br Results br Discussion Current attempts
Discussion Current attempts to obtain a definitive diagnosis of AD through specific amyloid neuroimaging fall short since β-amyloid is frequently found in brains of cognitively normal older adults.12, 13 In contrast, using available neuroimaging techniques to detect the characteristic association of cholinesterases with AD pathology may provide a more definitive diagnosis during life. The development of ligands that bind tightly to cholinesterases and that are also amenable to efficient radiolabelling, with isotopes like 18F for PET imaging, may offer new opportunities to reveal AD pathology in the living ON123300 receptor and enable more timely diagnosis and monitoring of treatment for the disease. Trifluoroacetophenones are good candidates for such ligands. Certain acetophenones have been shown to inhibit AChE through covalent interaction between the carbonyl functionality of the ketone inhibitor and the active site serine of the enzyme (Scheme 6). This interaction is facilitated by enhancing the electrophilicity of the ketone carbonyl carbon with adjacent electron-withdrawing groups such as a trifluoromethyl group (e.g., Compounds 1 and 2; Fig. 1). Replacement of hydrogen atoms with small electronegative fluorine atoms produces a bioisostere with comparable structure but enhanced ability to form a hemiketal intermediate that blocks substrate access to the enzyme. Trifluoromethyl ketones are known to be potent inhibitors of AChE.41, 42, 43 The nucleophile reacting with the ketone is the activated alkoxide of the catalytic serine residue of the enzyme (Ser203 in human AChE), that results in the formation of the hemiketal-enzyme complex (Scheme 6).44, 45 The present study sought to determine if chlorodifluoroacetophenones, such as 3 and 4, were amenable to a radiolabelling methodology to generate potential 18F imaging agents with high cholinesterase affinity. A number of procedures have been reported for the synthesis of trifluoromethyl ketones, including oxidation of α-trifluoromethyl alcohols, electrophilic addition of trifluoroacetic anhydride, alkylation and decarboxylation of fluorinated β-ketoesters or, as described here, through the use of organometallic intermediates. The latter approach (Scheme 1) was chosen as it offered a rapid direct method to obtain the desired m-substituted chlorodifluoroacetophenones to be precursors for fluoride exchange reactions. Since the chlorodifluoro derivatives were not commercially available, these were synthesized (Scheme 2) using an organolithium/Weinreb amide strategy. This approach also enabled the direct synthesis of trifluoroacetophenones for proof of identity with the halogen exchange reaction products and for the assessment of cholinesterase affinities (Table 1, Table 2). Due to the relatively short half-life of 18F (t1/2∼110min), the fluorination reaction to produce the radiolabelled trifluoroacetophenone ligand and all subsequent purification steps must be performed rapidly to maximize the specific activity of the radiolabelled product. It is also important to rapidly separate the desired product from remaining precursor, especially given the structural similarities of the chlorodifluoro precursors and trifluoro products. Purification by C18 reverse phase HPLC allowed for rapid separation of product from the precursor. The halogen exchange reaction with 19F isotope, and purification, can be performed within one hour and with reasonable yields. Considering time to synthesize and yields, the preparation of trifluoroacetophenone compounds from the chlorodifluoroacetophenone precursors represents a viable method for incorporation of 18F radioisotope for PET imaging of cholinesterase activity associated with AD pathology. The ability to detect this association by PET imaging is dependent on the affinity of the radioligand for cholinesterase and the length of time it remains in the enzyme-inhibitor hemiketal complex.