β-Indolyloxy Functionalized Aspartate Analogs as Inhibitors of the Excitatory Amino Acid Transporters (EAATs)

Abstract

The excitatory amino acid transporters (EAATs) mediate uptake of the major excitatory neurotransmitter l-glutamate (Glu). The essential functions governed by these transporters in regulating the central Glu level make them interesting therapeutic targets in a wide range of neurodegenerative and psychiatric disorders. l-Aspartate (Asp), another EAAT substrate, has served as a privileged scaffold for the development of EAAT inhibitors. In this study, we designed and synthesized the first β-indolyloxy Asp analogs 15a-d with the aim to probe a hitherto unexplored adjacent pocket to the substrate binding site. The pharmacological properties of 15a-d were characterized at hEAAT1-3 and rEAAT4 in a conventional [³H]-d-Asp uptake assay. Notably, thiophene analog 15b and the para-trifluoromethyl phenyl analog 15d were found to be hEAAT1,2-preferring inhibitors exhibiting IC₅₀ values in the high nanomolar range (0.21-0.71 μM) at these two transporters versus IC₅₀ values in the low micromolar range at EAAT3,4 (1.6-8.9 μM). In summary, the results presented herein open up for further structure-activity relationship studies of this new scaffold.

Figure 1

Chemical structures: (A) Endogenous EAAT substrates Glu and Asp; (B) EAAT inhibitors with l-Asp scaffold and pharmacological properties at EAAT1–4.^,

Figure 2

EAAT1 substrate binding pocket. (a) Overlay of crystal structures of EAAT1_cryst (white cartoon) in complex with l-Asp (PDB: 5llm, cyan stick) and EAAT1_cryst (gray cartoon) in complex with TFB-TBOA (PDB: 5mju, pink stick). Ile423 (red stick) of HP2 (red) in TFB-TBOA-bound complex is moved ∼12 Å away from its closed position (green stick) in the l-Asp-bound complex (green). (b) Excess space of TFB-TBOA binding pocket (PDB: 5mju). TFB-TBOA depicted as green and the ligand surface depicted as blue. Protein and protein surface depicted as gray (truncated at 7.0 Å from ligand).

Figure 3

Rational design of the first β-indolyloxy Asp analogs 15a–d.

Figure 4

Modeling of ligands into EAAT1_cryst (green stick) or into hmEAAT1 (white stick), and outline of the key residues (pink line) involved in binding the ligands and hydrogen bonds (yellow dash). (a) Overlay of binding pose of ligands (green) into hmEAAT1 and TFB-TBOA (PDB: 5mju, white) in EAAT1_cryst. (b) Binding mode of TFB-TBOA with EAAT1_cryst (PDB: 5mju). (c–g) Binding mode of compound 15a–d from induced-fit docking into hmEAAT1. Compound numbers are labeled on the respective figures.

Scheme 1. Retrosynthetic Analysis of Newly Designed EAAT Inhibitors 15a–d

Scheme 2. Synthesis of Asp Fragment 7 from d-Tartaric Acid 2

Reagents and conditions: (a) SOCl₂, MeOH, 0 °C to reflux, 4 h, quant.; (b) (i) HBr/AcOH, 0 °C to rt, overnight, (ii) AcCl, MeOH, 0 °C to reflux, 4 h, 74%; (c) NaN₃, DMF, rt, overnight, 68%; (d) H₂,10% Pd/C, MeOH, 45 °C, 3 h, 36%; (e) (Boc)₂O, Et₃N, DCM, 10 h, 62%.

Scheme 3. Synthesis of Compounds 15a–d from Methyl 6-Indolecarboxylate 8

Reagents and conditions: (a) NBS, THF, −78 °C, 2 h, 80%; (b) TsCl, NaH, THF, 0 °C to rt, 30 min, 98% (11a), 90% (10); (c) RB(OH)₂, Pd(dppf)Cl₂·CH₂Cl₂, Cs₂CO₃, dioxane/H₂O (4:1), 110 °C (M.W.), 15 min, 73% (11b), 69% (11c), 79% (11d); (d) LiAlH₄, THF, 0 °C to rt, 30 min, 86% (12a), 84% (12b), 80% (12c), 87% (12d); (e) MsCl, Et₃N, LiBr, THF, 0 °C to rt, 4 h, 68% (13a), 73% (13b), 69% (13c), 79% (13d); (f) compound 7, NaH, DMF, −15 °C, 4 h; (g) (i) TFA, DCM, 0 °C to rt, 2 h, (ii) LiOH·H₂O, THF/H₂O (1:1), 5 h, prep. HPLC, 8% (15a), 6% (15b), 5% (15c), 3% (15d) over 3 steps.

Figure 5

Concentration-inhibition curves for dl-TBOA, l-Glu, and compounds 15a–d at stable hEAAT1-HEK293, hEAAT2-HEK293, hEAAT3-HEK293, and rEAAT4-tsA201 cell lines in the [³H]-d-Asp uptake assay. Data are from representative experiments (out of a total of 3 independent experiments) and given as mean ± SD values in counts per min (CPM) based on duplicate determinations.