Synthesis and evaluation of α-haloketo toloxatone derivatives as covalent monoamine oxidase A inhibitors

Monoamin oksidaza (MAO) je mitohondrijski membranski encim, ki katalizira reakcijo oksidativne deaminacije in se nahaja v različnih tkivih človeškega telesa. MAO znižuje koncentracije živčnih prenašalcev in je tako povezana z nastankom in potekom bolezni, kot sta Parkinsonova in Alzheimerjeva bolezen ter številne druge bolezni živčnega sistema. Poznani sta dve izoobliki encima, MAO-A in MAO-B, ki se med seboj razlikujeta v obliki aktivnega mesta in v aminokislinski zanki pri vhodu v aktivno mesto. Glede na strukturne razlike med aktivnima mestoma humane (h)MAO-B, ki je ploščato in podolgovato, in hMAO-A, ki je okroglo, lahko načrtujemo selektivne zaviralce posameznega izoencima. V magistrski nalogi smo načrtovali in sintetizirali tarčne kovalentne zaviralce hMAO-A, ki bi se s kovalentno vezjo vezali na aminokislinska ostanka Cys321 oziroma Cys323. Tarčne kovalentne zaviralce najpogosteje načrtujemo na osnovi že znanega zaviralca izbrane biološke tarče, ki mu na ustrezno mesto v strukturi dodamo elektrofilno bojno glavo. Kot osnovno strukturo za uvajanje elektrofilnih bojnih glav smo izbrali reverzibilen zaviralec hMAO-A toloksaton in mu na meta oziroma para pozicijo benzenovega obroča, glede na oksazolidin-2-on, uvedli različne α-halometilketone oziroma oksoaldehid. Spojinam smo v biokemijskem testu določili zaviralno aktivnost na obeh izooblikah MAO in posredno preko časovne odvisnosti zaviranja ter testa reverzibilnosti preverili ali spojine kažejo značilnosti kovalentnega zaviranja. Sintetizirali smo 12 spojin, z različnimi spektroskopskimi oziroma kromatografskimi tehnikami potrdili njihovo identiteto in čistost ter jih ovrednotili v sklopljenem biokemijskem testu, ki je pokazal, da 11 spojin zavira vsaj eno izoobliko encima MAO, kar 9 spojin pa ima boljše zaviralne aktivnosti v primerjavi z izhodnim toloksatonom. Glede odnosa med strukturo in delovanjem lahko trdimo, da so para derivati boljši zaviralci v primerjavi z ustreznimi meta analogi. Rezultati testa reverzibilnosti ter odsotnost časovno-odvisnega zaviranja hMAO so pokazali, da so spojine z veliko verjetnostjo reverzibilni in nekovalentni zaviralci tako hMAO-A kot tudi hMAO-B encima. Z izbiro alternativnih kovalentnih bojnih glav, ki se tipično uporabljajo za ciljanje cisteinskih ostankov, bi lahko z uporabo računalniških pristopov načrtovali dodatne tarčne kovalentne zaviralce na osnovi strukture toloksatona.Monoamine oxidase (MAO) is a mitochondrial membrane enzyme that catalyses the oxidative deamination reaction and is found in various tissues of the human body. MAO decreases the concentration of neurotransmitters and is therefore associated with the development and progression of diseases, e.g., Parkinson\u27s and Alzheimer\u27s diseases and numerous other neurodegenerative disorders. Two isoforms of the enzyme are known, MAO-A and MAO-B, which differ in the shape of the active site and in the amino acid loop at the entrance to the active site cavity. Considering the structural differences of the active sites in human (h)MAO-B, which is flat and elongated, and hMAO-A, which is spherical, we can design selective inhibitors for a particular isoenzyme. In our Master’s thesis, we designed and synthesised targeted covalent hMAO-A inhibitors that could potentially covalently bind to amino acid residues Cys321 or Cys323. Targeted covalent inhibitors are usually designed based on an already known inhibitor for a selected biological target and have an electrophilic warhead attached to an appropriate site in the structure. As a structural basis for attaching electrophilic warheads, we chose the reversible inhibitor of hMAO-A, toloxatone, and bound various α-halomethyl ketones and oxoaldehyde to the meta and para positions of benzene, relative to oxazolidin-2-one. In the biochemical assays, we determined the inhibitory potencies of the compounds for both MAO isoforms and indirectly verified whether the compounds exhibited features of covalent inhibition by the time-dependent inhibition and reversibility assay. We synthesized 12 compounds, confirmed their identity and purity using various spectroscopic and chromatographic techniques, and evaluated them in a coupled biochemical assay. This showed that 11 compounds inhibited at least one isoform of MAO, with 9 compounds having more potent inhibitory activity compared with parent toloxatone. In terms of structure-activity relationship, we can claim that para derivates are more potent inhibitors than their meta analogues. The results of the reversibility assay and the absence of time-dependent inhibition of hMAO indicate that the compounds are most likely reversible and noncovalent inhibitors of both, hMAO-A/B enzymes. By selecting alternative covalent warheads typically used for targeting cysteine residues, we can by using computational approaches, design additional targeted covalent inhibitors on the structural basis of toloxatone