DK151263B - PROCEDURE FOR THE PREPARATION OF 9- (BETA-D-ARABINOFURANOSYL) -ADENINE, 5'-PHOSPHATE - Google Patents

Description

151263

The present invention relates to a process for the preparation of the ester product 9- (3-D-arabinofuranosyl) -adenine, 5'-phosphate by phosphorylation of 9- (β-D-arabinofuranosyl) -adenine and hydrolysis of the phosphorylated product.

In a known process for preparing the 5'-phosphate ester of a nucleoside (U.S. Patent No. 3,703,507), the corresponding nucleoside is reacted with phosphorus oxychloride (POClg). in acetic acid in the presence of pyridine. In another known method (U.S. Patent No. 3,413,382), the corresponding nucleoside is reacted with POCl., Or diphosphoryl chloride (P ^^ l ^) in the presence of trialkyl. phosphate solvent, the reaction product is hydrolyzed and neutralized and the desired nucleotide product is isolated by adsorption and elution techniques using activated charcoal or ion exchange resin. The known methods involve undesirable time-consuming manipulations and process steps, and they also use costly adsorbent media and elution solvents.

The process of the present invention for the preparation of the ester product 9- (3-D-arabinofuranosyl) -adenine, 51-phosphate is characterized in that 9- (3-D-arabinofuranosyl) -adenine is converted to a 5'-monophosphorylated intermediate with a phosphorylating agent in the presence of trialkyl phosphate solvent, the reaction mixture is subjected to aqueous hydrolysis, the pH of the aqueous hydrolysis mixture is adjusted sufficiently upward to cause aqueous and non-aqueous liquid phase separation, the trialkyl phosphate solvent is removed from the aqueous mixture, and residual aqueous mixture is maintained at a pH at which the ester product is insoluble to cause this ester product to precipitate from the aqueous mixture and said ester product is isolated.

The process of the invention has the advantage of leading to good yields of the desired product in crystalline form. The process also provides a favorable relationship between required volume and product yield, allowing for increased batch sizes. Product reprocessing is simple and requirements for both work and materials are kept to a minimum.

The process of the invention can be varied considerably. The starting material, 9- (3-D-arabinofuranosyl) -adiene, can be used either as a monohydrate or in anhydrous form, with the anhydrous nucleoside being preferred. The phosphorylating agent used may be any suitable agent, such as phosphorus oxyhalide, in particular POClg, POBr ^ or P203C14. The phosphorylating agent is used in an amount of approx. 1 to approx. 5 mol, and preferably about 5 mol. 1.5 to approx. 2 moles, for each mole of 9- (3-D-arabinofuranosyl) adenine. Phosphorus oxychloride is a preferred phosphorylation agent. The trialkylphosphate solvent used is an ester of phosphoric acid with an aliphatic (- - ()) alcohol such as trimethylphosphate or triethylphosphate). A ratio of about 15 to 1 is preferred in order to achieve the best results.The phosphorylation is generally carried out at temperatures of -30 to 50 ° C. The preferred range is between -10 and + 10 ° C, in which the reaction is completed in about 2 to 5 hours, at -20 ° C the reaction time is about 6 to 10 hours and at 30 ° C about 10 to 30 minutes. having the lidate or trichlorodiphosphate intermediate hydrolyzed to provide the ester product, this can be done by combining ice and / or water with the reaction mixture The pH of the resulting aqueous mixture is straight upwards by the addition of base, such as sodium hydroxide solution, in sufficient quantity to cause partitioning into an aqueous liquid phase and a non-aqueous liquid phase, appropriate to a value within the pH range of about 10 1 to approx. 2.5 or higher. The resulting mixture forming the two liquid phases is extracted with an inert, water-immiscible solvent such as dichloromethane or diethyl ether to remove the trialkyl phosphate. Since the desired product, 9- (3-D-arabinofuranosyl) -adiene, 5'-phosphate, is water-soluble at relatively low pH and at relatively high pH, the extracted aqueous phase is maintained, if necessary after further adjustment, at a pH value at which the product is insoluble (depending on conditions appropriate within the pH range of about 1.3 to about 2.5) and the mixture is recommended, preferably in the cold and grafted, to bring it desired ester product to precipitate as a solid phase from the aqueous mixture. The product obtained in pure crystalline form is isolated in free acid form in the usual manner, such as by filtration or centrifugation. The product may, if desired, be recrystallized or converted into saline form in solution by usual means, for example by adjusting the pH value.

The product, 9- (β-D-arabinofuranosyl) -adenine, 5'-phosphate, is useful as a pharmacological agent, especially as an antiviral agent, being active against the Herpes simplex virus, as described in the above-mentioned U.S. patent. No. 3,703,507, the disclosure of which is hereby incorporated by reference.

The process according to the invention is further described by the following examples.

151263 4

Example 1

A stirred mixture of 10.69 g (0.04 mole) of anhydrous 9- (β-D-ara-binofuranosyl) -adiene in 100 ml (0.586 mole) of triethyl phosphate was cooled to 0-2 ° C in an ice bath. To this stirred sludge was added dropwise 7.05 g (0.046 mole) of phosphorus oxychloride over a period of 2 hours, continuing to cool in an ice bath. The mixture was then stirred cold for a further 1 hour to give a clear to slightly misty solution poured onto 70 g of ice. The reaction vessel was rinsed with 10 ml of ice water and this was added to the hydrolysis mixture, which was then stirred in an ice bath for approx. 15 minutes. The mixture was stirred with cooling while the pH was adjusted from 0.6 to 2 using 50% sodium hydroxide solution. Another liquid phase separated and the temperature rose to approx. 10 ° C during this step. The mixture was stirred for a further 15 minutes while maintaining the pH at 2 by adding more base.

The mixture was then transferred to a separatory funnel and extracted with 100 ml of dichloromethane. The lower phase (non-aqueous, ca.

200 ml) was removed and the aqueous layer was again extracted using 50 ml of dichloromethane. The layers were again separated and the upper (aqueous) phase was reset to pH 2 using a few drops of base. This solution was seeded with a few crystals of 9- (β-D-arabinofuranosyl) -adiene, 5'-phosphate and stirred at room temperature until precipitation was well underway (about 10 minutes). Stirring was then stopped and the mixture was left to stand in the cold overnight. The resulting white crystalline mass was broken up by stirring and left to stand overnight in the cold. The mixture was then filtered with suction and the crystalline product, 9- (β-D-arabinofuranosyl) -adiene, 5'-phosphate, was washed successively with ice water (22 ml), cold 50% aqueous ethanol (35 ml) and cold. absolute ethanol (25 ml). After drying at 40 ° C under vacuum for 16 hours, the product weighed 10.59 g (76.2% of theory) and showed the following analysis:

Ion exchange column chromatography - UV sample:

Fraction I (unreacted nucleoside + adenine): 1.57%

Fraction II (desired 5'-phosphate product): 91.2%

Fraction III (disphosphates): 1.98% K.Fisher - Water; 5.62% total = 100.37%

Therefore, the corrected yield of 9- (β-D-arabinofuranosyl) -adenine, 5'-phosphate was 69.5% of theory.

The product analysis was performed by the following method, which is typical:

Ion exchange column chromatography UV sample

A chromatographic column (9 mm x 15 cm) is packed with 0.5 g of anion exchange resin (QAE-Sephadex A-25, Cl) which has been suspended in distilled water overnight. Ca. Weigh exactly 20 mg of sample and dissolve in 1.0 ml of 0.1N NaOH and transfer this solution to the column. The column is then eluted with water until a total of 25 ml is collected in a volumetric flask. This fraction contains unreacted 9- {β-D-arabinofuranosyl) -adiene and adenine. The column is then eluted with 0.1M phosphate buffer (pH 7.0) until 50 ml is collected. This second fraction contains the desired 5'-phosphate product. A third fraction containing disphosphate esters is recovered by eluting with 0.4M phosphate buffer (pH 7.0) until a total of 50 ml is collected.

The UV spectrum for each fraction is taken at 320-220 nm. (Fractions 1 and 3 without dilution and fraction 2 after 20-fold dilution). From the absorption at 260 nm (A ^ 26q), the content of each fraction is calculated as follows: a

mg of component = - x 10 x V x D

a (1%, 1 om) χ260 where V = volume (ml) of each fraction D = dilution a (1%, 1 ^) ^ 260 = f 9- (β-D-arabinofuranosyl) - adenine (Fraction I ) = 437 for the 51-phosphate product (Fraction II) = 361 for the diphosphates (Fraction III)

Based on the amount of each component, the percentage for each is then calculated.

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Example 2

To 100 ml of triethyl phosphate was added 10.69 g (0.04 mol) of anhydrous 9- (β-D-arabinofuranosyl) -adiene in one portion with stirring. This mixture was cooled in an ice bath to 2 ° C, and 9.20 g (0.06 mole) of phosphorus oxychloride was then added over a period of three minutes, with the temperature rising to ca. 4 ° C. The reaction mixture was stirred in the cold for 2 hours and 30 minutes, after which the clear solution was poured into 80 g of ice. This mixture was stirred in an ice bath to maintain a temperature below 10 ° C while adjusting the pH to 2 using 50% sodium hydroxide solution.

The resulting cloudy mixture was extracted twice using aliquots of 100 ml and 50 ml of dichloromethane, and the aqueous layer was again adjusted to pH 2 using additional base. After inoculation, this solution was placed in a refrigerator overnight, resulting in a dense white precipitate. This was stirred with a glass rod and again placed overnight in the cold. The white product was filtered off and washed using 22 ml of ice water, 35 ml of cold 50% ethanol and 25 ml of cold absolute ethanol. After drying in vacuo at 40 ° C, 9.60 g (69.1% of theory) of the product, 9- (β-D-arabinofuranosyl) -adiene, 5'-phosphate, were obtained, showing the following analysis:

Ion exchange column chromatography - UV sample:

Fraction I (unreacted nucleoside + adenine): 0.17%

Fraction II (desired 5'-phosphate product): 89.9%

Fraction III (diphosphates): 4.54%

Therefore, the corrected yield of 9- (β-D-arabinofuranosyl) -adenine, 5'-phosphate was 62.1% of theory.

Example 3

To an ice-cooled solution of 0.72 ml (0.04 mole) of water in 100 ml of triethyl phosphate was slowly added 12.27 g (0.08 mole) of phosphorus oxychloride at 0-5 ° C. Anhydrous 9- (β-D-arabinofuranosyl) -adenine (10.69 g, 0.04 mole) was then added in one portion with stirring to give an instantaneous temperature rise from 1 ° C to 9 ° C. The reaction mixture was ready in 30 minutes and after 2 hours and 30 minutes it was poured onto 100 g of ice and the pH was adjusted to 2 using 50% sodium hydroxide solution. After extraction twice using 200 ml aliquots and 100 ml of diethyl ether, the pH was again adjusted using additional base. The aqueous solution was then seeded with crystals of 9- (β-D-arabinofuranosyl) -adiene, 5'-phosphate and cooled in the refrigerator to precipitate the product, 9- (β-D-arabinofuranosyl) -adiene, 5'-phosphate , as described in previous examples. After filtration, washing and drying, the white product weighed 10.12 g (72.9% of theory) and had the following analysis:

Ion exchange column chromatography - OV sample:

Exercise I (unreacted nucleoside + adenine). 4.08%

Fraction II (desired 5'-phosphate product): 81.1%

Fraction III (diphosphates): 1.51%

The corrected yield of 9- (β-D-arabinofuranosyl) -adenine, 5'-phosphate was 59.1%.

Example 4

To a stirred slurry of 10.69 g (0.04 mole) of 9- (3-D-arabinofuranoyl) -adiene in 100 ml of triethyl phosphate cooled to 2 ° C in an ice bath was added over a period of 2 hours. , 58 g (0.046 mol) of diphosphoryl chloride ^ OgCl ^. The white reaction mixture was stirred in the cold for an additional 1 hour, during which time it became a clear solution. This solution was then poured onto 80 g of ice and 50% sodium hydroxide solution was added to this stirred, ice-bath, ac-cooled mixture to adjust the pH to 2. After extraction with dichloromethane, pH adjustment and inoculation, the aqueous solution was cooled . The product, which separated as a white crystalline solid, 9- (β-D-arabinofuranosyl) -adiene, 5'-phosphate, was collected by filtration, washed and dried, as described in the previous Examples to give 8.26 g ( 59.5% of theory) having the following analysis:

Ion exchange column chromatography - UV sample:

Fraction I (unreacted nucleoside + adenine): 0.24%

Fraction II (desired 5'-phosphate product): 94.3%

Fraction III (diphosphates): 2.0% K. Fischer - Water: 5.77% total = 102.3%

The corrected yield of 9- (β-D-arabinofiiranosyl) -adenine.

Therefore, 5'-phosphate was 56.1% of theory.

Claims (8)

151263 A process for preparing the ester product 9- (β-JD-arabinofuranosyl) -adiene, 5'-phosphate, characterized in that 9- (3-D-arabinofuranosyl) -adiene is converted to a 5'-monophosphorylated intermediate with a phosphorylating agent in the presence of trialkylphosphate solvent, the reaction mixture is subjected to hydrolysis, the pH of the aqueous hydrolysis mixture is adjusted sufficiently upward to cause aqueous and non-aqueous liquid phase separation, the trialkylphosphate solvent is removed from the aqueous mixture, aqueous mixture is maintained at a pH at which the ester product is insoluble to cause this ester product to precipitate from the aqueous mixture and said ester product is isolated. Process according to claim 1, characterized in that the phosphorylating agent is used in an amount of 1.15 to 2 moles for each mole of 9- (β-D-arabinofuranosyl) -adiene. Process according to claim 1 or 2, characterized in that the phosphorylating agent is phosphorus oxychloride. Process according to any one of claims 1-3, characterized in that the solvent is triethyl phosphate. Process according to any one of claims 1-4, characterized in that the molar ratio of trialkyl phosphate to 9- (β-D-arabinofuranosyl) -adiene is approx. 15 to 1. Process according to any one of claims 1-5, characterized in that the phosphorylation is carried out at temperatures between -10 and + 10 ° C. A process according to any one of claims 1-6, characterized in that the aqueous hydrolysis mixture is adjusted to a pH within the range of from approx. 1 to approx. 2.5. Process according to any one of claims 1-7, characterized in that the residual aqueous mixture is maintained at a pH within the range of from approx. 1.3 to approx. 2.5.