Design and Synthesis of Potent Quinazolines as Selective β-Glucocerebrosidase Modulators

Abstract

Gaucher's disease is a common genetic disease caused by mutations in the β-glucocerebrosidase (GBA1) gene that have been also linked to increased risk of Parkinson's disease and Lewy body dementia. Stabilization of misfolded mutant β-glucocerebrosidase (GCase) represents an important therapeutic strategy in synucleinopathies. Here we report a novel class of GCase quinazoline inhibitors, obtained in a high throughput screening, with moderate potency against wild-type GCase. Rational design and a SAR study of this class of compounds led to a new series of quinazoline derivatives with single-digit nanomolar potency. These compounds were shown to selectively stabilize GCase when compared to other lysosomal enzymes and to increase N370S mutant GCase protein concentration and activity in cell assays. To the best of our knowledge, these molecules are the most potent noniminosugar GCase modulators to date that may prove useful for future mechanistic studies and therapeutic approaches in Gaucher's and Parkinson's diseases.

Figure 1

Structures of GCase inhibitors

Figure 2

Rational design of a new series of potent quinazoline inhibitors

Figure 3

Fluorescent thermal shift analysis of selected compounds. Compound 9a, 9b, 11d, 11f, 11g, and IFG showed their ability to stabilize wild-type GCase in a dose-dependent manner. Data represent the results of three independent experiments performed with three replicates per sample.

Figure 4

Selectivity of inhibitor 11g with related hydrolases. 11g was tested on GCase, acid α-glucosidase (GAA), and α-galactosidase A (GLA). Data represent the results of three independent experiments performed with three replicates per sample.

Figure 5

Lineweaver–Burk plots of the enzyme kinetics of GCase inhibitors (A) 4 and (B) 11g. Each inhibitor was tested in triplicate in two independent assays at the concentrations shown in the legend box of each graph, with (○) indicating the absence of inhibitor. (A) Compounds 4 and (B) Compound 11g showed an increase in K_m and a decrease in V_max, indicating linear mixed inhibition.

Figure 6

Modulator 11g increases GCase protein levels in patient-derived Gaucher’s disease fibroblasts. Top: Western blot of N370S fibroblast treated with 11g or IFG for 3 days. Middle: Western blot of endoH digestion of N370S fibroblast after a 3-day treatment of 11g, IFG, or DMSO; endoH resistance of proteins indicates their post-ER localization; Bottom: Western blot of PNGase F digestion of N370S fibroblast after a 3-day treatment of 11g, IFG, or DMSO. The GAPDH signal was used for the loading control, n = 3.

Scheme 1

Synthesis of 4, 6a-6i, 7a-7i, 8a-8g, and 9a-9f with substituents on the secondary amine Reagents and conditions: (a) (i) sulfolane (ii) PCl₅; (b) RNH₂, K₂CO₃, DMF

Scheme 2

Synthesis of 11a-11h with modifications at the 2-position of the quinazoline ring Reagents and conditions: (a) 2,3-dihydro-1H-inden-2-amine, K₂CO₃, DMF; (b) RB(OH)₂, Pd(PPh₃)₄, K₂CO₃, 1,4-dioxane, H₂O