20.500.12556/RUL-121041 Ločba enantiomerov in encimsko vrednotenje zaviralcev butirilholin-esteraze Separation of enantiomers and evaluation of butyrylcholinesterase inhibitors Butirilholin-esteraza je encim, katerega koncentracija se z napredovanjem Alzheimerjeve bolezni značilno zviša, zaradi česar predstavlja pomembno tarčo za zdravljenje poznih stadijev te bolezni. S pomočjo visoko-tlačne tekočinske kromatografije smo razvili pogoje, ki so omogočili ločbo racemnih zmesi selektivnih zaviralcev butirilholin-esteraze do njihovih čistih enantiomerov. Izbrana kolona ima omejeno število dovoljenih mobilnih faz. Med izbranimi bazičnimi mobilnimi fazami, raztopina trietilamina in fosfatni pufer nista omogočali ločbe racemnih zmesi zaviralcev. Ločba z boratnim pufrom pa je bila uspešna in zato smo metodo iz analitske kolone prenesli na semi-preparativno kolono. Izoliranim miligramskim količinam enantiomerov N-((1-(2,3-dihidro-1H-inden-2-il)piperidin-3-il)metil)-N-(2-metolsietil)-2-nafamida in N-((1-benzilpiperidin-3-il)metil)-N-(2-metoksietil)naftalen-2-sulfonamida smo izmerili čistost in specifično sučnost. Z encimskim testom smo zaznali različno stopnjo stereoselektivnosti čistih enantiomerov. Določili smo srednjo zaviralno koncentracijo tako izoliranim čistim enantiomerom kot tudi njihovim racemnim zmesem. Ugotovili smo, da ima najaktivnejši enantiomer (+)-1 s 13,4 nM vrednostjo IC50 več kot 10-krat močnejšo afiniteto do aktivnega mesta butirilholin-esteraze kot distomer (-)-1. Pri sulfonamidnemu analogu (±)-2 pa je stereoselektivnost čistih enantiomeorov izrazito manjša. Evtomeru (-)-2 smo določili 4,5 nM vrednost IC50. Butyrylcholinesterase is an enzyme whose levels increase significantly with the progress of the Alzheimer's disease, which makes it a promising drug target in the late stages of the disease. Using High-Performance Liquid Chromatography, we have developed conditions that allowed separation of racemic mixtures of selective butyrylcholinesterase inhibitors into their pure enantiomers. Selected column has a limited number of appropriate mobile phases. Among the selected alkaline mobile phases, a solution of triethylamine and a phosphate buffer did not allow separation of the racemic mixture of inhibitors. Given the fact that separation with borate buffer was successful on analytical coloumn, the method was transferred to semi-preparative column. Milligram amounts of the enantiomers of N-((1-(2,3-dihydro-1H-inden-2-yl)piperidin-3-yl)methyl)-N-(2-methoxyethyl)-2-naphthamide and N-((1-benzylpiperidin-3-yl)methyl)-N-(2-methoxyethyl)naphthalene-2-sulfonamide were isolated. Further, their optical purity and specific rotation were measured. Using the enzyme assays different level of stereoselectivity of the pure enantiomers was observed. The median inhibitory concentration of pure enantiomers, as well as their racemic mixtures was determined. The most active enantiomer (+)-1 with 13.4 nM IC50 value has more than 10-times stronger affinity for the butyrylcholinesterase active site if compared to distomer (-)-1. In the case of sulfonamide analogue (±)-2, the stereoselectivity of pure enantiomers is markedly reduced, whereby 4.5 nM IC50 value was determined for eutomer (-)-2. butiril-holin esteraza kiralnost zaviralci butirilholin-esteraze enantiomeri ločba z boratnim pufrom encimski test true false false [M. Černigoj] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2020-09-29 10:51:18 2020-09-29 10:51:18 2022-09-01 04:01:16 0000-00-00 00:00:00 2015 0 Ljubljana 2015 Ljubljana V, 45 f. 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 543.057:616.894(043.3) 3885169 MAG_Cernigoj_Matevz_2015.pdf MAG_Cernigoj_Matevz_2015.pdf 1 15F30631C409A8A32D7B07C310EDEB0A f508d6f894b38d6e99087dcdb1d914776ffcad5510738c0bc6e7e6c7b26767da 35b8c78b-a1ba-11eb-a523-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=135889 Fakulteta za farmacijo 0 0 0