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Vitamin E je generičko ime koje obuhvata dve klase jedinjenja, tokoferole i tokotrienole, od kojih svaka klasa sadrži 4 izomera (α, β, γ, δ). Analozi vitamina E putem različitih mehanizama mogu ispoljiti snažnu citotoksičnu aktivnost na više tipova tumorskih ćelija, a da pritom ne remete u velikoj meri aktivnost zdravih ćelija. Ovu doktorsku disertaciju čine tri celine čiji će rezultati imati potencijalni značaj za primenu derivata vitamina E u terapiji malignih bolesti. Prva celina obuhvata studije kvantitativnog odnosa strukture i dejstva (Quantitative Structure-Activity Relationship, QSAR) derivata vitamina E i određivanje strukture farmakofore. U drugoj celini opisani su: (1) sinteza estara α-tokoferola sa aminokiselinama lizinom, prolinom, glutaminom, asparaginom i estara γ-tokotrienola sa lizinom, prolinom, glutaminom; (2) in vitro ispitivanja biološke aktivnosti sintetisanih estara na MCF-7 i MDA-MB 231 ćelijskim linijama tumora dojke, A549 ćelijskoj liniji tumora pluća i MRC-5 zdravoj ćelijskoj liniji fetalnih fibroblasta pluća; (3) ispitivanje ćelijskih mehanizama antiproliferativne aktivnosti i sinergističkog efekta najaktivnijeg sinetisanog jedinjenja sa doksorubicinom na multirezistentnoj MDA-MB 231 ćelijskoj liniji; (4) studije stabilnosti u humanoj plazmi; (5) pretraživanje ChEMBL baze bioaktivnih molekula primenom metode virtelnog skrininga zasnovanog na strukturi liganda. Treću celinu predstavlja dizajn novih analoga vitamina E sa potencijalno snažnijim antitumorskim dejstvom... Vitamin E is a generic name comprising of two classes of compounds, tocopherols and tocotrienols, each class containing 4 isomers (α, β, γ, δ). Analogs of vitamin E, via different mechanisms, may exhibit strong cytotoxic activity to many types of malignant cells, while not disrupting in a greater extent activity of healthy cells. This doctoral thesis includes three sections whose results will have potential importance for the use of vitamin E derivatives in the treatment of malignant diseases. The first section includes Quantitative Structure-Activity Relationship (QSAR) study of vitamin E analogues and pharmacophore structure determination. The second section covers: (1) synthesis of α-tocopherol esters with amino acids lysine, proline, glutamine, asparagine, and γ-tocotrienol esters with lysine, proline, glutamine; (2) in vitro testing of biological activities of the synthesized esters on the MCF-7 and MDA-MB 231 breast cancer cell lines, A549 lung cancer cell line, and the MRC-5 healthy fetal lung fibroblasts cell line; (3) testing of cellular mechanisms which underlie the antiproliferative activity and synergistic effect of the most active compound with doxorubicin on multidrug-resistant MDA-MB 231 cell line; (4) stability study in human plasma; (5) filtering of the ChEMBL database of bioactive molecules using the ligand based virtual screening protocol. The third section represents design of new analogues of vitamin E with enhanced antitumor activity. 2D- and 3D-QSAR studies were conducted on a series of vitamin E analogues with experimentally determined antiproliferative activity on MCF7 cell line, using PLS statistical method...

Vitamin E je generičko ime koje obuhvata dve klase jedinjenja, tokoferole i tokotrienole, od kojih svaka klasa sadrži 4 izomera (α, β, γ, δ). Analozi vitamina E putem različitih mehanizama mogu ispoljiti snažnu citotoksičnu aktivnost na više tipova tumorskih ćelija, a da pritom ne remete u velikoj meri aktivnost zdravih ćelija.Ovu doktorsku disertaciju čine tri celine čiji će rezultati imati potencijalni značaj za primenu derivata vitamina E u terapiji malignih bolesti.Prva celina obuhvata studije kvantitativnog odnosa strukture i dejstva (Quantitative Structure-Activity Relationship, QSAR) derivata vitamina E i određivanje strukture farmakofore.U drugoj celini opisani su: (1) sinteza estara α-tokoferola sa aminokiselinama lizinom, prolinom, glutaminom, asparaginom i estara γ-tokotrienola sa lizinom, prolinom, glutaminom; (2) in vitro ispitivanja biološke aktivnosti sintetisanih estara na MCF-7 i MDA-MB 231 ćelijskim linijama tumora dojke, A549 ćelijskoj liniji tumora pluća i MRC-5 zdravoj ćelijskoj liniji fetalnih fibroblasta pluća; (3) ispitivanje ćelijskih mehanizama antiproliferativne aktivnosti i sinergističkog efekta najaktivnijeg sinetisanog jedinjenja sa doksorubicinom na multirezistentnoj MDA-MB 231 ćelijskoj liniji; (4) studije stabilnosti u humanoj plazmi; (5) pretraživanje ChEMBL baze bioaktivnih molekula primenom metode virtelnog skrininga zasnovanog na strukturi liganda.Treću celinu predstavlja dizajn novih analoga vitamina E sa potencijalno snažnijim antitumorskim dejstvom... Vitamin E is a generic name comprising of two classes of compounds, tocopherols and tocotrienols, each class containing 4 isomers (α, β, γ, δ). Analogs of vitamin E, via different mechanisms, may exhibit strong cytotoxic activity to many types of malignant cells, while not disrupting in a greater extent activity of healthy cells.This doctoral thesis includes three sections whose results will have potential importance for the use of vitamin E derivatives in the treatment of malignant diseases.The first section includes Quantitative Structure-Activity Relationship (QSAR) study of vitamin E analogues and pharmacophore structure determination.The second section covers: (1) synthesis of α-tocopherol esters with amino acids lysine, proline, glutamine, asparagine, and γ-tocotrienol esters with lysine, proline, glutamine; (2) in vitro testing of biological activities of the synthesized esters on the MCF-7 and MDA-MB 231 breast cancer cell lines, A549 lung cancer cell line, and the MRC-5 healthy fetal lung fibroblasts cell line; (3) testing of cellular mechanisms which underlie the antiproliferative activity and synergistic effect of the most active compound with doxorubicin on multidrug-resistant MDA-MB 231 cell line; (4) stability study in human plasma; (5) filtering of the ChEMBL database of bioactive molecules using the ligand based virtual screening protocol.The third section represents design of new analogues of vitamin E with enhanced antitumor activity.2D- and 3D-QSAR studies were conducted on a series of vitamin E analogues with experimentally determined antiproliferative activity on MCF7 cell line, using PLSstatistical method...

U okviru ove doktorske disertacije je sintetisana serija aminokiselinskih derivata seskviterpenskog hinona avarona, izolovanog iz morskog sunđera Dysidea avara, kao i model jedinjenja tert-butilhinona. Određena je in vitro antitumorska aktivnost svih jedinjenja prema humanim tumorskim ćelijskim linijama (HeLa, A549, Fem-X, K562, MDA-MB-453) i zdravoj MRC-5 ćelijskoj liniji. Utvrđeno je da aminokiselinski derivati avarona pokazuju jače dejstvo nego odgovarajući aminokiselinski derivati tert-butilhinona, sa IC50 vrednostima pojedinih derivata nižim od 10 μM. Rezultati analize ćelijskog ciklusa na HeLa ćelijama ukazuju da je apoptoza jedan od mogućih mehanizama dejstva derivata. Ispitan je uticaj kaspaza 3, 8 i 9 na ćelijsku smrt korišćenjem specifičnih kaspaznih inhibitora. Ispitano je i antimikrobno dejstvo derivata prema nizu Gram-pozitivnih i Gramnegativnih bakterija, kao i na tri soja gljivica, pri čemu je zabeležena vrlo dobra aktivnost derivata avarona. Ispitane su interakcije derivata sa biomakromolekulima. SDS gel-ektroforezom i masenom spektrometrijom je potvrđeno da sintetisana jedinjenja ne modifikuju lizozim. Spektrofotometrijskim ispitivanjima je zabeležena pojava hiperhromizma kao posledica interakcija između hinona i CT-DNA. Utvrđeno je da dolazi do smanjenja intenziteta fluorescencije interkalatora etidijum-bromida i boje Hoechst 33258 koja se vezuje u malu brazdu u kompleksu derivata sa CT-DNA. Spektri cirkularnog dihroizma su ukazali na izostanak interkalacije hinona u CT-DNA. Rezultati molekulskog modelovanja su sugerisali da se derivati smeštaju u malu brazdu DNA. Elektroforetska ispitivanja su potvrdila da ne dolazi do cepanja plazmida pUC19 u prisustvu derivata. Na kraju je utvrđen stepen antioksidativne aktivnosti dobijenih aminokiselinskih derivata hinona. In this doctoral dissertation, a series of amino acid derivatives of the sesquiterpene quinone avarone, isolated from the marine sponge Dysidea avara, as well as the model compund tert-butylquinone has been synthesized. In vitro cytotoxic activity of all compounds toward human cancer cell lines (HeLa, A549, Fem-X, K562, MDA-MB-453) and normal MRC-5 cell line was determined. It was found that the amino acid derivatives of avarone exhibit a stronger activity than the corresponding amino acid derivatives of tert-butylquinone, with IC50 values of some derivatives less than 10 μM. The results of the cell cycle analysis of HeLa cells indicate that apoptosis is one of the possible mechanisms of action of derivatives. The effect of caspases 3, 8, and 9 on cell death by using specific caspase inhibitors was examined. The antimicrobial effect of the derivatives on the panel of Gram-positive and Gram-negative bacteria, as well as on three fungal species, were investigated, whereby very good activity of avarone derivatives was observed. Interactions of the derivatives with biomacromolecules were studied. SDS gelelectrophoresis and mass spectrometry confirmed the absence of modifications of lysozyme by any of the synthesized compounds. Spectrophotometric studies have recorded the occurrence of hyperchromism as a result of interactions between the quinone and CT-DNA. The quenching of the fluorescence of the intercalator ethidium bromide and of the minor groove binder Hoechst 33258 in the complex with CT-DNA by the derivatives was determined. Circular dichroism spectra demonstrated a nonintercalative binding mode of the quinone derivatives to CT-DNA. The results of molecular modeling suggested that the derivatives bind to the minor groove. The electrophoretic pattern showed no cleavage of the pUC19 plasmid in the presence of any derivatives. Finally, the degree of antioxidant activity of the obtained amino acid derivatives of the quinones was determined.

In this work the synthesis of 4,6-dimethyl-3-cyano-2-pyridone catalyzed by amino acids was studied. In the first part of the work, the optimization was carried out by varying the initial concentration of glycine, the molar ratio of the reactants (acetylacetone and cyanoacetamide) and the reaction time. It has been found that it is necessary to heat the reaction mixture and that the optimum concentration of the amino acid is 0.2 mmol cm-3. It was also found that the optimal concentration of cyanoacetamide is 3.0 mmol cm-3 (molar ratio cyanoacetamide: acetyl acetone = 3:1), and that the reaction time is 24 h. In the second part of the work, the influence of the structure of 19amino acids on the yield of 4,6- dimethyl-3-cyano-2-pyridone was investigated, using the optimal reaction conditions. It was found that the highest yield was achieved with arginine (almost 90%) and than with histidine (about 85%). With the most of other amino acid the yield was 50-60%. . U okviru rada prikazana je sinteza 4,6-dimetil-3-cijano-2-piridona katalizovana aminokiselinama. U prvom delu izvršena je optimizacija reakcije sinteze piridona varirajući početnu koncentraciju glicina, molski odnos reaktanata (acetilacetona i cijanoacetamida) i reakciono vreme. U drugom delu rada ispitan je uticaj strukture još 18 aminokiselina na prinos 4,6-dimetil-3-cijano-2-piridona. Najveći prinosi ostvareni su sa argininom (90%) i histidinom (85%) kao katalizatorima. .

U okviru ove doktorske disertacije je sintetisana serija aminokiselinskihderivata seskviterpenskog hinona avarona, izolovanog iz morskog sunđera Dysideaavara, kao i model jedinjenja tert-butilhinona.Određena je in vitro antitumorska aktivnost svih jedinjenja prema humanimtumorskim ćelijskim linijama (HeLa, A549, Fem-X, K562, MDA-MB-453) i zdravojMRC-5 ćelijskoj liniji. Utvrđeno je da aminokiselinski derivati avarona pokazuju jačedejstvo nego odgovarajući aminokiselinski derivati tert-butilhinona, sa IC50 vrednostimapojedinih derivata nižim od 10 μM. Rezultati analize ćelijskog ciklusa na HeLa ćelijamaukazuju da je apoptoza jedan od mogućih mehanizama dejstva derivata. Ispitan je uticajkaspaza 3, 8 i 9 na ćelijsku smrt korišćenjem specifičnih kaspaznih inhibitora.Ispitano je i antimikrobno dejstvo derivata prema nizu Gram-pozitivnih i Gramnegativnihbakterija, kao i na tri soja gljivica, pri čemu je zabeležena vrlo dobraaktivnost derivata avarona.Ispitane su interakcije derivata sa biomakromolekulima. SDS gel-ektroforezom imasenom spektrometrijom je potvrđeno da sintetisana jedinjenja ne modifikuju lizozim.Spektrofotometrijskim ispitivanjima je zabeležena pojava hiperhromizma kao posledicainterakcija između hinona i CT-DNA. Utvrđeno je da dolazi do smanjenja intenzitetafluorescencije interkalatora etidijum-bromida i boje Hoechst 33258 koja se vezuje umalu brazdu u kompleksu derivata sa CT-DNA. Spektri cirkularnog dihroizma suukazali na izostanak interkalacije hinona u CT-DNA. Rezultati molekulskogmodelovanja su sugerisali da se derivati smeštaju u malu brazdu DNA. Elektroforetskaispitivanja su potvrdila da ne dolazi do cepanja plazmida pUC19 u prisustvu derivata.Na kraju je utvrđen stepen antioksidativne aktivnosti dobijenih aminokiselinskihderivata hinona. In this doctoral dissertation, a series of amino acid derivatives of thesesquiterpene quinone avarone, isolated from the marine sponge Dysidea avara, as wellas the model compund tert-butylquinone has been synthesized.In vitro cytotoxic activity of all compounds toward human cancer cell lines(HeLa, A549, Fem-X, K562, MDA-MB-453) and normal MRC-5 cell line wasdetermined. It was found that the amino acid derivatives of avarone exhibit a strongeractivity than the corresponding amino acid derivatives of tert-butylquinone, with IC50values of some derivatives less than 10 μM. The results of the cell cycle analysis ofHeLa cells indicate that apoptosis is one of the possible mechanisms of action ofderivatives. The effect of caspases 3, 8, and 9 on cell death by using specific caspaseinhibitors was examined.The antimicrobial effect of the derivatives on the panel of Gram-positive andGram-negative bacteria, as well as on three fungal species, were investigated, wherebyvery good activity of avarone derivatives was observed.Interactions of the derivatives with biomacromolecules were studied. SDS gelelectrophoresisand mass spectrometry confirmed the absence of modifications oflysozyme by any of the synthesized compounds. Spectrophotometric studies haverecorded the occurrence of hyperchromism as a result of interactions between thequinone and CT-DNA. The quenching of the fluorescence of the intercalator ethidiumbromide and of the minor groove binder Hoechst 33258 in the complex with CT-DNAby the derivatives was determined. Circular dichroism spectra demonstrated a nonintercalativebinding mode of the quinone derivatives to CT-DNA. The results ofmolecular modeling suggested that the derivatives bind to the minor groove. Theelectrophoretic pattern showed no cleavage of the pUC19 plasmid in the presence ofany derivatives.Finally, the degree of antioxidant activity of the obtained amino acid derivativesof the quinones was determined.

Cilj ovog istraživanja bio je ispitivanje uticaja visokih koncentracija komercijalnog deterdženta „Merix“, (Henkel, Kruševac) na rast, razvoj i biohemijske karakteristike testiranih gljiva koje su izolovane iz kanalizacionih i industrijskih otpadnih voda. Gljive su gajene u tečnoj hranljivoj podlozi po Čapeku (kontrola) i istoj podlozi sa dodatkom deterdženta u koncentraciji 0,3% i 0,5%. U oglednom periodu od 3. do 16. dana ispitivane su promene: pH i redoks potencijala; količine proteina, slobodnih i ukupnih organskih kiselina; kvantitativni I kvalitativni sastav aminokiselina, kao i aktivnost određenih enzima u fermentacionoj tečnosti. Istovremeno je ispitivana i koncentracija deterdženta (anjonskih komponenti deterdženta) u fermentacionoj tečnosti pomoću MBAS metode. Sve gljive su razgradile deterdžent 0,3% koncentracije, a M. racemosus je razgradila i deterdžent 0,5% koncentracije. Deterdžent je uticao na inhibiciju biomase gljiva u različitom procentu u zavisnosti od vrste gljiva i mesta izolovanja. Kod gljive M. racemosus deterdžent je delovao stimulativno na biomasu, tako da je veća biomasa izmerena u podlozi sa 0,5% deterdžentom. Prisustvo deterdženta u hranljivoj podlozi i njegovih produkata degradacije tokom fermentacije gljiva uticalo je na promene svih ispitivanih biohemijskih parametara i enzimske aktivnosti. The aim of this study was to investigate the effect of high concentrations of commercial detergent "Merix" (Henkel, Krusevac) on growth and development and biochemical characteristics of the tested fungi were isolated from sewage and industrial wastewater. Fungi were grown in a liquid Czapek nutrient medium (control) and in the same medium with the addition of 0.3% and 0.5% detergent. The changing of pH values and redox potential; amounts of protein, free and total organic acids, the qualitative and quantitative composition of the amino acids, as well as the activity of certain enzymes were investigated in the fermentation liquid during the experimental period from 3 to 16 days. At the same time, concentration of detergent (anionic components of the detergent) in the fermentation broth was examined using MBAS method. All of the tested fungi were degrade detergent in concentration of 0.3%, but only M. racemosus decompose detergent in concentration of 0.5%. Detergent influenced the inhibition of the fungal biomass in varying percentages depending on the type of fungus and isolation. Detergent has a stimulating effect on biomass of fungus M. racemosus, so a greater biomass is measured in a medium containing 0.5% detergent. The presence of detergent in the nutrient medium and its degradation products during fermentation of fungi influenced the change of all the examined biochemical parameters and enzyme activities.