Discovery of Lacosamide Affinity Bait Agents That Exhibit Potent Voltage-Gated Sodium Channel Blocking Properties

Abstract

Lacosamide ((R)-1) is a recently marketed, first-in-class, antiepileptic drug. Patch-clamp electrophysiology studies are consistent with the notion that (R)-1 modulates voltage-gated Na(+) channel function by increasing and stabilizing the slow inactivation state without affecting fast inactivation. The molecular pathway(s) that regulate slow inactivation are poorly understood. Affinity baits are chemical reactive units, which when appended to a ligand (drug) can lead to irreversible, covalent modification of the receptor thus permitting drug binding site identification including, possibly, the site of ligand function. We describe, herein, the synthesis of four (R)-1 affinity baits, (R)-N-(4″-isothiocyanatobiphenyl-4'-yl)methyl 2-acetamido-3-methoxypropionamide ((R)-8), (S)-N-(4″-isothiocyanatobiphenyl-4'-yl)methyl 2-acetamido-3-methoxypropionamide ((S)-8), (R)-N-(3″-isothiocyanatobiphenyl-4'-yl)methyl 2-acetamido-3-methoxypropionamide ((R)-9), and (R)-N-(3″-acrylamidobiphenyl-4'-yl)methyl 2-acetamido-3-methoxypropionamide ((R)-10). The affinity bait compounds were designed to interact with the receptor(s) responsible for (R)-1-mediated slow inactivation. We show that (R)-8 and (R)-9 are potent inhibitors of Na(+) channel function and function by a pathway similar to that observed for (R)-1. We further demonstrate that (R)-8 function is stereospecific. The calculated IC50 values determined for Na(+) channel slow inactivation for (R)-1, (R)-8, and (R)-9 were 85.1, 0.1, and 0.2 μM, respectively. Incubating (R)-9 with the neuronal-like CAD cells led to appreciable levels of Na(+) channel slow inactivation after cellular wash, and the level of slow inactivation only modestly decreased with further incubation and washing. Collectively, these findings have identified a promising structural template to investigate the voltage-gated Na(+) channel slow inactivation process.

Dissecting the role of the CRMP2–neurofibromin complex on pain behaviors

“…CNRP1 inhibited total calcium influx with an IC 50 of 5.3 µM; at least 40% of this was due to inhibition of Cav2.2. This is within the same range as other strategies we used previously to target the CRMP2/Cav2.2 interplay: ( S )-lacosamide, 55,63,71 a molecule inhibiting CRMP2 phosphorylation, 54–56 had an IC 50 of 1.25 µM 54 ; tat-CBD3, a peptide directly inhibiting CRMP2/CaV2.2 interaction, had an IC 50 of 12.1 µM 5 ; and a membrane-tethered version of the same peptide presented an IC 50 of 2.8 µM. 23 These IC 50 values suggest that CNRP1 functions through the same signaling pathway as our previous CRMP2-targeting strategies.…”

Substituted N-(Biphenyl-4′-yl)methyl (R)-2-Acetamido-3-methoxypropionamides: Potent Anticonvulsants That Affect Frequency (Use) Dependence and Slow Inactivation of Sodium Channels

“…Compounds ( R )- 4 –( R )- 16 were prepared by similar routes (Schemes –)­ using the mixed anhydride coupling (MAC) method and employing ( R )- N - tert -butoxycarbonylserine (( R )- 45 ), ( R )-2-acetamido-3-hydroxypropionic acid (( R )- 69 ), or ( R )-2-acetamido-3-methoxypropionic acid ,, (( R )- 70 ) as the carboxylic acid component. The choice of the carboxylic acid was dictated by the ease of purification of the intermediate products and the need to avoid competing side reactions in the latter stages of the synthesis.…”

“…Deprotection of the tert -butoxycarbonyl group in ( R )- 56 –( R )- 65 with acid (HCl/dioxane) followed by acetylation (AcCl, Et 3 N) gave the desired products ( R )- 4 –( R )- 13 , respectively. For ( R )- 14 and ( R )- 15 , we coupled ( R )- 69 with (biphenyl-4-yl)­methylamines 37 and 38 (Scheme ), and for ( R )- 16 , we combined ( R )- 70 ,, with (3″- N -( tert -butoxycarbonyl)­aminobiphenyl-4′-yl)­methylamine ( 39 ) (Scheme ). To facilitate the preparation of ( R )- 14 –( R )- 16 , we first developed convenient synthetic procedures for ( R )- 69 and ( R )- 70 (Scheme ).…”