Synthesis, In Silico and Kinetics Evaluation of N-(β-d-glucopyranosyl)-2-arylimidazole-4(5)-carboxamides and N-(β-d-glucopyranosyl)-4(5)-arylimidazole-2-carboxamides as Glycogen Phosphorylase Inhibitors

Recently studied N-(β-d-glucopyranosyl)-3-aryl-1,2,4-triazole-5-carboxamides have proven to be low micromolar inhibitors of glycogen phosphorylase (GP), a validated target for the treatment of type 2 diabetes mellitus. Since in other settings, the bioisosteric replacement of the 1,2,4-triazole moiety with imidazole resulted in significantly more efficient GP inhibitors, in silico calculations using Glide molecular docking along with unbound state DFT calculations were performed on N-(β-d-glucopyranosyl)-arylimidazole-carboxamides, revealing their potential for strong GP inhibition. The syntheses of the target compounds involved the formation of an amide bond between per-O-acetylated β-d-glucopyranosylamine and the corresponding arylimidazole-carboxylic acids. Kinetics experiments on rabbit muscle GPb revealed low micromolar inhibitors, with the best inhibition constants (Kis) of ~3–4 µM obtained for 1- and 2-naphthyl-substituted N-(β-d-glucopyranosyl)-imidazolecarboxamides, 2b–c. The predicted protein–ligand interactions responsible for the observed potencies are discussed and will facilitate the structure-based design of other inhibitors targeting this important therapeutic target. Meanwhile, the importance of the careful consideration of ligand tautomeric states in binding calculations is highlighted, with the usefulness of DFT calculations in this regard proposed.


Introduction
Diabetes mellitus is a growing pandemic which poses many challenges for global health and economics, of which type 2 diabetes mellitus (T2DM) is the most common.The global prevalence of T2DM is projected to increase to 7079 people per 100,000 by 2030 and reflects a continuing trend across all the world [1].It is important to also note that there has been a concerning rise in its prevalence in lower-income countries.T2DM is documented as a metabolic disorder, attributed to abnormal insulin production and/or peripheral resistance to the action of insulin, advancing the development of hyperglycaemia [2].While there are different classes of approved antihyperglycaemic drugs [3], these treatments do not achieve the required degree of glucose control for a large number of patients [4].Several complications may arise in individuals suffering from T2DM when their blood glucose levels are inadequately regulated, including cardiovascular disease, loss of vision, neuropathy and nephropathy [5].Overall, there is an urgent need for new and more effective therapeutic options, combined with clinical preventive measures.
The glycogenolysis pathway has a direct impact on blood glucose levels; as glycogen phosphorylase (GP; EC 2.4.1.1)plays a fundamental role in this pathway, it is therefore an attractive target for the treatment of T2DM.GP exists in three isoforms: the liver, muscle and brain.More specifically, the liver isoform is of interest as it catalyses the breakdown of glycogen into glucose in the liver, the inhibition of which has potential to reduce hyperglycaemia in T2DM patients [6].Additionally, GP is also considered of promise as a target for other conditions such as myocardial and cerebral ischemias [7,8] and different cancers such as glioblastoma [9][10][11].Seven different GP ligand-binding sites have been discovered to date: catalytic, allosteric, new allosteric, glycogen storage, inhibitor, benzimidazole and quercetin-binding sites, with the catalytic site being a focal point of research to date [12][13][14][15].Physiological inhibitors of GP's catalytic site are typically glucose analogues such as α-D-glucose, with an inhibition constant (K i ) of 1.7 mM, and its anomer, β-D-glucose, which binds with a K i of 7.4 mM [16,17].The most potent glucose analogue inhibitors of GP are based on β-D-glucose and possess a carefully designed linker group attached to the β-position of the anomeric C-1 atom of the glucose ring and an extension of the linker, usually with a planar aromatic group, exploiting the favourable interactions in the so-called β-cavity of the GP catalytic site [12,18,19].The most efficient nanomolar inhibitors are exemplified in Table 1, representing the best three scaffolds to date: N-(β-D-glucopyranosyl)-N'-aroyl ureas (e.g., I) [15], glucopyranosylidene-spiro-heterocycles (e.g., II, III) [20] and C-β-D-glucopyranosyl heterocycles (e.g., IV, V) [21].A comparison of the inhibitory efficiency of IV and V shows the beneficial effect of switching to the imidazole V from the 1,2,4-triazole IV. neuropathy and nephropathy [5].Overall, there is an urgent need for new and more effective therapeutic options, combined with clinical preventive measures.
The glycogenolysis pathway has a direct impact on blood glucose levels; as glycogen phosphorylase (GP; EC 2.4.1.1)plays a fundamental role in this pathway, it is therefore an attractive target for the treatment of T2DM.GP exists in three isoforms: the liver, muscle and brain.More specifically, the liver isoform is of interest as it catalyses the breakdown of glycogen into glucose in the liver, the inhibition of which has potential to reduce hyperglycaemia in T2DM patients [6].Additionally, GP is also considered of promise as a target for other conditions such as myocardial and cerebral ischemias [7,8] and different cancers such as glioblastoma [9][10][11].Seven different GP ligand-binding sites have been discovered to date: catalytic, allosteric, new allosteric, glycogen storage, inhibitor, benzimidazole and quercetin-binding sites, with the catalytic site being a focal point of research to date [12][13][14][15].Physiological inhibitors of GP's catalytic site are typically glucose analogues such as α-D-glucose, with an inhibition constant (Ki) of 1.7 mM, and its anomer, β-D-glucose, which binds with a Ki of 7.4 mM [16,17].The most potent glucose analogue inhibitors of GP are based on β-D-glucose and possess a carefully designed linker group attached to the β-position of the anomeric C-1 atom of the glucose ring and an extension of the linker, usually with a planar aromatic group, exploiting the favourable interactions in the so-called β-cavity of the GP catalytic site [12,18,19].The most efficient nanomolar inhibitors are exemplified in Table 1, representing the best three scaffolds to date: N-(β-Dglucopyranosyl)-N'-aroyl ureas (e.g., I) [15], glucopyranosylidene-spiro-heterocycles (e.g., II, III) [20] and C-β-D-glucopyranosyl heterocycles (e.g., IV, V) [21].A comparison of the inhibitory efficiency of IV and V shows the beneficial effect of switching to the imidazole V from the 1,2,4-triazole IV.
The glycogenolysis pathway has a direct impact on blood glucose levels; as glycogen phosphorylase (GP; EC 2.4.1.1)plays a fundamental role in this pathway, it is therefore an attractive target for the treatment of T2DM.GP exists in three isoforms: the liver, muscle and brain.More specifically, the liver isoform is of interest as it catalyses the breakdown of glycogen into glucose in the liver, the inhibition of which has potential to reduce hyperglycaemia in T2DM patients [6].Additionally, GP is also considered of promise as a target for other conditions such as myocardial and cerebral ischemias [7,8] and different cancers such as glioblastoma [9][10][11].Seven different GP ligand-binding sites have been discovered to date: catalytic, allosteric, new allosteric, glycogen storage, inhibitor, benzimidazole and quercetin-binding sites, with the catalytic site being a focal point of research to date [12][13][14][15].Physiological inhibitors of GP's catalytic site are typically glucose analogues such as α-D-glucose, with an inhibition constant (Ki) of 1.7 mM, and its anomer, β-D-glucose, which binds with a Ki of 7.4 mM [16,17].The most potent glucose analogue inhibitors of GP are based on β-D-glucose and possess a carefully designed linker group attached to the β-position of the anomeric C-1 atom of the glucose ring and an extension of the linker, usually with a planar aromatic group, exploiting the favourable interactions in the so-called β-cavity of the GP catalytic site [12,18,19].The most efficient nanomolar inhibitors are exemplified in Table 1, representing the best three scaffolds to date: N-(β-Dglucopyranosyl)-N'-aroyl ureas (e.g., I) [15], glucopyranosylidene-spiro-heterocycles (e.g., II, III) [20] and C-β-D-glucopyranosyl heterocycles (e.g., IV, V) [21].A comparison of the inhibitory efficiency of IV and V shows the beneficial effect of switching to the imidazole V from the 1,2,4-triazole IV.
Non-classical bioisosteric replacements [22,23] of the highlighted amide moiety in I with several oxadiazole-type heterocycles were previously studied (Table 1, VI-VIII), which, however, resulted in no better inhibitors than I.Meanwhile, in silico docking calculations have been performed on N-(β-D-glucopyranosyl)-3-substituted-1,2,4-triazole-5carboxamide analogues IX with aryl substitutions: phenyl (a), 1-naphtyhl (b) and 2-naphthyl (c) [24].The phenyl-substituted IXa was reported as the most potent of the three analogues acting at the catalytic site, with a Ki of 1 µM for rabbit muscle glycogen phosphorylase b (rmGPb).neuropathy and nephropathy [5].Overall, there is an urgent need for new and more effective therapeutic options, combined with clinical preventive measures.
The glycogenolysis pathway has a direct impact on blood glucose levels; as glycogen phosphorylase (GP; EC 2.4.1.1)plays a fundamental role in this pathway, it is therefore an attractive target for the treatment of T2DM.GP exists in three isoforms: the liver, muscle and brain.More specifically, the liver isoform is of interest as it catalyses the breakdown of glycogen into glucose in the liver, the inhibition of which has potential to reduce hyperglycaemia in T2DM patients [6].Additionally, GP is also considered of promise as a target for other conditions such as myocardial and cerebral ischemias [7,8] and different cancers such as glioblastoma [9][10][11].Seven different GP ligand-binding sites have been discovered to date: catalytic, allosteric, new allosteric, glycogen storage, inhibitor, benzimidazole and quercetin-binding sites, with the catalytic site being a focal point of research to date [12][13][14][15].Physiological inhibitors of GP's catalytic site are typically glucose analogues such as α-D-glucose, with an inhibition constant (Ki) of 1.7 mM, and its anomer, β-D-glucose, which binds with a Ki of 7.4 mM [16,17].The most potent glucose analogue inhibitors of GP are based on β-D-glucose and possess a carefully designed linker group attached to the β-position of the anomeric C-1 atom of the glucose ring and an extension of the linker, usually with a planar aromatic group, exploiting the favourable interactions in the so-called β-cavity of the GP catalytic site [12,18,19].The most efficient nanomolar inhibitors are exemplified in Table 1, representing the best three scaffolds to date: N-(β-Dglucopyranosyl)-N'-aroyl ureas (e.g., I) [15], glucopyranosylidene-spiro-heterocycles (e.g., II, III) [20] and C-β-D-glucopyranosyl heterocycles (e.g., IV, V) [21].A comparison of the inhibitory efficiency of IV and V shows the beneficial effect of switching to the imidazole V from the 1,2,4-triazole IV.
Non-classical bioisosteric replacements [22,23] of the highlighted amide moiety in I with several oxadiazole-type heterocycles were previously studied (Table 1, VI-VIII), which, however, resulted in no better inhibitors than I.Meanwhile, in silico docking calculations have been performed on N-(β-D-glucopyranosyl)-3-substituted-1,2,4-triazole-5carboxamide analogues IX with aryl substitutions: phenyl (a), 1-naphtyhl (b) and 2-naphthyl (c) [24].The phenyl-substituted IXa was reported as the most potent of the three analogues acting at the catalytic site, with a Ki of 1 µM for rabbit muscle glycogen phosphorylase b (rmGPb).V X = CH (0.031) [21] Bioisosteric replacements in I and compounds studied neuropathy and nephropathy [5].Overall, there is an urgent need for new and more effective therapeutic options, combined with clinical preventive measures.
The glycogenolysis pathway has a direct impact on blood glucose levels; as glycogen phosphorylase (GP; EC 2.4.1.1)plays a fundamental role in this pathway, it is therefore an attractive target for the treatment of T2DM.GP exists in three isoforms: the liver, muscle and brain.More specifically, the liver isoform is of interest as it catalyses the breakdown of glycogen into glucose in the liver, the inhibition of which has potential to reduce hyperglycaemia in T2DM patients [6].Additionally, GP is also considered of promise as a target for other conditions such as myocardial and cerebral ischemias [7,8] and different cancers such as glioblastoma [9][10][11].Seven different GP ligand-binding sites have been discovered to date: catalytic, allosteric, new allosteric, glycogen storage, inhibitor, benzimidazole and quercetin-binding sites, with the catalytic site being a focal point of research to date [12][13][14][15].Physiological inhibitors of GP's catalytic site are typically glucose analogues such as α-D-glucose, with an inhibition constant (Ki) of 1.7 mM, and its anomer, β-D-glucose, which binds with a Ki of 7.4 mM [16,17].The most potent glucose analogue inhibitors of GP are based on β-D-glucose and possess a carefully designed linker group attached to the β-position of the anomeric C-1 atom of the glucose ring and an extension of the linker, usually with a planar aromatic group, exploiting the favourable interactions in the so-called β-cavity of the GP catalytic site [12,18,19].The most efficient nanomolar inhibitors are exemplified in Table 1, representing the best three scaffolds to date: N-(β-Dglucopyranosyl)-N'-aroyl ureas (e.g., I) [15], glucopyranosylidene-spiro-heterocycles (e.g., II, III) [20] and C-β-D-glucopyranosyl heterocycles (e.g., IV, V) [21].A comparison of the inhibitory efficiency of IV and V shows the beneficial effect of switching to the imidazole V from the 1,2,4-triazole IV.
Non-classical bioisosteric replacements [22,23] of the highlighted amide moiety in I with several oxadiazole-type heterocycles were previously studied (Table 1, VI-VIII), which, however, resulted in no better inhibitors than I.Meanwhile, in silico docking calculations have been performed on N-(β-D-glucopyranosyl)-3-substituted-1,2,4-triazole-5carboxamide analogues IX with aryl substitutions: phenyl (a), 1-naphtyhl (b) and 2-naphthyl (c) [24].The phenyl-substituted IXa was reported as the most potent of the three analogues acting at the catalytic site, with a Ki of 1 µM for rabbit muscle glycogen phosphorylase b (rmGPb).neuropathy and nephropathy [5].Overall, there is an urgent need for new and more effective therapeutic options, combined with clinical preventive measures.
The glycogenolysis pathway has a direct impact on blood glucose levels; as glycogen phosphorylase (GP; EC 2.4.1.1)plays a fundamental role in this pathway, it is therefore an attractive target for the treatment of T2DM.GP exists in three isoforms: the liver, muscle and brain.More specifically, the liver isoform is of interest as it catalyses the breakdown of glycogen into glucose in the liver, the inhibition of which has potential to reduce hyperglycaemia in T2DM patients [6].Additionally, GP is also considered of promise as a target for other conditions such as myocardial and cerebral ischemias [7,8] and different cancers such as glioblastoma [9][10][11].Seven different GP ligand-binding sites have been discovered to date: catalytic, allosteric, new allosteric, glycogen storage, inhibitor, benzimidazole and quercetin-binding sites, with the catalytic site being a focal point of research to date [12][13][14][15].Physiological inhibitors of GP's catalytic site are typically glucose analogues such as α-D-glucose, with an inhibition constant (Ki) of 1.7 mM, and its anomer, β-D-glucose, which binds with a Ki of 7.4 mM [16,17].The most potent glucose analogue inhibitors of GP are based on β-D-glucose and possess a carefully designed linker group attached to the β-position of the anomeric C-1 atom of the glucose ring and an extension of the linker, usually with a planar aromatic group, exploiting the favourable interactions in the so-called β-cavity of the GP catalytic site [12,18,19].The most efficient nanomolar inhibitors are exemplified in Table 1, representing the best three scaffolds to date: N-(β-Dglucopyranosyl)-N'-aroyl ureas (e.g., I) [15], glucopyranosylidene-spiro-heterocycles (e.g., II, III) [20] and C-β-D-glucopyranosyl heterocycles (e.g., IV, V) [21].A comparison of the inhibitory efficiency of IV and V shows the beneficial effect of switching to the imidazole V from the 1,2,4-triazole IV.
Non-classical bioisosteric replacements [22,23] of the highlighted amide moiety in I with several oxadiazole-type heterocycles were previously studied (Table 1, VI-VIII), which, however, resulted in no better inhibitors than I.Meanwhile, in silico docking calculations have been performed on N-(β-D-glucopyranosyl)-3-substituted-1,2,4-triazole-5carboxamide analogues IX with aryl substitutions: phenyl (a), 1-naphtyhl (b) and 2-naphthyl (c) [24].The phenyl-substituted IXa was reported as the most potent of the three analogues acting at the catalytic site, with a Ki of 1 µM for rabbit muscle glycogen phosphorylase b (rmGPb).neuropathy and nephropathy [5].Overall, there is an urgent need for new and more effective therapeutic options, combined with clinical preventive measures.
The glycogenolysis pathway has a direct impact on blood glucose levels; as glycogen phosphorylase (GP; EC 2.4.1.1)plays a fundamental role in this pathway, it is therefore an attractive target for the treatment of T2DM.GP exists in three isoforms: the liver, muscle and brain.More specifically, the liver isoform is of interest as it catalyses the breakdown of glycogen into glucose in the liver, the inhibition of which has potential to reduce hyperglycaemia in T2DM patients [6].Additionally, GP is also considered of promise as a target for other conditions such as myocardial and cerebral ischemias [7,8] and different cancers such as glioblastoma [9][10][11].Seven different GP ligand-binding sites have been discovered to date: catalytic, allosteric, new allosteric, glycogen storage, inhibitor, benzimidazole and quercetin-binding sites, with the catalytic site being a focal point of research to date [12][13][14][15].Physiological inhibitors of GP's catalytic site are typically glucose analogues such as α-D-glucose, with an inhibition constant (Ki) of 1.7 mM, and its anomer, β-D-glucose, which binds with a Ki of 7.4 mM [16,17].The most potent glucose analogue inhibitors of GP are based on β-D-glucose and possess a carefully designed linker group attached to the β-position of the anomeric C-1 atom of the glucose ring and an extension of the linker, usually with a planar aromatic group, exploiting the favourable interactions in the so-called β-cavity of the GP catalytic site [12,18,19].The most efficient nanomolar inhibitors are exemplified in Table 1, representing the best three scaffolds to date: N-(β-Dglucopyranosyl)-N'-aroyl ureas (e.g., I) [15], glucopyranosylidene-spiro-heterocycles (e.g., II, III) [20] and C-β-D-glucopyranosyl heterocycles (e.g., IV, V) [21].A comparison of the inhibitory efficiency of IV and V shows the beneficial effect of switching to the imidazole V from the 1,2,4-triazole IV.
Non-classical bioisosteric replacements [22,23] of the highlighted amide moiety in I with several oxadiazole-type heterocycles were previously studied (Table 1, VI-VIII), which, however, resulted in no better inhibitors than I.Meanwhile, in silico docking calculations have been performed on N-(β-D-glucopyranosyl)-3-substituted-1,2,4-triazole-5carboxamide analogues IX with aryl substitutions: phenyl (a), 1-naphtyhl (b) and 2-naphthyl (c) [24].The phenyl-substituted IXa was reported as the most potent of the three analogues acting at the catalytic site, with a Ki of 1 µM for rabbit muscle glycogen phosphorylase b (rmGPb).neuropathy and nephropathy [5].Overall, there is an urgent need for new and more effective therapeutic options, combined with clinical preventive measures.
The glycogenolysis pathway has a direct impact on blood glucose levels; as glycogen phosphorylase (GP; EC 2.4.1.1)plays a fundamental role in this pathway, it is therefore an attractive target for the treatment of T2DM.GP exists in three isoforms: the liver, muscle and brain.More specifically, the liver isoform is of interest as it catalyses the breakdown of glycogen into glucose in the liver, the inhibition of which has potential to reduce hyperglycaemia in T2DM patients [6].Additionally, GP is also considered of promise as a target for other conditions such as myocardial and cerebral ischemias [7,8] and different cancers such as glioblastoma [9][10][11].Seven different GP ligand-binding sites have been discovered to date: catalytic, allosteric, new allosteric, glycogen storage, inhibitor, benzimidazole and quercetin-binding sites, with the catalytic site being a focal point of research to date [12][13][14][15].Physiological inhibitors of GP's catalytic site are typically glucose analogues such as α-D-glucose, with an inhibition constant (Ki) of 1.7 mM, and its anomer, β-D-glucose, which binds with a Ki of 7.4 mM [16,17].The most potent glucose analogue inhibitors of GP are based on β-D-glucose and possess a carefully designed linker group attached to the β-position of the anomeric C-1 atom of the glucose ring and an extension of the linker, usually with a planar aromatic group, exploiting the favourable interactions in the so-called β-cavity of the GP catalytic site [12,18,19].The most efficient nanomolar inhibitors are exemplified in Table 1, representing the best three scaffolds to date: N-(β-Dglucopyranosyl)-N'-aroyl ureas (e.g., I) [15], glucopyranosylidene-spiro-heterocycles (e.g., II, III) [20] and C-β-D-glucopyranosyl heterocycles (e.g., IV, V) [21].A comparison of the inhibitory efficiency of IV and V shows the beneficial effect of switching to the imidazole V from the 1,2,4-triazole IV.
Non-classical bioisosteric replacements [22,23] of the highlighted amide moiety in I with several oxadiazole-type heterocycles were previously studied (Table 1, VI-VIII), which, however, resulted in no better inhibitors than I.Meanwhile, in silico docking calculations have been performed on N-(β-D-glucopyranosyl)-3-substituted-1,2,4-triazole-5carboxamide analogues IX with aryl substitutions: phenyl (a), 1-naphtyhl (b) and 2-naphthyl (c) [24].The phenyl-substituted IXa was reported as the most potent of the three analogues acting at the catalytic site, with a Ki of 1 µM for rabbit muscle glycogen phosphorylase b (rmGPb).neuropathy and nephropathy [5].Overall, there is an urgent need for new and more effective therapeutic options, combined with clinical preventive measures.
The glycogenolysis pathway has a direct impact on blood glucose levels; as glycogen phosphorylase (GP; EC 2.4.1.1)plays a fundamental role in this pathway, it is therefore an attractive target for the treatment of T2DM.GP exists in three isoforms: the liver, muscle and brain.More specifically, the liver isoform is of interest as it catalyses the breakdown of glycogen into glucose in the liver, the inhibition of which has potential to reduce hyperglycaemia in T2DM patients [6].Additionally, GP is also considered of promise as a target for other conditions such as myocardial and cerebral ischemias [7,8] and different cancers such as glioblastoma [9][10][11].Seven different GP ligand-binding sites have been discovered to date: catalytic, allosteric, new allosteric, glycogen storage, inhibitor, benzimidazole and quercetin-binding sites, with the catalytic site being a focal point of research to date [12][13][14][15].Physiological inhibitors of GP's catalytic site are typically glucose analogues such as α-D-glucose, with an inhibition constant (Ki) of 1.7 mM, and its anomer, β-D-glucose, which binds with a Ki of 7.4 mM [16,17].The most potent glucose analogue inhibitors of GP are based on β-D-glucose and possess a carefully designed linker group attached to the β-position of the anomeric C-1 atom of the glucose ring and an extension of the linker, usually with a planar aromatic group, exploiting the favourable interactions in the so-called β-cavity of the GP catalytic site [12,18,19].The most efficient nanomolar inhibitors are exemplified in Table 1, representing the best three scaffolds to date: N-(β-Dglucopyranosyl)-N'-aroyl ureas (e.g., I) [15], glucopyranosylidene-spiro-heterocycles (e.g., II, III) [20] and C-β-D-glucopyranosyl heterocycles (e.g., IV, V) [21].A comparison of the inhibitory efficiency of IV and V shows the beneficial effect of switching to the imidazole V from the 1,2,4-triazole IV.
Non-classical bioisosteric replacements [22,23] of the highlighted amide moiety in I with several oxadiazole-type heterocycles were previously studied (Table 1, VI-VIII), which, however, resulted in no better inhibitors than I.Meanwhile, in silico docking calculations have been performed on N-(β-D-glucopyranosyl)-3-substituted-1,2,4-triazole-5carboxamide analogues IX with aryl substitutions: phenyl (a), 1-naphtyhl (b) and 2-naphthyl (c) [24].The phenyl-substituted IXa was reported as the most potent of the three analogues acting at the catalytic site, with a Ki of 1 µM for rabbit muscle glycogen phosphorylase b (rmGPb).As a continuation of the bioisosteric replacement studies in I, and based on the experiences with IV and V, computational, synthetic and enzyme kinetic investigation of imidazole-containing compounds was undertaken in this work (Table 1, target compounds 1 and 2).Docking predictions for the bound states and DFT calculations on the unbound states revealed potential for potent GP inhibition, consistent with that observed previously for the N-(β-D-glucopyranosyl)-3-substituted-1,2,4-triazole-5-carboxamides analogues.Synthesis and subsequent kinetic evaluation of GP inhibition revealed this to be the case.As a continuation of the bioisosteric replacement studies in I, and based on the experiences with IV and V, computational, synthetic and enzyme kinetic investigation of imidazole-containing compounds was undertaken in this work (Table 1, target compounds 1 and 2).Docking predictions for the bound states and DFT calculations on the unbound states revealed potential for potent GP inhibition, consistent with that observed previously for the N-(β-D-glucopyranosyl)-3-substituted-1,2,4-triazole-5-carboxamides analogues.Synthesis and subsequent kinetic evaluation of GP inhibition revealed this to be the case.As a continuation of the bioisosteric replacement studies in I, and based on the experiences with IV and V, computational, synthetic and enzyme kinetic investigation of imidazole-containing compounds was undertaken in this work (Table 1, target compounds 1 and 2).Docking predictions for the bound states and DFT calculations on the unbound states revealed potential for potent GP inhibition, consistent with that observed previously for the N-(β-D-glucopyranosyl)-3-substituted-1,2,4-triazole-5-carboxamides analogues.Synthesis and subsequent kinetic evaluation of GP inhibition revealed this to be the case.As a continuation of the bioisosteric replacement studies in I, and based on the experiences with IV and V, computational, synthetic and enzyme kinetic investigation of imidazole-containing compounds was undertaken in this work (Table 1, target compounds  1 and 2 As a continuation of the bioisosteric replacement studies in I, and based on the experiences with IV and V, computational, synthetic and enzyme kinetic investigation of imidazole-containing compounds was undertaken in this work (Table 1, target compounds 1 and 2).Docking predictions for the bound states and DFT calculations on the unbound states revealed potential for potent GP inhibition, consistent with that observed previously for the N-(β-D-glucopyranosyl)-3-substituted-1,2,4-triazole-5-carboxamides analogues.Synthesis and subsequent kinetic evaluation of GP inhibition revealed this to be the case.
As a continuation of the bioisosteric replacement studies in I, and based on the experiences with IV and V, computational, synthetic and enzyme kinetic investigation of imidazole-containing compounds was undertaken in this work (Table 1, target compounds 1 and 2).Docking predictions for the bound states and DFT calculations on the unbound states revealed potential for potent GP inhibition, consistent with that observed previously for the N-(β-D-glucopyranosyl)-3-substituted-1,2,4-triazole-5-carboxamides analogues.Synthesis and subsequent kinetic evaluation of GP inhibition revealed this to be the case.

Computational 2.1.1. Unbound State Calculations
For the ligands, using Ar = phenyl as a prototype for the calculations, we first computed the preferred unbound state ionisation/tautomer states and the conformations of the prototype target ligands 1a and 2a, which would have a considerable impact on the binding potential for GP inhibition [18,24,27].The potential for the protonation of the ligands was determined by using DFT and Jaguar pK a calculations, revealing that the ligands would be in a neutral charge state.
The predicted pK a s for the protonated +1-charged compound 1a considering the deprotonation of N2(H) and N4(H) were 4.03 and 2.96, respectively, while for compound 2a, the corresponding values for N1(H) and N4(H) were 2.21 and 1.95, respectively.Considering the unbound tautomeric states of 1a and 2a (with the previously studied IXa also calculated for comparison), Monte Carlo Multiple Minima (MCMM) conformational search calculations were performed and post-processed using DFT (M06-2X/6-31+G*) gasand solution-phase (SM8) energy calculations.Previous successful applications of this approach to deciphering the tautomeric state preferences of β-D-glucose-heterocyclic-type compounds and their influence on binding have been reported [18,27].The results of these calculations are shown in Figure 1.For the IXa and 1a ligands, the gas-and solution-phasefavoured tautomers were consistently t1.For ligand IXa, t1 was predicted to be more favourable in solution by 1.9 kcal/mol, and for ligand 1a, t1 was more strongly favoured, by 4.8 kcal/mol.In the case of 2a, the t1 and t2 tautomers were similar in their gas-phase energies; however, the t1 tautomeric state was more favourable by 1.6 kcal/mol in solution.Solution-phase energies assume priority in terms of the favoured unbound states, and hence t1 was favoured for all three of the prototype ligands IXa, 1a and 2a.A study of the tautomeric preferences of the ligands in the solved PDB complexes revealed that the most stable water solution-phase tautomer was predominantly favoured for binding, depending on the free energy difference between the relevant tautomers [28].

Bound State Calculations
For the bound states, all possible tautomeric states (Table 2) were considered in terms of the binding to GP, with the docking calculations performed using Glide in standard precision (SP) mode.The Jaguar pK a s of the protonated (+1-charged) target prototype ligands 1a (pK a s~3-4) and 2a (pK a s~2) were quite low, as mentioned above.Nevertheless, within the core of a protein matrix, the pH may substantially differ compared to in solvent alone [29].With this in mind, an initial test of the prototype ligands 1a and 2a docked in their protonated state revealed that they did not outscore the neutral tautomers.Only the neutral tautomers, therefore, were considered from hereon.The results of the docking predictions in terms of their Glidescores and docking scores are shown in Table 2. Docking scores incorporate the approximate tautomeric state penalties into their final scores (an adjusted Glidescore) based on the LigPrep (unbound state)-predicted tautomeric state preferences.However, the unbound tautomeric state Monte Carlo/DFT calculations (alternative method) assume greater accuracy and priority in the analysis, as outlined below.
i. 2024, 25, x FOR PEER REVIEW 4 of 21 solution.Solution-phase energies assume priority in terms of the favoured unbound states, and hence t1 was favoured for all three of the prototype ligands IXa, 1a and 2a.A study of the tautomeric preferences of the ligands in the solved PDB complexes revealed that the most stable water solution-phase tautomer was predominantly favoured for binding, depending on the free energy difference between the relevant tautomers [28].

Bound State Calculations
For the bound states, all possible tautomeric states (Table 2) were considered in terms of the binding to GP, with the docking calculations performed using Glide in standard precision (SP) mode.The Jaguar pKas of the protonated (+1-charged) target prototype ligands 1a (pKas~3-4) and 2a (pKas~2) were quite low, as mentioned above.Nevertheless, within the core of a protein matrix, the pH may substantially differ compared to in solvent alone [29].With this in mind, an initial test of the prototype ligands 1a and 2a docked in their protonated state revealed that they did not outscore the neutral tautomers.Only the neutral tautomers, therefore, were considered from hereon.The results of the docking predictions in terms of their Glidescores and docking scores are shown in Table 2. Docking scores incorporate the approximate tautomeric state penalties into their final scores (an adjusted Glidescore) based on the LigPrep (unbound state)-predicted tautomeric state As mentioned, ligands IXa-c were used as a benchmark for comparison.For IXa-c, while the Glidescores were relatively similar for the three tautomers t1-t3, a preference for t1 binding was clearly predicted by the docking scores, which were in the range of −9.42-−10.71for the three aromatic substituents.This is consistent with our DFTcalculated preference for t1 in the unbound state.For 1a-c, the docking scores (and Glidescores) were similar to those of ligands IXa-c.However, the docking scores for 1a-c were comparable for the tautomers t1 (−9.17-−10.33)and t2 (−9.30-−10.72),as were the Glidescores.Given that the t1 tautomer was most favourable in the solution phase by 4.8 kcal/mol, it can be proposed that the compound binds as t1.The binding of the three analogues 1a-c is shown in Figure 2A-C, where we can see that the ligands exploit similar interactions at the GP catalytic site, with the predicted binding poses close to superimposable, apart from the differing aromatic groups.The standard glucose moiety hydrogen bonds involving the hydroxyl groups with the Asn284, His377, Asn484, Glu672 and Gly675 residues were expected and observed.The 2-substituted-imidazole-4(5)-carboxamide is involved in important hydrogen bonding interactions from N(3')H with the His377 backbone's CO (interactions with this group are recognised as crucial to the good inhibitory potential of glucose analogues [12,15]) and close to (~3.2 Å) the hydrogen bonding distance of the ligand O(1') with the Leu136 backbone's NH.The Asn284 backbone's NH is able to exploit a favourable interaction (~2.6 Å) with imidazole N(3).Protein-ligand interaction analysis using the Maestro program [30] reported π-π interactions between the ligand (1a and 1c) imidazoles and the His341 side-chain.For the inhibitor aromatic groups, there were also π-π interactions for all three analogues 1a-c with His341, but cation-π interactions with the +1-charged Arg292 side-chain were best positioned for the bound 1c (2-naphthyl) analogue.
For ligands 2a-c, the docking scores and Glidescores were again quite similar to those of IXa-c.Considering the two tautomers of 2a-c, the docking scores were again similar for t1 (−9.29-−10.59)and t2 (−9.18-−10.55),as were the Glidescores.From the Monte Carlo/DFT unbound state calculations, t1 was the most stable unbound state tautomer in solution by 1.6 kcal/mol, and it can be proposed that the ligands bind in this state.The predicted binding of t1 for ligands 2a-c is shown in Figure 2D-F, where, again, we can see that the three analogues bind in a similar manner.Indeed, the ligand poses are close to superimposable with the respective 1a-c ligands (i.e., including the aromatic groups).The protein-ligand interactions include the aforementioned standard glucose moiety hydroxyl interactions at the binding site, described above for 1a-c.The 4(5)-substituted-imidazole-2carboxamide is involved in hydrogen bond interactions with the His377 backbone's CO through ligand N(3 ′ )H.The O(1 ′ ) is close to hydrogen bonding distance from the NH in Leu136 (~3.1 Å), as also observed for 1a-c.However, for 2a-c, the imidazole N(1)H also is directed towards the His377 backbone's CO, although the distances range between 3.3 and 3.6 Å in the predicted models.As with 1a-c, the ligand aromatic groups are predicted to form π-π interactions with the His341 sidechain, and in the case of 1c, cation-π interactions involving the Arg292 side-chain with the 2-naphthyl ring.
are predicted to form π-π interactions with the His341 sidechain, and in the case of 1c, cation-π interactions involving the Arg292 side-chain with the 2-naphthyl ring.

Kinetics
Inhibitory constants (K i ) were determined for rabbit muscle glycogen phosphorylase.Enzyme activity was assayed in the direction of glycogen synthesis, as previously published [39].The experiments were performed using the rabbit muscle phosphorylase b (dephosphorylated, GPb) isoform.Kinetic data for the inhibition of GPb by the compounds were obtained in the presence of 10 µg/mL of the enzyme, varying the concentrations of α-D-glucose-1-phosphate (4-40 mM) and using constant concentrations (1%) of glycogen and AMP (1 mM).The enzymatic activities were presented in the form of a double reciprocal plot (Lineweaver-Burk).The plots were analysed using a non-linear data analysis program.The inhibitor constants (K i ) were determined using secondary plots, replotting the slopes from the Lineweaver-Burk plot against the inhibitor concentrations.The means of the standard errors for all calculated kinetic parameters averaged to less than 10% [40].
The results of the kinetics experiments for the target and synthesised compounds 1a-c and 2a-c are shown in Table 5.All the ligands were revealed as low micromolar potent inhibitors of rmGPb (≤16 µM), but with 2a being slightly less potent (K i = 67.4µM).The means of the standard errors for all calculated kinetic parameters averaged to less than 10% [40].
The results of the kinetics experiments for the target and synthesised compounds 1ac and 2a-c are shown in Table 5.All the ligands were revealed as low micromolar potent inhibitors of rmGPb (≤16 µM), but with 2a being slightly less potent (Ki = 67.4µM).The better potency of the 2b-c ligands (Kis~3-4 µM) compared to their 1b-c (Kis~13-16 µM) counterparts is likely due to the potential of additional interactions from imidazole N(1)H with the His377 backbone's CO (predicted as slightly longer than hydrogen bonding, Figure 2), in combination with those from carboxamide N(3')H.It is unclear, however, regarding the degree of the 2a ligands' lesser potency, except that it is unable to exploit the same interactions as the more extended 1-and 2-naphthyl groups in the GP catalytic site β-cavity.This, however, was not the case for 1a (Ki = 10.4 µM).The previously reported benchmark ligand IXa had a Ki of 1 µM in comparison; however, both 1c (Ki = 12.8 µM) and 2c (Ki = 3.3 µM) were similar or more potent, respectively, compared to the related IXc (Ki = 9.2 µM) compound.

Conclusions
Glycogen phosphorylase is an important therapeutic target for predominant conditions such as T2D and cancer.The design of effective glucose analogue inhibitors targeting the catalytic site is strongly dependent on the structure of the linker group, linking the glucose moiety with different (mainly aromatic) groups that extend into the catalytic site's β-cavity.In this study, we investigated the potential of ligands 1 and 2 with imidazole-4(5)-carboxamide (1) and imidazole-2-carboxamide (2)-type linkers in terms of their GP inhibitory potential, as a follow-up study to the previously reported 1,2,4-triazole-5-carboxamide (IX) linkers [24].Monte Carlo and DFT calculations on the unbound states of the ligands considering the different tautomers and Glide docking calculations for the prediction of the protein-ligand binding modes to GP predicted that both linkers may have at least similar effectiveness to the 1,2,4-triazole-5-carboxamide (IX), and ligands 1a-replotting the slopes from the Lineweaver-Burk plot against the inhibitor concentrations.The means of the standard errors for all calculated kinetic parameters averaged to less than 10% [40].
The results of the kinetics experiments for the target and synthesised compounds 1ac and 2a-c are shown in Table 5.All the ligands were revealed as low micromolar potent inhibitors of rmGPb (≤16 µM), but with 2a being slightly less potent (Ki = 67.4µM).The better potency of the 2b-c ligands (Kis~3-4 µM) compared to their 1b-c (Kis~13-16 µM) counterparts is likely due to the potential of additional interactions from imidazole N(1)H with the His377 backbone's CO (predicted as slightly longer than hydrogen bonding, Figure 2), in combination with those from carboxamide N(3')H.It is unclear, however, regarding the degree of the 2a ligands' lesser potency, except that it is unable to exploit the same interactions as the more extended 1-and 2-naphthyl groups in the GP catalytic site β-cavity.This, however, was not the case for 1a (Ki = 10.4 µM).The previously reported benchmark ligand IXa had a Ki of 1 µM in comparison; however, both 1c (Ki = 12.8 µM) and 2c (Ki = 3.3 µM) were similar or more potent, respectively, compared to the related IXc (Ki = 9.2 µM) compound.

Conclusions
Glycogen phosphorylase is an important therapeutic target for predominant conditions such as T2D and cancer.The design of effective glucose analogue inhibitors targeting the catalytic site is strongly dependent on the structure of the linker group, linking the glucose moiety with different (mainly aromatic) groups that extend into the catalytic site's β-cavity.In this study, we investigated the potential of ligands 1 and 2 with imidazole-4(5)-carboxamide (1) and imidazole-2-carboxamide (2)-type linkers in terms of their GP inhibitory potential, as a follow-up study to the previously reported 1,2,4-triazole-5-carboxamide (IX) linkers [24].Monte Carlo and DFT calculations on the unbound states of the ligands considering the different tautomers and Glide docking calculations for the prediction of the protein-ligand binding modes to GP predicted that both linkers may have at least similar effectiveness to the 1,2,4-triazole-5-carboxamide (IX), and ligands 1a-replotting the slopes from the Lineweaver-Burk plot against the inhibitor concentrations.The means of the standard errors for all calculated kinetic parameters averaged to less than 10% [40].
The results of the kinetics experiments for the target and synthesised compounds 1ac and 2a-c are shown in Table 5.All the ligands were revealed as low micromolar potent inhibitors of rmGPb (≤16 µM), but with 2a being slightly less potent (Ki = 67.4µM).The better potency of the 2b-c ligands (Kis~3-4 µM) compared to their 1b-c (Kis~13-16 µM) counterparts is likely due to the potential of additional interactions from imidazole N(1)H with the His377 backbone's CO (predicted as slightly longer than hydrogen bonding, Figure 2), in combination with those from carboxamide N(3')H.It is unclear, however, regarding the degree of the 2a ligands' lesser potency, except that it is unable to exploit the same interactions as the more extended 1-and 2-naphthyl groups in the GP catalytic site β-cavity.This, however, was not the case for 1a (Ki = 10.4 µM).The previously reported benchmark ligand IXa had a Ki of 1 µM in comparison; however, both 1c (Ki = 12.8 µM) and 2c (Ki = 3.3 µM) were similar or more potent, respectively, compared to the related IXc (Ki = 9.2 µM) compound.

Conclusions
Glycogen phosphorylase is an important therapeutic target for predominant conditions such as T2D and cancer.The design of effective glucose analogue inhibitors targeting the catalytic site is strongly dependent on the structure of the linker group, linking the glucose moiety with different (mainly aromatic) groups that extend into the catalytic site's β-cavity.In this study, we investigated the potential of ligands 1 and 2 with imidazole-4(5)-carboxamide (1) and imidazole-2-carboxamide (2)-type linkers in terms of their GP inhibitory potential, as a follow-up study to the previously reported 1,2,4-triazole-5-carboxamide (IX) linkers [24].Monte Carlo and DFT calculations on the unbound states of the ligands considering the different tautomers and Glide docking calculations for the prediction of the protein-ligand binding modes to GP predicted that both linkers may have at least similar effectiveness to the 1,2,4-triazole-5-carboxamide (IX), and ligands 1a- analysis program.The inhibitor constants (Ki) were determined using secondary plots, replotting the slopes from the Lineweaver-Burk plot against the inhibitor concentrations.
The means of the standard errors for all calculated kinetic parameters averaged to less than 10% [40].
The results of the kinetics experiments for the target and synthesised compounds 1ac and 2a-c are shown in Table 5.All the ligands were revealed as low micromolar potent inhibitors of rmGPb (≤16 µM), but with 2a being slightly less potent (Ki = 67.4µM).The better potency of the 2b-c ligands (Kis~3-4 µM) compared to their 1b-c (Kis~13-16 µM) counterparts is likely due to the potential of additional interactions from imidazole N(1)H with the His377 backbone's CO (predicted as slightly longer than hydrogen bonding, Figure 2), in combination with those from carboxamide N(3')H.It is unclear, however, regarding the degree of the 2a ligands' lesser potency, except that it is unable to exploit the same interactions as the more extended 1-and 2-naphthyl groups in the GP catalytic site β-cavity.This, however, was not the case for 1a (Ki = 10.4 µM).The previously reported benchmark ligand IXa had a Ki of 1 µM in comparison; however, both 1c (Ki = 12.8 µM) and 2c (Ki = 3.3 µM) were similar or more potent, respectively, compared to the related IXc (Ki = 9.2 µM) compound.

Conclusions
Glycogen phosphorylase is an important therapeutic target for predominant conditions such as T2D and cancer.The design of effective glucose analogue inhibitors targeting the catalytic site is strongly dependent on the structure of the linker group, linking the glucose moiety with different (mainly aromatic) groups that extend into the catalytic site's β-cavity.In this study, we investigated the potential of ligands 1 and 2 with imidazole-4(5)-carboxamide (1) and imidazole-2-carboxamide (2)-type linkers in terms of their GP inhibitory potential, as a follow-up study to the previously reported 1,2,4-triazole-5-carboxamide (IX) linkers [24].Monte Carlo and DFT calculations on the unbound states of the ligands considering the different tautomers and Glide docking calculations for the prediction of the protein-ligand binding modes to GP predicted that both linkers may have at least similar effectiveness to the 1,2,4-triazole-5-carboxamide (IX), and ligands 1a- analysis program.The inhibitor constants (Ki) were determined using secondary plots, replotting the slopes from the Lineweaver-Burk plot against the inhibitor concentrations.
The means of the standard errors for all calculated kinetic parameters averaged to less than 10% [40].
The results of the kinetics experiments for the target and synthesised compounds 1ac and 2a-c are shown in Table 5.All the ligands were revealed as low micromolar potent inhibitors of rmGPb (≤16 µM), but with 2a being slightly less potent (Ki = 67.4µM).The better potency of the 2b-c ligands (Kis~3-4 µM) compared to their 1b-c (Kis~13-16 µM) counterparts is likely due to the potential of additional interactions from imidazole N(1)H with the His377 backbone's CO (predicted as slightly longer than hydrogen bonding, Figure 2), in combination with those from carboxamide N(3')H.It is unclear, however, regarding the degree of the 2a ligands' lesser potency, except that it is unable to exploit the same interactions as the more extended 1-and 2-naphthyl groups in the GP catalytic site β-cavity.This, however, was not the case for 1a (Ki = 10.4 µM).The previously reported benchmark ligand IXa had a Ki of 1 µM in comparison; however, both 1c (Ki = 12.8 µM) and 2c (Ki = 3.3 µM) were similar or more potent, respectively, compared to the related IXc (Ki = 9.2 µM) compound.

Conclusions
Glycogen phosphorylase is an important therapeutic target for predominant conditions such as T2D and cancer.The design of effective glucose analogue inhibitors targeting the catalytic site is strongly dependent on the structure of the linker group, linking the glucose moiety with different (mainly aromatic) groups that extend into the catalytic site's β-cavity.In this study, we investigated the potential of ligands 1 and 2 with imidazole-4(5)-carboxamide (1) and imidazole-2-carboxamide (2)-type linkers in terms of their GP inhibitory potential, as a follow-up study to the previously reported 1,2,4-triazole-5-carboxamide (IX) linkers [24].Monte Carlo and DFT calculations on the unbound states of the ligands considering the different tautomers and Glide docking calculations for the prediction of the protein-ligand binding modes to GP predicted that both linkers may have at least similar effectiveness to the 1,2,4-triazole-5-carboxamide (IX), and ligands 1a- The better potency of the 2b-c ligands (K i s~3-4 µM) compared to their 1b-c (K i s~13-16 µM) counterparts is likely due to the potential of additional interactions from imidazole N(1)H with the His377 backbone's CO (predicted as slightly longer than hydrogen bonding, Figure 2), in combination with those from carboxamide N(3')H.It is unclear, however, regarding the degree of the 2a ligands' lesser potency, except that it is unable to exploit the same interactions as the more extended 1-and 2-naphthyl groups in the GP catalytic site β-cavity.This, however, was not the case for 1a (K i = 10.4 µM).The previously reported benchmark ligand IXa had a K i of 1 µM in comparison; however, both 1c (K i = 12.8 µM) and 2c (K i = 3.3 µM) were similar or more potent, respectively, compared to the related IXc (K i = 9.2 µM) compound.

Conclusions
Glycogen phosphorylase is an important therapeutic target for predominant conditions such as T2D and cancer.The design of effective glucose analogue inhibitors targeting the catalytic site is strongly dependent on the structure of the linker group, linking the glucose moiety with different (mainly aromatic) groups that extend into the catalytic site's β-cavity.In this study, we investigated the potential of ligands 1 and 2 with imidazole-4(5)-carboxamide (1) and imidazole-2-carboxamide (2)-type linkers in terms of their GP inhibitory potential, as a follow-up study to the previously reported 1,2,4-triazole-5-carboxamide (IX) linkers [24].Monte Carlo and DFT calculations on the unbound states of the ligands considering the different tautomers and Glide docking calculations for the prediction of the protein-ligand binding modes to GP predicted that both linkers may have at least similar effectiveness to the 1,2,4-triazole-5-carboxamide (IX), and ligands 1a-c and 2a-c with phenyl, 1-naphthyl and 2-naphthyl groups were considered for subsequent synthesis.The key step in the syntheses of the target compounds 1a-c and 2a-c was the formation of an amide bond between per-O-acetylated β-D-glucopyranosylamine and the corresponding arylimidazole-carboxylic acids.Enzyme activity was assayed in the direction of glycogen synthesis using the rabbit muscle glycogen phosphorylase b (dephosphorylated, GPb) isoform.These kinetic data revealed that all the synthesised compounds are low micromolar inhibitors of rmGPb (≤16 µM), but with 2a being slightly less potent.Novel potent inhibitors of GP have been reported which can be considered in cellular models of disease, as well as facilitating the structure-based design of new glucose analogue GP inhibitors.For example, their effects on glycogenolysis at the cellular level could next be probed [18]; also, the effectiveness of glucose analogue GP inhibitors against glioblastoma would be of interest [11].The importance of consideration of the tautomeric preferences of ligands in protein-ligand binding studies has again been highlighted in this study, as well as the usefulness of DFT calculations for this purpose [27].

Protein Preparation
Using the co-crystallised complex of rmGPb with a 3-(β-D-glucopyranosyl)-5substituted-1,2,4-triazole fluorene derivative (PDB ID: 6F3R) [41], Schrodinger's 'Protein Preparation Wizard' [30] was employed to prepare the GPb receptor structure for the calculations.Water molecules within 5 Å of the cognate ligand were initially retained (deleted for subsequent docking), missing hydrogen atoms added, bond orders assigned and the tautomer/ionisation states of the protein residues established according to pK a predictions using the PROPKA program [42] at a pH of 7. Minimisation was performed using the OPLS3e forcefield [43] to alleviate steric clashes and restrain heavy atoms to root mean square deviation (RMSD) within 0.3 Å of their crystallographic locations.

Ligand Preparation
All the ligands (IXa-c, 1a-c and 2a-c) were prepared for calculations using Schrodinger's Maestro and the LigPrep 5.6 program with Epik.[30] Favourable minimised tautomeric/ ionisation states were produced for each ligand with a target pH of 7 ± 2. The 1,2,4-triazole (IX) linker produced three tautomeric forms; the imidazole ligands 1 and 2 both produced two tautomers, as shown in Figure 1.To check the potential for protonation (+1 charge) of the imidazole heterocycles, Jaguar pK a calculations [30] were performed on 1a (pK a ~3-4) and 2a (pK a ~2), indicating these ligand states are not favoured in the free state and are unlikely in the bound state.
To calculate the unbound state tautomeric preferences, Monte Carlo Multiple Minima (MCMM) conformational searches using MacroModel 13.0 [30] were initially performed on the unbound prototype IXa, 1a and 2a ligands in their different tautomeric states.A total of 20,000 Monte Carlo steps were used, each followed by 200-step minimisation (truncated Newton conjugate gradient method); the OPLS3e forcefield was used together with an energy window of 7.5 kcal/mol above the global minimum to store the conformations.Redundant conformations were considered using an RMSD of 1.0 Å.The generated conformations were post-processed using DFT gas-phase (M06-2X/6-31 + G*) [ [44][45][46] optimisations, followed by solution-phase single-point energy calculations at the gas-phase optimum geometries (most stable conformations determined).For this purpose, M06-2X/6-31 + G* together with the SM8 continuum model [47] for water solvation were employed.The DFT calculations were performed using Jaguar 11.0.[30]

Docking Details
For the docking calculations using Glide 8.9 [30], the GPb catalytic binding site's shape and properties were mapped onto grids with dimensions of 27.6 Å × 27.6 Å × 27.6 Å centred on the native co-crystallised ligand (PDB code: 6F3R).The default parameters were applied, including van der Waals radius scaling (by 0.8).Positional constraints (1.0 Å) were applied to the four glucose hydroxyl O atoms to retain the β-D-glucopyranosyl moiety in its well-established crystallographic location.The docking calculations were performed in standard precision (SP) mode, including rewards for intra-molecular hydrogen bonds, enhanced planarity for conjugated π-groups and post-docking minimisation with strain correction, with up to 10 poses per ligand saved.

General Methods
The solvents were purified by distillation.Dichloromethane (DCM) was refluxed and distilled from P 4 O 10 and stored over 4 Å molecular sieves.Methanol was refluxed with magnesium turnings and iodine for a couple of hours and was distilled.Further, 1,4dioxane was distilled from sodium sand and stored over sodium wires.TLC was performed using DC Kieselgel 60 F 254 (Merck, Rahway, NJ, USA) plates, developed under 254 nm UV light and/or sprayed with EtOH/cc.H 2 SO 4 /p-anisaldehyde (96:5:1) and heated to 150 • C. For column chromatography, the Kieselgel 60 (Merck, particle size 0.063-0.200mm) was used.Optical rotations were determined using a Jasco P-2000 polarimeter at 25 • C. The NMR spectra were recorded using a Bruker Avance 400 MHz (400/100 MHz for 1 H/ 13 C) spectrometer at 298 ± 0.1 K.Chemical shifts are referenced to TMS or the residual solvent peaks.Chemical shifts are reported in ppm.All the compounds were characterised according to their one-( 1 H and 13 C) and two-dimensional (COSY, HSQC, HMBC) NMR spectra.High-resolution mass spectra were recorded using a Bruker maXis II UHR ESI-TOF MS instrument in positive mode.The 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosylamine (9) [48], 2-amino-1-arylethan-1-one hydrochlorides [33] and ethyl 2-bromo-1H-imidazole-4(5)-carboxylate (3) [31] were synthesised according to the procedures in the literature.3) (1 equiv.),arylboronic acid (2 equiv.)and Pd(amphos)Cl 2 (0.05 equiv.)were dissolved in dry 1,4-dioxane (2 mL/mmol as the starting materials), and then dry TEA (2 equiv.) was added to the solution.The mixture was stirred under an inert atmosphere (argon) at 140 • C for 2 h using MW irradiation.The reaction mixture was diluted with water and extracted with EtOAc.The combined organic layers were washed with water and brine, dried over MgSO 4 and filtered, and the solvent was removed under reduced pressure.The crude product was purified using column chromatography.

General Procedure B for the Synthesis of Ethyl 4(5)-aryl-1H-imidazole-2carboxylate Derivatives
To the solution of ethyl thiooxamate (1 equiv.) in dry DCM (2 mL/mmol), a solution of triethyloxonium tetrafluoroborate (1 equiv.) in dry DCM (2 mL/mmol) was added dropwise, and the resulting mixture was stirred at room temperature for 16 h and then evaporated to dryness.The residue was dissolved in glacial acetic acid (2 mL/mmol) and anhydrous NaOAc (2 equiv.),2-amino-1-arylethan-1-one hydrochloride (1 equiv.) was added to the solution and the mixture was stirred at 70 • C under an inert atmosphere (argon) for 2 h.The reaction mixture was concentrated under reduced pressure.Then, water was added, and the mixture was neutralised with saturated NaHCO 3 solution and extracted with EtOAc.The combined organic layers were washed with water and brine, dried over MgSO 4 and filtered, and the solvent was removed under reduced pressure.The crude product was purified using column chromatography.

General Procedure C for the Ester Hydrolysis
The corresponding ethyl aryl-1H-imidazole-carboxylate (1 equiv.) was dissolved/ suspended in EtOH (2 mL), and a solution of LiOH • H 2 O (3 equiv.) in water (2 mL) was added to it.The resulting mixture was stirred overnight at 50 • C. Subsequently, the pH was set to 5-6 with 1M HCl solution; then, the solvent was removed under reduced pressure, and the residue was purified using column chromatography.To the solution of 2-aryl-1H-imidazole-4(5)-carboxylic acid (1 equiv.) in dry DMF (1 mL), DIPEA (5 equiv.)and HATU (1.2 equiv.)were added, and the mixture was stirred for 15 min; then, a solution of 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl amine (9) (1.5 equiv.) in dry DMF (1 mL) was added to it.The reaction mixture was stirred overnight at room temperature.Then, the reaction mixture was diluted with water and extracted with EtOAc.The combined organic layers were washed with water and brine, dried over MgSO 4 and filtered, and the solvent was removed under reduced pressure.The crude product was purified using column chromatography.To the solution of a 4(5)-aryl-1H-imidazole-2-carboxylic acid (1 equiv.) in dry DMF (2 mL), a 50% solution of T3P in DMF (3 equiv.)and dry TEA (3 equiv.)were added, and the resulting mixture was stirred for 15 min.Then, a solution of 2,3,4,6-tetra-O-acetylβ-D-glucopyranosyl amine (1 equiv.) in dry DMF (1 mL) was added to it.The reaction mixture was stirred overnight at room temperature.The reaction mixture was diluted with water and extracted with EtOAc.The combined organic layers were washed with water and brine, dried over MgSO 4 and filtered, and the solvent was removed under reduced pressure.The crude product was purified using column chromatography.

21 VIa
). Docking predictions for the bound states and DFT calculations on the unbound states revealed potential for potent GP inhibition, consistent with that observed previously for the N-(β-D-glucopyranosyl)-3-substituted-1,2,4-triazole-5-carboxamides analogues.Synthesis and subsequent kinetic evaluation of GP inhibition revealed this to be the case.Target compounds in this study 1 Int.J. Mol.Sci.2024, 25, x FOR PEER REVIEW 3 of Inhibition data refer to rabbit muscle glycogen phosphorylase b (rmGPb).b Calculated from the IC50 values using the Cheng-Prusoff equation: Ki = IC50/(1 + [S]/Km).

Figure 1 .
Figure 1.A comparison of the various tautomeric forms of prototype benchmark IXa and prototype target 1a and 2a ligands with their predicted relative energies (kcal mol −1 ) calculated using DFT.Gas-phase energies were from M06-2X/6-31+G* optimisations, with solution-phase energies (M06-2X/6-31+G* and SM8 model of water solvation) calculated as single-point energies at the gas-phase optimised geometries.

Figure 1 .
Figure 1.A comparison of the various tautomeric forms of prototype benchmark IXa and prototype target 1a and 2a ligands with their predicted relative energies (kcal mol −1 ) calculated using DFT.Gas-phase energies were from M06-2X/6-31+G* optimisations, with solution-phase energies (M06-2X/6-31+G* and SM8 model of water solvation) calculated as single-point energies at the gas-phase optimised geometries.

Figure 2 .
Figure 2. Predicted GP-binding interactions of target ligands 1a-c in their respective panels (A-C) and 2a-c shown in their respective panels (D-F).Binding poses were based on Glide SP docking calculations, as described in the text.Hydrogen bonds are shown as black dash lines; π-π interactions as dashed cyan lines; cation-π interactions as dashed green lines.

Figure 2 .
Figure 2. Predicted GP-binding interactions of target ligands 1a-c in their respective panels (A-C) and 2a-c shown in their respective panels (D-F).Binding poses were based on Glide SP docking calculations, as described in the text.Hydrogen bonds are shown as black dash lines; π-π interactions as dashed cyan lines; cation-π interactions as dashed green lines.

Table 5 .
Inhibition constants (K i s in µM) for the synthesised N-(β-D-glucopyranosyl) imidazolecarboxamides a .Compound Ar replotting the slopes from the Lineweaver-Burk plot against the inhibitor concentrations.

Table 5 . 2 a
Inhibition constants (Kis in µM) for the synthesised N-(β-D-glucopyranosyl) imidazolecar-All Ki calculations were carried out using the GraphPad Prism5 (GraphPad Software, La Jolla, CA, USA) software.The results of kinetic experiments were calculated from at least three independent experiments.The standard deviations were calculated based on the average values for the independent experiments.

Table 5 . 2 a
Inhibition constants (Kis in µM) for the synthesised N-(β-D-glucopyranosyl) imidazolecar-All Ki calculations were carried out using the GraphPad Prism5 (GraphPad Software, La Jolla, CA, USA) software.The results of kinetic experiments were calculated from at least three independent experiments.The standard deviations were calculated based on the average values for the independent experiments.

Table 5 . 2 a
Inhibition constants (Kis in µM) for the synthesised N-(β-D-glucopyranosyl) imidazolecar-All Ki calculations were carried out using the GraphPad Prism5 (GraphPad Software, La Jolla, CA, USA) software.The results of kinetic experiments were calculated from at least three independent experiments.The standard deviations were calculated based on the average values for the independent experiments.

Table 5 . 2 a
Inhibition constants (Kis in µM) for the synthesised N-(β-D-glucopyranosyl) imidazolecar-All Ki calculations were carried out using the GraphPad Prism5 (GraphPad Software, La Jolla, CA, USA) software.The results of kinetic experiments were calculated from at least three independent experiments.The standard deviations were calculated based on the average values for the independent experiments.

Table 5 . 2 a
Inhibition constants (Kis in µM) for the synthesised N-(β-D-glucopyranosyl) imidazolecar-All Ki calculations were carried out using the GraphPad Prism5 (GraphPad Software, La Jolla, CA, USA) software.The results of kinetic experiments were calculated from at least three independent experiments.The standard deviations were calculated based on the average values for the independent experiments.

2 a
All K i calculations were carried out using the GraphPad Prism5 (GraphPad Software, La Jolla, CA, USA) software.The results of kinetic experiments were calculated from at least three independent experiments.The standard deviations were calculated based on the average values for the independent experiments.

Table 1 .
Selected glucose-derived inhibitors of glycogen phosphorylase (K i [µM]) a and the target compounds of this study.
Submicromolar Inhibitors with Diverse Scaffolds

Table 1 .
Selected glucose-derived inhibitors of glycogen phosphorylase (Ki [µM]) a and the target compounds of this study.

Table 1 .
Selected glucose-derived inhibitors of glycogen phosphorylase (Ki [µM]) a and the target compounds of this study.

Table 1 .
Selected glucose-derived inhibitors of glycogen phosphorylase (Ki [µM]) a and the target compounds of this study.

Table 2 .
Bound state calculation Glide SP docking results with Glidescores and docking scores (in parentheses) for each ligand tautomer of the target ligands 1a-c and 2a-c.Corresponding results for the previously studied IX scaffold[24]are given for comparison.