-prion acitivity. Other endogenous polysaccharides
interactions [14] and are related to their anti-prion acitivity. Other endogenous polysaccharides
including heparin, chondroitin sulfate (CS) and dermatan sulfate (DS), have also shown to bind
including heparin, chondroitin sulfate (CS) and dermatan sulfate (DS), have also shown to bind
prions[15,16],andtoinhibittheneurotoxicityofamyloidfibrils[17].
prions [15,16], and to inhibit the neurotoxicity of amyloid fibrils [17].

Figure 1. (A): Heparan mimetic representative fragment, abbreviated as HMCSX where n: molecular
Figure 1. (A): Heparan mimetic representative fragment, abbreviated as HMCSX where n:
length, S: degree of sulfate substitution, C: carboxylate substitution and X: hydrophobic group substituent;
molecularlength,S:degreeofsulfatesubstitution,C:carboxylatesubstitutionandX:hydrophobic
(B): Pentosan polysulfate representative fragment; (C): Heparan sulfate/heparin representative fragment.
groupsubstituent;(B):Pentosanpolysulfaterepresentativefragment;(C):Heparansulfate/heparin
represBenastaetdiv oenf rtahgemsee nfitn.dings, Ouidja and co-workers [18] reported a library of HMs of different
molecular sizes, containing various sulfation and carboxylation levels, and substituted, or not, by
Bdaisfefedreonnt htyhdersoephfionbdici cnogrse,s wOiuthid aj ageannerdal cfoor-mwuolrak HerMsC[S1X8.] (rFeigpuorret 1e,d starulcitburrae rAy).o SftuHdiMess ono fthdeiirf ferent
capacity to inhibit the accumulation of the abnormal protease-resistant prion conformation (PrPres) in
molecular sizes, containing various sulfation and carboxylation levels, and substituted, or not, by
chronically infected cells (ScGT1-7) and on their PrPrec binding ability showed that an optimal size and
differenthydrophobiccoreswithageneralformulaHMCSX.(Figure1,structureA).Studiesontheir
sulfation degree were needed for optimum activity. The incorporation of hydrophobic moieties
capacitytoinhibittheaccumulationoftheabnormalprotease-resistantprionconformation(PrPres)
increased compound efficacy while the presence of carboxymethyl moieties decreased it. It is important
in chronically infected cells (ScGT1-7) and on their PrPrec binding ability showed that an optimal
Molecules2017,22,864 3of46
size and sulfation degree were needed for optimum activity. The incorporation of hydrophobic
moieties increased compound efficacy while the presence of carboxymethyl moieties decreased it.
ItisimportanttoaddthatPrPres isadisease-promotingPrPSc-likeprionproteingeneratedinvivo,
followMinolgecuPlersP 20S1c7-,i 2n2d, 8u64c edmisfoldingofrecombinantPrPC[19]. 3 of 45
Ouidjaetal.[18]alsoobservedthatincreasingpolyanionssizeincreasesproductefficacy. When
to add that PrPres is a disease-promoting PrPSc-like prion protein generated in vivo, following
consideringthesamedegreeofsulfatesubstitution,amaximumeffectwasfoundat250gly/chain
PrPSc-induced misfolding of recombinant PrPC [19].
incellassays,withthepartiallysulfatedHM S (EC =0.01µMinScGT1-7cells). Theproduct
Ouidja et al. [18] also observed that incre2a5s0in1g. 0polya5n0ions size increases product efficacy. When
substitcuontesdidewriinthg tahep hsaemney ldaelgarneien oef msuelftahtye lsuesbtsetirtu(Xtio=n,P ah mMaexi)mwuimth effofercmt wualas fHouMn2d5 0aCt 205.50S g0l.5yX/c0h.3aiinn icnr eased
theabicleitlly atsosainyhs,i bwititPhr Pthrees paacrctuiamllyu lsautlifoanteidn HScMG2T501S-1.70 c(EeClls50 c=o m0.0p1a rμeΜd wini tShcGitsT1n-o7n c-ehlylsd).r oTphhe opbriozdeudcta nalog
(EC50=su0b.s6titvust.ed> 1w0itµhM a p).hTenhyelamlaonrienev moleuthmyiln eostuesr (aXn =d PahrMome) awtiicthP fhorMmeuldae HriMva25t0iCv0e.5Sw0.5aXs0.3t hinecroenaseepd rtehsee nting
ability to inhibit PrPres accumulation in ScGT1-7 cells compared with its non-hydrophobized analog
thebesteffect,suggestingthatitoffersahydrophobicbindingpointtotheC-terminalfragmentofthe
PrPC,(rEicCh50i =n 0h.6y vdsr.o >p10h oμbΜic). rTehseid muoerse. voluminous and aromatic PhMe derivative was the one presenting
the best effect, suggesting that it offers a hydrophobic binding point to the C-terminal fragment of
On the other hand heparin analogs may be structurally based on saccharide units whose
the PrPC, rich in hydrophobic residues.
disaccharideprecursorGlcNS6S-IdoA2S1ispresentedinScheme1. Inanattempttoknowwhether
On the other hand heparin analogs may be structurally based on saccharide units whose
thisdidsaiscacchchaarirdidee upnreictuhrasosrs GulfcfiNcSie6nS-tIadnoAti-2pSr 1i oisn parecsteivnitteyd iinn Spcrhieomn-ei n1.f Ienc taend atcteemllsp,tr teoc kennotlwy, wTheeruthyear tehtisa l.[20]
havecdhiasarcacchtearriidzee dunthit ehbasin sduifnfigciepnrto afinltei-porfiothne arcetipvriteys einn tpartiiovne-isntfreuctcetdu rceelolsf, urencpenrtolyte, cTteerduyGal ceNt aSl.6 S[2-0I]d oA2S
1 to PhrPa.veT chhearsayctnetrhizeesdi sthoe fbidnidsiancgc phraorfiilde eof1 thhea rsepbreeseenntdateivsec rsitbruecdtubrey oSf auintporoatnecdtecdo G-wlcNorSk6Se-rIsdo[A212]S using
glucur1o ntoo -P6r,P3.- lTahcteo snyenathnedsisg loufc doissaamcchinaeridhey d1 rhoacsh lboereind edaesscsritbaerdti nbgy mSaaittoe raianlds ctoo-owbotrakienrst h[2e1d] iussaicncgh aride
glucurono-6,3-lactone and glucosamine hydrochloride as starting materials to obtain the disaccharide
buildingblockbyreactionofthesugaracceptor2withtheconvenientlyprotected2-azidoglycosyl
building block by reaction of the sugar acceptor 2 with the conveniently protected 2-azido glycosyl
donor 3 catalysed by tert-butyldimethylsilyl trifluoromethanosulfonate (TBDMSOTf). Acceptor 2
donor 3 catalysed by tert-butyldimethylsilyl trifluoromethanosulfonate (TBDMSOTf). Acceptor 2 and
and ddoonnoorr 33 wweerer esysnythnetshizeesdiz oevder o1v5 estrep1s5 frsotmep Ds-gflruocmuroDn-og-6l,u3-cluacrtoonneo, -a6n,d3 -olavecrt o9 nstee,pas nfrdomo gvleurco9sasmteinpes from
glucoshaymdrinocehhloyrdidreo, crheslopericdtiev,elrye s(Spcehcetmivee l1y). (Scheme1).

SchemSech1e.mSey n1.t hSyesnitsheosfisd oisf adcicsahcacrhiadreid1e. 1R. eRaegaegnentstsa anndd ccoonnddiittiioonnss: :(a()a T)BTDBMDSMOSTOf, TDfC,DMC, MMS, 4M ÅS, 4−2Å0 ,°C− 20◦C
→r.t.,→18 r.ht.,, 7187 %h,. 77%.
Teruya et al. described a significant binding of unprotected disaccharide GlcNS6S-IdoA2S 1 with
Terercuoymabeintaanl.t dperisocnr ipbreodteains (iPgrnPi)fi (cKaDn =t b9.i7n dμiMn)g, tohfaut nwparso ctoencftierdmeddis bayc cchoamrpideteitGivlec NinhSi6bSit-iIodno uAs2inSg1 with
recomhbeipnaarnint oprr pioenntposraontee ipnol(yPsruPlf)a(tKe by =su9r.f7acµe Mpla),smthoant rwesaosncaoncnefi. Hrmowedevbeyr, cthoem dpiseatcicthivareidine hstirbuicttiuorne using
D
heparidnido rnopte enxthoisbaitn aenpti-oplryisoun lafacttievibtyy isnu prrfiaocne-ipnlfaecstmedo cnelrles.s o nance. However,thedisaccharidestructure
didnotexhGilbyictosaanmtii-nporgiloyncaancs ti(vGiAtyGisn) parreio nbi-oinmfoelcetceudlesc eollfs .interest for prion-related diseases playing
important roles in prion formation and infection [22]. GAG involvement in prion disease pathogenesis
Glycosaminoglycans (GAGs) are biomolecules of interest for prion-related diseases playing
and PrPC conversion into PrPSc has been widely reported [15,23–25]. Yagamuchi et al. [26] described,
importantrolesinprionformationandinfection[22]. GAGinvolvementinpriondiseasepathogenesis
for the first time, polymers of acrylamide decorated, via an amide bond, with regioselectively sulfated
andPrPCconversionintoPrPSchasbeenwidelyreported[15,23–25]. Yagamuchietal.[26]described,
2-acetamido-2-deoxyglycopyranosyloxyphenyl moieties (Figure 2), and tested them, as well as the
forthep-finristrtotpimheen,ypl osluylfmateerds golfycaocsriydlea mpriedcuerdsoercso mraitmedic,kviniag aGnAaGms imdoenboomnedrs,,w toit shcrreeegni ofoser ltehcetiirv aeblyilistyu lfated
2-acetatom inidhiob-i2t -PdrePoScx foyrgmlyactioopn yinr apnrioosny-ilnofxeyctpehd ecnelylsl. moieties(Figure2),andtestedthem,aswellasthe
p-nitropheAnymlosnugl ftahtee dsuglflaytecdo sgidlyecoppryercaunrossoidressm animd itchkei npgolyGmAeGrics cmomonpoomunedrss ,etxoamscinreeedn, tfhoer 4t-hsuelifraatebdi lityto
inhibitdPerriPvaStcivfoe r6m, aantdi otwnoin glpycroiopno-lyinmfeercst,e 1d0 caneldl s1.1 (Figure 2), inhibited PrPSc formation with 50% effective
Admosoens gbetlhoews 2u0l fμagt/emdLg (lEyDco50p =y 1r0a, n4o asnidd e9s μagn/mdLt,h reesppoelcytimveelyri)c. Aco cmompboiunantdiosne oxfa amn iNn-eadce,ttyhl egr4o-uspu lfated
at C-2 and a sulfate group at either C-4 or C-6 on glucopyranoside might be involved in the inhibition
derivative6,andtwoglycopolymers,10and11(Figure2),inhibitedPrPScformationwith50%effective
of PrPSc formation. Furthermore, PrPSc formation was inhibited by polymeric compound 11 but not
dosesbelow20µg/mL(ED =10,4and9µg/mL,respectively). AcombinationofanN-acetylgroup
by the glucoside 7, sugg5e0sting the importance of a polyvalent structure.
Molecules2017,22,864 4of46
atC-2andasulfategroupateitherC-4orC-6onglucopyranosidemightbeinvolvedintheinhibition
ofPrPScformation. Furthermore,PrPScformationwasinhibitedbypolymericcompound11butnot
bytheglucoside7,suggestingtheimportanceofapolyvalentstructure.
Molecules 2017, 22, 864 4 of 45
Molecules 2017, 22, 864 4 of 45
OH OH
HOHO OHO O -OH3OSO OOH O
HOHO ONHAOc NO2 -OH3OSO NOHAcO NO2
NH4Ac NO2 5NHAc NO2
O4H OSO53-
-O-3OS3OHSOOHO OHOONHAOcO NO2 HHOHOHOO OONSOHOA3c-OO NO2
N6HAc NO2 7NHAc NO2
OS6O3- OH OSO73-
HO O O
HOHO OSOOO3H- O NO2 HOOH OOSOHO3-O NO2
HO 8 O HO 9 O
OH NO2 OH NO2
8 9
OH OSO3-
-O3SOHO O O HOHO O O
-O3S-OHOO3SOHOOHOHONHOAcHOONHNAHcAOc NHCONNHHCCOO HOHO HOHOOOSONSOHO3A-3c-OONSONHOHA3cA-cO NHCONHNCHOCO
-O3SOHO O O10 HOHO O O11
NHAc NHCO NHAc NHCO
Figure2F.igCuroem 2.p Cooumnpdosutnedsst etedstf1e0odr fothr ethien hinihbiibtiitoionn ooff PPrrPPSSc cfofromrmatiaot1ni1 oinn pinriopnr-iionfne-citnedfe ccetlelsd: scuellflast:eds u lfated
p-nitrophenyl glycopyranosides, and glycopolymers composed by a polyacrylamide cha in decorated
p-nitrophenylglycopyranosides,andglycopolymerscomposedbyapolyacrylamidechaindecorated
Figwuirteh 2su. lCfaotemdp 2o-aucnedtasm tiedsote-2d- dfeoorx tyh-eβ -Din-hgliubcitoipoynr aonf oPsyrPloSxc yfporhmenaytli omno iient ipesr ivoina -ainmfiedcete bdo ncdel (las:d asputlefadt ed
withsulffraotme d[226]-)a. cetamido-2-deoxy-β-D-glucopyranosyloxyphenylmoietiesviaamidebond(adapted
p-nitrophenyl glycopyranosides, and glycopolymers composed by a polyacrylamide chain decorated
from[26]).
with sulfated 2-acetamido-2-deoxy-β-D-glucopyranosyloxyphenyl moieties via amide bond (adapted
The synthesis of the p-nitrophenyl glycosides sulfated at 3, 4 or 6 position is depicted in Scheme 2
from [26]).
[27]. Position 6 of pNP-β-D-GlcNAc was selectively sulfated with a sulfur trioxide complex (Me3N·SO3)
The synthesis of the p-nitrophenyl glycosides sulfated at 3, 4 or 6 position is depicted in
affording compound 7 in 52% yield. In order to generate sulfated glucosides at position 3 and 4,
Schemete2rTt[-h2be7u ]ts.yylPndotihpsehisteiinso ynolfs 6itlhyoelf cpph-NnloitPrrio-dβpe -h(DTen-BGyDll PcgSNlCyAcl)o cswiwdaesa scs shsuoelsfleaentce ttdiov afeti lr3ys,t l4syu oplrfr a6ot tpeeocdts iwptiooistniht iiosa nds eu6p.l ifAcutfertedtr r iisnou Sxlfciadhtieeomnc eo m2 plex
(Me N[27·wS].O iPtho )ssiuatilfoffunor r6 dt roiionf xpgiNdcePo -cmβo-mpDop-Gluelnxc NdboA7thci nw5 aa5ns2 ds%e 6le yrceitegivlidoeil.syoI nsmueolfrrasd tweeder rwteo iotghbte aan isneuerldafu tienr ts6ru3io%lxf aiodtveee drcaoglmll yupiceleoldxs i(adMnedes 3tNhaet·SnpO o3)s ition
3 3
affording compound 7 in 52% yield. In order to generate sulfated glucosides at position 3 and 4,
3 and 4s,etpearrta-bteudt byyld coipluhmenn ychlsroilmylatcohglroaprihdye. (TBDPSCl) was chosen to firstly protect position 6. After
tert-butyldiphenylsilyl chloride (TBDPSCl) was chosen to firstly protect position 6. After sulfation
sulfationwithsulfurtrioxidecomplexboth5and6regioisomerswereobtainedin63%overallyield
with sulfur trioxide complex both 5 and 6 regioisomers were obtained in 63% overall yield and then
andtsheepnarsaetpeda rbayt ecdolubmynco clhurmomnactohgroramphayto. graphy.

Scheme 2. Synthesis of compounds 5–7. Reagents and Conditions: (a) Me3N·SO3, DMF, 40 °C, 3 h,
Dowex (Na+), 52%; (b) TBDPSCl, py, DMAP, r.t., 24 h, 95%; (c) Me3N·SO3, DMF, 40 °C, 10 h, Dowex
(Na+), 63% (5/6 = 1.4/1); (d) TBAF, THF, r.t., 24 h, 98%.

SchemScehe2m. eS y2.n Sthyensthisesoisf coof mcopmopuonudnsds5 –57–.7. RReeaaggeennttss aanndd CCoonndidtiitoinosn: s(:a)( aM)eM3Ne·SNO3·,S ODM,FD, M40 F°,C4, 03 ◦hC, ,3h,
3 3
DoweDxow(Nexa +(N),a5+2),% 52;%(b; )(bT)B TDBPDSPCSCl,lp, py,yD, DMMAAPP,, rr.t.t..,, 2244 hh,, 9955%%; ;((cc) )MMe3eNN·SO·S3O, DM,DFM, 4F0, °4C0, 1◦0C h,,1 D0ohw,Dexo wex
3 3
(Na+), 63% (5/6 = 1.4/1); (d) TBAF, THF, r.t., 24 h, 98%.
(Na+),63%(5/6=1.4/1);(d)TBAF,THF,r.t.,24h,98%.
Molecules2017,22,864 5of46
Molecules 2017, 22, 864 5 of 45
MorerecentlyNishizawaetal. introducedthep-methoxyphenylaminoglucoside13(Figure3)
Molecules 2017, 22, 864 5 of 45
More recently Nishizawa et al. introduced the p-methoxyphenyl aminoglucoside 13 (Figure 3)
asanewtypeofanti-prioncompound,commerciallyavailable,andabletoinhibitabnormalprion
as a new type of anti-prion compound, commercially available, and able to inhibit abnormal prion
proteinfoMrmoraet iroenceinntlpy rNioinsh-iinzafewcat eedt anle. uinrtorobdlauscteodm thaec epl-lmseitnhoaxpyrpihoennsytlr aamini-niongdleupcoesniddee n1t3 m(Faignunreer ,3)w hen
protein formation in prion-infected neuroblastoma cells in a prion strain-independent manner, when
thecealsl sa wneewre tytrpeea toefd anfotir-pmrioonre cothmapnou1ndda, yc.oTmhmee5rc0i%allyin ahviabiiltaibolne, danodse afboler tPor Pinrhesibfiot rambnaotriomnali spr5i.o3n6 and
the cells were treated for more than 1 day. The 50% inhibition dose for PrPres formation is 5.36 and
protein formation in prion-infected neuroblastoma cells in a prion strain-independent manner, when
3.33(3µ.3g3/ (mμgL/)minL)p inri opnri-oinn-fiencfteecdtedS cSNcN2a2ac ceellllss aanndd NN116677 cceelllsl,s ,rersepsepcetcivtievlye l[y28[]2. 8].
the cells were treated for more than 1 day. The 50% inhibition dose for PrPres formation is 5.36 and
3.33 (μg/mL) in prion-infected ScN2a cells and N167 cells, respectively [28].

FigFuigreur3e. 3M. Moloelceuculalarrs sttrruuccttuurree ooff tthhee aammininog olgulcuocsoidseid 1e3.1 3.
Figure 3. Molecular structure of the aminoglucoside 13.
In 2009, Charvériat and co-workers presented two different families of prion replication
In 2009, Charvériat and co-workers presented two different families of prion replication
inhibitors [29]. One of them is based on 3-aminosteroid structures and the other on erythromycin A
In 2009, Charvériat and co-workers presented two different families of prion replication
inhibditeorrivsa[t2iv9e].s Obenaerinogf tshacecmhairsidbea rseesdidounes3 a-nadm ainn oosxtiemreo ifdunscttriuonctaulirtye s(Faingdurteh 4e).o Tthhee erioghnt ecroymthprooumndysc inA
inhibitors [29]. One of them is based on 3-aminosteroid structures and the other on erythromycin A
derivianthivibeistebde ParrPinregs ascaccucmhualraitdioenr eins itdwuoe csealln cdulatunreosx (iSmNe5f6u anncdt iGonT1a lcietyll (liFniegsu) rinef4e)c.teTdh beye tihgeh Cthcoanmdpleoru nds
derivatives bearing saccharide residues and an oxime functionality (Figure 4). The eight compounds
inhibsitcerdapPier Pstrreasina,c acnudm buyl amtioounsei-nadtwapotecde lslccraupltieu rsetrsai(nS N225 L6, arnesdpeGcTtiv1eclyel. l lines)infectedbytheChandler
inhibited PrPres accumulation in two cell cultures (SN56 and GT1 cell lines) infected by the Chandler
scrapiestrain,andbymouse-adaptedscrapiestrain22L,respectively.
scrapie strain, and by mouse-adapted scrapie strain 22 L, respectively.

Figure 4. Chemical structure of inhibitors belonging to distinct chemical families: 3-aminosteroids 14–20
and erythromycin A derivative 21 [29].
Figure 4. Chemical structure of inhibitors belonging to distinct chemical families: 3-aminosteroids 14–20
Figure4. Chemicalstructureofinhibitorsbelongingtodistinctchemicalfamilies: 3-aminosteroids
and erythromycin A derivative 21 [29].
14–20Daonsde–erreystphoronmsey ccinurAvedse, riwvaitthiv eth21e [2id9]e.ntification of IC50 by densitometry, confirmed the
concentration-dependent PrPres inhibitory activity of compounds 14–21 on Chandler-infected SN56
Dose–response curves, with the identification of IC50 by densitometry, confirmed the
Dccoeolnlssce e[–n2rt9er]as. tpCiooonnm-sdpeeopuecnnuddr ve1ne7ts a,PnrdPw reeistr hiynthhtirhboiemtoyriycdi nea ncdttieivfiriitcvyaa totiifvo ecno 2m1o pfsohuoICnwd5e0sd 1 bt4h–ye2 1ldo owenne ssCtih tIoaCmn5d0 el(et0rr.y4-i, nafnecdcot ne0d.fi3 r SμmNMe5)d6, the
respectively. Interestingly, they found that compound 21 interacted directly with PrPres stability
concecneltlrsa [t2io9]n. -Cdoempepnodunedn t1P7 raPnrdes eirnyhthibroitmoryycinac dtievriivtyatoivfec 2o1m sphoowunedd sth1e4 –lo2w1eosnt ICCh50a (n0.d4l eanr-din 0f.e3c μteMd),S N56
cellsr[o2ebs9sp]e.ercvCtiinovgem layp .c ohIunatnnegdree s1itn7in taghnleyd s, hetahrpeyyet hofrfo outhmned yT ctmih ncautd rcveoermi ivnpa otthiuven efdlu 221o1rs eihsncoteewnrcaeecd-tbetdahs eeddilr otehwcetlreyms twaIli Ctshh ifPt(r 0aPs.r4sesa aysnt. adTbhi0lei.st3ye µM),
results indicated that the interaction between PrPC and compound 21 leads to a peculi5a0r conformation
respeocbtisverevlyin.gI an ctehraensgtein ing ltyh,et shheaypef oouf nthde Tthma cturcvoem inp othuen fdluo2r1esicnetnecrea-cbtaesdedd thireercmtlayl swhiiftth asPsraPy.r eTshsetsaeb ility
of PrPC that could induce alteration of its amyloidogenic properties, and consequently lead to the
results indicated that the interaction between PrPC and compound 21 leads to a peculiar conformation
obserivnihnibgitaiocnh oafn cgoenivnertshioens htoa PprePoresf [t2h9e]. Tmcurveinthefluorescence-basedthermalshiftassay. These
of PrPC that could induce alteration of its amyloidogenic properties, and consequently lead to the
resultsindMicoartee rdectehnattlyth oethienrt enraatuctriaol nglbyectowsiedeens wPirtPhC anatni-dprcioomn apcotiuvnitdy h2a1vlee abdeesnt odeascpreibceudli ianr lcitoenraftourrme,a tion
inhibition of conversion to PrPres [29].
ofPrPnaCmtehlayt bcaocousliddei-nAd (u2c2e, Faigltuerrea 5ti)o. Tnhoisf nitastuarmal ytrlioteidrpoegneen gilcycporsoidpee brteiaersin,ga na dtricsoacncsheaqruideen mtloyielteya hdatso the
More recently other natural glycosides with anti-prion activity have been described in literature,
inhibbiteieonn roefcecnotnlyv feorusinodn toto ePxhrPibriets a[n2t9i-]a.myloid properties [30]. It is an active principle of the medicinal
namely bacoside-A (22, Figure 5). This natural triterpene glycoside bearing a trisaccharide moiety has
plant Bacopa monniera, whose components include amphiphilic compounds structurally based on
Mbeoenre rreececnentltyl yfooutnhde rton eaxthuirbailt ganlytic-oamsidyleosidw pirthopaenrttiie-sp r[3io0]n. Iatc itsi vaint yachtiavvee pbrienecnipdlee socf rtihbee mdeindilciitnearla ture,
sterol glycosides, and is used in traditional Indian medicine to treat various nervous disorders, and
nameplylanbta cBoasciodpea -mAo(n2n2ie,rFai, gwuhreos5e) .cTomhipsonnaetnutrsa ilntcrliutedrep aemnephgilpyhciolisci dceombepaoruinngdsa sttrriuscatcucrhaallryi dbeasmedo ioenty has
to promote memory enhancement [31,32].
beensrteecreonl gtllyycfoosuidneds, taonedx ihs iubsietda nint it-raamdiytilooniadl pInrdoipaenr mtieesdi[c3in0e]. tIot tirseaatn vaacrtioivues pnerrinvociupsl deiosofrtdheersm, aenddi cinal
planttoB pacroopmaomte omnenmieorary, wenhhoasnececmoemntp [o3n1,e3n2]t.s include amphiphilic compounds structurally based on
sterolglycosides,andisusedintraditionalIndianmedicinetotreatvariousnervousdisorders,andto

promotememoryenhancement[31,32].
Molecules2017,22,864 6of46
Molecules 2017, 22, 864 6 of 45
HO H
Molecules 2017, 22, 864 6 of 45
H C
3
HO H
CH CHH C
3 3 3
O
OH CH CH
3 3
O
O
HO O
OHO O OHO H3C CH3
O
O O
HOOH
HOHOOHO O OH O H3C CH223
HOH C O
OH 2 OHOH 22
HOHO
Figure 5. Molecular structure of bacoside A (22).
HFOigHureC5.MolecularstructureofbacosideA(22).
2 OH
Recent studies have suggested that bacoside-A might exhibit therapeutic effects against amyloid
Figure 5. Molecular structure of bacoside A (22).
Rdiesceeansetss, tsuudchie ass hAalvzheesimugegr’es sdteisdeatshea (tAbDa)c [o3s3i,d34e]-.A Mmorieg rhetceenxthlyi bMitatlhisehreavp eetu atli. chaevffee icntvseasgtiagiantesdt athmey loid
diseaisnetse,rascutciohnas sbAetlwzeheenim baecro’ssiddeis-eAa saend(A thDe) 2[13-3r,e3s4id].uMe aomreylroeicdeongtelnyicM daeltiesrhmeivnaentta ol.f hthaev epriinovne pstriogtaeitne dthe
Recent studies have suggested that bacoside-A might exhibit therapeutic effects against amyloid
interaPdcritPsie1o0a6n–s1se26s b,b esyut wcshpe eaecsnt Arbolsazcchooepismyid eaern’-sdA d maisniecadrsoetsh c(AeopD2y1) -[er3xe3ps,3eid4ri]um. eMenaotmrse y[r3el5oc,ei3dn6ot]l.yg T eMhneaiycli sdehveeatvel ureamt taeildn. hatanhvtee o cifonntvhseeesqtipugreaintoecnde tpohrfe o tein
PrP10b6ina–c1te2or6saibdctyeio-Asnps/P ebcrePttr(wo10se6ce–on1 pb2ya6c)a oinsnidtdeemr-aAcict iaronondssc touhppe yo2n1e -xmrepseeimdriubmera eannmet ysbli[ol3iad5yo,e3gr6se] n.(iFcTi ghdueertyeer em6v)i.an lTaunhate t oeefdx tphteher eipmcrioeonnnts apel qrdoutaeetiann c eof
bacosrPeidvrPeea1-0Al6e–1d/26 a PbcrcyPe l(se1pr0ae6tcit–or1no 2soc6fo )PpirynP 1ta0e6n–r1da26 c fmtibiiorcinrl ofsoscuromppaoytni oenxm pinee mrtihmbe erpnartnesse e[n3bc5ie,l3 ao6yf] .el irpTsihde( Fbyii lgeavuyaerlreus.a6 It)me.dpT othhrteea nectoxlynp,s etehrqieumye fneocnuetn aodlf data
that the enhanced fibrillation of the peptide, induced by bacoside-A, went in parallel with significant
revealbeadcoascidcee-lAer/aPtriPo(n10o6f–P12r6P) interacfitibornisl fuopromna mtioenmibnratnhee bpirlaeyseernsc (eFoigfulripe i6d).b Tilhaey eerxsp.eIrmimpeonrttaal ndtalyta, they
reduced membrane intera1c0ti6o–n12s6 and bilayer disruption, demonstrating a direct relationship between
foundretvheaatletdh aecceenlehraatniocne dof fiPrbPr1i0l6–l1a26t ifoibnrilo ffortmheatipoenp inti tdhee, pirnedseunccee dof bliypidb abciloayseidrse. -IAm,powrteannttlyin, thpeayr faolulenldw ith
externally-induced accelerated fibrillation and inhibition of membrane interactions (Figure 6), indicating
that the enhanced fibrillation of the peptide, induced by bacoside-A, went in parallel with significant
significantreducedmembraneinteractionsandbilayerdisruption,demonstratingadirectrelationship
bacoside-A (22) as a potential therapeutic agent for TSE and amyloid diseases.
reduced membrane interactions and bilayer disruption, demonstrating a direct relationship between
betweenexternally-inducedacceleratedfibrillationandinhibitionofmembraneinteractions(Figure6),
externally-induced accelerated fibrillation and inhibition of membrane interactions (Figure 6), indicating
indicatingbacoside-A(22)asapotentialtherapeuticagentforTSEandamyloiddiseases.
bacoside-A (22) as a potential therapeutic agent for TSE and amyloid diseases.

Figure 6. Schematic model for the activity of Bacoside-A; up: PrP106–126 alone forms abundant pre-fibril
aggregates which interact with, and disrupt membrane bilayers; down: when PrP106–126 is pre-i ncubated
with bacoside-A, fibrillation is accelerated resulting in lower abundance of membrane-active species
Figur(eFadig6au.prteeS d6c. h fSreocmhmea m[t3ia0ct]i)cm. mooddeell ffoorr tthhee acatcivtiitvyi toyf BoafcoBsaidceo-sAid; uep-A: P;ruP1p06:–12P6 raPlo1n0e6 –f1o2r6mas laobnuendfoanrmt psrea-fbiburnild ant
aggregates which interact with, and disrupt membrane bilayers; down: when PrP106–126 is pre-incubated
pre-fibrilaggregateswhichinteractwith, anddisruptmembranebilayers; down: whenPrP
with bacoside-A, fibrillation is accelerated resulting in lower abundance of membrane-active specie10s 6–126
i2s.2p. Arer-oimncautibc aStceadffowldist hwitbha Ncoitsriodgee-nA C,ofinbtariinlliantgio Snix-iMs eamccbeelreerda Rteindg rHeestuelrtoicnygcleisn lower abundance of
(adapted from [30]).
membrane-activespecies(adaptedfrom[30]).
2.2.1. Pyridine Dicarbonitrile Derivatives
2.2. Aromatic Scaffolds with Nitrogen Containing Six-Membered Ring Heterocycles
2.2. AromPatyircidSicnaeff odlidcsarwbiotnhitNriilter odgeernivCatoivnetas inwienrge Sfiirxs-tMly epmubbelrisehdeRdi ning 2H0e0t0e raosc yac nleesw class of rationally
d2e.2si.1g.n Peydr ildeaindes Dwicitahr baocntiivtritiyle aDgeariinvsatt iPvrePsS c replication in PrPSc-infected mouse neuroblastoma cells
2.2.1.(SPcyNr2ida)i n[3e7D]. iIcna prbaortniciutrlialre cDomerpivouatnidv e2s3 (Figure 7) was reported to inhibit PrPSc formation in ScN2a
Pyridine dicarbonitrile derivatives were firstly published in 2000 as a new class of rationally
P dyersiidgninede dleiacdasr bwointhit ariclteivditeyr aivgaatinivste sPrwPSec rreepfilricsatltyionp uinb lPisrPhSec-dinifnec2te0d0 0maosusae nneewurocblalassstoomfar acteilolsn ally
design(SecdN2leaa) d[3s7]w. Iinth paarcttiicvuiltayr acogmaipnosutnPdr P23S c(Friegpulriec a7t)i ownasi nrePporPrtSecd- itnof einchteibdit mProPuScs feornmeuatrioonb lians StocNm2aa cells
(ScN2 a)[37]. Inparticularcompound23(Figure7)wasreportedtoinhibitPrPScformationinScN2a
Molecules2017,22,864 7of46
Molecules 2017, 22, 864 7 of 45
cellsinadose-dependentmanner,withanIC of18.0µMinScN2acells,andlowlevelsoftoxicity[37].
Molecules 2017, 22, 864 50 7 of 45
cells in a dose-dependent manner, with an IC50 of 18.0 μM in ScN2a cells, and low levels of toxicity [37].
IthasbeenproposedthatitmaybindtoachaperonerequireditselftobindtoPrPforitsconversion
It has been proposed that it may bind to a chaperone required itself to bind to PrP for its conversion
intoPcerPllsS cin, cao dnosseeq-dueepnetnlydeinnth mibaintinnegr, twhiitshc aonn IvCe50r osifo 1n8.0[3 μ7M,3 8in]. ScN2a cells, and low levels of toxicity [37].
into PrPSc, consequently inhibiting this conversion [37,38].
It has been proposed that it may bind to a chaperone required itself to bind to PrP for its conversion
into PrPSc, consequently inhibiting this conversion [37,38].

FiguFriegu7r.eT 7h. rTeherepey pryidriidniened diciacarrbboonniittrriillee ddeerriivvaatitvivees swwithit hini vnitvroit raontai-nptrii-opnr iaocntivaictyti.v i ty.
Figure 7. Three pyridine dicarbonitrile derivatives with in vitro anti-prion activity.
In 2006, Reddy and co-workers focused on improving the activity of compound 23 through the
Idnev2e0lo0p6m, Renetd adnyd aenvdalucaot-iwono rokf ear ssfmoaclul sleibdraoryn oimf ppyrorivdiinneg dthicearabcotnivitirtilye odferciovmatipveosu n[3d9].2 3Atftherro ugh
In 2006, Reddy and co-workers focused on improving the activity of compound 23 through the
thedpeevrefolormpminge ndtocaknindge svtuadluieas twiointh oaf parospmosaeldl lbiibnrdairnyg osiftep myoriddeiln foerd ciocmarpboounndit rsielleecdtieorni,v 4a5t sivtreusct[u3r9a]ll.yA fter
development and evaluation of a small library of pyridine dicarbonitrile derivatives [39]. After
perfodrmiveinrsge dcoomckpionugndstsu wdeieres swynitthheasipzreodp boys ae donbei-npdoti nthgreseit-ecommopdonelenfot rcocuopmlipngo urenadctisoenle, hcteiroenin, i4ll5ussttrrautcetdu rally
performing docking studies with a proposed binding site model for compound selection, 45 structurally
diverisne Scchoemmpe o3u fonrd csomwpeoruensdy 2n4t,h tehsei mzeodst bpyromaisoinneg- cpoomtptohurnede- icno tmhips ostnuednyt. coupling reaction, herein
diverse compounds were synthesized by a one-pot three-component coupling reaction, herein illustrated
illustratedinScheme3forcompound24,themostpromisingcompoundinthisstudy.
in Scheme 3 for compound 24, the most promising compound in this study.

Scheme 3. (A) One-pot synthesis of pyridine dicarbonitrile derivative 24; (B) Proposed rea ction
mechanism. Reagents and conditions: (a) EtOH, reflux piperidine, 3 h, then air-exposed overnight (19%) [39].
Scheme 3. (A) One-pot synthesis of pyridine dicarbonitrile derivative 24; (B) Proposed reaction
Scheme 3. (A) One-pot synthesis of pyridine dicarbonitrile derivative 24; (B) Proposed reaction
mechanism. Reagents and conditions: (a) EtOH, reflux piperidine, 3 h, then air-exposed overnight (19%) [39].
mechanism. Reagents and conditions: (a) EtOH, reflux piperidine, 3 h, then air-exposed overnight
( 19%)[39].
Molecules2017,22,864 8of46
Reactionoptimizationinvolvedthestudyoftheoptimumaldehyde/thiol/malononitrileratio,
whichwasfoundtobe1:1:2,aswellasthebestsolvent,thebestcatalyticbaseandtheidealreaction
duratMioolnec.ulMes 2e0c1h7,a 2n2,i 8s6t4i cally,asdepictedinScheme3B,duringthefirst3hofreactioninethan8 oofl 4u5 nder
reflux,theinitialbase-catalyzedattackofamalononitrileaniontothealdehydefollowedbyelimination
Reaction optimization involved the study of the optimum aldehyde/thiol/malononitrile ratio, which
ofwatergivesanadduct,whichsubsequentlysufferstheadditionofbenzylthioltogiveaniminonitrile
was found to be 1:1:2, as well as the best solvent, the best catalytic base and the ideal reaction duration.
intermediate. Then,asecondmoleculeofmalononitrileisaddedtopromoteacyclizationreaction
Mechanistically, as depicted in Scheme 3B, during the first 3 h of reaction in ethanol under reflux, the
that affords the central dihydropyridine core; from this point, air-derived O is required for the
initial base-catalyzed attack of a malononitrile anion to the aldehyde followed by elim2ination of water
aromatizationintothefinalpyridineringbychemicaloxidation. Eventhoughtheyieldsobtainedfor
gives an adduct, which subsequently suffers the addition of benzylthiol to give an iminonitrile
thisone-potreactionwereupto52%,compound24wasachievedinonly19%yield.
intermediate. Then, a second molecule of malononitrile is added to promote a cyclization reaction
Cthoamt apfofourndds t2h4e scuecncterasls fduilhlyydirnotpeyraricdtiende wcoirteh; afrllotmh rteheisP prPoiCntf,o arimr-sdeursiveeddi nOt2h iiss rsetquudirye:dh ufomr athne PrPC
(huPraProCm; batiinzdatiinong ianftfio nthitey fiantal4 p0yµriMdi,ne% rRinUg mbyax ch=e5m5i.c5a)l, otxriudnactiaotne.d Evheunm thaonugPhr PthCe (yti-ehldusP orbPtCai;n%edR fUorm ax =
28.8)tahnisd onmeu-proint reeaPcrtPioCn (wmeoreP urPp Cto; %52R%U, comp=ou5n2d.6 2)4. wThase aacnhaielvoegdp ino sosnelsys 1in9%g yaie3l-dc.h lorophenylthiolat
max
C-4hadaCsoimgnpiofiucnadn t2l4y shuicgcehsesrfualflyfi ninitteyratoctweda rwdisthP arlPl tChr(eheu PPrrPPCC fo%rmRsU used =in1 t2h3is. 6s;tut-dhyu: PhurPmCan% PRrPUC =
max max
91.8;(mhuoPPrrPPC;C b%inRdiUng affi=ni7ty2 .a2t) 4;0h μoMw,e %veRrU,imtaxw =a 5s5.f5o)u, tnrudntcoatbede htouxmica.n NPrePvCe (rtt-hhuelPersPsC,; c%oRmUpmoaxu =n 2d8.284) was
max
moreapnodt menutritnhea nPrtPhCe (mleoaPdrPcoC;m %pRoUumnaxd =2 532.i6n). PTrhPeS ac-nianlfoegc tpeodssmesosiunsge ab 3r-acihnlomroepshoedneyrlmthiaoll (aStM C-B4) hcaedll as ,and
significantly higher affinity towards PrPC (huPrPC %RUmax = 123.6; t-huPrPC %RUmax = 91.8; moPrPC
presentedahighassociation/dissociationresponseratio,whichisidealwhenlookingforapotential
%RUmax = 72.2); however, it was found to be toxic. Nevertheless, compound 24 was more potent than
newdrugcandidate.
the lead compound 23 in PrPSc-infected mouse brain mesodermal (SMB) cells, and presented a high
Overall,mostpyridinedicarbonitrilederivativespossessingthebenzylsulfanylmoietydisplayed
association/dissociation response ratio, which is ideal when looking for a potential new drug candidate.
someleveOlvoefraalflfi, mniotsyt ptoywridairndes dPicraPrbCo.nTithrirloe udgerhivtahteiveevs aplousasetsiosinngo tfhael lbesnyznytlhsueslfiazneydl manoaieltoyg dsi,sRpleadyeddy and
co-wosorkmeer slecvoenl oclfu adffeindittyh taotwtharedssu PbrsPtCit. uTehnrotuagthC t-h4eo efvtahlueacteionnt roafl aplly sryidntihneescizoerde aonfaploygrsid, Rineedddyic aanrdb ocon-itriles
doeswnoortkeinrsfl cuoennclcuedtehde thbaitn tdhien sgubtsotitPurePnCt aat sCm-4 uocf hthae scetnhterasl upbysritditiunee ncotraet oCf p-6y.ridIninef adcitc,artbhoisnihtraildes been
predidcoteeds ninot pinrfilourendcoec tkhien bginpdoinsegs tow PhrePrCe asth meutchhi oaes tthhee rsumbostiietutyenwt aats Cp-6la. cIne dfacdte, tehpisi hnasdid beeethn eprheydpicottehde tical
in prior docking poses where the thioether moiety was placed deep inside the hypothetical pocket,
pocket,whereasthesubstituentatC-4wasgenerallyplacedtowardstheopeningofthecavity.
whereas the substituent at C-4 was generally placed towards the opening of the cavity.
Compound25(Figure7)waspublishedinasubsequentreportbyMayetal.presentingathorough
Compound 25 (Figure 7) was published in a subsequent report by May et al. presenting a
structure-activityrelationshipstudyonpyridinedicarbonitrilederivativesbearingaminesubstituents
thorough structure-activity relationship study on pyridine dicarbonitrile derivatives bearing amine
atC-6andfuranorhalobenzenesubstituentsatC-4[38]. Thesyntheticapproachtowardsdesigned
substituents at C-6 and furan or halobenzene substituents at C-4 [38]. The synthetic approach towards
derivdaetsivigensedw daesriqvuaititveeds iwstaisn qcutiftreo dmisttinhcet fprroemv itoheu sproenvieo,uasn odnei,s ahnder ies ihnerileliuns itllruastterdatefdo rfocro cmomppoouunndd 25in
Schem25e in4 .Scheme 4.

SchemScehe4m. eS y4n. tShyensthisesoifs pofy rpiydriindeinde idciacrabrboonnititrriillee ddeerriivvaattiivvee 2255. .RReaegaegnetsn tasnda ncdoncdointiodnitsi:o (nas): β(-aa)laβn-inalea, nine,
EtOH, r.t., 72 h; (b) 2-cyanoacetamide, piperidine, EtOH, reflux, 8 h; (c) 10% KOH, DMF, r.t., 1 min;
EtOH,r.t.,72h;(b)2-cyanoacetamide,piperidine,EtOH,reflux,8h;(c)10%KOH,DMF,r.t.,1min;(d)
(d) (diethylamino) alkyl halide, r.t., 5 h [38].
(diethylamino)alkylhalide,r.t.,5h[38].
Firstly, a β-alanine-catalysed Knoevenagel condensation afforded the arylidene malononitrile 26,
Fwihrsicthly w,aasβ su-ablsaenquinene-tlcya ctoanlyvseertdedK inntoo einvteenrmagedeilacteo 2n7d beyn rseaatcitoinng awfiftohr ad medolethcuelea orfy 2l-icdyeanneoamceatalomnidoen, itrile
26, winh tihceh prwesaesncse uobf speipqeuriednintley, ucnodnerv reerftluexd. Tihnetno, aifntetre rtrmeaetmdieantte w2it7h 1b0y% KreOaHct iinn gdimweitthhylfaormmaomleicdue le of
2-cya(nDoMacFe),t atmhei dhea,loinamthineep wreasse nadcedeodf ptoi pfeorrimd inthee, udnesdireerdr esflulufaxn.yTl hmeno,ieatfyt,e trhturse aatfmfoerdnitnwg itthhe1 f0in%alK OH
compound 25. Although the yields for each reaction step are not specified, final compounds were
indimethylformamide(DMF),thehaloaminewasaddedtoformthedesiredsulfanylmoiety,thus
obtained in 10–70% overall yield.
affordingthefinalcompound25. Althoughtheyieldsforeachreactionsteparenotspecified,final
Most of these pyridine dicarbonitrile derivatives were not toxic up to 25 μM and exhibited EC50
compoundswereobtainedin10–70%overallyield.
values below 15 μM in ScNa2 cells. Moreover, they were generally more active than compound 24
Mostofthesepyridinedicarbonitrilederivativeswerenottoxicupto25µMandexhibitedEC
(Figure 7) which, in this study, presented no activity up to 25 μM. Compounds with tertiary alkyl 50
valueasmbineelos wat 1C5-6µ Mwerine gSecnNeraa2llyc emllso.reM acotriveeo vtehra,nt htheoysew beeraerignegn perriamllayrym aomreinea cmtivoieettiehsa, nwchoemreapso und
24 (Fmigeutar-esu7b)stwituhteicdh h,ailnobtehnizsensetus daty C, -p4r weseerne tfeodunndo toa bceti vpriteyferuapblet oto2 f5urµanM-s.uCbsotimtupteodu cnodmspowuinthds toerr tiary
to the corresponding para-substituted analogs. Among all synthesized pyridine dicarbonitrile derivatives,
Molecules2017,22,864 9of46
alkylaminesatC-6weregenerallymoreactivethanthosebearingprimaryaminemoieties,whereas
meta-substitutedhalobenzenesatC-4werefoundtobepreferabletofuran-substitutedcompounds
Molecules 2017, 22, 864 9 of 45
or to the corresponding para-substituted analogs. Among all synthesized pyridine dicarbonitrile
derivatives, compound 25 was found to be the most promising, with an EC value of 5.5 µM in
compound 25 was found to be the most promising, with an EC50 value of 5.5 μ5M0 in ScNa2 cells.
Molecules 2017, 22, 864 9 of 45
ScNaM2coerellosv.eMr, iot rperoesveenrt,eidt hpirgehs ewnatteedr sholiugbhilwitya atenrd sao glouobdi lpiteyrmaneadbialitgyo roadte pine ar mstaenadbairlidt yparraatleleil naratifsictaianl dard
paralmcleoelmmaprbotruiafinncdei ap2le5rm mweeamasb bfiloriutaynn ades stpaoye br(mPe AtehMaeb PimAlio)t ys(lto apgsrPsoeam y= i−s( iP6n.Ag0 ,±M w0P.i1tA)h, )ian(ndlo iEcgaCPti5en0 gv= aal −guoeo6 od.0f p5±h.5a r0mμ.1Ma)c, oiinkn idnSecicNtiacat p2in rocgefilalles..g ood
pharmHMaoocwroeeokvvienerre,, tniitoc p pirner sovefiinvltoee d.d Hhatioag hwa rweev aateverra, isnloaolbulibeni ulvitpiyv taoon tddh aaist g apooaoirdne tp.a ev rmaielaabbilleityu pratteo inth ai sstpanodinartd. parallel artificial
membrane permeability assay (PAMPA) (logPe = − 6.0 ± 0.1), indicating a good pharmacokinetic profile.
2.2.2.2P.2i.p2.e PraipzeinraezDinee rDivearitvivateisves
However, no in vivo data are available up to this point.
BasedBaosnedt hoen pthree vpiroeuvisoluysdlye dscersicbriebdedn enueuroropprrootteeccttiivvee aaccttivivitiyty anadn dabaiblitiyli toyf othfet h2,e5-2d,i5k-edtiokpeiptoepraizpienrea zine
2sc.2a.f2f.o Pldip teor amziondeu Dlaetrei vpartoivteeisn -protein interactions [40], Bolognesi and co-workers have generated a
scaffoldtomodulateprotein-proteininteractions[40],Bolognesiandco-workershavegeneratedasmall
librarsymoafllBc olaismberdap rooyun o ntfhd ceso pmbrepeavoriuionnugdslsdy bidfefeaesrrciernnibgte dadir fonfeemruearnotitpc aroarotnemdctaihvteiect eaacrnotidva irhtoyem taenraodtia carobsmuilibtayst itcoit fsu utehbnes tt2si,t5iun-ednpiktoses tiointpi opipnoessriat3izoainnnesd 6of
3sc aanffdo l6d o tfo a mceondturalal t3e,6 p-droimteeinth-pyrlpotipeienr aiznitneera-2c,t5io-dniso n[4e0 c],o rBeo [l4o1g]n. eTshi ea naudt hcoo-rws eonrkveisriso nheadv et hgaetn ae rpaltaenda ra,
acentral3,6-dimethylpiperazine-2,5-dionecore[41].Theauthorsenvisionedthataplanar,symmetrical
ssymmamll leitbrricaarly m oof lceocumlep owuitnhd asr obmeaartiincg e nddif fgerroeunpt sa wroomualdti bc ea indde ahle ttoe oropatirmomizae ttihc es duebssitrietude annttsi -iang gproesgitaitoinngs
moleculewitharomaticendgroupswouldbeidealtooptimizethedesiredanti-aggregatingproperties
p3 raonpde r6t ioefs a a cnedn,t rianl 32,061-d0,i mperethseynlpteipde rcaozminpeo-u2,n5d-d 2io8n (eF cigoruer e[4 81)] . aTsh ea anuetwho arsn etin-pvrisioionn eleda dth afot ra pfulartnhaerr,
and,idsnyemv2e0ml1oe0pt,rmipcearnle mts.e onletecudlec owmithp aoruonmdat2ic8 e(nFdi gguroruep8s) waosualdn beew idaenalt it-op orpiotinmliezea dthfeo drefsuirretdh aenrtdi-aegvgerleogpamtinegn t.
properties and, in 2010, presented compound 28 (Figure 8) as a new anti-prion lead for further
development.

FigFuirgeur8e. 8F.o Fuorupr ippiperearazzininee ddeerriivvaattiivveess wwitihth anatni-tpi-rpiornio anctaivcittiyv.i ty.

Compound 28 inhibited PrPSc accumulation in PrPSc-mouse hypothalamus (ScGT1) cells with an
CECom50 pvaoluune dof2 84.i1n μhMibF,i tiwegduhriePl e8r .P mFSoacuianr ctpacipiunemirnagzui lnaaept dipoerrnoivxiainmtivPaertseP wlySic t-h7m 5a%not ui-cpseerlilo hnvy iaapcbtoiivltihittyya .l aatm thuiss (cSocnGceTn1t)racteiollns. with
anECM5e0cvhCaaonlumisetpiooc ufsnt4ud.d1 2ieµ8s Mi nfoh,ciwubishtienidlge PomrnP aSacig nagctrcaeuignmaituniolganta ikopinnp eirntoi cPxsri PmreScva-meteaolleyuds7 et5 hh%aytp ctohetilhsl amvlaioamlbeucislui (tlSey caGacttTst1 hb) iycs eilcnlsot enwrcaitechtni natrnga tion.
MechdEaCinre5i0cs ttvliyac luswteui todhf i er4es.c1fo omμcMubis,n inawnght iolpen rimaognag inprteraogintaeitinnigo n(arepkcpiPnrroePxt)ii cmtsoar teienvlyhe iab7l5iet% di tstch eclalo tnvtvihaeibrssilimiotyno laientc tutoh lietsh aecc otpnsacbtehynotilronagttieiocrnaal.c ting
direcPMtlryePcSwhc-laiinkthies ctirocen csftoourmdmibeeisrn afaonncdut, sipnintregiro eonsnt iapnggrgolyrt,ee wginahtii(loern et hckPein rceoPtr)ircests orpeoivnnedhailinebgdi t 3th-i taasnt dtch o4is-np mvyeroirldseyicolu nalen aianlcottgos sb wtyh eiernetp erroaautchgtoihnllgyo gical
PrPScdt-elinirke tecitmclyoe nsw fleoitsrhsm arecetrcivoaemn adbg,ianiniantnsettr PepsrrtPiioSncn ga lcpycr,uowmtehuinilla et(irtoehnceP, ccrooPmr) rpteoso puionnhndidsb ipintr geits3se -ncatoinnndgv eb4re-spnioyznern idieny, tfloua rtnahanel, otphgaisothwpoheleornegeirc ooarul g hly
tentimqPurePinsSco-llelioksnsee ca omcntoifvoieertmiaeegsr ad aiinndds nt, oiPntr teePxrSehcsitbainictcg aulnym,y w uahclatiiltvei oitthnye., ccoormrepspoounnddinsgp 3r-e asnedn t4i-npgyrbideynlz aennaelo,gfus rwaenr,et rhoiuogphhleyn eor
quinotelonn teiTmhmeeso sliteersatsiie gashcttdfiovirdew anagoradti nessyxtnh PtihrbPeisStci saa ncocfy ucamocmutiplvaotiuitoynn.d, c2o8m inpvooulvnedds opnrelys eonntein rge abcetniozne nstee,p f u(Sracnh,e mtheio 5p)h. Ienn eth oisr
rqeuaicntoiolno,n etw moo ieeqtuieisv adliedn tnso ot fe xphiciboilti naanldy eahcytidvei tyw. ere mixed with one equivalent of the commercially
The straightforward synthesis of compound 28 involved only one reaction step (Scheme 5).
availaTbhlee s1t,r4a-digihactfeotyrwlpaiprde rsayzninthee-2s,i5s- odfi ocnoem dpiossuonlvde 2d8 iinn vDoMlvFe,d i no nthlye opnrees reenaccet ioofn t rsiteetph y(Slacmheimnee. 5A) . dInou tbhlies
Inthisreaction, two equivalents of picolinaldehyde were mixed with one equivalent of the
areldacotli-ocno,n tdweon seaqtiuoinv aalte nrotso mof tepmicopleinraatludereh ygdaev ew ceorme pmoiuxnedd 2w8i tihn 6o4n%e eyqieulidv,a aleffnot rdofi ntgh ee xccolumsmiveerlcyi atlhlye
commdaveeasriiclraieabdll le(y Z1,,4Za-v)d-aiisaioclaemtbyellrep. ip1e,r4a-zdiniaec-2e,t5y-dlpioinpee rdaiszsionlvee-2d, 5in- dDiMonFe, ind tihses oplrveesedncien ofD trMietFh,ylianmitnhee. Ap dreosuebnlec e of
triethylamine. Adoublealdol-condensationatroomtemperaturegavecompound28in64%yield,
aldol-condensation at room temperature gave compound 28 in 64% yield, affording exclusively the
afforddiensgireedx c(Zlu,Zsi)v-iesolymtehre. desired(Z,Z)-isomer.

Scheme 5. Synthesis of diketopiperazine 28. Reagents and conditions: (a) TEA, DMF, r.t., 16 h (64%) [41].

The isopropylpiperazine derivative 29 (Figure 8) was later published by Li et al. as the most
ScheSmchee5m.eS 5y.n Styhnetshisesoisf odfi kdeiktoetpoippieprearzazininee2 288.. RReeaaggeennttss aanndd ccoonndidtiitoinosn: s(:a)( aT)ETAE, AD,MDFM, rF.t,.,r .1t6., h1 6(6h4%(6) 4[%41)].[ 41].
promising compound of a small library of optimized arylamides with anti-prion activity, which were
The isopropylpiperazine derivative 29 (Figure 8) was later published by Li et al. as the most
promising compound of a small library of optimized arylamides with anti-prion activity, which were
Molecules2017,22,864 10of46
The isopropylpiperazine derivative 29 (Figure 8) was later published by Li et al. as the
most promising compound of a small library of optimized arylamides with anti-prion activity,
which were developed based on a moderately active piperazine arylamide derivative [42]. Here,
Molecules 2017, 22, 864 10 of 45
the piperazine ring is placed in one of the extremities of the molecule, thus contrasting with
Bologdneveeslio’sperda btiaosneda loen ian mwodheircahtelyth aectidveik peitpoepraizpienrea azriynlaemwidae sdetrhiveatcivoer e[42m]. oHieertey, thaec tpiinpgeraazsinteh reingli nker
is placed in one of the extremities of the molecule, thus contrasting with Bolognesi’s rationale in which
betweentwoidenticalpyridinegroups. ThesynthesisofthisamidebearingtheN-alkylpiperazine
the diketopiperazine was the core moiety acting as the linker between two identical pyridine groups.
ring is very straightforward and involves only one reaction step, namely the standard coupling
The synthesis of this amide bearing the N-alkyl piperazine ring is very straightforward and involves
between a carboxylic acid and an arylamine carried out in the presence of the uranium salt
only one reaction step, namely the standard coupling between a carboxylic acid and an arylamine carried
O-(7-azabenzotriazol-1-yl)-N,N,N(cid:48),N(cid:48)-tetramethyluroniumhexafluorophosphate(HATU)asanacid
out in the presence of the uranium salt O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium
activahteixnagflrueoargopenhotsapnhdatNe ,(NH-AdTiiUso) pasr oapn yalceidth aycltaivmatiinneg (rDeaIgPeEnAt a),nidn Na,nNh-yddiisroopursopDyMletFh,yaltamroionme (DteImPEpAe)r,a ture
gaveicno amnhpyodurnodus2 D9MinF6, 6at% royoimel dtem(Spchereamtuere6 g).ave compound 29 in 66% yield (Scheme 6).

SchemScehe6m.Se y6n. Sthyensthisesoifs pofi ppeipraezrainziened edreirvivaatitvivee 2299.. RReeaaggeenntst sanand dcocnodnitdiiotnios:n (sa:) (Ha)AHTUA,T DUIP,DEAIP, EanAh,yadnrohuysd rous
DMF, r.t., 12 h (66%) [42].
DMF,r.t.,12h(66%)[42].
The initially conducted structure-activity relationship studies revealed that the biphenyl group
Theinitiallyconductedstructure-activityrelationshipstudiesrevealedthatthebiphenylgroup
linked to the carbonyl of the amide functionality led to more potent compounds than those bearing only
linkedonteo atrhoemcaatricb orinnygl. Tohfeth peipaemraizdineef umnociteitoyn walaist yrelgeadrdtoedm aos raenp imotpeonrttacnotm fepaotuurned fosrt hthaen mthaionsteenbaenacrei nogf only
oneacroommpaotuicnrdi nsogl.uTbhilietyp aipnder tahzuisn ewmaso kieeptyt twhraosurgehgoaurtd tehdis asesraiens iomf dpeorritvaantitvfees.a Ftuurrethfeorrmtohree,m 4-abiinptheennaynl ceof
compaonuanlodgss owluebrei liptyreafenrdabtlhe utso wthaesirk 3e-pbtipthhreonuylg hcoonugtetnheirsss, earnieds coofmdpeoriuvnadtsiv ceosn.tFauinritnhge rlmarogere N,4-a-blkipylh enyl
analomgosiewtieerse inp trheef epripaebrlaezitnoe trhinegir w3e-rbei mphoeren pyoltceonnt tgheanne trhso,sae ncodntcaoimninpgo sumnadlslerc oNn-atalkiynli ngrgoulaprsg. eWNith-a lkyl
moieatine sECin50 otfh oenplyi p22e rnaMzi angeairnisnt gPrwPSec raeccmumourleatpioont einn itnt PhraPnSc-tinhfoecsteedc omnotuasien ninegurosbmlaasltloemraN (S-acNlk2yal-cgl3r)o ups.
cells, compound 29 stood out among this series of derivatives and was found to be 100-fold more
WithanEC ofonly22nMagainstPrPSc accumulationininPrPSc-infectedmouseneuroblastoma
50
potent than the original lead compound. It displayed acceptable metabolic stability in mouse liver
(ScN2a-cl3)cells,compound29stoodoutamongthisseriesofderivativesandwasfoundtobe100-fold
microsomes (t1/2 > 60 min) and good brain exposure upon oral administration in mice, thus coming
morepotentthantheoriginalleadcompound. Itdisplayedacceptablemetabolicstabilityinmouse
across as a promising lead for further development. However, the in vivo evaluation of this
livercmomicproousonmd teos c(otn1/fi2rm> w60hemthienr )thaen ndangoomodolabrr raainngee xppootesnucrye oubpseornveodr ianl vaitdrom iisn inisdteraedti otrnanisnpomsaicbele, thus
comintog TaScEr oins sviavsoa mpordoemls iissi nstgilll elaacdkifnogr. furtherdevelopment. However,theinvivoevaluationofthis
compounCdotmopcoounnfidr m30 w(Fihgeutrhee 8r),t ohne tnhea notohmero hlaanrdr,a wngase ppuobtleisnhceyd obyb sLeeridveeld anind vcoitllreoagisueins dase ae dpitprearnazsipnoe sable
toTSdEeirnivvatiivvoe wmiothd aecltsivisitys tiinll PlraPcSkc-iinngfe.cted mice [43]. This molecule was identified after high-throughput
Cscoremenpiongu nofd 103,000(0F ciogmupreou8n)d,so inn StMheB coetlhlse, rwhhearned fo,uwr paispepruazbilnies hdeedrivbatyivLese eidmeelrgaendd asc ao nlleewag culaesss as a
piperoafz PirnPeresd ceornivveartsiivone/awmitphlifaiccattiivoint yinihnibPitroPrsS.c N-iontfeewctoerdthym, aic seig[n4if3i]c.anTt hdiescrmeaosele icnu PlrePrwes afosrmidaetniotnifi weads after
not achieved by compound 30 in a cell-free assay but it was shown to be very potent when tested in
high-throughput screening of 10,000 compounds in SMB cells, where four piperazine derivatives
emermgeodusaes naeunroebwlasctloamssa o(Nf2Par)P creelslsc, odnisvpelarysiinogn /ana mICp50l iofifc 0a.t4i oμnMi. nMhoibreiotovresr., pNrooteteaswoomret hayc,tivaitsyi gwnaisfi cant
not affected in its presence, indicating that it does not influence cellular trafficking or processing of
decreaseinPrPresformationwasnotachievedbycompound30inacell-freeassaybutitwasshown
PrPC. Taken together, these results suggest an indirect inhibitory mechanism of action for compound
to be3v0,e irny wphoicthe nitt pwrohbeabnlyt etasrtgeedtsi nmimsfooludsinegn oer uagrogbrelgaasttioomn cao-(fNac2toar)s cperlelsse,ndt iisnp thlaey cienllg-baasnedIC as5s0aoy,f b0u.t4 µM.
Moreaobvseenr,t pwrohetena usosimnge oancltyiv tihtye wpuarsifnieodt parfifoenc tpedrotieninit. sApltreersneanticvee,lyin, dit icmaatiyn gactt hbayt iitndduoceisngn ocetlilnulflaur e nce
cellulParrPtrersa cflfiecakrainncge.o Arnpyrhoocwes,s tihnigs opfipPerrPazCin.eT adkeerinvatotivgee tshuecrc,etshsfeuslelyr epsruollotsngsuedg gthees tinacnuibnadtiiorne cttimineh iinb itory
mechParnPiSscm-inofefcatecdti oCn57foBrLc/6o mmipcoe ufrnodm3 104,4i ntow 15h7i cdhayitsp wrohbena baldymtainrgisetetsremd ivsifao aldni inngtroarpeargigtorneegaalt iinojnecctoio-nfa, ctors
presewnthiilne tah deirceecltl -abnaasloegd waistsha ay ,lobwuetra IbCs5e0 nantdw bheeanrinugs ian 3g-cohnlolyrotphheepnyulr mifioeidetyp,r iinosntepadro otfe tihne. 2A-pltyerrindaytl ively,
it magyroaucpt, bdyidi nnodtu. cing cellular PrPres clearance. Anyhow, this piperazine derivative successfully
This study succeeded to illustrate in vivo effects of piperazine derivatives against TSEs, and
prolongedtheincubationtimeinPrPSc-infectedC57BL/6micefrom144to157dayswhenadministered
provided important hints on the potential anti-prion mechanism of action of these types of compounds.
viaanintraperitonealinjection,whileadirectanalogwithalowerIC andbearinga3-chlorophenyl
It is relevant to note that another piperazine derivative, compound 5301 (Figure 8), was found to
moiety,insteadofthe2-pyridylgroup,didnot.
enhance the clearance of PrPSc in ScN2a cells, with an IC50 of 0.1 μM [44]. Interestingly, this compound
Tish ai swsetlul-dknyoswunc ctyereodseinde tkoinialslue sintrhaitbeitoinr ivnidviocaetefdfe fcotrs tohfe ptriepaetmraeznitn oef dcherroivnaict imveyseloaigda lienusktaTemSEias,, and
proviadnedd wimasp osrhtoawntn htion tsstroonngthlye apcotitveanttiinagl athneti -lpyrsioosnommael cdheagnriasdmatoiofna cotfi oPnrPoSfc,t hweistheotuytp easffoecftcinogm tphoeu nds.
Itisreblieovsyanntthteosins ootre ltohcaatliazantoiothne orfp PirpPeCr.a Tzhineesed reersiuvlatst iavree, ccoonmsipsoteunnt dan3d1 c(Flairgifuyr eth8e) ,fiwndasinfgosu rnedgatrodeinngh ance
thecltehaer maneccheaonfisPmr PoSf cacintioSnc oNf 2thaec peilplse,rawziinthe daneriIvCativoe f300.. 1µM[44]. Interestingly,thiscompoundisa
50