Several C-β-D-glucopyranosyl azoles have recently been unravelled among the most potent glycogen phosphorylase (GP) catalytic site inhibitors discovered to date. Towards further exploring their translational potential, ex-vivo experiments have been performed for their effectiveness in reduction of glycogenolysis in hepatocytes. New compounds for these experiments were predicted in silico where, for the first time, effective ranking of GP catalytic site inhibitor potencies using the MM-GBSA method has been demonstrated. For a congeneric training set of 27 ligands, excellent statistics in terms of Pearson (Rp) and Spearman (Rs) correlations (both 0.98), predictive index (PI = 0.99) and AU-ROC (0.99) for predicted versus experimental binding affinities were obtained, with ligand tautomeric/ionization states additionally considered using DFT calculations. Seven 2-aryl-4(5)-(β-D-glucopyranosyl)imidazoles and 2-aryl-4-(β-D-glucopyranosyl)-thiazoles were subsequently synthesized and kinetics experiments against rabbit muscle GPb revealed new potent inhibitors with best Ki-s in the low µM range (5c = 1.97 µM; 13b = 4.58 µM). Ten C-β-D-glucopyranosyl azoles were then tested ex-vivo in mouse primary hepatocytes. Four of these (5a-c and 9d) demonstrated significant reduction of glucagon stimulated glycogenolysis (IC50 = 30-60 µM). Structural and predicted physicochemical properties associated with their effectiveness was analysed, with permeability related parameters identified as crucial factors. The most effective ligand series 5 contained an imidazole ring and the calculated pKa (Epik: 6.2; Jaguar 5.5) for protonated imidazole suggests that cellular permeation through the neutral state is favoured, while within the cell there is predicted more favourable binding to GP in the protonated form.
3-(β-d-Glucopyranosyl)-5-substituted-1,2,4-triazoles have been revealed as an effective scaffold for the development of potent glycogen phosphorylase (GP) inhibitors but with the potency very sensitive to the nature of the alkyl/aryl 5-substituent (Kun et al., Eur. J. Med. Chem. 2014, 76, 567). For a training set of these ligands, quantum mechanics-polarized ligand docking (QM-PLD) demonstrated good potential to identify larger differences in potencies (predictive index PI = 0.82) and potent inhibitors with K's < 10 μM (AU-ROC = 0.86). Accordingly, in silico screening of 2335 new analogues exploiting the ZINC docking database was performed and nine predicted candidates selected for synthesis. The compounds were prepared in O-perbenzoylated forms by either ring transformation of 5-β-d-glucopyranosyl tetrazole by N-benzyl-arenecarboximidoyl chlorides, ring closure of C-(β-d-glucopyranosyl)formamidrazone with aroyl chlorides, or that of N-(β-d-glucopyranosylcarbonyl)arenethiocarboxamides by hydrazine, followed by deprotections. Kinetics experiments against rabbit muscle GPb (rmGPb) and human liver GPa (hlGPa) revealed five compounds as potent low μM inhibitors with three of these on the submicromolar range for rmGPa. X-ray crystallographic analysis sourced the potency to a combination of favorable interactions from the 1,2,4-triazole and suitable aryl substituents in the GP catalytic site. The compounds also revealed promising calculated pharmacokinetic profiles.
The first systematic study on the synthesis of 2-C-(β-d-glycopyranosyl)pyrimidines either from amidine A or glycosyl cyanides B and 1,3-dicarbonyl compounds.
Large scale (up to 20 g) preparation ofIandIIallowed the best inhibitors of glycogen phosphorylaseIIIto be synthesized in close to 60% overall yields fromI.
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