Design, synthesis and evaluation of potential drugs for treatment of Alzheimer\u27s disease with multiple mechanisms of action

Nevrodegenerativna obolenja zaradi staranja prebivalstva in posledično večjega števila bolnikov predstavljajo pereč zdravstveni problem. Med najbolj prepoznanimi je Alzheimerjeva bolezen, napredujoča motnja centralnega živčnega sistema, ki vodi v propad predelov možganov, posledično izgubo spomina ter motenj ostalih kognitivnih sposobnosti. Točen mehanizem nastanka in poteka bolezni še vedno ni pojasnjen, znano pa je, da do klinične slike vodi več patoloških procesov na molekularnem in celičnem nivoju. Izrazite poškodbe holinergičnega sistema in upad koncentracije živčnega prenašalca acetil holina privedejo do tipičnih motenj v spominu, ki se lajšajo z zaviralci holin esteraz. Encim butirilholin esteraza (BChE) opravlja v nekaterih predelih možganov podporno vlogo pri zaključevanju holinergičnega prenosa, z napredovanjem bolezni pa lahko ta vloga celo prevlada, saj encim opravi glavnino hidrolazne aktivnosti. Zaradi povečane aktivnosti in izražanja v poznih stadijih boleznih predstavlja BChE pomembno tarčo za lajšanje simptomov v teh stopnjah Alzheimerjeve bolezni. Poleg opisanega igrajo pri razvoju bolezni pomembno vlogo tudi številni drugi procesi. Nastanek in agregacija amiloida beta do amiloidnih plakov je ena od najbolj izrazitih patoloških sprememb, opaženih pri bolnikih. Različne zvrsti amiloida beta povezujejo z nevrotoksičnostjo, h kateri pa pomembno prispevajo tudi povečan oksidativni stres in motnje v porazdelitvi biološko pomembnih dvovalentnih kovinskih ionov. Trenutna simptomatska terapija Alzheimerjeve bolezni nima velikega vpliva na potek bolezni. Za učinkovito zdravljenje in izboljšanje simptomov bi bilo zato potrebno delovati na več tarč hkrati. Razvoj potencialnih učinkovin, ki bi lahko vzročno vplivale na nastanek in potek bolezni, se je tako usmeril v načrtovanje učinkovin z multiplim mehanizmom delovanja (multifunkcionalne spojine). Selektiven zaviralec BChE 1 (IC50 = 21,3 nM), ki je bil rezultat uspešnega strukturno podprtega virtualnega rešetanja, in njegovo kristalno strukturo v kompleksu s človeško BChE, smo uporabili kot osnovo za načrtovanje multifunkcionalnih ligandov predstavljenih v tem delu. Spojina 1 zavira spontano agregacijo amiloida beta do toksičnih oligomerov in fibrilov, zaradi česar deluje nevroprotektivno. Ob upoštevanju pomembne vloge kovinskih ionov v nastanku Alzheimerjeve bolezni, smo naftalen spojine 1 zamenjali z nitroksolinom, ki deluje kot kelator dvovaletnih kovinskih ionov. Spojina 8g z nanomolarno zaviralno aktivnostjo na človeški BChE (IC50 = 215 nM) selektivno kompleksira bakrove(II) ione in zavira spontano agregacijo amiloida beta, hkrati pa ima tudi šibek antioksidativni učinek. Kristalna struktura te spojine v kompleksu s človeško BChE je razkrila vezavo nitroksolina v acil vezavni žepek in s tem pokazala, da je ta del vezavnega mesta sposoben vezati tudi od naftalena večje fragmente. Derivate nitroksolina tako smatramo kot dobro izhodišče za nadaljnjo optimizacijo učinkovin za terapijo Alzheimerjeve bolezni z multiplim mehanizmom delovanja. Vzporedno z razvojem multifunkcionalnih ligandov je potekala tudi optimizacija zaviralne aktivnosti spojine zadetka 1 ter raziskovanje odnosa med strukturo in zaviralnim delovanjem analogov. Velika večina sprememb v strukturi zaviralca je poslabšala zaviralno aktivnost. Z nadomestitvijo metoksietilne verige spojine 1 z 2-(dimetilamino)etilno verigo pa smo uspeli izkoristiti še dodatno interakcijo s Trp82 človeške BChE, kar je povečalo moč zaviranja, ki se pri spojini 3 giblje v pikomolarnem območju. Spojina je ohranila način vezave v aktivno mesto BChE in šibek nevroprotektiven učinek. Glede na pomembno vlogo oksidativnega stresa in motenj v porazdelitvi kovinskih ionov, smo v molekulo najboljšega reverzibilnega zaviralca BChE s piperidinsko strukturo 3 uvajali različne fragmente s sposobnostjo kompleksacije kovinskih ionov in antioksidativnimi lastnostmi. Spojini 8 in 11 z IC50 vrednostima za človeško BChE 13,8 ± 0,6 oziroma 11,1 ± 0,6 nM, spadata med najboljše zaviralce iz te serije. Kristalna struktura kompleksa spojine 11 in človeške BChE je, podobno kot za nitroksolinski derivat, pokazala vezavo 8-hidroksikinolinskega fragmenta v acil vezavni žep encima. Spojina je v kokristalu vezana tudi na površini med dvema monomeroma encima, kjer vezavno mesto tvorita dva plitva žepa. Alternativna vezava po vsej verjetnosti nima vpliva na aktivnost encima, spojina pa bi lahko bila potencialno uporabna kot spodbujevalec kristalizacije človeške BChE. 8-hidroksikinolinska derivata 8 in 11 kažeta tudi dobre antioksidativne lastnosti in kompleksacijo bakrovih(II) ionov, ki po vezavi v kompleks niso več redoks aktivni. Kljub temu, da spojini ne zavirata procesa spontane agregacije amiloida beta, spojina 11 ščiti nevroblastomske SH-SY5Y celice pred toksičnostjo fibrilov amiloida beta. Poleg navedenega oba analoga znižata znotrajcelične koncentracije reaktivnih kisikovih zvrsti, s pasivno difuzijo prehajata membrane v Caco2 celičnem modelu ter nista substrata za prenašalne proteine (primer P-glikoprotein). Derivata 8 in 11 zaradi dobrega profila dodatnih aktivnosti v primerjavi z nitroksolinskimi analogi in spodbudnih rezultatov na celičnih testih predstavljata naslednjo generacijo izboljšanih multifunkcionalnih ligandov za terapijo Alzheimerjeve bolezni. Poleg že vseh naštetih faktorjev je pri Alzheimerjevi bolezni opazna tudi povečana aktivnost monoamin oksidaze v določenih predelih možganov. Povečana oksidativna deaminacija zniža koncentracijo živčnih prenašalcev, kar privede do motenj v vedenju. Pri metabolizmu nastane tudi več toksičnih produktov, ki še dodatno prispevajo k oksidativnemu stresu in nevrodegeneraciji, zato predstavlja monoamin oksidaza dobro tarčo, preko katere lahko vplivamo tudi na vzroke nastanka Alzheimerjeve bolezni. Zelo pogosto se zaviralna aktivnost na monoamin oksidazi združi z zaviranjem ene ali obeh holin esteraz. V tem primeru govorimo o t.i. dualnih zaviralcih, ki jih je v literaturi opisanih kar nekaj, in jih lahko razdelimo na tri razrede: derivati propargilamina, kumarini in derivati naravnih spojin. Najbolj raziskana je zagotovo prva skupina, kjer spojina ASS234 in ladostigil kažeta dobre rezultate v predkliničnih testiranjih, slednji pa tudi v kliničnih fazah testiranj. Preko uvedbe propargilne skupine na piperidinski dušik analoga spojine zadetka 1 smo pri spojini 6 dosegli dualno mikromolarno zaviranje dveh encimov, BChE (IC50 = 2,600 ± 0,348 μM) in monoamin oksidaze B (IC50 = 53,904 ± 4,781 μM). Zaviranje monoamin oksidaze B je ireverzibilno in časovno odvisno, kar sovpada z literaturno znanimi podatki o zaviralcih propargilaminskega tipa. Spojina 6 v testu prehajanja s sistemom vzporednih umetnih membran kaže velik potencial za prehajanje krvno možganske pregrade, za nevroblastomske celice ni citotoksična, sočasno pa jih ščiti tudi pred toksičnostjo amiloida beta. N-propargilpiperidinske dualne zaviralce BChE in monoamin oksidaze B lahko tako smatramo kot dobro izhodišče za nadaljnjo optimizacijo in razvoj multiplih ligandov za terapijo Alzheimerjeve bolezni.Due to the aging of the population and the concomitant increase in the number of people with neurodegenerative diseases, these disorders are becoming an enormous health care problem. Among the most renowned is the Alzheimer\u27s disease, a progressive disorder of central nervous system leading to dysfunctions in certain areas of the brain, and subsequent loss of memory and disturbances in other cognitive functions. The exact cause of the disease is still unknown, yet several pathological processes on molecular and cellular level lead to the observed clinical picture. Alterations in the cholinergic system and reduced levels of neurotransmitter acetylcholine lead to typical memory loss, which can be alleviated by cholinesterase inhibitors. Butyrylcholinesterase (BChE) helps with the termination of the cholinergic neurotransmission in some parts of the brain. With the progression of the disease its hydrolytic function becomes more important and can even take over the cholinesterase activity. Due to the increased activity and expression in latter stages of Alzheimer\u27s disease, BChE represents an important target for the alleviation of symptoms in the late stages of Alzheimer\u27s disease. Several other factors significantly contribute to the development of Alzheimer\u27s disease. Aggregation of amyloid beta peptide and the formation of senile plaques are one of the hallmark pathological changes. Apart from various amyloid beta species, increased oxidative stress and disturbances in bivalent metal ion distribution are also related to neurotoxicity. Current symptomatic management of Alzheimer\u27s disease does not have any significant effect on the disease progression, thus several pathological changes should be targeted simultaneously to address the underlying cause. The development of new drugs has therefore shifted towards multi-target-directed ligands (multifunctional ligands) capable of interacting with the factors related to the disease development and progression. Selective BChE inhibitor 1 (IC50 = 21.3 nM), a result of a successful structure based virtual screening, and its crystal structure in complex with human BChE was used as the basis for the design of multifunctional ligands presented herein. This compound also inhibits self-induced aggregation of amyloid beta to oligomers and fibrils and has a neuroprotective activity. Taking into the account the important role of metal ions in the disease pathogenesis, the naphthalene moiety of 1 was replaced with nitroxoline, a known chelating agent of bivalent metal ions, to yield the multifunctional ligand 8g. Compound 8g, with nanomolar inhibitory potency against human BChE (IC50 = 250 nM), also selectively chelates copper(II) ions, inhibits amyloid beta aggregation, and has weak antioxidant activity. The co-crystal structure of 8g with human BChE reveals the binding of the nitroxoline into the acyl binding pocket and proves that this part of the active site gorge can indeed accommodate fragments, bigger than naphthalene. This nitroxoline derivative can therefore be considered as a good starting point for further optimization of multi-target-directed ligands against Alzheimer\u27s disease. In parallel to the development of multifunctional ligands, the optimization of compound 1 was performed in order to increase the inhibitory potency and to elucidate the structure-activity relationships. The majority of structural modifications led to diminished inhibitory potency. On the other hand, the replacement of methoxyethyl side chain of 1 with 2-(dimethylamino)ethyl moiety sequestered an additional interaction with Trp82 of human BChE, which led to increased inhibitory potency. Compound 3 is a picomolar BChE inhibitor with the same binding mode into the BChE active site gorge as observed for 1. Derivative 3 also shows weak neuroprotective activity. The structure of the most potent reversible piperidine-based inhibitor 3 was used to design another series of multifunctional ligands with metal chelating and antioxidant properties. Compounds 8 and 11 with IC50 values for human BChE 13.8 ± 0.6 nM and 11.1 ± 0.6 nM, respectively, are among the best inhibitors in the series. Resolved crystal structure of compound 11 in human BChE has once again confirmed the binding of 8-hydroxyquinoline moiety in the acyl binding pocket as observed before for the nitroxoline analog. The compound is also bound to the interface of monomeric units of BChE where the binding site is composed of shallow pockets, one on each monomer. This alternative binding probably does not affect the enzyme activity, nonetheless the compound could be useful as an inductor of human BChE crystallization. 8-Hydroxyquinoline derivatives 8 and 11 are also good antioxidants and chelators of copper(II) ions. The complexes of compounds 8 and 11 with copper(II) are not redox active. No effect on amyloid beta self-induced aggregation has been observed, however compound 11 protects neuroblastoma SH-SY5Y cell from toxic amyloid beta fibrils. Both analogs reduce the intracellular levels of reactive oxygen species, penetrate the membrane in the Caco2 cell model via passive diffusion, and are not substrates of any efflux systems (e.g. P-glycoprotein) in the mentioned cell line. These derivatives with additional activities in comparison to the nitroxoline analogs, and good results in the cell based assays represent the next generation of multifunctional ligands for the treatment of Alzheimer\u27s disease. In addition to all of the described factors of Alzheimer\u27s disease, increased activity of monoamine oxidase is detected in particular areas of the brain. Higher level of oxidative deamination diminishes the concentrations of neurotransmitters and this further leads to disturbances in the behavior. In the metabolic reactions of monoamine oxidase additional toxic products are generated, which augment the observed oxidative stress and neurodegeneration. This enzyme thus represents another target through which the effect on the cause of the Alzheimer\u27s disease can be achieved. Often the inhibitory activity on monoamine oxidase is combined with cholinesterases inhibition. Three main classes of dual inhibitors are described in the literature: propargylamine derivatives, coumarins and derivatives of natural compounds. The first group is the most studied one, where compound ASS234 and ladostigil display promising results in preclinical studiesthe latter is also in clinical trials. The attachment of the propargyl group onto the piperidine nitrogen of the hit compound 1 resulted in compound 6, a dual micromolar BChE (IC50 = 2.600 ± 0.348 μM) and monoamine oxidase B (IC50 = 53.904 ± 4.781 μM) inhibitor. The mechanism of monoamine oxidase B inhibition is time-dependent and irreversible, which is in accordance with the literature data on propargylamine based inhibitors. Compound 6 crosses the blood brain barrier as determined in vitro in parallel artificial membrane permeability assay, is not toxic to neuroblastoma cells, and protects them against amyloid beta induced toxicity. N-propargylpiperidine based dual BChE/monoamine oxidase B inhibitors represent a solid ground for additional optimization and development of multifunctional anti-Alzheimer\u27s disease agents