Lead identification of 1,4-benzoxazines as anti-tuberculosis agents

描述（由申请人提供）：需要新类别的抗结核药物来对抗现有药物的耐药性并缩短治疗时间。本修订提案探讨了1,4苯并恶嗪作为抗结核药物的潜力。筛选程序鉴定出6种1,4苯并恶嗪对结核分枝杆菌具有亚微克/毫升mic，未检测到哺乳动物细胞毒性，结果选择性指数为bbb100。还揭示了几种构效关系。这些化合物在低微克/毫升水平下对小鼠巨噬细胞中的结核分枝杆菌也有活性。极性表面积计算预测口服给药时吸收良好，初步结果表明口服给药对小鼠毒性低。新的初步数据表明，缺乏谷胱甘肽加合物的形成，对耐药菌株的活性以及（通过表达谱）现有药物所不具有的作用机制。为了从这类化合物中鉴定出一个先导化合物，我们将采用一种策略，以最小的合成努力在所有可能的位置上覆盖取代，合成一个大约150个新的1,4苯并恶嗪的集中文库。经典合成和平行合成将通过聚羰基化合物（2,4二酮酸和四酮）与氨基酚反应制备4-二氢- 2h -1,4-苯并恶嗪。另外的类似物将通过氯化产生，然后与N， O和S亲核试剂反应，产生3-乙烯基- 2h -1,4苯并恶嗪。根据“利平斯基规则”，除了低极性表面积和可旋转键的数量外，这些化合物被预测为类似药物并且可口服。所有化合物将在低氧孵育下评估其对对数相结核分枝杆菌的活性以及对非复制结核分枝杆菌的活性。所有化合物还将评估其对哺乳动物细胞系的毒性。药效（MIC<1 ug/ml）和选择性（选择性指数bbb100）化合物对耐药菌株的抗结核活性和对小鼠巨噬细胞的活性将进行评估。将评估活性化合物在人微粒体存在下的稳定性，P-450介导的细胞毒性和蛋白质结合。具有最佳体外特征的化合物将被评估其在小鼠体内的药代动力学特性，包括口服生物利用度和肺组织浓度。将评估最佳候选药物通过CACO-2细胞与牛脑上皮细胞系的比较传代，以及活性谱和耐药频率。作用机制将通过转录谱分析以及利用基于微阵列的筛选和测序鉴定耐药突变体的基因突变来研究。这一探索性R21提案有望确定这类药物产生临床有用的结核病药物的潜力，并为进一步优化和临床前开发提供先导化合物。

DESCRIPTION (provided by applicant): New classes of anti-tuberculosis agents are needed to combat resistance to existing agents and to shorten the duration of therapy. This revised proposal explores the potential of 1,4 benzoxazines as anti-tuberculosis agents. A screening program identified six 1,4 benzoxazines with sub-microgram/ml MICs against M. tuberculosis and no detectable mammalian cell cytotoxicity with resulting selectivity indices of >100. Several structure-activity relationships were also revealed. These compounds were also active at low microgram/ml levels against M. tuberculosis in murine macrophages. Polar surface area calculations predict good absorption when administered orally and preliminary results suggest low toxicity in mice upon oral administration. New preliminary data suggests a lack of formation of glutathione adducts, activity against drug-resistant strains and (via expression profiling) a mechanism of action not shared by existing agents. In order to identify a lead compound from this class a focused library of approximately one hundred and fifty new 1,4 benzoxazines will be synthesized using a strategy that covers substitution at all possible position with minimal synthetic effort. Both classical and parallel synthesis will be used to produce 4-dihydro-2H-1,4-benzoxazines via reaction of polycarbonyl compounds (2,4 diketo acids and tetraketones) with oaminophenols. Additional analogs will be produced by chlorination and then reaction with N, O- and S nucleophilic reagents resulting in the production of 3-vinyl-2H-1,4 benzoxazines. These compounds are predicted to be drug-like and orally available based on "Lipinski rules" in addition to low polar surface area and numbers of rotatable bonds. All compounds will be assessed for activity against both logarithmic phase M. tuberculosis as well as against non-replicating M. tuberculosis using low oxygen incubation. All compounds will also be assessed for toxicity to a mammalian cell line. Potent (MIC<1 ug/ml) and selective (selectivity indices>100) compounds will be assessed for anti-TB activity against drug-resistant strains, and activity in murine macrophages. Active compounds will be assessed for stability in the presence of human microsomes, P-450 mediated cytotoxicity and protein binding. Compounds with the best in vitro profiles will be evaluated for pharmacokinetic properties in mice including oral bioavailability and lung tissue concentrations. The best candidates will be assessed for comparative passage through CACO-2 cells vs. a bovine brain epithelial cell line, as well as for spectrum of activity and frequency of resistance. Mechanism of action will be investigated by transcriptional profiling as well as identification of gene mutations in resistant mutants using microarray-based screening and sequencing. This exploratory R21 proposal is expected to determine the potential of this class to yield a clinically useful agent for tuberculosis and to provide a lead compound(s) for further optimization and pre-clinical development.