Discovery,Optimization And Biological Evaluation Of Novel Topoisomerase Inhibitors

DNA topoisomerases (Tops) are essential enzymes that regulate the cellular processes such as replication, transcription, recombination, chromatin and chromosomal segregation by altering DNA topology. DNA Tops can be classified into two types, topoisomerase Ⅰ (Top1) and topoisomerase Ⅱ (Top2). They catalyze the breakage and religation of DNA, maintaining the topological changes of DNA and various DNA metabolic processes. Due to their important role in DNA metabolism, the ability to interfere with the functions of Tops or generating Top-mediated DNA damage is an effective strategy for cancer chemotherapy. Tops have been considered as the most important targets for tumor chemotherapy. Clinically, approximately50%of the chemotherapy regimens contain at least one Top inhibitor.The present dissertation aims to identify novel Top inhibitors as antitumor agents. A series of novel Top1and Top2inhibitors were identified by structure-based virtual screening. The structure-activity relationships, antitumor activity and mechanism of action of evodiamine derivatives and4-substituted anthra[2,1-c][1,2,5]thiadiazole-6,11-dione analogs were investigated, which led to the discovery of highly potent antitumor agents.1. Discovery and biological evaluation of novel topoisomerase I inhibitors by structure-based virtual screeningStructure-based virtual screening was applied to the discovery of structurally diverse Top1inhibitors. From23compounds selected by virtual screening from the Specs database, a total of14compounds were found to be topoisomerase I inhibitors. Five hits (compounds Al, A14, A20, A21, and A23) also showed moderate to good in vitro antitumor activity. Compound Al showed excellent cytotoxicity with IC50values in the range of0.5-25μM. Compounds Al and A20(evodiamine) were considered as good starting points for the development of new antitumor lead compounds.2. Design, synthesis and antitumor mechanism of novel evodiamine derivativesEvodiamine is a quinazolinocarboline alkaloid isolated from the fruits of traditional Chinese herb Evodiae fructus. Previously, evodiamine was identified as potent topoisomerase I inhibitor by structure-based virtual screening and showed moderate in vitro antitumor activity. It was also considered as a new antitumor lead compound. Herein,51novel evodiamine derivatives bearing various substitutions or modified scaffold were synthesized. Among them, a number of evodiamine derivatives (B4, B5, B38and B39) showed substantial increase of the antitumor activity, with GI50values lower than3nM. Moreover, these highly potent compounds (B4, B38, B39, B41and B45) can effectively induce the apoptosis and G2/M arrest of A549cells. Interestingly, further computational target prediction calculations in combination with biological assays confirmed that the evodiamine derivatives acted by dual inhibition of topoisomerases Ⅰ and Ⅱ. Moreover, several hydroxyl derivatives, such as10-hydroxyl evodiamine (B38) and3-amino-10-hydroxyl evodiamine (B45), also showed good in vivo antitumor efficacy with low toxicity and good tolerance. At the dose of1mg/kg or2mg/kg, B38and B45reduced44.43%and52.71%tumor growth, respectively. They represent promising candidates for the development of novel antitumor agents.3.4-Substituted anthra[2,1-c][1,2,5]thiadiazole-6,11-dione derivatives: design, synthesis and antitumor potency.Previously,4-tosylanthra[1,2-c][1,2,5]thiadiazole-6,11-dione (Al) was identified as potent topoisomerase I inhibitor by structure-based virtual screening and showed good antitumor activity in vitro. Based on the structure of Al,54novel analogs were designed and synthesized, which can be classed into two types,4-thio and4-amine substituted derivatives. The former series exhibited weak antitumor activity because of their poor solubility. Only few compounds (D14and D15) showed moderate to good in vitro antitumor activity. The latter series were proved to have stronger antitumor activity with better solubility. Three compounds (El, E6and E19) showed high cytotoxicity similar to that of camptothecin.4. Discovery and biological evaluation of novel topoisomerase Ⅱ inhibitors by structure-based virtual screeningTop2catalytic inhibitors have the advantages of modulating the cytotoxic effects of topoisomerase II poisons and overcoming the multidrug resistance (MDR). ATP binding site (ATPase) binders represent an important class of Top2catalytic inhibitors. On the basis of the crystal structures of ATPase, docking-based virtual screening was applied to the discover novel Top2inhibitors. From37compounds selected by virtual screening, a total of19compounds were found to be Top2inhibitors. Six hits (compounds F2, F8, F15, F17, F26and F27) were more active than etoposide at the concentration of100μM. In particular, compound F8exhibited better antitumor activity than etoposide in A549cancer cells. Compound F20showed good antitumor activity against all the three cancer cells. The biological assay indicated that compounds F8and F20could stabilize the Top2-DNA cleaved complex slightly. Molecular docking predicted that compounds F8and F20might bind at the ATP binding site. They were considered as good starting points for the development of new antitumor lead compounds targeting Top2.In summary, structure-based virtual screening were successfully used to discover novel Top1and Top2inhibitors with a hit rate of60.9%and51.4%, respectively. On the basis of the identified lead compounds, a total of105new derivatives were designed and synthesized. Among them, several highly active antitumor compounds were identified which showed excellent activity at molecular, cellular and in vivo level. Target identification and structural optimization of traditional Chinese medicine evodiamine were performed for the first time using the modern drug design techonologies. Evodiamine derivatives were proved to be dual Top1/Top2inhibitors and several of them showed excellent antitumor potency with low toxicity, which provided basis for the development of novel antitumor agents.