| The genus podophyllum is one of the important members of the lignane class of natural products derived from plants, which possesses the ability to antitumor, antiviral, antifungal and insect resistant. In recent years, hundreds of derivatives have been designed and synthesized, such as etoposide (VP-16) and teniposide (VM-26), which are more potent antitumor agents, and they were officially approved respectively for clinical use against various types of cancers including testicular cancer, breast cancer, and small-cell lung cancer. Despite of their wide clinic application, several limitations hindered their use, such as poor bioavailability, the serious side effect and drug resistance which has inspired to further search for new effective antitumor agents based on this scaffold.To obtain better therapeutic agents with high activities and less toxic to natural cell, a series of C4-N-substituted podophyllum derivatives were synthesized based on the scaffolds of PTOX and DMEP, making using of theories including bioisosterism, combination principles and local modification. Then we further to discuss mechanisms of action of podophyllum derivatives. The main points can be listed as follows:In the first part, Medical industry broadly uses2-aminopyrimidine and2-aminopyridine as intermediates, so it is possible that these two compounds could generate more potent derivatives when they are substituted in the C-4position by N linkage. Thus, we aimed to have successfully synthesized four C4-N-arylamino substituted podophyllum derivatives. The structures of final products were confirmed by their1H NMR,13C NMR, ESI-MS spectral properties and elemental analyses, and purity estimation was performed with HPLC. To explore the structure activity relationship (SAR), the cytotoxicity of four N linkage podophyllum derivates (compounds lg-2g and compounds lh-2h) was evaluated in vitro by a MTT growth inhibition assay, which was carried out with a panel of five tumor cell lines: HeLa, BGC-823, A549, Huh7and MCF-7. The results showed that, compared to compounds1g and1h, compounds2g and2h were more potency against tumor cells, which implied that2-aminopyridine could be considered as a better substituent in the C-4positions of PTOX and DMEP by N linkage.On the basis of these SAR analysis results, we subsequently aimed to explore the influence of a chlorine substituent in the heterocyclic ring of2-aminopyridine in the second part. Therefore, eight N linkage podophyllum derivates containing chlorine atom were synthesized by the synthetic route. The structures of the final products were confirmed by their1H NMR,13C NMR, ESI-MS spectral properties and elemental analyses. The results of in vitro cytotoxicity using the same five human tumor cell lines showed that all eight compounds exhibited cytotoxicity. Several important SARs could be deduced from these results. Firstly, we observed that the introduction of a chlorine atom into the nitrogen heterocycle caused a considerable improvement in activity. Secondly, we found that changing the position of the chlorine atom in the nitrogen heterocycle caused a major alteration, with the order3-position>4-position>5-position>6-position. Thirdly, podophyllum derivatives with the4’-position demethylated were more important to the antitumor activity. Fourthly, we observed that the trend in cell potency was maintained regardless of whether PTOX or DMEP was substituted.According to the mentioned results, further biological evaluations had been focused on the highest potency compounds3g and3h substituted by2-amino-3-chloropyridine in PTOX and DMEP, and the study was carried out to determine their antitumor mechanisms in the third part. Upon further study, we found that compounds3g and3h exhibited the strongest antitumor potency, arrested HeLa cells in the G2/M phase of the cell cycle, and caused apoptosis in HeLa cells. The latter effect may be due to increased P53expression resulting in the up-regulation of Bax, the down-regulation of Bcl-2, and the activation of caspase-3. The response occurred in a dose-dependent mannerIn the fourth part,(1) for further insight into the biological effects of compound3g on cellular microtubule arrangement, we investigated microtubule structure and distribution in HeLa cells by indirect immunofluorescence (IF) using an anti α-tubulin antibody;(2) To further understand the antitumor mechanisms of compound3h substituted in the scaffold of DMEP, we evaluated the catalytic activity of topoisomerase II using a kDNA decatenation assay. The results provide strong evidence that compound3g definitively inhibits microtubule polymerization and compound3h exhibits antiproliferative properties against human tumor cells by inhibiting the activity of topoisomerase II, and does so in a dose dependent manner.In this study, we synthesized a series of N linkage podophyllum derivates followed by SAR analysis and biological studies. The presented data provide insights into the modification of podophyllum derivatives. This work also adds to the current body of knowledge detailing the mechanisms of action of podophyllum derivatives. In addition, it provides useful information for structural optimization studies in general, as well as for studies focused on the discovery and development of effective antitumor agents from natural parent compounds. |
PDF Full Text Request