Novel Group of AChE Reactivators—Synthesis, In Vitro Reactivation and Molecular Docking Study

16 Pages·2017·2.28 MB·English

molecules
Article
Novel Group of AChE Reactivators—Synthesis,
In Vitro Reactivation and Molecular Docking Study
DavidMalinak1,2,EugenieNepovimova1,DanielJun2,3,KamilMusilek1,2,* and
KamilKuca1,*
1 DepartmentofChemistry,FacultyofScience,UniversityofHradecKralove,
Rokitanskeho62,50003HradecKralove,CzechRepublic;[email protected](D.M.);
[email protected](E.N.)
2 UniversityHospitalinHradecKralove,Sokolska581,50005HradecKralove,CzechRepublic;
[email protected]
3 DepartmentofToxicologyandMilitaryPharmacy,FacultyofMilitaryHealthSciences,
UniversityofDefence,Trebesska1575,50001HradecKralove,CzechRepublic
* Correspondence:[email protected](K.M.);[email protected](K.K.);Tel.:[phone](K.K.)
(cid:1)(cid:2)(cid:3)(cid:1)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:1)
(cid:1)(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)
Received:15August2018;Accepted:5September2018;Published:7September2018
Abstract:Theacetylcholinesterase(AChE)reactivators(e.g.,obidoxime,asoxime)becameanessential
partoforganophosphorus(OP)poisoningtreatment,togetherwithatropineanddiazepam. Theyare
referredtoasacausaltreatmentofOPpoisoning,becausetheyareabletosplittheOPmoietyfrom
AChEactivesiteandthusrenewitsfunction. Inthisapproach,fifteennovelAChEreactivatorswere
determined. TheirmoleculardesignoriginatedfromformerK-oximecompoundsK048andK074
withremainingoximepartofthemoleculeandmodifiedpartwithheteroareniummoiety. Thenovel
compounds were prepared, evaluated invitro on human AChE (HssAChE) inhibited by tabun,
paraoxon,methylparaoxonorDFPandcomparedtocommercialHssAChEreactivators(pralidoxime,
methoxime, trimedoxime, obidoxime, asoxime) or previously prepared compounds (K048, K074,
K075,K203). SomeofpresentedoximereactivatorsshowedpromisingabilitytoreactivateHssAChE
comparableorhigherthantheusedstandards. Themolecularmodellingstudywasperformedwith
one compound that presented the ability to reactivate GA-inhibited HssAChE. The SAR features
concerningtheheteroareniumpartofthereactivator’smoleculearedescribed.
Keywords: organophosphate;acetylcholinesterase;reactivation;oxime;invitro;moleculardocking
1. Introduction
Organophosphorusagents(OP)areamongthemosttoxiccompoundsdevelopedbyhumans.
Theywereoriginallydiscoveredaspesticides(e.g.,paraoxon,methylparaoxon;Figure1),butthey
werefurtherintroducedasnerveagents(e.g.,tabun,sarin,soman,VX;Figure1),withhighlylethal
effects[1]. SomeOPsarealsoutilizedasindustrialagents(e.g.,flameretardants). Theirmechanism
of action is based on the irreversible inhibition of cholinesterases [2]. The toxic effect is caused by
inhibitionofacetylcholinesterase(AChE),whileinhibitedbutyrylcholinesterase(BChE)takespart
as an OP bioscavenger [3]. The inhibited AChE cannot fulfil its native function of degrading the
neurotransmitteracetylcholine.Thus,acetylcholineisaccumulatedinthesynapses,causingpermanent
stimulationofthecholinergic(muscarinicornicotinic)receptors.Forthisreason,cholinergicsymptoms
(e.g.,miosis,salivation,lacrimation,breathdepression)occurthatmayworsentocholinergiccrisis
ordeathfromthemalfunctionofbreathingmusclesandthedepressionofthebreathingcenterinthe
centralnervoussystem[4].
Molecules2018,23,2291;doi:10.3390/molecules23092291 www.mdpi.com/journal/molecules
MMoolleeccuulleess 22001188,, 2233,, 2x2 F9O1R PEER REVIEW 22o off1 167
Molecules 2018, 23, x FOR PEER REVIEW 2 of 17

Figure 1. Selected organophosphorus compounds.
FFiigguurree 11.. SSeelleecctteedd oorrggaannoopphhoosspphhoorruuss ccoommppoouunnddss..
OP intoxication is usually managed using two treatment strategies. The first pre-treatment
OOPP iinnttooxxiiccaattiioonn iiss uussuuaallllyy mmaannaaggeedd uussiinngg ttwwoo ttrreeaattmmeenntt ssttrraatteeggiieess.. TThhee ffiirrsstt pprree--ttrreeaattmmeenntt
strategy for first responders (e.g., soldiers, medical staff) consists in the protection of the organism
ssttrraatteeggyy foforr firfisrts rtesrpesopnodnedrse r(se.g(.e, .gso.,ldsioerlds,i emrse,dimcael dsitcaaffl) sctoanffs)istcso inns itshtes pinrottehcetiopnr ootfe tchteio onrgoafnitshme
against OP attack. Pre-treatment can be performed using reversible AChE inhibitors (e.g.,
aogrgaainnsits mOaPg aaitntastckO. PParett-atrceka.tmPernet- trceaantm been tpcearnfobrempeder fuorsminegd ruevsienrgsibrelev eArsCibhleE AinChhiEbitionrhsi b(ieto.gr.s,
pyridostigmine chloride), stoichiometric scavengers (e.g., recombinant BChE) or catalytic
(pey.gri.d, opsytirgidmoisnteig mchinloericdhel)o,r idsteo),icshtiooimcheitormic etsrcicavsecnagveernsg e(res.g(.e, .gr.,ecroemcobminbainnta ntBCBhCEh)E )oorr ccaattaallyyttiicc
bioscavengers (e.g., phosphotriesterase, paraoxonase) [5,6]. The second post-treatment strategy
bbiioossccaavveennggeerrss (e(.eg.g.,.p, hpohsopshpohtroitersiteesrtaesrea,spea, rpaoaxraoonxaosen)a[s5e,)6 ].[5T,6h]e. sTechoen dsepcoosntd-t repaotsmt-etnretasttmraetengty sctorantseisgtys
consists of managing already-OP-intoxicated organisms. The symptomatic post-treatment is focused
coofnmsiasntsa goifn mgaanlraegaidnyg -aOlrPe-aindtyo-xOicPa-tinedtooxricgaatneids morsg.aTnhisemsys.m Tphteo smyamtipctopmosatt-itcr epaotmst-etnretaitsmfoecnut sies dfoocnustehde
on the amelioration of the OP intoxication symptoms [2]. Atropine (parasympatholytic drug) and
oamn ethlieo raamtioenlioorfatthioenO oPf itnhteo xOicPa tiinontosxyicmatpiotonm ssy[m2]p.tAomtros p[i2n]e. A(ptarorapsiynme p(paathraoslyytmicpdartuhgo)lyatnicd ddriuagze) paanmd
diazepam (anticonvulsive drug), connected to supportive care, are usually used for this purpose [4].
(dainatziecponamvu (lasinvteicdornuvgu)l,scivone ndercutged), tcoonsunpecptoerdt itvoe scuaprep,oarrteivues cuaarlely, aursee udsfuoarlltyh iusspeudr fpoors teh[i4s ]p.uTrhpeocsaeu [s4a]l.
The causal post-treatment is provided by oxime reactivators (e.g., pralidoxime, asoxime, obidoxime,
pTohset -ctareuastaml penotsti-strperaotvmideendt ibsy porxoivmideerde abcyti voaxtiomrse (ree.agc.,tipvraatloidrso x(eim.ge.,, parsaolxidimoxe,imobei,d aosxoixmime,et,r iombieddooxxiimmee,,
trimedoxime, methoxime; 1–5; Figure 2) via nucleophilic attack of the oximate towards the
tmriemtheodxoixmime;e1, –5m;eFtihgouxriem2e);v i1a–n5;u cFliegouprhei li2c) atvtiaac knoufctlheoepohxiilmic ataetttaocwk arodf stthhee OoxPi-mAaCthe Etocowmarpdlesx t[h7e].
OP-AChE complex [7]. Such drugs are able to split the OP moiety from the AChE active site and
OSuPc-hAdCrhuEg scoamrepalbelxe [t7o].s pSulicthth derOugPsm aoreie atyblfer otom stphleitA thCeh EOPac mtivoeiestiyte fraonmd rtehset oAreCihtsE naacttiivvee fsuintec taionnd.
restore its native function. The reactivation depends on many factors, e.g., OP dose, type of
Trehsetorreea cittisv antaiotinved efpuenncdtisoonn. mThaen yrefaaccttiovrast,ieo.ng .,dOepPednodsse ,otny pmeoafnrye afcatcitvoartso, rea.ngd., iOtsPp odtoesnec,y taygpaei nosft
reactivator and its potency against certain OP, delay of reactivation treatment [8]. In fact, there is not
rceeartcatiivnaOtoPr, adnedla iytso pforteeancctyiv aagtiaoinnsttr eceartmtaienn Ot[P8,] .dIenlafya cotf, rtehaecrteivisatnioont atresiantmgleenrte [a8c]t.i vIna tfoarcta, vthaeilraeb ilse nfoort
a single reactivator available for a broad variety of OP compounds. The treatment is made even
aa bsirnogalde vraeraicettiyvaotfoOr Pavcaoimlabploeu fnodr sa. Tbhroeatdre avtamrieentyt iosfm OaPd ecoevmepnomunodres.c Tomhep tlirceaattemdebnyt tihs empardoec eesvseonf
more complicated by the process of aging [9]. The OP moiety bound to the cholinesterase active site
magoinreg c[o9m].pTlihceatOedP bmyo tiheety pbroocuenssd otfo atghiengch [o9]l.i nTehsete OraPs emaociteitvye bsoituenids ftuo rtthhee rchcooloinrdeisnteartaesde vaicatilvoes ssiotef
is further coordinated via loss of one ester moiety (O-dealkylation). Negatively charged oxygen
iosn feuersttheerrm coooiertdyin(Oat-edde avlkiay llaotsiso no)f. Noneeg aetsivteerl ymcohiaertgye d(Oo-xdyegaleknyalaptpioena)r.s ,Naengdattihveelnyu cclheaorpgheidli cooxxyigmene
appears, and the nucleophilic oxime reactivator is further ineffective. The aging process is well
arepapcetiavrast, oarnids futhreth neruicnleeoffpehctiilvice .oTxhimeea grienagctpivroactoesr siiss fwuertlhldero ciunmeffeenctteivdef.o Trhnee ravgeiangge nptrso(coevsse risa twimelel
documented for nerve agents (over a time scale from minutes to hours), but some pesticides (e.g.,
dscoacluemfroenmtemd infourt ensetroveh oaugresn),tsb u(otvseorm ae tpimeseti csicdaeles (fero.gm., mmeitnhuatmesi dtoo phhoousr)sm), abyuat lssoombee dpeeasltkicyildaetes d(ein.g.a,
methamidophos) may also be dealkylated in a similar manner [10].
msimetihlaarmmidaonpnheors[)1 0m].ay also be dealkylated in a similar manner [10].

FFiigguurree 22.. CCoommmmeerrcciiaallllyy aavvaaiillaabbllee AACChhEE rreeaaccttiivvaattoorrss..
Figure 2. Commercially available AChE reactivators.
Thoughtheasoxime(2)wasformerlyfoundtobethemostbroad-spectrumoximereactivator
Though the asoxime (2) was formerly found to be the most broad-spectrum oxime reactivator
fornTerhvoeuagghe tnhtep aosiosoxnimineg (s2,)i twisasn ofotrambelerltyo fcoouunndt etroa cbte, eth.ge. ,mtaobsut nbrooraOd-Psppeecsttriucimde oixnitmoxe irceaaticotinva[1to1]r.
for nerve agent poisonings, it is not able to counteract, e.g., tabun or OP pesticide intoxication [11].
Tfohru ns,ermvaen aygsecnite nptoifiiscotneianmgss, airt eist rnyoint gabtoled teov ceoloupntreeraacctitv, aet.go.r,s tfaobrunne rovre OaPg epnets-itnichidibei tiendtoAxCichaEtioann d[1/1o]r.
Thus, many scientific teams are trying to develop reactivators for nerve agent-inhibited AChE
TOhPups,e smticaindye pscoiiesnotnifinicg .teRaemcesn talrye, btorythinqgu atote rdneavreyloapn drenaocnt-ivqautaotresr nfaorry nAeCrvheE aregaecntti-vinathoirbsitheadv eAbCeheEn
and/or OP pesticide poisoning. Recently, both quaternary and non-quaternary AChE reactivators
aconnds/iodre OrePd fpoersptiocisdseib lpeomisoannaingge.m ReencteonftlOy,P biontthox qicuaattioernn,ainryc luanddin gnotanb-quunaetxerpnoasruyr eA[C12h,1E3 r].eactivators
have been considered for possible management of OP intoxication, including tabun exposure [12,13].
have been considered for possible management of OP intoxication, including tabun exposure [12,13].
Molecules2018,23,2291 3of16
MMoolleeccuulleess 22001188,, 2233,, xx FFOORR PPEEEERR RREEVVIIEEWW 33 ooff 1177
222... DDDeeesssiiigggnnn aaannnddd SSSyyynnnttthhheeesssiiisss MMMooonnnooo---OOOxxxiiimmmeee BBBiiissspppyyyrrriiidddiiinnniiiuuummm RRReeeaaaccctttiiivvvaaatttooorrrsss
TTThhheee dddeeevvveeelllooopppmmmeeennnttt ooofff mmmooonnnooo--o-ooxxxiimmimeee AAACCChhhEEE rreeraaecacttciitvviavattaootrorssr swwwiitthhit htteettterraatrmmameetthehtyyhllyeennleeen lleiinnlkkineekrr eiirss didseedssccerrsiicbbreeiddb eiidnn
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rrreeeaaaccctttiiivvvaaatttooorrrsss wwwiiittthhh (((EEE)))---bbbuuuttt---222---eeennn---111,,,444---dddiiiyyylll (((666–––777))) aaannnddd bbbuuuttt---111,,,444---dddiiiyyylll (((888–––999))) llliiinnnkkkaaagggeee,,, wwwhhhiiiccchhh ssshhhooowwweeeddd sssooommmeee
ppprrrooommmiiisssiiinnnggg cccooommmpppooouuunnndddsss fffooorrr fffuuurrrttthhheeerrr ssstttuuudddyyy bbbyyy iiinnn vvviiitttrrrooo aaannnddd iiinnn vvviiivvvooo eeexxxpppeeerrriiimmmeeennntttsss ( ((FFFiiiggguuurrreee 3 33))) [ [[111444,,1,11555]]]...

FFFiiiggguuurrreee 333... PPPrrrooommmiiisssiiinnnggg fffooorrrmmmeeerrrlllyyy dddeeevvveeelllooopppeeeddd AAACCChhhEEE rrreeeaaaccctttiiivvvaaatttooorrrsss...
TTThhheee nnneeerrrvvveee aaagggeeennnttt tttaaabbbuuunnn (((GGGAAA))) rrreeemmmaaaiiinnnsss ttthhheee mmmaaaiiinnn OOOPPP ooofff rrreeeaaaccctttiiivvvaaatttiiiooonnn iiinnnttteeerrreeesssttt,,, fffooollllllooowwweeeddd bbbyyy ttthhheee
ssseeellleeecccttteeeddd OOOPPP pppeeessstttiiiccciiidddeeesss... TTThhheee ppprrreeevvviiiooouuusss ssstttrrruuuccctttuuurrraaalll fiffiinnndddiiinnngggsss ssshhhooowwweeeddd ttthhhaaattt mmmooonnnooo---oooxxxiiimmmeee bbbiiissspppyyyrrriiidddiiinnniiiuuummm
cccooommmpppooouuunnndddsss (((666,,, 999))) mmmaaayyy hhhaaavvveee sssiiimmmiiilllaaarrr rrreeeaaaccctttiiivvvaaatttiiiooonnn aaabbbiiillliiitttiiieeesss aaagggaaaiiinnnsssttt tttaaabbbuuunnn---iiinnnhhhiiibbbiiittteeeddd AAACCChhhEEE aaasss ttthhheee kkknnnooowwwnnn
bbbiiisss---oooxxxiiimmmeeesss (((333–––444))) [[11[616,6,11,717]7]..] F.FuurrFtthuheertrrhmmeoormrree,o, trthhe,ee ctchooennnnceeoccnttiinooennc tlliiinonnkkaaglgieen kaaatt gaae lleeanntggttahh looeffn 33g––th44 CCo--fCC3 bb–oo4nndCdss- C bbeebttwwoneeedenns
tbtwwetoow ppeyeynrriiddtwiinnoiiuupmmy rmmidooiniieeitutiimeess mwwoaasise tcciooennssswiiddaeesrreecddo nttsooi dbbeeer eoodpptttiiommbaaell offooprrt iGmGAAal rrfeeoaarccGttiivAvaattriieooannc t[[i1v188a]]t..i oFFnoorr[ 1tt8hh]ii.ss Frroeeraasstoohnnis,,
prperraoosppo--n11,,,33p--ddroiiyypll-,, 1,bb3uu-dtt--i11y,,l44,--ddbiiuyytll- 1aa,4nn-ddd iy((EEl))a--nbbudutt--(22E--)ee-nnb--u11t,,-442---ddeiinyy-ll1 ,ll4iinn-dkkieeyrrlss liwwnkeeerreers ppwrreeevrveiioopuurssellyyv iouuussseelddy u[[11s44e,,d1155[]]1.. 4T,T1hh5ee].
Tbbuhutet--11b,,u44--tdd-1iiy,y4ll- dlliiinnykklaalggineek wwagaaess wtthhaees sstpphaaeccseeprr attchheaartt tbbheeassttt bffeuuslltffiifllluleelddfi lttlhhedee fftoohuuennfdodu llneenndgglttehhn gccrrtihitteecrrriiiootnenr.. iAoAndd.ddAiittidioodnniaatilllolyyn,, attlhhlyee,
btbhiises--booixxsii-mmoxeei mccooemmcpopmoouupnnoddu nwwdiittwhh ibbthuutbt--u11,t,44-1--dd,4ii-yydlli ylliilnnlkkineekrr e((r77))( 7ww)awassa ppsrrpeervveiivooiuuossullsyyl yffoofuuounndndd ttooto bbbeee aaa pppooottteeennnttt rrreeeaaaccctttiiivvvaaatttooorrr ooofff
GGGAAA---iiinnnhhhiiibbbiiittteeeddd AAACCChhhEEE,,, aaalllttthhhooouuuggghhh iiittt rrreeesssuuulllttteeeddd iiinnn iiinnncccrrreeeaaassseeeddd tttoooxxxiiiccciiitttyyy ddduuurrriiinnnggg ttthhheee iiinnn vvviiivvvooo ssstttuuudddiiieeesss [ [[111999,,2,22000]]]...
FFFooorrr ttthhheee aaabbbooovvveee---mmmeeennntttiiiooonnneeeddd rrreeeaaasssooonnnsss,,, bbbiiisssqqquuuaaattteeerrrnnnaaarrryyy mmmooonnnooo---oooxxxiiimmmeee cccooommmpppooouuunnndddsss wwwiiittthhh bbbuuuttt---111,,,444---dddiiiyyylll
llliiinnnkkkaaagggeee aaannndddv avvraayrriyyniignnggs e cssoeecncoodnnpddy rppidyyirrniiddiuiinnmiiuurmmin grrioinnrggm ooorrie tmmyoooiineetttyyh eoosnne cttohhneed sspeeyccrooindnddin ipupymyrriirddiniinngiiuuwmme r erriidnneggs igwwneeerrdee
(dd1ee0ss–ii2gg4nn;eeFddi g((11u00r–e–22444);;. FFiigguurree 44))..

FFFiiiggguuurrreee 444... DDDeeesssiiigggnnn ooofff bbbiiisssqqquuuaaattteeerrrnnnaaarrryyy mmmooonnnooo---oooxxxiiimmmeee rrreeeaaaccctttiiivvvaaatttooorrrsss wwwiiittthhh bbbuuuttt---111,,,444---dddiiiyyylll llliiinnnkkkaaagggeee...
Thissecondmoietywasdesignedinthe4-positionofthesecondpyridiniumringtoallowthe
TThhiiss sseeccoonndd mmooiieettyy wwaass ddeessiiggnneedd iinn tthhee 44--ppoossiittiioonn ooff tthhee sseeccoonndd ppyyrriiddiinniiuumm rriinngg ttoo aallllooww tthhee
reactivatormoleculetopenetrateintothenarrowAChEactivesite. Thenon-oximemoietieswere
rreeaaccttiivvaattoorr mmoolleeccuullee ttoo ppeenneettrraattee iinnttoo tthhee nnaarrrrooww AACChhEE aaccttiivvee ssiittee.. TThhee nnoonn--ooxxiimmee mmooiieettiieess wweerree
chosenasrepresentativesofhydrophilic(e.g.,hydroxymethyl,methylcarbonyl,carboxyl,ethylcarboxyl,
cchhoosseenn aass rreepprreesseennttaattiivveess ooff hhyyddrroopphhiilliicc ((ee..gg..,, hhyyddrrooxxyymmeetthhyyll,, mmeetthhyyllccaarrbboonnyyll,, ccaarrbbooxxyyll,,
nitrile,amidoxime;18–24)andhydrophobic(e.g.,methyl,tert-butyl,phenyl,benzyl;14–17)nature,
eetthhyyllccaarrbbooxxyyll,, nniittrriillee,, aammiiddooxxiimmee;; 1188––2244)) aanndd hhyyddrroopphhoobbiicc ((ee..gg..,, mmeetthhyyll,, tteerrtt--bbuuttyyll,, pphheennyyll,, bbeennzzyyll;;
orthewholepyridiniummoietywasreplacedbyanotherheteroareniumring(e.g., pyridazinium,
1144––1177)) nnaattuurree,, oorr tthhee wwhhoollee ppyyrriiddiinniiuumm mmooiieettyy wwaass rreeppllaacceedd bbyy aannootthheerr hheetteerrooaarreenniiuumm rriinngg ((ee..gg..,,
quinolinium,isoquinolinium;10–13). Theselectedchangesweredesignedtoestablishmorepossible
ppyyrriiddaazziinniiuumm,, qquuiinnoolliinniiuumm,, iissooqquuiinnoolliinniiuumm;; 1100––1133)).. TThhee sseelleecctteedd cchhaannggeess wweerree ddeessiiggnneedd ttoo eessttaabblliisshh
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Molecules 2018, 23, x FOR PEER REVIEW 4 of 17
more possible interactions between reactivator and AChE, whether the π–cationic, π-π or hydrogen
interactions between reactivator and AChE, whether the π–cationic, π-π or hydrogen bonding
bonding interactions were the most common ones [21].
interactionswerethemostcommonones[21].
The mono-oxime reactivators with tetramethylene linker were synthesized via a standard
Themono-oximereactivatorswithtetramethylenelinkerweresynthesizedviaastandard2-step
2-step strategy (Scheme 1) [15]. The monoquaternary compound were prepared in 5 molar excess of
strategy (Scheme 1) [15]. The monoquaternary compound were prepared in 5 molar excess of the
the alkylating agent (1,4-dibromobutane). The monoquaternary compound was further separated
alkylatingagent(1,4-dibromobutane). Themonoquaternarycompoundwasfurtherseparatedfromthe
from the side bisquaternary side product by crystallization from acetonitrile (MeCN), where the
sidebisquaternarysideproductbycrystallizationfromacetonitrile(MeCN),wherethebisquaternary
bisquaternary side product was almost insoluble. Secondly, the synthesis of mono-oxime reactivator
sideproductwasalmostinsoluble. Secondly,thesynthesisofmono-oximereactivatorwascompleted
was completed by the addition of appropriate heterocyclic agent (1.5 molar excess). The
bytheadditionofappropriateheterocyclicagent(1.5molarexcess). ThecrystallizationofMeCNwas
crystallization of MeCN was used again to give non-soluble bisquaternary reactivator (10–24) in a
usedagaintogivenon-solublebisquaternaryreactivator(10–24)inasatisfactorypurityandyield.
satisfactory purity and yield.

ScShcehmemee1 .1.S Syynnththeessisiso off mmoonnoo--ooxxiimmee rreeaaccttiivvaattoorrss wwiitthh tteettrraammeeththyylelennee lilninkkere.r .
3.3R. eRseuslutsltas nadndD Disicsucusssisoionn
3.1. HssAChEReactivationResults
3.1. HssAChE Reactivation Results
ThereactivationexperimentswereperofrmedonhumanrecombinantAChE(HssAChE)inhibited
The reactivation experiments were perofrmed on human recombinant AChE (HssAChE)
byintahbibuinte(dG bAy) ,tpabaurano (xGoAn)(,P pOaXra)o,xmoent h(PyOlpXa)r,a moxeothny(lMpaerPaOoxXo)no (rMdieiPsoOpXr)o opry dlfliiusoorporpohpoyslfpluhoarteop(DhoFsPp)huastien g
the(DsFtaPn) duasrindgE tlhlme satnanpdraortdo cEolllm[2a2n] .pGroAtowcoals [2se2l]e. cGtAed waasst hseelencetrevde aas gtehne tnoefrvme aaigneinntt oerf emsta.iTn hinetcehreosste. n
OPThpee scthicoisdeens O(PPO pXes,tMiciedPeOs X(P)OaXnd, MtheePOmXim) aicndag tehne tm(DimFiPc) awgeenret (uDsFedP), awsetrhee uysaedre, adsi stthineyct amree dmisbteinrscto f
themOemPbfaemrs iolyf twhiet hOrPe mfaaminiliyn gwditihm reetmhyailn-(iMnge PdOimXe)t,hdyile-t(hMyelP-(OPOX)X, )doierthdyiils-o(PpOroXp)y ol-r( DdiFisPo)psrcoapfyfol-l(dDsFaPft)e r
ACschaEffoinldhsi baifttieorn A.CThhEe rineahcibtiivtiaotnio. Tnhree sruealtcstifvoartiaolnl treesstueldts cfoomr apllo tuesntdeds acroemlpisoteudndins aTraeb lliest1e.d in Table 1.
Thereactivationresultsfortabun-inhibitedHssAChEshowedextensivedifferences,eveninthe
groupofcommercialandknowncompounds[23]. Amongthese,pralidoxime(1),asoxime(2)and
methoxime(5)showedpoorreactivationability[23]. Ontheotherhand,obidoxime(3),trimedoxime
(4)andcompounds6–9displayedincreasedreactivation,withthebestresultsbeingobtainedforK203
(9;48%)at100µMandK074(7;25%)at10µMconcentration. Takentogetherwithtoxicityissues,some
previouslyknownAChEreactivators(e.g.,9)haveimprovedreactivationabilitytowardstabunboth
invitroandinvivo[24]. Thenewlypreparedcompoundswerepreparedwiththeideaofimproved
tabun-inhibitedHssAChEreactivation. However,thisobjectivewasnotfulfilledonthebasisofthe
invitrodataforbothtestedconcentrations,withtheexceptionofcompound23. Reactivator23(13%)
showedbetterresultsthancommercialcompound3(8%)at10µMconcentration,whichtendstobe
attainableinbloodafterpossibleinvivoadministration,butitsactivitywasfoundtobeworsethan
previouslyknowncompounds4(17%),7–9(25%,18%,21%)[25].
Molecules2018,23,2291 5of16
Table1.Reactivationresultsofcommercial,formerlypublishedandnewlypreparedAChEreactivators.
Non-Reactivating GA-HssAChEReactivation POX-HssAChE MePOX-HssAChE DFP-HssAChE HssAChE
Moiety (%±SD) Reactivation(%±SD) Reactivation(%±SD) Reactivation(%±SD) IC50±SD
Compound 100µM 10µM 100µM 10µM 100µM 10µM 100µM 10µM µM
1(2-PAM) - 3.3±0.5 2.4±0.2 10.7±0.3 2.1±0.1 30.2±0.3 22.4±0.7 1.3±0.6 0.1±0.4 878±171
2(HI-6) CONH2 0.9±0.6 0.8±0.3 6.2±0.6 1.7±0.1 13.6±0.2 17.9±0.4 0.7±0.1 1.4±0.1 203±39
3(obidoxime) CH=NOH 15.1±0.9 7.9±0.5 59.7±1.0 22.4±0.4 61.7±0.3 45.3±0.9 7.6±0.7 3.3±0.4 577±113
4(trimedoxime) CH=NOH 31.5±1.2 16.9±0.2 44.3±0.6 22.5±1.3 51.4±0.9 59.5±0.7 10.0±0.5 2.7±0.3 167±33
5(methoxime) CH=NOH 2.9±0.01 2.1±0.5 16.1±0.5 1.8±0.3 14.2±0.1 14.3±0.2 2.4±0.1 0.6±0.3 2010±391
6(K048) CONH2 27.4±1.1 10.0±0.6 25.7±0.7 12.5±0.2 54.4±0.9 29.1±0.4 3.8±0.1 1.4±0.1 349±68
7(K074) CH=NOH 32.1±0.6 24.6±0.2 31.1±0.4 13.9±0.8 17.9±0.1 21.2±0.4 1.4±0.5 1.4±0.2 29±6
8(K075) CH=NOH 19.5±1.0 18.1±0.2 28.5±1.1 14.1±0.3 19.2±0.9 22.8±0.2 1.1±0.3 1.5±0.2 80±16
9(K203) CONH2 48.1±1.5 21.2±0.3 39.3±0.4 13.1±0.4 55.9±0.5 41.1±0.1 4.4±0.1 2.1±0.4 566±110
10 pyridinium 3.9±0.1 4.5±0.3 16.6±1.0 3.5±0.3 14.3±0.8 13.2±0.1 2.4±0.5 0.4±0.3 -
11 pyridazinium 1.5±0.5 4.1±0.2 6.3±0.8 3.0±0.3 7.1±0.7 11.8±0.4 1.2±0.7 1.0±0.4 -
12 quinolinium 0.6±0.4 3.1±0.3 0.6±0.5 2.0±0.1 2.2±0.5 8.6±0.4 0.2±1.0 0.8±0.2 -
13 isoquinolinium 3.5±0.7 6.0±0.2 9.1±0.3 8.7±0.3 8.2±0.8 16.9±0.4 0.7±0.2 1.1±0.2 -
14 Me 3.4±0.4 6.3±0.3 24.5±0.3 7.6±0.6 9.5±0.5 9.4±0.2 1.1±0.4 0.8±0.3 -
15 tBut 1.7±0.4 3.9±0.3 18.7±0.2 58.7±4.0 4.0±0.5 7.6±0.5 1.7±0.5 0.8±0.3 24±6
16 Ph 0.7±0.4 3.5±0.3 6.9±0.4 8.9±0.5 1.7±0.3 6.2±0.4 0.7±0.2 1.0±0.3 6±1
17 Bn 0.3±0.2 1.9±0.6 1.2±0.1 4.7±0.3 0.0±0.1 3.8±0.4 0.0±0.5 0.8±0.2 -
18 CH2OH 2.6±0.3 4.0±0.3 20.6±0.1 8.0±0.1 6.7±0.3 8.1±0.3 3.8±1.0 1.4±0.4 -
19 COMe 1.7±0.3 3.1±0.1 11.9±1.0 3.9±0.3 5.2±0.4 7.5±0.1 1.5±0.3 1.0±0.2 -
20 COOH 3.3±0.5 6.2±0.3 14.5±0.6 4.2±0.4 9.5±0.3 8.8±0.4 1.1±0.5 0.4±0.1 -
21 COOEt 1.7±0.5 4.1±0.2 7.1±0.7 4.0±0.6 3.8±0.4 7.2±0.2 0.8±0.3 1.1±0.2 -
22 CN 3.6±0.4 5.2±0.2 10.1±0.8 42.6±0.1 11.1±0.1 13.8±0.3 1.7±0.5 0.4±0.2 45±9
23 C(NH2)=NOH 12.8±0.9 13.1±0.4 19.1±0.8 7.4±0.3 20.2±1.0 20.7±0.4 3.1±0.8 2.0±0.2 90±17
24 3,4-CONH2 1.8±0.6 4.5±0.2 2.0±0.4 2.2±0.8 4.2±0.5 10.1±0.2 0.7±0.4 0.7±1.0 -
Molecules2018,23,2291 6of16
Furthermore,POXreactivationwastested. Notsurprisingly,thecommercialcompounds1–2and
5werefoundnottobeeffectiveinthiscase[26]. Theothercommercialcompounds3–4presented
increasedreactivationofPOX-inhibitedHssAChEatbothdeterminedconcentrations(around22%
at 10 µM). The previously known compounds 6–9 showed lower reactivation ability up to 14% at
humanattainableconcentration. Thenewlypreparedcompoundswerefoundtobepoororaverage
reactivatorsofPOX-inhibitedHssAChE,withtheexceptionofcompounds15(59%)and22(43%)at
10µM.Thesetwocompoundsexceededallcommerciallyandpreviouslyknownreactivatorstestedin
thisstudyagainstPOX-inducedinhibition.
The MePOX reactivation showed some similarities with POX reactivation. The commercial
compounds 2 and 5 were found to be poor reactivators. However, surprisingly, the reactivation
of compound 1 was found to be better at both concentrations (up to 30%) compared to POX
reactivation(upto11%). Nevertheless,commercialcompounds3–4showedenhancedreactivation
of MePOX-inhibited HssAChE at human attainable concentration (45 and 60% at 10 µM) [15].
Thepreviouslyknowncompounds6–9alsoasappearedtobepromisingMePOXreactivators,with
a maximum of 41% at 10 µM for compound 9. The newly prepared oximes showed decreased
reactivationofMePOX-inhibitedHssAChEcomparedtothestandards,withtwocompounds,13(17%)
and23(21%),displayedsomeeffectat10µMconcentration.
ThepoorreactivationofDFP-inhibitedHssAChEwasexpected[15]. Thepreviousexperiments
were confirmed by all standards 1–9, where only compound 4 had some effective invitro
reactivation[27]. Thenewlypreparedcompoundsalsofulfilledthesefindingswithminororpoor
reactivationofDFP-inhibitedHssAChE.
Whenthereactivationdataatvariousconcentrationsweretakenintoaccount,somecompounds
(e.g., 15, 16, 22) were shown to be better reactivators at lower concentration (10 µM), compared
to their reactivation data at higher concentration (100 µM). These reactivation results can be
explained by mixed reactivation-inhibition behavior of newly prepared compounds at various
concentrations[28]. Forthispurpose,theinhibitoryeffecttowardsHssAChEofselectedreactivators
(1–9,15–16and22–23)wasdetermined(Table1). Thecommercialcompounds1–5wereshowntobe
poor HssAChE inhibitors, with the best inhibitory ability exhibited by trimedoxime (4; 167 µM).
Two previously known reactivators, 7 (29 µM) and 8 (80 µM), showed even better HssAChE
inhibition. Subsequently, compounds 4 and 7–8, as the best AChE inhibitors among the
standards,showedtheabove-mentionedmixedreactivation-inhibitioneffectforMePOXreactivation.
Similarly, some newly prepared compounds (15–16, 22) were found to be potent HssAChE
inhibitors. In accordance with our hypothesis, compound 16 (IC 6 µM) was shown to be a
50
weaker reactivator of POX-inhibited HssAChE at a concentration of 100 µM (7%) than at 10 µM
(9%), while it greatly inhibited HssAChE at the 100 µM concentration (screening concentration
100µM>>IC 6µM). At 10 µM, compound 16 was found to be a slightly better reactivator
50
(9%), while its inhibition ability decreased ten-fold, and it was closer to its IC value (6 µM).
50
Similarly,compound15(screeningconcentration100µM>>IC 24µM)andcompound22(screening
50
concentration100µM>>IC 45µM)confirmedthemixedreactivation-inhibitionhypothesisduring
50
POX reactivation, while compound 23 (screening concentration 100 µM ≈ IC 90 µM) did not
50
accuratelyfulfilthistrend.
3.2. MolecularModellingResultsandSARDiscussion
The molecular modelling study was performed with oximes 3, 9 and 23 to rationalize their
interactionswithGA-inhibitedHssAChE[29]. Oxime3waschosenasaknowncommercialstandard
thatisabletoreactivateGA-inhibitedAChE[30]. Additionally,oxime9wasselectedfromamong
thepreviouslypreparedcompoundsasoneofthebestreactivatorsofGA-inhibitedHssAChE[17].
Oxime23wasselectedasthebestreactivatorofGA-inducedtoxicityinvitrofromamongthenewly
preparedcompounds.
Molecules2018,23,2291 7of16
The molecular modelling study was performed to better understand the invitro results.
The crystal structure of HssAChE in complex with fasciculin 2 (pdb 1b41) was chosen as a crystal
structurewithgoodstructuralresolution(2.76Å)[31]. Thefasciculinmoleculewasdeleted,whilethe
moleculeofGAwasmanuallyattachedtothecatalytictriadresidueSer203,andthewholemoleculeof
GA-inhibitedHssAChEwasminimizedusingAutodockTools(ADT)[32]. Theimportantaromatic
residueswithintheHssAChEactivesitewereselectedasflexibleresidues,includingtheGA-Ser203
moiety. ThemoleculesofthreereactivatorswerealsominimizedbyADT.Themolecularmodelling
wasperformedbyAutodockVina[33].
Compound3(thelowestbindingenergy−5.7kcal/mol)displayedtypicalinteractionswithAChE
aromatic residues (Figure 5). The oxime-pyridinium ring was attached to Trp86 (3.6 Å), while the
distance between oximate oxygen and GA phosphorus atom (6.8 Å) was found to be longer than
previouslyobservedresults. Thesecondpyridiniumringwasstabilizedviainteractionswitharomatic
residuesoftheperipheralaromaticsite(PAS),namelyPhe338(3.4Å),Trp286(4.2Å),Tyr124(4.0Å)
andTyr341(3.5Å).Theoxygenatomintheconnectinglinkerseemedtobestabilizedbyinteractions
withphenolicmoietiesofTyr124(2.6Å),Tyr337(3.8Å)andTyr341(3.7Å).Compound9(thelowest
binding energy −5.5 kcal/mol) showed similar results to compound 3, with some exceptions in
termsofflexibleresidues(Figures5and6). Inthiscase,theoximepyridiniumringwasstackedto
Trp86(3.6Å)andthedistancebetweentheoximeandtheGAphosphorusatom(5.1Å)wasbetterthan
previouslypublishedresults. Notsurprisingly,thenon-oximepyridiniumringwasstabilizedinPAS
byPhe338(3.6Å),Trp286(3.6Å),Tyr124(4.3Å)andTyr341(3.6Å),wheretheflexibleresidueTyr341
washighlydistortedcomparedtothesameresidueinthecaseofcompound3. Thedoublebondinthe
linkerseemedtobestabilizedbyπ-πinteractionwithTyr337(4.0Å).Thecarbamoylmoietydisplayed
importanthydrogenbondswithTrp286(2.2Å)andPhe295(2.9Å).Compound23(thelowestbinding
energy−6.4kcal/mol)presenteddifferentbindingwhentheamidoximepyridiniumringwasattached
toTrp86(3.6Å),andamidoximemoietywasnotlocatedfarfromGAphosphorus(5.3Å;Figure6).
ThesecondringbearingoximemoietywasstabilizedinPASbyPhe338(3.5Å),Trp286(3.7Å),Tyr124
(3.6Å)andTyr341(3.6Å).TheamidoximemoietywasfurtherhydrogenbondedwithGly120(3.3Å)
andGlu202(2.1Å).
Molecules 2018, 23, x FOR PEER REVIEW 9 of 17

Figure 5. The molecular modelling results with selected flexible residues of compound 3 (in green)
Figure5.Themolecularmodellingresultswithselectedflexibleresiduesofcompound3(ingreen)and
and compound 9 (in magenta).
compound9(inmagenta).

Figure 6. The molecular modelling results with selected flexible residues of compound 9 (in
magenta) and compound 23 (in yellow).
Taken together, compound 9 showed more important interactions with GA-inhibited HssAChE
active sites compared to compounds 3 and 23 [29]. This might be a reason for its increased
reactivation ability in the case of GA-induced in vitro inhibition. The lower reactivation efficacy of
compound 3 might be explained by its different interactions within the GA-HssAChE active site with
inappropriate reactivator accommodation between Trp86 and PAS residues. Conversely, the lower
in vitro reactivation efficacy of compound 23 might be related to the favorable structure with
stabilized amidoxime moiety positioned close to GA-Ser203, while the spatial structure with oxime
moiety positioned close to GA-Ser203 was found to be less favorable.
Concerning the SAR of oxime reactivators for GA or OP pesticide-inhibited HssAChE, several
important attributes should be considered. Firstly, the bisquaternary mono-oximes were already
Molecules 2018, 23, x FOR PEER REVIEW 9 of 17

MolecuFliegsu20r1e8 5,.23 T,2h2e9 1molecular modelling results with selected flexible residues of compound 3 (in green)8 of16
and compound 9 (in magenta).

Figure 6. The molecular modelling results with selected flexible residues of compound 9 (in
Figure6.Themolecularmodellingresultswithselectedflexibleresiduesofcompound9(inmagenta)
magenta) and compound 23 (in yellow).
andcompound23(inyellow).
TTaakkeenn ttooggeetthheerr,, ccoommppoouunndd 99 sshhoowweedd mmoorree iimmppoorrttaanntt iinntteerraaccttiioonnss wwiitthh GGAA--iinnhhiibbiitteedd HHssssAACChhEE
aaccttiivvee ssiitteess ccoommppaarreedd ttoo ccoommppoouunnddss 33 aanndd 2233 [[2299]].. TThhisis mmiigghhtt bbee aa rreeaassoonn ffoorr iittss iinnccrreeaasseedd
rreeaaccttiivvaattiioonn aabbiilliittyy iinn ththee ccaasese oof fGGAA-i-nidnduucecde dini nvivtirtor oinihnihbiibtiiotino.n T.hTeh leowloewr ererarcetaivcatitvioanti oenffiecfaficcya coyf
coofmcopmoupnodu n3d m3igmhitg bhet ebxepelaxipnleadin beyd itbsy diitfsfedriefnfetr ienntetrianctteiroancst iwonitshwini tthhien GthAe-HGsAsA-HCshsEA aCchtiEvea cstiitve ewsiitthe
iwniatphpinroapprpiraotep rrieaatectrievaacttoivr aatcocroamccmomodmatoidoant iboentwbeetewne TenrpT8r6p 8a6ndan PdAPSA rSerseisdiudeuse.s C.Conovnevresresleyly, ,tthhee lloowweerr
iinn vviittrroo rereaactcitvivataitoinone ffiefcfaiccaycoyf coofm cpoomupnodu2n3dm 2i3g hmtbigehrte labtee dretloatthede fatov otrhaeb lfeasvtoruracbtulere swtriuthctsutraeb iwliziethd
satmabidiliozxeidm aemmidooiextiympe omsiotiioentye dpocsliotsioenteodG cAlo-sSee tro2 0G3A, w-Sheril2e03th, ewshpilaet itahles strpuactitaulr estrwuictthuroex wimiteh moxoiimetey
mpoosiiettioyn peodsictlioosneetdo cGloAse-S teor 2G0A3-wSears2f0o3u wndast ofobuenlde stso fbaev olersasb flea.vorable.
CCoonncceerrnniinngg ththee SASAR Rof oofxiomxeim reeacrteivaacttiovrast foorrs GfoAr oGr AOPo presOtiPcidpee-sintihciibdiet-eidn hHibssiAteCdhHE,s ssAevCehraEl,
ismevpeorratlainmt paotrttraibnuttaetstr isbhuotueslds hboeu lcdobnesicdoenrseidd.e rFeidrs.tFlyir, sttlhye, thbiesbqiusaqtueartnearrnya rmymonoon-oo-xoixmimese swwereere aallrreeaaddyy
foundtobesatisfactoryreactivatorsofGA-orpesticide-inhibitedAChEcomparedtobis-oximes[34].

The 4-positioned oxime moiety on the pyridinium scaffold has been used and proposed many
times as being optimal for reactivation due to its pKa [35]. Furthermore, the connecting linkage
betweenheteroareniumringsinthelengthof3–4C-CatomswashighlightedinthecaseofGA-or
pesticide-inhibitedAChE[36]. Inthiscase,thenewlypreparedcompoundswith4-memberedlinkers
showedresultsequivalenttopreviouslypreparedserieswith3-memberedlinkerswithanalogous
non-oximemoieties. Additionally,thehereroareniumpartofthemoleculewithoutoximemoietywas
found to be very important for proper reactivation effects in the case of various OPs. The second
heteroareniumringwasdeterminedtobeanimportantmoietyforinteractionswitharomaticamino
acidresiduesinthePASregion. Itsmodificationbypyridazinium,quinoliniumorisoquinolinium
moieties(11–13)didnotleadtoenhancedreactivationofGAorOPpesticideinhibitedHssAChE[15].
However, its modification by various moieties bound in the 4-position of pyridinium scaffold
showed better reactivation compared to known and analogous bis-oxime 7. Namely, some newly
preparedcompoundswithhydrophobic(15-tertbutyl)orhydrophilicmoieties(22-carbonitrileand
23-amidoxime)werefoundtobepromisingreactivatorsofselectedOPpesticideinhibitedHssAChE.
TheirincreasedreactivationabilitymightbeexplainedbyadditionalinteractionswithPASamino
acidresidues,wheretertbutyl(15)wasproventointeractwitharomaticresidues, andcarbonitrile
(22)oramidoxime(23)areavailableforhydrogenbonding[37,38]. Fortheabove-mentionedreasons,
Molecules2018,23,2291 9of16
thesecondheteroareniumpartofthemoleculeistheessentialmolecularfragment, andshouldbe
properlydesignedforenhancedreactivationofOPinhibitedHssAChE[34].
4. ExperimentalSection
4.1. ChemicalPreparation
Solvents (acetone, DMF, MeCN) and reagents were purchased from Sigma-Aldrich (Prague,
Czech Republic) and used without further purification. Reactions were monitored by TLC using
DC-AlufolienCelluloseF(Merck,Darmstadt,Germany)andmobilephaseBuOH-CH COOH-H O
3 2
5:1:2,detectionbysolutionofDragendorffreagent(solutioncontaining10mLCH COOH,50mLH O
3 2
and 5 mL of basic solution prepared by mixing of two fractions—fraction A: 850 mg Bi(NO ) ,
3 3
40mLH O, 10mLCH COOH; fraction B: 8 g KI, 20 mL H O). Melting points were measured on
2 3 2
microheatingstagePHMK05(VEBKombinatNagema,Radebeul,Germany)andwereuncorrected.
NMRspectraweregenerallyrecordedatVarianGemini300(1H300MHz,13C75MHz,Varian,
Palo Alto, CA, USA). In all cases, the chemical shift values for 1H spectra are reported in ppm (δ)
relativetoresidualDMSO(δ2.50)orD O(δ4.79),shiftvaluesfor13Cspectraarereportedinppm(δ)
2
relativetosolventpeakdimethylsulfoxide—d δ39.43. Signalsarequotedass(singlet),d(doublet),
6
t(triplet)andm(multiplet).
Themassspectra(MSrespectivelyMSn)weremeasuredonaLCQFLEETiontrapandevaluated
using Xcalibur v 2.5.0 software (both Thermo Fisher Scientific, San Jose, CA, USA). The sample
wasdissolvedindeionizedwater(Goro,s.r.o.,Prague,CzechRepublic),andinjectedcontinuously
(8µL/min)byHamiltonsyringeintoelectrosprayionsource. Theparametersofelectrospraywereset
upasfollows: sheathgasflowrate20arbitraryunits,auxgasflowrate5arbitraryunits,sweepgas
flowrate0arbitraryunits,sprayvoltage5kV,capillarytemperature275◦C,capillaryvoltage13V,
tubelens100V.
4.2. PreparationofBisquaternarySalts
Initially the monoquaternary semi-product was prepared: a solution of the
4-hydroxyiminomethylpyridine(4.0g,32.8mM)and1,4-dibromobutane(19.6mL,163.8mM)inacetone
(30mL)wasstirredatrefluxfor8h[15]. Thereactionmixturewascooledtoroomtemperatureandthe
crystallinecrudeproductcollectedbyfiltrationandwashedwithacetone(3×20mL).Thesolidcrude
productwascrystalizedfromMeCN(1gofcrudeproductper100mLofMeCN),wheretheboiling
mixturewasfilteredunderareducedpressure. Thefiltratewasevaporatedunderareducedpressure
to produce the pure monoquaternary salt of 1-(4-bromobutyl)-4-hydroxyiminomethylpyridinium
bromide(75%).
Secondly,thesynthesisofthebisquaternarysaltwascompleted.Asolutionofthemonoquaternary
salt (0.50 g, 1.5 mM) and selected heteroaromatic compound (3.0 mM) in DMF or MeCN (10 mL)
wasstirredat70–80◦Cfor8h. Thereactionmixturewascooledtoroomtemperatureandportioned
withacetone(80mL);thecrystallinecrudeproductwascollectedbyfiltration,washedwithacetone
(3×20mL)andcrystallizedfromboilingMeCN.Thepurebisquaternarysaltwasobtainedasthe
solidcompoundinsolubleinboilingMeCNasthemixturesofcis/transoximeisomers.
4.3. PreparedMonoquaternarySalt
1-(4-Bromobutyl)-4-((hydroxyimino)methyl)pyridiniumbromide. M.p. 154–155◦C.Yield91%. 1H-NMR
(300 MHz, DMSO d ): δ (ppm) 12.82 (s, 1H, NOH), 9.08 (d, 2H, J = 6.5 Hz, H-2,6), 8.44 (s, 1H,
6
-CH=NOH), 8.25 (d, 2H, J = 6.4 Hz, H-3,5), 4.64 (t, 2H, J = 7.2 Hz, N-CH -), 3.57 (t, 2H, J = 6.6 Hz,
2
CH -Br),2.08–2.01(m,2H,-CH -),1.86–1.80(m,2H,-CH -). 13C-NMR(75MHz,DMSOd ): δ(ppm)
2 2 2 6
148.32,145.01,144.88,124.04,59.25,33.91,29.29,28.56. EA:calculated35.53%C,4.17%H,8.29%N;
found35.58%C,4.12%H,8.25%N.ESI-MS:m/z257.0[M]+(calculated[C H BrN O]+257.03).
10 14 2
Molecules2018,23,2291 10of16
4.4. PreparedBisquaternarySalts
4-Hydroxyiminomethyl-1,1(cid:48)-(but-1,4-diyl)-bispyridiniumdibromide(10)[39]. M.p. 243–244◦C.Yield48%.
1H-NMR(300MHz,DMSOd ): δ(ppm)12.84(s,1H,NOH),9.18(d,2H,J=6.2Hz,H-2,6),9.12(d,2H,
6
J =6.2Hz,H-2(cid:48),6(cid:48)),8.67-8.59(m,1H,H-4(cid:48)),8.46(s,1H,-CH=NOH),8.25(d,2H,J =6.2Hz,H-3,5),
8.22–8.15(m,2H,H-3(cid:48),5(cid:48)),4.80–4.62(m,4H,N-CH -),1.97(m,4H,N-CH -CH -). 13C-NMR(75MHz,
2 2 2
DMSO d ): δ (ppm) 148.30, 145.53, 145.01, 144.98, 144.75, 128.05, 124.00, 59.63, 59.19, 27.06, 26.95.
6
EA:calculated43.19%C,4.59%H,10.07%N;found43.29%C,4.75%H,10.08%N.ESI-MS:m/z128.6
[M/2]2+(calculated[C H N O/2]2+128.58).
15 19 3
4-Hydroxyiminomethyl-1,1(cid:48)-(but-1,4-diyl)-1-pyridinium-1(cid:48)-pyridaziniumdibromide(11). M.p. 222–224◦C.
Yield 71%. 1H-NMR (300 MHz, DMSO d ): δ (ppm) 10.05 (d, 1H, J = 5.8 Hz, H-6(cid:48)), 9.65 (d, 1H,
6
J=4.8Hz,H-3(cid:48)), 9.11 (d, 2H, J = 6.0 Hz, H-2,6), 8.80–8.73 (m, 1H, H-4(cid:48)), 8.67–8.60 (m, 1H, H-5(cid:48)),
8.45(s,1H,-CH=NOH),8.26(d,2H,J =6.0Hz,H-3,5),4.91(t,2H,J =6.3Hz,N’-CH -),4.68(t,2H,
2
J=6.3Hz,N-CH -),2.10–1.96(m,4H,N-CH -CH -). 13C-NMR(75MHz,DMSOd ): δ(ppm)154.47,
2 2 2 6
150.12, 148.34, 145.03, 136.58, 136.01, 124.00, 63.44, 59.23, 26.98, 25.73. EA: calculated 40.21% C,
4.34% H, 13.40% N; found 40.09% C, 4.49% H, 13.27% N. ESI-MS: m/z 129.1 [M/2]2+ (calculated
[C H N O/2]2+129.08).
14 18 4
4-Hydroxyiminomethyl-1,1(cid:48)-(but-1,4-diyl)-1-pyridinium-1(cid:48)-quinoliniumdibromide(12)[39].M.p.177–179◦C.
Yield21%. 1H-NMR(300MHz,DMSOd ): δ(ppm)9.68(d,1H,J=5.8Hz,H-2(cid:48)),9.34(d,1H,J=8.4Hz,
6
H-8(cid:48)), 9.12 (d, 2H, J = 6.2 Hz, H-2,6), 8.71 (d, 1H, J = 8.8 Hz, H-4(cid:48)), 8.53 (d, 1H, J = 8.1 Hz, H-5(cid:48)),
8.45(s,1H,-CH=NOH),8.33–8.17(m,4H,H-3,3(cid:48),5,7(cid:48)),8.11–8.02(m,1H,H-6(cid:48)),5.17(t,2H,J=7.0Hz,
N’-CH -),4.71(t,2H,J=7.0Hz,N-CH -),2.20–1.93(m,4H,N-CH -CH -). 13C-NMR(75MHz,DMSO
2 2 2 2
d ): δ(ppm)149.71,148.29,147.41,145.01,137.33,135.58,130.70,129.81,129.66,123.99,122.16,119.00,
6
59.33,58.35,27.36,25.88. EA:calculated48.85%C,4.53%H,8.99%N;found48.41%C,4.63%H,9.06%
N.ESI-MS:m/z153.6[M/2]2+(calculated[C H N O/2]2+153.59).
19 21 3
4-Hydroxyiminomethyl-1,1(cid:48)-(but-1,4-diyl)-1-pyridinium-1(cid:48)-isoquinolinium dibromide (13) [39]. M.p.
205–206 ◦C. Yield 26%. 1H-NMR (300 MHz, DMSO d ): δ (ppm) 10.26 (s, 1H, H-1(cid:48)), 9.12 (d, 2H,
6
J=6.2Hz,H-2,6),8.87(d,1H,J=6.7Hz,H-3(cid:48)),8.64(d,1H,J=6.7Hz,H-4(cid:48)),8.51(d,1H,J=8.2Hz,
H-8(cid:48)),8.44(s,1H,-CH=NOH),8.38(d,1H,J=8.2Hz,H-5(cid:48)),8.32–8.21(m,3H,H-3,5,7(cid:48)),8.12–8.04(m,1H,
6(cid:48)),4.83(t,2H,J=6.7Hz,N-CH -),4.70(t,2H,J=6.7Hz,N’-CH -),2.18–1.94(m,4H,N-CH -CH -).
2 2 2 2
13C-NMR(75MHz,DMSOd ): δ(ppm)150.08,148.30,145.02,136.92,136.82,134.85,131.09,130.33,
6
127.19,127.13,125.80,124.99,59.67,59.29,27.03,26.81. EA:calculated48.85%C,4.53%H,8.99%N;
found48.53%C,4.75%H,8.78%N.ESI-MS:m/z153.6[M]2+(calculated[C H N O]2+153.59).
19 19 3
4-Hydroxyiminomethyl-4(cid:48)-methyl-1,1(cid:48)-(but-1,4-diyl)-bispyridinium dibromide (14). M.p. 190–192 ◦C.
Yield47%. 1H-NMR(300MHz,D O):δ(ppm)8.83(d,2H,J=6.2Hz,H-2,6),8.65(d,2H,J=6.0Hz,
2
H-2(cid:48),6(cid:48)), 8.39 (s, 1H, -CH=NOH), 8.21 (d, 2H, J = 6.0 Hz, H-3,5), 7.89 (d, 2H, J = 6.0 Hz, H-3(cid:48),5(cid:48)),
4.70–4.58 (m, 4H, N-CH -,N’-CH -), 2.23 (s, 3H, -CH ), 2.15–2.08 (m, 4H, N-CH -CH -). 13C-NMR
2 2 3 2 2
(75MHz, D Od ): δ(ppm)161.06, 149.47, 146.87, 145.09, 143.67, 129.43, 125.60, 61.09, 60.50, 30.89.
2 6
EA:calculated44.57%C,4.91%H,9.75%N,found44.13%C,5.00%H,9.61%N.ESI-MS:m/z270.0
[M2+ −H](calculatedfor[C H N O2+ −H]270.17).
16 21 3
4-Hydroxyiminomethyl-4(cid:48)-(1,1-dimethylethyl)-1,1(cid:48)-(but-1,4-diyl)-bispyridinium dibromide (15). M.p.
146–148◦C.Yield75%. 1H-NMR(300MHz,D O):δ(ppm)8.83(d,2H,J=6.2Hz,H-2,6),8.70(d,2H,
2
J = 6.2 Hz, H-2(cid:48),6(cid:48)), 8.38 (s, 1H, -CH=NOH), 8.21 (d, 2H, J = 6.2 Hz, H-3,5), 8.08 (d, 2H, J=6.2Hz,
H-3(cid:48),5(cid:48)), 4.68–4.59 (m, 4H, N-CH -,-N’-CH -), 2.14–2.08 (m, 4H, N-CH -CH -), 1.40 (s, 9H, -CH ).
2 2 2 2 3
13C-NMR(75MHz,D Od ): δ(ppm)159.71,158.49,157.67,155.86,154.35,134.61,134.25,70.23,70.06,
2 6
39.88,36.84. EA:calculated48.22%C,5.75%H,8.88%N,found47.91%C,6.13%H,8.54%N.ESI-MS:
m/z156.5[M2+/2](calculatedfor[C H N O2+/2]156.61).
19 27 3