Discovery of Novel Imidazopyridine GSK-3β Inhibitors Supported by Computational Approaches

Abstract

The interest of research groups and pharmaceutical companies to discover novel GSK-3β inhibitors has increased over the years considering the involvement of this enzyme in many pathophysiological processes and diseases. Along this line, we recently reported on 1H-indazole-3-carboxamide (INDZ) derivatives 1-6, showing good GSK-3β inhibition activity. However, they suffered from generally poor central nervous system (CNS) permeability. Here, we describe the design, synthesis, and in vitro characterization of novel imidazo[1,5-a]pyridine-1-carboxamide (IMID 1) and imidazo[1,5-a]pyridine-3-carboxamide (IMID 2) compounds (7-18) to overcome such liability. In detail, structure-based approaches and fine-tuning of physicochemical properties guided the design of derivatives 7-18 resulting in ameliorated absorption, distribution, metabolism, and excretion (ADME) properties. A crystal structure of 16 in complex with GSK-3β enzyme (PDB entry 6Y9S) confirmed the in silico models. Despite the nanomolar inhibition activity, the new core compounds showed a reduction in potency with respect to INDZ derivatives 1-6. In this context, Molecular Dynamics (MD) and Quantum Mechanics (QM) based approaches along with NMR investigation helped to rationalize the observed structure activity relationship (SAR). With these findings, the key role of the acidic hydrogen of the central core for a tight interaction within the ATP pocket of the enzyme reflecting in good GSK-3β affinity was demonstrated.

Keywords: 1H-indazole-3-carboxamide core (INDZ); CNS permeability; NMR characterization; X-ray crystallography; glycogen synthase kinase-3β (GSK-3β); imidazo[1,5-a]pyridine-1-carboxamide core (IMID 1); imidazo[1,5-a]pyridine-3-carboxamide core (IMID 2); molecular docking; molecular dynamics (MD); quantum mechanics (QM).

Figure 1

Exploration of IMID 1 and IMID 2 derivatives 7–18 starting from previous INDZ 1–6 to improve CNS penetration.

Scheme 1

Synthesis of imidazo[1,5-a]pyridine-1-carboxamide derivatives 7–12.

Scheme 2

Synthesis of imidazo[1,5-a]pyridine-3-carboxamide derivatives 13–18.

Figure 2

(a) Generic binding mode of the INDZ core in the ATP binding domain of GSK-3β enzyme extracted from crystallographic studies discussed elsewhere [16,17,19]. The 2D structure of the INDZ core with explicit Ha and Hb atoms is also showed; (b) X-ray co-crystal structure of inhibitor 2 in complex with GSK-3β enzyme (PDB entry 6Y9R). The deep, central and outer H-bond interactions are represented as red, green, and orange dashed lines, respectively. The most relevant residues of the ATP binding site are showed in light blue (for clarity, light transparency is used for some amino acids). Relevant water molecules around inhibitor 2 are also displayed. Both figures were prepared with VMD 1.9.4 [24].

Figure 3

Binding poses of INDZ, IMID 1, and IMID 2 derivatives, reported as yellow, cyan, and orange ball and sticks representation, respectively, in complex with GSK-3β. Each panel represents a triad bearing the same R₁ and R₂ groups, (a) 1-7-13, (b) 2-8-14, (c) 3-9-15, (d) 4-10-16, (e) 5-11-17, (f) 6-12-18. The most relevant residues of the ATP binding site are also showed in grey. Deep, central, and outer H-bonds are represented in red, green, and orange, respectively, while dark dashed lines refer to other interactions involving Phe67, Lys85, and Gln185.

Figure 4

RMSD evolution of (a) protein and (b) ligand with respect to the protein, for each 100 ns MD simulation using frame 0 as reference. The RMSD ranges are 0–2.66 Å in a and 0–4.25 Å in b (dark green-yellow-white color scale corresponds to 0 to 4.25Å values).

Figure 5

Overlap of the docking poses (yellow) versus co-crystal structure (green) of 16 in the complex with GSK-3β enzyme.

Figure 6

Distance of the deep (red), central (green) and outer (orange) H-bond interactions across 100 ns MD simulation of derivatives 1–18. The plots are ordered per INDZ-IMID 1-IMID 2 triplet. Dashed lines represent the mean value of the H-bond distance.

Figure 7

D-H-A angles (degrees) of the deep H-bond interaction across 100 ns simulation of 1–18 systems. The plots are ordered per INDZ-IMID 1-IMID 2 triplet and colored as yellow, cyan, and orange, respectively. Dashed lines correspond to the mean value of the D-H-A angle.

Figure 8

Temperature coefficient calculated for N-Hb of compound 14 (IMID 2).