N-Hydroxypyrazolyl glycine derivatives as selective N-methyl-D-aspartic acid receptor ligands

Abstract

A series of analogues based on N-hydroxypyrazole as a bioisostere for the distal carboxylate group of aspartate have been designed, synthesized, and pharmacologically characterized. Affinity studies on the major glutamate receptor subgroups show that these 4-substituted N-hydroxypyrazol-5-yl glycine (NHP5G) derivatives are selectively recognized by N-methyl- d-aspartic acid (NMDA) receptors and that the ( R)-enantiomers are preferred. Moreover, several of the compounds are able to discriminate between individual subtypes among the NMDA receptors, providing new pharmacological tools. For example, 4-propyl NHP5G is an antagonist at the NR1/NR2A subtype but an agonist at the NR1/NR2D subtype. Molecular docking studies indicate that the substituent protrudes into a region that may be further exploited to improve subtype selectivity, thereby opening up a design strategy for ligands which can differentiate individual NMDA receptor subtypes.

Figure 1

Structures of the excitatory amino acids glutamic acid and aspartic acid and bioisosteric analogues of these compounds with activity at the glutamate receptors.

Figure 2

Two chiral HPLC chromatograms of (S)- and (R)-6d, respectively, showing the elution order on an analytical Crownpak CR(−) column. The order is reversed on a preparative Crownpak CR(+) column.

Figure 3

Electronic Circular Dichroism spectra (A) of compounds (R)- and (S)-6f at a concentration of 0.325 mg/mL in H₂O compared with a TD-DFT simulated spectra (B) of a weighted average of eight low energy conformers as well as a single low energy conformer of (S)-6f in dilute acid.

Figure 4

Mean concentration–response curves for racemic compounds Et- (6c), Pr- (6d) and Cl-NHP5G (6f) determined using two-electrode voltage-clamp recordings on Xenopus oocytes expressing NR1 in combination with NR2A-D. The curves are normalized to the maximal current response (I_max) in the same recording. Data points are represented as mean ± SEM. All EC₅₀-values are listed in Table 2.

Figure 5

A) Comparison of maximal currents induced by Et- (6c), Pr- (6d) and Cl-NHP5G (6f) relative to Glu at NR1/NR2A-D subtypes. All bars are represented as mean ±SEM. All relative I_max-values are listed in Table 2. B) Representative current responses obtained from a two-electrode voltage-clamp recording on a Xenopus oocyte expressing NR1/NR2A receptors. Currents were evoked by Glu alone or co-application of Glu and Pr-NHP5G at the concentrations indicated above each response.

Figure 6

A) Mean concentration–response curves for compounds (R)-6a-d,f (R=H, Me, Et, n-Pr, Cl, respectively) determined using two-electrode voltage-clamp recordings on Xenopus oocytes expressing NR1/NR2B receptors. The curves are normalized to the maximal current response (I_max) to Glu in the same recording. Data points are represented as mean ± SEM. All EC₅₀-values are listed in Table 3. B) Bar graph showing the maximal currents (I_max) of the ligands relative to the maximal current induced bu Glu. C) Representative current responses obtained from a two-electrode voltage-clamp recording on a Xenopus oocyte expressing NR1/NR2B receptors. Currents were evoked by Glu alone or co-application of Glu and (R)-6e at the concentrations indicated above each response.

Figure 7

A) Structure of a homology model of the ABD of NR2B (cyan) containing n-Pr-NHP5G ((R)-6d, blue) showing the residues (red) lining a crevice to the agonist binding site. Previous studies have shown these residues to be important determinants of agonist efficacies of several compounds. B) Et-NHP5G ((R)-6c, pink) and n-Pr-NHP5G ((R)-6d, blue) docked in the homology model. The distances from the amino group of Lys485 to the n-propyl group and the peptide carbonyl between Arg712 and Gly713 are indicated in Ångstroms. Water molecules are shown as red asterisks.

Scheme 1

Reagents and conditions: a) I-Cl; b) 1. i-PrMgCl, −78°C, THF; 2. RC=OR′; c) TFA, Et₃SiH; d) SO₂Cl₂ (for 10e); e) 1. n-BuLi, −78°C; 2. Diethyl N-Boc iminomalonate; f) H₂, Pd/C, MeOH; g) 1. LiOH; 2. HCl