In vitro activity: SA4503 protects motor neuron NSC34 cells against superoxide dismutase 1 and serum free neurotoxicity. It upregulates the phosphorylation levels of Akt and extracellular signal-regulated kinase (ERK) 1/2.The sigma receptor might be involved in several diseases in the central nervous system. SA4503, a potent σ1receptor agonist, has 103-fold higher affinity for σ1 (IC50=17.4 nM) than σ2 (IC50=1,784 nM) sites in guinea pig brain membranes. SA4503 is 14-fold selective for σ1 (Ki=4.6 nM) over σ2 (Ki=63.1 nM) sites in guinea pig brain homogenates.

Kinase Assay: SA4503, a potent sigma(1) receptor agonist, is reported as having 103-fold higher affinity for sigma(1) (IC(50) = 17.4 nM) than sigma(2) (IC(50) = 1,784 nM) sites in guinea pig brain membranes. Modest structural changes appear to have major effects on sigma(1)/sigma(2) selectivity. The fluoroethyl analog, FE-SA4503, is described as having high primary affinity for sigma(2) sites (IC(50) = 2.11 nM) and a weaker interaction with sigma(1) sites (IC(50) = 6.48 nM). SA4503 and FE-SA4503 have been radiolabeled for PET studies, and both bind selectively to sigma(1) receptors in animal and human brain in vivo. We prepared SA4503 and FE-SA4503 as reference compounds for radioligand development efforts. In our hands, SA4503 is 14-fold selective for sigma(1) (K(i) = 4.6 nM) over sigma(2) (K(i) = 63.1 nM) sites in guinea pig brain homogenates. Further, FE-SA4503 exhibits the same 14-fold selectivity for sigma(1) (K(i) = 8.0 nM) over sigma(2) (K(i) = 113.2 nM) receptors. The main differences from previously reported values stem from sigma(2) affinity determinations. This protocol, displacement of [(3)H]DTG binding to sigma(2) sites using (+)-pentazocine (200 nM) to mask sigma(1) sites, was validated by the proper rank order of sigma(2) inhibitory potencies shown by a panel of additional ligands: ifenprodil> haloperidol> DTG>> (+)-pentazocine. Robust Pearson correlation (r = 1.0, P = 0.002; slope = 0.97) was observed for our pK(i) values against those from a prior study by others. The findings have bearing on structure-activity relationships for this active series, and on conclusions that might be drawn from experiments relying upon defined sigma(1)/sigma(2) binding selectivity.

Cell Assay:The NSC34 cells are seeded at a density of 7000 cells per well into 96-well plates with D-MEM and transfected using Lipofectamine 2000 mixed with 2 μg /mL of plasmid vector in D-MEM for 6 h. After 6 h, the cell-culture medium is replaced with fresh D-MEM and culture and allowed to proceed for a further 42 h. The cells are then transferred to serum-free D-MEM and immediately treated with SA4503 at a final concentration of 1, 3, or 10 nM.