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

描述(申请人提供)：尽管以神经元损伤和变性为特征的疾病对公共健康和经济有巨大的影响，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，阻止或减缓神经变性的药物是非常可取的。一个新出现的靶点是神经元型一氧化氮合酶(NNOS)，这是一种产生信号分子NO的酶。虽然高水平的NO是正常神经功能所必需的，但高水平的NO与慢性神经退行性病变(如帕金森氏病)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的理想方法。然而，由于大多数神经型一氧化氮合酶抑制剂模仿天然底物L-精氨酸，其治疗实用性因其过高的极性和碱性而降低，这些特性导致胃肠道摄取不良和血脑屏障通透性低。此外，必须注意不要抑制相关的一氧化氮合酶亚型eNOS和iNOS，否则可能会产生危险的副作用。本文详细描述了设计和优化生物可用nNOS抑制剂的几种策略。首先，初步数据表明，N-苄基苯乙胺核是一种具有很强的nNOS活性和~100倍的异构体选择性的支架。在目标1中，通过简单的合成路线将一个低pKA杂环嫁接到这个支架上，应该会得到一个不那么基本的精氨酸模拟物，分子模拟提供了证据，证明这些化合物应该以类似于已报道的nNOS抑制剂的方式结合。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，将实施纳入卤素和含卤素基团的战略，这一战略已被证明在提高许多类中枢神经系统药物的脑渗透率方面是有效的。在目标2b中，氧杂环烷基将被引入到苯乙链中，以降低仲胺的高pKa，这一修饰预计将在没有空间障碍的情况下保持这些胺的氢键能力。最后，在目标3中，将在大肠杆菌中表达并纯化nNOS及其异构体。化合物将通过血红蛋白捕获分析来对抗酶进行检测。此外，选定的化合物将在基于细胞的nNOS测试中进行测试，分析它们的代谢稳定性，并在Caco-2模型中测试渗透性(以估计它们的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).