Introduction: Finding agents with potential anticancer effect is imperative in modern oncology. It is increasingly noted that certain chemical structures have specific toxic effect on cancer cells. Artemisinin discovery marked the beginning of peroxide research as a potential replacement for traditional antimalarial drugs, and the structurally simple class of peroxides that emerged from these studies was the 1,2,4,5-tetraoxanes, which was soon shown that in addition to antimalarial show strong antiproliferative activity . Malignant cells have a disruption in the regulatory mechanisms that govern cellular proliferation and homeostasis. The ability of tumor cell populations to increase the number of cells is determined not only by the intensity of cell proliferation, but also with rate of the removal of cells. Programmed cell death - apoptosis, is the main source of this removal. The aim of this study was to determine the level of the cytotoxic effect of six mixed tetraoxanes to various human malignant cells, as well as to determine the coefficient of selectivity in their activity in comparison to healthy immunocompetent cells. In order to gain insight into the mechanism of action of investigated compounds the type of cell death induced by tetraoxanes will be examined, as well as production of reactive oxygen species in HeLa cells. The goal of 3D QSAR studies on antiproliferative activities of thirty three 1,2,4,5-tetraoxane derivatives against HeLa and Fem-x tumor cell lines has been to determine which are most important pharmacophore of steroid tetraoxanes that affect the potency of the compounds against HeLa and Fem x-tumor cell lines. Material and Methods: The cytotoxic effect of tetraoxanes DO-122 - DO-124 and DO-126 - DO-128, against five tumor cell lines were examined with a standard MTT assay. In order to determine the mode of HeLa cell death induced by the investigated compounds, morphological analysis by microscopic examination of acridine orange and ethidium bromide stained cells was performed. Analysis to determine the cell cycle phase distribution of HeLa cells stained with propidium iodide was performed on the flow cytometer. Intracellular production of reactive oxygen species (ROS) was measured using the fluorescent color 2,7-Dichlorodihydrofluorescein diacetate. The dependence of the structure and function of the thirty-three 1,2,4,5-tetraoxane derivatives against HeLa and Fem-x cell lines was demonstrated with a 3D QSAR study. Results: The tested tetraoxanes show dose-dependent antiproliferative activity in micromolar concentrations to target tumor cells with good selectivity in activities to tumor cells, compared to normal immunocompetent cells. treatment of HeLa cells for 24-hour with all tested tetraoxanes induce the typical morphological features of late apoptosis (condensed and/or fragmented nuclei). After 24 and 48 hours of incubation with tetraoxanes, the number of HeLa cells in G1 phase was significantly increased. After treatment of HeLa cells with examined tetraoxanes, the level of reactive oxygen species increased significantly indicating a possible oxidative stress. Using GRIND methodology revealed that the pharmacophore which may account for the most significant effect was amide NH of primary or secondary amides and acetoxy fragments at positions 7 and 12 of the steroid nucleus, which together with the tetraoxane ring moiety is common to all compounds. Conclusions: The tested tetraoxanes show dose-dependent antiproliferative activity in micromolar concentrations to the target tumor cells. The results show that the tested tetraoxanes induce apoptosis in HeLa cells, stop cell cycle in the G1 phase, and potently generate ROS pointing to possible oxidative stress. 3D QSAR study revealed that the pharmacophore of steroid tetraoxanes which may account for the most significant effect is amide NH of primary or secondary amides and acetoxy fragments at positions 7 and 12 of the steroid nucleus, which together with the tetraoxane ring moiety is common to all compounds. Uvod: Pronalaženje agenasa sa potencijalnim antitumorskim dejstvom je imperativ u modernoj onkologiji. Pri tome se sve više zapaža da određene hemijske strukture imaju specifičnije toksično dejstvo na maligne ćelije. Otkriće artemizinina je označilo početak istraživanja peroksida kao potencijalne zamene za tradicionalne antimalarijske lekove, a iz ovih istraživanja nastala je i strukturno jednostavna klasa peroksida - 1,2,4,5-tetraoksani, za koje je ubrzo pokazano da pored antimalarijskog pokazuju i snažan antiproliferativni efekat. Maligne ćelije imaju poremećaje u regulatornim mehanizmima koji upravljaju ćelijskom proliferacijom i homeostazom. Sposobnost tumorskih ćelijskih populacija da povećaju broj ćelija je određena ne samo intenzitetom ćelijske proliferacije, već i brzinom uklanjanja ćelija. Programirana ćelijska smrt - apoptoza, predstavlja glavni izvor ovog uklanjanja. Cilj rada je bio da se odredi nivo citotoksičnog dejstva grupe mešovitih tetraoksana prema različitim humanim malignim ćelijama, kao i da se odredi koeficijenat selektivnosti u njihovom dejstvu u odnosu na zdrave imunokompetentne ćelije. U cilju dobijanja uvida u mehanizam dejstva ispitivanih jedinjenja odrediće se tip ćelijske smrti koju indukuju ispitivani tetraoksani, kao i produkcija reaktivnih kiseoničnih vrsta u Hela ćelijama. Cilj 3D QSAR studije o antiproliferativoj aktivnosti trideset tri 1,2,4,5-tetraoksanska derivata prema Hela i Fem-x tumorskim ćelijskim linijama, je bio da se utvrdi koje su najvažnije farmakofore steroidnih tetraoksana koje utiču na potenciju ispitivanih jedinjenja prema HeLa i Fem-x tumorskim ćelijskim linijama. Materijal i metode: Citotoksično dejstvo tetraoksana DO-122 - DO-124 i DO-126 - DO-128, prema pet tumorskih ćelijskih linija je ispitivano standardnim MTT testom. U cilju određivanja tipa ćelijske smrti indukovane tretmanom ispitivanim tetraoksanima načinjena je morfološka analiza HeLa ćelija obojenih smešom akridin oranža i etidijum bromida. Analiza određivanja distribucije faza ćelijskog ciklusa HeLa ćelija obojenih propidijum jodidom, urađena je na protočnom citometru. Produkcija intraćelijskih reaktivnih vrsta kiseonika (ROS) je merena fluorometrijski pomoću fluorescentne boje 2’,7’-dihlorodihidrofluorescein diacetata. Zavisnost strukture i funkcije trideset tri 1,2,4,5-tetraoksanskih derivata prema HeLa i Fem-x ćelijskim linijama pokazana je 3D QSAR studijom. Rezultati: Ispitivani tetraoksani pokazuju dozno-zavisnu antiproliferativnu aktivnost u mikromolarnim koncentracijama prema ciljnim tumorskim ćelijama, uz dobru selektivnost u aktivnosti prema tumorskim ćelijama, u odnosu na normalne imunokompetentne ćelije. 24-časovni tretman HeLa ćelija sa svim ispitivanim tetraoksanima indukuju tipične morfološke karakteristike kasne apoptoze (kondenzovana i/ili fragmentisana jedra). Posle 24 i 48 časova inkubacije sa tetraoksanima, broj HeLa ćelija u G1 fazi se značajno uvećava. Nakon tretmana HeLa ćelija ispitivanim tetraoksanima, nivo reaktivnih kiseoničnih vrsta je značajno porastao ukazujući na mogući oksidativni stres. Korišćenjem GRIND metodologije utvrđeno je da su farmakofore kojima se može pripisati najzna?ajnije dejstvo, amidni NH primarnih ili sekundarnih amida i acetoksi fragmenti na pozicijama 7 i 12 steroidnog jezgra, koji su zajedno sa tetraoksanskim prstenom, zajednički za sva ispitivana jedinjenja. Zaključci: Ispitivani tetraoksani pokazuju dozno-zavisnu antiproliferativnu aktivnost u mikromolarnim koncentracijama prema ciljnim tumorskim ćelijama. Rezultati pokazuju i da ispitivani tetraoksani indukuju apoptozu i zaustavljaju HeLa ćelije u G1 fazi ćelijskog ciklusa i potentno generišu ROS ukazujući na mogući oksidativni stres. U 3D QSAR studiji utvrđeno je da su farmakofore steroidnih tetraoksana kojima se može pripisati najznačajnije dejstvo, amidni NH primarnih ili sekundarnih amida i acetoksi fragmenti na pozicijama 7 i 12 steroidnog jezgra, koji su zajedno sa tetraoksanskim prstenom, zajednički za sva ispitivana jedinjenja.