Abstract
Muscarinic acetylcholine receptors (mAChRs) are a pivotal constituent of the central and peripheral nervous system. Yet, therapeutic and diagnostic applications thereof are hampered by the lack of subtype selective ligands. Within this work, we synthesized and chemically characterized three different stereoisomers of hydrobenzoin esters of arecaidine by NMR, HR-MS, chiral chromatography, and HPLC-logP. All compounds are structurally eligible for carbon-11 labeling and show appropriate stability in Dulbecco’s phosphate-buffered saline (DPBS) and F12 cell culture medium. A competitive radioligand binding assay on Chinese hamster ovary cell membranes comprising the human mAChR subtypes M1-M5 showed the highest orthosteric binding affinity for subtype M1 and a strong influence of stereochemistry on binding affinity, which corresponds to in silico molecular docking experiments. Ki values toward M1 were determined as 99 ± 19 nM, 800 ± 200 nM, and 380 ± 90 nM for the (R,R)-, (S,S)-, and racemic (R,S)-stereoisomer, respectively, highlighting the importance of stereochemical variations in mAChR ligand development. All three stereoisomers were shown to act as antagonists toward mAChR M1 using a Fluo-4 calcium efflux assay. With respect to future positron emission tomography (PET) tracer development, the (R,R)-isomer appears especially promising as a lead structure due to its highest subtype selectivity and lowest Ki value.
Highlights
Muscarinic acetylcholine receptors are G protein-coupled receptors, which are strongly involved in the parasympathetic nervous system’s signal transduction. mAChRs appear as five subtypes, which differ in their expression pattern and downstream signaling [1]
The stability of 1, 2, and 3 in buffer and cell culture medium was investigated because their disintegration could impair biological testing. 1, 2, and 3 show a comparable rate of decomposition in Dulbecco’s phosphate-buffered saline (DPBS) and Ham’s F12 cell culture medium at 37 ◦C over a time period of 60 h (Figure 3), which is slow enough to allow for biological testing and drug development
The variable affinity toward the other subtypes highlights the relevance of chiral ligands in the quest for subtype selectivity
Summary
Muscarinic acetylcholine receptors (mAChRs) are G protein-coupled receptors, which are strongly involved in the parasympathetic nervous system’s signal transduction. mAChRs appear as five subtypes, which differ in their expression pattern and downstream signaling [1]. MAChRs appear as five subtypes, which differ in their expression pattern and downstream signaling [1] Their conserved orthosteric binding units hamper subtype selective molecular targeting [2]. MAChR agonists have been proposed as promising drugs for the treatment of Alzheimer’s disease symptoms, hitherto, no compound passed clinical trial phase 3 [6]. Considering the insufficient penetration of the blood–brain barrier as well as unsuitable pharmacokinetics and -dynamics in treatment regimens of e.g., ParkinPhsaormna’cseudticiaslse2a02s0e, ,13t,hxeFsOeR dPErEuRgRsEVoIfEtWen merely have peripheral effects on disease sym2potfo12ms. As they do not act on the disease origin [9], they do not have the ability to alter disease progression. Diphenylmethyl esters of arecaidine (Caonacnluddibng, ,Ftihgeurerpee1rt)oiwreeorfemrAecCehnRtliygadnidsscfoovr ebroethdthaesraoprythanodstdeiraigcnomsiAs iCs chhRaralicgtearnizdeds with low nanomolarbyaffia lnaciktyoaf nhidghparfofinnoituy nanceddsiMmu1ltsaenleeocutsilvyistyu.btUypnefoserlteuctnivaetecloym, pstoruonndgs. nDoipnhsepneyclmifiecthbyilnedstienrsg observed in the precnolfainanoriemccaaoilldaeirnveaaf(flaiunaaitnytdioabnn, dFriegpsurtorrenao1iu)nnwecdeerdethrMeec1iernstuellyseecdtiiasvcsiotyvP.eErUeTdnftoarsratoucrnetahrtoseslyt[e1,r3isc]tr.monCAgCohnnRosnildsigpeaernciidnfsigcwbtihitnhadltionwngonspecific binding isoobfsteervnedcaiun stehde pbryechlinyidcarloepvhaloubatiiconinrteesrtraacintieodntshe[i1r4u],sethaissPwETortkracaeirms s[1t3o]. sCyonntshideesriinzge tahnatd evaluate similar conmonpsopuecnifdicsbwinditinhgriesdouftecnedcaulispedopbyhihlyicdirtoyp.hoIbnicthinitsersatcutiodnys, [w14]e, tihnistrwoodrukcaeimtshteo syynntthheseiszeis of chiral hydrobenzaonidneevsaltueartseosifmairlaerccaoimdipnoeu,nwdshwicithh raerdeufcuedllylipcohpahrilaiccittye.rIinzethdisbsytundyu,cwleeainrtmrodaugcneetthiecsryenstohensaisnce (NMR) spectroscoorpefsyochnaianrnadclehh(yNigdMrhoRbr)eesnspzoeolciutnrtoiesosctnoep-rmsy aoanfsdsahrseipgcaheidcretinrsooe,lmuwtieohtnirc-yhm(aaHsrseRsfp-uMelclytSro)cm,haaerntardyct(etHrhiRzee-MdpuSb)ry,iatnnyudwctlheaeasrpaumsraistgeynswestaeicsd by chiral and reversaesdsepsshedaseb-yhicghhirpalerafnodrmraevnecreseldiquphidasce-hhrioghmapteorfgorrampahnyce(HliqPuLidC)c. hPrhomysaitcoogrcahpehmy ic(HalPpLCar).ameters as well as staPbhilyistiyc,ocahffiemniictayl, paanradmfeutenrcstaios nwaeliltays tsotawbialirtyd, maffAinCityh, RansdwfuenrectiaosnsaelistysetdowbayrdinmvAiCtrhoRsmweetrheods for all synthesizeadssceossmedpboyuinnvdistr.oImneothrdodesrftoor aullnsdynetrhsetsaiznedd cthome pdoiuffnedrse.nInceoridnerbtionudnidnegrsataffindntihteiedsi,fftehreenbceininding poses of the novbsetiluncddoiienmsg. pafofiunnitidess, wtheerbeinadsisnegsspeodsesbyofitnhesinliocvoeldcoomckpionugndsstuwdeireesa.ssessed by in silico docking
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