| Abstract | U prvom dijelu istraživanja ovog doktorskog rada sintetizirano je četrdeset harmicina amidnoga tipa (7a-h, 13a-h, 19a-h, 22a-h, 25a-f, i-p), hibridnih spojeva harmina/srodnih β-karbolinskih alkaloida i cimetne kiseline/odabranih derivata cimetne kiseline (DCK-a) međusobno povezanih amidnom vezom. U tu svrhu pripravljeni su amini 6, 12, 18, 21 i 24, čime je primarna amino skupina uvedena u pet položaja β-karbolina: 1, 3, 6, 7 i 9. U idućem koraku sintetizirani su harmicini amidnoga tipa reakcijama povezivanja dobivenih amina s cimetnom kiselinom/DCK-ima korištenjem standardnih reakcijskih uvjeta (HATU/DIEA). Harmicini su karakterizirani spektroskopskim (1H, 13C NMR, IR) i spektrometrijskim (MS) tehnikama te im je određeno talište. Svim spojevima ispitano je antiplazmodijsko djelovanje in vitro na eritrocitnu fazu životnog ciklusa dva soja Plasmodium falciparum (Pf3D7 i PfDd2), hepatocitnu fazu životnog ciklusa P. berghei i citotoksičnost na humanu staničnu liniju hepatocelularnog karcinoma (HepG2).
U drugom dijelu istraživanja provedena je analiza kvantitativnog odnosa strukture i djelovanja (engl. quantitative structure-activity relationship, QSAR). Antiplazmodijsko djelovanje harmicina na eritrocitnu fazu Pf3D7 povezano je s pripadajućim izračunatim molekulskim deskriptorima te je primjenom metode višestruke linearne regresije dobiven prediktivni model ovisnosti log IC50 o dvije varijable: izoelektričnoj točki (pI) i Abrahamovom deskriptoru E (Abr E). Model je validiran unutarnjom 10-strukom unakrsnom validacijom.
U trećem dijelu istraživanja, za provedbu vanjske validacije modela predložene su strukture 356 novih harmicina te su im, prema dobivenom modelu, izračunate predviđene IC50 vrijednosti. Potom je odabrano, sintetizirano i karakterizirano ukupno 19 harmicina: šest harmicina amidnoga tipa (25k-p), 11 harmicina karbamatnog tipa (28a-k) i dva harmicina ureidnog tipa (32b,c) u položaju N-9 β-karbolina, te je ispitano njihovo antiplazmodijsko djelovanje in vitro na eritrocitnu fazu životnog ciklusa plazmodija (Pf3D7 i PfDd2). Na temelju dobivenih rezultata izračunate su razlike između eksperimentalnih i predviđenih vrijednosti i srednja apsolutna pogreška predviđanja. Najjače djelovanje, gotovo dva reda veličine jače od harmina, pokazali su harmicini amidnoga tipa u položaju N-9 β-karbolina (25b-e,i,j).
Rezultati ovog doktorskog rada predstavljaju temelj za daljnji razvoj novih harmicina amidnoga tipa s pojačanim antiplazmodijskim djelovanjem. |
| Abstract (english) | The first part of this thesis includes the synthesis of forty amide-type harmicines (7a-h, 13a-h, 19a-h, 22a-h, 25a-f, i-p), hybrid compounds in which harmine/β-carboline alkaloids and cinnamic acid or cinnamic acid derivatives (CADs) are linked via an amide bond. First, the primary amino group was introduced in five positions of the β-carboline ring: 1, 3, 6, 7 and 9, which resulted in the synthesis of amines 6, 12, 18, 21 and 24. Subsequently, the coupling reaction of amines and cinnamic acid/CADs were preformed using standard conditions (HATU/DIEA). The synthesized compounds were characterized by spectroscopic (1H, 13C NMR, IR) and spectrometric (MS) techniques, as well as determination of their melting points. Finally, we evaluated biological activities of the prepared harmicines, i.e. in vitro antiplasmodial activity against the erythrocytic stage of the Plasmodium falciparum life cycle (Pf3D7 and PfDd2) and hepatic stage of the P. berghei life cycle, as well as cytotoxicity against human hepatocellular carcinoma cell line (HepG2).
In the second part of this thesis quantitative structure-activity relationship (QSAR) analysis was performed using a multiple linear regression technique. A set of molecular descriptors underwent the forward stepwise regression procedure and the predictive model was built by selecting two molecular descriptors: isoelectric point (pI) and Abraham descriptor E (Abr E). The constructed model was internally validated using 10-fold cross-validation.
For the purpose of external validation of the constructed model, in the third part of this thesis 356 novel harmicines were designed and their predicted logIC50 values calculated, followed by the synthesis and characterization of six amide-type harmicines (25k-p), eleven carbamate type-harmicines (28a-k) and two ureido-type harmicines (32b,c) in the position N-9 of the β-carboline. Next, we evaluated in vitro antiplasmodial activity of novel harmicines against the erythrocytic stage of the P. falciparum life cycle (Pf3D7 and PfDd2). Based on the obtained results, the absolute differences between the experimental and predicted values as well as mean absolute error were calculated. The most active compounds, amide-type harmicines in the position N-9 of the β-carboline (25b-e,i,j), displayed at least two orders of magnitude stronger activities than the parent compound, harmine. The results of this doctoral thesis represent the basis for the further development of novel amide-type harmicines with enhanced antiplasmodial activity. |
