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

描述（由申请人提供）：将氟掺入小分子药物候选物中是改善药物性质的有效方法。目前，后期阶段将氟原子引入到活性候选药物支架上对合成化学家来说是一个重大挑战。该提案描述了一种新的策略，以解决通过选择性酶促芳香族羟基化获得酚的重大挑战。该酶促步骤之后将使用最近开发的化学方法并在其基础上进行改进，将所得酚化学转化为芳基氟化物。该策略将允许化学-酶促芳香族C-H-吡喃用于药物先导物的后期修饰。该策略的成功实施将填补生物催化芳香族羟基化方面的现有空白，并促进快速获得有价值的复杂含氟药物和候选药物。该项目的具体目标是：1）设计细胞色素P450酶以进行芳香族羟基化，2）扩大酶促芳香族羟基化并完成芳基氟的化学引入，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。利用定向进化，细胞色素P450 BM 3将被开发为不仅高产率地进行芳香羟基化，而且具有前所未有的区域选择性。由于邻位、Meta和帕拉羟基化都是可能的，因此将工程化具有每种类型的区域选择性的酶。为了帮助这一工程努力，一种新的高通量筛选（HTS）协议将开发酶活性，与一个独特的组合，揭示反应的区域选择性的测定。从定向进化努力中产生的酶将用于在药物氟比洛芬上进行芳香族羟基化，以克为单位。从这些羟基化反应中分离出的复杂酚类将转化为芳基三氟甲磺酸酯，并最终转化为广受欢迎的芳基氟化物，这是最近在简单芳族分子上证明的化学方法。将对芳基氟化物进行分析，以证明药物性质的改善。为氟比洛芬芳基羟基化而进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，酚类产物将转化为芳基氟化物并测试其增强的性质。 公共卫生相关性：氟原子存在于许多药物中，因为它可以使药物更有效，副作用更少。我们提出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.