Chemo-enzymatic Approach to Late-stage Aromatic Fluorination of Drug Scaffolds

说明(申请人提供)：将氟掺入小分子候选药物是改善药物性能的有效方法。目前，将氟原子引入活性候选药物支架的后期阶段对合成化学家来说是一个巨大的挑战。这项建议描述了一种新的策略，以解决通过选择性酶促芳香族羟化来获取苯酚的重大挑战。在这一酶促反应步骤之后，将利用最近发展起来的化学方法将所得的酚类化合物化学转化为芳基氟化物，并加以改进。这一策略将允许化学-酶促芳香族C-H氟化用于药物先导化合物的后期修饰。这一战略的成功实施将填补生物催化芳香族羟化方面的现有空白，并有助于快速获得有价值的复杂氟化药物和候选药物。该项目的具体目标是1)改造细胞色素P450酶进行芳香族羟基化，2)放大酶促芳香族羟化反应并完成芳基氟化物的化学引入，3)将目标1和2中开发的策略扩展到药物华法林和双氯芬酸。通过定向进化，细胞色素P450 BM3将被开发用于芳香族羟基化反应，不仅产率高，而且具有前所未有的区域选择性。由于邻位、间位和对位羟基化都是可能的，因此将设计出具有每种区域选择性的酶。为了帮助这一工程工作，将开发一种新的高通量筛选(HTS)方法来检测酶的活性，并采用独特的分析组合来揭示反应的区域选择性。定向进化工作产生的酶将用于对药物氟比洛芬进行克级别的芳香族羟基化反应。从这些羟基化反应中分离出来的复杂酚将转化为三氟代芳烃，最后利用最近在简单芳香分子上展示的化学方法转化为广受欢迎的芳基氟化物。将对芳基氟化物进行测试，以证明改进的药物性能。用于氟比洛芬芳基羟化反应的酶将用于重要药物华法林和双氯芬酸的选择性羟化反应，酚类产品将转化为芳基氟化物，并进行增强性能测试。 与公共健康相关：在许多药物中发现了氟原子，因为它可以使它们更有效，副作用更少。我们建议的策略将允许制药研究人员通过生物和化学相结合的方法更快地制造具有氟原子的候选药物。这种方法有望缩短将新药带给患者所需的时间。

DESCRIPTION (provided by applicant): The incorporation of fluorine into small molecule pharmaceutical candidates is a powerful method to improve drug properties. Currently, the late-stage introduction of fluorine atoms onto active drug-candidate scaffolds is a significant challenge for synthetic chemists. This proposal describes a novel strategy to address the significant challenge of accessing phenols by selective enzymatic aromatic hydroxylation. This enzymatic step will be followed by chemical transformation of the resulting phenols to aryl fluorides using, and improving upon, recently developed chemistry. This strategy will allow chemo-enzymatic aromatic C-H fluorination to be used for the late-stage modification of drug leads. Successful implementation of this strategy will fulfill an existing gap in biocatalytic aromatic hydroxylation, as well as facilitate rapid access to valuable complex fluorinated drugs and drug candidates. The specific aims of this project are 1) to engineer cytochrome P450 enzymes to conduct aromatic hydroxylation, 2) to scale up enzymatic aromatic hydroxylation and complete the chemical introduction of the aryl fluoride, and 3) to expand the strategy developed in aims 1 and 2 to the drugs warfarin and diclofenac. Using directed evolution, cytochrome P450 BM3 will be developed to carry out aromatic hydroxylation not only in high yield, but also with unprecedented regioselectivity. As ortho, meta, and para hydroxylation are all possible, enzymes with each type of regioselectivity will be engineered. To aid in this engineering effort, a novel high-throughput screening (HTS) protocol will be developed for enzyme activity, with a unique combination of assays that reveal the regioselectivites of the reactions. The enzymes resulting from directed evolution efforts will be used to carry out aromatic hydroxylations on a drug, flurbiprofen, on a gram scale. The complex phenols isolated from these hydroxylations will be transformed to aryl triflates, and finally to the sought-after aryl fluorides using chemistry recently demonstrated on simple aromatic molecules. The aryl fluorides will be assayed to demonstrate improved drug properties. The enzymes evolved for flurbiprofen aryl hydroxylation will be used for the selective hydroxylation of the important drugs warfarin and diclofenac, and the phenolic products will be converted to aryl fluorides and tested for enhanced properties. PUBLIC HEALTH RELEVANCE: The fluorine atom is found in a number of drugs because it can make them much more effective with fewer side-effects. Our proposed strategy will allow pharmaceutical researchers to make drug candidates with fluorine atoms faster by a combined biological and chemical method. This method is expected to shorten the time required to bring new drugs to patients.