The overaccumulation of glycogen appears as a hallmark in various glycogen storage diseases (GSDs), including Pompe, Cori, Andersen, and Lafora disease. Accumulating evidence suggests that suppression of glycogen accumulation represents a potential therapeutic approach for treating these GSDs. Using a fluorescence polarization assay designed to screen for inhibitors of the key glycogen synthetic enzyme, glycogen synthase (GS), we identified a substituted imidazole, (rac)-2-methoxy-4-(1-(2-(1-methylpyrrolidin-2-yl)­ethyl)-4-phenyl-1H-imidazol-5-yl)­phenol (H23), as a first-in-class inhibitor for yeast GS 2 (yGsy2p). Data from X-ray crystallography at 2.85 Å, as well as kinetic data, revealed that H23 bound within the uridine diphosphate glucose binding pocket of yGsy2p. The high conservation of residues between human and yeast GS in direct contact with H23 informed the development of around 500 H23 analogs. These analogs produced a structure–activity relationship profile that led to the identification of a substituted pyrazole, 4-(4-(4-hydroxyphenyl)-3-(trifluoromethyl)-1H-pyrazol-5-yl)­pyrogallol, with a 300-fold improved potency against human GS. These substituted pyrazoles possess a promising scaffold for drug development efforts targeting GS activity in GSDs associated with excess glycogen accumulation.

The regiospecific Mannich aminomethylation of 7-hydroxyisoflavonoids using bis(N,N-dimethylamino)methane afforded C-8 substituted N,N-dimethylaminomethyl adducts, and the regioselective aminomethylation of 5-hydroxy-7-methoxyisoflavonoids afforded predominantly the C-6 substituted N,N-dimethylaminomethyl adducts. Acetylation of these C-6 or C-8 Mannich bases with potassium acetate in acetic anhydride provided access to the corresponding acetoxymethyl derivatives that were subsequently converted to hydroxymethyl- and methoxymethyl-substituted 5-hydroxy- or 7-hydroxyisoflavonoids related to naturally occurring flavonoids. The C-8 acetoxymethyl, hydroxymethyl or methoxymethyl-substituted isoflavonoids possessed promising inhibitory potency in the low micromolar range in a prostate cancer PC-3 cell proliferation assay.

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