Design, Synthesis, and Biology of Inhibitors of Neuronal Nitric Oxide Synthase

描述（由申请人提供）：尽管以神经元损伤和变性为特征的疾病具有巨大的公共卫生和经济影响，但这些疾病的治疗通常仅限于姑息治疗和减缓症状进展。因此，非常需要停止或减缓神经变性的药物。一个新兴的目标是神经元型一氧化氮合酶（nNOS），一种产生信号分子NO的酶。尽管正常神经元功能需要NO，但高水平的NO与慢性神经退行性疾病（如帕金森病）以及中风、偏头痛和其他疾病有关。因此，抑制这种酶可能是治疗这些疾病所需的。尽管如此，由于大多数nNOS抑制剂模拟天然底物L-精氨酸，它们的治疗实用性因其过度的极性和碱性而降低，达特性质导致GI吸收差和血脑屏障通透性低。此外，必须注意不要抑制相关的NOS亚型eNOS和iNOS，否则可能导致危险的副作用。本文详述的工作描述了用于设计和优化生物可利用的nNOS抑制剂的几种策略。首先，初步数据表明，N-苄基苯乙胺核心是一个支架，赋予有效的nNOS活性和~100倍的异构体选择性。在目标1中，通过简单的合成途径将低pKa杂环接枝到该支架上应导致碱性较低的精氨酸模拟物，并且分子建模提供了这些化合物应以类似于报道的nNOS抑制剂的方式结合的证据。然后将对含有有效替代杂环的化合物进行额外的优化。在目标2a中，将进行卤素和含卤素基团的掺入，这是一种已被证明可有效增强许多类别CNS药物的脑渗透的策略。在目标2b中，氧杂环丁烷基团将被引入苯乙基链中以降低仲胺的高pKa，预测这种修饰可以保持这些胺的氢键能力而没有空间位阻。最后，在目标3中，nNOS及其同种型将在E. coli中表达并纯化。将通过血红蛋白捕获试验针对酶测定化合物。此外，将在基于细胞的nNOS试验中检测选定化合物，测定其代谢稳定性，并在Caco-2模型中检测渗透性（以估计其GI和CNS摄取）。

DESCRIPTION (provided by applicant): Although diseases characterized by neuronal damage and degeneration have an enormous public health and economic impact, treatment of these disorders is often limited to palliative care and slowing of symptom progression. Therefore, drugs that stop or slow neurodegeneration are highly desirable. One emerging target is neuronal nitric oxide synthase (nNOS), an enzyme that produces the signaling molecule NO. Although required for normal neuronal function, high levels of NO have been implicated in chronic neurodegenerative pathologies (such as Parkinson's disease) as well as stroke, migraines, and other disorders. Ergo, inhibition of this enzyme could be desirable for treatment of these diseases. Nonetheless, as most nNOS inhibitors mimic the natural substrate L-arginine, their therapeutic practicality is diminished by their excessive polarity and basicity, properties tat cause poor GI uptake and low blood-brain barrier permeability. Additionally, care must be taken to not inhibit the related NOS isoforms eNOS and iNOS, or dangerous side effects could result. The work detailed herein describes several strategies for the design and optimization of bioavailable nNOS inhibitors. First, preliminary data indicates that the N-benzylphenethylamine core is a scaffold that confers potent nNOS activity and ~100-fold isoform selectivity. In Aim 1, grafting a low-pKa heterocycle onto this scaffold via facile synthetic routes should result in a less basic arginine mimetic, and molecular modeling provides evidence that these compounds should bind in a manner similar to reported nNOS inhibitors. Additional optimizations will then be performed on compounds containing effective alternative heterocycles. In Aim 2a, incorporation of halogens and halogen- containing groups, a strategy that has proven effective at enhancing brain penetration for many classes of CNS drugs, will be performed. In Aim 2b, oxetane groups will be introduced into the phenethyl chain to decrease the high pKa of the secondary amines, a modification that is predicted to preserve the hydrogen-bonding capability of these amines without steric encumbrance. Finally, in Aim 3, nNOS and its isoforms will be expressed in E. coli and purified. Compounds will be assayed against the enzymes by the hemoglobin capture assay. In addition, select compounds will be tested in a cell-based nNOS assay, assayed for their metabolic stability, and tested for permeability in a Caco-2 model (to estimate both their GI and CNS uptake).