Načrtovanje, sinteza in vrednotenje derivatov 4'-O-benziltiramina kot kovalentnih zaviralcev monoamin oksidaze B

Kavaš, Vid (author), Knez, Damijan (mentor), Meden, Anže (co-mentor)

Tarčni kovalentni zaviralci so spojine, ki se kovalentno vežejo na specifično tarčo in zavrejo njeno biološko delovanje. Zaželeno je, da se del molekule, odgovoren za selektivno nekovalentno vezavo z visoko afiniteto, veže v aktivno mesto encima, da lahko šibko elektrofilna glava molekule tvori vez z nukleofilom v bližini ter tako kovalentno zavre encim. Tarčni kovalentni zaviralci običajno ciljajo tiolno skupino cisteina, vendar zaradi nizke zastopanosti le-tega v proteinih razvijajo tarčne kovalentne zaviralce, ki ciljajo druge nukleofilne ostanke, najpogosteje lizin, serin, treonin, aspartat in glutamat. Lizin vsebuje primarni amin, ki lahko reagira z elektrofilno bojno glavo, vendar je v fizioloških pogojih skoraj vedno protoniran in tako ni na voljo kot nukleofil. Da lahko uspešno ciljamo lizinske preostanke, morajo le-ti imeti dovolj nizko vrednost pKa, da so vsaj v določenem deležu v neprotonirani obliki. V sklopu magistrske naloge smo se osredotočili na razvoj selektivnih zaviralcev monoamin oksidaze B (MAO-B). Cilj je bil razvoj tarčnih kovalentnih zaviralcev, ki ciljajo nekatalitični lizin 296 v aktivnem mestu encima, saj ima ε-amino skupina Lys296 izračunano vrednost pKa enako 7,3. Izhajali smo iz 4`-O-benzil tiramina, ki vsebuje benziloksi farmakofor, značilen za selektivne zaviralce MAO-B. Med sintezo načrtovanih zaviralcev MAO-B smo uporabili klasične pristope za sintezo organskih molekul. Elektrofilne bojne glave smo vezali na osnovni skelet z alkiliranjem amina ali pa z uporabo aktiviranih oblik kislin (karboksilne ali sulfonske) in tako tvorili sečnine, amide, estre in tioestre. Pripravili smo 31 derivatov 4`-O-benzil tiramina in z biokemijskimi testi preverili njihovo zaviralno delovanje na MAO-A in MAO-B. Spojine 12, 24, 28a, 34, 42, 43b, 46 in 47b so zavirale MAO-B z IC50 vrednostmi, nižjimi od 100 nM. Kot pomembne bojne glave smo prepoznali karbamoil in sulfamoil fluoride, nitrile, imidazolide in 1,2,3-triazolide. Čeprav so nekateri pripravljeni derivati močneje zavirali MAO-B, je za nadaljnjo optimizacijo najbolj obetavna spojina 28a, saj ima od spojin, ki nakazujejo na kovalentno vezavo (24, 28a, 28b), najnižjo vrednost IC50.

• Language: Slovenian
• Keywords: monoamin oksidaza B, tarčni kovalentni zaviralci usmerjeni proti lizinu, derivati 4`-O-benzil tiramina, elektrofilne bojne glave
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2022
• PID: 20.500.12556/RUL-139736
• Publication date in RUL: 07.09.2022

Secondary language

• Language: English
• Title: Design, synthesis and evaluation of 4'-O-benzyltyramine derivatives as covalent monoamine oxidase B inhibitors

Abstract

Targeted covalent inhibitors are compounds that form covalent bonds with their target and suppress its function. Preferably, the non-covalent core of the molecule binds with high affinity to the active site of the target, whereupon the weakly electrophilic warhead forms a covalent bond with a nucleophilic residue, resulting in inhibition. The nucleophile of interest is usually the thiol group of cysteine residues, but because of their low abundance in proteins, lysine, serine, threonine, aspartate, and glutamate have also been explored recently. The primary ε-amine of lysine can react with the electrophilic warhead, yet protonation of the amine at physiological pH substantially decreases its nucleophilicity. For lysine to be suitable as a target nucleophile, the pKa value of the ε-amine should be low enough for the amine to be at least partially in unprotonated form. In this master's thesis, we focused on selective inhibitors of monoamine oxidase B (MAO-B). Our goal was to develop targeted covalent inhibitors for lysine 296 near the active site, which we identified as a suitable target because the predicted pKa value of the ε-amine group is 7,3. We started with 4`-O-benzyl tyramine containing the benzyloxy pharmacophore archetypal for selective MAO-B inhibitors. To synthesize the designed MAO-B inhibitors, we used classical organic chemistry approaches. Electrophilic warheads were attached to the core structure by amine alkylation or by the use of activated acid derivatives (carboxylic or sulfonic) to give urea, amide, ester, and thioester derivatives. We synthesized 31 4`-O-benzyl-tyramine derivatives and assayed for their inhibition of monoamine oxidase A and B. Compounds 12, 24, 28a, 34, 42, 43b, 46, and 47b inhibited MAO-B with IC50 values lower than 100 nM. The optimal warheads were carbamoyl and sulfamoyl fluorides, nitriles, imidazolides, and 1,2,3-triazolides. Regardless of the potent MAO-B inhibition by various derivatives, the most promising compound for further optimization is 28a, the most potent MAO-B inhibitor among the compounds (24, 28a, 28b) for which covalent inhibition is proposed.

• Keywords: monoamine oxidase B, lysine targeting covalent inhibitors, 4`-O-benzyl tyramine derivates, electrophilic warheads