Chemical space exploration of p38ɑ MAP kinase and butyrylcholinesterase dual inhibitors with piridazine scaffold

Alzheimerjeva bolezen je kronična nevrodegenerativna motnja, ki vodi v upad kognitivnih funkcij in nastanek nevropsihiatričnih simptomov. Poznamo več možnih patofizioloških mehanizmov nastanka bolezni, ki kažejo na smiselnost raziskovanja novih zdravilnih učinkovin z multiplim delovanjem. Aktualni terapevtski tarči sta butirilholin esteraza (BChE), ki jo povezujemo s poslabšanjem kognitivnih funkcij, ter kinaza p38α MAP (p38ɑ MAPK), ki je povezana z nevrovnetjem in prispeva k propadanju nevronov. Na osnovi spojine MW150, selektivnega zaviralca p38α MAPK, smo načrtovali in sintetizirali dvojne zaviralce p38ɑ MAPK in BChE s piridazinskim, 5-aminopirazolnim in morfolin-3-onskim ogrodjem. Pri sintezi derivatov piridazina smo izhajali iz Weinrebovega amida, ki smo ga preko nukleofilne adicije organolitijevega reagenta pretvorili v keton. Sledila je reakcija nukleofilne substitucije z etil bromoacetatom, kjer smo dobili 4-ketoester, ki smo ga pretvorili do cikličnega hidrazida in nato oksidirali do piridazin-6-ona. Slednjega smo s fosforil kloridom pretvorili v 6-kloropiridazin, na katerega pa smo z namenom raziskovanja odnosa med strukturo in delovanjem pripenjali različne amine. Derivate 5-aminopirazola smo sintetizirali preko aktiviranega N-hidroksisukcinimidnega estra, ki smo ga pretvorili v 2-cianoketon, tega nadalje v β-kloroakrilonitril, sledila pa je kondenzacija do 5-aminopirazola 35. Sinteza morfolin-3-ona 44 je potekala v treh stopnjah, najprej smo z nukleofilno adicijo nitrita na mesto α ketona uvedli oksim, ki smo ga v naslednji stopnji reducirali do amina in nato v dveh zaporednih stopnjah zaprli do morfolin-3-onskega obroča. Sintetizirali smo 15 spojin, ki smo jim s spektroskopskimi in kromatografskimi metodami potrdili identiteto in čistost ter ovrednotili vpliv strukturnih modifikacij na zaviralno delovanje na človeški acetilholin esterazi (hAChE), hBChE in p38α MAPK. Večina sintetiziranih derivatov je zavirala katalitično aktivnost kinaze p38α MAPK, medtem ko je hBChE zaviralo manj spojin. Najmočnejše in uravnoteženo zaviralno delovanje na obeh tarčah je imel piridazin 15 [IC50(hBChE) = 39,9 ± 4,8 nMIC50(p38α MAPK) = 127 ± 12 nM].Alzheimer\u27s disease is a chronic neurodegenerative disease that leads to a decline in cognitive function and the development of neuropsychiatric symptoms. Several possible pathophysiological mechanisms for the onset of the disease are described, which justified the development of drugs with multiple mechanisms of action. Therapeutic targets of interest are butyrylcholinesterase (BChE), which is related to cognitive decline, and p38α MAPK, which is associated with neuroinflammation and contributes to neuronal degeneration. Based on the structure of selective p38α MAPK inhibitor MW150, we designed and synthesized pyridazine, 5-aminopyrazole, and morpholin-3-one derivatives as dual inhibitors of p38ɑ MAPK and BChE. The synthesis of pyridazine derivatives began with the corresponding Weinreb amide, which was converted to a ketone by nucleophilic addition of an organolithium reagent. This was followed by a nucleophilic substitution reaction with ethyl bromoacetate to form 4-keto ester, which was further converted to cyclic hydrazide, and then oxidized to pyridazin-6-one. The latter was converted to a 6-chloropyridazine derivative using phosphoryl chloride, and various amines were attached to study the structure-activity relationship. 5-Aminopyrazole derivatives were synthesized via activated N-hydroxysuccinimide ester, which was first converted to 2-cyanoketone, then to β-chloroacrylonitrile and finally via condensation to 5-aminopyrazole 35. The synthesis of the morpholin-3-one derivative was carried out in three steps. First, oxime was formed by nucleophilic addition of nitrite to the α-position of ketone, which was reduced to amine and then converted to morpholin-3-one 44 in two successive steps. We synthesized 15 compounds, confirmed their identity and purity by various spectroscopic and chromatographic methods, and evaluated the effects of structural changes on inhibitory activity on human acetylcholinesterase (h)AChE, hBChE, and p38α MAPK. Majority of synthesized derivatives inhibited the catalytic activity of the kinase, while only a few compounds inhibited hBChE. Pyridazine 15, was the most potent and balanced dual inhibitor [IC50(hBChE) = 39.9 ± 4.8 nM, IC50(p38α MAPK) = 127 ± 12 nM]