Abstract
PurposeA neuropathological hallmark of Alzheimer’s disease (AD) is the presence of amyloid-β (Aβ) plaques in the brain, which are observed in a significant number of cognitively normal, older adults as well. In AD, butyrylcholinesterase (BChE) becomes associated with Aβ aggregates, making it a promising target for imaging probes to support diagnosis of AD. In this study, we present the synthesis, radiochemistry, in vitro and preliminary ex and in vivo investigations of a selective, reversible BChE inhibitor as PET-tracer for evaluation as an AD diagnostic.ProceduresRadiolabeling of the inhibitor was achieved by fluorination of a respective tosylated precursor using K[18F]. IC50 values of the fluorinated compound were obtained in a colorimetric assay using recombinant, human (h) BChE. Dissociation constants were determined by measuring hBChE activity in the presence of different concentrations of inhibitor.ResultsRadiofluorination of the tosylate precursor gave the desired radiotracer in an average radiochemical yield of 20 ± 3 %. Identity and > 95.5 % radiochemical purity were confirmed by HPLC and TLC autoradiography. The inhibitory potency determined in Ellman’s assay gave an IC50 value of 118.3 ± 19.6 nM. Dissociation constants measured in kinetic experiments revealed lower affinity of the inhibitor for binding to the acylated enzyme (K2 = 68.0 nM) in comparison to the free enzyme (K1 = 32.9 nM).ConclusionsThe reversibly acting, selective radiotracer is synthetically easily accessible and retains promising activity and binding potential on hBChE. Radiosynthesis with 18F labeling of tosylates was feasible in a reasonable time frame and good radiochemical yield.
Highlights
Alzheimer’s disease (AD) is an incurable, progressive neurodegenerative disorder and the most frequent cause of dementia with high prevalence and a long asymptomatic phase [1, 2]
During purity control by liquid chromatography/mass spectrometry (LCMS), we found a slow side reaction due to intramolecular ring closure by nucleophilic attack of the piperidine nitrogen (Fig. 3)
In our additional synthetic approach towards central building block 3 (Fig. 2), we found that both amide groups of TBDMS-protected compound 9 can be reduced by lithium aluminum hydride and the crude product can directly be coupled to naphthalene-2-sulfonyl chloride to obtain deprotected alcohol 3
Summary
Alzheimer’s disease (AD) is an incurable, progressive neurodegenerative disorder and the most frequent cause of dementia with high prevalence and a long asymptomatic phase [1, 2]. This could be shown for the pseudoirreversible BChE inhibitor phenyl 4-123I-iodophenylcarbamate (123I-PIP), which accumulated in Aβ plaques with ChE activity in human AD brain tissue [29] This tracer has been investigated very recently as a potential diagnostic and treatment monitoring tool for BChE activity changes in multiple sclerosis showing promising preliminary results [30]. Investigations into the influence of the carbamate structure that is transferred to BChE were made by altering spacer lengths and attached heterocyclic moieties This resulted in sets of inhibitors with short, medium and long duration of action and pronounced neuroprotectivity in an AD mouse model, showing the therapeutic potential of such inhibitors [31]. We chose to incorporate 18F as radioisotope to take advantage of a long half-life and high positron yields in combination with a good spatial resolution due to relatively low positron energies in comparison to other radioisotopes commonly used for PET studies (e.g., 18F: 0.65 MeV, 68Ga: 1.90 MeV) [34, 35]
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