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U okviru ove disertacije dizajnirano je 30 anilidopipredina, analoga jakog -opioidnog agonista i kliničkog analgetika fentanila. Razvijen je efikasan sintetički put za njihovo dobijanje, a sintetisana jedinjenja mogu se podeliti u dve serije. U okviru prve serije sintetisano je 13 novih analoga fentanila sa različitim azotnim grupama u položaju C3 piperidinskog prstena. Takođe je optimizovana sinteza dva prethodno sintetisana biciklična analoga fentanila. Jedinjenja prve serije predstavljaju prve poznate analoge fentanila sa azotnim grupama u položaju C3. U okviru druge serije sintetisano je 15 novih analoga fentanila, koji su u položaju N1 povezani sa pojedinim N-arilpiperazinima preko linearnih alkil mostova različitih dužina. Jedinjenja druge serije dizajnirana su da budu potencijalni heterobivalenti ligandi, jer sadrže anilidopiperidinsko jezgro fentanila kao -opioidnu agonističku farmakoforu, a N-arilpiperazin kao D2-dopaminergičku, agonističku farmakoforu. Analgetička aktivnost svih 30 jedinjenja ispitana je in vivo. Samo četiri jedinjenja, sva iz prve serije, pokazala su analgetičku aktivnost. Najaktivnije jedinjenje, oko 1,8 puta manje aktivno od fentanila, karakterišu brzo i kratko dejstvo analgetičkog efekta, pa se stoga može potencijalno iskoristiti u različitim farmacutskim formulacijama za tretman bola. Afinitet vezivanja jedinjenja druge serije za dopaminergčke D2 receptore ispitan je in vitro testovima kompeticije koristeći 3H spiperon kao radioaktivni ligand. Odsustvo analgetičke aktivnosti i slab afinitet vezivanja za dopaminergičke D2 receptore, ukazuje da jedinjenja druge serije ne mogu poslužiti kao heterobivalentni ligandi. Preliminarna doking analiza urađena je za jedinjenja koja su pokazala farmakološku aktivnost. This PhD thesis encompasses the design of 30 anilidopiperidines, as analogues of potent -opioid agonist and clinical analgesic, fentanyl. All compounds were prepared by the novel and effcient synthetic routes, and can be divided in two series. In the first series, 13 new analogues of fentanyl with various nitrogen groups in the C3 position of the piperidine ring were synthesized. In addition, the synthesis of two bicyclic analogues of fentanyl, previously synthesized by our group, was optimized. The compounds of the first series represent the first known fentanyl analogues possessing any nitrogen substituent at C3 position of the piperidine ring. The second series involved preparation of 15 new compounds consisting of fentanyl moiety and several arylpiperazine groups joined by the respective secondary nitrogens, via the variable-length alkyl linkers. The compounds were designed to be potential heterobivalent ligands, since they include both the fentanyl core, as the -opioid agonist pharmacophore, and N-arylpiperazine moiety as the D2-dopaminergic agonist pharmacophore. The analgesic activity of 30 compounds was tested in vivo. Only four compounds, all from the first series, showed analgesic activity. The most potent of them was 1.8 times less potent analgesic than fentanyl, with fast onset and short duration of analgesic effect, which makes this compound potentially suitable for different pharmacological formulation in the pain treatment. Binding affinity of the compounds of the second series towards dopaminergic D2 receptors was determined by in vitro competitive assay, using 3H spiperon as radioactive ligand. Compounds of the second series were found to be inactive as analgesics and with low affinity towards dopaminergic D2 receptors. Therefore, the compounds do not represent heterobivalent ligands. Preliminary docking analysis was performed for the pharmacologically active compounds.

Poslednjih godina sve više ljudi oboleva od šizofrenije, depresije i drugih neuroloških poremećaja. Njihi izazivaju promene na nivou dopaminergičkog i serotonergičkog sistema u mozgu. Dizajn i sinteza dopaminergičkih i serotonergičkih liganada koji bi našli primenu u lečenju bolesti izazvanih disfunkcijom ovih receptorskih sistema u CNS, a čije korišćenje ne bi izazivalo negativne efekte je primaran zadatak naučnika u oblasti medicinske hemije. Poznavanje strukture receptora, odnosno mesta vezivanja liganada u receptoru, doprinelo bi bržem razvoju potencijalnih lekova. U okviru ove doktorske disertacije uspešno je sintetisano i okarakterisano 46 novih liganada. Ligandi su razvrstani u dve serije: ligandi sa piperidinskim prstenom u mostu između glave (benzil, benzoil, fenetil, fenacetil grupe sa odgovarajućim supstituentima) i repa (2-metoksifenilpiperazin) i ligandi sa različitom dužinom alkil mosta između glave (supstituisani benzimidazoli) i repa (2-metoksifenilpiperazin). Za sve novosintetisane ligande određen je farmakološki profil vezivanja za dopaminske D2, serotoninske 5HT2a i adrenergičke α1 receptore u testovima kompetitivnog vezivanja sa radioaktivno obeleženim ligandima. U seriji liganada sa piperidinskim prstenom u mostu između glave (benzil, benzoil, fenetil, fenacetil grupe sa odgovarajućim supstituentima) i repa (2-metoksifenilpiperazin) uočeno je da supstituisani ligandi pokazuju veći afinitet prema dopaminskim D2 receptorima nego nesupstituisani ligandi. Pored toga, ligandi sa većim stepenom fleksibilnosti (metilenski most između piperidinskog i piperazinskog prstena) pokazali su veći afinitet od rigidnih liganada (piperazinski prsten je direktno vezan za piperidinski prsten). Ova serija liganada pokazala je odsustvo ili slab afinitet prema serotoninskim 5HT2a receptorima. U seriji liganada sa različitom dužinom alkil mosta između benzimidazolskog dela i 2-metoksifenilpiperazina utvrđeno je da najveći afinitet prema dopaminskim D2 redceptorima pokazuju ligandi sa pet i šest ugljenikovih atoma u mostu. Izuzev dva liganda, ostala jedinjenja iz ove serije poseduju umeren afinitet vezivanja za serotoninske 5HT2a receptore. U radu je putem doking analize, pored ortosternog mesta vezivanja ispitano i mesto vezivanja dopaminskog D2 receptora koje čini druga ekstracelularna petlja (ecl2). Doking analiza predviđa da ligandi pored toga što ostvaruju aromatične interakcije sa aminokiselinskim ostacima u hidrofobnom džepu receptora Phe 386 (6.44), Trp 390 (6.48), Tyr 420 (7.43), formiraju i hidrofobne interakcije sa aminokiselinskim ostacima Phe 393 i His 397 koji se nalaze u ecl2. Takođe, preliminarna doking analiza liganada sa različitom dužinom alkil mosta između glave (supstituisani benzimidazoli) i repa (2-metoksifenilpiperazin), ukazuje na postojanje hidrofobnih interakcija između liganda i Ile 166, Leu 170, Ile 184, Phe 189, Val 111, Ile 398, His 397 iz regiona druge ekstracelularne petlje. Schizophrenia, depression and related neurological disorders are modern day diseases, caused by dopaminergic and serotonergic imbalances in the brain. Design and synthesis of dopaminergic and serotonergic ligands without side effects, which could find application in the treatment of these CNS disorders, is one of the main objectives of medicinal chemistry. Improved understanding of dopamine D2 receptor binding site would contribute to faster development of potential drugs. As a part of this thesis, 46 new ligands were synthesized and characterized. The ligands were grouped into two sets: ligands with a piperidine ring in the bridge between the head (benzyl, benzoyl, phenethyl, and phenylacetyl groups with different substituents) and the tail (2-methoxyphenylpiperazine) and ligands with different lengths of the alkyl bridge between the head (substituted benzimidazoles) and the tail (2-methoxyphenylpiperazine). All newly synthesized ligands were evaluated for D2, 5HT2a and α1 affinity in an in vitro competitive displacement assay using radiolabeled ligands. Among ligands with the piperidine ring in the bridge between the head (benzyl, benzoyl, phenethyl, phenylacetyl groups with different substituents) and the tail (2-methoxyphenylpiperazine), substituted ligands exhibited a higher affinity for dopamine D2 receptors. In addition, ligands with a greater degree of flexibility (a methylene bridge between the piperidine and piperazine rings) demonstrated a higher affinity compared to rigid ligands (the piperazine ring is bound directly to the piperidine ring). This series of ligands displayed no or low affinity for the serotonin 5HT2a receptors. Among ligands with different lengths of the alkyl bridge between the benzimidazole and 2-methoxyphenylpiperazine part of the molecule, highest affinity for the dopamine D2 receptor was demonstrated by ligands with five or six carbon atoms in the bridge. Compounds in this series, with the exception of two ligands, had a moderate binding affinity for the serotonin 5HT2a receptors. The docking analysis was used to examine the D2 orthosteric binding site and the alternative binding site formed by the second extracellular loop (ecl2). Preliminary docking analysis predicts that ligands, in addition to the interactions with Phe 386 (6.44), Trp 390 (6.48) and Tyr 420 (7.43) in the hydrophobic pocket of the orthosteric binding site, form hydrophobic interactions with Phe 393 and His 397, located in the ecl2. Likewise, the preliminary docking analysis of ligands with the different lengths of the alkyl bridge between the head (substituted benzimidazoles) and the tail (2-methoxyphenyl piperazine), indicated the existence of hydrophobic interactions between the ligands and Ile 166, Leu 170, Ile 184, Phe 189, Val 111, Ile 398 and His 397, located in the second extracellular loop (ecl2).