Synthesis, characterization and biological activity of harmicines, harmiquins and harmicenes

Rak i malarija su smrtonosne bolesti koje predstavljaju značajan globalni javnozdravstveni problem. Učinkovitost postojećih citostatika i antimalarika opada uslijed pojave rezistencije, zbog čega je potrebno kontinuirano istraživati nove potencijalne lijekove. Popularan pristup u razvoju novih lijekova je molekulska hibridizacija, odnosno kovalentno povezivanje dva bioaktivna spoja s ciljem poboljšanja njihovog djelovanja. U okviru ovog doktorskog rada sintetizirani su hibridni spojevi harmina, -karbolinskog alkaloida s izraženim antimalarijskim i protutumorskim djelovanjem, i: 1) derivata cimetne kiseline (harmicini), 2) klorokina (harmikini), 3) ferocena (harmiceni). U pripravi harmicina korištena je bakrom(I) katalizirana azid-alkin cikloadicija, odnosno klik-reakcija koja je rezultirala harmicinima triazolskog tipa. Harmikini i harmiceni pripravljeni su korištenjem klik-reakcije te reakcije povezivanja amina i karboksilnih kiselina, dajući harmikine i harmicene triazolskog i amidnog tipa. Za potrebe klik-reakcija sintetizirani su odgovarajući alkini i azidi - karbolina, derivata cimetne kiseline, 7-klorkinolina te ferocena, dok su amini i karboksilna kiselina harmina te karboksilna kiselina i amini 7-klorkinolina pripravljeni za potrebe reakcija povezivanja. Novi spojevi karakterizirani su uobičajenim analitičkim i spektroskopskim metodama te je ispitano njihovo antiproliferativno i antimalarijsko djelovanje in vitro. U seriji harmicina najjače antiproliferativno djelovanje ostvarili su triazoli 49ae, dok su u seriji harmikina najcitotoksičniji bili triazoli 54 i 57. Najsnažnije antiproliferativno djelovanje među harmicenima prema svim ispitivanim staničnim linijama pokazao je amid 82, dok su harmiceni triazolskog tipa 67, odnosno 66 i 69 pokazali značajno i selektivno djelovanje prema MCF-7 i HCT116. Najbolje antimalarijsko djelovanje na eritrocitnu fazu životnog ciklusa svih ispitanih sojeva P. falciparum, u nanomolarnim koncentracijama, pokazali su harmikini. Spoj 63 ispoljio je 5,5 puta snažniji učinak u odnosu na klorokin (CQ) (IC50 = 2 ± 0,3 nM), dok je spoj 65 bio najučinkovitiji prema sojevima plazmodija rezistentnima na postojeće antimalarike. Temeljem dobivenih rezultata može se zaključiti da harmikini predstavljaju spojeve uzore za razvoj novih potencijalnih antimalarika, dok je harmicine i harmicene potrebno razvijati kao potencijalne protutumorske lijekove.Cancer and malaria are life-threatening diseases that pose a permanent public health problem. Existing cytostatic and antimalarial drugs are progressively losing their efficacy due to the development of drug resistance. Thus, there is a constant need for the development of novel and effective drugs. One of the possible approaches is molecular hybridization, i.e. covalent linking of two bioactive moieties into a single molecule with improved properties. In this dissertation, hybrids comprising harmin, a -carboline alkaloid with pronounced antimalarial and antitumor activity, and: 1) cinnamic acid derivatives (harmicines), 2) chloroquine (harmiquins), 3) ferrocene (harmicenes) were prepared. The harmicines were synthesized by a copper(I)-catalyzed azide-alkyne cycloaddition ("click" reaction) leading to triazole-type hybrids. On the other hand, harmiquins and harmicenes were prepared by both "click" and coupling reactions, giving triazole- and amide-type harmiquins and harmicenes. To obtain the required starting compounds for the "click" reaction, synthetic routes to -carboline-, cinnamic acid derivative-, 7-chloroquinoline-, and ferrocene-based alkynes and azides were developed. For the purpose of coupling reactions, 7-chloroquinoline- and harmine-based amines and acids were prepared. The novel compounds were characterized by standard methods (1H and 13C NMR, IR, MS). The antimalarial activity of the prepared compounds was evaluated in vitro against the erythrocytic and hepatic stages of the Plasmodium life cycle, as well as the antiproliferative activity against a panel of human tumor cell lines. Among the harmicines, triazoles 49a-e exhibited the strongest antiproliferative activity, whereas in the harmiquins series, triazoles 54 and 57 were the most cytotoxic compounds. Among the harmicenes, amide 82 showed the strongest cytotoxic activity against all tested cell lines (IC50 < 10 M), whereas triazole-type harmicenes 67, 66, and 69 displayed significant and selective activity against MCF-7 and HCT116. Harmiquins have shown the best antimalarial activity against the erythrocytic phase of chloroquine-sensitive and -resistant P. falciparum strains (IC50 in low nanomolar concentrations). Compound 63 showed a 5.5-fold stronger effect than chloroquine (IC50 = 2 ± 0.3 nM), while compound 65 was the most effective against resistant Plasmodium strains. The results suggest that harmiquins represent novel antimalarial hits, whereas further studies including harmicines and harmicenes should focus on their anticancer properties