Amino acid anthranilamide derivatives as a new class of glycogen phosphorylase inhibitors

A series of amino acid anthranilamide derivatives identified from a high-throughput screening campaign as novel, potent, and glucose-sensitive inhibitors of human liver glycogen phosphorylase a are described. A solid-phase synthesis using Wang resin was also developed which provided efficient access to a variety of analogues, and resulted in the identification of key structure–activity relationships, and the discovery of a potent exemplar (IC₅₀ = 80 nM). The SAR scope, synthetic strategy, and in vitro results for this series are presented herein.     

Synthesis of some derivatives of C-(1-deoxy-1-N-substituted-D-glucopyranosyl)formic acid (D-gluco-hept-2-ulopyranosonic acid) as potential inhibitors of glycogen phosphorylase

Per-O-benzoylated derivatives (amide, methyl ester and glycinamide) of C-(1-azido-1-deoxy-alpha-D-glucopyranosyl)formic acid obtained by azide substitution in the corresponding C-(1-bromo-1-deoxy-beta-D-glucopyranosyl)formic acid derivatives were debenzoylated by the Zemplén-protocol. Per-O-benzoylated C-(1-azido-1-deoxy-alpha-D-glucopyranosyl)formamide was dehydrated by oxalyl chloride-DMF to give the corresponding nitrile, while from its reduction mixture obtained by Raney-nickel or sodium hydrogentelluride C-(1-amino-1-deoxy-beta-D-glucopyranosyl)formamide could be isolated. Acetylation of this amino-amide by Ac2O/Py and subsequent debenzoylation gave C-(1-acetamido-1-deoxy-beta-D-glucopyranosyl)formamide. Applying the same conditions to the crude reduction mixture allowed the alpha-anomer to be isolated as a minor component. An alternative pathway to produce the above beta-anomer appeared in the reaction of C-(1-bromo-1-deoxy-beta-D-glucopyranosyl)formamide with CH3CN in the presence of Ag2CO3 to yield 1-acetamido-2,3,4,6,-tetra-O-benzoyl-1-deoxy-beta-D-glucopyranosyl cyanide, which was hydrated, in the presence of TiCl4, to the formamide. Some of the new compounds were shown to be weak inhibitors of muscle glycogen phosphorylase b.     

Synthesis of glucoconjugates of oleanolic acid as inhibitors of glycogen phosphorylase

Synthesis and biological evaluation of glucoconjugates of oleanolic acid, linked by either a triazole moiety or an ester function, as novel inhibitors of glycogen phosphorylase have been described. Several triterpene-glycoside conjugates exhibited moderate-to-good inhibitory activity against rabbit muscle GPa. Compound 12 showed the best inhibition with an IC₅₀ value of 1.14 μM. Structure-activity relationship (SAR) analysis of these inhibitors is also discussed. Possible binding modes of compound 12 were explored by molecular docking simulations.     

Synthesis of N-(beta-D-glucopyranosyl) monoamides of dicarboxylic acids as potential inhibitors of glycogen phosphorylase

O-peracetylated N-(beta-D-glucopyranosyl)imino trimethylphosphorane obtained in situ from 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl azide and PMe3 was reacted with saturated and unsaturated aliphatic and aromatic dicarboxylic acids, or their anhydrides, or monoesters to give the corresponding N-(beta-D-glucopyranosyl) monoamides of dicarboxylic acids or derivatives. The acetyl protecting groups were removed according to the Zemplén protocol to give a series of compounds which showed moderate inhibitory effects against rabbit muscle glycogen phosphorylase b. The best inhibitor was 3-(N-beta-D-glucopyranosyl-carbamoyl)propanoic acid (7) with Ki = 20 microM.     

Synthesis and biological evaluation of novel pyrazolo[4,3-b]oleanane derivatives as inhibitors of glycogen phosphorylase

Eighteen pyrazolo[4,3-b]oleanane derivatives have been synthesized and biologically evaluated as inhibitors of rabbit muscle GPa. Key compound 5 was readily obtained in four steps starting from oleanolic acid (OA; 1). Further modification based on pyrazolo triterpene 5 resulted in 17 novel pyrazolo pentacyclic triterpenes. All of the synthesized pyrazolo[4,3-b]oleanane derivatives were biologically assayed against rabbit muscle GPa. Within this series of compounds, pyrazole triterpene 19 (IC(50)=9.9 microM) exhibited more potent activity than the parent compound 1. Preliminary structure-activity relationship analysis of the pyrazolo[4,3-b]oleanane derivatives as GPa inhibitors is discussed.     

Synthesis,in vitro ADME profiling and in vivo pharmacological evaluation of novel glycogen phosphorylase inhibitors

A small set of indole-2-carboxamide derivatives identified from a high-throughput screening campaign has been described as a novel, potent, and glucose-sensitive inhibitors of glycogen phosphorylase a (GPa). Among this series of compounds, compound 2 exhibited moderate GP inhibitory activity (IC₅₀ = 0.29 μM), good cellular efficacy (IC₅₀ = 3.24 μM for HepG2 cells and IC₅₀ = 7.15 μM for isolated rat hepatocytes), together with good absorption, distribution, metabolism, and elimination (ADME) profiles. The in vivo animal study revealed that compound 2 significantly inhibited an increase of fasting blood glucose level in adrenaline-induced diabetic mice.     

Molecular docking analysis of glycogen phosphorylase with inhibitors from Cissampelos pareira Linn.

Cissampelos pareira Linn. is a climbing herb known in Indian traditional medicine as laghupatha. It belongs to the Menispermaceae family. The enzyme glycogen phosphorylase (GP) is a promising target for the treatment of type-2 diabetes (T2DM). A variety of natural product inhibitors with both pharmaceutical and nutraceutical potential have been reported in the search for powerful, selective and drug-like GP inhibitors that could lead to hypoglycemic medicines. Therefore, it is of interest to document the molecular docking analysis data of glycogen phosphorylase with compounds from Cissampelos pareira Linn. We report the optimal binding features of 4 compounds namely Trans-N-feruloyltyramine, Coclaurine, Magnoflorine, and Curine with the target protein for further consideration in the context of T2DM.     

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 2.5 mM).     

Skeletal muscle glycogen phosphorylase is irreversibly inhibited by mercury: Molecular,cellular and kinetic aspects

Muscle glycogen phosphorylase (GP) plays an important role in muscle functions. Mercury has toxic effects in skeletal muscle leading to muscle weakness or cramps. However, the mechanisms underlying these toxic effects are poorly understood. We report that GP is irreversibly inhibited by inorganic (Hg²⁺) and organic (CH₃Hg⁺) mercury (IC₅₀ = 380 nM and k_inact = 600 M⁻¹ s⁻¹ for Hg²⁺ and IC₅₀ = 43 μM and k_inact = 13 M⁻¹ s⁻¹ for CH₃Hg⁺) through reaction of these compounds with cysteine residues of the enzyme. Our data suggest that the irreversible inhibition of GP could represent one of the mechanisms that contribute to mercury-dependent muscle toxicity.     

Synthesis of new modified truncated peptides and inhibition of glycogen phosphorylase

The first solution state structural analysis (NMR) of the C‐terminal sequence of human G_L that binds to glycogen phosphorylase a (GPa), PEWPSYLGYEKLGPYY‐NH₂ ( 1 ), showed it to be in a random coil conformation. This was supported by molecular dynamics simulation (modelled in solution) using NAMD 2.6. The conformational ambiguity of the peptide makes the structural arrangement of the peptide (and internal residues) strongly dependent on the environment. Thirteen tetra‐peptide fragments of the C‐terminal sequence, YEKLG‐NH₂, and the corresponding tri‐ and di‐peptide sequences were used in a fragment screen against GPa. Compound 2 (H‐GPYY‐NH₂) did not give an IC₅₀ value, whereas PEWPSYLGYEKLGPYY‐NH₂ ( 1 ) displayed an IC₅₀ of 34 µM against GPa. Truncated peptides derived from 1 , (EKL‐NH₂, EKLG‐NH₂, and AcEKNH₂) inhibited GPa (21%, 32%, 63%, respectively at 22 mM ). These studies suggest key residues within the peptide chain have additional molecular interactions with GPa. The interaction of intra‐sequence residues in combination with the terminal residues of PEWPSYLGYEKLGPYY with GPa may form the basis for the design of new inhibitors of GPa. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis of new glycosyl biuret and urea derivatives as potential glycoenzyme inhibitors

O-Peracetylated 1-(β-d-glucopyranosyl)-5-phenylbiuret was prepared in the reaction of O-peracetylated β-d-glucopyranosylisocyanate and phenylurea. The reaction of O-peracetylated N-β-d-glucopyranosylurea with phenylisocyanate furnished the corresponding 1-(β-d-glucopyranosyl)-3,5-diphenyl- as well as 3-(β-d-glucopyranosyl)-1,5-diphenyl biurets besides 1-(β-d-glucopyranosyl)-3-phenylurea. O-Peracetylated 1-(β-d-glucopyranosyl)-5-(β-d-glycopyranosyl)biurets were obtained in one-pot reactions of O-peracetylated β-d-glucopyranosylamine with OCNCOCl followed by a second glycopyranosylamine of β-d-gluco, β-d-galacto and β-d-xylo configurations. O-Acyl protected 1-(β-d-glucopyranosyl)-3-(β-d-glycopyranosylcarbonyl)ureas were obtained from the reaction of β-d-glucopyranosylisocyanate with C-(glycopyranosyl)formamides of β-d-gluco and β-d-galacto configurations. The O-acyl protecting groups were removed under acid- or base-catalyzed transesterification conditions, except for the N-acylurea derivatives where the cleavage of the N-acyl groups was faster than deprotection. Some of the new compounds exhibited moderate inhibition against rabbit muscle glycogen phosphorylase b and human salivary α-amylase.     

Probing the β-pocket of the active site of human liver glycogen phosphorylase with 3-(C-β-d-glucopyranosyl)-5-(4-substituted-phenyl)-1, 2, 4-triazole inhibitors

Human liver glycogen phosphorylase (hlGP), a key enzyme in glycogen metabolism, is a valid pharmaceutical target for the development of new anti-hyperglycaemic agents for type 2 diabetes. Inhibitor discovery studies have focused on the active site and in particular on glucopyranose based compounds with a β-1 substituent long enough to exploit interactions with a cavity adjacent to the active site, termed the β-pocket. Recently, C-β-d-glucopyranosyl imidazoles and 1, 2, 4-triazoles proved to be the best known glucose derived inhibitors of hlGP. Here we probe the β-pocket by studying the inhibitory effect of six different groups at the para position of 3-(β-d-glucopyranosyl phenyl)-5-phenyl-, 1, 2, 4-triazoles in hlGP by kinetics and X-ray crystallography. The most bioactive compound was the one with an amine substituent to show a K_i value of 0.43 μM. Structural studies have revealed the physicochemical diversity of the β-pocket providing information for future rational inhibitor design studies.     

Altered membrane association of glycogen phosphorylase in the dystrophic chicken

The subcellular distribution of glycogen phosphorylase in pectoralis muscle from normal and dystrophic chickens was determined as a function of age. A substantially larger proportion of the total activity was associated with membranes cellular organelles, both mitochondria and sarcoplasmic reticulum, in preparations from dystrophic birds. The difference could be detected as early as 2 weeks ex ovo. Interaction of phosphorylase with cellular membranes may provide a probe for the underlying membrane defect in this dystrophyl model.     

Crystallographic studies on acyl ureas, a new class of glycogen phosphorylase inhibitors, as potential antidiabetic drugs

Acyl ureas were discovered as a novel class of inhibitors for glycogen phosphorylase, a molecular target to control hyperglycemia in type 2 diabetics. This series is exemplified by 6-{2,6-Dichloro- 4-[3-(2-chloro-benzoyl)-ureido]-phenoxy}-hexanoic acid, which inhibits human liver glycogen phosphorylase a with an IC(50) of 2.0 microM. Here we analyze four crystal structures of acyl urea derivatives in complex with rabbit muscle glycogen phosphorylase b to elucidate the mechanism of inhibition of these inhibitors. The structures were determined and refined to 2.26 Angstroms resolution and demonstrate that the inhibitors bind at the allosteric activator site, where the physiological activator AMP binds. Acyl ureas induce conformational changes in the vicinity of the allosteric site. Our findings suggest that acyl ureas inhibit glycogen phosphorylase by direct inhibition of AMP binding and by indirect inhibition of substrate binding through stabilization of the T' state.     

Kinetic and crystallographic studies of glucopyranose spirohydantoin and glucopyranosylamine analogs inhibitors of glycogen phosphorylase

Glycogen phosphorylase (GP) is currently exploited as a target for inhibition of hepatic glycogenolysis under high glucose conditions. Spirohydantoin of glucopyranose and N-acetyl-beta-D-glucopyranosylamine have been identified as the most potent inhibitors of GP that bind at the catalytic site. Four spirohydantoin and three beta-D-glucopyranosylamine analogs have been designed, synthesized and tested for inhibition of GP in kinetic experiments. Depending on the functional group introduced, the K(i) values varied from 16.5 microM to 1200 microM. In order to rationalize the kinetic results, we determined the crystal structures of the analogs in complex with GP. All the inhibitors bound at the catalytic site of the enzyme, by making direct and water-mediated hydrogen bonds with the protein and by inducing minor movements of the side chains of Asp283 and Asn284, of the 280s loop that blocks access of the substrate glycogen to the catalytic site, and changes in the water structure in the vicinity of the site. The differences observed in the Ki values of the analogs can be interpreted in terms of variations in hydrogen bonding and van der Waals interactions, desolvation effects, ligand conformational entropy, and displacement of water molecules on ligand binding to the catalytic site.     

New pentacyclic triterpenes from Gypsophila oldhamiana and their biological evaluation as glycogen phosphorylase inhibitors

The three new pentacyclic triterpenes 1-3 were isolated and elucidated from the roots of Gypsophila oldhamiana, together with seven known triterpenes (4-10). These triterpenes were evaluated for their glycogen phosphorylase inhibitory activity and preliminary structure-activity trends are suggested.     

Synthesis and pharmacological evaluation of bis-3-(3,4-dichlorophenyl)acrylamide derivatives as glycogen phosphorylase inhibitors

During our research using a high-throughput screening system for discovery of a new class of human liver glycogen phosphorylase a (hLGPa) inhibitors, a series of 3-(3,4-dichlorophenyl)acrylamide derivatives were synthesized, and their inhibitory activities toward hLGPa were evaluated. Among the derivatives, (2E,2'E)-N,N'-pentane-1,5-diylbis[3-(3,4-dichlorophenyl)acrylamide] (6c) inhibited hLGPa with an IC(50) value of 0.023 microM. An X-ray crystallographic study of the enzyme-6c complex showed that the inhibitor is bound at the dimer interface site, where the 3,4-dichlorophenyl moiety interacts hydrophobically with the enzyme.     

Synthesis and antiprotozoal activity of novel 2-{[2-(1H-imidazol-1-yl)ethyl]sulfanyl}-1H-benzimidazole derivatives

A series of 19 new 2-{[2-(1H-imidazol-1-yl)ethyl]sulfanyl}-1H-benzimidazole derivatives was synthesized starting from the properly substituted 1,2-phenylendiamine. These compounds have hydrogen or methyl at position 1; while hydrogen, chlorine, ethoxy or methoxycarbonyl group is at position 5 and/or 6. The novel compounds were tested against protozoa Trichomonas vaginalis, Giardia intestinalis and Entamoeba histolytica. Experimental evaluations revealed strong activity for all tested compounds, having IC₅₀ values in the nanomolar range, which were even better than metronidazole, the drug of choice for these parasites.