Phenanthroline Derivatives Targeting Quadruplex DNAs:Design, Synthesis And Evaluation Of Biological Activity

Six novel1,10-phenanthroline derivatives (1-6) were designed and synthesized based on2,9-dimethyl-1,10-phenthroline. Interactions of these compounds with human telomere G-quadruplex, i-motif DNAs and oncogene promoter c-kit2and c-myc G-qudruplex DNAs were studied in detail. Furthermore, effects of the compounds on the telomerase activity and on the transcriptional level of c-kit2and c-myc oncogene were investigated. Finally, their effects on the proliferation and cell cycle of HeLa and HepG2cells were measured. The main results are as following:Six novel1,10-phenanthroline derivatives (1-6) were synthesized and characterized by means of IR,1H NMR,13C NMR, ESI-MS and element analysis methods.CD spectra showed that compounds1-4could induce human telomere G-rich sequence (HTG21) to fold into an antiparallel G-quadruplex structure from a random or hybrid structure. In combination with FRET-melting experiment and PCR stop assay, these compounds could stabilize antiparallel G-quadruplex structure even with the existence of25times excess of duplex competitor (ds26). TRAP-LIG assay indicated that2μM compounds1-3could completely inhibit the telomerase activity.Binding stoichiometry and affinity between compounds1-3and human telomere G-quadruplex and i-motif DNAs were evaluated through Job plot, UV visible absorption titration and competitive dialysis methods. Binding stoichiometries of the compounds to G-quadruplex and i-motif DNAs are2:1and1:1, respectively. Results indicated that these compounds exhibited higher binding affinities for G-quadruplex and i-motif DNAs than ct DNA. UV-melting results indicated that compounds1-3could stabilize the i-motif structure with melting temperature increase (△Tm) of7.2-10.1℃, respectively. The thermodynamic parameters (△G,△H and△S) results indicated that binding processes of the compounds to either quadruplex or duplex DNAs were entropically driven.The conformation effect, stabilization capability and binding affinities of compounds1-3on c-kit2and c-myc G-quadruplexes DNAs were investigated by means of CD spectra, FRET-melting assay, PCR stop assay, UV-visible absorption titration and FID assay. The additions of compounds1-3perturbed the base-stacking structure of c-kit2G-quadruplex slightly, while no obvious effect was observed on c-myc G-quadruplex. The three compounds could moderately stabilize both the c-kit and c-myc G-quadruplexes and exhibit higher G-quadruplex selectivity over duplex. Stabilization potential of the compounds for c-kit2G-quadruplex was larger than for c-myc G-quadruplex. The affinities of the compounds to G-quadruplex were higher than to ct DNA, and these compounds also showed higher affinity to c-myc than to c-kit2G-quadruplex. Real time RT-PCR results indicated that compound1could decrease the transcription level of c-kit2and c-myc.MTT assay indicated that compounds1-4could inhibit the proliferation of both HeLa and HepG2cancer cells when incubated for72h or20days. The IC50values of HeLa cells were about2μM for compounds1-3incubating for72h, respectively, while the IC50values of HepG2cells were0.5-4.4μM for compounds1-4, respectively. In the long-time cell cytotoxicity experiment (incubating for20days),0.1μM compounds1-3could completely inhibit the proliferation of HepG2cancer cells after16days. In the flow cytometric assay, the HeLa cells were all arrested in G0/G1phase by the compounds,. On the contrary, compounds1-3had a slight decrease effect on G0/G1-phase cell population for HepG2cells.