以吡咯并喹唑啉为母核的新型结核分枝杆菌二氢叶酸还原酶选择性抑制剂的设计、合成与评价

Dihydrofolate reductase (mt-DHFR) of Mycobacterium tuberculosis (M.tb) is an important enzyme in the folate metabolize pathway. By blocking this target, it will lead M.tb to the thymineless death. Currently p-aminosalicylic acid (PAS) is the only one in the first and second line anti-tuberculosis drugs, and it functions as the prodrug of mt-DHFR inhibitor. However, as PAS causes serious gastrointestinal diseases, and the PAS resistant M.tb strains have been developed, it is urgent to develop novel mt-DHFR selective inhibitors. In our previous publications, we reported a chemical compound with the core of 1,3-diamino-7H-pyrrolo[3,2-f]quinazoline as the mt-DHFR inhibitor. The M,tb inhibition ability of this hit compound is in the same level of PAS, and it contains a novel chemical structure, which will significantly expand the chemical space of mt-DHFR inhibitors. More recently, we have designed and synthesized a series of compounds, which are analogs of the previous hit compound, and they show strong anti M.tb effects with a reasonable selectivity against human DHFR in vitro. In the current project, based on our previous work, the lead optimization will be performed to improve the activity and selectivity of the hit compounds both in vitro and in vivo, and the pharmaceutical properties will be optimized at the mean time to make them more drug like, by using computer aided drug design and chemical synthesis. Through the study, we are aiming to obtain 2-3 drug-like leading compounds, and the biological mechanism of these compounds will be studied by analyzing the ligand-protein interactions. We believe that the successful development of the current project will build a solid foundation for the discovery of novel anti-TB drugs.

二氢叶酸还原酶（mt-DHFR）是结核分枝杆菌（M.tb）叶酸代谢途径中重要节点，阻断该靶点，将导致M.tb无胸腺嘧啶死亡。目前临床主要抗结核药物仅有对氨基水杨酸（PAS）为mt-DHFR抑制剂前药，但易引起严重的胃肠道疾病，且其耐药结核菌株已出现，因此亟需研发新型mt-DHFR选择性抑制剂。我们前期报道了以1,3-二氨基-7H-吡咯并[3,2-f]喹唑啉为母核的mt-DHFR抑制剂，其体外活性与PAS相当，且具有全新结构骨架，此发现将极大拓展该靶点抑制剂的化学空间。近期，我们合成了一系列该类抑制剂，显著提高了其体外活性并兼具较好的选择性。本项目将在前期基础上，拟采用多种药物设计技术对该类分子进行结构优化，旨在提高其选择性、体内外活性和成药性，以期发现2-3个先导结构，此外通过深入研究先导结构与靶点的相互作用，解析其分子作用机理。本项目的顺利开展，将为发现新型抗结核药物奠定坚实的基础。

结核分枝杆菌和革兰氏阴性菌的治疗急需新型药物的研发。本课题组以前期发现一类吡咯喹唑啉类化合物为基础，通过计算机辅助药物设计得到了一系列结构衍生物，发现部分化合物对H37Ra的活性高于利福平、对结核分枝杆菌标准株活性与异烟肼和利福平相当、并对多药耐药菌株(MDR-TB)以及巨噬细胞感染模型的活性与利福平相当。进一步发现部分化合物具有广谱抗菌活性，特别对于革兰氏阴性菌（G-）（包括大肠埃希菌、肺炎克雷伯菌、鲍曼不动杆菌）和革兰氏阳性菌（G+菌）（包括金黄色葡萄球菌、粪肠球菌）具有很强的活性。联合抗菌实验结果发现此类化合物与磺胺甲噁唑存在显著协同作用。2个化合物在大肠埃希菌ATCC 25922感染的小鼠模型中表现出良好的保护作用。在化合物活性机制研究中，通过酶抑制实验、分子互作实验，细胞水平代谢产物回补实验、靶蛋白过表达实验，确定了二氢叶酸还原酶为该类化合物的主要作用靶标，并且发现胸腺嘧啶合酶是该类的次要作用靶标，从而确认了该类化合物具有叶酸通路双靶点抑制作用，解释了其与磺胺甲噁唑超强的联用效果来源于叶酸通路三靶点阻断的机理。后续优选化合物与二氢叶酸还原酶共结晶实验首次阐明了该类化合物与其主要靶标的结合模式，为后续研究奠定了基础。综上所述，本项目首次在抗菌药物研发领域，揭示并深入研究了吡咯喹唑啉类化合物的叶酸通路双靶点抗菌分子机制，为新型细菌叶酸拮抗剂的研发提供了优秀的先导化合物。