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Kako maligne ćelije vremenom mutiraju i postaju rezistentne na postojeće lekove postoji stalna potreba za novim antitumorskim lekovima. Steroidni molekuli igraju vrlo važne uloge u biološkim sistemima. Usled hidrofobnosti nesmetano prolaze kroz ćelijske membrane pa su pogodan polazni materijal za dizajn novih lekova. Trasformacijama krutog tetracikličnog steroidnog skeleta nastaju biološki aktivni molekuli sa novim fiziološkim funkcijama. Sa druge strane, hidrazoni sadrže heteroatome i smatraju se značajnim intermedijerima pri sintezi heterocikličnih jedinjenja. Stoga je sintetisano i okarakterisano pedeset novih derivata steroidnih hidrazona, a zatim je određena njihova citotoksičnost i ispitani su mehanizmi citotoksičnog delovanja. Prema testiranim malignim ćelijskim linijama (HeLa, K562, MDA-MB-361, MDA-MB-453, LS174, A549), naročito prema HeLa ćelijama adenokarcinoma cerviksa i prema K562 ćelijama hronične mijeloidne leukemije, nova jedinjenja su ispoljila selektivnu citotoksičnost zavisnu od koncentracije na mikrogramskoj skali, u mnogim slučajevima bolju od hemioterapeutika cisplatina. Nasuprot tome, pokazala su vrlo slabu citotoksičnost na humane PBMC, što se ogleda u koeficijentima selektivnosti daleko višim od vrednosti 2,5. Morfološkom analizom HeLa ćelija tretiranih novim derivatima utvrđeno je njihovo pro-apoptotsko dejstvo što je potvrđeno rezultatima protočne citometrije. Određivanjem ciljnih kaspaza ustanovljeno je da do apoptoze dolazi posredstvom kaspaze-3, -8 i -9. Svi ispitivani derivati su pokazali sposobnost da inhibiraju angiogenezu ćelija EA.hy926 in vitro. Testirani tiazolidin-4-oni nisu prouzrokovali značajne promene u nivoima ekspresije gena MMP2, MMP9 i VEGFA u tretiranim HeLa ćelijama. Rezultati sprovedenih in vitro istraživanja mogu da ukažu na značajan antitumorski potencijal što ova jedinjenja ubraja u potencijalne kandidate za in vivo ispitivanja.

Usled stalne potrebe za novim antitumorskim lekovima, počevši od androstanskog bis(semikarbazona), sintetisan je novi bis(karbazatni) estar (bisKBZ) za koji je određena antimikrobna aktivnost, citotoksična aktivnost na tri maligne ćelijske linije, i urađen je brine shrimp test toksičnosti. Novo jedinjenje se nije pokazalo kao dobar antimikrobni agens, ispoljilo je umerenu citotoksičnost prema testiranim ćelijskim linijama i pokazalo se kao pet puta manje toksično od cisplatina za račiće Artemia salina što je u dobroj korelaciji sa citotoksičnošću prema HeLa ćelijskoj liniji., Due to the constant need for new antitumor drugs, starting from androstane bis(semicarbazone), a new bis(carbazate) ester (bis-KBZ) was synthesized; antimicrobial activity and cytotoxicity against three malignant cell lines were determined, and the brine shrimp toxicity test was performed. The new compound did not prove to be a good antimicrobial agent, it showed moderate cytotoxicity to the tested cell lines, and proved to be five times less toxic than cisplatin to Artemia salina, which correlates well with the cytotoxicity to HeLa cell line.

In this work, Pt(II) complexes of previously synthesized steroidal thiosemicarbazones were synthesized and characterized. The ligands and their metal complexes were studied by analytical and spectroscopic data (elemental analysis, IR, 1D NMR and 2D NMR, HSQC, HMBC, NOESY, COSY), the analysis of which enabled complete 1H and 13C assignments of each compound including E and Z isomers. All the synthesized ligands and complexes were screened for their cytotoxic and antimicrobial activity. The results demonstrate that new steroidal thiosemicarbazone complexes were significantly less cytotoxic than corresponding steroidal thiosemicarbazones. Also, complexes show lower antimicrobial activity than the standard drugs, similar to the activity of the starting thiosemicarbazones., Почевши од претходно синтетисаних стероидних тиосемикарбазона, у овом раду су синтетисани и окарактерисани комплекси платине(II). Лиганди и њихови метални комплекси проучавани су аналитичким и спектроскопским методама (елементална анализа, ИЦ, 1D NMR и 2D NMR, HSQC, HMBC, NOESY, COSY). Анализом добијених података омогућена је потпуна 1H и 13C асигнација свих једињења укључујући Е и Z изомере. За синтетисане лиганде, као и њихове комплексе испитивана је цитотоксична и антимикробна активност. Резултати указују на то да нови стероидни тиосемикарбазонски комплекси испољавају значајно нижу цитотоксичност од одговарајућих стероидних тиосемикарбазона. Поред тога, комплекси поседују антимикробну активност сличну активности полазних тиосемикарбазона, a нижу од стандардних лекова

U potrazi za biološki aktivnim jedinjenjima, počev od progesterona i 3-okso-α,β-nezasićenih androstenskih steroida, u okviru ove disertacije sintetisano je i potpunookarakterisano pedeset novih derivata steroidnih hidrazona, od kojih po jedanaesttiosemikarbazona, tijadiazolina i semikarbazona, dvanaest tiazolidin-4-ona i petkarbazatnih estara.Po prvi put je urađena detaljna analiza stereohemije steroidnih hidrazona u položajimaC-3/17 androstenskih, odnosno C-3/20 progesteronskih derivata. Struktura istereohemija potvrđene su rezultatima rendgenske strukturne analize za tijadiazolin 7a,prvo okarakterisano steroidno jedinjenje koje sadrži šestočlani ugljenični prstenkondenzovan sa spiro-tijadiazolinskim prstenom, i tiazolidinon 9b-E, prvi steroidniderivat sa poznatom konfiguracijom dvostruke veze u položaju C-3 hidrazonotiazolidin-4-onskog fragmenta.Sintetisana jedinjenja su ispoljila najjaču citotoksičnost prema HeLa ćelijamaadenokarcinoma cerviksa i prema K562 ćelijama hronične mijeloidne leukemije, a posvojoj aktivnosti istakli su se tiosemikarbazoni 2a, 2b, 2c i 2f, tijadiazolini 8a i 8e itiazolidin-4-oni 9a i 10a. Pritom su koeficijenti selektivnosti u antikancerskom dejstvuprema malignim ćelijama u odnosu na normalne humane PBMC, kako na nestimulisanetako i na mitogenom stimulisane, bili daleko veći od vrednosti 2,5 što ova jedinjenjasvrstava u potencijalne kandidate za in vivo ispitivanja. Sumporni derivati bili su dalekoaktivniji od kiseoničnih. Najaktivniji derivati indukovali su apoptozu posredstvomkaspaza-3, -8 i -9 i inhibirali angiogezu in vitro zbog čega se smatra da posedujuznačajan antikancerski potencijal., Searching for biologically active compounds, within this doctoraldissertation fifty new steroidal hydrazone derivatives, of which 11 thiosemicarbazones,11 thiadiazolines, 12 thiazolidin-4-ones, 11 semicarbazones and 5 carbazate esters, weresynthesized starting with progesterone and 3-oxo-α,β-unsaturated androstene steroids,and fully characterized.For the first time, detailed stereochemistry analyses of steroidal hydrazones in the C-3/17 positions of androstene derivatives, or C-3/20 positions of progesterone derivativeswas done. Structure and stereochemistry were confirmed by the results of X-rayanalyses for thiadiazoline 7a, the first characterized steroid compound that contains sixmemberedcarbon ring condensed with the spiro-thiadiazoline ring, and thiazolidinone9b-E, the first steroidal derivative with known configuration of double bond in C-3position of the hydrazono-thiazolidin-4-one fragment.Synthesised compounds manifested the best cytotoxicity towards HeLa cervixadenocarcinoma cells, and K562 cells of chronic myeloide leukemia, the best activitybeing showed by thiosemicarbazones 2a, 2b, 2c and 2f, thiadiazolines 8a and 8e, andthiazolidin-4-ones 9a and 10a. All of these compounds exhibited considerably higherintensities of cytotoxic action against malignant cells when compared with normalhuman PBMC, both resting and mitogen-stimulated, with coefficient of selectivityhigher than 2.5, which makes these compounds potential candidates for in vivoexperiments. Sulfur derivatives were much more active than oxygen derivatives. Themost active derivatives induced apoptosis mediated by caspase-3, -8 and -9, and theyinhibited angiogenesis in vitro, because of what they are considered to have significantanticancer potential.

This study aimed to describe quality and potential health benefits of three Vranac wines obtained from new grape clones (CI, CII and CIII) recently developed and recognised. In this aim, their phenolic profiles, anti-2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical activities along with metal compositions were analyzed. Compared with the commercial one, CII and CIII Vranac wines were found to contain both higher contents of total phenolics, flavonoids and monomeric anthocyanins as well as anti-DPPH radical activities. The most abundant phenolics were gallic acid (8.88-16.36 mg/L), catechin (12.42-24.35 mg/L) and epicatechin (4.30-7.79 mg/L). Finally, the metal content of all the analyzed samples was within the toxicological safety limits. Taken all together, Montenegrin Vranac wines represent a rich source of both phenolics and minerals exhibiting promising antioxidant potential. CII and CIII wines can be considered for commercialising due to their high amounts of gallic acid, catechin and epicatechin, significant K/Na ratios and favourable contents of essential metals making them products with health added values.

In this study, the polyphenol profile and antioxidant activity of the hydro-ethanolic extract of the fennel fruit were examined in order to investigate the possibility of its application as a potential functional food additive. Total phenols were analyzed by the method of Folin-Ciocalteu, while total flavonoids were determined by the aluminum chloride colorimetric method. The separation and quantification of phenolic compounds were performed by LC-MS/MS analysis, using a multiple reaction monitoring (MRM) mode. The antioxidant capacity was determined by FRAP and DPPH assays. The high values of total phenolics and flavonoids were found, as well as high antioxidant activity which amounted to 9023.33 ± 38.19 µmol Fe(II)/l and 3.73 ± 0.04 mmol TE/l, tested by FRAP and DPPH assays, respectively. Among the identified phenolic compounds, p-hydroxybenzoic and chlorogenic acids were detected as predominant. The obtained results indicated that the hydro-ethanolic extract of the fennel fruit can be used in food industry as a potential natural antioxidant., U ovom radu određivan je sadržaj polifenola i antioksidativna aktivnost vodeno-etanolnog ekstrakta morača, sa ciljem ispitivanja mogućnosti njegove primene kao potencijalnog funkcionalnog aditiva. Ukupni fenoli su analizirani metodom po Folin-Ciocalteu, dok je ukupan sadržaj flavonoida određen kolorimetrijskom metodom primenom aluminijum hlorida. Razdvajanje i kvantifikacija fenolnih jedinjenja postignuti su upotrebom LC-MS/MS metode u režimu koji omogućava istovremeno praćenje više jonskih prelaza. Antioksidativni kapacitet je određivan primenom testova FRAP i DPPH. U testiranom ekstraktu dobijene su visoke vrednosti za ukupne fenole i flavonoide, a dobijena je i visoka vrednost antioksidativne aktivnosti, koja je iznosila 9023.33 ± 38,19 mmol Fe(II)/l i 3,73 ± 0,04 mmol TE/l, računato primenom testa FRAP odnosno testa DPPH. Među fenolnim jedinjenjima, phidroksibenzoeva i hlorogena kiselina su pronađene kao dominantne. Dobijeni rezultati ukazuju na to da se ekstrakt morača može primenjivati u prehrambenoj industriji kao potencijalni prirodni antioksidans.

The synthesis and cytotoxic activities determination of new steroidal mono- and bis(thiazolidin-4-ones) 4a-f and 5a-f have been performed. Their anticancer action was also evaluated in comparison to previously synthesized and reported corresponding steroidal thiosemicarbazones. All compounds were obtained as stereoisomeric mixtures with different configuration (E or Z) in the hydrazone moiety at the C-3 position. After several consecutive crystallizations diastereomerically pure major (5)-isomers of mono-thiazolidin-4-ones were isolated. The structure and stereochemistry of 2,4-thiazolidinedione,2-[(17-oxoandrost-4-en-3-ylidene)hydrazone] were confirmed by X-ray analysis. A pathway for the formation of thiazolidin-4-one ring was proposed. The steroid thiazolidinone derivatives examined in this study exerted selective concentration-dependent cytotoxic activities on six tested malignant cell lines. Ten out of twelve examined compounds exhibited strong cytotoxic effects on K562 cells (IC50 values from 8.5 mu M to 14.9 mu M), eight on HeLa cells (IC50 values ranging from 8.9 mu M to 15.1 mu M) while against MDA-MB-361 cells six compouds exerted similar or even higher cytotoxic action (IC50 values from 12.7 mu M to 25.6 mu M) than cisplatin (21.5 mu M) which served as a positive control. Eight of these ten compounds showed high selectivity in the cytotoxic action against HeLa and K562 cancer cell lines when compared with normal human fibroblasts MRC-5 and normal human PBMC. The study of mechanisms of the anticancer activity of the two selected compounds, mono- and bis(thiazolidin-4-one) derivatives of 19-norandrost-4-ene-3,17-dione 4a and 5a, revealed that both of these compounds induced apoptosis in HeLa cells through extrinsic and intrinsic signalling pathways. Treatment of EA.hy926 cells with sub-toxic concentrations of these compounds led to the inhibition of cell connecting and sprouting, and tube formation. The synthesized compounds exhibited poor antioxidant activ

The composition and content of individual phenolic compounds in various fruit wines and overall antioxidant properties of these fruit wines were studied. Phenolic compounds were separated by reverse phase (RP) HPLC techniques, and their content was determined by means of mass spectrometer (MS) with triple quadrupole (TQ) analyser, which recorded specific precursor-product transitions. Antioxidant activity was monitored by the following spectrophotometric methods: DPPH, FRAP and Folin-Ciocalteu assay, respectively. Four types of berries (raspberry, blackberry, chokeberry and blueberry), one type of pome (apple) and one type of stone fruit (cherries) were used for the production of fruit wines. Corresponding fruit wines were produced by microvinification with or without adding sugar into the must before fermentation. Increase in alcohol level was responsible for the increased content of phenolic compounds in fruit wine due to improved extraction conditions. Produced fruit wines were preserved by adding SO2 which interferes with the determination of antioxidant activity of phenolic compounds. In this case, the development of a correlation method based on FRAP assay was introduced. Fruit wines are a rich source of substances which show beneficial effects on human health. Depending on the fruit type, different antioxidant compounds were predominant in wine samples.

The composition and content of individual phenolic compounds in various fruit wines and overall antioxidant properties of these fruit wines were studied. Phenolic compounds were separated by reverse phase (RP) HPLC techniques, and their content was determined by means of mass spectrometer (MS) with triple quadrupole (TQ) analyser, which recorded specific precursor-product transitions. Antioxidant activity was monitored by the following spectrophotometric methods: DPPH, FRAP and Folin-Ciocalteu assay, respectively. Four types of berries (raspberry, blackberry, chokeberry and blueberry), one type of pome (apple) and one type of stone fruit (cherries) were used for the production of fruit wines. Corresponding fruit wines were produced by microvinification with or without adding sugar into the must before fermentation. Increase in alcohol level was responsible for the increased content of phenolic compounds in fruit wine due to improved extraction conditions. Produced fruit wines were preserved by adding SO2 which interferes with the determination of antioxidant activity of phenolic compounds. In this case, the development of a correlation method based on FRAP assay was introduced. Fruit wines are a rich source of substances which show beneficial effects on human health. Depending on the fruit type, different antioxidant compounds were predominant in wine samples.

A series of new steroidal mono- and bis(thiosemicarbazones) (2a-e and 3a-e) and corresponding mono- and bis(1,3,4-thiadiazolines) (4a-e and 5a-e) was synthesized, characterized and evaluated for their anticancer activity. Detailed NMR analysis of the mono-and bis(thiosemicarbazones) revealed the presence of two stereoisomers (Z and E) with different configurations in the hydrazone moiety at the C-3 position, where the substituents on the C(3)]=N double bond in the main isomers adopted the E configuration. The configurations at C-3 and C-17 in thiadiazolines 4a-e and 5a-e were deduced by detailed NMR analysis as well as by the examination of Dreiding molecular models and X-ray analysis of 3-thiadiazoline 4a, which confirmed the structure and absolute configuration at C-3. The synthesized compounds were tested against six cancer cell lines (HeLa, K562, MDA-MB-361, MDA-MB-453, LS174 and A549), the normal human cell line MRC-5 and peripheral blood mononuclear cells (PBMC) isolated from healthy donors. The best activity was exhibited by 3-thiosemicarbazones 2a, 2b, 2c and 2e and 3,17-bis(thiadiazolines) 5a and 5d. Examination of the mechanisms of cytotoxicity on cervical adenocarcinoma HeLa cells revealed the pro-apoptotic action of these compounds, which triggered both extrinsic and intrinsic apoptotic pathways. These compounds also showed the ability to decrease angiogenesis in vitro. In addition, 3,17-bis(thiadiazolines) 5a and 5d showed high selectivity in anticancer activity against all the examined malignant cell lines. Compound 5a displayed prominent anticancer potential. The tested compounds showed poor antimicrobial activity.

A series of new steroidal mono- and bis(thiosemicarbazones) (2a-e and 3a-e) and corresponding mono- and bis(1,3,4-thiadiazolines) (4a-e and 5a-e) was synthesized, characterized and evaluated for their anticancer activity. Detailed NMR analysis of the mono-and bis(thiosemicarbazones) revealed the presence of two stereoisomers (Z and E) with different configurations in the hydrazone moiety at the C-3 position, where the substituents on the C(3)]=N double bond in the main isomers adopted the E configuration. The configurations at C-3 and C-17 in thiadiazolines 4a-e and 5a-e were deduced by detailed NMR analysis as well as by the examination of Dreiding molecular models and X-ray analysis of 3-thiadiazoline 4a, which confirmed the structure and absolute configuration at C-3. The synthesized compounds were tested against six cancer cell lines (HeLa, K562, MDA-MB-361, MDA-MB-453, LS174 and A549), the normal human cell line MRC-5 and peripheral blood mononuclear cells (PBMC) isolated from healthy donors. The best activity was exhibited by 3-thiosemicarbazones 2a, 2b, 2c and 2e and 3,17-bis(thiadiazolines) 5a and 5d. Examination of the mechanisms of cytotoxicity on cervical adenocarcinoma HeLa cells revealed the pro-apoptotic action of these compounds, which triggered both extrinsic and intrinsic apoptotic pathways. These compounds also showed the ability to decrease angiogenesis in vitro. In addition, 3,17-bis(thiadiazolines) 5a and 5d showed high selectivity in anticancer activity against all the examined malignant cell lines. Compound 5a displayed prominent anticancer potential. The tested compounds showed poor antimicrobial activity.

An ultra-performance liquid chromatographic triple quadrupole mass spectrometric (UPLC-QqQ-MS/MS) method with a multiple reactions monitoring mode (MRM) was developed and validated for the quantification of vanillin and syringaldehyde in plum brandy. The method showed good linearity (0.05 to 10 mg L-1) and low limits of detection and quantification (the LOD and LOQ values were 11.6 and 38.2 mu g L-1 for vanillin, and 12.7 and 42.0 mu g L-1 for syringaldehyde, respectively). The overall intra-day and inter-day variations were less than 4.21 % and the overall recovery was over 93.0 %. The correlation coefficients (R-2) of the calibration curves were higher than 0.9999. In order to evaluate whether the method was suitable for use as a routine analytical tool, vanillin and syringaldehyde were determined in 31 samples of Serbian plum brandy.

Medlar is the fruit of Mespilus germanica L. in the family of Rosaceae. The fruit can be eaten only if "bletted" (softened by frost or longer storage). The effect of the maturation stages on the volatile compounds of the medlar fruit was investigated during two different stages. Volatile flavour substances were isolated from the minced pulp of unripe and full ripe medlar fruits by simultaneous steam distillation extraction (SDE) with methylene chloride as the extracting solvent. The concentrate was analyzed by GC-FID-MS. Hexanoic and hexadecanoic acids were the predominant acids, hexanal and (E)-2-hexenal were the predominant aldehydes, (Z)-3-hexenol and hexanol were the predominant alcohols, with p-cymene, terpinen-4-ol, and γ-terpinene (the terpenes responsible for the characteristic medlar flavour) being also present. The C6 aliphatic compounds, such as hexanal and (E)-2-hexenal, were observed as the major volatile constituents in the green stage. In contrast, hexanol and (Z)-3-hexenol were the main volatiles in ripe fruits., Mušmula (Mespilus germanica L) je voće iz porodice ruža (Rosaceae). Zeleni plodovi mušmule nisu jestivi već treba da ugnile (omekšaju usled mraza ili tokom čuvanja) pre nego što se koriste u ishrani. Efekat procesa sazrevanja na isparljive komponente ploda mušmule ispitivan je u dve različite faze njegove zrelosti. Isparljiva jedinjenja su izolovana iz izmrvljene pulpe nezrelih i zrelih plodova mušmule pomoću simultane destilacije vodenom parom i ekstrakcije metilen-hloridom. Koncentrovani uzorak je analiziran kombinacijom metoda GC-FID-MS. Heksanska i palmitinska kiselina su najzastupljenije kiseline, heksanal i (E)-2-heksenal su najzastupljeniji aldehidi, (Z)-3-heksenol i heksanol su najzastupljeniji alkoholi i p-cimen, terpinen-4-ol i γ-terpinen, terpeni odgovorni za karakterističnu aromu ploda mušmule, takođe su prisutni. C6 alifatična jedinjenja, kao što su heksanal i (E)-2-heksenal su nađeni kao glavne isparljive komponente zelenih mušmula. Za razliku od njih, heksanol, (Z)-3-heksenol su glavne komponente zrelih plodova mušmule.

Medlar is the fruit of Mespilus germanica L. in the family of Rosaceae. The fruit can be eaten only if "bletted" (softened by frost or longer storage). The effect of the maturation stages on the volatile compounds of the medlar fruit was investigated during two different stages. Volatile flavour substances were isolated from the minced pulp of unripe and full ripe medlar fruits by simultaneous steam distillation extraction (SDE) with methylene chloride as the extracting solvent. The concentrate was analysed by GC-FID-MS. Hexanoic and hexadecanoic acids were the predominant acids, hexanal and (E)-2-hexenal were the predominant aldehydes, (Z)-3-hexenol and hexanol were the predominant alcohols, with p-cymene, terpinen-4-ol, and gamma-terpinene (the terpenes responsible for the characteristic medlar flavour) being also present. The C6 aliphatic compounds, such as hexanal and (E)-2-hexenal, were observed as the major volatile constituents in the green stage. In contrast, hexanol and (Z)-3-hexenol were the main volatiles in ripe fruits., Mušmula (Mespilus germanica L) je voće iz porodice ruža (Rosaceae). Zeleni plodovi mušmule nisu jestivi već treba da ugnile (omekšaju usled mraza ili tokom čuvanja) pre nego što se koriste u ishrani. Efekat procesa sazrevanja na isparljive komponente ploda mušmule ispitivan je u dve različite faze njegove zrelosti. Isparljiva jedinjenja su izolovana iz izmrvljene pulpe nezrelih i zrelih plodova mušmule pomoću simultane destilacije vodenom parom i ekstrakcije metilen-hloridom. Koncentrovani uzorak je analiziran kombinacijom metoda GC-FID-MS. Heksanska i palmitinska kiselina su najzastupljenije kiseline, heksanal i (E)-2-heksenal su najzastupljeniji aldehidi, (Z)-3-heksenol i heksanol su najzastupljeniji alkoholi i p-cimen, terpinen-4-ol i γ-terpinen, terpeni odgovorni za karakterističnu aromu ploda mušmule, takođe su prisutni. C6 alifatična jedinjenja, kao što su heksanal i (E)-2-heksenal su nađeni kao glavne isparljive komponente zelenih mušmula. Za razliku od njih, heksanol, (Z)-3-heksenol su glavne komponente zrelih plodova mušmule.