Discovery of Potent AChE-MAO B Inhibitors through the Dec

15 Pages·2016·1.48 MB·English

molecules
Article
Searching for Multi-Targeting Neurotherapeutics
against Alzheimer’s: Discovery of Potent AChE-MAO
B Inhibitors through the Decoration of the
2H-Chromen-2-one Structural Motif
LeonardoPisani1,RobertaFarina1,RamonSoto-Otero2,NunzioDenora1,
GiuseppeFeliceMangiatordi1,OrazioNicolotti1,EstefaniaMendez-Alvarez2,
CosimoDamianoAltomare1,MarcoCatto1,*andAngeloCarotti1,*
1 DipartimentodiFarmacia—ScienzedelFarmaco,UniversitàdegliStudidiBari“AldoMoro”,
viaE.Orabona,4,I-70125Bari,Italy;[email protected](L.P.);[email protected](R.F.);
[email protected](N.D.);[email protected](G.F.M.);[email protected](O.N.);
[email protected](C.D.A.)
2 GrupodeNeuroquimica,DepartamentodeBioquimicayBiologiaMolecular,FacultaddeMedicina,
UniversidaddeSantiagodeCompostela,SanFranciscoI,E-15782SantiagodeCompostela,Spain;
[email protected](R.S.-O.);[email protected](E.M.-A.)
* Correspondence:[email protected](M.C.);[email protected](A.C.);
Tel.:[phone](M.C.);[phone](A.C.);Fax:[phone](M.C.&A.C.)
AcademicEditors:MichaelDeckerandDiegoMuñoz-Torrero
Received:15February2016;Accepted:10March2016;Published:17March2016
Abstract: The need for developing real disease-modifying drugs against neurodegenerative
syndromes,particularlyAlzheimer’sdisease(AD),shiftedresearchtowardsreliabledrugdiscovery
strategiestounveilclinicalcandidateswithhighertherapeuticefficacythansingle-targetingdrugs.
By following the multi-target approach, we designed and synthesized a novel class of dual
acetylcholinesterase(AChE)-monoamineoxidaseB(MAO-B)inhibitorsthroughthedecorationofthe
2H-chromen-2-oneskeleton. Compoundsbearingapropargylaminemoietyatposition3displayed
thehighestinvitroinhibitoryactivitiesagainstMAO-B.Withinthisseries,derivative3hemerged
as the most interesting hit compound, being a moderate AChE inhibitor (IC = 8.99 µM) and a
50
potentandselectiveMAO-Binhibitor(IC =2.8nM).Preliminarystudiesinhumanneuroblastoma
50
SH-SY5Y cell lines demonstrated its low cytotoxicity and disclosed a promising neuroprotective
effectatlowdoses(0.1µM)underoxidativestressconditionspromotedbytwomitochondrialtoxins
(oligomycin-Aandrotenone). InaMadin-Darbycaninekidney(MDCK)II-MDR1cell-basedtransport
study,Compound3hwasabletopermeatetheBBB-mimickingmonolayeranddidnotresultina
glycoprotein-p(P-gp)substrate,showinganeffluxratio=0.96,closetothatofdiazepam.
Keywords: Alzheimer’s disease; cholinesterase inhibitors; coumarins; MAO inhibitors;
multi-target-directedligands
1. Introduction
Nowadays,agrowingshareoftheelderlypopulationexperiencestheheavyburdenofcognitive
declineassociatedwithsevereneurodegenerativedisorders(NDs). Inthiscontext,acentralroleis
occupiedbyAlzheimer’sdisease(AD),beingthefirstcauseofage-relateddementiawithaworrisome
projectionofaround100millionpatientsworldwideby2050. InpeoplesufferingfromAD,learning
deficits along with the impairment of memory and language skills result from the disruption of
cholinergictransmissioninhippocampalareas[1]. Asaconsequenceoftheso-called“cholinergic
hypothesis”, up to date, the pharmacological therapy of AD has been essentially based on the
Molecules2016,21,362;doi:10.3390/molecules21030362 www.mdpi.com/journal/molecules
Molecules2016,21,362 2of15
restoration of adequate levels of acetylcholine (ACh). Thus, the majority of registered drugs for
the curing of AD belongs to the class of acetylcholinesterase (AChE) inhibitors (i.e., rivastigmine,
galantamineanddonepezil)thatareabletocounteractAChdepletionbyreducingitsdegradation
catalyzed by AChE [2]. Apart from AChE blockade, a drug with a different mechanism of action
(memantine),whichhasantagonismtoNMDAreceptors,hasmorerecentlybeenintroducedforthe
curing of moderate to severe forms of AD [3]. However, none of these medicines is able to slow
downorreversetheneuronaldegeneration,andallofthemexertonlysymptomaticrelief. Despite
hugeeffortsperformedbyresearchers,thecuringofADstillrepresentsachallengingtask,andareal
disease-modifyingtherapyisstillneeded. AsformanyNDs,convergingandrisingevidencesshed
lightonthemultifacetedetiologyofAD[4].Thismeansthatsimultaneousaberrations,includingmetal
unbalance,proteinmisfolding/aggregationandproductionoftoxicradicalsamongothers,trigger
and/orsustaintheneurotoxiccascadeultimatelyleadingtoneuronaldeath. Bymeansofmodulating
twoormoretargetsinvolvedintheonsetand/orprogressionofthedisease,themulti-targetstrategy
hasbeenregardedasaninnovativetoolfordiscoveringneurotherapeuticsagainstAD[5]. Inrecent
years,programshittingbothproductionandaggregationofβ-amyloidandhyperphosphorylatedtau
proteinfailedtoidentifyagentswiththerapeuticefficacy[6]. Thus,theattentionhasbeenshiftedto
otherrelevantpathophysiologicalmechanisms. Inparticular,oxidativestresshasbeenconsidered
adownstreameventanticipatingthedepositionofamyloidplaquesandneurofibrillarytangles[7].
Deregulation of endogenous detoxification redox systems and over-production of radical species
lead to lipid peroxidation and nucleic acid mutations. In this context, a neuroprotective activity
againstoxidativestresshasbeenclaimedfornewchemicalentitiesabletoinhibitmonoamineoxidases
(MAOs)[8]. TheseFAD-dependentenzymescatalyzetheoxidativedeaminationofaminesderiving
fromdiet(e.g.,tyramine),physiologicalneuronalpathways(e.g.,severalneurotransmitters,suchas
catecholamines) or drug assumption. The catalytic cycle produces hydrogen peroxide, a harmful
by-productcapableoftriggeringreactiveoxygenspecies(ROS)release. Twoisoforms(MAO-Aand-B)
differinginsensitivitytosubstrateandinhibitorsareknown[9]. Inparticular,theMAO-Bisoform
hasreceivedgreatattention. Infact,itpredominatesinCNS,andmanystudiesreporteditsincreased
activityinbrainsaffectedbyAD[10]. Thus,thesimultaneousinhibitionofMAO-B,aswellasAChE
enzymaticactivityrepresentsaninnovativetherapeuticweapon[8]andmultifunctionalmoleculesable
toreduceradicals’formationandtotackleAChdecreasemaybebeneficialtohampertheneurotoxic
cascadeinAD.
Coumarin(2H-chromen-2-one)isanature-friendlyheterocycle,easytobefunctionalizedwitha
highdegreeofpotentialchemicaldiversity[11]. Aswerecentlyreported,itsplanarbackbonecanbe
efficientlylodgedintotheMAO-Bcatalyticsite[12,13]andisabletointeractwiththeperipheralanionic
site(PAS)ofAChE[14]. AimingatidentifyingnovelmultipotentcompoundswithdualAChE-MAO-B
inhibitoryactivity[15],herein,weinvestigatedthechemicaldecorationofthe2H-chromen-2-onecore
aroundthelessexploredposition3,byintroducingvariously-substitutedprotonatablebasicmoieties.
Ontheotherside,alowervariabilitywasexploredatpositions6and7bystudyingtheeffectofmethoxy
groups[16]. InvitroenzymaticscreeningtowardsChEsandMAOsledustoidentifyadualinhibitor
(derivative3h)endowedwithapotentandselectiveMAO-Binhibitorypotencyinthenanomolar
range(IC =2.8nM)togetherwithamoderateAChEaffinity(IC =8.99µM).HitCompound3hwas
50 50
submittedtotransportstudiesinMadin-Darbycaninekidney(MDCK)-IIpermeabilitymodelinorder
toevaluateitsabilitytocrossBBBandpenetrateintoCNS.Furthermore,cell-basedassayswereusedto
assesstheneuroprotectiveeffectagainstdifferentoxidativeinsults(hydrogenperoxide,oligomycin-A
androtenone)inhumanneuroblastomaSH-SY5Ycelllines. Therefore,coumarin-based3hemergedas
apromisingneuroprotectiveagentwithamulti-targetprofile,deservingadeeperinvestigationasa
potentialanti-ADtherapeuticagent.
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Molecules 2016, 21, 362 3 of 15
22.. RReessuullttss aanndd DDiissccuussssiioonn
2. Results and Discussion
2.1. Chemistry
2.1. Chemistry
2.1. Chemistry
TThhee ssyynnththeetitcicp patahthwwayayto tofi nfainlaml umltui-lttair-gtaertgceot ucmouarminasri3nas– m3a–amnd a8nids d8e pisic dteedpiincteSdch ienm Secsh1emaneds 21.
The synthetic pathway to final multi-target coumarins 3a–m and 8 is depicted in Schemes 1
3a-nMd e2t.h 3y-lM-2eHth-cyhl-r2oHm-echnr-2o-monenes-2-1obn,cesw 1ber,ce woebrtea ionbetdaintherdo tuhgrhouagho an eo-npeo-tpoptr pocroedceudruerest satratrintigngw witihth aa
and 2. 3-Methyl-2H-chromen-2-ones 1b,c were obtained through a one-pot procedure starting with a
WWiittttiigg--ccoonnddeennssaattiioonn ffoolllloowweedd bbyy iinnttrraammoolleeccuullaarr ccyycclliizzaattiioonn iinn rreeflfluuxxiinngg NN,,NN--ddiieetthhyyllaanniilliinnee [[1177]]..
Wittig-condensation followed by intramolecular cyclization in refluxing N,N-diethylaniline [17].
BBrroommiinnaattiioonn oof f1b1,bc ,pcropmroomteodt ebdy Nby-brNom-borosumcocisnuimcciidneim (NidBeS)( aNnBdS b)enanzodylb peenrzooxyidlep (eDrBoPxi)d aes t(hDeB raPd)icaasl
Bromination of 1b,c promoted by N-bromosuccinimide (NBS) and benzoyl peroxide (DBP) as the radical
tihneitiraatodric aaflfoirnditeidat ionrtearfmfoerddieadtesin 2tae–rcm tehdaita wteesre2 aco–ucptlheadt wwitehre thceo suupilteadblew aimthinteh etos yuiietladb Cleoammpionuendtos
initiator afforded intermediates 2a–c that were coupled with the suitable amine to yield Compounds
y33aiae––lmdm C((SSoccmhhepemmouee n 11d))s.. AA3ass– mddeepp(Siicccttheededm iinen 1SS)cc.hheeAmmsee d22e,, pAAicllCtCelld33--mmineeddSiicaahtteeeddm erreeg2g,iiooAsseellCleeclc3tti-ivmveee dbbiiiass-t-deddeemmreeetgthhiyoylslaaettliieoocnnti vooeff
b2i,s4-,d5-etmrimetehtyhloaxtiyobnenozfa2l,d4e,5h-ytrdime efuthronxisyhbeedn zaaldldeehhyyddee 4f u[r1n8i]s,h wedhiaclhd ewhyads eh4ea[t1e8d], awt hriechfluwx aws hiteha ttehde
2,4,5-trimethoxybenzaldehyde furnished aldehyde 4 [18], which was heated at reflux with the
aatprperfloupxriwatiet hphthoespahpoprruosp yrliiadtee ipnh oo-sxpyhleonreu,s thyulisd oebitnaion-ixnygle cnoeu,mthaurisno dbetraiivnaintigvec o5.u Amfaterrin pdroetreicvtaintigv eth5e.
appropriate phosphorus ylide in o-xylene, thus obtaining coumarin derivative 5. After protecting the
Apfhteenroplrioc tgercotiunpg, tthhee prehseunlotilnicg gbreonuzpo,atthee ersetesur l6t iunngdbeernwzeonatt ea ersatderic6alu bnrdoemrwineantitoan rwaditihca NlbBrSo amnidn aDtiBoPn.
phenolic group, the resulting benzoate ester 6 underwent a radical bromination with NBS and DBP.
wFiinthalN coBuSpalnindgD oBf P7. wFiintha lNc-omueptlhinyglporofp7awrgityhlaNm-imnee tihny tlhper oppraesrgenyclaem ofin aeni enxtcheessp oref speontcaessoiufman ceaxrcbeosnsaotfe
Final coupling of 7 with N-methylpropargylamine in the presence of an excess of potassium carbonate
paolltoawsseiudm thcea rrebmonoavtael aolfl othwee bdetnhzeoraetme gorvoaulpo fatnhde tbheen pzoreapteargartoiuonp oafn 8d. thepreparationof8.
allowed the removal of the benzoate group and the preparation of 8.

SScchheemmee 11.. SSyynntthheessiiss ooff ccoouummaarriinnss 33aa––mm.. RReeaaggeennttss aanndd ccoonnddiittiioonnss:: ((ii)) NN--bbrroommoossuucccciinniimmiiddee,, bbeennzzooyyll
Scheme 1. Synthesis of coumarins 3a–m. Reagents and conditions: (i) N-bromosuccinimide, benzoyl
pppeeerrroooxxxiiidddeee,,, CCCCCClll444,,, rrreeefflflluuuxxx,,, 222–––777 hhh;;; (((iiiiii))) sssuuuiiitttaaabbbllleee aaammmiiinnneee,,, tttrrriiieeettthhhyyylllaaammmiiinnneee (((fffooorrr 333aaa,,,bbb,,, 333ddd–––eee,,, 333ggg,,, 3 33ll–l––mmm))) o oorrr K KK22C2CCOOO333
((ffoorr 33cc,, 33ff,, 33hh––kk)),, DDMMFF aatt 7700 ˝°CC ((ffoorr 33aa,, 33ll––mm)) oorr TTHHFF aatt rroooomm tteemmppeerraattuurree( (33bb––kk)),,3 3.5.5––2244h h..
(for 3c, 3f, 3h–k), DMF at 70 °C (for 3a, 3l–m) or THF at room temperature (3b–k), 3.5–24 h.

Scheme 2. Synthesis of coumarin 8. Reagents and conditions: (i) (carbethoxyethylidene)triphenylphosphorane,
SScchheemmee 22.. SSyynntthheessiiss ooff ccoouummaarriinn 88.. RReeaaggeennttss aanndd ccoonnddiittiioonnss:: ((ii)) ((ccaarrbbeetthhooxxyyeetthhyylliiddeennee))ttrriipphheennyyllpphhoosspphhoorraannee,,
o-xylene, reflux, 12 h; (ii) triethylamine, benzoyl chloride, dry THF, 5 h; (iii) N-bromosuccinimide, benzoyl
oo--xxyylleennee,, rreeflfluuxx, 1,21 2h;h (i;i)( itir)iettrhieytlhamylianme,i nbeen,zboeynlz cohylolrcihdleo, rdirdye ,TdHrFy, 5T hH; F(,iii5) Nh;-b(irioi)mNos-burcocimniomsuidcec,i bneimnziodyel,
bppeeenrrozoxoxiyiddleep,, eCCrCoCxll4i4,d, rreee,ffClluuCxxl,,4 11,22r e hhfl;; u ((ixivv,))1 NN2-h-mm;(eeittvhh)yyNllpp-rmrooepptaahrrygglyypllraaommpiainnrege,,y KKla22mCCOiOn33e,, , ddKrry2yC TTOHH3FF,,,d oorvyveeTrrnHniigFg,hhott.v. ernight.
2.2. Biological Evaluation
22..22.. BBiioollooggiiccaall EEvvaalluuaattiioonn
Limited structural modifications were investigated at positions 6 and 7 of the coumarin backbone
LLiimmiitteedd ssttrruuccttuurraall mmooddiifificcaattiioonnss wweerree iinnvveessttiiggaatteedd aatt ppoossiittiioonnss 66 aanndd 77 ooff tthhee ccoouummaarriinn bbaacckkbboonnee
with the introduction of methoxy groups. On the other side, a higher diversity was explored in the
wwiitthh tthhee iinnttrroodduuccttiioonn ooff mmeetthhooxxyy ggrroouuppss.. OOnn tthhee ootthheerr ssiiddee,, aa hhiigghheerr ddiivveerrssiittyy wwaass eexxpplloorreedd iinn tthhee
flexible basic moiety where hydrophilic hydrogen-bonding, propargyl and bulkier benzylic groups
flfleexxiibbllee bbaassiicc mmooiieettyy wwhheerree hhyyddrroopphhiilliicc hhyyddrrooggeenn--bboonnddiinngg,, pprrooppaarrggyyll aanndd bbuullkkiieerr bbeennzzyylliicc ggrroouuppss
were inserted. Since cation-π interactions stabilize the complexes of AChE with natural substrate
wweerree iinnsseerrtteedd.. SSiinnccee ccaattiioonn--ππ iinntteerraaccttiioonnss ssttaabbiilliizzee tthhee ccoommpplleexxeess ooff AACChhEE wwiitthh nnaattuurraall ssuubbssttrraattee
(ACh) and positively-charged inhibitors (e.g., quaternary ammonium salts) at the level of both binding
((AACChh)) aanndd ppoossiittiivveellyy--cchhaarrggeedd iinnhhiibbiittoorrss ((ee..gg..,, qquuaatteerrnnaarryy aammmmoonniiuumm ssaallttss)) aatt tthhee lleevveell ooff bbootthh bbiinnddiinngg
sites (PAS and CAS), a methylene spacer linked the amino group to the heterocyclic scaffold in order
sites (PAS and CAS), a methylene spacer linked the amino group to the heterocyclic scaffold in order
to obtain a protonatable base. A ligand-based approach inspired the introduction of propargylamines,
to obtain a protonatable base. A ligand-based approach inspired the introduction of propargylamines,
a typical structural feature of potent and irreversible MAO-B inhibitors rasagiline [19] and selegiline.
a typical structural feature of potent and irreversible MAO-B inhibitors rasagiline [19] and selegiline.
Molecules2016,21,362 4of15
sites(PASandCAS),amethylenespacerlinkedtheaminogrouptotheheterocyclicscaffoldinorder
toobtainaprotonatablebase. Aligand-basedapproachinspiredtheintroductionofpropargylamines,
atypicalstructuralfeatureofpotentandirreversibleMAO-Binhibitorsrasagiline[19]andselegiline.
Molecules 2016, 21, 362 4 of 15
2H-Chromen-2-ones 3a–m and 8 were evaluated as inhibitors of electric eel AChE (eeAChE),
equin2eHs-Cerhurmombeunt-y2r-oynlcehso 3lian–emst earnadse 8( ewsBeCreh eEv)aalunadterda taMs iAnhOisbi(torMrsA oOf -eAlecatrnidc ereMl AAOC-hBE) .(eDeAatCahaEr)e,
ielqluusitnrea tesderiunmT abbulety1r.yIlnchvoitlrioneesntzeyramsea ti(cesaBsCsahyEs)o annCdh rEast wMerAeOasc c(ormMpAliOsh-Aed athnrdo urMghAtOhe-Bw).e lDl-aktnao warne
silpluecsttrraotpehdo itno mTaebtrleic 1.E Ilnlm viatrno’ senmzyemthaotdic a[2s0sa].ys oMnA COhEisn whiebriet oarcycoamcptilvisithieesd twhreoreugmh ethaseu wreedll-konnowrant
bspraeicntrmopihtoocthoomnedtrriica lEhllommaong’se nmateethso[d21 []2.0P].r oMpAarOg yilnahmibiniteo-rbye aarcitnivgitiinehs iwbietorer sm3ec,as3uf,re3dh oann dra8t bwrearine
pmriet-oicnhcounbdartieadl hwomithogtehneateens z[y21m].e Pprorpeparagryaltaiomninfeo-rbe3a0rinmgi ninhbiebfiotorers t3hce, 3afd, 3dhit aionnd o8 fwkeyren uprrae-minicnuebaatnedd
dweittehr mthien aetniozynmofe MprAepOaaracttiiovnit yf.oRr e3s0u mltsina rbeerfeopreo rttheed ainddTiatbiolen 1ofa skIyCnur(aµmMin)eo ranads tdheetperemrciennattaiogne ooff
50
iMnhAibOi taioctnivaitty1.0 RµeMsu.lts are reported in Table 1 as IC50 (µM) or as the percentage of inhibition at 10 µM.
TTaabbllee 11.. MMoonnooaammiinnee ooxxiiddaassee ((MMAAOO)) aanndd cchhoolliinneesstteerraassee ((AACChhEE,, BBCChhEE)) iinnhhiibbiittiioonn ddaattaa ooff CCoommppoouunnddss
33aa––mm aanndd 88..
R R
1 N 3
R
4
R O O
2
IC50(µM) or % Inhibition at 10 µM a
Comp. R1 R2 R3 R4 MAO-A b IC50(µM)MorA%OI-nBh bibitionat10AµCMhEa c BChE d
C3oam p. HR 1 RH2 HR 3 CH2CRO4NH2 MAO-<A5%b MAO-Bb<5% AChEc12 ± 1 B6C.2h%E d± 0.3%
3b3 ea HH HH MHe CH32′-CFOBNnH 2 <5%<5% <5% <5% 12˘61.8 ± 0.1 6.2%1˘5%0. 3±% 2%
33cb e HH HH MMee CH312-CFB≡nCH <75.%0 ± 1.4 f <05.%377 ± 0.014 f 6.8˘08.1.4 ± 0.5 15%˘<25%%
3d33d c HHH OOHMMee MMMeee CH332′1--CFF”BBnnC H 6.70.%06.˘0˘%10. 4.±8 f%0.8% 0.377<˘5%0.01<45%f 86..46˘˘006..51.6 ± 0.1 8.5%8<.5˘5%%2 .±4 %2.4%
3e3 e HH OOMMee MMee 33′1--CCNNBBnn 9.2%9.2˘%1 .±0 %1.0% <5% <5% 8.4˘08.3.4 ± 0.3 <5%<5%
3f33 gfe ee OHHM e OOOMMMeee MMMeee CCHH3122CCCl”B≡CnCHH 0.676.009.%6%6˘˘9%10. .1±0%1 00.0f10 f 0.214<˘05.%201.040 8± f0.008 f 41.44˘˘11.114 ± 1 1139%%1˘˘3%12 %%± 1%
3g3h e e OOMMee OOMMee MMee CH3′2CCl”BCnH 7<.50%%f ± 1.1% 0.0028˘0.00<054%f 9.0˘04.5.4 ± 1.1 <159%% ± 2%
3h3 ie e OOMMee OOMMee MHe CH2MCe≡CH <5%<5% f 0<.50%028 ± 0.0004 f 13˘91.0 ± 0.5 10%˘<25%%
3i3 3ekj OOOMMMeee OOOMMMeee HHH 41CMBNneB n 20%<5˘%<35%% <<55%% <5% 28..84˘˘10..1213 ± 1 1237%%10˘˘%21 %%± 2%
3j3 l OOMMee OOMMee HH CH2CBOn NH2 <5%<5% <5% <5% 8.0˘12.2.8 ± 0.2 10%27˘%1 %± 1%
3k3m OOMMee OOMMee HH CH4′2CCNOBNnH Me <250%% ± 3% <5% <5% 7.7˘08.4.4 ± 1.1 11%13˘%1 %± 2%
3l8 OOMMee OOMHe HM e CCHH22CCO”NCHH2 35%˘<25%%f 11%˘1%<f5% 9.3˘08.4.0 ± 1.2 <150%% ± 1%
3m OMe ORDMaosneae gpileiznielH CH2CONHMe 0.412+-0<.152%3 g 0.0044+-0.00<059%g 0.020˘- 07.0.70 2± 0.4 2.31˘1-%0. 1± 1%
8 aValOueMsea remOeaHn ofthMreee indepCeHn2dCe≡nCtHex perimen3t5s;%b ±fr 2o%m fr atbrainh1o1m%o ±g e1n%a tfe s;cfrom9e.3le ±c t0r.i4c eel;dfrom<5%
equineserum;Deotnesetpeedzails hydrochloridesalt;fdetermin-e dafter30minpre-i-n cubation;g0d.0a2ta0 t±a k0.e0n02fr om[129.3] .± 0.1
Rasagiline 0.412 + 0.123 g 0.0044 + 0.0009 g - -
a Values are mean of three independent experiments; b from rat brain homogenates; c from electric
Concerning ChEs’ activities, all tested compounds showed no activity or very low inhibitory
eel; d from equine serum; e tested as hydrochloride salt; f determined after 30 min pre-incubation;
potencytowardsBChE,and3jwasthemostactiveinhibitor(27%inhibitionat10µM).Fortheentire
g data taken from [19].
series, the affinity towards AChE was moderate, and IC values were in the range 2.78–14.4 µM,
50
clearlyindicatingthatthereportedstructuralmodificationsbothonthecoumarinskeletonandonthe
Concerning ChEs’ activities, all tested compounds showed no activity or very low inhibitory
basicmoietyexertedalimitedinfluenceontheactivity. Withinbothsmallseriesofunsubstitutedand
potency towards BChE, and 3j was the most active inhibitor (27% inhibition at 10 µM). For the entire
7-monomethoxycompounds(3a–cand3d–f,respectively),themorehinderedandlipophilicbenzylic
series, the affinity towards AChE was moderate, and IC50 values were in the range 2.78–14.4 µM,
groupwastoleratedbyAChEbetterthanthepropargylandhydrophilicamidegroups(3b>3aand
clearly indicating that the reported structural modifications both on the coumarin skeleton and on
3c,3d,3e>3f). Thepresenceofthehydrogenbondingbenzylaminegroupyieldedthemostpotent
the basic moiety exerted a limited influence on the activity. Within both small series of unsubstituted
AChEinhibitor(3j,IC =2.78µM)belongingtotheclassof6,7-dimethoxyderivatives. Regarding
and 7-monomethoxy c5o0mpounds (3a–c and 3d–f, respectively), the more hindered and lipophilic
MAOsaffinity,withtheexceptionofpropargylamines3c,3fand3h,theinhibitoryactivitiesdisplayed
benzylic group was tolerated by AChE better than the propargyl and hydrophilic amide groups
bycompoundshereinreportedweresurprisinglyverylow,irrespectiveoftheelectronicandlipophilic
(3b > 3a and 3c, 3d, 3e > 3f). The presence of the hydrogen bonding benzylamine group yielded the
featuresofthebasicchainandcoumarinsubstituents. Aninterestingeffectonactivityandisoform
most potent AChE inhibitor (3j, IC50 = 2.78 µM) belonging to the class of 6,7-dimethoxy derivatives.
selectivitycanbeobservedformethoxysubstituentsatpositions6and7,withMAO-Baffinityrising
Regarding MAOs affinity, with the exception of propargylamines 3c, 3f and 3h, the inhibitory
fromunsubstitutedtomono-anddi-methoxycompounds(3c<3f<3h).Interestingly,anoppositetrend
activities displayed by compounds herein reported were surprisingly very low, irrespective of the
wasperformedtowardsMAO-A(3h<3f<3c),wheretheaffinitydropdeterminedtheoutstanding
electronic and lipophilic features of the basic chain and coumarin substituents. An interesting effect
selectivitydisplayedby3h. Thedramaticlossofaffinityfor6-methoxy-7-hydroxycoumarin8might
on activity and isoform selectivity can be observed for methoxy substituents at positions 6 and 7,
beascribedtothepossibilityofforminganintramolecularhydrogenbondresponsibleforthelackof
with MAO-B affinity rising from unsubstituted to mono- and di-methoxy compounds (3c < 3f < 3h).
polarinteractions,whichnegativelyinfluencedthebindingof8withbothMAOisoforms.
Interestingly, an opposite trend was performed towards MAO-A (3h < 3f < 3c), where the affinity
drop determined the outstanding selectivity displayed by 3h. The dramatic loss of affinity for
6-methoxy-7-hydroxycoumarin 8 might be ascribed to the possibility of forming an intramolecular
hydrogen bond responsible for the lack of polar interactions, which negatively influenced the
binding of 8 with both MAO isoforms.
Molecules2016,21,362 5of15
On the basis of the enzymatic screening, Compound 3h came into light as a promising dual
AChE-MMoAlecOule-sB 20i1n6,h 21ib, 3i6t2o rdeservingdeeperinvestigations. Therefore,apreliminaryevalu5 aoft i1o5 nofthe
cytotoxicityandneuroprotectiveefficacyofthishitcompoundwasundertakeninthehumanSH-SY5Y
Molecules 2016, 21, 362 5 of 15
On the basis of the enzymatic screening, Compound 3h came into light as a promising dual
neuroblastomacelllinebyfollowingthe3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide
AChE-MAO-B inhibitor deserving deeper investigations. Therefore, a preliminary evaluation of the
On the basis of the enzymatic screening, Compound 3h came into light as a promising dual
(MTT)assay[22]. TheviabilityofSH-SY5Ycellswasmeasuredafter24hoursincubationwithdifferent
cytotoxicity and neuroprotective efficacy of this hit compound was undertaken in the human SH-SY5Y
AChE-MAO-B inhibitor deserving deeper investigations. Therefore, a preliminary evaluation of the
concentnreautrioobnlassotofm3ah creall nlignien bgy ffroollmow0in.1g– t5h0e 3µ-(M4,5,-adnimdeuthnytltrheiaazteodl-2c-yell)l-s2,5w-deirpeheunsyel-dteatrsaztohleiucmo nbrtoromlid(Fe igure1).
cytotoxicity and neuroprotective efficacy of this hit compound was undertaken in the human SH-SY5Y
Thecom(MpToTu)n adsssahy o[2w2]e. dThae nvieagbliliigtyib olfe ScHy-tSoYt5oYx icceiltlsy waalss omaetastuhreedh iagfthere s2t4 dhoousress inecmubpaltoioyne dwiitnh dthifefeerexnpt eriment.
neuroblastoma cell line by following the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide
Inthesca(oMmncTeeTnS)t Harass-tiaSoyYn [s52 Yo2]f. c 3Thehl rela lvniingaibeni,lgit thfyr eoomfn S e0Hu.1-rS–oY50p5 Yµro Mctee,l lcasnt widva uesn amtcreetaaisvtueidrtey cdeo allffst 3ewrh e2ra4e gh uaosiuenrdss atinst chtuhrbee acetoiopnntrr owo-li at(hFp idogiupffrteeor 1eti)nc. t insults
The compound showed a negligible cytotoxicity also at the highest doses employed in the experiment.
(hydrogceonnpceenrtoraxtiidones, roof t3ehn roanngeinagn dfroomli g0.o1m–5y0 cµiMn-,A an)dw uanstrsetauteddi ecdellisn wseerpe aursaetde atse tshtes c(oFnigtruolr e(F2ig)u.rAe l1l).o fthese
In the same SH-SY5Y cell line, the neuroprotective activity of 3h against three pro-apoptotic insults
modelsTohfen ceoumrpoonuanldd sahmowaegde ai nnedgulicgeibolex ciydtaottoivxiecistytr aelssso cato tnhde ihtiigohnessta dnodseos vemerp-lporyoedd uinc tthioe nexopferriamdeincta. lswith
(hydrogen peroxide, rotenone and oligomycin-A) was studied in separate tests (Figure 2). All of
In the same SH-SY5Y cell line, the neuroprotective activity of 3h against three pro-apoptotic insults
differenthtemsee mchoadneliss omf ns.euInrofnaacl td,aHm2aOge2 inmdauycea ocxtidaastiaver astdreicssa cloinnditiitaiotnosr ,anpdro odveurc-pinrogdoutchtieorn hofa rramdifcuallsr eactive
(hydrogen peroxide, rotenone and oligomycin-A) was studied in separate tests (Figure 2). All of
oxygenwtshiptehes ecd imieffsoe.rdeeOnltsn mofte hncheeuaonrotishnmaeslr .d Ihanam fnaacdgt,e, H bin2oOdtu2h cmera ooytx eaidnctao tainsv eae sr[at2rd3ei]scsaa lc noindnitdioaitltioiogrn,o spm aronyddcu oicnvine-gAr -optbrhoeedhr uhacavtriemonaf usolf m rreaaidtcoitcivcahels o ndrial
toxins bowxyyitgihme dnpi fsafpeirerecininetg sm. Oaectnh dathnifiefs emortseh. neInrt hflaaecnvt,d eH,l sb2Oott2h hme raoryte easncpot inarsea at[ 2or3ar]dy aicncadhl ianoiilntiigaoatomnr,yd pcirtnoh-dAeu cAbineThgP aovtphe eraros h dmaurimctotfciuohl onrne.adcArtiiavtle a 1 µM
toxins by impairing at different levels the respiratory chain and the ATP production. At a 1 µM
concentroaxtyigoenn, 3sphecdieissp. Olany ethde noothienr chraenads,e boofthc erloltesnuornveiv [a23l.] Aanmd ooldigeormatyecinne-Au rboephraovtee casti omnitoacghaoinndsrtiarol tenone
concentration, 3h displayed no increase of cell survival. A moderate neuroprotection against rotenone
andoligtoomxinysc ibny- Aimwpaairsinogb saet rdvifefdereantta lelvoewlse trhceo rnecspeinratrtoartyio cnha(0in.1 aµndM t)h.e ATP production. At a 1 µM
and oligomycin-A was observed at a lower concentration (0.1 µM).
concentration, 3h displayed no increase of cell survival. A moderate neuroprotection against rotenone
and oligomycin-A was observed at a lower concentration (0.1 µM).

FigureF1i.guVriea 1b.i Vlitiaybioliftyh oufm huamnanne nueruorobblalassttoommaa SSHH-S-YS5YY5 cYellcse ilnlscuibnactuedb afotre 2d4 fho wri2th4 Chowmpitoh unCdo 3mh paot und3h
different concentrations (range 0.1–50 µM). Untreated cells were used as the control. Results are
atdifferFeingturceo 1n. cVeinabtrilaittyio onf shu(rmaanng nee0u.r1o–b5la0stµomMa) .SHU-nSYtr5eYa tceedlls cinecllusbwateedr efour s2e4d h awsitthh Ceocmopnoturonld. 3Rhe astu ltsare
expressed as the percentage of viable cells observed after treatment with Compound 3h vs. untreated
expresseddiffaesretnhte cponecrecnetnratatigoneso (fravnigaeb l0e.1c–e5l0ls µoMb)s.e Urvnetrdeaateftde rcetlrles awtmeree nutswedi tahs Cthoem copnoturonl.d R3ehsuvltss. aurne treated
control cells (100%) and shown as the mean ± SD (n = 3).
expressed as the percentage of viable cells observed after treatment with Compound 3h vs. untreated
controlcells(100%)andshownasthemean˘SD(n=3).
control cells (100%) and shown as the mean ± SD (n = 3).

Figure 2. Neuroprotection effect of 3h on the viability of human neuroblastoma SH-SY5Y cells.
VFiiagbuilriety 2 w. Nase mureoapsruorteedct iaofnte re f2f4e cht coof -i3nhc uobna ttihoen vofia SbHili-tSyY 5oYf hcuelmlsa wn itnhe uthroe bnlaesutroomtoax iScH in-SsuYl5tY ( Hc2eOlls2 .
Figure219.5N µeMu,r oolpigroomteycctiino-An e3f0f eµcMt oofr 3rohteonnonteh e75v µiaMb)i laintyd oCfohmupmouannd n3heu art odbiflfaesretontm coanScHen-tSraYti5oYnsc (e1l lµsM.V iability
wasmeaa1Vn9sid5au b0µri.e1lMid tµy, Mo awlfi)gta eoosrr m mi2nye4 catihnsheu-A race bo3ds-0 eia nµnftcMceeur o ob2fra4 3r tohhito e(ccnnooo-noinntferc ouS7l5bH e aµxt-MpiSoeYn)r ia5monYfde Sn cCHteso-l)mSl. sYRp5weoYsuui ntclhtdesl l3tashhr we ea etint xdhpei frutfheerseros eentndoet u xcaroisocn ttchoienexn ispctur eairlnttcisoe(unnHltsta 2((g1HOe µ 22oOMf1 2 95µM,
oligomvyicaibnle-A cel3ls0, aµnMd doartar roetpernesoennet t7h5e mµMean)sa ±n SdDC (no =m 3p).o und3hatdifferentconcentrations(1µMand
and 0.1 µM) or in the absence of 3h (control experiments). Results are expressed as the percentage of
0.1 µM)voirabinle tchelelsa, bansden dcaetao rfe3phres(ecnotn tthreo mleexapnse r±i mSDe n(nt s=) 3.)R. esultsareexpressedasthepercentageofviable
cell s,anddatarepresentthemeans˘SD(n=3).
Molecules2016,21,362 6of15
CNS-active molecules should be able to enter the brain after permeating the blood brain
barrier(BBB)bypassivediffusionandshouldbedevoidofinteractionswithglycoprotein-P(P-gp),
which limits brain uptake and serves as a defensive efflux mechanism of xenobiotics for the CNS.
Brain permeation and P-gp interaction were estimated in a cell-based invitro method using the
Madin-Darby canine kidney (MDCK) cell line [24,25]. After transfection with the human MDR1
cDNA(MDCKII-MDR1),thislineshowsahighexpressionofP-gp(MDR1)andverytightcellular
junctions,thusrepresentingareliableBBBinvitromodel. Bidirectionaltransportfor3hwasevaluated
inapical-to-basal(AP)andbasal-to-apical(BL)directions,andthemeasuredapparentpermeability
values(P AP=2.27ˆ10´5cm/s,P BL2.18ˆ10´5cm/s)weresuperiortothoseofdiazepam
app app
(P AP = 1.46 ˆ 10´5 cm/s, P BL 1.23 ˆ 10´5 cm/s), used as positive CNS-permeant control
app app
(Table2). Aneffluxratio(ER)equalto0.96andmuchlowerthanthethresholdvalue(ER=2)indicates
that 3h is not likely to be a substrate for P-gp. Taken together, these results pointed out that 3h
mightbeconsideredapromising,non-cytotoxicandneuroprotectivehitcompoundendowedwitha
multi-targetprofilebasedonapotentMAO-BinhibitionandamoderateAChEaffinity. Derivative3h
canberegardedasaCNSactiveagentabletorapidlypermeatethetightmonolayerwithoutinteracting
witheffluxsystemsanddeservesfurtherstudiesasananti-Alzheimeragentinanimalmodels.
Table2. Bi-directionaltransportof3handcontrolcompoundsacrossMadin-Darbycaninekidney
(MDCK)II-MDR1cells.
Compd. PappAP(cm/s) PappBL(cm/s) ERaPappBL/PappAP
3h 2.27ˆ10´5 2.18ˆ10´5 0.96
diazepam 1.46ˆ10´5 1.23ˆ10´5 0.84
FD-4 1.03ˆ10´6 2.08ˆ10´7 0.20
aEffluxratio(ER)wascalculatedusingthefollowingequation:ER=PappBL/PappAP,wherePappBListhe
apparentpermeabilityofbasal-to-apicaltransportandPappAPistheapparentpermeabilityofapical-to-basal
transport. Aneffluxratiogreaterthan2indicatesthatatestcompoundislikelytobeasubstrateforP-gp
transport.Fluoresceinisothiocyanate-dextran(FD4)anddiazepamwereusedasparacellularandtranscellular
markers,respectively,ofcellmonolayers’integrityandasaninternalcontroltoverifytightjunctionintegrity
duringtheassay.
3. MaterialsandMethods
3.1. Chemistry
Startingmaterials,reagentsandanalytical-gradesolventswerepurchasedfromSigma-Aldrich
(Milan,Italy).Thepurityofalloftheintermediates,checkedby1H-NMRandHPLC,wasalwaysbetter
than95%. Allofthenewly-preparedandtestedcompoundsshowedanHPLCpurityhigherthan98%.
ColumnchromatographywasperformedusingMercksilicagel60(0.063–0.200mm,70–230mesh).
FlashchromatographicseparationswereperformedonaBiotageSP1purificationsystem(Biotage
SwedenAB,Uppsala,Sweden)usingflashcartridgesprepackedwithKP-Sil32–63µm,60Åsilica.
All reactions were routinely checked by TLC using Merck Kieselgel 60 F aluminum plates and
254
visualizedbyUVlightoriodine.Regardingthereactionrequiringtheuseofdrysolvents,theglassware
wasflame-driedandthencooledunderastreamofdryargonbeforeuse. Elementalanalyseswere
performedonaEuroEA3000analyzer(Eurovector,Milan,Italy)onlyonthefinalcompoundstested
asMAOsandChEsinhibitors. ThemeasuredvaluesforC,HandNagreedtowithin˘0.40%ofthe
theoreticalvalues. Nuclearmagneticresonance(NMR)spectrawererecordedonaVarianMercury
300instrumentat300MHz(Varian,Milan,Italy)atambienttemperatureinthespecifieddeuterated
solvent. Chemicalshifts(δ)arequotedinpartspermillion(ppm)andarereferencedtotheresidual
solventpeak. ThecouplingconstantsJaregiveninHertz(Hz). Thefollowingabbreviationswereused:
s(singlet),d(doublet),dd(doubletofdoublet),t(triplet),q(quadruplet),m(multiplet),brs(broad
signal);signalsduetoOHandNHprotonswerelocatedbydeuteriumexchangewithD O.Melting
2
pointsweredeterminedbythecapillarymethodonaStuartScientificSMP3electrothermalapparatus
(BibbyScientific,Stone,UK)andareuncorrected.
Molecules2016,21,362 7of15
7-Methoxy-3-methyl-2H-chromen-2-one (1b): 7-Hydroxy-3-methyl-2H-chromen-2-one [15] (3.80 g,
20.0mmol)wasdissolvedinanhydrousDMF(70mL),andpotassiumcarbonate(2.76g,20.0mmol)
wasadded. Afterstirringfor1h,methyliodide(1.49mL,24.0mmol)wasdroppedviasyringe. The
mixturewasstirredovernightatroomtemperatureandthenpouredontocrushedice. Afterextraction
withethylacetate(3ˆ250mL),theorganicphaseswerecollectedanddriedoverNa SO . Evaporation
2 4
ofthesolventyieldedthedesiredcoumarin1,whichwasusedwithoutfurtherpurification. Yield:
61%. Spectroscopicandanalyticdataagreewiththosereportedintheliterature[26].
2-Hydroxy-4,5-dimethoxybenzaldehyde: 2,4,5-Trimethoxybenzaldehyde(3.92g,20.0mmol)wasdissolved
indryCH Cl (20mL)beforeaddingdropwiseborontribromide(1.0NinCH Cl ,20mmol,20mL)
2 2 2 2
whilecoolingat0˝C.Thereactionmixturewasslowlywarmedatroomtemperatureandleftunder
vigorousmagneticstirringfor20h. Water(250mL)wascarefullyaddedwithsimultaneouscooling
withanexternalice-bath. Thesuspensionwasstirredfor1handthenpartitionedwithanadditional
amount of CH Cl (80 mL). After extraction with CH Cl (3 ˆ 100 mL), the organic layers were
2 2 2 2
collectedanddriedoverNa SO . Themixturewasconcentratedtodrynessandpurifiedthroughflash
2 4
chromatography(gradienteluent: ethylacetateinn-hexane10%Ñ50%),yieldingayellowsolid. Yield:
78%. 1H-NMR(DMSO-d )δ: 3.71(3H,s,OCH ),3.80(3H,s,OCH ),6.53(1H,s,H-3),7.11(s,1H,H-6),
6 3 3
10.00(s,1H,CHO),10.69(s,1H,disappearingwithD O,OH).
2
6,7-Dimethoxy-3-methyl-2H-chromen-2-one(1c): Toasolutionof2-hydroxy-4,5-dimethoxybenzaldehyde
(3.29 g, 18.0 mmol) in N,N-diethylaniline (60 mL), (carbethoxyethylidene) triphenylphosphorane
(7.18g,19.8mmol)wasadded. Themixturewasstirredatroomtemperaturefor2handthenrefluxed
for3h. Aftercoolingatroomtemperature, HCl(4.0Ninwater, 150mL)wasslowlyaddedwhile
coolingat0˝C,andtheresultingsuspensionwasextractedwithethylacetate(3ˆ80mL).Theorganic
layerswerecollected,driedoverNa SO andconcentratedtodrynessunderrotaryevaporation. The
2 4
resultingcrudeoilresiduewaspurifiedthroughflashchromatography(gradienteluent: ethylacetate
in n-hexane 0%Ñ60%), thus furnishing the desired coumarin 1c as an off-white solid. Yield: 80%.
1H-NMR(DMSO-d )δ: 2.04(3H,s,CH ),3.77(3H,s,OCH ),3.82(3H,s,OCH ),7.03(1H,s,H-8),7.13
6 3 3 3
(s,1H,H-5),7.74(s,1H,H-4).
3-(Bromomethyl)-2H-chromen-2-one(2a): 3-Methyl-2H-chromen-2-one[27]1a(1.60g,10.0mmol)was
suspendedinCCl (10mL),andthenN-bromosuccinimide(1.78g,10.0mmol)andbenzoylperoxide
4
(0.484g,1.00mmol)wereadded. Themixturewasrefluxedfor3.5h,andthesuccinimideresidue
wasfilteredoff. Thedesiredbromidecrystallizedfromthehotsolution. Anadditionalamountof2a
wasobtainedasfollows. Themotherliquorwasconcentratedtodryness, thusfurnishingacrude
solidthatwastreatedwithmethanolandfiltered,yielding2aasayellowsolidwithsatisfactorypurity.
Yield: 76%. 1H-NMR(DMSO-d )δ: 4.55(2H,s,CH Br),7.37–7.40(1H,m,H-6),7.43–7.45(1H,m,H-8),
6 2
7.63–7.66(1H,m,H-7),7.74(1H,dd,J =7.8Hz,J =1.5Hz,H-5),8.30(1H,s,H-4).
1 2
3-(Bromomethyl)-7-methoxy-2H-chromen-2-one (2b): To a stirred suspension of 7-methoxy-3-methyl-
2H-chromen-2-one(1b,1.82g,9.60mmol)inCCl (9.6mL),N-bromosuccinimide(2.05g,11.5mmol)
4
andbenzoylperoxide(0.465g,1.92mmol)wereadded. Afterheatingatrefluxfor2h,thehotreaction
mixturewasfilteredtoremovethesuccinimideby-product,andbromo-derivative2bcrystallizedfrom
CCl asayellowishsolid. Yield: 62%. 1H-NMR(CDCl )δ: 3.89(3H,s,OCH ),4.43(2H,s,CH Br),
4 3 3 2
6.85(1H,d,J=1.9Hz,H-8),6.88(1H,dd,J =8.3Hz,J =2.5Hz,H-6),7.40(1H,d,J=8.8Hz,H-5),
1 2
7.80(1H,s,H-4).
3-(Bromomethyl)-6,7-dimethoxy-2H-chromen-2-one (2c): 6,7-Dimethoxy-3-methyl-2H-chromen-2-one
(1c,1.98g,9.00mmol)wasdissolvedinhotCCl (9mL).N-Bromosuccinimide(1.69g,9.50mmol)was
4
addedfollowedbybenzoylperoxide(0.218g,0.90mmol),whileheating. Thereactionmixturewas
refluxedfor7h. Aftercoolingatroomtemperature,thesolventwasremovedunderreducedpressure.
Theresultingcrudesolidwastreatedwithmethanol,filteredandwashedwithhotethanol,yielding
Molecules2016,21,362 8of15
2casayellowsolid. Yield: 77%. 1H-NMR(CDCl )δ: 3.92(3H,s, OCH ), 3.96(3H,s, OCH ), 4.44
3 3 3
(2H,s,CH Br),6.85(1H,s,H-8),6.86(1H,s,H-5),7.78(1H,s,H-4).
2
Generalprocedureforthesynthesisofamines3a,3land3m: GlyNH ¨HCl(for3a,3l,0.464g,
2
4.20 mmol) or GlyNHCH ¨HCl (for 3m, 0.523 g, 4.20 mmol) were suspended in DMF (4 mL) with
3
triethylamine(0.585mL,4.20mmol)undervigorousmagneticstirringwhileheatingat70˝C.The
suitable bromide 2a (0.143 g, 0.60 mmol) or 2c (0.179 g, 0.60 mmol), previously dissolved in DMF
(2mL),wasaddedportion-wisewithin3.5h(0.250mLevery30min). Themixturewasstirredat70˝C
foranadditional4h. Aftercoolingatroomtemperature,brine(50mL)wasadded,andthemixture
wasextractedwithethylacetate(3ˆ30mL).Theorganiclayerswerecollected,washedwithbrine
(3ˆ20mL),driedoverNa SO andconcentratedunderreducedpressure. Thecrudemixtureswere
2 4
furtherpurifiedasindicatedbelow.
N2-[(2-oxo-2H-Chromen-3-yl)methyl]glycinamide (3a): Purification through flash chromatography
(gradienteluent: methanolinchloroform0%Ñ5%). Yield: 62%. Mp: 149(dec.),159–160˝C.1H-NMR
(DMSO-d )δ: 2.62(1H,s,NH;dis. withD O),3.11(2H,s,CH CO),3.54(2H,s,CH NH),7.07(1H,s,
6 2 2 2
NH;dis. withD O),7.33–7.36(2H,m,NH,H-6;1Hdis. withD O),7.40(1H,d,J=8.3Hz,H-8),7.57
2 2
(1H,t,J=7.8Hz,H-7),7.70(1H,d,J=7.3Hz,H-5),8.00(1H,s,H-4). Anal. C62.41,H5.26,N12.00%,
calcd. forC H N O ,C62.06,H5.21,N12.06%.
12 12 2 3
N2-[(6,7-Dimethoxy-2-oxo-2H-chromen-3-yl)methyl]glycinamide (3l): Purification through flash
chromatography(gradienteluent: methanolinchloroform0%Ñ15%)affordedasolidthatwasfurther
crystallizedfromhotethanol95˝. Yield: 57%. Mp: 176–178˝C.1H-NMR(DMSO-d )δ: 3.09(2H,s,
6
CH CO),3.49(2H,s,CH NH),3.79(3H,s,OCH ),3.84(3H,s,OCH ),7.05(1H,s,H-8),7.06(1H,s,
2 2 3 3
NH; dis. with D O), 7.23 (1H, s, H-5), 7.34 (1H, s, NH; dis. with D O), 7.90 (1H, s, H-4); NH not
2 2
detected. Anal. C57.86,H5.27,N9.24%,calcd. forC H N O ,C57.53,H5.52,N9.58%.
14 16 2 5
N2-[(6,7-Dimethoxy-2-oxo-2H-chromen-3-yl)methyl]-N1-methylglycinamide (3m): Purification through
flashchromatography(gradienteluent: methanolinchloroform0%Ñ10%)affordedasolidthatwas
furthercrystallizedfromhotethanol. Yield: 65%. Mp: 147–149˝C.1H-NMR(DMSO-d )δ: 2.59(3H,d,
6
J=4.7Hz,CH NH),3.10(2H,s,CH CO),3.48(2H,s,CH NH),3.79(3H,s,OCH ),3.84(3H,s,OCH ),
3 2 2 3 3
7.06(1H,s,H-8),7.21(1H,s,H-5),7.82(1H,brq,J=4.7Hz,CH NH;dis. withD O),7.89(1H,s,H-4);
3 2
NHnotdetected. Anal. C59.07,H5.65,N8.93%,calcd. forC H N O ,C58.82,H5.92,N9.15%.
15 18 2 5
General procedure for the synthesis of amines 3b, 3d, 3e and 3g: Triethylamine (0.335 mL,
2.40mmol) and suitable substituted N-benzylmethylamine hydrochlorides [15] (0.72 mmol) were
addedtoasolutionoftheappropriatebromo-intermediate2a–c(0.60mmol)inTHF(5mL).After
stirring at room temperature for 24 h, the inorganic residue was filtered-off, and the solvent was
evaporatedundervacuum. Flashchromatographypurificationaffordedthedesiredcompoundswith
differentelutingconditionsillustratedasfollows.
3-{[(3-Fluorobenzyl)(methyl)amino]methyl}-2H-chromen-2-one hydrochloride (3b): Purification through
flashchromatography(gradienteluent: ethylacetateinn-hexane0%Ñ70%)affordedanoilthatwas
transformed into the corresponding hydrochloride by treatment with the commercially available
solutionofHCl4.0Nindioxane. Yield: 60%. Mp: 236–237˝C.1H-NMR(DMSO-d )δ: 2.71(3H,s,
6
NCH ), 4.16–4.26 (2H, m, CH ), 4.36–4.39 (1H, m, Ar-CHa), 4.51–4.54 (1H, m, Ar-CHb), 7.30–7.33
3 2
(1H, m, H-6), 7.37–7.53 (5H, m, C H , H-8), 7.71 (1H, t, J = 7.3 Hz, H-7), 7.77 (1H, d, J = 7.3 Hz,
6 4
H-5),8.33(1H,s,H-4),10.02(1H,s,NH+;dis. withD O).Anal. C65.02,H4.96,N4.00%,calcd. for
2
C H FClNO ,C64.77,H5.13,N4.20%.
18 17 2
3-{[(3-Fluorobenzyl)(methyl)amino]methyl}-7-methoxy-2H-chromen-2-one(3d): Purificationthroughflash
chromatography (gradient eluent: ethyl acetate in dichloromethane 0%Ñ20%). Yield: 74%. Oil
compound. 1H-NMR(DMSO-d )δ: 2.17(3H,s,NCH ),3.38(2H,s,CH ),3.61(2H,s,Ar-CH ),3.84
6 3 2 2
(3H, s, OCH ), 6.94 (1H, dd, J = 8.8 Hz, J = 2.5 Hz, H-6), 6.98 (1H, d, J = 1.9 Hz, H-8), 7.03–7.07
3 1 2
Molecules2016,21,362 9of15
(1H, m, Ar-CHa), 7.19–7.20 (2H, m, Ar-CHb, Ar-CHc), 7.33–7.37 (1H, m, Ar-CHd), 7.68 (1H, d,
J = 8.8 Hz, H-5), 7.98 (1H, s, H-4). Anal. C 70.03, H 5.59, N 4.39%, calcd. for C H FNO , C
19 18 3
69.71,H5.54,N4.28%.
3-{[[(7-Methoxy-2-oxo-2H-chromen-3-yl)methyl](methyl)amino]methyl}benzonitrile (3e): Purification
throughflashchromatography(gradienteluent: ethylacetateindichloromethane0%Ñ20%)afforded
anoilthatwastreatedwithdiethyletherandfurnishedthedesiredproductasayellowsolid. Yield:
88%. Mp: 123–125 ˝C. 1H-NMR (DMSO-d ) δ: 2.16 (3H, s, NCH ), 3.39 (2H, s, CH ), 3.65 (2H, s,
6 3 2
Ar-CH ), 3.84 (3H, s, OCH ), 6.94 (1H, dd, J = 8.8 Hz, J = 2.4 Hz, H-6), 6.98 (1H, d, J = 2.4 Hz,
2 3 1 2
H-8), 7.53 (1H, t, J = 7.3 Hz, H-5a), 7.67 (1H, d, J = 8.8 Hz, H-5), 7.70–7.72 (2H, m, H-4a, H-6a),
7.81(1H,s,H-2a),7.98(1H,s,H-4). Anal. C72.09,H5.38,N8.15%,calcd. forC H N O ,C71.84,H
20 18 2 3
5.43,N8.38%.
3-{[(3-Chlorobenzyl)(methyl)amino]methyl}-6,7-dimethoxy-2H-chromen-2-onehydrochloride(3g): Purification
throughflashchromatography(gradienteluent: ethylacetateinn-hexane0%Ñ60%)affordedanoil
thatwasdissolvedindioxaneandtransformedintothecorrespondinghydrochloridebytreatment
with the commercially available solution of HCl 4.0 N in dioxane. Yield: 96%. Mp: 229–230 ˝C.
1H-NMR(DMSO-d )δ: 2.65(3H,brs,NCH ),3.81(3H,s,OCH ),3.87(3H,s,OCH ),4.14–4.18(2H,m,
6 3 3 3
CH ),4.29–4.35(1H,m,Ar-CHaN),4.47–4.51(1H,m,Ar-CHbN),7.15(1H,s,H-8),7.23(1H,s,H-2a),
2
7.45–7.58(3H,m,H-4a,H-5a,H-6a),7.73(1H,s,H-5),8.24(1H,s,H-4),10.35(1H,brs,NH+;dis. with
D O).Anal. C58.09,H5.08,N3.27%,calcd. forC H Cl NO ,C58.55,H5.16,N3.41%.
2 20 21 2 4
Generalprocedureforthesynthesisofamines3c,3fand3h:Appropriatebromide2a–c(0.6mmol)
wasdissolvedinTHF(3mL),andK CO (0.083g,0.6mmol)andN-methylpropargylamine(0.10mL,
2 3
1.2mmol)wereadded. Afterstirringfor6hatroomtemperature,theinorganicresiduewasfiltered
off,andtheresultingmixturewaspurifiedasdescribedbelow.
3-{[Methyl(prop-2-yn-1-yl)amino]methyl}-2H-chromen-2-one(3c): Purifiedthroughcrystallizationfrom
ethanol/water. Yield: 46%. Mp: 77–78˝C.1H-NMR(acetone-d )δ: 2.38(3H,s,NCH ),2.75(1H,t,
6 3
J = 2.3 Hz, C”CH), 3.45 (2H, d, J = 2.3 Hz, CH -C”CH), 3.50 (2H, s, CH -N(CH )-CH -C”CH),
2 2 3 2
7.33–7.36(2H,m,H-6,H-8),7.55–7.61(1H,m,H-7),7.70(1H,dd,J =8.2Hz,J =1.8Hz,H-5),7.92
1 2
(1H,s,H-4). Anal. C74.36,H5.55,N6.01%,calcd. forC H NO ,C73.99,H5.77,N6.16%.
14 13 2
7-Methoxy-3-{[methyl(prop-2-yn-1-yl)amino]methyl}-2H-chromen-2-one hydrochloride (3f): Purification
through column chromatography (gradient eluent: ethyl acetate in n-hexane 40%Ñ70%) afforded
anoilthatwastransformedintothecorrespondinghydrochlorideasfollowswiththecommercially
availablesolutionofHCl4.0Nindioxane. Yield: 66%. Mp: 203–205˝C(dec.). 1H-NMR(DMSO-d )
6
δ: 2.76(3H,s,NCH ),3.86(4H,brs,C”CH,OCH ),4.11–4.14(4H,m,CH -NH+-CH ),6.99(1H,dd,
3 3 2 2
J =8.8Hz,J =2.3Hz,H-6),7.07(1H,d,J=2.3Hz,H-8),7.68(1H,d,J=8.2Hz,H-5),8.22(1H,s,H-4),
1 2
10.50(1H,brs,NH+;dis. withD O).Anal. C61.49,H5.49,N4.49%,calcd. forC H ClNO ,C61.33,
2 15 16 3
H5.49,N4.77%.
6,7-Dimethoxy-3-{[methyl(prop-2-yn-1-yl)amino]methyl}-2H-chromen-2-onehydrochloride(3h): Purification
throughflashchromatography(gradienteluent:ethylacetateinn-hexane20%Ñ70%)affordedayellow
solidthatwastransformedintothecorrespondinghydrochloridewiththecommerciallyavailable
solutionofHCl4.0Nindioxane. Yield: 92%. Mp: 223–225˝C.1H-NMR(DMSO-d )δ: 2.76(3H,s,
6
NCH ),3.81(3H,s,OCH ),3.87(3H,s,OCH ),4.11(4H,brs,CH -NH+-CH ),7.15(1H,s,H-8),7.25
3 3 3 2 2
(1H,s,H-5),8.19(1H,s,H-4),10.52(1H,s,NH+;dis. withD O);C”CHnotdetected. Anal. C59.58,H
2
5.41,N4.26%,calcd. forC H ClNO ,C59.35,H5.60,N4.33%.
16 18 4
General procedure for the synthesis of amines 3i, 3j and 3k: The appropriate amine (for 3i:
commerciallyavailablemethylamine2.0NinTHF,1.5mL,3.0mmol;for3j: benzylamine,0.328mL,
3.0mmol;for3k: 4-(aminomethyl)benzonitrilehydrochloride[15],0.506g,3.0mmol)wasdilutedor
suspendedinTHF(2.2mL),andK CO (0.138g,1.0mmol)wasadded. Themixturewaskeptatroom
2 3
Molecules2016,21,362 10of15
temperatureundermagneticstirring,andbromide2c(0.155g,0.50mmol),previouslydissolvedin
THF(2.8mL),wasaddedinsmallaliquotsevery30min(7portions,0.40mLeach). Themixturewas
stirredforanadditional30min. Theinorganicresiduewasfiltered,andtheresultingsolutionwas
concentratedundervacuumandpurifiedthroughflashchromatographyasdetailedbelow.
6,7-Dimethoxy-3-[(methylamino)methyl]-2H-chromen-2-onehydrochloride(3i): Purificationthroughflash
chromatography (gradient eluent: methanol in ethyl acetate 0%Ñ5%) afforded an oil that was
transformedintothecorrespondinghydrochloridewiththecommerciallyavailablesolutionofHCl
1.25Ninethanol. Yield: 92%. Mp: 215–217˝C.1H-NMR(DMSO-d )δ: 2.59(3H,s,NCH ),3.81(3H,s,
6 3
OCH ),3.87(3H,s,OCH ),3.99(2H,s,NCH ),7.15(1H,s,H-8),7.25(1H,s,H-5),8.11(1H,s,H-4),
3 3 2
8.76(2H,brs,NH +;dis. withD O).Anal. C54.90,H5.36,N4.77%,calcd. forC H ClNO ,C54.65,
2 2 13 16 4
H5.64,N4.90%.
3-[(Benzylamino)methyl]-6,7-dimethoxy-2H-chromen-2-one(3j):Purificationthroughflashchromatography
(gradient eluent: methanol in ethyl acetate 0%Ñ5%). Yield: 88%. Mp: 112–114 ˝C. 1H-NMR
(DMSO-d ): δ2.62(1H,brs,NH),3.49(2H,s,NCH ),3.74(2H,s,Ar-CH ),3.79(3H,s,OCH ),3.83
6 2 2 3
(3H,s,OCH ),7.05(1H,s,H-8),7.27(1H,s,H-5),7.19–7.38(5H,m,Ph),7.92(1H,s,H-4). Anal. C70.23,
3
H5.72,N4.17%,calcd. forC H NO ,C70.14,H5.89,N4.31%.
19 19 4
4-({[(6,7-Dimethoxy-2-oxo-2H-chromen-3-yl)methyl]amino}methyl)benzonitrile(3k): Purificationthrough
flash chromatography (gradient eluent: methanol in ethyl acetate 0%Ñ5%). Yield: 93%. Mp:
146–148˝C. 1H-NMR(DMSO-d )δ: 3.49(2H,s,NCH ),3.79(3H,s,OCH ),3.83–3.85(5H,m,Ar-CH ,
6 2 3 2
OCH ),7.06(1H,s,H-8),7.25(1H,s,H-5),7.58(2H,d,J=8.3Hz,H-3a,H-5a),7.78(2H,d,J=8.3Hz,
3
H-2a,H-6a),7.90(1H,s,H-4);NHnotdetected. Anal. C68.88,H5.06,N7.94%,calcd. forC H N O ,
20 18 2 4
C68.56,H5.18,N8.00%.
2,4-Dihydroxy-5-methoxybenzaldehyde (4) [18]: In a flame-dried round-bottom flask, dry AlCl
3
(8.00 g, 60.0 mmol) was suspended in dry dichloromethane (30 mL), and a solution of
2,4,5-trimethoxybenzaldehyde(1.96g,10.0mmol)indrydichloromethane(20mL)wasaddeddropwise
whilecoolingat0˝Cwithanexternalicebath. Themixturewasthenrefluxedfor4handpoured
ontocrushedice(100g). ConcentratedHCl(10mL)wascarefullyadded,andtheresultingsuspension
was vigorously stirred for additional 15 min. After extraction with dichloromethane (3 ˆ 50 mL),
the organic layers were collected and dried over Na SO . The solvent was removed under rotary
2 4
evaporation,andtheresultingcrudewascrystallizedfromhottoluene. Yield: 75%. Spectroscopicand
analyticdataagreewiththosereportedintheliterature[18].
7-Hydroxy-6-methoxy-3-methyl-2H-chromen-2-one(5): Aldehyde4(0.841g,5.00mmol)wasdissolved
indryo-xylene(25mL)beforetheadditionof(carbethoxyethylidene)triphenylphosphorane(2.72g,
7.50 mmol). After heating at reflux for 12 h, the solvent was evaporated under vacuum. Column
chromatography purification (eluent: ethyl acetate in n-hexane 40%, v/v) afforded the desired
coumarin. Yield: 56%. 1H-NMR(acetone-d )δ: 2.09(3H,s,CH ),3.90(3H,s,OCH ),6.79(1H,s,H-8),
6 3 3
7.12(1H,s,H-5),7.65(1H,s,H-4);OHnotdetected.
6-Methoxy-3-methyl-2-oxo-2H-chromen-7-yl benzoate (6): Intermediate 5 (0.516 g, 2.50 mmol), was
dissolvedindryTHF(20mL),andtriethylamine(0.522mL,3.75mmol)wasadded. Thesolutionwas
cooledat0˝C,andbenzoylchloride(0.435mL,3.75mmol)wasaddeddropwise. After10min,the
coolingbathwasremoved,andthemixturewasstirredatroomtemperaturefor5h. Theresulting
suspensionwasdilutedwithdichloromethane(100mL)andwashedwithsaturatedaqueousNaHCO
3
(3ˆ40mL).TheorganicphasewasdriedoverNa SO andfiltered,andthesolventwasevaporated
2 4
undervacuum. Theresiduepurifiedthroughflashchromatography(gradienteluent: ethylacetatein
n-hexane0%Ñ40%). Yield: 59%. 1H-NMR(CDCl )δ: 2.84(3H,s,CH ),3.87(3H,s,OCH ),7.33(1H,s,
3 3 3
H-8),7.61(2H,t,J=7.8Hz,H-3a,H-5a),7.73–7.77(2H,m,H-5,H-4a),7.96(1H,s,H-4),8.18(2H,d,
J=7.3Hz,H-2a,H-6a).