Sinteza in vrednotenje derivatov toloksatona z vinilsulfonom kot kovalentno bojno glavo

Rajk, Luka (author), Sosič, Izidor (mentor), Knez, Damijan (co-mentor)

Tarčni kovalentni zaviralci so spojine, ki s kovalentno vezavo na encim zavirajo njegovo delovanje. Kovalentna vez se vzpostavi med nekatalitičnim aminokislinskim ostankom v encimu in elektrofilno funkcionalno skupino kovalentnega zaviralca. Tarčne kovalentne zaviralce običajno načrtujemo s pripenjanjem elektrofilnih bojnih glav na že znane reverzibilne zaviralce. Monoamin oksidaza (MAO) je flavoencim, ki se nahaja na zunanji mitohondrijski membrani in katalizira deaminacijo aminov. Pri ljudeh poznamo dve izoformi MAO, in sicer MAO-A in MAO-B, ki imata 70 % strukturno podobnost, razlikujeta pa se po velikosti in obliki aktivnega mesta ter substratih, ki jih metabolizirata. Selektivno zaviranje encima MAO-A želimo doseči predvsem pri zdravljenju depresivnih stanj, ki so posledica pomanjkanja seratonina in noradrenalina. V sklopu magistrske naloge smo načrtovali in sintetizirali ireverzibilne selektivne zaviralce encima MAO-A, pri čemer smo kot osnovni skelet uporabili strukturo učinkovine toloksaton, ki je znan reverzibilen zaviralec MAO-A. S pripenjanjem vinil sulfonskega fragmenta kot elektrofilne bojne glave na ogrodje toloksatona smo želeli doseči kovalentno ireverzibilno vezavo s cisteinoma Cys321 in Cys323, ki se nahajata v bližini aktivnega mesta encima MAO-A in sta dokazano dobra nukleofila. Za pripravo derivatov toloksatona smo uporabili večstopenjsko sintezo, ki je obsegala šest sinteznih korakov, pri kateri sta bila ključna ciklizacija do oksazolidin-2-onskega obroča in oksidacija sulfida. Sintetizirali smo meta in para položajne izomere ter jim nato v biokemijskem testiranju na izoliranih rekombinantnih humanih MAO-A in MAO-B določili zaviralno aktivnost. Uspešno smo sintetizirali 6 končnih spojin (10, 14, 16, 18, 21 in 22). Najboljšo zaviralno aktivnost na MAO-A med načrtovanimi potencialno kovalnetnimi zaviralci je pokazala spojina 10 (IC50 = 32,66 ± 3,03 µM), ki v svoji strukturi vsebuje vinil sulfonski fragment na para mestu glede na oksazolidin-2-onski obroč. Dobre zaviralne lastnosti sta pokazali tudi spojini 21 in 22, ki pa imata elektrofilni fragment nadomeščen z metilno skupino. Za spojino 10 smo s testoma časovne odvisnosti zaviranja in testom reverzibilnosti pokazali, da ne deluje kot kovalenten, ampak kot nekovalenten reverzibilen zaviralec MAO-A.

• Language: Slovenian
• Keywords: tarčni kovalentni zaviralci, MAO-A, derivati toloksatona, vinil sulfonska bojna glava, ireverzibilni zaviralci.
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2021
• PID: 20.500.12556/RUL-124495
• Publication date in RUL: 27.01.2021

Secondary language

• Language: English
• Title: Synthesis and evaluation of toloxatone derivatives with vinyl sulfone as a covalent warhead

Abstract

Targeted covalent inhibitors are compounds which inhibit the activity of a given target by binding covalently to it. The covalent bond is formed between a non-catalytic amino acid residue of the enzyme and the electrophilic functional group of covalent inhibitor. Targeted covalent inhibitors are commonly designed by attaching the electrophilic warheads to known reversible inhibitors. Monoamine oxidase (MAO) is a flavoenzyme located at the outside of the mitochondrial membrane, and catalyses the deamination of amines. In humans, two isoforms of MAO are known, namely MAO-A and MAO-B, which have 70% structural similarity. Nonetheless, they differ in the size and shape of active site and the substrate specificity. Selective inhibition of the MAO-A enzyme is to be achieved primarily in the treatment of depressive conditions resulting from serotonin and norepinephrine deficiency. In the Master's thesis presented herein, we attempted to designe and synthesize irreversible selective inhibitors of MAO-A based on the structure of toloxatone, a known reversible MAO-A inhibitor. By attaching the vinyl sulfone fragment as electrophilic warhead to the toloxatone core, we sought to achieve covalent irreversible binding to cysteines Cys321 and Cys323 located near the active site of MAO-A enzyme that are proven to be good nucleophiles. A sequential synthetic path consisting of six steps was used to prepare toloxatone derivatives, in which cyclization to the oxazolidin-2-one ring and sulfide oxidation were two curcial reactions. Meta and para positional isomers were synthesized and evaluated biochemically on isolated recombinant human MAO-A and MAO-B. We successfully synthesized 6 final compounds (10, 14, 16, 18, 21 and 22). Compound 10 (IC50 = 32.66 ± 3.03 µM), containing the vinyl sulfone fragment at the para position relative to oxazolidin-2-one ring, was the most potent MAO-A inhibitor among the synthesized compounds. Compounds 21 and 22 that possessed the methyl group instead of the electrophilic moieties also displayed low micromolar inhibition of MAO-A. Despite bearing the electrophilic warhead, compound 10 inhibited MAO-A in a reversible and non-time-dependent manner as demonstrated by reversibility and inhibition time-dependency assays, respectively.

• Keywords: targeted covalent inhibitors, MAO-A, toloxatone derivatives, vinyl sulfone warhead, irreversible inhibitors.