由于耐药性的出现，研究新作用机制、新结构的抗肿瘤药物具有重要意义。DNA拓扑异构酶I（Top1）是一个有效的肿瘤治疗靶点。本课题前期研究发现了一类新机制的、特异性作用于Top1的中氮茚并氮杂萘醌类（Azan）抑制剂。其作用机制不同于传统的拓扑毒剂，Azan作用于Top1的Asn421残基附近的空腔，干扰了Top1与DNA的结合，从而抑制了Top1断裂DNA的活性，并选择性将肿瘤细胞阻滞在G2/M期。本项目计划在前期构效关系和计算机辅助药物设计等手段的指导下，对活性分子进行系统的结构优化、设计合成新化合物库；通过抗肿瘤活性及Top1抑制活性研究，获得高活性、低毒的先导化合物；深入研究先导化合物的抗肿瘤谱及其与Top1的相互作用，探讨其作用位点、Top1抑制活性及抗肿瘤活性之间的关联性和分子机制；初步评价其体内抗肿瘤活性、急性毒性；系统分析其构效关系，为研发新机制的、新型抗肿瘤药物奠定基础。

Research and discovery of antitumor drug with new mechanism of action or new chemical structure is important due to the emergence of drug resistance. DNA topoisomerase I (Top1) is a validated target for cancer treatment. In our previous studies, we have designed and synthesized a series of aza-naphthindolizinedione derivatives (Azan), a kind of Top1 specific inhibitors with new mechanism of action. Unlike the typical Top1 poison, such as Camptothecin, Azan may bind to the pocket around residue Asn421, which prevent the DNA from bending or moving significantly, and resulting in decreased cleavage activity of Top1. As a result, Azan has shown strong antiproliferative activity specifically against cancer cells, and arrested the cancer cell cycle at the G2/M phase. Based on these findings, in this project, we plan: 1) to modify the active molecules and establish a new library of compounds for antitumor drug screening based on our previous studies including structure-activity relationship and computer aided drug design; 2) to obtain highly active leading compounds with low toxicity in vivo through anticancer activity and Top1 inhibitory activity studies; 3) to study the antitumor spectrum of the leading compounds and their interactions with Top1; 4) to study the relationship among the binding site, Top1 inhibitory activity and antitumor activity, as well as the inhibition mechanism for these compounds; 5) to study their in vivo antitumor activitiy and the acute toxicity; 6) to analyze the structure-activity relationship of all Azan derivatives. This study will set up a basis for the discovery of Azan derivatives as potential antitumor drugs with new mechanism of action.

研究新结构、新作用机制的抗肿瘤药物是对抗临床耐药性的重要手段。本项目计划对前期发现的Azan类Top1催化型抑制剂进行系统的结构优化和活性研究，期望获得更高活性的先导化合物，并初步研究其成药性。基于这一研究计划，本项目设计、合成了四个系列、共230多个新类似物。经过系统的酶抑制活性和体外细胞毒活性研究，发现了8个具有高酶抑制活性和细胞毒活性的先导化合物，深入的机制研究证实，其中6个先导化合物为Top1催化型抑制剂、2个为Top1毒剂。为了深入研究先导化合物活性，分别选择催化型抑制剂I-2和Top1毒剂III-96为代表性分子进行深入研究。一）利用基因突变细胞株为对象，研究发现I-2在细胞内的主要作用靶点为Top1，其作用位点不同于经典的Top1毒剂CPT；另外，I-2不是药物外排蛋白Pgp的底物。这些研究表明，该类分子是一类新作用机制的活性物质，对临床常见的耐药肿瘤细胞有良好的杀伤作用。体内活性研究发现，I-2的毒副作用低，具有显著的抑瘤效果，在20 mg/kg剂量下，抑瘤指数（WTI）为38.3%。但是，与体外细胞毒性相比，其体内抑瘤效果较弱。药代动力学结果提示，这可能是I-2的生物利用度（10.2%）较低所致。这一结果提示我们，未来该类物质的优化应以提高生物利用度为主。二）III-96可造成显著的DNA损伤，诱导肿瘤细胞凋亡；III-96体内急性毒性较低，在192 mg/kg的剂量下，未发现明显毒性；其体内抑瘤效果明显，并具有显著的浓度依赖性；在10 mg/kg剂量下，WTI为59.6%。以上研究阐明了Top1抑制剂，尤其是催化型抑制剂的酶抑制活性、体外细胞毒性和体内抗肿瘤活性的相关性，探讨了其作为抗肿瘤药物先导的可行性，为发现新作用机制的创新药物奠定了实验基础，具有重要的科学意义。