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

描述（由申请人提供）：在小分子候选药物中掺入氟是一种改善药物性能的有效方法。目前，在活性候选药物支架的后期引入氟原子对合成化学家来说是一个重大挑战。该建议描述了一种新的策略，以解决通过选择性酶促芳香羟基化获得酚的重大挑战。这一酶促步骤之后，将利用和改进最近发展的化学方法，将所得到的酚化学转化为芳基氟化物。这一策略将允许化学酶芳族C-H氟化用于药物先导物的后期修饰。这一战略的成功实施将填补生物催化芳羟基化方面的现有空白，并促进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)设计细胞色素P450酶以进行芳香羟基化，2)扩大酶促芳香羟基化并完成芳酰氟的化学引入，以及3)将目标1和2中制定的策略扩展到药物华法林和双氯芬酸。利用定向进化技术，细胞色素P450 BM3不仅可以高产地进行芳香羟基化反应，而且具有前所未有的区域选择性。由于邻位、间位和对羟基化都是可能的，因此将设计具有每种类型区域选择性的酶。为了帮助这项工程工作，将开发一种新的高通量筛选（HTS）方案，用于酶活性，并结合独特的检测方法来揭示反应的区域选择性。由定向进化努力产生的酶将用于对一种药物氟比洛芬进行芳香羟基化，以克为单位。从这些羟基化分离的复杂酚将转化为芳基三氟酸酯，最后利用最近在简单芳香分子上证明的化学反应转化为广受欢迎的芳基氟化物。将对芳基氟化物进行分析，以证明改进的药物性能。用于氟比洛芬芳基羟基化的酶将用于重要药物华法林和双氯芬酸的选择性羟基化，酚类产品将转化为芳基氟化物并测试其增强性能。

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.