RU2133754C1 - Method of phosphonation of 2′,3′-didesoxynucleosides - Google Patents

Abstract

FIELD: synthesis of nucleosides and nucleotides. SUBSTANCE: method comprises preparing phosphonating PCl₃- based agent using tert-butanol and pyridine, reacting it with 2′,3′-didesoxynucleoside and subsequently adding water and isolating the desired product by separating inorganic phosphonation products in the form of ammonium salts in the presence of trialkyl phosphate. Desired products, namely 5H-phosphonates of 2′,3′-didesoxy nucleosides, are prepared in the form ammonium salts thereof which are then transformed into alkali and alkaline-earth metal salts. Yield is 70-80%. EFFECT: simplified process which is industrially applicable.

Description

 The invention relates to the field of synthesis of nucleosides and relates to an improved method of phosphonation of 2 ', 3'-dideoxynucleosides.

 Known methods for phosphonization of nucleosides involve the use of complex methods of chromatographic isolation and purification of target products and are mainly focused on obtaining small amounts of 5'-H-phosphonates 2 ', 3'-dideoxynucleosides or their derivatives.

 Therefore, the urgent task is to develop a simplified method of phosphonation of 2 ', 3'-dideoxynucleosides, implemented in standard industrial equipment, without the use of preparative chromatography.

A known method of phosphonization of nucleosides [5'-Hydrogenphosphonates and 5'-methylphosphonates of sugarmodified pyrimidine nucleosides as potential anti-HIV-1 agents / Kraevsky AA, Tarusova NB, Zhu Q. et al. // Nucleosides and Nucleotides. - 1992, v. 11, N 2-4, p. 177-196] using the tert-butylammonium salt of phosphorous acid as the phosphonating agent. Phosphonation is carried out in the presence of dicyclohexylcarbodiimide in pyridine at 0-4 ^o C for 8-9 hours. After adding water to the reaction mass, the target product is isolated on a column (Dowex, gradient eluent HN ₄ HCO ₃ 0-0.5 M, then 0.5 M NH ₄ HCO ₃ with 90% alcohol), purified again on a column (DEAE- cellulose, eluent NH ₄ HCO ₃ 0-0.3 M) and dried with distillation of absolute alcohol. Yield 75%.

 In addition to the disadvantages noted above, this method uses dicyclohexylcarbodiimide, a powerful allergen, the use of which on an industrial scale is problematic.

In another method [A convinient and general approach to the synthesis of properly protected D-nucleoside-3'-hydrogenphosphonates via phosphite intermediates / Marugg JE, Tromp M., E. Kuyl-Yeheskiely et. al.// Tetrahedron Lett. -1986, v. 27, N 23, p. 2661-2664] for the synthesis of compounds containing an H-phosphonate group, 2-chloro-5,6-benzo-1,3,2-dioxophospharin-4-one (salicylchlorophosphite) is used. This reagent is stable and easily obtained in solid form by the interaction of salicylic acid with PCl ₃ . The use of salicylchlorophosphite for phosphonation of 2 ', 3'-dideoxynucleosides, followed by column chromatography to isolate the desired products, leads to the production of 5'-H-phosphonates of the corresponding nucleosides in 50-55% yield, which also does not allow the use of this phosphonization method on an industrial scale .

Closest to the claimed is a method of phosphonation of 2 ', 3'-dideoxynucleosides described in USSR patent N 1548182, C 07 H 19/073. In this method, tris (imidazolyl) phosphate is used as the phosphonizing agent, which is obtained by reacting PCl ₃ with imidazole in the presence of triethylamine in acetonitrile at 0 ^° C. Phosphonation is carried out by reacting 2 ′, 3′-dideoxynucleoside with the obtained phosphonating agent (without isolation from the reaction mass) in acetonitrile at a temperature of 5-20 ^o C. In this case, up to 200 ml of acetonitrile is consumed per 1 g of nucleoside. Purification of the target product is also carried out by column chromatography (Toyerl, gradient eluent-ammonium bicarbonate 0-0.3 M). Excess salt is removed by repeated evaporation with water. The residue is lyophilized from water. The target products (5'-H-phosphonates-2 ', 3'-dideoxynucleosides) are obtained in the form of ammonium salts with a yield of 50-80%.

 However, the use of preparative chromatography, large volumes of solvent (acetonitrile) and expensive imidazole make this method of phosphonization of nucleosides technically difficult and uneconomical, which complicates its implementation on an industrial scale.

 The present invention allows to simplify the process of phosphonation of 2 ', 3'-dideoxynucleosides and makes possible its implementation on an industrial scale.

The proposed method for phosphonation of 2 ', 3'-dideoxynucleosides is the preparation of a phosphonating agent based on PCl ₃ using tert-butanol and pyridine, its interaction with 2', 3'-dideoxynucleoside, followed by the addition of water and the isolation of the target product by separation of inorganic phosphonation products in the form of ammonium salts in the presence of trialkyl phosphate.

In the proposed method for the preparation of 2 ', 3'-dideoxynucleosides, another phosphonating agent based on PCl _{3 is used} , which is obtained using tertbutanol and pyridine, and the target product is isolated without using preparative column chromatography by separating the inorganic products formed during phosphonation in the form ammonium salts.

 The target products — 5′-H-phosphonates of 2 ′, 3′-dideoxynucleosides — are obtained in the form of their ammonium salts, which are then transformed into alkali or alkaline earth metal salts.

The use of the agent obtained by the interaction of PCl ₃ with tert-butanol in the presence of pyridine for the phosphonation of nucleosides has not been known to date. The use of this phosphonizing agent can significantly reduce the volume of the solvent used in the synthesis (acetonitrile), which simplifies the phosphonation process itself and the isolation of the target products.

 The ammonium salts of inorganic compounds formed during phosphonation (primarily ammonium chloride and ammonium phosphite) are not soluble in trialkyl phosphates, and the ammonium salts of 5'-H-phosphonates 2 ', 3'-dideoxynucleosides are soluble. This allows you to separate the target products from inorganic phosphonation products by simple filtration without the use of preparative chromatography.

 The method of phosphonation of 2 ', 3'-dideoxynucleosides according to the invention is as follows.

Tert-butanol and pyridine are successively added to a solution of PCl ₃ in acetonitrile. The reaction mass is cooled to 0-5 ^o C, add to it a solution of the corresponding 2 ', 3'-dideoxynucleoside in acetonitrile and incubated for 5 to 20 hours. Then water and chloroform are added, the layers are separated, and aqueous ammonia is added to the aqueous layer. Evaporate the water in vacuo in the presence of trialkyl phosphate and filter out the ammonium salts of the inorganic phosphonation products. An alkali metal or alkaline earth metal hydroxide solution is added to a solution of the ammonium salt of 5'-H-phosphonate 2 ', 3'-dideoxynucleoside, water is evaporated under vacuum, filtered, the precipitate is washed with acetone and dried under vacuum. The expected products are obtained with a yield of 75 - 80% based on the starting 2 ', 3'-dideoxynucleoside. The purity and structure of the target products are confirmed by NMR and UV spectroscopy and HPLC.

NMR spectra were recorded on a 250 MHz Brucker spectrometer. UV spectra were determined on a Specord UV-vis M-40 spectrophotometer. High performance liquid chromatography was performed using a Nucleosil 120-7 NH ₂ column, linear gradient KH ₂ PO ₄ (0.05-1 M), flow rate 1 ml / min.

 For a better understanding of the invention, the following are examples of its specific implementation.

 Example 1

To a solution of 7.6 ml (87 mmol) of PCl ₃ in 60 ml of acetonitrile, 4 ml (42.5 mmol) of tert-butanol and 8 ml (99 mmol) of pyridine are successively added. The reaction mass is cooled to 0-5 ^o C and add to it 10 g (37.5 mmol) of 3'-azido-2 ', 3'-dideoxythymidine (AZT) in 100 ml of acetonitrile. After adding the entire amount of AZT carry out the exposure at 20 ^o C for 4-5 hours. Then add 60 ml of water, after which the reaction mass is treated with 160 ml of chloroform, the salts are separated and 60 ml of a 25% ammonia solution are added to the aqueous layer. The mixture is kept for 30 minutes. After evaporation of water under vacuum in the presence of 80 ml of tributyl phosphate, inorganic salts are filtered off, which are washed with 20 ml of tributyl phosphate. To a solution of the ammonium salt of 5'-H-phosphonate AZT in tributyl phosphate was added 36 ml of a 1N sodium hydroxide solution, the mixture was kept for 1 hour. After evaporation of water under vacuum, the product is separated by filtration, after adding 180 ml of acetone. After washing with acetone, it is dried under vacuum at a temperature of 40 ^o C. Receive 10.6 g of product by physicochemical characteristics of the identical sodium salt of 5'-H-phosphonate AZT (RU 2106353 C1, C 07 H 19/073), yield 80% .

 Example 2

The phosphonation process is carried out analogously to example 1 with the difference that 7 ml of PCl ₃ , 3.75 ml of tert-butanol and 7 ml of pyridine are used. Obtain 9.5 g of sodium salt of 5'-H-phosphonate AZT, yield 70%.

 Example 3

 The phosphonation process is carried out analogously to example 2 with the difference that the shutter speed after adding the entire amount of AZT is carried out for 20 hours. Obtain 9 g of sodium salt of 5'-H-phosphonate AZT, yield 66%.

 Example 4

The phosphonation process is carried out analogously to example 2 with the difference that the shutter speed after adding the entire amount of AZT is carried out at 30 ^o C, and the amount of tert-butyl phosphate used is up to 120 ml. Obtain 9 g of sodium salt of 5'-H-phosphonate AZT, yield 66%.

 Example 5

 The phosphonation process is carried out analogously to example 1 with the difference that triethyl phosphate is used instead of tributyl phosphate. Obtain 9 g of sodium salt of 5'-H-phosphonate AZT, yield 66%.

 Example 6

The phosphonation process is carried out analogously to example 1 with the difference that 100 ml of acetonitrile is used to prepare the PCl ₃ solution, and the AZT solution is prepared in 150 ml of acetonitrile. Obtain 10.6 g of sodium salt of 5'-H-phosphonate AZT, yield 80%.

 Example 7

 The phosphonation process is carried out analogously to example 1 with the difference that the translation of the ammonium salt of 5'-H-phosphonate AZT into sodium is carried out by removing tributyl phosphate by extraction with 100 ml of chloroform in the presence of 60 ml of water. After separation of the layers in the reaction with NaOH, an aqueous solution is used. Before evaporating water, 50 ml of tributyl phosphate are added to the mixture. Obtain 10 g of sodium salt of 5'-H-phosphonate AZT, yield 75%.

 Example 8

 The phosphonation process is carried out analogously to example 5 with the difference that the translation of the ammonium salt of 5'-H-phosphonate AZT into sodium is carried out by adding 60 ml of a 0.5N solution of sodium hydroxide in methanol, which is removed under vacuum. Obtain 9 g of sodium salt of 5'-H-phosphonate AZT, yield 66%.

 Example 9

 The phosphonation process is carried out analogously to example 1 with the difference that instead of sodium hydroxide solution, 36 ml of a 0.5N calcium hydroxide solution (suspension) are used. 10.5 g of product are obtained according to the physicochemical characteristics of the identical calcium salt of AZT 5'-H-phosphonate (RU 2106353 C1, C 07 H 19/073), yield 80%.

 Example 10

 The phosphonation process is carried out analogously to example 1 with the difference that instead of sodium hydroxide solution, 36 ml of 0.5N barium hydroxide solution (suspension) are used. Obtain 11.9 g of product by physico-chemical characteristics of the identical barium salt of 5'-H-phosphonate AZT (RU 2106353 C1, C 07 H 19/073), yield 80%.

 Example 11

 The phosphonation process is carried out analogously to example 1 with the difference that instead of sodium hydroxide solution, 36 ml of a 1N potassium hydroxide solution are used. Obtain 11 g of the product according to the physicochemical characteristics of the identical potassium salt of 5'-H-phosphonate AZT (RU 2106353 C1, C 07 H 19/073), yield 80%.

 Example 12

The phosphonation process is carried out analogously to example 1 with the difference that 1 kg of AZT is processed at a time in the synthesis, for which 760 ml of PCl ₃ , 16 l of acetonitrile, 400 ml of tert-butanol, 800 ml of pyridine, 6 l of water, 16 l of chloroform are used 6 l of 25% ammonia, 10 l of tributyl phosphate, 18 l of acetone, and instead of an aqueous solution of sodium hydroxide, 6 l of a 0.5N solution of NaOH in methanol are used. Obtain 1 kg of sodium salt of 5'-H-phosphonate AZT, yield 75%.

Claims (1)

The method of phosphonation of 2 ', 3'-dideoxynucleosides, including the preparation of a phosphonating agent based on PCl ₃ , its interaction with 2', 3'-dideoxynucleoside in acetonitrile, followed by the addition of water and the isolation of the target product, characterized in that the preparation of the phosphonating agent is carried out using tert-butanol and pyridine, and the selection of the target product is carried out by separating the inorganic phosphonation products in the form of ammonium salts in the presence of trialkyl phosphate.