CA1088927A - PROCESS FOR THE PRODUCTION OF 9-(.beta.-D- ARABINOFURANOSYL)ADENINE, 5'-PHOSPHATE - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

Process for the production of an ester product, 9-(.beta.-D-arabinofuranosyl)adenine, 5'-phosphate, in which 9-(.beta.-D-arabinofuranosyl)adenine is converted to a 5'-mono-phosphorylated intermediate product in the presence of a trialkyl phosphate solvent, the reaction mixture is then treated with a non-aqueous diluent in which the intermediate product is insoluble, the solid intermediate product is isolated and subjected to aqueous hydrolysis, the pH of the hydrolysis mixture being maintained so as to precipitate the ester product from the aqueous mixture, and the ester product is isolated.

Description

.,, ,,--'\ , ; ' ' 'l?

~889~7 This application is a division of copending Canadian application Serlal No. 2gO,047, filed Novelrber 2, 1977.

.... .. _ . . _ .. _ ... . . .. .. .
SU~5MARY AND DETAILED DESC~IPTION
.
The present invention relates to a process ~or the . p~oduc~ion o~ the ester produc~ 9~ D-arabinouranosyl)-adenine, 5'~phosphate, by phosphorylation o~ 9-(Q D-arabino~uranosyl)adenine and hydrolysis o~ the phosphorylated product. More particularly, the invention rela~es to such process in which ~he desired es~er produc~ is obtained .
in crystalline form directly from the reaction mixtureO
. . In one prior ar~ method ~or preparing the S'-phospha~e ,o es~e~ of a nucleoside (U.S. Patent No. 3,703,507), ~he corresponding nucleoside is reacted wi~h phosphorus oxychloride (POC13) in acetic acid in the presence of pyridine. In ano~her prior art method tU.S. Paten~
- No~ 3,413j282), the corresponding nucleoside is reacted wi~h POcl3 or diphosphoryl chloride (P203C14) in the presence of trialkyl phosphate solvent, the reac~ion product . ;
hydrolyzed and neutralized, and the desired nucleotide product is isolated by adsorption and elution techniqaes using activated carbon or ion exchange resin~ The prior so art methods undesirably invo~ve time-consuming manipulations and processing s~eps; ehey also use costly adsorbing m~dia and ~lution sol~ent~.

.
~ . ~ 2 -- ~ .

, ' , ' ! ' .

' .: '. ' , ' , ' : , .' ': ' "' ' : ' . .

~08B5~27 The present invention, in a process for the production of the ester product 9~ D-arabinofuranosyl)adenine, 5'-phosphate, comprises the steps of reacting 9-~-D-arabino-furanosyl)adenine with a phosphorylalting agent in the presence of trialkyl phosphate solvent, subjecting the reaction mixture to aqueous hydrolysis, adjus~ing the pH of the aqueous hydrolysis mixture upward sufficiently to cause separa~ion into aqueous and ;-non-aqueous liquid phases, removing the trialkyl phosphate sol~ent from the aqueous mixture, main~aining the residual aqueous mixture at a pH at which the ester product is insoluble to cause said ester product to precipitate as a solid phase from the aqueous mixture, and isolating said product. The process of the invention advantageously provides good yields of the desired product in crystalline form. The process also provides a fa~orable ratio of required volume to product yield thereby permitting increased batch sizes; ~he product work-up is simple and labor and material needs are minimized.
The process of the invention is subject to considerable variation. The starting material~ 9~ D-arabinofuranosyl~-~o adenine can be used ei~her as the monohydrate or in the anhydrousfonm, the anhydrous nucleoside being preferredO The phosphorylating agent emplo~ed is any suitable agent such as a phosphorous oxyhalide, in particular POCl39 POBr3 and P203C14. The .'. ' ' ~ , ' ~ 7 phosphorylating agent ls used in a ratio of about 1 to 5 moles, and preferably about 1.15 to 2 moles, for each mole of 9~(~-D-arabinofuranosyl)adenine. Phospho~ls oxychloride ~s a pref~rred phosphorylating agent. The trialkyl phosphate solvent used is an ester of phosphoric acid with a Cl to C4 aliphatic alcohol, such as trimethyl phosphate or triethyl phosphate. Triethyl phosphate is a preferred solvent. The molar ratio of trialkyl phosphate solven~ to nucleoside in the process is at least 5 to 1. A ratio of about 15 to 1 is preferred for best results. The phosphorylation is ordinarily carried out at temperatures from -30 to 50 C. The preferred range is be~ween -10 to +10 C. at which the reaction is complete in about 2 to 5 hoursO At -20 C r the reaction time is about 6 to 10 hours and at 30 C. about 10 to 30 minutes. Following the reaction, the phosphorodihalidate or trichlorodiphosphate intermediate is hydrolyzed to provlde the ester product. This can be done by com~ining ice and/or water with the reaction mixture. The pH of the resulting aqueous ;
mixture is according to the invention adjusted upwardly by ad-dition of base such as sodium hydroxide solution in sufficient ~o amount to cause separation into a~ aqueous liquid phase and a non-aqueous liquid phase, suitably to a value in the pH range from about 1 to about 2.5 or higher, Th~ resulting mixture, which forms the ~wo liquid phases, ls extracted with an inert wa~er-immiscible solvent such as dichloromethane or diethyl ether to remove the trialkyl phosphateO Since the desired produ~t, 9~ D-~ 4 ~

. .
,: . . . : . .

1CU389 ~

arabinofuranosyl)adenine, 5'-phosphate, is water-soluble at -relatively low pH and at relative~y high pH, the extrac~ed aqueous phase is maintained according to the in~ention, if necessary af~er fur~her ad~us~ment, at a pH at which the product is insoluble (depending on conditlons suitably in the pH range from about 1.3 to about 2.5~ and the mixture is held, pre~erably in the cold and with seeding, to cause the 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 by conventional means such as filtra~ion or centrifuga-tion. The product can be recrystallized, if desired, or converted to salt form in solu~ion by conven~ional means, for example, by appropriate adjustment of the pH. In an optional procedure~
instead of hydrolyzing the reaction mixture directly in water as described, one can hydrolyze the in~ermediate phosphoro-dihalidate or trichlorodiphosphate precipitate obtaincd by adding the reaction mixture to a non-aqueous liquid (such as dichloro-methane or diethyl ether) in which the intermediate is insoluble.
- The product, 9-(~-D-arabinofuranosyl)adenine, 5'phosphate, is useful as a pharmacological agent, especially as an anti-viral agent, ~eing active against Herpes ~implex virus, as de~cribed in ~he above-mentioned U.S. Paten~ No. 3,703,507~

The invention is illustrated by the following examples.

.

o 5 ~

.

~82Z7 Example 1 A stirred mixture of 10.69 g. (0.04 mole) o~ anhydrous 9~ D-arabinofuranosyl)adenine in 100 ml. (0.586 mole) o~
triethyl phosphate was cooled to 0-2 C. in an ice bath.
To this stirred slurry was then added 7.05 g. (0.046 mole) of phosphorus oxychloride dropwis~ during a two hour period while maintaining ice bath cooling. The mixture was then stirred cold for another hour to give a clear to slightly hazy solution which was poured onto 70 g. of ice. The reaction ~lask 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 about 15 minutes. T~e mixture was stirred with cooling while the pH was adjusted from 0.6 to 2 using 50% sodium hydroxide solution. A second liquid phase separated and the temperature rose to about 10~ C. during this step. The mixture was stirred for another 15 minutes while the pH was held at 2 b~ the addition of more base.
The mixture was then transferred to a separatory funnel ~ -and extracted with 100 ml. of dichloromethane. The lower phase (non-aqueous, about 200 ml.) was removed and the aqueous layer was again extracted using 50 ml. of dichloro~
methane. The layers were again separated and the upper (agueous) phase was readjusted to pH 2 uslng a ~ew drops of base. This solution was seeded with a few crystals of "

: . ~ ;. . .
j. . .

~0!38927 9~ D-arabinofuranosyl)adenine 9 5'-phosphate, and stirred at room ~empera~ure until precipita~ion was well underway (about 10 minutes). Stirring was then stopped and the mixture was allowed to stand in the cold overnight. The resulting white crystalline mass was broken up by stirring and again allowed to stand overnigh~ in the cold. The mixture was then filtered with suction, and the crystalline product, 9~ D-arabinofuranosyl)adenine, 5'-phosphate, was washed successively with ice water (22 ml.), cold S0% aqueous ethanol (35 ml.)~ and cold absolute ethanol ~25 ml.~. After drying at 40 C. in ~acuo for 16 hours the product weighed 10.59 g.
(76.2% of theory) and analyzed as ~ollows:

Ion Exchange Column Chromato~raphy - W Assay:
Fraction I (unreacted nucleoside ~ adenine): 1~57%
Fraction II ~desired 5'-phosphate product): 91.2 %
Fraction III (diphosphates): 1.98%

K. Fisher Water: 5 62%
Total =100.37%
The corrected yield of 9-(~-D-arabinofuranosyl)adenine, 5'-phosphate was therefore 69~5% of theoryO
The produc~ analysis is carried out by the following procedure which is typical:

.:

~8~3~27 Ion Exchan~,e Column Ch ~ W Assay A chromatographic column (9 mmO x 15 cm.) is packed with 0.5 g. of anion exchange resin (QAE-Sephadex A-25, Cl which has been suspended in distilled wa~er overnight. Ap-proximately 20 mg. o~ sample is accurately weighed and dissolved in 1.0 ml. o~ 0.lN NaOH and this solution is transferred to the column. The column is then eluted with water until a total of 25 ml. has been collected in a ~olume~ric flask. This ~raction con~ains unreacted 9-(~-D-ID arabinofuranosyl3adenine and adenine. The column is theneluted with O.lM phosphate buffer (pH 7.0) until 50 ml. has been collected. This second fraction contains the desired 5'-phosphate product. A third fraction containing diphosphate esters is obtained by eluting with 0.4M phosphate buffer (pH 7.0) until a total of 50 ml. has been collected. ;~
The W spectrum of each ~raction is run at 320-220 nm.
(Fractions 1 and 3 wi~hout dilution and fraction 2 after a 20-fold dilution). From the absorption at 260 nm.(A ~260)~ the contents o~ each fraction are calculated as follows:

m8. of component = ~260 x 10 x V x D

a (1%9 1 cm.) ~ 260 where V = volume (ml.) o each fraction D = dilution - a (1%5 1 cm.~260 ~ 569 for-9-(~-D-arabinofuranosyl)-adenine (Fraction I) 437 for the 5'-phosphate product (Fraction II) 361 for the diphosphates (~rac~ion - III) From the amount o~ each component, the percent of each is then calculated.
- 7a -.

1 ~ 889 ~7 ExampLe 2 To 100 ml. of stirred~ cold (2 C.) triethyl phosphate was added 9.20 g. (0.06 mo~e) o phosphorus oxychloride within a two minute period. To this stirred solution was then added 10.69 g. (0.04 mole) of anhydrous 9~ D-arabinofuranosyl)adenine in one portion with a resultant reaction temperature rise to about 4 C. The mixture was stirred in the ice bath for 2 hours 10 minutes, produ~ing a clear solution about 1 hour 50 minutes after the addition.
The reaction solution was then poured into 800 ml. of cold diethyl ether and stirred for one hour in an ice bath.
This white suspension was filtered in the cold and the hygroscopic solid was washed twice with 100-ml. portions of cold sol~ent, then dissolved in 80 ml. of water. After separating an ether layer, the aqueous solution was adjus~ed to pH 2 using 50% sodium hydroxide solution, then seeded with pure 9~ D-arabinofuranosyl)adenine, 5'-phosphate, and placed in the refrigerator to cool for two days with occasional stirring. The precipitate was then filtered, washed in turn with 35 ml. of cold 50% ethanol and 25 ml. of cold absoLute ethanol, and finally, dried in vacuo at 40 C.
to give 8.88 g. ~63.9V~o of theory) of 9~ D-arabinofuranosyl)-adenineg 5'-phosphate, assaying as follows: -Ion Exchange CoLumn ChromatographY -UV assa Fraction I tunreacted nucleoside ~ adenine): 0.38%
Fractlon II (desired 5l-phosphate product): 92.8%

Fractlon III (diphosphates): 4.03%

The corrected yield of 9~ D-arabinofuranosyl)adenine, S'-phosphate, was ~herefore 59.3% of theory.

:, -: ;, .

~0889Z7 Example 3 To lOO ml o trie~hyl phosphate was added 10.69 g.
~0.04 mole) of anhydrous 9~ D-arabino~uranosyl)adenine 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 during a three-minute period, the temperature rising to about 4 C. The reaction mixture was stirred in the cold for 2 hours 30 minutes~ a~ter which the clear solution was poured into 80 g. of ice. This mixture ,o was stirred in an ice bath to maintain a ~emperature below 10 C. while the pH was adjusted to 2 using 50% sodium , hydroxide solution~ The resulting turbid mixture was extracted twice using 100-ml. and 50-mlO portions of dichloromethane and the aqueous layer was again adjusted to pH 2 using additional caustic. After seeding, this solution was placed in the refrigerator overnight to give a dense w~ite precipitate. This was stirred with a glass rod and again al~owed to stand overnight in the cold. The white product was filtered and washed using 22 ml. of ice water~ 35 ml. of cold L~ 50% ethanol and 25 ml. of cold absolute ethanol. A~ter drying in vacuo at 40 C., 9.60 g. (69.1% of theory) of the produc~, 9~ D-arabinofuranosyl)adenine9 5'~phosphate, was ob~ained which assayed as follows:
Ion Exchan~e Col~n Chromato~raphy - W assay:

Fraction I (~mreac~ed nucleoside + adenine): 0.17%
Fraction II (desired 5'-phosphate product): 89.9%
Fraction III (diphosphates): 4.54%
,, 9 ' ' . ' ' ' .. ' . , ~. :

The corrected yield o 9-(~-D~arabinofuranosyl3adenine, 5'-phosphate was thereore 62.1% of theory.

Example 4 To an ice bath-cooled solution o~ 0O72 ml. (0.0~ mole~ : :
of water in 100 ml. of triethyl phospha~e was slowly added 12.27 g. (0.08 mole) of phosphorus oxychloride at 0-5 C.
Anhydrous 9-(~-D-arabinofuranosyl)adenine (10.6g g., 0;04 mole) was then added in one portion wi~h stirring to gi~e an im-mediate temperature rise from 1 to 9 C. The reaction mixture was clear in 30 minutes and after 2 hours 30 minutes it was poured onto 100 g. o~ ice and ~he pH was adjusted to 2 using 50% sodium hydroxide solution. After ex~racting twice using 200-ml. and 100-ml. portions of diethyl ether, the pH was again adjusted using additional base. The aqueous solution was then seeded with crystals of 9~ D-arabino-furanosyl~adenine, 5'-phosphate, and cooled in the refrigerator to precipitate the product, 9-(~-D-arabino~uranosyl)adenine, 5'-phosphate, as described in previous examples. After filtering, washing, and drying~ the white product weig~ed 10.12 g. (72.g% of theory) and assayed as ollows:

Ion Exchan~e Columm~ Chromato~aphy - W Assay Fraction I (unreacted nucleoside ~ adenine): 4.08%
Fraction II (desired S'-phosphate produc~): 81.1% :.
Fraction III (diphosphates): 1.51%
The corrected yield of 9~ D-arabinofu*anosyl)adenine, 5'-phosphate, was 59.1%.

~o ~0 .

~ 8~Z~7 Example 5 , To a stirred slurry of 10.69 g. (0.04 mole) of 9~ D-arabinofuranosyl)adenine in 100 ml. of triethylphosphate cooled to 2 C. in an ice bath was added during a 2-hour period 11.58 g. (0.046 mole) o~ diphosphoryl chloride (P203C14). The white reaction mixture was stirred in the cold for an additional hour, becoming a clear solution during this time. This solution was then poured onto 80 g.
o~ ice and 50% sodium hydroxide solution was added to this ,0 stirred, ice bath-cooled mix~ture to adjust the pH to 2.
After extracting with dichloromethane~ readjusting the pH and seeding, the aqueous solution was cooled. The product which separated as a white crystalline solid, 9~ D-arabinofuranosyl)adenine, 5'-phosphate, was collected by ~iltration and washed and dried, as described in previous examples to give 8.26 g. (59 . 5~/o of theory) which assayed as follows:
Ion Exchange Column Chromato~raphy - W Assay:
Fraction I tunreacted nucleoside ~ adenine): 0.24%
~o Fraction II (desired S'-phosphate product): 94.3 %
~raction III (diphosphates): 2.0 %
K~ Fisher Water: 5,77%
_ Total =10~.3 %

The corrected yield of 9-(~-D~arabinofuranosyl)adenine, 5'-phosphate was therefore 56.1% of theory.

Claims (7)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. In a process for the production of the ester product 9-(.beta.-D-arabinofuranosyl)adenine, 5'-phosphate, the steps comprising converting 9-(.beta.-D-arabinofuranosyl)adenine to a 5'-monophosphorylated intermediate product in the presence of trialkyl phosphate solvent, treating the reaction mixture with a non-aqueous diluent in which the intermediate product is insoluble to cause said intermediate product to separate as a solid, isolating the solid and subjecting it to aqueous hydrolysis, maintaining the aqueous hydrolysis mixture at a pH at which the ester product is insoluble to cause said ester product to pre-cipitate from the aqueous mixture, and isolating said ester product.

2. Process according to claim 1 in which the phos-phorylating agent is used in a ratio of 1.15 to 2 moles for each mole of 9-(.beta.-D-arabinofuranosyl)adenine.

3. Process according to claim 1 in which the phos-phorylating agent is phosphorous oxychloride.

4. Process according to claim 1 in which the solvent is triethyl phosphate.

5. Process according to claim 1 in which the molar ratio of trialkyl phosphate to 9-(.beta.-D-arabinofuranosyl)-adenine is about 15 to 1.

6. Process according to claim 1 in which the phos-phorylation is carried out at temperatures between -10 to +10°C.

7. Process according to claim 1 in which the aqueous hydrolysis mixture is maintained at a pH in the range from about 1.3 to about 2.5.