The present study sought to determine if chlorodifluoroaceto
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 monoamine oxidase inhibitor 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. Previous studies have mostly focused on the interaction of trifluoromethyl ketones with AChE.29, 30, 45, 46 Here, BuChE is also considered since it represents a major enzyme component associated with AD pathology.15, 16, 17, 18 Two trifluoroacetophenones, compounds 1 and 2, were examined as representative potential radioligands. For comparison, the immediate chlorodifluoro precursors (3 and 4, respectively) were also evaluated kinetically for any significant effects on enzyme inhibition due to exchanging halogen species. The ability of fluorinated acetophenones to interact with the catalytic serine of cholinesterases and form a hemiketal complex is indicated by measurable time-dependent inhibition of the enzyme and determined primarily by the magnitude of the first order dissociation rate constant kd for the enzyme-inhibitor complex. Lower kd values are correlated with higher affinity for enzyme as measured by the experimental Ki (Table 1) and slower attainment of steady state equilibrium (Scheme 4). tert-Butyl trifluoroacetophenone (2) showed the highest affinity for AChE, with a Ki of 0.4nM (Table 1). Earlier reported Ki values for 2 with eel and torpedo AChE (1.9 and 3.6pM, respectively), and mouse AChE (3.7–3.8pM).30, 47 appear to indicate much higher affinities than that reported here for 2 with human AChE (Table 1). However, typical of most ketones and aldehydes, acetophenones are subject to a hydration equilibrium in aqueous solution (Scheme 6), and these previously reported values were adjusted to take into account the hydration equilibrium constant Khyd to reflect only the presence of the active ketone form of the molecule. Khyd has been reported as ∼500 for 2. Before adjustment for Khyd, the earlier Ki values for 2 become 0.9 and 1.8nM with eel and torpedo AChE, respectively, and 1.9nM for mouse AChE. These values are only slightly higher than the 0.4nM value in Table 1. Compound 1 also has been reported to show time-dependent inhibition of eel and torpedo AChE with respective Ki values of 4nM and 28nM and of mouse AChE with a Ki of 15nM, all prior to correction for hydration, in reasonable agreement with the Ki value of 7nM for human AChE in Table 1. The Ki values reported in Table 1, Table 2 were not adjusted for hydration to better reflect the actual activity of these molecules in living systems where both the hydrated and nonhydrated forms will be present during imaging studies.