非可逆靶向抑制剂在肿瘤的治疗中发挥着重要的作用，此类药物多数含有迈克尔受体，与酶中的亲核成分发生共价结合。然而，迈克尔受体的存在也使分子表现出较强的毒性。因此，开发既能与酶非可逆结合，毒性又小的基团，对非可逆抑制剂的发展具有重要的意义。申请人在前期研究中发现，含有2-硝基咪唑的分子在肿瘤低氧环境下能够被还原活化，并与亲核成分发生共价结合，而在正常组织中，由于2-硝基咪唑未被活化，不发生共价结合而无毒，因此，它是开发非可逆抑制剂的理想基团。本项目拟将2-硝基咪唑引入非可逆抑制剂结构中，替换其迈克尔受体，设计一系列分子，并在虚拟筛选的基础上对其合成；分别在低氧和常氧下，评价目标分子的细胞毒及酶活性，分析构效关系；深入验证目标分子与靶蛋白的非可逆结合状况，评价其作用机制；最后，评价目标分子的体内抗肿瘤活性，系统验证以2-硝基咪唑为共价结合基团发展非可逆抑制剂的可行性。

Irreversible inhibitors play an important role in the treatment of cancers, and most of these drugs contain Michael acceptors so as to covalently bind to the nucleophiles of enzymes. However, the presence of Michael receptors makes molecules exhibit strong toxicity. Therefore, it is of great significance for the development of irreversible inhibitors to explore groups which can not only covalently bind to the receptors, but also have no toxicity to normal tissues. In previous studies, we found that 2-nitroimidazole-contained molecules could be reductive activation and covalently bind to nucleophiles in tumor hypoxia environment. In addition, 2-nitroimidazole was inactive and had no toxicity in normal tissues, so it was an ideal group for developing irreversible inhibitors. In this project, we intend to design a series of compounds through introducing 2-nitroimidazole groups into the structures of irreversible inhibitors to replace their Michael acceptors, and synthesize them on the basis of computer-aided virtual screening. Then the structure-activity relationships of these molecules will be concluded after evaluation of the cytotoxicities and enzyme inhibitory activities both under normoxia and hypoxia. In addition, the irreversible bindings between molecules and enzymes are meant to be verified and the mechanism of actions of molecules is going to be assessed. Finally, the molecules’ in vivo antitumor activities will be tested. Based on these results, we will comprehensively evaluate the feasibility of developing 2-nitroimidazole-contained irreversible inhibitors.