3-Glucosylated 5-amino-1,2,4-oxadiazoles: synthesis and evaluation as glycogen phosphorylase inhibitors

Summary Glycogen phosporylase (GP) is a promising target for the control of glycaemia. The design of inhibitors binding at the catalytic site has been accomplished through various families of glucose-based derivatives such as oxadiazoles. Further elaboration of the oxadiazole aromatic aglycon moiety is now reported with 3-glucosyl-5-amino-1,2,4-oxadiazoles synthesized by condensation of a C-glucosyl amidoxime with N,N’-dialkylcarbodiimides or Vilsmeier salts. The 5-amino group introduced on the oxadiazole scaffold was expected to provide better inhibition of GP through potential additional interactions with the enzyme’s catalytic site; however, no inhibition was observed at 625 µM.


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General methods. All reagents and solvents used for syntheses were commercial and used without further purification. Solvents were distilled over Mg/I 2 (MeOH), or purchased dry.
Reactions were performed under argon atmosphere. NMR spectra were recorded at 293 K, unless stated otherwise, using a 300 MHz, a 400 MHz or a 500 MHz spectrometer. Shifts are referenced relative to deuterated solvent residual peaks. Low and high resolutions mass spectra were recorded using a Bruker MicrOTOF-Q II XL spectrometer. Thin-layer chromatography (TLC) was carried out on aluminum sheets coated with silica gel 60 F 254 (Macherey-Nagel). TLC plates were inspected by UV light (λ = 254 nm) and developed by treatment with a solution of 10% H 2 SO 4 in EtOH/H 2 O (1:1 v/v) followed by heating. Silica gel column chromatography was performed with silica gel Si 60 (40-63 μm). Thick-layer chromatography was performed to purify compounds on a 20-50 mg scale using standard silica coated TLC plates (thickness 200 µm) presented above. The compound was desorbed from the silica gel with CH 2 Cl 2 /MeOH 9:1. Optical rotations were measured using a Perkin Elmer polarimeter and values are given in 10 −1 deg·cm 2 ·g −1 . Melting points were measured on a Büchi apparatus and are uncorrected. Ureas 1a and 1e were purchased from commercial sources.

General procedure A for the synthesis of urea (2b-d):
To a solution of amine (5-10 equiv) in anhydrous dichloromethane (25 mL) at 0 °C was added dropwise a solution of triphosgene (1 equiv) in anhydrous dichloromethane (5 mL).
The reacting mixture was stirred at 0 °C for 1 h and then stirred at room temperature for additional 4 h. After completion, water (100 mL) was added to dissolve the precipitate. The aqueous layer was extracted with dichloromethane (2 × 25 mL). The combined organic layers were washed with 1 N HCl (25 mL), saturated NaHCO 3 (25 mL) and brine (25 mL). The solution was dried (Na 2 SO 4 ) and concentrated to give the desired product of satisfying purity (>95%) for use in the next step.

dialkylamino-1,2,4-oxadiazoles (4a-e):
Oxalyl chloride (1.2 equiv) was added dropwise to a solution of urea 2a-e (1 equiv) in anhydrous toluene or anhydrous dichloromethane (10 mL) at 0 °C. The resulting mixture was stirred at room temperature overnight. After completion, the solvent was removed under vacuum and the solid was washed with cold ethyl acetate (3 × 5 mL) to give the desired Vilsmeier salt intermediate of sufficient purity for use in the next step.
A solution of 3-(2,3,4,6-tetra-O-benzoyl-β-D-glucopyranosyl)-formamidoxime 3 (1 equiv) and triethylamine (4 equiv) in anhydrous dichloromethane (10 mL) was added dropwise to a solution of the freshly prepared Vilsmeier salt in anhydrous dichloromethane (10 mL). The resulting mixture was stirred at room temperature overnight. After completion, the solvent was evaporated under vacuum and the crude product purified by silica gel column chromatography to afford compounds 4a-e.

General procedure C for debenzoylation (5a-e and 7a,b):
Benzoylated compound 4a-e and 6a,b was dissolved in methanol/dichloromethane (3:1) and sodium methoxide was added until pH reached 9. The resulting mixture was stirred overnight at room temperature. After completion, the crude product was purified by silica gel column chromatography or thick layer chromatography.

3-(β-D-Glucopyranosyl)-5-(N-methyl-N-benzylamino)-1,2,4-oxadiazole (5b)
Obtained as colorless oil (8 mg, 0.022 mmol, 62%) following general procedure C: 3-(2,3,4,6-Tetra-O-benzoyl-β-D-glucopyranosyl)-5-(N-methyl-N-benzylamino) -1,2,4-oxadiazole (4b, 29 mg, 0.037 mmol) and purified by thick-layer chromatography (CH 2 Cl 2 /MeOH, 80 /20, v/v Methods Enzymol. 1962, 5, 369-373) using dithiothreitol instead of L-cysteine, and recrystallized at least three times before use with a specific activity of 55 U/mg protein. Kinetic data for the inhibition of rabbit skeletal muscle glycogen phosphorylase were collected using different concentrations of -D-glucose-1-phosphate (2 and 20 mM), constant concentrations of glycogen (1% w/v) and AMP (1 mM), and various concentrations of inhibitor. Inhibitor was dissolved in dimethyl sulfoxide (DMSO) and diluted in the assay buffer (50 mM triethanolamine, 1 mM EDTA and 1 mM dithiothreitol) so that the DMSO concentration in the assay should be lower than 1%. The enzymatic activities were presented in the form of doublereciprocal plots (Lineweaver-Burk) applying a nonlinear data analysis program. The means of standard errors for all calculated kinetic parameters averaged to less than 10%. IC 50 values were determined in the presence of 4 mM -D-glucose-1-phosphate, 1 mM AMP, 1% glycogen, and varying concentrations of the inhibitor. The stock solution of GP inhibitor was made by DMSO as a solvent and the concentration was 250 mM. The final concentrations of the compounds in the assay were 6.25, 12. 5, 31.25, 62.5, 125, 312.5 and 625 M (this is the highest because the DMSO concentration was 0.25% in the system and has no effect on the enzyme activity).