Synthesis, Kinetic and Conformational Studies of 2-Substituted-5-(ß-d-glucopyranosyl)-pyrimidin-4-ones as Potential Inhibitors of Glycogen Phosphorylase.

Dysregulation of glycogen phosphorylase, an enzyme involved in glucose homeostasis, may lead to a number of pathological states such as type 2 diabetes and cancer, making it an important molecular target for the development of new forms of pharmaceutical intervention. Based on our previous work on the design and synthesis of 4-arylamino-1-(ß-d-glucopyranosyl)pyrimidin-2-ones, which inhibit the activity of glycogen phosphorylase by binding at its catalytic site, we report herein a general synthesis of 2-substituted-5-(ß-d-glucopyranosyl)pyrimidin-4-ones, a related class of metabolically stable, C-glucosyl-based, analogues. The synthetic development consists of a metallated heterocycle, produced from 5-bromo-2-methylthiouracil, in addition to protected d-gluconolactone, followed by organosilane reduction. The methylthio handle allowed derivatization through hydrolysis, ammonolysis and arylamine substitution, and the new compounds were found to be potent (µM) inhibitors of rabbit muscle glycogen phosphorylase. The results were interpreted with the help of density functional theory calculations and conformational analysis and were compared with previous findings.

Synthesis of New C- and N-ß-d-Glucopyranosyl Derivatives of Imidazole, 1,2,3-Triazole and Tetrazole, and Their Evaluation as Inhibitors of Glycogen Phosphorylase.

The aim of the present study was to broaden the structure-activity relationships of C- and N-ß-d-glucopyranosyl azole type inhibitors of glycogen phosphorylase. 1-Aryl-4-ß-d-gluco-pyranosyl-1,2,3-triazoles were prepared by copper catalyzed azide-alkyne cycloadditions between O-perbenzylated or O-peracetylated ß-d-glucopyranosyl ethynes and aryl azides. 1-ß-d-Gluco-pyranosyl-4-phenyl imidazole was obtained in a glycosylation of 4(5)-phenylimidazole with O-peracetylated α-d-glucopyranosyl bromide. C-ß-d-Glucopyranosyl-N-substituted-tetrazoles were synthesized by alkylation/arylation of O-perbenzoylated 5-ß-d-glucopyranosyl-tetrazole or from a 2,6-anhydroheptose tosylhydrazone and arenediazonium salts. 5-Substituted tetrazoles were glycosylated by O-peracetylated α-d-glucopyranosyl bromide to give N-ß-d-glucopyranosyl-C-substituted-tetrazoles. Standard deprotections gave test compounds which were assayed against rabbit muscle glycogen phosphorylase b. Most of the compounds proved inactive, the best inhibitor was 2-ß-d-glucopyranosyl-5-phenyltetrazole (IC50 600 µM). These studies extended the structure-activity relationships of ß-d-glucopyranosyl azole type inhibitors and revealed the extreme sensitivity of such type of inhibitors towards the structure of the azole moiety.

Glucopyranosylidene-spiro-iminothiazolidinone, a new bicyclic ring system: synthesis, derivatization, and evaluation for inhibition of glycogen phosphorylase by enzyme kinetic and crystallographic methods.

The reaction of thiourea with O-perbenzoylated C-(1-bromo-1-deoxy-ß-D-glucopyranosyl)formamide gave the new anomeric spirocycle 1R-1,5-anhydro-D-glucitol-spiro-[1,5]-2-imino-1,3-thiazolidin-4-one. Acylation and sulfonylation with the corresponding acyl chlorides (RCOCl or RSO2Cl where R=tBu, Ph, 4-Me-C6H4, 1- and 2-naphthyl) produced the corresponding 2-acylimino- and 2-sulfonylimino-thiazolidinones, respectively. Alkylation by MeI, allyl-bromide and BnBr produced mixtures of the respective N-alkylimino- and N,N'-dialkyl-imino-thiazolidinones, while reactions with 1,2-dibromoethane and 1,3-dibromopropane furnished spirocyclic 5,6-dihydro-imidazo[2,1-b]thiazolidin-3-one and 6,7-dihydro-5H-thiazolidino[3,2-a]pyrimidin-3-one, respectively. Removal of the O-benzoyl protecting groups by the Zemplén protocol led to test compounds most of which proved micromolar inhibitors of rabbit muscle glycogen phosphorylase b (RMGPb). Best inhibitors were the 2-benzoylimino- (Ki=9µM) and the 2-naphthoylimino-thiazolidinones (Ki=10 µM). Crystallographic studies of the unsubstituted spiro-thiazolidinone and the above most efficient inhibitors in complex with RMGPb confirmed the preference and inhibitory effect that aromatic (and especially 2-naphthyl) derivatives show for the catalytic site promoting the inactive conformation of the enzyme.

Synthesis of tartaric acid analogues of FR258900 and their evaluation as glycogen phosphorylase inhibitors.

Di-O-cinnamoylated, -p-coumaroylated, and -feruloylated d-, l- and meso-tartaric acids were synthesized as analogues of the natural product FR258900, a glycogen phosphorylase (GP) inhibitor with in vivo antihyperglycaemic activity. The new compounds inhibited rabbit muscle GP in the low micromolar range, and bound to the allosteric site of the enzyme. The best inhibitor was 2,3-di-O-feruloyl meso-tartaric acid and had Ki values of 2.0µM against AMP (competitive) and 3.36µM against glucose-1-phosphate (non-competitive).

Design and synthesis of novel photoaffinity probes for study of the target proteins of oleanolic acid.

To explore the molecular mechanisms of oleanolic acid, two novel photoaffinity probes were synthesized based on the structure-activity relationship reported previously. Their potency were evaluated in an enzyme inhibition assay against rabbit muscle glycogen phosphorylase a (RMGPa), a known target protein of oleanolic acid. The inhibitory activity of probe 2 was only about two-fold less potent than the mother compound oleanolic acid. The photoaffinity labeling experiments were also performed and two proteins were specifically tagged by probe 2. The results suggest that the synthesized probes could be used as powerful tools to isolate and identify the target proteins of oleanolic acid.

N-(4-Substituted-benzoyl)-N'-(ß-d-glucopyranosyl)ureas as inhibitors of glycogen phosphorylase: Synthesis and evaluation by kinetic, crystallographic, and molecular modelling methods.

N-(4-Substituted-benzoyl)-N'-(ß-d-glucopyranosyl) ureas (substituents: Me, Ph, Cl, OH, OMe, NO(2), NH(2), COOH, and COOMe) were synthesised by ZnCl(2) catalysed acylation of O-peracetylated ß-d-glucopyranosyl urea as well as in reactions of O-peracetylated or O-unprotected glucopyranosylamines and acyl-isocyanates. O-deprotections were carried out by base or acid catalysed transesterifications where necessary. Kinetic studies revealed that most of these compounds were low micromolar inhibitors of rabbit muscle glycogen phosphorylase b (RMGPb). The best inhibitor was the 4-methylbenzoyl compound (K(i)=2.3µM). Crystallographic analyses of complexes of several of the compounds with RMGPb showed that the analogues exploited, together with water molecules, the available space at the ß-pocket subsite and induced a more extended shift of the 280s loop compared to RMGPb in complex with the unsubstituted benzoyl urea. The results suggest the key role of the water molecules in ligand binding and structure-based ligand design. Molecular docking study of selected inhibitors was done to show the ability of the binding affinity prediction. The binding affinity of the highest scored docked poses was calculated and correlated with experimentally measured K(i) values. Results show that correlation is high with the R-squared (R(2)) coefficient over 0.9.

C-Glucosylated malonitrile as a key intermediate towards carbohydrate-based glycogen phosphorylase inhibitors.

Glycogen utilization involves glycogen phosphorylase, an enzyme which appears to be a potential target for the regulation of glycaemia, as the liver isoform is a major player for hepatic glucose output. A single C-glucosylated malonitrile allowed for the synthesis of three glucose-based derivatives namely bis-oxadiazoles, bis-amides and a C-glucosylated tetrahydropyrimidin-2-one. When evaluated as glycogen phosphorylase inhibitors, two of the synthesized compounds displayed inhibition in the sub-millimolar range. In silico studies revealed that only one out of the bis-amides obtained and the C-glucosylated tetrahydropyrimidin-2-one may bind at the catalytic site.

Synthesis of 1-(D-glucopyranosyl)-1,2,3-triazoles and their evaluation as glycogen phosphorylase inhibitors.

1-(D-Glucopyranosyl)-1,2,3-triazoles were prepared from per-O-acetylated alpha- and beta-D-glucopyranosyl azides as well as per-O-benzoylated (beta-D-gluco-hept-2-ulopyranosylazide)onamide and onic acid methylester by using azide-alkyne cycloaddition catalysed by in situ generated Cu(I) under aqueous conditions. The O-acyl protecting groups were removed by the Zemplén protocol. The test compounds were assayed against rabbit muscle glycogen phosphorylase b to show that the beta-D-glucopyranosyl derivatives were superior inhibitors as compared to the two other series of triazoles.

The binding of ß-d-glucopyranosyl-thiosemicarbazone derivatives to glycogen phosphorylase: A new class of inhibitors.

Glycogen phosphorylase (GP) is a promising target for the treatment of type 2 diabetes. In the process of structure based drug design for GP, a group of 15 aromatic aldehyde 4-(ß-d-glucopyranosyl)thiosemicarbazones have been synthesized and evaluated as inhibitors of rabbit muscle glycogen phosphorylase b (GPb) by kinetic studies. These compounds are competitive inhibitors of GPb with respect to α-d-glucose-1-phosphate with IC(50) values ranging from 5.7 to 524.3µM. In order to elucidate the structural basis of their inhibition, the crystal structures of these compounds in complex with GPb at 1.95-2.23Å resolution were determined. The complex structures reveal that the inhibitors are accommodated at the catalytic site with the glucopyranosyl moiety at approximately the same position as α-d-glucose and stabilize the T conformation of the 280s loop. The thiosemicarbazone part of the studied glucosyl thiosemicarbazones possess a moiety derived from substituted benzaldehydes with NO(2), F, Cl, Br, OH, OMe, CF(3), or Me at the ortho-, meta- or para-position of the aromatic ring as well as a moiety derived from 4-pyridinecarboxaldehyde. These fit tightly into the ß-pocket, a side channel from the catalytic site with no access to the bulk solvent. The differences in their inhibitory potency can be interpreted in terms of variations in the interactions of the aldehyde-derived moiety with protein residues in the ß-pocket. In addition, 14 out of the 15 studied inhibitors were found bound at the new allosteric site of the enzyme.

Inactivation of rabbit muscle glycogen phosphorylase b by peroxynitrite revisited: does the nitration of Tyr613 in the allosteric inhibition site control enzymatic function?

There is increasing evidence that sequence-specific formation of 3-nitrotyrosine (3-NT) may cause functional changes in target proteins. Recently, the nitration of Tyr residues in glycogen phosphorylase b (Ph-b) was implicated in the age-associated decline of protein function [Sharov et al., Exp. Gerontol. 41 (2006) 407-416]; in another report, the nitration of one specific residue, Tyr613, located in the allosteric inhibition site was hypothesized as a rationale for peroxynitrite inactivation [Dairou et al., J. Mol. Biol. 372 (2007) 1009-1021]. In this study, we have optimized the analysis of in-gel Ph-b digests by high performance liquid chromatography-electro spray ionization-tandem mass spectrometry, in order to achieve a quantitative analysis of nitration of individual Tyr residues at a high coverage of Tyr-containing sequences (92%). Our data do not confirm the role of Tyr613 nitration in the control of enzymatic function. Furthermore, we show here that the enzymatic activity of Ph-b does not directly correlate with the protein nitration levels, and that the modification of Cys and, potentially, other amino acid residues can better rationalize Ph-b inactivation by peroxynitrite.

Synthesis and structure-activity relationships of C-glycosylated oxadiazoles as inhibitors of glycogen phosphorylase.

A series of per-O-benzoylated 5-beta-D-glucopyranosyl-2-substituted-1,3,4-oxadiazoles was prepared by acylation of the corresponding 5-(beta-D-glucopyranosyl)tetrazole. As an alternative, oxidation of 2,6-anhydro-aldose benzoylhydrazones by iodobenzene I,I-diacetate afforded the same oxadiazoles. 1,3-Dipolar cycloaddition of nitrile oxides to per-O-benzoylated beta-D-glucopyranosyl cyanide gave the corresponding 5-beta-D-glucopyranosyl-3-substituted-1,2,4-oxadiazoles. The O-benzoyl protecting groups were removed by base-catalyzed transesterification. The 1,3,4-oxadiazoles were practically inefficient as inhibitors of rabbit muscle glycogen phosphorylase b while the 1,2,4-oxadiazoles displayed inhibitory activities in the micromolar range. The best inhibitors were the 5-beta-D-glucopyranosyl-3-(4-methylphenyl- and -2-naphthyl)-1,2,4-oxadiazoles (K(i)=8.8 and 11.6 microM, respectively). A detailed analysis of the structure-activity relationships is presented.

Glucose-based spiro-heterocycles as potent inhibitors of glycogen phosphorylase.

Glucopyranosylidene-spiro-1,4,2-oxathiazoles were prepared in high yields by NBS-mediated spiro-cyclization of the corresponding glucosyl-hydroximothioates. In an effort to synthesize analogous glucopyranosylidene-spiro-1,2,4-oxadiazolines, with a nitrogen atom instead of the sulphur, attempted cyclizations resulted in aromatization of the heterocycle with opening of the pyranosyl ring. Enzymatic measurements showed that some of the glucose-based inhibitors were active in the micromolar range. The 2-naphthyl-substituted 1,4,2-oxathiazole displayed the best inhibition against RMGPb (K(i)=160 nM), among glucose-based inhibitors known to date.

Synthesis and glycogen phosphorylase inhibitory activity of N-(beta-D-glucopyranosyl)amides possessing 1,4-benzodioxane moiety.

A series of N-(beta-D-glucopyranosyl)amides 5d-i were synthesized by PMe(3) mediated Staudinger reaction of O-peracetylated beta-D-glucopyranosyl azide (1) followed by acylation with carboxylic acids 3d-i and subsequent Zemplén deacetylation. The new compounds were tested for their inhibitory activity against rabbit muscle glycogen phosphorylase and the structure-activity relationships of these compounds are also discussed in this paper.

Glycogen phosphorylase a inhibitors with a phenethylphenylphthalimide skeleton derived from thalidomide-related alpha-glucosidase inhibitors and liver X receptor antagonists.

Novel glycogen phosphorylase a (GPa) inhibitors with a phenethylphenylphthalimide skeleton were prepared based on alpha-glucosidase inhibitors and liver X receptor (LXR) antagonists derived from thalidomide. Their structure-activity relationships were analyzed. Some of the compounds thus prepared showed potent inhibitory activity against rabbit muscle GPa with more than 10-fold greater efficacy than a typical GPa inhibitor, 1,4-dideoxy-1,4-imino-D-arabinitol.

Studies on the reversible enzyme reaction of rabbit muscle glycogen phosphorylase b using isothermal titration calorimetry.

Glycogen phosphorylase enzymes (GP) catalyse reversible reactions; the glucose transfer from glycogen to inorganic phosphate (Pi, phosphorolysis) or the reverse glucose transfer from glucose-1-phosphate (G-1-P) to glycogen (synthesis). Rabbit muscle GPb (rmGPb) was used as a model enzyme to study the reversible enzyme reaction. To follow both directions of this reversible reaction, we have developed a novel isothermal titration calorimetry (ITC) method for the determination of the direct reaction rate. The preference of forward or reverse reaction was ensured by the 0.1 or 10 concentration ratios of G-1-P/Pi, respectively. Substrate specificity was studied using different maltooligosaccharides and glycogen. Based on the KM values, glycogen and 2-chloro-4-nitrophenyl maltoheptaoside (CNP-G7) were found to be analogous substrates, which allowed to optimize the method by taking advantage of the CNP chromophore being detectable in HPLC. In case of CNP-G7, substrate inhibition was observed and characterised by Ki of 23 ±â€¯7 mM. Inhibition of human GP is a promising strategy for the treatment of diabetes. Our ITC measurements have confirmed that caffeine and glucopyranosylidene-spiro-thiohydantoin (GTH), as known GPb inhibitors, inhibit the rmGPb-catalysed reversible reaction in both directions. Ki values obtained in the direction of synthesis (1.92 ±â€¯0.14 mM for caffeine and 11.5 ±â€¯2.0 µM for GTH) have been shown to be in good agreement with the Ki values obtained in the direction of phosphorolysis (4.05 ±â€¯0.26 mM for caffeine and 13.8 ±â€¯1.6 µM for GTH). The higher difference between the inhibition constants of caffeine was explained by the non-competitive mechanism. The described ITC method using the developed experimental design and reaction conditions is suitable for activity measurements of different phosphorylase enzymes on various substrates and is applicable for inhibition studies as well.

Synthesis of C-ß-d-glucopyranosyl derivatives of some fused azoles for the inhibition of glycogen phosphorylase.

Annulated C-ß-d-glucopyranosyl heterocycles were synthesized and tested as inhibitors of glycogen phosphorylase. 2-(ß-d-Glucopyranosyl)-1H-imidazo[4,5-b]pyridine was formed by ring-closure of O-perbenzoylated C-ß-d-glucopyranosyl formic acid with 2,3-diaminopyridine in the presence of triphenylphosphite. Cyclisations of bromomethyl 2,3,4,6-tetra-O-benzoyl-ß-d-glucopyranosyl ketone with a set of 2-aminoheterocycles resulted in constitutionally reversed C-ß-d-glucopyranosyl imidazoles fused by pyridine, pyrimidine, thiazole, 1,3,4-thiadiazole, benzothiazole and benzimidazole. O-Debenzoylation of the above compounds was effected by standard transesterification to get the test compounds. The 1H-imidazo[4,5-b]pyridine proved to be a low micromolar inhibitor (Ki = 21.1 µM) of rabbit muscle glycogen phosphorylase b, while the other heterocycles displayed weak or no inhibition against the same enzyme.

Synthesis of a C-glucosylated cyclopropylamide and evaluation as a glycogen phosphorylase inhibitor.

The synthesis of carbohydrate-based glycogen phosphorylase inhibitors is attractive for potential applications in the treatment of type 2 diabetes. A titanium-mediated synthesis led to a benzoylated C-glucosylated cyclopropylamine intermediate, which underwent a benzoyl migration to afford the corresponding 2-hydroxy-C-glycoside. X-ray crystallographic studies revealed a unit cell composed of four molecules as pairs of dimers connected through two hydrogen bonds. The deprotection of the benzoate esters under Zemplén conditions afforded a glycogen phosphorylase inhibitor candidate displaying weak inhibition toward glycogen phosphorylase (16% at 2.5mM).

Evaluation of novel hyphodermin derivatives as glycogen phosphorylase a inhibitors.

The lipophilicity, permeability, solubility, polar surface area and 'rule-of-five' properties were assessed, using QikProp v2.5 (Schrödinger, Inc.) and ALOGPS 2.1 calculations, for 25 Hyphodermin derivatives. These compounds obeyed the 'rule-of-five', and the calculated physicochemical values were generally within desired limits. All compounds were tested against Glycogen Phosphorylase a (GPa). Four phenyl and benzyl substituted 2-oxo-hexahydro and tetrahydrobenzo[cd]indole carboxylic acids were identified as novel inhibitors of GPa with estimated IC(50) values in the range 0.8-1.3mM. Molecular modelling of these novel inhibitors was used to obtain the main structural features of this class of molecule for future structure-activity relationship studies.

Assessment of synthetic methods for the preparation of N-beta-d-glucopyranosyl-N'-substituted ureas, -thioureas and related compounds.

Preparation of O-peracetylated N-beta-d-glucopyranosyl-N'-acyl urea derivatives resulted in the formation of anomeric mixtures under the following conditions: acylation of O-peracetylated beta-d-glucopyranosyl urea by acyl chlorides in the presence of ZnCl(2) in refluxing CHCl(3); addition of O-peracetylated beta-d-glucopyranosylamine to acyl isocyanates in acetonitrile at rt; addition of carboxamides to in situ prepared O-peracetylated beta-d-glucopyranosyl isocyanate in refluxing toluene. Deprotection of O-peracetylated N-beta-d-glucopyranosyl-N'-acyl ureas either under base (NaOMe in MeOH at or below rt) or under acid (KHSO(4) or AcCl in MeOH at rt) catalyzed transesterification conditions resulted in unavoidable partial cleavage of the N'-acyl moieties. Reaction of beta-d-glucopyranosylammonium carbamate with an isocyanate, isothiocyanate or isoselenocyanate in dry pyridine at rt appears as a general method for the preparation of the corresponding beta-d-glucopyranosyl ureas, -thio- and -selenoureas, respectively, inclusive N'-acyl derivatives.

Mechanism of aggregation of UV-irradiated glycogen phosphorylase b at a low temperature in the presence of crowders and trimethylamine N-oxide.

To characterize the initial stages of protein aggregation, the kinetics of aggregation of UV-irradiated glycogen phosphorylase b (UV-Phb) was studied under conditions when the aggregation proceeded at a low rate (10°C, 0.03M Hepes buffer, pH6.8, containing 0.1M NaCl). Aggregation of UV-Phb was induced by polyethylene glycol and Ficoll-70, acting as crowders, or a natural osmolyte trimethylamine N-oxide (TMAO). It has been shown that the initial rate of the stage of aggregate growth is proportional to the protein concentration squared, suggesting that the order of aggregation with respect to the protein is equal to two. It has been concluded that the aggregation mechanism of UV-Phb at 10°C in the presence of crowders includes the nucleation stage and stages of protein aggregate growth (the basic aggregation pathway). The aggregation mechanism is complicated in the presence of TMAO, and the stage of aggregate-aggregate assembly induced by TMAO should be added to the basic aggregation pathway. It has been shown that the ability of TMAO at a low concentration (0.05M) to induce aggregation of UV-Phb is due to the decrease in the absolute value of zeta potential of the protein in the presence of TMAO.