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.

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 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.

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.

Synthesis of C-xylopyranosyl- and xylopyranosylidene-spiro-heterocycles as potential inhibitors of glycogen phosphorylase.

New derivatives of d-xylose with aglycons of the most efficient glucose derived inhibitors of glycogen phosphorylase were synthesized to explore the specificity of the enzyme towards the structure of the sugar part of the molecules. Thus, 2-(ß-d-xylopyranosyl)benzimidazole and 3-substituted-5-(ß-d-xylopyranosyl)-1,2,4-triazoles were obtained in multistep procedures from O-perbenzoylated ß-d-xylopyranosyl cyanide. Cycloadditions of nitrile-oxides and O-peracetylated exo-xylal obtained from the corresponding ß-d-xylopyranosyl cyanide furnished xylopyranosylidene-spiro-isoxazoline derivatives. Oxidative ring closure of O-peracetylated ß-d-xylopyranosyl-thiohydroximates prepared from 1-thio-ß-d-xylopyranose and nitrile-oxides gave xylopyranosylidene-spiro-oxathiazoles. The fully deprotected test compounds were assayed against rabbit muscle glycogen phosphorylase b to show moderate inhibition for 3-(2-naphthyl)-5-(ß-d-xylopyranosyl)-1,2,4-triazole (IC50=0.9mM) only.

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 2-(ß-D-glucopyranosylamino)-5-substituted-1,3,4-oxadiazoles for inhibition of glycogen phosphorylase.

Aromatic aldehyde 4-(2,3,4,6-tetra-O-acetyl-ß-d-glucopyranosyl)semicarbazones were synthesized by the addition of different hydrazones onto O-peracetylated ß-d-glucopyranosyl isocyanate. Oxidative transformations of these precursors gave O-protected 2-(ß-d-glucopyranosylamino)-5-substituted-1,3,4-oxadiazoles. Removal of the O-acetyl protecting groups under Zemplén conditions gave test compounds to show low micromolar inhibition against rabbit muscle glycogen phosphorylase b. Best inhibitors of these series were 4-(ß-d-glucopyranosyl)semicarbazones of 4-nitrobenzaldehyde (Ki=4.5µM), 2-naphthaldehyde (Ki=5.5µM) and 2-(ß-d-glucopyranosylamino)-5-(4-methylphenyl)-1,3,4-oxadiazole (Ki=12µM).

Synthesis of 2-(ß-D-glucopyranosyl)-5-(substituted-amino)-1,3,4-oxa- and -thiadiazoles for the inhibition of glycogen phosphorylase.

O-Perbenzoylated 4-phenyl-[C-(ß-d-glucopyranosyl)formaldehyde]semicarbazone was prepared in the reaction of O-perbenzoylated ß-d-glucopyranosyl cyanide and 4-phenylsemicarbazide in the presence of Raney-Ni. Acylation of O-perbenzoylated C-(ß-d-glucopyranosyl)formaldehyde semicarbazone furnished the corresponding 4-acyl-[C-(ß-d-glucopyranosyl)formaldehyde]semicarbazones. The reaction of O-perbenzoylated C-(ß-d-glucopyranosyl)formaldehyde semicarbazone with the corresponding thiosemicarbazide resulted in O-perbenzoylated C-(ß-d-glucopyranosyl)formaldehyde thiosemicarbazone and its 4-phenyl derivative. Acylation of O-perbenzoylated C-(ß-d-glucopyranosyl)formaldehyde thiosemicarbazone provided the corresponding 4-acyl-2-acylamino-5-(ß-d-glucopyranosyl)-Δ(2)-1,3,4-thiadiazolidines. Oxidative transformations of these precursors gave O-protected 2-(ß-d-glucopyranosyl)-5-substituted-amino-1,3,4-oxa- and -thiadiazoles. The O-benzoyl protecting groups were removed under base-catalysed transesterification conditions. The C-glucopyranosyl heterocyclic compounds proved inactive against rabbit muscle glycogen phosphorylase b, however, the semicarbazones showed moderate inhibition (best inhibitor was 4-phenyl-[C-(ß-d-glucopyranosyl)formaldehyde]semicarbazone (Ki=29µM).

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).

Synthesis of substituted 2-(ß-D-glucopyranosyl)-benzimidazoles and their evaluation as inhibitors of glycogen phosphorylase.

Microwave assisted condensation of O-perbenzoylated C-(ß-d-glucopyranosyl)formic acid with 1,2-diaminobenzenes in the presence of triphenylphosphite gave the corresponding O-protected 2-(ß-d-glucopyranosyl)-benzimidazoles in moderate yields. O-Perbenzoylated C-(ß-d-glucopyranosyl)formamide and -thioformamide were transformed into the corresponding ethyl C-(ß-d-glucopyranosyl)formimidate and -thioformimidate, respectively, by Et3O·BF4. Treatment of the formimidate with 1,2-diaminobenzenes afforded O-protected 2-(ß-d-glucopyranosyl)-benzimidazoles in good to excellent yields. Similar reaction of the thioformimidate gave these compounds in lower yields. The O-benzoyl protecting groups were removed by the Zemplén protocol. These test compounds were assayed against rabbit muscle glycogen phosphorylase (GP) b, the prototype of liver GP, the rate limiting enzyme of glycogen degradation. The best inhibitors were 2-(ß-d-glucopyranosyl)-4-methyl-benzimidazole (Ki=2.8µM) and 2-(ß-d-glucopyranosyl)-naphtho[2,3-d]imidazole (Ki=2.1µM) exhibiting a ∼3-4 times stronger binding than the unsubstituted parent compound.

Synthesis and biological evaluation of novel pentacyclic triterpene derivatives as potential PPARγ agonists.

Synthesis and biological evaluation of a novel series of substituted pentacyclic triterpene derivatives as potential PPARγ agoinsts and glycogen phosphorylase inhibitors have been described. Compounds 11 and 17 showed potent PPARγ agonistic activity and activated the transcription activity of PPARγ in a dose-dependent manner. On the other hand, eleven compounds exhibited moderate inhibitory activity against rabbit muscle glycogen phosphorylase a (RMGPa), and triterpene 10 was the best one. Structure-activity relationship (SAR) is also discussed.

Discovery of novel dual-action antidiabetic agents that inhibit glycogen phosphorylase and activate glucokinase.

Dual-target-directed agents simultaneously inhibiting glycogen phosphorylase (GP) and activating glucokinase (GK) could decelerate the inflow of glucose from glycogenolysis and accelerate the outflow of glucose in the liver, therefore allow for a better control over hyperglycaemia in a synergetic manner. A series of hybrid compounds were designed by structure-assisted and ligand-based strategies. In vitro bioassays found two novel compounds (1j, 6g) worthy of further optimization on balance of dual action to GP and GK. In addition, for single-target activity, two compounds exhibited more potent GP inhibitory activity and four compounds showed better GK activation than their corresponding references.

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.

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.

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.

Synthesis of variously coupled conjugates of D-glucose, 1,3,4-oxadiazole, and 1,2,3-triazole for inhibition of glycogen phosphorylase.

5-(O-Perbenzoylated-ß-D-glucopyranosyl)tetrazole was obtained from O-perbenzoylated-ß-D-glucopyranosyl cyanide by Bu(3)SnN(3) or Me(3)SiN(3)-Bu(2)SnO. This tetrazole was transformed into 5-ethynyl- as well as 5-chloromethyl-2-(O-perbenzoylated-ß-D-glucopyranosyl)-1,3,4-oxadiazoles by acylation with propiolic acid-DCC or chloroacetyl chloride, respectively. The chloromethyl oxadiazole gave the corresponding azidomethyl derivative on treatment with NaN(3). These compounds were reacted with several alkynes and azides under Cu(I) catalysed cycloaddition conditions to give, after removal of the protecting groups by the Zemplén protocol, ß-D-glucopyranosyl-1,3,4-oxadiazolyl-1,2,3-triazole, ß-D-glucopyranosyl-1,2,3-triazolyl-1,3,4-oxadiazole, and ß-D-glucopyranosyl-1,3,4-oxadiazolylmethyl-1,2,3-triazole type compounds. 5-Phenyltetrazole was also transformed under the above conditions into a series of aryl-1,3,4-oxadiazolyl-1,2,3-triazoles, aryl-1,2,3-triazolyl-1,3,4-oxadiazoles, and aryl-1,3,4-oxadiazolylmethyl-1,2,3-triazoles. The new compounds were assayed against rabbit muscle glycogen phosphorylase b and the best inhibitors had inhibition constants in the upper micromolar range (2-phenyl-5-[1-(ß-D-glucopyranosyl)-1,2,3-triazol-4-yl]-1,3,4-oxadiazole 36: K(i)=854µM, 2-(ß-D-glucopyranosyl)-5-[1-(naphthalen-2-yl)-1,2,3-triazol-4-yl]-1,3,4-oxadiazole 47: K(i)=745µM).

Identification of pentacyclic triterpenes derivatives as potent inhibitors against glycogen phosphorylase based on 3D-QSAR studies.

Naturally occurring pentacyclic triterpenes (PT), a novel class of inhibitors against glycogen phosphorylase (GP), hold promise for the treatment of type-2 diabetes and other diseases with disorders in glycogen metabolism. To identify novel and more potent GP inhibitors, the receptor-based comparative molecular field analysis (CoMFA) and comparative molecular similarity analysis (CoMSIA) approaches were performed to investigate the quantitative structure-activity relationships (QSAR) among 106 PT analogues. The validated models demonstrated that the elongated or bulky substitutions in C17 position and/or C2, C3 positions are favorable. Then based on the structural information extracted from these models, 56 derivatives were synthesized and biochemically tested in this study. The IC50 value of the most potent compound P50 was found to be 1.1 µM.

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.

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.