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Cilj rada je prikazati sintezu i evaluaciju konjugata fenilboronične i cimetne kiseline. Fenilboronična kiselina pripada derivatima borne kiseline. Poznata je po svojim mogućim medicinskim svojstvima te je istraživana kao sredstvo u liječenje raka. Cimetna kiselina je široko zastupljena u biljnom svijetu, a izvor je većine derivata hidroksicimetne kiseline poput kumarinske, kafeinske, ferulinske i sinapinske kiseline. U ovom radu opisane su fenilboronična i cimetna kiselina općenito, njihova kemijska svojstva i strukture, upotreba njihovih derivata, te konačno sinteza fenilboronične i cimetne kiseline. Za potrebe rada korištena je relevantna literatura koja se odnosi na stručne članke i knjige koji su u skladu sa temom rada. The aim of this work is to present the synthesis and evaluation of the conjugate of phenylboronic acid and cinnamic acid. Phenylboronic acid belongs to the derivatives of boric acid. It is known for its potential medicinal properties and has been explored as a treatment for cancer. Cinnamic acid is widely distributed in the plant world and is the source of most hydroxycinnamic acid derivatives such as coumarin, caffeic acid, ferulic acid, and sinapic acid. In this paper, phenylboronic and cinnamic acids in general, their chemical properties and structures, the use of their derivatives and finally the synthesis of phenylboronic and cinnamic acids are described. Relevant literature was consulted for the paper, referring to technical articles and books corresponding to the subject of the paper.

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.

Harmin i derivati cimetne kiseline su prirodni produkti koji pokazuju širok spektar bioloških djelovanja, među kojima su antimalarijsko i citostatsko djelovanje. Njihovim kovalentnim povezivanjem u hibridne spojeve, harmicine, nastoji se postići poboljšano djelovanje, smanjenje neželjenih učinaka i interakcija te smanjenje rezistencije na postojeću farmakoterapiju. U okviru ovog diplomskog rada sintetizirani su O-harmicini triazolskog tipa, 6a-e, korištenjem Cu(I) katalizirane alkin-azid cikloadicije. Usto, sintetizirani su i polazni spojevi, azidi derivata cimetne kiseline 3a-e i alkin 5. O-harmicini 6a-e su karakterizirani uobičajenim analitičkim i spektroskopskim metodama (talište, IR, 1H i 13C NMR, MS). U daljnjim istraživanjima sintetiziranim spojevima će biti ispitano citostatsko djelovanje na nizu tumorskih staničnih linija. Harmine and cinnamic acid derivatives are natural products with a broad spectrum of biological activities, which includes antimalarial and cytotoxic effects. By their covalent binding to hybrid molecules, harmicines, improved action, reduction of side effects, drug interactions and resistance to existing pharmacotherapy might be achieved. In this thesis, O-harmicines of the triazole type 6a-e were synthesised using the Cu(I)-catalyzed dipolar alkyne-azide cycloaddition. Starting compounds, cynnamyl azides 3a-e and alkyne 5 were synthesized as well. Structures of O-harmicines were confirmed using conventional analytical and spectroscopic methods (melting point, IR, 1H and 13C NMR, MS). Further studies will focus on the evaluation of their cytotoxic activity against several cancer cell lines.

U ovom radu sintetizirana su i strukturno karakterizirana četiri nova harmicina 15-18, hibridna spoja β-karbolina i derivata cimetnih kiselina, povezana 1,2,3-1H-triazolom, kao i prekursori potrebni za njihovu sintezu, cimetni azidi 7-10 i alkin β-karbolina 14. Cimetni azidi sintetizirani su iz odgovarajućeg CAD u 4 reakcijska koraka. U reakciji s tionil-kloridom nastali su kiselinski kloridi. Slijede esterifikacija i redukcija estera. Nastali primarni alkoholi s ADMP uz DBU u THF-u daju cimetne azide 7-10. Sinteza alkina u poloţaju C6 β-karbolinskog prstena započinje iz 5-metoksitriptamina Pictet-Spenglerovom kondenzacijom, nakon koje slijedi aromatizacija tetrahidro-β-karbolina. Nastali β-karbolin podvrgnut je kiseloj hidrolozi uz nastanak fenola koji s propargil-bromidom daje alkin 14.Za povezivanje cimetnih azida 7-10 i β-karbolinskog alkina 14 u hibridne spojeve, harmicine 15-18, korištena je bakrom(I) katalizirana azid-alkin cikloadicija koja rezultira uvođenjem triazolskog prstena, bioizostera amidne skupine, u strukturu hibridnog spoja. Strukture sintetiziranih spojeva potvrđene su uobičajenim spektroskopskim metodama (1H i 13C NMR, MS, IR). Sva četiri spoja zadovoljavaju Lipinskijeva i Veberova pravila, a dobru oralnu bioraspoloţivost predviđa im i alat SwissADME. Sintetiziranim derivatima će u daljnjim istraţivanjima biti ispitano biološko djelovanje. Four novel harminines 15-18, hybrid compounds consisting of β-carboline and cinnamic acid derivative, linked via 1,2,3-triazole, have been synthesized and structurally characterized, as well as their precursors, cinnamic acid azides 7-10 and β-carboline alkyne 14. Cinnamyl azides 7-10 were sythesized from corresponding CAD in a four step procedure. Reaction with thionyl chloride, followed by esterification and ester reduction led to formation of alcohols. In the final step, alcohols and ADMP formed cinnamic acid azides 7-10. Synthesis of alkyne 14 started from 5-methoxytryptamine by Pictet-Spengler condensation, followed by aromatization of tetrahydro-β-carboline ring. Generated β-carboline was exposed to acid hydrolisis resulting in the formation of a phenol, which was alkylated with propargyl bromide, to form an alkyne 14.The copper(I)-catalyzed azide-alkyne cycloaddition was used to link alkyne 14 and cinnamyl azides 7-10 into novel hybrid compounds, harmicines 15-18. As a result, triazole ring was formed, which is a bioisostere of the amide group. Structures of the newly prepared compounds were confirmed using conventional analytical and spectroscopic methods (1H i 13C NMR, MS, IR). All four novel derivatives meet all of the Lipinski's and Veber's criteria for good oral bioavailability of potential drugs. SwissADME tool predicts good oral bioavailability of all four derivatives. Further studies will investigate biological activity of the prepared harmicines.

Ovaj rad dio je istraživanja provedenog na Zavodu za farmaceutsku kemiju Farmaceutsko-biokemijskog fakulteta Sveučilišta u Zagrebu koji obuhvaća dizajniranje, sintetiziranje i biološku evaluaciju derivata harmina s antimalarijskim djelovanjem. Cilj ovog rada bila je sinteza i karakterizacija dosad neopisana četiri 1,2,3-triazolna derivata harmina koji u svojoj strukturi sadrže derivate cimetne kiseline. Sinteza je započeta prevođenjem derivata cimetne kiseline do odgovarajućih azida (3a,b) prema dolje navedenoj shemi, a harmin i harmol, alkohol nastao demetiliranjem harmina, prevedeni su u odgovarajuće alkine (5 i 6) u reakciji s propargil bromidom. Bakar (I)-katalizirajućom azid-alkin ciklizacijom u kojoj su početni reaktanti bili azidi derivata cimetne kiseline (3a,b) i alkini (5 i 6) dobiveni su konačni produkti 7a,b i 8a,b. Strukture svih sintetiziranih spojeva potvrđene su uobičajenim spektroskopskim metodama (IR, 1H i 13C NMR, MS). Čvrstim produktima određeno je talište. Sva četiri spoja zadovoljavaju Lipinskijeva i Veberova pravila, a dobru oralnu bioraspoloživost predviđa im i alat SwissADME. Sintetiziranim derivatima će u daljnjim istraživanjima biti ispitano biološko djelovanje. This paper is a part of ongoing reaserch at the Department of Medicinal Chemistry, Faculty of Pharmacy and Biochemistry, University of Zagreb. Main focus of the research is design, synthesis and biological evaluation of novel harmine derivates as potential antimalarial drugs. The aim of this diploma thesis was the synthesis and characterization of four novel harmine 1,2,3-triazole derivates containing cinnamic acid derivates in their structure. Synthesis began with transformation of cinnamic acid derivates to azides (3a,b) in a four step procedure. On the other hand, harmine and harmole (4), an alcohol generated by demethylation of harmine, were transformed in alkynes 5 and 6, in the reaction of alkylation using propargyl bromide. The copper(I)-catalyzed azidealkyne cycloaddition, in which cinnamic acid derivates azides (3a,b) and alkynes (5 and 6) were used, resulted in the formation of final compounds 7a,b (N-derivatives) and 8a,b (O-derivatives). Structures of the newly prepared compounds were confirmed using conventional analytical and spectroscopic methods (IR, 1H and 13C NMR, MS). Melting points of the solid compounds were also determined. All four novel derivatives meet all of the Lipinski and Veber criteria for good oral bioavailability of potential drugs. SwissADME web tool predicts good oral bioavailability of all four derivatives. Further studies will investigate antimalarial activity of the new harmine derivatives.

Pregled literature o prirodnim spojevima koji posjeduju antimalarijsko djelovanje pokazao je da harmin, β-karbolinski alkaloid ima potvrđeno in vivo i in vitro antimalarijsko djelovanje. Mogući mehanizam djelovanja harmina je inhibicija P. falciparum Hsp90, koji ima važnu ulogu u razvoju rezistencije na liječenje. U ovom radu opisana je sinteza i karakterizacija novih derivata β-karbolina i cimetne kiseline povezanih 1,2,3-triazolskim mostom s ciljem optimizacije β-karbolinske strukture dobivanja derivata poboljšanog djelovanja. Kao početni spoj u sintezi korišten je metilni ester triptofana, koji je Pictet-Spenglerovom reakcijom u mikrovalnom reaktoru preveden u tetrahidro-β-karbolinski spoj 5. Njegovom oksidacijom uz Pd/C i Li2CO3 dobiven je ester 6 koji je redukcijom s LiAlH4 preveden u odgovarajući alkohol (7). Dobiveni β-karbolinski alkohol je u reakciji s propargil bromidom preveden u N-propargilirani derivat 8. U klik reakciji azid-alkin cikloadicije katalizirane bakrom N-propargiliranog derivata 8 i prethodno sintetiziranih azida cimetne 1, odnosno p-metoksicimetne kiseline 4 dobivena su dva 1,2,3-triazolska derivata (9, 10). Strukture sintetiziranih spojeva potvrđene su uobičajenim analitičkim i spektroskopskim metodama (tt, MS, IR, 1H i 13C NMR). Sintetiziranim derivatima β-karbolina 9, 10 će u daljnim istraživanjima biti ispitano biološko djelovanje. The literature survey revealed a natural product found in several plants, β-carboline alcaloid harmine, to possess in vivo and in vitro antimalarial activity. Assumed mechanism of action is inhibition of P. falciparum Hsp90, which has an important role in the development of antimalarial resistance. This research describes synthesis and characterization of two new derivates of β-carboline and cinnamic acid linked with 1,2,3-triazole bridge, with the aim of optimizing β-carboline structure in order to achieve increased and more selective antimalarial activity. The initial compound in the synthesis sequence was tryptophan methyl ester, which was converted into tetrahydro-β-carboline 5 compound in Pictet-Spengler reaction. Further oxydation with Pd/C and Li2CO3 gave the appropriate ester 6, which was reduced to corresponding alcohol 7. The β-carboline alcohol 7 was then converted into N-propargilated derivate 8 in the reaction with propargyl bromide. Cooper-catalyzed alkyne-azide cycloadition click reaction of the N-propargilated derivate 8 and previously synthethized azides of cinnamic (1) and p-methoxy cinnamic acid (4) gave the final compounds 1,2,3-triazole derivates (9, 10). Structures of synthetized compounds were confirmed the usual analytical and spectroscopic methods (mp, MS, IR, 1H and 13C NMR). Antimalarial activity of synthetized harmine derivates 9 and 10 will be tested.

Novija istraživanja pokazuju da su epigenetske modifikacije jednako bitne u procesu razvoja tumora kao i mutacije gena. Posebno se ističu acetilacije, odnosno deacetilacije jer utječu na strukturu kromatina čime određuju gensku ekspresiju. U tumorskih stanica uočena je prekomjerna aktivnost histonskih deacetilaza (HDAC) koje uklanjaju ε-acetilnu skupinu iz bočnih aminokiselinskih ogranaka lizina. Dolazi do utišavanja tumorsupresorskih gena te aktivacije gena koji potiču preživljavanje i proliferaciju stanica tumora. Lijekovi inhibitori HDAC kompleksiraju ione cinka iz aktivnog mjesta enzima. Prema kemijskoj strukturi dijelimo ih u četiri skupine, od kojih su najzanimljivije hidroksamske kiseline zbog visoke sposobnosti keliranja iona metala i inhibicije HDAC-a. Cilj ovoga diplomskog rada bila je sinteza hibridnog derivata primakina i cimetne kiseline s terminalnom hidroksamskom kiselinom, za kojeg se pretpostavlja da bi, zbog svojih strukturnih karakteristika, mogao imati antimalarijsko i/ili citostatsko djelovanje. Isprobane su tri sintetske metode, no unatoč tome, sinteza ciljne molekule nije bila uspješna. Recent research shows that carcinogenesis cannot be explained only by genetic alterations, but also involves epigenetic modifications. In particular acetylation and deacetylation affect the structure of chromatin which determines gene expression. In tumor cells excessive activity of histone deacetylases (HDAC), enzymes that remove the ε-acetyl group from the lateral lysine amino acid residues, was observed. It results in suppression of tumor-suppressor genes and activation of genes that stimulate survival and proliferation of tumor cells. HDAC inhibitors complex zinc ions found in the enzyme's active site. According to the chemical structure, they are divided into four groups, of which hydroxamic acids are the most interesting ones, due to their high metal ion chelating ability. The goal of this diploma thesis was the synthesis of a hybrid derivative primaquine and cinnamic acid with terminal hydroxamic acid moiety, which might, due to the structural characteristics, exert cytostatic and/or antimalarial activity. Although three synthetic methods were employed, synthesis of the target compound was not achieved.

Ovaj rad se nadovezuje na višegodišnja istraživanja provedena na Zavodu za farmaceutsku kemiju Farmaceutsko-biokemijskog fakulteta Sveučilišta u Zagrebu koja obuhvaćaju dizajniranje, sintezu i biološku evaluaciju derivata primakina s antiproliferativnim djelovanjem. Rezultati tih istraživanja, uz literaturne izvore o biološkim djelovanjima derivata cimetne kiseline, temelj su dizajna novih derivata primakina i cimetne kiseline kao potencijalnih citostatika. Cilj ovog rada bila je sinteza i karakterizacija šest novih acilsemikarbazidnih derivata primakina koji u svojoj strukturi sadrže derivate cimetne kiseline. Kao početni spoj u sintezi korišten je klorid 1-benzotriazolkarboksilne kiseline, BtcCl (1). Primakin je reakcijom s BtcCl preveden u primakinski benzotriazolid (2), a zatim pomoću hidrazina u primakinski semikarbazid (3). Derivati cimetne kiseline prevedeni su u kiselinske kloride 4a-f reakcijom s tionil-kloridom. Reakcijom primakinskog semikarbazida (3) s kloridom odgovarajućeg derivata cimetne kiseline 4a-f dobiveni su acilsemikarbazidni derivati 5a-f koji nisu do sad opisani u literaturi. Strukture svih sintetiziranih spojeva potvrđene su uobičajenim spektroskopskim metodama (IR, 1H i 13C NMR, MS). Čvrstim produktima određeno je talište. Sintetiziranim derivatima će u daljnjim istraživanjima biti ispitano biološko djelovanje. In the last few years, a research group at the Department of Medicinal Chemistry has focused on primaquine derivatization. This includes design, synthesis and biological evaluation of novel primaquine derivatives with antiproliferative activity. The purpose of this research was synthesis and characterization of six novel primaquine derivatives that include cinnamid acid derivatives (CADs) in their structure. The reaction of 1- benzotriazolecarboxylic acid chloride (BtcCl, 1) with primaquine (PQ) gave primaquine benzotriazolide (2). Primaquine semicarbazide (3) was prepared in the reaction of primaquine benzotriazolide (2) with hydrazine. Cinnamic acid derivatives (CAD) gave acyl chlorides 4a-f in the reaction with thyonil chloride. Final products, acylsemicarbazide CAD-PQ derivatives 5a-f were finally obtained in the reaction of primaquine semicarbazide (3) and corresponding acyl chlorides 4a-f. Structures of newly prepared primaquine derivatives were confirmed by IR, 1H and 13C NMR spectroscopy and MS. Melting point of solid compounds were also determined. Antiproliferative activity of synthesized primaquine derivatives will be tested in further analysis.

Cimetna i klorogenska kiselina polifenolni su spojevi koji su pronađeni voću, povrću, cvijeću i medu. Cilj je ovog rada bio odrediti UV/VIS svojstva cimetne i klorogenske kiseline u ovisnosti o primijenjenom pH. Snimanjem UV/VIS spektra cimetne i klorogenske kiseline u rasponu od 200 do 800 nm uočeno je kako obje kiseline pokazuju jedan vrh apsorbancije karakterističan za sve polifenole, a osim ovog vrha cimetna je kiselina pokazivala jedan dodatni vrh apsorbancije, a klorogenska kiselina čak tri dodatna vrha apsorbancije. Intenzitet i položaj, kao i pomak položaja vrhova specifičnih za pojedinu ispitanu kiselinu pokazali su se ovisnim o primjenjenom pH što je najvjerojatnije uzrokovano disocijacijom funkcionalnih skupina polifenola. Cinnamic and chlorogenic acid belongs to polyphenolic compounds found in fruit, vegetables, flowers and honey. The aim of this study was to determine pH dependence of cinnamic and chlorogenic acid UV/VIS properties. UV/VIS spectra recording from 200 to 800 nm revealed existence of one unique absorption peak characteristic for all polyphenols. Additionally, cinnamic acid showed existence of another peak characteristic for this polyphenol, and chlorogenic acid existence of three absorption peaks. Intensity and positions, as well as absorption peaks shift, showed dependence on pH applied, what can be attributed to dissociation of functional groups.