Discovery of a new class of ionotropic glutamate receptor antagonists by the rational design of (2S,3R)-3-(3-carboxyphenyl)-pyrrolidine-2-carboxylic acid

Abstract

The kainic acid (KA) receptors belong to the class of glutamate (Glu) receptors in the brain and constitute a promising target for the treatment of neurological and/or psychiatric diseases such as schizophrenia, major depression, and epilepsy. Five KA subtypes have been identified and named GluK1-5. In this article, we present the discovery of (2S,3R)-3-(3-carboxyphenyl)-pyrrolidine-2-carboxylic acid (1) based on a rational design process. Target compound 1 was synthesized by a stereoselective strategy in 10 steps from commercially available starting materials. Binding affinities of 1 at native ionotropic Glu receptors were determined to be in the micromolar range (AMPA, 51 μM; KA, 22 μM; NMDA 6 μM), with the highest affinity for cloned homomeric KA receptor subtypes GluK1,3 (3.0 and 8.1 μM, respectively). Functional characterization of 1 by two electrode voltage clamp (TEVC) electrophysiology at a nondesensitizing mutant of GluK1 showed full competitive antagonistic behavior with a K(b) of 11.4 μM.

Keywords: Glutamate receptors; antagonist; kainic acid receptors; rational design.

Figure 1

Superimposition of X-ray structures of the LBD of GluK1 cocrystallized with ATPO (black; 1VSO in type code) and with LY466195 (purple, 2QS4 in green) with an indication of the two binding domains D1 and D2. The two antagonists occupy distinct spatial positions with respect to their distal functional groups. Furthermore, the two residues important for the binding of the distal acid group, Ser141 and Thr142, are being forced out by ATPO by 2.85 Å and 1.55 Å, respectively.

Figure 2

Chemical structures of iGluR agonist kainic acid (KA) and KA with the incorporation of a chemical spacer (KA-CS) leading to the rational design of potential iGluR antagonist 1.

Figure 3

Superimposition of the low-energy conformation of 1 (type code) with the binding conformations of ATPO (black), UBP310 (green), and LY466195 (purple) obtained from X-ray crystal structures with PDB codes 1VSO, 2QS4, and 2F34, respectively.

Scheme 1. Synthetic Pathway toward 1

Reagents and conditions: (a) TBSCl, Et₃N, CH₂Cl₂; (b) BOC₂O, Et₃N, DMAP, CH₂Cl₂ (87% two steps); (c) LHMDS, THF, −78 °C, then PhSeCl; (d) H₂O₂, EtOAc, 0 °C to RT (65% two steps); (e) 5, t-BuLi, CuCN, TMSCl, THF, −50 °C, then 4 (90%); (f) BH₃·THF, reflux, then NaOH, H₂O₂, 0 °C to rt (71%); (g) TBAF, THF, rt (94%); (h) RuCl₃, NaIO₄, H₂O, MeCN, EtOAc (75%); (i) HCl/dioxane, rt, then recrystallization from AcOH (44%); (j) TBSCl, imidazole, DMF (96%).

Figure 4

Inhibition of Glu responses by 1 at the nondesensitizing mutants of GluK1 and GluK3, expressed in X. laevis oocytes and measured by TEVC electrophysiology. Shown are the pooled data normalized to the control response in the absence of antagonist from 5 to 6 experiments conducted in duplicate. GluK1, Glu = 100 μM, IC₅₀ = 18.6 μM; GluK3, Glu = 5 mM, IC₅₀ = 323 μM. Inset: traces from one oocyte expressing the GluK3 mutant. Stimulations in duplicate at increasing concentrations of 1 (in μM): 0, 5, 10, 20, 50, 100, 200, 300, 1000; (V_h = −90 mV). Scale bars: 50 nA and 1 min.