Alkyl chain replacement on position 1 of indazole dual inhibitors of butyrylcholinesterase and mitogen-activated kinase p38α

Alzheimerjeva bolezen je kronična progresivna nevrodegenerativna bolezen, pri kateri so v ospredju znaki demence. Poleg kognitivnih motenj alzheimerjevo bolezen spremljajo tudi nevropsihiatrični simptomi. Etiologija bolezni še ni popolnoma pojasnjena, venar pa med možne vzroke štejejo senilne lehe, nevrofibrilarne pentlje, moteno holinergično transmisijo, nevrovnetje in oksidativni stres. Trenutni terapevtski pristopi za zdravljenje alzheimerjeve bolezni vključujejo zaviralce acetilholinesteraz, zaviralce N-metil-D-aspartatnih receptorjev in protitelesa proti amiloidu beta. Registrirane učinkovine se uporabljajo predvsem z namenom upočasnitve upadanja kakovosti življenja in ne vplivajo na proces bolezni, ob čemer se pojavlja nuja po razvoju izboljšanih učinkovin za zdravljenje alzheimerjeve bolezni. Potencialno tarčo za zdravljenje bolezni predstavljata butirilholin esteraza, katere encimska aktivnost je povečana v možganih bolnikov z alzheimerjevo boleznijo in z mitogenom aktivirana protein kinaza p38α, ki prispeva k nevrovnetju in apoptozi nevronskih celic, mikroglije in astrocitov. Zato smo v magistrski nalogi sintetizirali štiri nove dvojne zaviralce butirilholin esteraze in z mitogenom aktivirane protein kinaze p38α. Izhajali smo iz že znane spojine ARRY-371797, znanega selektivnega zaviralca p38α, ki zavira tudi butirilholin esterazo in vitro. Ohranili smo osrednji 5,6-disubstituiran indazolni skelet, pri čemer je substituent na mestu 5 vedno predstavljala 2,4-difluorofenoksi skupina, za katero predvidevamo, da tvori pi-pi interakcije v acil-vezavnem žepu BChE in ugodno zasede hidrofobno regijo I p38α. Na mestu 6 smo ohranili N-(2-(dimetilamino)etil)amidni substituent, za katerega predvidevamo, da tvori močne kation-pi interakcije s Trp82 holin-vezavnega žepa butirilholin esteraze, v aktivnem mestu p38α pa naj bi bil usmerjen proti topilu in tako ne bi motil vezave liganda v ATP-vezavno mesto. Z namenom, da bi dosegli močnejše zaviralno delovanje na obe tarči in proučili vpliv različnih verig na aktivnost molekule, smo na mestu 1 indazolnega obroča zamenjali izobutilno skupino z drugimi alkilnimi verigami. Z metodo po Ellmanu in ADP-Glo testom smo ovrednotili aktivnost sintetiziranih spojin 7A-E na oba encima in vitro in ugotovili, da spojina 7D z izopentilno alkilno verigo najmočneje zavira encim butirilholin esterazo, najmočnejše zaviralno delovanje na z mitogenom aktivirano protein kinazo p38α pa smo dosegli s spojino 7E, pri kateri cikloheksen najverjetneje dodatno rigidizira strukturo spojine, da zasede najboljšo lego v aktivnem mestu encima. Zaviralno delovanje spojine 7E (IC50 = 87,6 nM) je celo preseglo učinkovitost ARRY-371797 na z mitogenom aktivirano protein kinazo p38α. Dosegli smo selektivnost na butirilholin esterazo v primerjavi z acetilholinesterazo in učinkovito zaviralno delovanje na z mitogenom aktivirano protein kinazo p38α, kar predstavlja pomembno izhodišče za nadaljnji razvoj učinkovin za zdravljenje AB.Alzheimer\u27s disease is a chronic progressive neurodegenerative disease in which the signs of dementia are prominent and, in addition to cognitive disorders, disease is also accompanied by neuropsychiatric symptoms. The etiology of the disease is not yet fully understood, but the possible causes and processes of Alzheimer\u27s disease are associated with senile plaques, neurofibrillary tangles, disturbed cholinergic transmission, neuroinflammation and oxidative stress. Current therapeutic approaches for the treatment of Alzheimer\u27s disease include acetylcholinesterase inhibitors, inhibitors of N-methyl-D-aspartate receptors and anti-amyloid-beta antibodies. Registered active substances are primarily used to slow down the decline in quality of life and do not affect the disease process. Because of that there is a need to find and synthesize improved active substances for the treatment of Alzheimer\u27s disease. Butyrylcholinesterase, whose enzyme activity is increased in the brains of patients with Alzheimer\u27s disease, and mitogen-activated protein kinase p38α, which contribute to neuroinflammation and apoptosis of neurons, microglia and astrocytes, are potential targets for the treatment of the disease. Therefore, in our master\u27s thesis, we synthesized four new dual inhibitors of butyrylcholinesterase and mitogen-activated protein kinase p38α. We started from the already known compound ARRY-371797, which has an inhibitory effect on both enzymes. We kept the central 5,6-disubstituted indazole skeleton, whereby the substituent in position 5 was always represented by a 2,4-difluorophenoxy group, which we predict forms pi-pi interactions in the acyl-binding pocket of butyrylcholinesterase and favorably occupies the hydrophobic region I of p38α. The N-(2-(dimethylamino)ethyl)amide substituent was retained in position 6, which is predicted to form strong cation-pi interactions with Trp82 of the choline-binding pocket of butyrylcholinesterase, and in the active site of p38α, it is expected to be directed towards the solvent and thus would not interfere with ligand binding to the ATP-binding site. In order to achieve a stronger inhibitory effect on both targets and to study the influence of different alkyl chains on the activity of the molecule, we replaced the isobutyl group at position 1 of the indazole ring with other alkyl chains. Using the Ellman method and the ADP-Glo test, we evaluated the synthesized compounds 7A-E and found that compound 7D with an isopentyl alkyl chain most strongly inhibits the BChE enzyme, and the strongest inhibitory effect on p38α MAPK was achieved with compound 7E, in which cyclohexene most likely additionally rigidifies the structure of the compound and occupies the best position in the active site of the enzyme. The inhibitory activity of compound 7E (IC50 = 87,6 nM) was even better that activity of ARRY-371797 on mitogen-activated protein kinase p38α. We achieved selectivity for butyrylcholinesterase compared to acetylcholinesterase and effective inhibitory action on mitogen-activated protein kinase p38α which represents an important starting point for the further development of agents for the treatment of AD