C -( β - D -Glucopyranosyl) heterocycles as potential glycogen phosphorylase inhibitors

Per-O -acetylated and -benzoylated β - D -glucopyranosyl cyanides were transformed into the corresponding 5-( β - D -Glucopyranosyl)tetrazoles, 2-( β - D -glucopyranosyl)benzothiazoles, and, via the benzoylated C -( β - D -glucopyranosyl) ethyl thioformimidate, 2-( β - D -glucopyranosyl)- benzimidazoles. Acylation of the tetrazoles, either by acetic or trifluoroacetic anhydride, gave 5-( β - D -glucopyranosyl)-2-methyl- and -2-trifluoromethyl-1,3,4-oxadiazoles, respectively. Removal of the protecting groups furnished new inhibitors of glycogen phosphorylase exhibiting inhibitor constants in the micromolar range.


Introduction
Diabetes mellitus is a serious metabolic disease afflicting ~6 % of the population in Western societies.Its prevalence is dramatically increasing worldwide and the estimated number of diabetic patients is 220 million for the year 2010, indicating a 46 % enhancement in the world population during a decade. 1 This disease, characterized by chronically elevated blood glucose levels, is becoming one of the largest contributors to mortality especially due to its long term complications like retinopathy, neuropathy, and nephropathy, but first of all cardiovascular diseases. 2iabetes is divided into two main forms: type 1 (or insulin dependent diabetes mellitus), that is characterized by total insulin deficiency; and type 2, representing more than 90 % of all diagnosed cases, 1,3 that exhibits impaired insulin secretion and/or insulin resistance.While type 1 diabetics can be treated by the administration of exogeneous insulin, for type 2 patients generally diet, exercise, and oral hypoglycemic agents are prescribed, 2 however, the latter are inadequate for 30-40 % of the patients. 4Therefore, several new approaches have been investigated intensively in an attempt to find more appropriate treatments for type 2 diabetes. 5One of the emerging targets is the inhibition of glycogen phosphorylase (GP), the main regulatory enzyme in the liver, responsible for the control of blood sugar levels. 3,6mong small molecule inhibitors of these enzymes a large number of glucose derivatives have been investigated. 7The tested glucose analog inhibitors comprise glucosides, N-acylβ-Dglucopyranosylamines, thioglucosides, a 1-deoxy-D-gluco-heptulopyranose 2-phosphate, nojiritetrazole, 2,6-anhydro-heptonamides, and glucopyranosylidene-spiro-heterocycles. 8The strongest inhibition (K i values determined with rabbit muscle GPb enzyme) was achieved by a glucopyranosylidene-spiro-hydantoin 9,10 (Table 1, Entry 1: K i = 3-4 µM) and its thio analog 11,10 (Table 1, Entry 2: K i = 5 µM).In vivo hypoglycemic efficiency of the thiohydantoin derivative was also demonstrated. 12The recently synthesized N-2-naphthoyl-N'β-D-glucopyranosyl urea (Table 1, Entry 3: K i = 0.4 µM) proved to be the best glucose derived inhibitor of GP known to date. 8Most of these inhibitors target the so-called β-channel of the enzyme which is an empty pocket next to the highly glucose specific catalytic center surrounded by amino acid side chains of mixed character. 13Since no C-( β-D-glucopyranosyl) heterocycles have yet been investigated as GP inhibitors, we have decided to prepare such derivatives exhibiting acidic, basic, and neutral properties in the heterocyclic moieties, in the hope that favorable interactions may arise with the β-channel of the enzyme.
Deacylation of 5, 8, 10, and 12 under Zemplén conditions was straightforward to yield the deprotected derivatives 7, 9, 11, and 13, respectively.Deacetylation of 2 had to be carried out using a short reaction time with cooling, 19 otherwise an unseparable mixture of several unidentified products was obtained.Trifluoromethyloxadiazoles 14 and 15 did not give isolable deprotected compounds under several deacylation conditions (a similar anomaly was observed with other sugar trifluoromethyloxadiazoles 20 ).This work a Kinetic measurements were carried out as described before. 10eliminary kinetic measurements with rabbit muscle glycogen phosphorylase b enzyme were performed as described before, 10 and the data are collected in Table 1.The new compounds have inhibitor constants in the micromolar range or are not inhibitory at all (Entries 5-9).The presence of the CN group in 4 instead of CONH 2 (Entries 4 and 5) makes the inhibition stronger.
The tetrazole ring of slightly acidic character (Entry 6) is clearly unfavorable for the binding of 7 to the enzyme.The neutral aglycons in 11 and 13 result in moderate inhibitors (Entries 8 and 9).The most efficient inhibitor of this series was benzimidazole 9 (Entry 7), however, even this compound is less effective than the best glucose analog (Entry 3).Its stronger effect in comparison with 11 must be due to the amphoteric character of the heterocycle (compare Entries 7 and 8), however, the present data do not allow more precise conclusions to be drawn for the nature of its binding.Detailed kinetic studies and X-ray crystallographic evaluation of the binding modes of these compounds to GP enzymes are in progress and will be published elsewhere.

5-(2′,3′,4′,6′-Tetra-O-acetyl-β-D-glucopyranosyl)tetrazole (5).
To a solution of 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl cyanide 14 2 (1 g, 2.80 mmol) in abs.DMF (4 ml), ammonium chloride (275 mg, 5.14 mmol) and sodium azide (334 mg, 5.14 mmol) were added.The reaction mixture was refluxed for 3.5 hours, then cooled to room temperature, filtered, the residue washed with acetone, and the solvent evaporated in vacuo.The obtained syrup was placed into an ice bath and pyridine (0.12 ml) and acetic anhydride (0.24 ml) were added.The mixture was allowed to stand at room temperature for 16 hours, then it was diluted with chloroform and water.The aqueous phase was extracted with chloroform again, and the combined organic phases were succesively washed with saturated aqueous sodium hydrogen carbonate solution, 2 M hydrochloric acid, and water.The organic phase was dried over magnesium sulfate and concentrated in vacuo.(100 mg, 0.17 mmol) was dissolved in diethyl ether (15 ml) and ethanethiol (25 µl, 0.33 mmol) was added to the solution.The mixture was cooled down in an ice bath, and hydrogen chloride gas was introduced for 5 hours.The solvent was then evaporated, the residue (550 mg) dissolved in abs.pyridine (15 ml), and 1,2-benzenediamine (92.9 mg, 0.86 mmol) was added.The reaction mixture was stirred at room temperature for 24 hours.The solvent was then evaporated in vacuo and the obtained syrup was purified by column chromatography (eluent: hexane-ethyl acetate 3:2) to give

General procedure for the Zemplén deacylation
The acetylated and benzolated compounds were dissolved in abs.methanol and 1 M methanolic sodium methoxide solution was added to the solutions in catalytic amount.The reaction mixture was kept at room temperature for a given time and then neutralized with a cation exchange resin Amberlyst 15 (H + form).Filtration and removal of the solvent resulted in the corresponding deacylated sugar derivatives.

Table 1 .
Preliminary kinetic data a on the inhibition of rabbit muscle glycogen phosphorylase b by the new compounds 4