Structure-activity relationships of a small-molecule inhibitor of the PDZ domain of PICK1

Abstract

Recently, we described the first small-molecule inhibitor, (E)-ethyl 2-cyano-3-(3,4-dichlorophenyl)acryloylcarbamate (1), of the PDZ domain of protein interacting with Calpha-kinase 1 (PICK1), a potential drug target against brain ischemia, pain and cocaine addiction. Herein, we explore structure-activity relationships of 1 by introducing subtle modifications of the acryloylcarbamate scaffold and variations of the substituents on this scaffold. The configuration around the double bond of 1 and analogues was settled by a combination of X-ray crystallography, NMR and density functional theory calculations. Thereby, docking studies were used to correlate biological affinities with structural considerations for ligand-protein interactions. The most potent analogue obtained in this study showed an improvement in affinity compared to 1 and is currently a lead in further studies of PICK1 inhibition.

Fig. 1

Structure of lead compound 1 and the concordant generic structure together with its retrosynthetic analysis. Substituents explored in this SAR study are indicated R_1–3.

Fig. 2

Semi-quantitative pull-down assay of selected compounds towards PICK1. DMSO indicates the amount of protein pulled down when no compound is added but only the equivalent amount of DMSO. The columns represent the average values and the error bars represent standard deviations based on three individual experiments.

Fig. 3

Perspective drawing (ORTEP-3) of compound 41. Displacement ellipsoids of the non-hydrogen atoms are shown at the 50% probability level. Hydrogen atoms are represented by spheres of arbitrary size.

Fig. 4

The binding of compound 1 in the PDZ domain of PICK1. The protein is rendered in green and the ligand in stick representation with the chlorine atoms shown in deep blue, oxygens in red, carbons in purple, aromatic carbons in white and nitrogen atoms in blue. Panels (a)–(d) show magnifications of the corresponding regions indicated by the labelled frames in the main figure. These (a)–(d) framed regions contain the ligand moieties that contribute most to binding, i.e., the chloro substituents, the phenyl moiety, the cyano group and the alkyl tail, respectively. In panel (a) the positions of the 3-trifluoromethyl and the 4-methoxy moieties of compound 49 and 44, are shown in orange and cyan, respectively; in (b) the naphthyl and biphenyl analogues, compounds 25 and 27 are shown in yellow and wheat; in (c) the ethyl and benzyl analogues, compounds 38 and 40 are shown in white and orange; in panel (d) compounds 28 and 29 with the isopropyl substituents are shown in blue and pink.

Scheme 1

(a) POCl₃, DMF, toluene, 70 °C, 1.5 h (82%); (b) 3,4-dichlorobenzaldehyde, piperidine, AcOH, DMF, 2 h (43%).

Scheme 2

(a) H₂ (1 atm.), Pd/C (3.5 mol%), EtOAc, rt, 24 h (76%); (b) SOCl₂, reflux, 2 h (73%); (c) sodium (ethoxycarbonyl)amide, DMF, rt, 1 h (14%); (d) BuNH₂, MeOH, rt, 1 h (91%); (e) 3,4-dichlorobenzaldehyde, piperidinium acetate (cat.), DMF, rt, 48 h (47%); (f) acrylonitrile, DABCO, dioxane–water (1 : 1), rt, 24 h (68%); (g) PBr₃, Et₂O, rt, 2 h (69%); (h) NH₃, MeOH, rt, 2 h (12: 19%; 13: 65%); (i) EtOCOCl, Et₃N, THF, rt, 1 h (64%).

Scheme 3

(a) POCl₃, DMF, toluene, 70 °C, 1–2 h; (b) Cl₃CCONCO, CH₂Cl₂, 0 °C→23 °C, 2 h; (c) K₂CO₃,MeOH, 50 °C, 16 h (87%); (d) 2-cyanoacetic acid, POCl₃, DMF, toluene, 70 °C, 1.5 h; (e) Et₃N, THF, rt, 2 h