Design and synthesis of fluorescently labelled naphthalenesulfonamide inhibitors of butyrylcholinesterase

S staranjem prebivalstva se povečuje pogostnost za starostnike značilnih obolenj, kamor sodi tudi Alzheimerjeva bolezen, ki je napredujoča nevrodegenerativna bolezen in najpogostejši vzrok demence. Kognitivni propad bolnika kot posledica bolezni je za svojce in skrbnike fizično in psihično izčrpavajoč, zaradi česar je pogosto potrebna premestitev bolnika v oskrbo domov za starostnike. Zaradi neozdravljivosti in velikega socialno-ekonomskega bremena bolezni je iskanje novih učinkovin in diagnostičnih orodij v porastu. Holinergična hipoteza je prvi poskus razlage patogeneze Alzheimerjeve bolezni po kateri holinergična disfunkcija povzroča propad kognitivnih sposobnosti bolnika. Na osnovi te hipoteze so razvili zaviralce acetilholin-esteraze. Potencialna nova tarča za zdravljenje Alzheimerjeve bolezni je soroden encim butirilholin-esteraza, katerega aktivnost se z napredovanjem bolezni nasprotno kot pri acetilholin-esterazi poveča. Butirilholin-esteraza naj bi tako v moţganih bolnikov prevzela glavno vlogo hidrolize acetilholina, vključena pa naj bi bila tudi v nastajanje za Alzheimerjevo bolezen značilnih amiloidnih plakov. Za potrebe razvoja nove, enostavne in neposredne metode za določanje aktivnosti butirilholin-esteraze na moţganskih rezinah smo poskušali razviti selektiven fluorescenčno označen zaviralec omenjenega encima. Izhajali smo iz optimiziranega zaviralca 2, ki so ga razvili raziskovalci na Fakulteti za farmacijo, ga kemijsko modificirali in nanj s pristopi \u27klik\u27 kemije pripenjali rodamin B in NBD-Cl. Mesto uvajanja fluorofora smo izbrali glede na kristalno strukuro kompleksa butirilholin-esteraze z zaviralcem 2 ter dobro opisano povezavo med strukturo in aktivnostjo njegovih analogov. Flurofore smo pripenjali prek hidrofilnega distančnika, da bi se izognili teţavam nespecifične vezave zaviralca na lipofilno moţgansko rezino. Zaradi teţav povezanih s sintetiziranjem zaviralca označenega z rodaminom B, nam je uspelo sintetizirati samo z NBD označen zaviralec 24, kateremu smo določili in vitro sposobnost zaviranja obeh holin-esteraz. Spojina 24 je selektiven zaviralec butirilholin-esteraze s srednjo zaviralno koncentracijo 214,16 nM. Uvedba flurofora na zaviralec močno zmanjša aktivnost v primerjavi s spojino 2, zaradi česar spojina 24 ni bila ustrezna za barvanje moţganskih rezin.Frequency of geriatric diseases is increasing due to population ageing. One of the most common cause of dementia is Alzheimer\u27s disease, which is a progressive neurodegenerative disease. Cognitive deterioration as a consequence of Alzheimer\u27s disease is physically and mentally exhausting for caretakers and it often results in institutionalisation of the patient. Lack of cure and great socioeconomic burden of the disease make research of new medicine and diagnostic tools very important. The cholinergic hypothesis is the first attempt of explaining the pathogenesis of Alzheimer\u27s disease, according to which cholinergic dysfunction causes decline in patients cognitive ability. The hypothesis led to the discovery of cholinesterase inhibitors. A new promising drug target is butyrylcholinesterase as its activity increases in later stages of Alzheimer\u27s disease. On the contrary, activity of acetylcholinesterase decreases. Therefore, it is supposed that butyrylcholinesterase takes over the cholinesterase activity in advanced stages of the disease. Also, butyrycholinesterase is thought to be involved in formation of amyloid plaques, which are a pathophysiological hallmark of Alzheimer\u27s disease. For the development of a simple and direct method for detecting butyrylcholinesterase on brain slices we tried to develop a selective fluorescently labeled butyrylcholinesterase inhibitor. Our attempt was based on the structure of an inhibitor 2 that was developed by researchers at the Faculty of Pharmacy. Firstly, we chemically modified its structure to allow introduction of derivatives of rhodamine B and NBD fluorophores by means of click chemistry. The introduction site of fluorophores was choosen on the basis of the crystal structure of the inhibitor 2 in complex with butyrylcholinesterase and well described structure-activity relationships. Fluorophores were attached by a hydrophilic linker in order to minimize nonspecific binding of the inhibitor to the lipophilic brain slice. Because the synthesis of inhibitor labeled with rhodamine B turned out to be troublesome, we managed to synthesise only an inhibitor 24 labeled with NBD. In vitro inhibitory potency of compound 24 against both cholinesterases was determinedcompound is selective towards butyrylcholinesterase with half maximal inhibitory concentration of 214.16 nM. Attachment of the fluorophore caused a significant loss of binding capacity to the enzyme when compared to inhibitor 2 and as a consequence compound 24 was not appropriate for detection of butyrylcholinesterase on brain slices