RU2190620C1 - N-[3h]-acetyl-d-glucosamine and method of its synthesis - Google Patents

Abstract

FIELD: organic chemistry, chemical technology, radiolabeled compounds. SUBSTANCE: invention describes the compound N-[³H]-acetyl-D-glucosamine of the formula (I):

and a method of its synthesis. Method involves the condensation reaction of D-glucosamine hydrochloride with [³H]CH₃COOHNa in the presence of dicyclohexylcarbodiimide or metho-p-toluenesulfonate of 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide or carbonylditriazole. EFFECT: improved method of synthesis, expanded assortment of labeled physiologically active compounds based on recommendation of conditions of their synthesis. 2 cl

Description

 The invention relates to organic chemistry and may find application in analytical and biological chemistry, in applied medicine.

 When studying physiologically active compounds, their labeled analogues are used.

Данное известное соединение входит в состав хитина, мукопептидов и мукополисахаридов и его используют как субстрат при изучении ряда ферментов (P. C. Dunlop et al., J Biol Chem 255, 1542 (1980), G.E. Gibson, M.Shimada, Biochem. Pharmacol. 29, 167(1980), P Meindl, H.Tippy, Hoppe-Seyleler s Z. Physiol Chem 350, 1088 (1969), J. Garrell, C.M. Cuchillo, FEBS Lett 190, 329 (1985) [I].Known N-acetyl-D-glucosamine of the formula I

This known compound is a part of chitin, mucopeptides and mucopolysaccharides and is used as a substrate in the study of a number of enzymes (PC Dunlop et al., J Biol Chem 255, 1542 (1980), GE Gibson, M. Shimada, Biochem. Pharmacol. 29, 167 (1980), P Meindl, H. Tippy, Hoppe-Seyleler s Z. Physiol Chem 350, 1088 (1969), J. Garrell, CM Cuchillo, FEBS Lett 190, 329 (1985) [I].

 However, labeled analogues of this compound are not described, and accordingly, methods for their preparation are not described.

 The technical result achieved by the present invention is to expand the assortment of labeled physiologically active compounds due to the recommendations of the conditions for their preparation.

This technical result is achieved by the fact that N- [ ³ H] acetyl-D-glucosamine is obtained, and also by the fact that the method for its preparation consists in condensing D-hydrochloric acid glucosamine with [ ³ H] CH ₃ COONa in the presence of dicyclohexylcarbodiimide or meth 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide or carbonyldithriazole-toluenesulfonate.

The problem of acetylation of amines with unlabeled reagents is not difficult. The preparation of amides using acetic anhydride, acetic acid chloride, etc. is well known and occurs in high yield. However, in the synthesis of labeled analogues of these reagents, one encounters a number of difficulties that make these techniques unattractive. The main difficulty is the high volatility of these reagents. Only in a specially equipped laboratory, where the labeled vapors of these compounds are excluded from the atmosphere, is it possible to work with them. In addition, the high cost of these reagents prevents their widespread use. Unlike them, a label can be added directly to sodium acetate, it is non-volatile (melting point 324 ^o С). All this makes it extremely attractive for obtaining labeled amides. Currently, the synthesis of acetylated amines using labeled sodium acetate is not known. The essence of the method is to mix solutions of labeled sodium acetate with hydrochloric acid amine-containing compound, as a result of which labeled acetic acid and sodium chloride are formed. After adding a condensing agent, the ampoule is sealed and heated. After the reaction, the contents of the ampoule are frozen and all volatile products are separated by freezing in a special container, the residue is dissolved in methanol, acidified with hydrochloric acid and evaporated to remove traces of labeled acetic acid. Dicyclohexylcarbodiimide, meth-p-toluenesulfonate 1-cyclohexyl-3 (2-morpholinoethyl) carbodiimide and carbonyldithriazole were used as a condensing agent. The use of carbonyldithriazole was optimal. The synthesis takes place according to the scheme:
[ ³ H] CH ₃ COOH + carbonylditriazole = [ ³ H] CH ₃ CO-triazole + CO ₂ + triazole [ ³ H] CH ₃ CO-triazole + D-glucosamine = N- [ ³ H] acetyl-D-glucosamine + triazole.

The labeled amides were purified and analyzed on a 10x250 mm column, Silasorb NH ₂ 10 μm, v - 2.0 ml / min, acetonitrile-water (5: 1), retention time 16.24 min, on a 10x150 mm column, Kromasil 100 C ₁₈ , 7 μm, v - 2.0 ml / min, in a 0.1% aqueous trifluoroacetic acid system, retention time 4.12 min, on a 4x150 mm column, Zorbax SB-C ₈ , 3.5 μm, v - 0.8 ml / min, in a system of 5 mM aqueous sulfuric acid, the retention time for the α-isomer is 2.16 minutes, for the β-isomer 2.35 minutes. The following are examples of the implementation of the invention.

 Example 1

A) Obtaining [ ³ H] CH ₃ COONa
4 mg of CH ₃ COONa, loaded onto 40 mg of 5% Pd / C, were placed in the reaction vial. Then the ampoule was evacuated to a pressure of 0.1 Pa, filled with gaseous tritium to a pressure of 333 hPa and kept at a temperature of 240 ^{° C. for} 20 minutes. Excess tritium was removed by evacuation. After cooling, the reaction products were dissolved in methanol (6x1 ml), the catalyst was filtered off, and the filtrates were evaporated several times with methanol to remove labile tritium. The residue was purified by HPLC on a 4x150 mm column, Zorbax SB-C ₈ 3.5 μm, v - 1.0 ml / min, in a system of 5 mM aqueous sulfuric acid, retention time 3.45 min. The yield of [ ³ H] CH ₃ COONa is 80-85%, the molar radioactivity is 30-35 Ci / mmol, the radiochemical purity is 98-99%.

B) Condensation of [ ³ H] CH ₃ COONa with D-glucosamine.

1 mg of the obtained [ ³ H] CH ₃ COONa and 2.5 mg of hydrochloric acid glucosamine, which were dissolved in 1.0 ml of dimethylformamide, were placed in the reaction vial. After 5 minutes, 10 mg of dicyclohexylcarbodiimide was added, and after another 60 minutes, 20 μl of triethylamine. The reaction was carried out for 6 hours. After removal of volatile products, the residue was dissolved in 0.5 ml of 2 N. HCl and again evaporated to remove unreacted acetic acid. After purification by HPLC, the yield of N- [ ³ H] acetyl-D-glucosamine obtained was 2.5%.

 Example 2

After obtaining [ ³ H] CH ₃ COONa as described in Example 1, condensation was carried out as follows. To 0.3 ml of an aqueous solution of 3.5 mg of D-glucosamine hydrochloride and 0.5 mg of [ ³ H] CH ₃ COONa, 27.2 mg of 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate and conducted the reaction for 3 days at room temperature. After processing the reaction mixture as described in Example 1, the yield of [ ³ H] aiethyl-D-glucosamine was 8%.

 Example 3

After obtaining [ ³ H] CH ₃ COONa as described in Example 1, condensation was carried out as follows. To a 1.0 ml solution of 4.7 mg of D-glucosamine hydrochloride and 1.0 mg of [ ³ H] CH ₃ COONa in dimethylformamide, 6.0 mg of carbonyldithriazole was added, the ampoule was sealed and the reaction was carried out for 60 minutes at 30 ^° C, and then 180 min at 80 ^o C. After processing the reaction mixture as described in example 1, the yield of N- [ ³ H] acetyl-D-glucosamine was 14.5%.

Thus, a new method has been developed for the preparation of a new tritium-labeled compound -N- [ ³ H] acetyl-D-glucosamine.

Claims (1)

1. N- [ ³ H] Acetyl-D-Glucosamine of Formula I

2. A method of producing N- [ ³ H] acetyl-D-glucosamine, which consists in the condensation of hydrochloric acid D-glucosamine with [ ³ H] CH ₃ COONa in the presence of dicyclohexylcarbodiimide, or 1-cyclohexyl-3- metho-p-toluenesulfonate ( 2-morpholinoethyl) carbodiimide, or carbonylditriazole.