Identification of C-β-d-Glucopyranosyl Azole-Type Inhibitors of Glycogen Phosphorylase That Reduce Glycogenolysis in Hepatocytes: In Silico Design, Synthesis, in Vitro Kinetics, and ex Vivo Studies

Several C-β-d-glucopyranosyl azoles have recently been uncovered as among the most potent glycogen phosphorylase (GP) catalytic site inhibitors discovered to date. Toward 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 molecular mechanics-generalized Born surface area (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 area under the receiver operating characteristic curve (AU-ROC = 0.99) for predicted versus experimental binding affinities were obtained, with ligand tautomeric/ionization states additionally considered using density functional theory (DFT). 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 values in the low micromolar 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 were analyzed 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 favored, while within the cell, there is predicted more favorable binding to GP in the protonated form

A multidisciplinary study of 3-(β-d-glucopyranosyl)-5-substituted-1,2,4-triazole derivatives as glycogen phosphorylase inhibitors: Computation, synthesis, crystallography and kinetics reveal new potent inhibitors

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. [Abstract copyright: Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Synthesis of 4-amidomethyl-1-glucosyl-1,2,3-triazoles and evaluation as glycogen phosphorylase inhibitors.

International audienceGlycogen phosphorylase (GP) appears as a key enzyme for the control of hyperglycemia in the context of type 2 diabetes. In order to gain additional data for structure-activity studies of the inhibition of this enzyme, a series of eight GP inhibitor candidates were prepared from peracetylglucopyranosyl azide 1 by click-chemistry. The need for a N-Boc-protected propargylamine was identified in the CuAAC with azide 1 under Meldal's conditions, while Sharpless' conditions were better adapted to the CuAAC of azide 1 with propargyl bromide. Cycloaddition of Boc-propargylamine with azide 1 afforded the N-Boc precursor of a 4-aminomethyl-1-glucosyl-1,2,3-triazole which gave access to a series of eight amide and sulfonamide derivatives. After deacetylation, enzymatic studies revealed poor to moderate inhibitions toward this enzyme. The N-Boc-protected amine was the best inhibitor (IC50=620 μM) unexpectedly slightly better than the 2-naphthylamido substituted analogue (IC50=650 μM)

Design and Synthesis of 3-(β-<span style="font-variant: small-caps">d</span>-Glucopyranosyl)-4-amino/4-guanidino Pyrazole Derivatives and Analysis of Their Glycogen Phosphorylase Inhibitory Potential

Glycogen phosphorylase (GP) is a key regulator of glucose levels and, with that, an important target for the discovery of novel treatments against type 2 diabetes. β-d-Glucopyranosyl derivatives have provided some of the most potent GP inhibitors discovered to date. In this regard, C-β-d-glucopyranosyl azole type inhibitors proved to be particularly effective, with 2- and 4-β-d-glucopyranosyl imidazoles among the most potent designed to date. His377 backbone C=O hydrogen bonding and ion–ion interactions of the protonated imidazole with Asp283 from the 280s loop, stabilizing the inactive state, were proposed as crucial to the observed potencies. Towards further exploring these features, 4-amino-3-(β-d-glucopyranosyl)-5-phenyl-1H-pyrazole (3) and 3-(β-d-glucopyranosyl)-4-guanidino-5-phenyl-1H-pyrazole (4) were designed and synthesized with the potential to exploit similar interactions. Binding assay experiments against rabbit muscle GPb revealed 3 as a moderate inhibitor (IC50 = 565 µM), but 4 displayed no inhibition at 625 µM concentration. Towards understanding the observed inhibitions, docking and post-docking molecular mechanics—generalized Born surface area (MM-GBSA) binding free energy calculations were performed, together with Monte Carlo and density functional theory (DFT) calculations on the free unbound ligands. The computations revealed that while 3 was predicted to hydrogen bond with His377 C=O in its favoured tautomeric state, the interactions with Asp283 were not direct and there were no ion–ion interactions; for 4, the most stable tautomer did not have the His377 backbone C=O interaction and while ion–ion interactions and direct hydrogen bonding with Asp283 were predicted, the conformational strain and entropy loss of the ligand in the bound state was significant. The importance of consideration of tautomeric states and ligand strain for glucose analogues in the confined space of the catalytic site with the 280s loop in the closed position was highlighted

Synthesis of 1,2,3-triazoles from xylosyl and 5-thioxylosyl azides : evaluation of the xylose scaffold for the design of potential glycogen phosphorylase inhibitors

International audienceVarious acetylenic derivatives and acetylated β-D-xylopyranosyl azide or the 5-thio-β-d-xylopyranosyl analogue were coupled by Cu(I)-catalyzed azide alkyne 1,3-dipolar cycloaddition (CuAAC) to afford a series of 1-xylosyl-4-substituted 1,2,3-triazoles. Controlled oxidation of the endocyclic sulfur atom of the 5-thioxylose moiety led to the corresponding sulfoxides and sulfones. Deacetylation afforded 19 hydroxylated xylose and 5-thioxylose derivatives, found to be only sparingly water-soluble. Compared to glucose-based analogues, they appeared to be much weaker inhibitors of glycogen phosphorylase, as the absence of a hydroxymethyl group weakens their binding at the enzyme active site. However, such new xylose derivatives might be useful glycomimetics

Synthetic, enzyme kinetic, and protein crystallographic studies of C-β-D-glucopyranosyl pyrroles and imidazoles reveal and explain low nanomolar inhibition of human liver glycogen phosphorylase

C-β-D-Glucopyranosyl pyrrole derivatives were prepared in the reactions of pyrrole, 2-, and 3-aryl-pyrroles with O-peracetylated β-D-glucopyranosyl trichloroacetimidate, while 2-(β-D-glucopyranosyl) indole was obtained by a cross coupling of O-perbenzylated β-D-glucopyranosyl acetylene with N-tosyl-2-iodoaniline followed by spontaneous ring closure. An improved synthesis of O-perbenzoylated 2-(β-D-glucopyranosyl) imidazoles was achieved by reacting C-glucopyranosyl formimidates with α-aminoketones. The deprotected compounds were assayed with isoforms of glycogen phosphorylase (GP) to show no activity of the pyrroles against rabbit muscle GPb. The imidazoles proved to be the best known glucose derived inhibitors of not only the muscle enzymes (both a and b) but also of the pharmacologically relevant human liver GPa (Ki = 156 and 26 nM for the 4(5)-phenyl and -(2-naphthyl) derivatives, respectively). An X-ray crystallographic study of the rmGPb-imidazole complexes revealed structural features of the strong binding, and also allowed to explain the absence of inhibition for the pyrrole derivatives. © 2016 Elsevier Masson SA