Microwave-assisted Synthesis Of Metalloporphyrin Complexes And The Molecular Recognition With C-myc G4DNA

Because of its unique structure and property, especially the special affinity with the tumor cells, more and more attentions are focused on the porphyrin and metal porphyrin, a class of nature compounds, which has potential application prospect as antitumor drugs in clinical medicine and correlation diagnostic reagents. In recent years, a series of this type of complexes were designed and synthesized to apply in antitumor, and some entity with high antitumor activity have been reported. On the other hand, G-quadruplex DNA, which has been a potential target for small molecule drugs, will constructed for those G-rich DNA sequences via Hoogsteen hydrogen bonds in the presence of monovalent cations. More recently, quadruplex sequence of telomere and the original cancer gene have been become the most popular small molecule anticancer drug targets of the current research. It’s found that the promoter oncogene, which is over-expressed in up to85%of solid tumor, can also form a G-quadruplex conformation in the presence of potassium or sodium ion, and play a key role in the proliferation and apoptosis of tumor cells. Those complexes, which can stabilize the conformation of promoter oncogene G-quadruplex usually exhibit high inhibitory activity against tumor cells. There are a lot of reports about interaction of porphyrins and various G-quadruplex DNA, even some with meaningful conclusions, however, little information was obtained about the proto-oncogene sequence, which also can form quadruplex. As in this mind, in the paper a series of porphyrins and metal porphyrins were prepared under microwave-assisted and characterized by ESI-MS,1H-NMR,13C-NMR spectra and other spectroscopic methods. What’s more, as a positive control, those compounds were also synthesized by the conventional method. Furthermore, the inhibitory activity of metal porphyrins against cancer cells were evaluated by using MTT assay after a48h treatment. Besides, the binding behavior of metal porphyrins with c-myc G4DNA has been also investigated. The major results of this paper are listed in the following.1. Synthesis of5,10,15,20-tetra(4-methoxyphenyl) porphyrin with copper(II) and the molecular recognition with c-myc G4DNAFirstly,5,10,15,20-tetra(4-methoxyphenyl) porphyrin (TMOPP) was synthesized from p-methoxybenzaldehyde and pyrrole in propionic acid solution, and then5,10,15,20-tetra (4-methoxyphenyl) porphyrin copper(II)(CuTMOPP) was synthesized from TMOPP and cupric acetate in DMF solution, and purified by using column chromatograph. Both TMOPP and CuTMOPP have been characterized by1H NMR,13C NMR, ESI-MS, UV, AFS and IR spectra. Although there was little obvious difference in both methods in yields, but the reaction time was more shorten for microwave-assisted synthesis technique than that of conventional method, especially in the second step, which could increase the reaction efficiency to around15times.The interaction of CuTMOPP with c-myc G4DNA has been studied by using UV-vis, CD spectra, FRET experiments and PCR-Stop experiments, the results showed that CuTMOPP can bind with c-myc G4-DNA with high affinity. This was confirmed by the hypochromism and red shift in electronic spectra, decrease of fluorescence spectra. What’s more, the CD signal strength slightly decreased of c-myc G4DNA solution after adding the target compounds, suggesting that it could case little influence on the configuration of c-myc G4DNA. This was also confirmed by the FRET assay. Those results indicated that the target compound CuTMOPP may bind c-myc G4DNA by electrostatic binding mode, as a result, the the replication of Top polymerase was blocked, and an evaluation of biological activity of CuTMOPP is now in progress.2. Synthesis of5-[4-(4-bromobutoxy)phenyl]-10,15,20-trimethoxypheny-porphyrin with copper(II) and the molecular recognition with c-myc G4DNA5,10,15,20-tetra (4-methoxyphenyl) porphyrin (p-HTMOPP) was synthesized from p-hydroxy benzaldehyde,p-methoxy benzaldehyde and pyrrole in propionic acid solution. The crude product was purified through silica gel column. And then,5-[4-(4-bromobutoxy)phenyl]-10,15,20-trimethoxyphenyporphyrin (p-BrTMOPP) has been prepared from p-HTMOPP and1,4-dibromobutane in DMF solution. Lastly, the target compound p-CuBrTMOPP was synthesized from p-BrTMOPP and cupric acetate in DMF solution to purify raw product through column chromatography. All those complexes have been characterized by spectroscopy methods, including1H NMR,13C NMR, ESI-MS, UV, AFS and IR spectra. The yield of those target compounds though microwave-assised synthesis were not higher than that conventional synthesis method, but the difference was not significantly.The inhibitory activity of the target compound p-CuBrTMOPP against cancer cells were evaluated by using MTT assay after a48h treatment. It’s found that p-CuBrTMOPP could selectively inhibit the growth of human breast cancer cells (MCF-7) with inhibitory activity (IC50) about15.6μM, which was better than the positive control cisplatin. The interaction mechanism of the target compound p-CuBrTMOPP with c-myc G4DNA was further studied by thermal denaturation melting point and PCR-Stop experiments, as well as spectroscopy methods. Upon adding the target compound solution, obvious hypochro-mism and red shif were observed, which was also obtained in the fluorescence intensity, indicating that p-CuBrTMOPP could bind to c-myc G4DNA. From the CD spectra of c-myc G4DNA solution in the presence of p-CuBrTMOPP and the thermal denaturation melting point assay, it support that p-CuBrTMOPP could bind and stabilize the conformation of c-myc G4DNA vis electrostatic binding or groove binding. The results of PCR-Stop assay was further confirmed, which shows that the replication of DNA was blocked after treatment with the target compound.3. Synthesis of5-[4-(4-bromobutoxy) phenyl]-10,15,20-trimethoxypheny-porphyrin with zinc(Ⅱ) and the molecular recognition with c-myc G4DNAThe synthesis method of5-[4-(4-bromobutoxy) phenyl]-10,15,20-trimethoxy-phenyporphyrin with zinc(Ⅱ)(p-ZnBrTMOPP) and p-CuBrTMOPP are as the same method as above, but with cupric acetate replaced with zinc acetate. The target compounds have been characterized by spectroscopy methods. Besides, the target compounds have been also synthesized by conventional and microwave-assisted methods, with little obvious difference.The inhibitory activity of the target compounds against cancer cells were evaluated by using MTT assay. It’s found that p-ZnBrTMOPP has no inhibiting effect on the screening of tumor cell lines, And whether the activity of other tumor cell lines, still further screening.The binding behavior of p-ZnBrTMOP with c-myc G4DNA has been also investigated, suggesting that this type complex could bind and stabilize the conformation of c-myc G4DNA by electrostatic binding or groove binding mode, as a result, the replication of DNA was blocked, and an evaluation of biological activity is now in progress.