Chemical and biophysical characterization of novel potassium channel blocker 3-fluoro-5-methylpyridin-4-amine

4-aminopyridine (4AP) is a potassium (K+) channel blocker used clinically to improve walking in people with multiple sclerosis (MS). 4AP binds to exposed K+ channels in demyelinated axons, reducing the leakage of intracellular K+ and enhancing impulse conduction. Multiple derivatives of 4AP capable of blocking K+ channels have been reported including three radiolabeled with positron emitting isotopes for imaging demyelinated lesions using positron emission tomography (PET). Here, we describe 3-fluoro-5-methylpyridin-4-amine (5Me3F4AP), a novel K+ channel blocker with potential application in PET. 5Me3F4AP has comparable potency to 4AP and the PET tracer 3-fluoro-4-aminopyridine (3F4AP). Compared to 3F4AP, 5Me3F4AP is more lipophilic (logD = 0.664 ± 0.005 vs. 0.414 ± 0.002) and slightly more basic (p K a = 7.46 ± 0.01 vs . 7.37 ± 0.07). In addition, 5Me3F4AP appears to be more permeable to an artificial brain membrane and more stable towards oxidation by the cytochrome P450 enzyme family 2 subfamily E member 1 (CYP2E1), responsible for the metabolism of 4AP and 3F4AP. Taken together, 5Me3F4AP has promising properties for PET imaging warranting additional investigation. Significance Statement The PET tracer [18F]3-fluoro-4-aminopyridine ([18F]3F4AP) binds to K+ channels in demyelinated axons and has shown promise for imaging demyelinated lesions in animal models. However, its use in humans may be compromised due to rapid metabolism. Thus, a novel 3F4AP derivative amenable to labeling with fluorine-18 was designed and evaluated in vitro . The results indicate that 5-methyl-3F4AP exhibits high binding affinity, good physicochemical properties and slower oxidation by CYP2E1 than 3F4AP, making it a promising candidate for further PET studies. ### Competing Interest Statement PB has a financial interest in Fuzionaire Diagnostics and the University of Chicago. PB is a named inventor on patents related to 18F-3F4AP owned by the University of Chicago and licensed to Fuzionaire Diagnostics. Dr. Brugarolas interests were reviewed and are managed by MGH and Mass General Brigham in accordance with their conflict-of-interest policies. The other authors declare no conflict of interest. * 4AP : 4-aminopyridine; AUC : area under curve; BBB : blood-brain barrier; CI95 : 95% of confidence interval COVC : Cut-Open Voltage Clamp; CYP2E1 : cytochrome P450 family 2 subfamily E member 1; EGTA : ethylene glycol-bis( ß -aminoethyl ether)- N,N,N’,N’ -tetraacetic acid; 3F4AP : 3-fluoro-4-aminopyridine; HEPES : N -2-hydroxyethyl-piperazine- N’ -2-ethanesulfonic acid; HPLC : high-performance liquid chromatography; HRMS : high resolution mass spectrometry; IC50 : half maximal inhibitory concentration; 5Me3F4AP : 3-fluoro-5-methylpyridin-4-amine; MES : 2( N -morpholino)ethanesulfonic acid; MS : multiple sclerosis; NMDG : N -methyl-D-glucamine; 3OH4AP : 3-hydroxy-4-aminopyridine; PBS : phosphate buffered saline; PET : positron emission tomography; PPF : percent of parent fraction; RFUs : relative fluorescence units; s.d. : standard deviation.