Inhibition of N-Type Calcium Channels by Fluorophenoxyanilide Derivatives

Abstract

A set of fluorophenoxyanilides, designed to be simplified analogues of previously reported ω-conotoxin GVIA mimetics, were prepared and tested for N-type calcium channel inhibition in a SH-SY5Y neuroblastoma FLIPR assay. N-type or Cav2.2 channel is a validated target for the treatment of refractory chronic pain. Despite being significantly less complex than the originally designed mimetics, up to a seven-fold improvement in activity was observed.

Inhibition of human N‐ and T‐type calcium channels by an ortho‐phenoxyanilide derivative, MONIRO‐1

“…In this study, we examined the effects of an ortho ‐phenoxyanilide on the inhibition and biophysical properties of VGCCs. This ortho ‐phenoxyanilide, MONIRO‐1, is a low MW compound and was selected for its ability to inhibit VGCCs in a high‐throughput calcium influx assay (Gleeson et al, ). To ascertain its selectivity and mode of action, we examined its potency of block and biophysical properties on a variety of VGCCs.…”

“…Given that MONIRO‐1 inhibits depolarization‐activated calcium responses from SH‐SY5Y cells (Gleeson et al, ), we determined its efficacy and selectivity against several VGCCs expressed in HEK293 cells. After screening representative members of each VGCC family (L‐, N‐, P/Q‐, R‐ and T‐types), we established that MONIRO‐1 preferentially inhibits T‐type channels (Ca v 3.1, Ca v 3.2 and Ca v 3.3 channels), modestly interacts with N‐type Ca v 2.2 channels (with 5–20 times less affinity compared to T‐type calcium channels) and has poor efficacy for L‐type (Ca v 1.2 and Ca v 1.3 channels IC 50 > 100 μM).…”

“…Compound 2, N‐(2‐(4‐fluorophenoxy)phenyl)‐4‐(3‐guanidinopropoxy)benzamide hydrochloride, MONIRO‐1, was synthesized from 2‐(4‐fluorophenoxy)aniline in four steps, as reported previously (Gleeson et al ., ). 1 H NMR (400 MHz, CD 3 OD): δ 7.88–7.85 (m, 1H), 7.78–7.76 (m, 2H), 7.21–7.18 (m, 2H), 7.07–6.98 (m, 6H), 6.97–6.94 (m, 1H), 4.13 (t, J = 5.9 Hz, 2H), 3.41 (t, J = 6.8 Hz, 2H), 2.11–2.05 (m, 2H).…”

“…To develop these calcium channel inhibitors, we screened a small library of related derivatives, using FLIPR Tetra® high‐throughput cellular screening system, for their ability to inhibit Ca v 2.2‐mediated calcium responses in SH‐SY5Y cells using the fluorescent indicator Calcium 4 dye (Molecular Devices, Sunnyvale, CA, USA) (Gleeson et al, ). In this assay, MONIRO‐1 (compound 2 , Figure ) inhibited Ca 2+ responses elicited by KCl‐mediated depolarization with an IC 50 of 124 μM.…”

Bioactive Mimetics of Conotoxins and other Venom Peptides

“…For Ca v 2.2 inhibition studies, SH-SY5Y neuroblastoma cells that endogenously express human Ca v 2.2 channels in a physiologically relevant context, are briefly incubated with varying concentrations of the compounds of interest, in the presence of saturating concentrations of the L-type calcium channel blocker nifedipine. The Ca 2+ responses produced by KCl-depolarization are measured and dose-response curves enable calculation of IC 50 values [ 51 , 53 , 54 ]. The major drawbacks of the radioligand displacement assay—The requirement for radioisotopes and the fact that it is not a functional assay—are negated with the FLIPR assay.…”