Note: Descriptions are shown in the official language in which they were submitted.
~)77030
CANADA
1569A
2100.1316
SUMM~RY AND DETAILED DESCRIPTION
The present invention relates to a process for the
production of the ester product, 9-(~-D-arabinofuranosyl)-
adenine, 5'-phosphate, by phosphorylation of 9-(~-D-
arabinofuranosyl)adenine and hydrolysis of the phosphorylated
product. More particularly, the invention relates to such
process in which the desired ester product is obtained
in crystalline form directly from the reaction mixture.
In one prior art method for preparing the 5'-phosphate
ester of a nucleoside (U.S. Patent No. 3,703,507), the
corresponding nucleoside is reacted with phosphorus
oxychloride (POC13) in acetic acid in the presence of
pyridine. In another prior art method (U.S~ Patent
No. 3,413,282), the corresponding nucleoside is reacted
with POC13 or diphosphoryl chloride (P2O3C14) 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 techni~ues
using activated carbon or ion exchange resin. The prior
art methods undesirably involve time-consuming manipulations
and processing steps; they also use costly adsorbing media
and elution solvents.
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~n7703~
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 phosphorylating agent in the presence
of trialkyl phosphate solvent, subjecting the reaction mixture
to aqueous hydrolysis, adjusting the pH of the aqueous hydrolysis
mixture upward sufficiently to cause separation into aqueous and
non-aqueous liquid phases, removing the trialkyl phosphate
solvent from the aqueous mixture, maintaining 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
favorable ratio of required volume to product yield thereby
permitting increased batch sizes; the 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)-
adenine can be used either as the monohydrate or in the anhydrous
form, the anhydrous nucleoside being preferred. The phosphorylating
agent employed is any suitable agent such as a phosphorous
oxyhalide, in particular POC13, POBr3 and P203C14. The
1~77030
phosphorylating agent is 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. Phosphorus oxychloride is a preferred
phosphorylating a8ent. 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 solvent 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 between -10 to +10 C. at which
the reaction is complete 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. Following the reaction, the phosphorodihalidate or
trichlorodiphosphate intermediate is hydrolyzed to provide the r
ester product. This can be done by combining 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
amount to cause separation into an 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. The resulting mixture, which
forms 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-(~-D-
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1077030
arabinofuranosyl)adenine, 5'-phosphate, is water-soluble at
relatively low pH and at relatively high pH, the extracted
aqueous phase is maintained according to the invention, if
necessary after further ad~us~ment, at a pH at which the product
is insoluble (depending on conditions suitably in the pH range
from about 1.3 to about 2.5) and the mixture is held, preferably
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
,c acid form by conventional means such as filtration or centrifuga-
tion. The product can be recrystallized, if desired, or converted
to salt form in solution by conventional meansl 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 intermediate phosphoro- r
dihalidate or trichlorodiphosphate precipitate obtained 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, being active against Herpes simplex virus, as
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describèd in the above-mentioned U.S. Patent No. 3,703,507,
. ' ` '
The invention i5 illustrated by the following examples.
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~C~77030
Example 1
A stirred mixture of 10.69 g. (0.04 mole) of anhydrous
9-(~-D-arabinofuranosyl)adenine in 100 ml. (0.586 mole) of
triethyl phosphate was cooled to 0-2 C. in an ice bath.
To this stirred slurry was then added 7.n5 g. (0.046 mole)
of phosphorus oxychloride dropwise 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 flask 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. The 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 by 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
(aqueous) phase was readjusted to pH 2 using a few drops of
base. This solution was seeded with a few crystals of
1~77030
9-(~-D-arabinofuranosyl)adenine, 5'-phosphate, and stirred
at room temperature until precipitation 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 overnight in the cold. The mix~ure
was then filtered with suction, and the crystalline product,
9-(~-D-arabinofuranosyl)adenine, 5'-phosphate, was washed
successively with ice water (22 ml.), cold 50% aqueous ethanol
,o (35 ml.), and cold absolute ethanol (25 ml.). After drying
at 40 C. in vacuo for 16 hours the product weighed 10.59 g.
(76.2% of theory) and analyxed as follows: ~
Ion Exchan~e 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 theory.
~v The product analysis is carried out by the following
procedure which is typical:
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Ion Exchange Column Chromato~raphY - UV Assay
A chromatographic column (9 mm. x 15 cm.) is packed
with O . 5 g. of anion exchange resin (QAE-Sephadex A-25, Cl )
which has been suspended ln distilled water overnight. Ap-
proximately 20 mg. of sample is accurately weighed and
dissolved in 1,0 ml. of O.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
~olumetric flask. This fraction contains unreacted 9-(~-D-
arabinofuranosyl)adenine and adenine. The column is then
eluted 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 fraction is run at 320-220 nm.
(Fractions 1 and 3 without dilution and fraction 2 after a
20-fold dilutionj. From the absorption at 260 nm.(A ~260)~ the
contents of each fraction are calculated as follows:
mg. of component = 260 x 10 x V x D
a (1%, 1 cm.) 260
where V = volume (ml.) of each fraction
D ~ dilution
a (170, 1 cm.)~260 8 569 for 9-(~-D-arabinofuranosyl)-
adenine (Fraction I)
437 for the 5'-phosphate product
(Fraction II)
G 361 for the diphosphates (Fraction
III)
From the amount of each component, the percent of each is
th~n calculated. - 7a -
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1~77030
Example 2
To 100 ml. of stirred, cold (2 C.) triethyl phosphate
was added 9.20 g. (0.06 mole) of 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, producing
a clear solution about 1 hour 50 minutes after the addition. r
,o 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 solvent, then dissolved in 80 ml. of water. After
separating an ether layer, the aqueous solution was adjusted
to pH 2 using 50% sodium hydroxide solution, then seeded
with pure 9-(~-D-arabinofuranosyl)adenine, 5'-phosphat~,
and placed in the refrigerator to cool for two days with
occasional stirring. The precipitate was then filtered,
~D 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)-
adenine, 5'-phosphate, assaying as follows:
Ion Exchange Column Chromatography - W assay:
Fraction I (unreacted nucleoside + adenine): 0.38%
Fraction II (desired 5'-phosphate product): 92.8%
Fraction III (diphosphates): 4.03%
The corrected yield of 9-(~-D-arabinofuranosyl)adenine, 5'-
phosphate, was therefore 59~3V/o of theory.
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10~7030
Example 3
To 100 ml. of triethyl phosphate was added 10.69 g.
(0.04 mole) o~ anhydrous 9-(~-D-arabinofuranosyl)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 abou~ 4 C. The reaction mixture was
stirred in the cold for 2 hours 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 the pH was adjusted to 2 using 50% sodium
hydroxide solution. The resulting turbid mixture was
extracted twice using 100-ml. and 50-ml. 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 white
precipitate. This was stirred with a glass rod and again
allowed to stand overnight in the cold. The white product was
filtered 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~/o of theory) of the product,
9-(~-D-arabinofuranosyl)adenine, 5'-phosphate, was obtained
which assayed as follows:
Ion Exchan~e Column Chromato~raphy - ~ assay:
Fraction I (unreacted nucleoside + adenine): 0.17%
Fraction II (desired 5'-phosphate product): 89.9%
Fraction III (diphosphates): 4.54%
_ g _
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107~030
The corrected yield of 9~ D-arab-;nofuranosyl)adenine,
5'-phosphate was therefore 62.1% of theory.
Example 4
To an ice bath-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-(~-D-arabinofuranosyl)adenine (10.69 g., 0.04 mole)
was then added in one portion with stirring to give 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. of ice and the pH was adjusted
to 2 using 50% sodium hydroxide solution. After extracting
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-arabinofuranosyl)adenine,
5'-phosphate, as described in previous examples. After
filtering, washing, and drying, the white product weighed
10.12 g. (72.9% of theory) and assayed as follows:
Ion Exchange Column Chromatography - UV Assay:
Fraction I (unreacted nucleoside + adenine): 4.08%
Fraction II (desired 5'-phosphate product): 81.1%
Fraction III (diphosphates): 1. 51%
The corrected yield of 9-(~-D-arabinofuranosyl)adenine, 51 ~
phosphate, was 59~1%~
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1077030
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) of 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.
of ice and 50% sodium hydroxide solution was added to this
stirred, ice bath-cooled mixture 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-ara~inofuranosyl)adenine, 5'-phosphate, was collected
by filtration and washed and dried, as described in previous
examples to give 8.26 g. (59.5% of theory) which assayed
as follows:
Ion Exchange Column Chromato raphy - W Assay:
Fraction I (unreacted nucleoside + adenine): 0.24%
~D Fraction II (desired 5'-phosphate product): 94.3 %
Fraction III (diphosphates): 2.0 %
K. Fisher Water: 5.77%
Total = 102.3 %
The corrected yield of 9-(~-D-arabinofuranosyl)adenine,
5'-phosphate was therefore 56.1% of theory.
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