Stereoselectivity for the (R)‐Enantiomer of HA‐966 (l‐Hydroxy‐3‐Aminopyrrolidone‐2) at the Glycine Site of the N‐Methyl‐d‐Aspartate Receptor Complex

Abstract: HA‐966 (1‐hydroxy‐3‐aminopyrrolidone‐2) is an antagonist at the glycine allosteric site of the N‐methyl‐d‐aspartate receptor ionophore complex. Unlike presently known glycine antagonists, HA‐966 is chiral. We report stereoselectivity for the (R)‐enantiomer at the glycine antagonist site. In [3H]glycine binding, the (R)‐enantiomer has an IC50 of 4.1 ± 0.6 μM. The racemic mixture has an IC50 of 11.2 ± 0.5 μM, whereas (S)‐HA‐966 has an IC50 greater than 900 μM. In glycine‐stimulated [3H]l‐[1‐(2‐thienyl)cyclohexyl] piperidine binding, the (R)‐enantiomer inhibits with an IC50 of 121 ± 61μM, whereas the racemic mixture has an IC50 of 216 ± 113 μM and (S)‐HA‐966 is inactive. The inhibition by (R)‐HA‐966 can be prevented by the addition of glycine. (R)‐HA‐966 and racemic HA‐966, but not (S) HA‐966, also prevent N‐methyl‐d‐aspartate cytotoxicity in cortical cultures. The (R)‐enantiomer and, less potently, the (S)‐enantiomer inhibit N‐methyl‐d‐aspartate‐evoked [3H]norepinephrine release from rat hippocampal slices (IC50 values of about 0.3 mM and 1.6 mM, respectively), but only the inhibition by (R)‐HA‐966 is reversed by added glycine. In glutamate‐evoked contractions of the guinea pig ileum, (R)‐HA‐966 causes a glycine‐reversible inhibition (IC50 of about 150 μM), whereas (S)‐HA‐966 is much less potent (IC50 of greater than 1mM). These results demonstrate stereoselectivity of the glycine antagonist site of the N‐methyl‐d‐aspartate receptor complex in a variety of tissues and assays. The stereoselectivity also confirms the specificity of N‐methyl‐d‐aspartate receptors in glutamate‐evoked contractions of the guinea pig ileum, and supports their similarity to central N‐methyl‐d‐aspartate receptors.