Design, synthesis and biochemical evaluation of azine dual butyrylcholine esterase and mitogen activated protein kinase 14 inhibitors

Alzheimerjeva bolezen (AB) je progresivna nevrodegenerativna bolezen, za katero so značilne izgubljanje spomina in pešanje kognitivnih sposobnosti. Z mitogenom aktivirana protein kinaza MAPK 14 (tudi p38α MAPK) in butirilholinesteraza (BChE) po najnovejših dognanjih predstavljata tarči, ki bi lahko imeli eno ključnih vlog pri nastanku in napredovanju te bolezni. Signalne kaskade p38α MAPK svojo vlogo pri napredovanju AB izkazujejo pri procesih z mikroglijo in astrociti posredovanega nevrovnetja, pri pretirani fosforilaciji proteina tau in pri spremembi sinaptične plastičnosti v nevronih. BChE kot serinska hidrolaza katalizira hidrolizo acetilholina, ki ima pomemben vpliv v različnih kognitivnih procesih ter se nabira v amiloidnih plakih β in nevrofibrilarnih pentljah, kjer stimulira njihovo patološko delovanje. V magistrski nalogi smo načrtovali, sintetizirali in ovrednotili tri potencialne dvojne azinske zaviralce BChE in p38α MAPK. Za osnovo pri načrtovanju in optimizaciji spojin nam je služila spojina ARRY-371797. Ker predpostavljamo, da je za vezavo na obe tarči v strukturi zaviralcev nujna skupina, ki bo zasedla mesto v hidrofobnem žepu p38α MAPK in acil-vezavnem žepu BChE, smo v načrtovanih spojinah ohranili 2,4-difluorofenoksi skupino. Prav tako smo v strukturi ARRY-371797 ohranili -(CH2)2NMe2 skupino. Ta namreč vsebuje -NMe2 skupino, ki je pri zaviralcu BChE 1 tvorila, za zaviralno jakost nujne, kation-π interakcije s Trp82 v holin-vezavnem žepu encima. Predvidevamo tudi, da je za zaviranje p38α MAPK ključna interakcija med heterocikličnim dušikom in Met109 na sklepni regiji tarče. Da bi ohranili ali celo povečali jakost zaviranja p38α MAPK in BChE, smo v ARRY-371797 1-izobutil-1H-indazolni del zamenjali z različnimi azinskimi obroči, s čimer smo dosegli manjše in bolj fleksibilne molekule. Hkrati smo s tem želeli raziskati tudi točen vpliv mesta dušika v omenjenem heterociklu na jakost zaviranja encimov. Sintetizirane spojine 16-18 na podlagi vrednosti rezidualne aktivnosti niso imele zaviralnega delovanja na p38α MAPK in BChE. Kljub temu smo prišli do pomembnih ugotovitev. V strukturi zaviralcev je za vezavo na vezavno mesto p38α MAPK, zaradi dovolj velike razdalje med heterocikličnim dušikom in 2,4-difluorofenoksi skupino, potrebno večje, dvociklično ogrodje (npr. indazol). Za zaviranje BChE je pri zaviralcih namesto rigidnega azinskega »strukturnega jedra« potrebna večja fleksibilnost -le tega, kar bi povečalo možnosti za zgoraj omenjene vezave ostalih fragmentov v vezavnem mestu encima. Pri 1 namreč to strukturno jedro predstavlja gibljiv piperidinski obroč. Spojine 16-18 kljub slabi aktivnosti tako vseeno predstavljajo dobro osnovo za nadaljnji razvoj in optimizacijo učinkovin pri hkratnem zaviranju tarč p38α MAPK in BChE za zdravljenje AB.Alzheimer\u27s disease (AD) is a progressive neurodegenerative disease, characterized by impairment of memory and cognitive abilities. Mitogen-activated protein kinase 14 (or p38α MAPK) and butyrylcholin esterase (BChE), represent targets, which play key roles in the onset and progression of this disease. In progression of AD, MAPK\u27s signalling cascades demonstrate their role in processes of microglia and astrocyte-mediated neuroinflammation, in hyperphosphorylation of protein tau and in changes in alteration of synaptic plasticity in neurons. BChE, a serine hydrolase, catalyses the hydrolysis of acetylcholine, which has an important role in various cognitive processes and accumulates in amyloid plaques β and neurofibrillary tangles, where it stimulates their pathological activities. In this Master\u27s thesis, we synthesised and evaluated three potential dual azine BChE and p38α MAPK inhibitors. ARRY-371797, a known compound from Pfizer Inc. served us as the basis for synthesis of our compounds. In its structure, the 2,4-difluorophenoxy group, which we assumed interacts in the hydrophobic pocket of p38α MAPK\u27s and acyl-binding pocket of BChE\u27s binding site remained unchanged. -(CH2)2NMe2 functional group remained unchanged as well, since it includes -NMe2 functional group, which was important for the inhibitory potency of 1 through its cation-π interactions with Trp82 in the cholin-binding pocket of BChE’s binding site. When inhibiting p38α MAPK, we assumed that interaction between heterocyclic nitrogen and Met109 at the p38α MAPK\u27s hinge region is also important. To preserve or even enhance inhibiting potency, we replaced the 1-isobuthyl-1H-indazole part with different azine rings, to obtain smaller and more flexible molecules. We also wanted to compare the influence of the position of nitrogen atoms on the inhibiting potency of inhibitors. Residual activities of the synthesised compounds 16-18 did not show the desired potency. However, we still came to important conclusions. When binding to the binding site of p38α MAPK, large enough distance between nitrogen and 2,4-difluorophenoxy moiety is necessary. In that aspect, the most suitable core is heteroaromatic bicyclic core, for example indazole. When inhibiting BChE, instead of rigid azine structural core, some other core, with greater flexibility should be applied. This would enhance chances for the aforementioned interactions of other inhibitor’s fragments when binding to the binding site of BChE. In 1 for example, this core represents piperidine ring. Compounds 16-18, despite their poor activity, still represent a good basis for further development and optimization of dual inhibitors of p38α MAPK and BChE for the treatment of AB