Title of Invention

METHOD FOR PURIFYING 5' -PROTECTED 2' -DEOXYPURINE NUCLEOSIDES

Abstract A method for efficiently purifying 5' protected 2'-deoxypurine nucleosides, efficient production of which has previously been difficult. Impurities can be separated by obtaining the 5' protected 2'-deoxypurine nucleoside as an inclusion crystal including a solvent such as that having a nitrile structure in order to purify the 5' protected 2' -deoxypurine nucleoside at a high purity. This invention enables synthesis of highly purified protected deoxypurine nucleosides easily on a large scale, which has previously been performed by column chromatography method.
Full Text TITLE OF THE INVENTION
METHOD FOR PURIFYING 5'-PROTECTED 2'-DEOXYPURINE
NUCLEOSIDES
1.Field of the invention
The present invention relates to a method of purifying
5'-protected 2'-deoxypurine nucleoside and a derivative
thereof, as well as solvent-inclusion compounds thereof
obtained by the method. More specifically, the present
invention relates to a method of purifying 5'-protected
2' -deoxypurine nucleosides useful for producing them as well
as a solvent inclusion compounds of 5'-protected
2'-deoxypurine nucleosides which are obtained by the
purification method.
2.Description of the Related Art
5'-protected 2'-deoxy-b-purine nucleosides are
compounds useful as raw materials for antisense DNA or the
like, which has recently been developed.
In recent years, with developments in manufacturing
genomic drugs, antisense DNA drugs or the like have rapidly
been developed. Therewith, a DNA oligomer used as a raw
material, and further, protected deoxy nucleosides used as
raw materials for the oligomer are increasingly demanded.
Regarding the pharmaceutical uses, it is necessary to use an
extremely highly purified intermediate product to reduce
generation of by-products formed based on impurities to a
minimum.
As is clear from Japanese Patent Laid-Open Nos. 58-180500
and 63-179889, National Publication of International Patent
Application No. 6-507883, etc., 5'-protected deoxypurine
nucleosides have been purified by column chromatographic
method till now. By this method, separation of impurities
greatly different in their polarities or structures may be
carried out relatively easily, but elimination of impurities
having a similar structure is difficult. In particular, there
are many cases where it is difficult to eliminate a
3'-substituted isomer that is a especially problematic
impurity. In addition, since this method needs a large-scale
purification device, in view of mass production and mass supply
in the future, it cannot help saying that this method has a
large problem.
Up till now, studies regarding elimination of impurities
without using column chromatography have been made.
Specifically, purification by reprecipitation method is
disclosed in Japanese Patent Laid-Open No. 60-152495 and a
PCT application. WO200075154. The reprecipitation method is
a method in which, after a crude compound is dissolved in a
soluble solvent, the compound is compulsively reprecipltated
by addition of an insoluble solvent or dropping into an
insoluble solvent. Consequently, its purification ability
is basically low. Moreover, it is industrially difficult to
appropriately control the amount ratio between the soluble
solvent and the insoluble solvent. In addition, where the
amount ratio of these solvents is set inappropriately, it
easily results in oilification or generates a viscous
precipitate so that purification is apt to end in failure.
Actually, according to a method described in Japanese Patent
Laid-Open No. 60-152495, in some cases, the purified product
is obtained as a viscous syrupy substance, and, from an
industrial viewpoint, this is a problem. Although some
methods of forming amorphous by reprecipitation has been
disclosed till now, no methods for obtaining a crystal by
crystallization or recrystallization are known.
SUMMARY OF THE INVENTION
The present invention has been completed in view of the
conventional problems, and the object of the present invention
is to provide a purification method, which is efficient and
does not need special facilities by which extremely highly
purified 5'-protected 2'-deoxypurine nucleosides can be
obtained.
As a result of intensive studies by the present inventors
directed toward the above object, it has been found that, using
a nltrile solvent such as acetonitrile, 5'-protected
2' -deoxypurine nucleosides can be obtained as a crystal that
includes the solvent, and then it can be purified by a
purification method using crystallization or
recrystallization, thereby completing the present invention.

Thus, the present invention includes the following
embodiments:
(1) A method of purifying a 5'protected 2'-deoxypurine
nucleoside, which comprises the steps of:
obtaining a compound represented by the following formula
(1):
wherein R1 represents a 4-methoxytrityl group,
4,4'-dimethoxytrityl group or triphenylmethyl group, and B
represents a purine group wherein an amino group is protected;
in the form of inclusion crystals including a solvent, in a
liquid medium comprising the solvent for inclusion; and
recovering the inclusion crystals from the liquid medium.
(2) A method of purifying a 5'protected 2'-deoxypurine
nucleoside according to the above section (1), wherein the
solvent for inclusion is a nitrile compound substituted by
a lower alkyl group or an aryl group.
(3) A method of purifying a 5'protected 2'-deoxypurine
nucleoside according to the above section (2). wherein the
solvent for inclusion is acetonitrlle.
(4) A method of purifying a 5'protected 2'-deoxypurine
nucleoside according to any one of the above sections (1) to
(3), wherein a crude preparation comprising the compound of
the formula (1) and a compound of the following formula (2):
wherein R2 represents a hydrogen atom, 4-methoxytrityl group,
4.4'-dimethoxytrityl group or triphenylmethyl group, and R1
and B have the same definitions as stated above ;
is dissolved in the liquidmedium and the compound of the formula
(2) is removed into the liquid medium by recovering the compound
of the formula (1) in the form of inclusion crystals from the
liquid medium.
(5) A method of purifying a 5'protected 2'-deoxypurine
nucleoside according to any one of the above sections (1) to
(4), wherein the compound of the formula (1) is a compound
of the following formula (3)
or a compound of the following formula (4)
(6) A method of purifying a 5'protected 2'-deoxypurine
nucleoside according to any one of the above sections (1) to
(5), wherein the Inclusion crystals of the compound of the
formula (1) including the solvent for inclusion are
recrystalized from a liquid medium consisting of the solvent
for inclusion.
(7) A method of purifying a 5'protected 2'-deoxypurine
nucleoside according to any one of the above sections (1) to
(6), wherein the liquid medium consisting of a single solvent
for inclusion.
(8) An inclusion compound represented by the following
formula (5)
wherein Rl and B have the same definitions as stated above.
m and n are independently an integer, R3 is a lower alkyl group
or an aryl group.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention will be described in detail below.
The pusine group forming the B in the formula (1) means
a nucleic acid purine base of a natural or an unnatural
nuclcoside. Specific examples include adenine and guanine.
examples of a protecting group for the amino group of
the purine group include an alkyl group, an alkylacyl group
and e. benzoyl group.
The alkyl group may be straight chained or branched, or
another functional group may be added thereto, as far as the
function as the protecting group can be maintained. Examples
of the alkyl group include a methyl group, an ethyl group,
an n-propyl group, a 2-propyl group, an n-butyl group, an
iso-butyl group, etc.
The alkylacyl group may be straight chained or branched,
or may form a ring, or another functional group may be added
thereto, as far as the function as the protecting group can
be maintained. Examples of the alkylacyl group include an
acetyL group, a propionyl group, an n-butyryl group, an
iso-butyryl group, a pivaloyl group, an n-pentyloyl group,
an iso-pentyloyl group, a cyclopropylcarbonyl group, a
phenoxyacetyl group, etc.
The benzoyl group may not be substituted or may be
substituted as far as the function as the protecting group
can be maintained. One substituent may be at any one of
positions 2, 3 and 4 of a phenyl group. Moreover, the
substituent may be at a plurality of positions. Examples of
the substituents include an alkyl group such as a methyl group,
an ethyl group, a 2-propyl group, an n-butyl group or a
tert-butyl group; a hydroxyl group; an alkyloxy group such
as a methoxy group, an ethoxy group, an n-propyloxy group,
a 2-propyloxy group or an n-butyloxy group; a nitro group;
a halogen group such as a fluoro group, a chloro group, a bromo
group or an iodo group; an amino group; an alkylamino group
such as a methylamino group, an ethylamino group, an
n-propylamino group, a dimethylamino group, a diethylamino
group or a diisopropylamino group; an acyl group such as an
acetyl group, a propionyl group or a benzoyl group; a phenyl
group; a pyrldinyl group, etc.
Specific examples of the benzoyl groups include a benzoyl
group, a 2-chlorobenzoyl group, a 3-chlorobenzoyl group, a
4-chlorobenzoyl group, a 2-bromobenzoyl group, a
3-bromobenzoyl group, a 4-bromobenzoyl group, a
2-fluorobenzoyl group, a 3-fluorobenzoyl group, a
4-fluorobenzoyl group, a 2-methoxybenzoyl group, a
3-methoxybenzoyl group, a 4-methoxybenzoyl group, a
2-nitrobenzoyl group, a 3-nitrobenzoyl group, a
4-nitrobenzoyl group, a 2-aminobenzoyl group, a ,
3-aminobenzoyl group, a 4-aminobenzoyl group, a
2-methylaminobenzoyl group, a 3-methylaminobenzoyl group, a
4-methylaminobenzoyl group, a 2-dimethylaminobenzoyl group,
a 3-dlmethylaminobenzoyl group, a 4-dimethylaminobenzoyl
group, a 4-phenylbenzoylgroup, a 4-acetylbenzoyl group, etc.
Preferable Examples of a nitrile compound having a lower
alXyl group or an aryl group, as the solvent for formation
of the inclusion crystals, is those represented by the formula;
R3-CN, wherein R3 is a lower alkyl group or an aryl group.
Examples of the alkyl group include a methyl group, an ethyl
group, an n-propyl group, a 2-propyl group, an n-butyl group
and an iso-butyl group. Examples of the aryl group include
a phenylgroup, a4-methylphenyl group, a4-methoxyphenylgroup.
____Examples of the nitrile compounds having the lower alKyl
group or the aryl group include acetonitrile. propionitrile,
n-butyronitrila, iso-butyronitrile, n-pentanenitrile,
n-heaxanenitrile, benzonitrile, etc. At least one of these
nitrile compounds may be used.
____Regarding m and n in the formula (5), m may be preferably
an in teger selected from 1 to 5, and n may be preferably an
integer selected from 1 to 5.
The liquid medium for formation of the inclusion crystals
may be composed either only of the solvent to be included in
the crystals or of a mixture of the solvent for inclusion and
other solvent(s) which does not form inclusion crystals at
a ratio capable of mixing. Examples of the other solvents
capable of being mixed with the solvent for inclusion include
alcohols such as methanol, ethanol and isopropanol; esters
such as ethyl acetate and butyl acetate; ketones such as acetone,
methylathylketone and mathylisobutylketon; ethers such as
diethyl ether, diisopropylether, dioxane and tetrahydrofuran
(THF); aromatic hydrocarbons such as benzene, toluene, cumene,
xylena, mesitylene, diisopropylbenzene and
triisopropylbenzene; halogenated hydrocarbons such as
dichloromethane, chloroform and dichloroethane; pyridines
such has pyridine, lutidine and quinoline; tertiary amines such
as triethylamine and tributylamine; polar solvents such as
dimethylformamide (DMF), dimethyl imidazolidinone (DMI) and
dimethyl sulfoxide (DMSO); water etc. At least one of these
solvents may be used as the other solvent. The mixing ratio
of the above listed other solvent (s) is 100% by weight or less
with respect to the solvent for inclusion, preferably 20% by
weight or less, and more preferably 10% by weight or less.
The term "inclusion crystal" inclusing a solvent is herein
used to mean that a solvent plays an auxiliary role to form
a crystal structure, such that a crystal is formed in a form
wherein a solvent is taken up into a crystal lattice thereof,
or a complex is formed by a weak interaction between a crystal
and a solvent. The inclusion form and the crystal structure
are not particularly limited.
The amount of a nitrile solvent in crystallization and
recrystallization are not particularly limited, as far as the
amount is below the saturation solubility of a compound to
be purified to the solution, but desirably the amount of the
solvent is 5 times by weight or more to 150 times by weight
or less of the amount of the compound of the formula (1), and
further desirably it is 8 times by weight or more to 50 times
by weight or less of the amount of the compound of the formula
(1).
A temperature for crystallization and recrystallization
are not particularly limited, but a temperature within a range
from -10°C to the boiling point of a solvent or a liquid medium
is desired. Generally, purification can be performed more
sufficiently by a single time of recrystallization. but
purification at higher purity can also be realized by
performing recrystallization repeatedly. A preferable
liquid medium for recrystallization is that consisting of a
solvent for inclusion alone and it is more preferable to used
the same single solvent for inclusion in both of
crystallization and recrystallization.
As stated above, according to the present invention, it
becomes possible to efficiently purify protected 5' protected
2'-deoxypurine nucleosides.
Examples
The present invention will be further specifically
described in the following examples. The examples are not
intended to limit the scope of the invention.
Example 1
Production of a N2-lsobutyryl-5'-0-(4,4'-dimethoxytrityl)
-2'-deoxyguanosine and acetonitrile (2:1) complex
110 g (95% content) of N2-isobutyryl-2-deoxyguanosine
was subjected to azeotropic dehydration with pyridine, and
then dissolved in 2.4 L of pyridine. After adding 118.3 g
of 4,4'-dimethoxytrityl chloride at room temperature, the
mixture was stirred at room temperature for 3 hours. After
neutralizing hydrochloric acid with sodium bicarbonate, the
reaction solution was concentrated to about 400 g. After
adding 1.5 L of ethyl acetate and 1.5 L of water were added
thereto, the mixed solution was separated, and then washing
with water was repeatedly performed until no
N2-isobutyryl-2-deoxyguanosine as a raw material was found
in an organic layer thereof. After washing with 1 kg of 20%
sodium chloride solution, drying with sodium sulfate was
performed. After performing filtration, a solvent was
removed and ethyl acetate was then added so that the total
amount became 700 g.
This solution was dropped into 3,200 g of diisopropyl
ether, while intensively stirring, and thereafter the obtained
solution was stirred at room temperature for 2 hours. The
solid obtained by filtration was subjected to vacuum drying
at 5C0C, and after confirming that a constant weight was
obtained, it was dissolved in 4.4 L of acetonitrile at room
temperature. After stirring at room temperature for 4 hours.
the deposited solid was filtrated. The obtained product was
subjected to vacuum drying at room temperature for 4 hours
and then an NMR analysis. It was found by the analysis that
the obtained product included 2 molecules of acetonitrile per
one molecule of the target compound to be purified. Where
vacuum drying was performed at 40°C for 12 hours, it was found
by the subsequent NMR analysis that the obtained product
included one molecule of acetonitrile per one molecule of the
target compound to be purified.
Where vacuum drying was performed at 50°C for 15 hours,
it was found by the subsequent NMR analysis that the obtained
product included 0.67 molecule of acetonitrile perone molecule
of the target compound to be purified. Further, where vacuum
drying was performed at 55°C until a constant weight was
obtained (for 24 hours), it was found by the subsequent NMR
analysis that the obtained product included 0.5 molecule of
acetoritrile per one molecule of the target compound to be
purified. By X-ray diffraction (XRD). it was found that the
obtained product was a crystal in any of the above dry conditions.
Moreover, by TG-DTA analysis, it was found that, regarding
a crystal included 0.5 molecule of acetonitrile per one
molecule of the target compound to be purified, that was
obtained by drying until a constant weight was obtained, the
weight of the crystal was not reduced until a temperature was
raised to the temperature for endothermic reaction (81°C to
93°C), the crystal having no attached solvent. The yield was
168 g (yield rate 84.8%). As a result of an analysis by HPLC
(UV 254 nm). the purity was 99.7% by area. The largest impurity
was N2-isobutyryl-3',5'-O-bis(4,4'-dimethoxytrityl)-2'-
deoxyguanosine (0.17% by area).
NMR (DMSO-d6) d: 12.1 (s. 1H). 11.7 (s. 1H). 8.1 (s, 1H). 7.3
(m. 2H). 7.3-7.3 (m. 7H). 6.9-6.8 (m, 4H), 6.3 (t. J-6 Hz.
1H). 5.4 (a, 1H), 4.4 (m. 1H), 3.97 (m. 1H). 3.724 (s. 3H).
3.719 (s. 3H), 3.2 (m. 1H), 3.1 (m. 1H), 2.8 (m. 1H). 2.4 (m,
1H). 2.1 (s. 3/2H. acetonitrile). 1.1 (d. J-6.8 Hz, 6H).
1R(KBr) cm-1:3398, 3238, 2935; 2838, 1679. 1609. 1561, 1509.
1252. 1178. 1034. 830 (Absorption of acetonitrile derived from
nitrile was weak and was not observed.)
Example 2
Production of
N6-benzoyl-5' -0- (4,4' -dimsthoxytrltyl)-2' -deoxgadengsine
45.0 g (0.127 mol) of N6-benzoyldeoxyadenosine was
dissolved in 500 ml of pyridine followed by azeotropic
dehydration, and then the obtained product was dissolved in
500 ml. of pyridine. While stirring. 42.9 g (0.127 mol) of
4.4'-climethoxytrityl chloride was added thereto, and the
mixture was further stirred at room temperature for 2.5 hours.
After 12.8 g of sodium bicarbonate was added thereto and the
mixture was stirred at room temperature for 30 minutes, a
solvent was removed at reduced pressure. To the residue, 830
ml of methylisobutylketone was added, and while stirring 830
ml of water was further added thereto followed by stirring
for 10 minutes. Subsequently, an organic layer thereof was
collected, washed with a saturated saline solution and dried
with sodium sulfate, and thereafter a solvent was removed at
reduced pressure. The residue was dropped in 800 ml of
diiscpropyl ether that was intensively stirred, and the
genezated precipitate was collected by filtration.
The precipitate was recrystallized from 750 ml of
acetcnitrile, and the crystal product was collected by
filtration. As a result of an NMR analysis, the product
included one molecule of acetonitrile per one molecule of the
target compound to be purified. When the crystal product was
subjected to vacuum drying, acetonitrile was lost. The weight
was 66.7 g. When the obtained product was analyzed by high
performance liquid chromatography [ODS: octadecyl silica gel
column. acetonitrile/water (8:2)], using a UV detector (254
nm), the purity was 99.5%. The yield of
N6-benzoyl-5' -o- (4,4' -dimethoxytrityl) -2' -deoxyadenosine
was 79.4%. The largest impurity was
N6-benzoyl-3'. 5' -O-bis(4,4' -dimethoxytrityl) -2' -deoxyaden
osine (0.15% by area).
Refexonce Example 1
Production of N2-lsobutyryl-3', 5'-0-
bis (4,4' -dlmethoxytrltyl) -2' -deoxyguanoslne
After the filtrate obtained by the recrystallization in
Example 1 was concentrated, the concentrate residue was
purified by column chromatography (ethyl acetate/hexane).
The purified fraction was concentrated to obtained a yellow
oily product. The yellow oily product was then dropped into
diisopropyl ether and the precipitate thus formed was recovered
by filtration and dried, whereby the titled compound was
obtained as a light yellow powder.
1H NMR(400MHz,DMSO-d6) d l2.1(lH,s), 11.5(lH,s), 7.9(lH,s),
7.5-7.2(18H,m), 6.8(8H,m), 6.2(lH,m), 4.4(lH,m). 3.8(lH,m).
3.7(12H,s), 3.6(lH.m). 3.0(lH,m), 2.8( lH.m) , 2 .'4( lH,m) ,
1.8(lH,m), l.l(6H,m), 1.0(6H,m)
Reference Example 2
Production of N2-isobutyryl-5'-
O- (tert-butyldimethylsilyl) -2' -deoxyguanoslne
N2-isobutyryl-2'-deoxyguanoslne (20.5 g) was dissolved
in 200 ml of DMF. After 18.5 g of imidazole was dissolved
in the resultant solution by addition, 21.4 g of
tert-butyldimethylsilyl chrolide was then added. DMF (100
ml) was further added and the solution was stirred at room
temperature. After 8 hours, extraction using chloroform was
carried out and the organic layer was washed by a saturated
aqueous solution of sodium chloride and dried with anhydrous
magnesium sulfate. After concentration of the extract, the
target compound was separated by column chromatography
(methanol/chloroform). The fraction solution including the
target compound was prepared and concentrated to obtain 20.5
g of the titled compound (yield; 74%).
1H NMR(400MHz.CDCl3) d:12.4(lH,s). 10.9(lH.s), 8.0(lH,s),
6.0(lH,dd,J«6.4,6.4Hz), 4.6(2H,m), 4.1(lH,d,J=2.OHz),
3.8(2H,m), 2.9(lH,m), 2.4(2H,m), 1.3(6H,m), 0.8(9H,s),
0.02(6H,s)
Reference Example 3
Production of N2-isobutyryl-5'-0-
(tert-butyldlmethylsilyl)-3'-O-(4.4' -dimethoxytrityl) -2' -
deoxyguanosine
N2-isobutyryl-5'-O-(tert-butyldimethylsilyl)-2'-deox
yguanosine (19.8 g) was dissolved in 100 ml Of anhydrous
pyridine. 4,4'-dimethoxytrityl chloride (1.66 g) was added
to the resultant solution and 140 ml of anhydrous pyridine
was further added, follow by stirring at 40 °C. After the
reaction completed, the reaction mixture was neutralized by
sodium hydrogen carbonate and pyridine was distilled off.
Extraction using chloroform was carried out and the extract
was washed by a saturated aqueous solution of sodium chloride
and dried with anhydrous magnesium sulfate. The extract was
concentrated and purified by column chromatography (ethyl
acetate/hexane) to obtain 25.8g of the titled compound (yield:
78%).
1H NMR(400MHz,CDCl3) d: 11.9(lH.s). 8.1(lH,s). 7.8(lH.s).
7.5-7.2(9H,m). 6.8(4H.m),6.2(lH.dd.J=6.0,8.4Hz), 4.4(lH.m).
4.1(lH.m), 3.8(6H.s), 3.6(lH,dd.J=8.8.11.2Hz),
3.3(lH.dd,J=2.8,11.2Hz), 2.6(lH,m), 2.0-1.6(2H,m),
1.3(6H,m), 0.8(9H.s), 0.02(6H,s)
Reference Example 4
Production of N2-isobutyryl-3'-O-
(4.4'-dlmethoxytrityl)-2'-deoxyguanosin.
N2-isobutyryl)-5' -0- (tert-butyldimethylsilyl) -3' -O- (4
,4'-dlmethoxytrityl)-2'-deoxyguanosin (25 g) was dissolved
in 200 ml of dry THF. A THF solution (40 ml) of
tetrabutylammonium fluoride was added to the resultant
solution and 100 ml of dry THF was further added, followed
by stirring at room temperature. After 8 hours, extraction
using chloroform was carried out, and the extract was washed
by a saturated solution of sodium chloride and dried with
anhydrous magnesium sulfate. The extract was then
concentrated and purified by column chromatography (ethyl
acetate/hexane/methanol) to obtain 12.9 g of the titled
compound as a white powder (yield: 60%).
1H NMR ( 400MHZ, CDC13 ) d : 12.0(lH,s), 8.3(1H,S), 7.7(lH,s),
7.5-7.2(9H,m), 6.8(4H,m), 6.2(lH,dd,J-5.2, lOHz). 4.5(lH,m),
4.0(lH,m), 3.8(6H,s), 3.7(lH,m), 3.3(lH,m), 2.6(lH,m),
2.4(lH,m), 1.7(lH,m), 1.2(6H,m)
Comparative Example 1
In respect of an ability to eliminate impurities such
as the 3'-substituted isomer
[N2-isobutyryl-3' -O- (4,4' -dimethoxytrityl) -2' -deoxyguanos
ine] or the 3',5'-multiple substituted form
[N2-isobutyryl-3', 5' -O-bis( 4,4' -dimethoxytrityl) -2' -deoxy
guanosine], there was made a comparison between a method
involving recrystallization with an acetonitrile solvent and
a purification method involving reprecipitation using
dichloromethane as a soluble solvent, and hexane or toluene
as an insoluble solvent. In addition, the yield rate of a
product of interest,
[N2-isobutyryl-5' -0- (4,4' -dimethoxytrltyl) -2' -deoxyguanos
ine], was compared between both methods, and also thermal
analysis (DSC: endothermic peak, endothermic energy) of the
obtained crystal was carried out. The results thus obtained
are shown in Table 1, which include the contents of the 3' isomer
and the multi-substituted compound in the crude crystal
preparation, and the crystals after recrystalization using
each solvent.
As is clear from the results concerning DSC, it was
revealed that, since the endothermic peaks of the compounds
were different, the crystal types of the compounds were also
different. Regarding the large endothermic energy, it was
further revealed that the product obtained from acetnitrile
alone was formed as crystals.
Reference Example 5
Production of N6-benzoyl-3', 5' -0-
bis(4.4'-dimethoxytrltyl) -2'-deoxyadenosine
After the filtrate obtained by the recrystallization in
Example 2 was concentrated, the concentrate residue was
purified by column chromatography (ethyl acetate/hexane).
The purified fraction was concentrated to obtained a yellow
oily product. The yellow oily product was then dropped into
diisopropyl ether and the precipitate thus formed was recovered
by filtration and dried, whereby the titled compound was
obtained as a light yellow powder.
1H NMR(400MHz,DMSO-d6)d: 11.2(lH,br), 8.6(lH,s), 8.5(lH,s),
8.0(2H,m), 7.7-6.7(29H,m), 6.4(lH,m), 4.3(lH,m), 4.1(lH,m),
3.8(14H,s), 3.6(lH,m), 3.0(lH,m), 1.0(6H,m)
Reference Example 6
Production of N6-benzoyl-5'-0-
(tert-butyldimethylsilyl)-2' -deoxyadenosine
N6-benzoyl-2' -deoxyadenosine (20.95 g) was dissolved in
200 ml of DMF. Imidazole (15.7 g) was dissolved to the
resultant solution by addition. Then, 20.8 g of
tert-butyldimethylsilyl chloride was added. DMF (100 ml) was
further added and the solution was stirred at room temperature.
After 8 hours, extraction using chloroform was carried out
and the organic layer was washed by a saturated aqueous solution
of sodium chloride and dried with anhydrous magnesium sulfate.
After concentration of the extract, the target compound was
purified by column chromatography (methanol/chloroform) to
obtain 16.1 g of the titled compound (yield; 61%).
1H NMR(400MHz,CDCl3) d:9.1(lH,s), 8.8(lH,s), 8.3(lH,s),
8.0(2H,d,J=12.4Hz), 7.6-7.5(3H,m). 6.6(lH,dd,J=6.8,6.8Hz),
4.7(lH,m), 4.1(lH,m), 3.9(2H,m), 2.8(lH,m), 2.6(lH,m),
0.9(9H,s). 0.1(6H,s)
Reference Example 7
Production of N6-benzoyl-5'-0-
(tert-butyldimethylsilyl) -3' -O- (4.4' -dimethoxytrityl) - 2' -
deoxyadenosine
N6-bezoyl-5'-O-(tert-butyldimethylsilyl)-2'-deoxyade
nosine (16.1 g) was dissolved in 100 ml of anhydrous pyridine.
4,4' -dimethoxytrityl chloride (12.8 g) and anhydrous pyridine
(140 ml) were further added, follow by stirring at 45 °C. After
the reaction completed, the reaction mixture was neutralized
by sodium hydrogen carbonate, and pyridine was distilled off.
Extraction using chloroform was carried out and the extract
was washed by a saturated aqueous solution of sodium chloride
and dried with anhydrous magnesium sulfate. The extract was
concentrated and then purified by column chromatography (ethyl
acetate/hexane) to obtain 16.7 g of the titled compound (yield:
63%).
1H NMR(400MHz,CDC13) d :9.1(lH.s), 8.9(lH.s), 8.3(lH,s).
8.1(2H,d.J=7.2Hz). 7.7-7.3(12H.m), 6.9(4H,m).
6.7(lH,dd.J=5.6,8.4Hz), 4.5(lH,m), 4.2(2H,m), 3.9(6H.s),
3.7(lH,dd,J=2.4,11.2Hz). 3.4(lH.dd, J=8.4,11.2Hz).
2.2-2.1(2H,m), 2.0-1.6(2H,m), 1.3(1.5H,m), 0.9(9H,s),
O.O3(6H,S)
Reference Example 8
Production of N6-bezoyl-3'-O-
(4,4'-dlmethoxytrltyl)-2'-deoxyadenosine
N6-benzoyl-5'-O-(tert-butyldimethylsilyl)-3'-O-(4,4'
-dimethoxytrityl)-2'-deoxyadenosine (16.2 g) was dissolved
in 200 ml of dry THF. A THF solution (31 ml) of
tetrabutylammonium fluoride was added to the resultant
solution and 100 -ml of dry THF was further added, followed
by stirring at room temperature. After 8 hours, THF was
distilled off and extraction using chloroform was carried out.
The extract was then washed by a saturated solution of sodium
chloride and dried with anhydrous magnesium sulfate. The
extract was then concentrated and purified by column
chromatography (ethyl acetate/hexane/methanol) to obtain
13.8 g of the titled compound as a white powder (yield: 98%).
1H NMR(400MHz,CDC13) d:9.0(lH.br). 8.7(1H,S), 8.1(lH,s),
8.0(2H,m), 7.7-7.2(12H,m). 6.9(4H.m), 6.4(lH,m), 4.6(lH,m),
4.1(1.3H,m). 3.8(6H,s), 3.7(lH,m), 3.3(lH,m). 2.7(lH,m).
2.0(2H.m), 2.0-1.6(2H,m), 1.7(lH.m). 1.3(2H,m)
Comparative Example 2
In respect of an ability to eliminate impurities such
as the 3'-substituted isomer
(N6-benzoyl-3'-0-(4,4'-dimethoxytrityl)-2'-deoxyadenosine
) or the 3',5'-multiple substituted form
[N6-benzoyl-3'. 5' -O-bis(4.4' -dimethoxytrityl) -2 '-deoxyade
nosine), there was made a comparison between a method involving
recrystallization with an acetonitrile solvent and a
purification method involving reprecipitation with
dichloromethane as a soluble solvent and a mixed solution of
t-butylmethyl ether and hexane (1:2) as an insoluble solvent.
In addition, the yield rate of a product of interest,
[N6-benzoyl-5' -0- (4,4* -dimethoxytrityl) -2' -deoxyadenosine
], was compared between both methods, and also thermal analysis
(DSC: endothermic peak, endothermic energy) of the obtained
crystal was carried out.
The results thus obtained are shown in Table 2, which
include the contents of the 3' isomer and the multi-substituted
compound in the crude crystal preparation, and the crystals
after recrystalization using each solvent.
As is clear from the results concerning DSC it was
revealed that, since the endothermic peaks of the compounds
were different, the crystal types of the compounds were also
different. Regarding the large endothermic energy, it was
further revealed that the product obtained from acetnitrile
alone was formed as crystals.
The condition of HPLC for each compound is as follows:
(1) HPLC conditions in Comparative Examples 1 and 2 (analysis
of the amount of the multiple substituted form):
Column: Develosil TMS-UG-5
150 mm x ø4.6
Flow rate: 1.0 mL/min
t
Column temperature: 40°C
Detection wavelength: 254 nm
Mobile phase: gradient conditions
Time (rain) Liquid B (%)
0 20
15 70
35 100
40 100
45 20
60 STOP
[Liquid A]
100 mL of 100 mM triethylamlne-acetic acid (pH7)/880 mL
of water/20 mL of acetonitrile
[Liquid B]
100 mL of 100 mM triethylamine-acetic acid (pH7)/900 mL
of acetonitrile
(2) HPLC conditions in Comparative Examples 1 and 2 (analysis
of the amount of the 3'- isomer):
Column: Develosil TMS-UG-5
150 mm x ø4.6
Mobile phase: acetonitrile-water (55 : 45)
Flow rate: 1.0 mL/min
Column temperature: 40°C
Detection wavelength: 254 nm
Conditions for thermal analysis:
Apparatus: DSC-7 (PerkinElmer)
Rate of temperature rise: 10°C/min
Condition for XRD:
Apparatus: RAD-RVC (RIGAKU)
X-ray target: Cu 50kV 200 mA
According to the present invention, a method capable of
mass production enables production of highly purified
protected 2'-deoxypurine nucleosides more efficiently than
conventional methods.
WE CLAIM:
1. A method of purifying a 5' protected 2'-deoxypurine
nucleoslde, which comprises the steps of:
(a) preparing a solution of a compound represented by
the following formula (1):
wherein R1 represents a 4-methoxytrityl group, 4,4'-
dimethoxytrltyl group or triphenylmethyl group, and B
represents a purine group wherein an amino group is
protected;
in a liquid medium comprising a solvent for forming
inclusion crystals comprising said solvent and the
compound of formula (1);
(b) forming inclusion crystals comprising said solvent
and the compound of formula (1) in said solution by
treating said solution alone;
(c) recovering said inclusion crystals from the liquid
medium of the solution.
A method of purifying a 5'protected 2'-deoxypurine
nucleoslde according to claim 1, wherein the solvent for
Inclusion is a nitrile compound substituted by a lower alkyl
group or an aryl group.
A method of purifying a 5'protected 2'-deoxypurine
nudcosldc according to claim 2, wherein the solvent for
inclusion is acetonltrile.
. A method of purifying a 5'protected 2'-deoxypurine
nucleoslde according to any one of claims 1 to 3, wherein a
crude preparation comprising the compound of the formula
(1) and a compound of the following formula (2):
wherein R2 represents a hydrogen atom, 4-methoxytrityl group,
4,4'-dlmethoxytrityl group or triphenylmethyl group, and R1
and B have the same definitions as stated above ;
is dissolved in the liquid medium and the compound of the formula
(2) is removed into the liquid medium by recovering the compound
of the formula (1) in the form of inclusion crystals from the
liquid medium.
5. A method of purifying a 5' protected
2'-deoxypurine nucleoside according to any one of claims 1
to 4, wherein the compound of the formula (1) is a compound
of the following formula (3)
or a compound of the following formula (4)
6.A method of purifying a 5'protected 2'deoxypurine nucteoslde
according to any one of claims 1 to 5, wherein the inclusion
crystals of the compound of the formula (1) Including the
solvent for Inclusion are recrystalllzed from a liquid medium
consisting of the solvent for Inclusion.
7.A method of purifying a 5'protected 2'-deoxypurlne nudeoslde
according to any one of claims 1 to 6, wherein the liquid
medium consisting of a single solvent for Inclusion.
8.An inclusion compound of a 5'protected 2'-deoxypurine
nucleoside with a nitrite compound represented by the following
formula (5);
wherein Rl and B have the same definitions as stated above,
m and n are independently an integer, R3 is a lower alkyl group
or ar aiyl group.
A method for efficiently purifying 5' protected
2' -deoxypurine nucleosides, efficient production of which has
previously been difficult. Impurities can be separated by
obtaining the 5' protected 2'-deoxypurine nucleoside as an
inclusion crystal including a solvent such as that having a
nitrile structure in order to purify the 5' protected
2' -deoxypurine nucleoside at a high purity. This invention
enables synthesis of highly purified protected deoxypurine
nucleosides easily on a large scale, which has previously been
performed by column chromatography method.

Documents:

205-CAL-2002-FORM-27.pdf

205-cal-2002-granted-abstract.pdf

205-cal-2002-granted-claims.pdf

205-cal-2002-granted-correspondence.pdf

205-cal-2002-granted-description (complete).pdf

205-cal-2002-granted-examination report.pdf

205-cal-2002-granted-form 1.pdf

205-cal-2002-granted-form 18.pdf

205-cal-2002-granted-form 2.pdf

205-cal-2002-granted-form 3.pdf

205-cal-2002-granted-form 5.pdf

205-cal-2002-granted-gpa.pdf

205-cal-2002-granted-reply to examination report.pdf

205-cal-2002-granted-specification.pdf

205-cal-2002-granted-translated copy of priority document.pdf


Patent Number 222902
Indian Patent Application Number 205/CAL/2002
PG Journal Number 35/2008
Publication Date 29-Aug-2008
Grant Date 27-Aug-2008
Date of Filing 10-Apr-2002
Name of Patentee MITSUI CHEMICALS, INC.
Applicant Address 2-5, KASUMIGASEKI 3-CHOME, CHIYODA-KU, TOKYO
Inventors:
# Inventor's Name Inventor's Address
1 KOMATSU HIRONORI MITSUI CHEMICALS, INC., 1144, TOGO, MOBARASHI, CHIBA
2 KOUNO TOSHIYUKI MITSUI CHEMICALS, INC., 1144, TOGO, MOBARASHI, CHIBA
3 TSUCHIYA KATSUTOSHI MITSUI CHEMICALS, INC., 1144, TOGO, MOBARASHI, CHIBA
PCT International Classification Number C 07 H 19/173
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 2001-113835 2001-04-12 Japan