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|Title:||Triazaspiroalkenes as potential inhibitors of dihydrofolate reductase||Authors:||ONG PAULINE||Keywords:||Triazaspiroalkenes, high throughput screening, dihydrofolate reductase inhibitors, antiproliferative activity, cell cycle analysis.||Issue Date:||19-Aug-2009||Citation:||ONG PAULINE (2009-08-19). Triazaspiroalkenes as potential inhibitors of dihydrofolate reductase. ScholarBank@NUS Repository.||Abstract:||Previously, high throughput screening (HTS) has been adopted in the screening for dihydrofolate reductase (DHFR) inhibitors in 96-well plate. In this thesis, a sensitive and cost-effective HTS for rapid identification of murine DHFR inhibitors in 384-well microplate was developed. The absorbance of NADPH at two time points was measured as an indicator of the DHFR activity instead of the continuous monitoring of the rate of decrease of NADPH in real time. This method allowed more flexibility in the detection process. This is the first report of the miniaturization of DHFR assay into a 384-well microplate, using a final volume of 50 µL as the reaction mixture. Diamino-phenyl-triazaspiroalkenes were found to be biologically active yet were less studied in the past. It was hypothesized that diamino-phenyl-triazaspiroalkenes of a particular ring size, when appropriately substituted, would possess DHFR inhibitory activity as well as anticancer activity. A library of seventeen diamino-((1-phenylalkyl-1H-1,2,3-triazol-4-yl)methoxy)-phenyltriazaspiroalkenes (P-III) was synthesised using click chemistry where a triazole was formed from the Cu-(I) catalysed 1,3-polar addition of azide onto acetylene. P-III demonstrated dramatically improved rhDHFR inhibitory activity. Triazole possesses two nitrogen atoms, which are capable of forming hydrogen bonds with Asn64 in rhDHFR; thus providing strategic interaction points for the compound to the active site of rhDHFR. With the overall enhancement of flexibility and lipophilicity, diamino-(phenylalkyloxy)phenyl-triazaspiroalkene (P-IV) showed excellent antiproliferative activity against both A549 and MDA-MB-231 cell lines. Compounds with longer side chain showed the best antiproliferative activity, suggesting that lipophilicity was crucial for the penetration of cell membrane. 2,4-Diamino-5-phenyl-1,3,5-triazaspiro[5.5]undeca-1,3-dienes of P-IV did not inhibit rhDHFR. Cell cycle analysis suggested that diamino(phenylalkyloxy)phenyl-triazaspiro[5.5]undeca-1,3-dienes caused G1 phase arrest of the A549 cell cycle; meanwhile, S phase arrest was observed for MDA-MB-231 cell cycle. This observation proposed that there might be other biological target for diaminophenyltriazaspiro[5.5]undeca-1,3-dienes. In conclusion, the objective of the development of a miniaturised HTS in 384-well microplate for the identification of murine DHFR inhibitors was achieved. The hypothesis that diamino-phenyl-triazaspiroalkenes of a particular ring size when appropriately substituted in the phenyl ring would possess DHFR inhibitory activity as well as anticancer activity was proven to be true.||URI:||http://scholarbank.nus.edu.sg/handle/10635/18415|
|Appears in Collections:||Ph.D Theses (Open)|
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checked on Apr 19, 2019
checked on Apr 19, 2019
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