Wydział Chemii (WCh)/Faculty of Chemistry
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Browsing Wydział Chemii (WCh)/Faculty of Chemistry by Author "Baraniak, Dagmara Sara"
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Item Synteza koniugatów nukleozydów z alkaloidami kory chinowej z zastosowaniem metod chemii ”click”(2012-03-26T06:16:48Z) Baraniak, Dagmara Sara; Celewicz, Lech. PromotorNucleoside analogues have found successful application as antiviral and anticancer agents. Among them an important place occupy 2’,3’-dideoxynucleosides (ddNs), i.e. 3’-azido-3’-deoxythymidine (AZT). Notably, AZT has been originally developed as a potential antitumor drug, but later it has found a widespread use as an antiretroviral drug (anti-HIV), and recently it is used as an anticancer agent, especially in combination with cisplatin, methotrexate and 5-fluorouracil. In general, the mechanism of antiviral or cytostatic action of ddNs involves their intracellular conversion to an active form of the drug – the 5’-triphosphates, which are inhibitors of the relevant enzymes in the replication process (HIV reverse transcriptase or DNA polymerase). They act as competitive inhibitors or chain terminator of the nascent DNA strand due to lack of 3’-hydroxyl group of the relevant enzymes. In recent years, a number of attempts were undertaken to design prodrugs like ddNs that do not require the first step of phopshorylation. The subject of presented studies was the synthesis, cytotoxic activity and fluorescent properties of conjugates of nucleosides with Cinchona alkaloids obtained using the methods of click chemistry. At the first stage of the study the synthesis of the following azido-nucleosides was carried out: 3’-azido-3’-deoxythymidine, 5’-azido-5’-deoxythymidine (5’-AZT), 2-azidoadenosine (2-N3-A) and 2-azido-N6-methyladenosine (2-N3-N6-Me-A). Next the modified Cinchona alkaloids, i.e. 10,11-didehydro- and 9-O-propargyl derivatives of alkaloids were obtained, which were containing a terminal triple bond in their structure. The main aim of the study was to develop a general method of synthesis of nucleoside – alkaloid conjugates. For this aim, the Cu(I) catalyzed Huisgen reaction was used (CuAAC), employing as the azide component a azido-nucleoside analogue and as the alkyne component – a modified Cinchona alkaloid. The synthesized compounds were evaluated for cytotoxic activity in KB and MCF-7 cancer cells. For each conjugate was determined IC50 parameter (inhibitory concentration; concentration of compound causing 50% inhibition of tumor cell growth). The high cytotoxic activity against tumor cells showed following conjugates: 3’-azido-3’-deoxythymidine with 10,11-didehydroquinine (IC50 = 1,30 μg/mL), 5’-azido-5’-deoxythymidine with 9-O-propargylqunidine (IC50 = 1,70 μg/mL), 2-azido-N6-methyladenosine with 10,11-didehydroquinine (IC50 = 1,70 μg/mL), 5’-azido-5’-deoxythymidine with 10,11-didehydroqunidine (IC50 = 1,75 μg/mL). Because Cinchona alkaloids display significant fluorescence emission, it was also measured fluorescence emission (in acetonitrile and buffer acetate at pH = 4) for obtained nucleoside–alkaloid conjugates and the fluorescence quantum yield was calculated. The results showed that the position of the maximum emission bands depends on the solvents used and the structure of the compound, but mostly it was observed at about λ = 360 nm in acetonitrile. The acetate buffer was used to check, if the conjugates could be excite in a further range of the spectrum. It was found that emission of these conjugates recorded in acetate buffer are shifted to longer wavelength (400÷450 nm), it means that these compounds can be excited using longer wavelength. The research was also aimed to obtain conjugates of 5’-phosphoramidates of AZT with 10,11-didehydroquinine (DDQn). It is worth noting that nucleoside 5’-phosphoramidates more easily penetrate the phospholipid cell membrane, and after entering the cell are hydrolyzed to 5’-phosphates, bypassing first phosphorylation step. Hence the idea of synthesis of potential prodrugs with masked phosphate group, which include discussed conjugates. Based on this idea, the inverse concept was planned – the synthesis of phosphorylated alkaloid components, namely 9-O-phosphoramidates of DDQn, which underwent coupling with AZT and gave desired hybrids. In addition, the method of synthesis of 9-O-phosphoramidates of DDQn that was described, it has not been previously presented in the scientific literature and the obtained compounds are a new class of 10,11-didehydroquinine derivatives. The thesis presents the studies on the synthesis of conjugates of azidonucleosides with Cinchona alkaloids, using the copper(I) catalyzed 1,3-dipolar Huisgen cycloaddition reaction in accordance with a novel concept introduced by Sharpless and called click chemistry. Almost all tested conjugates, synthesized in this work, showed high or moderate cytotoxic activity. Moreover, because of the free hydroxyl group at the 3’- or 5’-position the obtained conjugates can be incorporated into synthesized DNA (RNA) respectively at the 5’ or 3’-end. What is more the aromatic quinoline ring of nucleoside alkaloid conjugates, because of its planar structure, can be inserted between the base pairs of the double helix of DNA (intercalation). It was determined that the synthesized conjugates show strong fluorescence emission therefore can be used as fluorescent probes for oligonucleotides or fragments of DNA. In conclusion, a general method for the synthesis of nucleoside – Cinchona alkaloid conjugates has been developed. What is more the antitumor studies have shown, that it is reasonable to search for determinants of biological activity within the nucleoside conjugates formed with other potentially active compounds such as Cinchona alkaloids. Based on our observation, it can be concluded, that the CuAAC reaction gives the possibility of broad application in the synthesis of conjugates between azidonucleosides and modified Cinchona alkaloids containing terminal triple bond.