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

1,4-苯并嗪作为抗结核药物的初步鉴定

• 批准号: 7146363
• 负责人: Scott G. Franzblau
• 金额: $ 23.25万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-20 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7146363
• 关键词: binding proteins; cell line; cytotoxicity; heterocyclic polycyclic compound; lead; library; macrophage; pharmacokinetics; protein binding; tuberculosis

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.

描述（由申请人提供）：需要新型抗结核药物来对抗现有药物的耐药性并缩短治疗持续时间。该修订提案探讨了 1,4 苯并恶嗪作为抗结核药物的潜力。筛选程序鉴定出六种 1,4 苯并恶嗪，其针对结核分枝杆菌的 MIC 为亚微克/毫升，且未检测到哺乳动物细胞的细胞毒性，最终选择性指数 > 100。还揭示了几种结构-活性关系。这些化合物在低微克/毫升水平下也对鼠巨噬细胞中的结核分枝杆菌具有活性。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后对小鼠的毒性较低。新的初步数据表明，缺乏谷胱甘肽加合物的形成、对抗耐药菌株的活性以及（通过表达谱）现有药物所不具备的作用机制。为了从此类中鉴定先导化合物，将使用涵盖以最小的合成工作量覆盖所有可能位置的取代的策略来合成大约一百五十个新的1,4苯并恶嗪的集中库。经典合成法和平行合成法都将用于通过多羰基化合物（2,4 二酮酸和四酮）与邻氨基苯酚的反应来生产 4-二氢-2H-1,4-苯并恶嗪。通过氯化，然后与 N、O- 和 S 亲核试剂反应，生成 3-乙烯基-2H-1,4 苯并恶嗪，从而生成其他类似物。除了低极性表面积和可旋转键数量之外，根据“利平斯基规则”，这些化合物预计具有类似药物和口服用途。将使用低氧孵育评估所有化合物针对对数期结核分枝杆菌以及针对非复制结核分枝杆菌的活性。所有化合物还将评估对哺乳动物细胞系的毒性。将评估有效（MIC <1 ug/ml）和选择性（选择性指数> 100）化合物针对耐药菌株的抗结核活性以及在鼠巨噬细胞中的活性。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。将评估具有最佳体外特征的化合物在小鼠中的药代动力学特性，包括口服生物利用度和肺组织浓度。将评估最佳候选者在 CACO-2 细胞与牛脑上皮细胞系中的比较传代情况，以及活性谱和耐药频率。将通过转录分析以及使用基于微阵列的筛选和测序鉴定抗性突变体中的基因突变来研究作用机制。这一探索性 R21 提案预计将确定此类药物产生临床上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。