JP2002371093A - Method for purifying 5'-protected 2'-deoxypurine nucleosides - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an efficient method for purifying protected deoxypurinenucleosides in which it has been difficult hitherto to carry out efficient purification. SOLUTION: Impurities can be separated by obtaining the deoxypurine nucleoside as an inclusion crystal including a solvent having a nitrile structure to purify protected deoxypurine nucleoside at a high purity. This method enables, easily on a large scale, synthesis of highly purified protected deoxypurine nucleosides, which has been impossible hitherto without using an advanced column chromatography method.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for purifying 5'-protected 2'-deoxy-purine nucleosides and derivatives thereof, and to a solvent-conjugated compound obtained by the purification method. More specifically, the present invention provides a purification method useful for producing 5′-protected 2′-deoxy-β-purine nucleosides and a 5′-protected 2′-deoxy-β- obtained by the purification method. It relates to purine nucleosides solvent-conjugated compounds.

[0002]

2. Description of the Related Art 5'-protected 2'-deoxy-β-purine nucleosides are useful compounds as raw materials for antisense DNAs and the like which have been recently developed.

[0003] In recent years, with the progress of genomic drug discovery, antisense DNA drugs and the like have been rapidly developed. Along with this, the demand for a DNA oligomer as a raw material and a protected deoxynucleoside as a raw material for the oligomer is increasing. On the other hand, for pharmaceutical applications, it is necessary to use an intermediate product of extremely high purity in order to minimize the generation of by-products due to impurities contained therein.

Until now, 5'-protected deoxypurine nucleosides have been disclosed in JP-A-58-180500, JP-A-63-179889 and JP-A-6-507883.
As is clear from the examples of the publications, it has been purified using column chromatography. In this method, it is relatively easy to separate impurities having significantly different polarities and structures, but it is often difficult to remove impurities having a similar structure. In particular, it is difficult to remove the 3'-substituted isomer, which is particularly problematic as an impurity. In addition, since a large-scale refining device is required, it must be said that there is a great problem in mass production and mass supply in the future.

[0005] In addition, studies have been made to remove impurities without using column chromatography. Specifically, regarding purification by the reprecipitation method,
0-152495 and PCT application WO200075
154 publication. What is the reprecipitation method?
This is a method in which a crude compound is dissolved in a soluble solvent in advance, and then the insoluble solvent is added or dropped into the insoluble solvent to forcibly reprecipitate. Therefore, the purification capacity is basically low. In addition, it is industrially difficult to appropriately adjust the ratio of the soluble solvent and the insoluble solvent, and if the ratio is incorrect, the oil is easily converted to oil or a viscous precipitate is formed, so that purification tends to fail. In fact, according to the method disclosed in JP-A-60-152495, a viscous candy may be obtained, which is industrially problematic. So far, a method of amorphization by reprecipitation has been disclosed, but a method of obtaining crystals by crystallization or recrystallization has not been known.

[0006]

SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned conventional problems, and has as its object to provide an extremely high-purity 5′-protected compound which is efficient and requires no special equipment. An object of the present invention is to provide a purification method capable of obtaining 2'-deoxypurine nucleosides.

[0007]

Means for Solving the Problems As a result of intensive studies on the above-mentioned problems, the present inventors have found that the use of acetonitrile and other nitrile solvents makes it possible to protect the 5′-protected 2′-position.
The present inventors have found that deoxypurine nucleosides can be obtained as crystals in which these solvents are conjugated, and can be purified by a purification method by crystallization or recrystallization, thereby completing the present invention. That is, the present invention includes the following embodiments.

(1) Equation [1]

[0009]

Embedded image (Wherein R1 represents a 4-methoxytrityl group, a 4,4′-dimethoxytrityl group or a triphenylmethyl group,
B represents a purine group in which an amino group is protected. A) obtaining the compound represented by the formula (1) as crystals in which the inclusion solvent is immersed in a solvent containing the inclusion solvent; and recovering the conjugated crystals from the solvent. For purifying 5′-protected 2′-deoxypurine nucleosides.

(2) The solvent for inclusion is R3-CN (R
3 represents a lower alkyl group or an aryl group. The method for purifying a 5'-protected 2'-deoxypurine nucleoside according to the above item (1), which is a nitrile compound represented by the formula (1). (3) The method for purifying 5′-protected 2′-deoxypurine nucleosides according to the above (2), wherein the conjugation solvent is acetonitrile. (4) The compound of the above formula (1) and the following formula (2)

[0011]

Embedded image (Wherein R2 is a hydrogen atom, a 4-methoxytrityl group,
4,4′-dimethoxytrityl group or triphenylmethyl group, R1 and B are as defined above. ) Is dissolved in the solvent, and the compound of the formula (1) is recovered from the solvent as crystals embracing the solvent, whereby the compound of the formula (2) is dissolved in the solvent. The method for purifying a 5'-protected 2'-deoxypurine nucleoside according to any one of the above items (1) to (3), which is separated in a solvent.

(5) The compound of the formula (1) is represented by the following formula (3)

[0013]

Embedded image

Or the following formula (4)

[0015]

Embedded image

The method for purifying a 5'-protected 2'-deoxypurine nucleoside according to any one of the above items (1) to (4). (6) From the solvent comprising the above-mentioned solvent for inclusion, the above formula (1)
The 5′-protected 2′- according to any one of the above items (1) to (5), which recrystallizes a solvent-conjugated crystal of the compound of the above (1).
A method for purifying deoxypurine nucleosides. (7) The method for purifying a 5′-protected 2′-deoxypurine nucleoside according to any one of the above items (1) to (6), wherein the solvent is a single solvent. (8) The following equation (5)

[0017]

Embedded image (Wherein m and n are each independently any integer, R
1 represents a 4-methoxytrityl group, 4,4'-dimethoxytrityl group or triphenylmethyl group, R3 represents a lower alkyl group or an aryl group, and B represents a purine group in which an amino group is protected. Conjugated compound represented by).

[0018]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.

In the above formula (1), the purine group constituting B represents a nucleic acid purine base of a natural or unnatural nucleoside. Specific examples include adenine and guanine.

Examples of the amino-protecting group of the purine group include an alkyl group, an alkylacyl group and a benzoyl group. The alkyl group may be linear or branched, and may have another functional group as long as the desired function as a 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, and an i-butyl group.

The alkylacyl group may be linear or branched, may form a ring, and may have another functional group as long as the desired function as a protecting group can be maintained. Good. Examples of the alkylacyl group include, for example,
Examples include an acetyl group, a propionyl group, an n-butyryl group, an i-butyryl group, a pivaloyl group, an n-pentyroyl group, an i-pentyloyl group, a cyclopropylcarbonyl group, a phenoxyacetyl group, and the like.

The benzoyl group may be unsubstituted or substituted, and the phenyl group may have a substituent at any of the 2-, 3- and 4-positions. Further, a substituent may be present at a plurality of positions. Examples of the substituent include a methyl group, an ethyl group, a 2-propyl group, an n-butyl group, and t.
Alkyl groups such as -butyl group, hydroxyl group, methoxy group, ethoxy group, n-propyloxy group, 2-propyloxy group, alkyloxy groups such as n-butyloxy group, nitro group, fluoro group, chloro group, bromo group , An amino group, an amino group, a methylamino group, an ethylamino group, an n-propylamino group, a dimethylamino group, a diethylamino group, an alkylamino group such as a diisopropylamino group, an acetyl group, a propionyl group, a benzoyl group, etc. Acyl group, phenyl group, pyridinyl group and the like.

More specifically, benzoyl, 2-chlorobenzoyl, 3-chlorobenzoyl, 4-chlorobenzoyl, 2-bromobenzoyl, 3-bromobenzoyl, 4-bromobenzoyl, 2-fluoro Benzoyl group, 3-fluorobenzoyl group, 4-fluorobenzoyl group, 2-methoxybenzoyl group, 3-methoxybenzoyl group, 4-methoxybenzoyl group, 2-nitrobenzoyl group, 3-nitrobenzoyl group, 4-nitrobenzoyl group , 2-aminobenzoyl, 3-aminobenzoyl, 4-aminobenzoyl, 2-methylaminobenzoyl, 3-methylaminobenzoyl, 4-methylaminobenzoyl, 2-dimethylaminobenzoyl, 3-dimethyl Aminobenzoyl group, 4-dimethylaminobenzoyl group 4-phenylbenzoyl group, 4-acetyl benzoyl, and the like.

As the solvent for forming the inclusion crystals, R3-C
N (R3 represents a lower alkyl group or an aryl group)
A nitrile compound represented by the following formula (1) can be preferably used.

The alkyl group as R3 is preferably an alkyl group having 1 to 6 carbon atoms, more preferably an alkyl group having 1 to 4 carbon atoms. Specific examples of the lower alkyl group include, for example, a methyl group, an ethyl group, an n-propyl group,
-Propyl group, n-butyl group, i-butyl group and the like. Examples of the aryl group as R3 include a phenyl group, a 4-methylphenyl group, and a 4-methoxyphenyl group.

Examples of the nitrile compound substituted with a lower alkyl group or an aryl group include acetonitrile, propionitrile, n-butyronitrile, i-butyronitrile, n-pentanenitrile, n-hexanenitrile, benzonitrile and the like. . At least one of these can be used.

In the formula (5), m and n each independently represent an arbitrary integer, and m is preferably an integer selected from 1 to 5, and n is an integer selected from 1 to 5. Is preferred.

The solvent for obtaining the solvent-impregnated crystal may be composed solely of a solvent to be impregnated with the crystal, or may be used by being mixed with another solvent in a miscible ratio. Solvents that can be used for mixing include alcohols such as methanol, ethanol, and isopropanol;
Esters such as ethyl acetate and butyl acetate, acetone,
Ketones such as methyl ethyl ketone and methyl isobutyl ketone, diethyl ether, diisopropyl ether,
Ethers such as dioxane and THF, aromatic hydrocarbons such as benzene, toluene, cumene, xylene, mesitylene, diisopropylbenzene and triisopropylbenzene, halogenated hydrocarbons such as dichloromethane, chloroform and dichloroethane; pyridine, lutidine and quinoline And tertiary amines such as triethylamine and tributylamine, polar solvents such as DMF, DMI and DMSO, and water. At least one of these can be used. The ratio at the time of mixing is 100% by weight or less with respect to the solvent for inclusion, but is preferably 2% by weight.
It is at most 0% by weight, more preferably at most 10% by weight.

A crystal in which a solvent is embraced is used to form a crystal in a form in which the solvent is incorporated into a crystal lattice, or to form a complex by weak interaction with the solvent. This refers to a state in which the solvent plays an auxiliary role, and there is no particular limitation on the morphology and crystal structure of the inclusion.

The amount of the nitrile compound in the solvent at the time of crystallization or recrystallization is not particularly limited as long as the amount of the compound of the formula (1) in the solvent is not more than its saturation solubility. It is desirable to use the nitrile compound in an amount of 5 to 150 times the compound 1 of the formula (1) on the basis of
More preferably, it is used at a ratio of 50 times or more.

The temperature at the time of crystallization or recrystallization is not particularly limited, but is preferably in the range of −10 ° C. to the solvent or the boiling point of the solvent. In general, sufficient purification can be achieved by a single recrystallization, but higher purity can be achieved by repeated recrystallization. As the solvent for recrystallization, a solvent consisting of only the inclusion solvent is preferable, and it is more preferable to use the same single inclusion solvent for both crystallization and recrystallization.

According to the present invention, the protected deoxypurine nucleosides can be efficiently purified.

[0033]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the invention is limited thereto.

Example 1 N2-isobutyryl-5'-O- (4,4'-dimethoxy
Citrityl) -2'-deoxyguanosine acetonit
Preparation of Ryl (2: 1) Complex N2-Isobutyryl-2-deoxyguanosine 110 g
(95% content) was azeotropically dehydrated with pyridine and further 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 baking soda, the reaction solution was concentrated to about 400 g. 1.5 L of ethyl acetate, water 1.
After adding 5 L, the mixture was separated, and the raw material N2-
Washing with water was repeated until isobutyryl-2-deoxyguanosine disappeared. 1 kg of 20% sodium chloride aqueous solution
And then dried over sodium sulfate. After filtration,
After the solvent was distilled off, ethyl acetate was added so that the total weight became 700 g.

This solution was dissolved in diisopropyl ether 320
After dripping into 0 g with vigorous stirring, the mixture was stirred at room temperature for 2 hours. The solid obtained by filtration was dried under reduced pressure at 50 ° C., and after confirming that the solid content became constant, the solid was dissolved in 4.4 L of acetonitrile at room temperature. After stirring at room temperature for 4 hours, the precipitated solid was filtered. Dry under reduced pressure at room temperature for 4 hours
R analysis revealed that two molecules of acetonitrile were conjugated to one molecule of the target compound. Further, the resultant was dried under reduced pressure at 40 ° C. for 12 hours and subjected to NMR analysis. As a result, one molecule of the target compound was conjugated with one molecule of acetonitrile.

After drying under reduced pressure at 50 ° C. for 15 hours, NM
As a result of R analysis, 0.67 molecule of acetonitrile was conjugated to one molecule of the target compound. Further, the product was dried under reduced pressure at 55 ° C. until the weight became constant (24 hours) and analyzed by NMR. As a result, 0.5 molecule of acetonitrile was conjugated to 1 molecule of the target compound. XRD measurement revealed that the crystals were crystalline in any dry state. TG-D
TA analysis revealed that the crystals containing 0.5 molecules of acetonitrile obtained by drying to constant weight did not lose weight to the temperature at which an endothermic reaction occurs (81-93 ° C.) and had no attached solvent. . Yield 168 g (84.8 yield)
%). Analysis by HPLC (UV 254 nm) revealed a purity of 99.7 area%. Maximum impurity is N2
-Isobutyryl-3 ', 5'-O-bis (4,4'-dimethoxytrityl) -2'-deoxyguanosine (0.
17 area%).

NMR (DMSO-d ₆ ) δ: 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 = 6Hz, 1H), 5.4 (m, 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 = 6.8H
z, 6H) IR (KBr) cm ^-1 : 3398,3238,2935,2838,1679,1609,1561,150
9, 1252, 1178, 1034, 830. (The absorption from nitrile of acetonitrile is weak and not observed).

Example 2 N6-benzoyl-5'-O- (4,4'-dimethoxy
Salicylic) -2'-deoxyadenosine prepared N6- benzoyl-deoxyadenosine 45.0 g (0.
127 mol) was dissolved in 500 ml of pyridine and azeotropically dehydrated. Then, 42.9 g (0.127 mol) of 4,4'-dimethoxytrityl chloride was dissolved in 500 ml of pyridine while stirring.
l) was added and the mixture was stirred at room temperature for 2.5 hours. After adding 12.8 g of sodium hydrogen carbonate and stirring at room temperature for 30 minutes, the solvent was distilled off under reduced pressure. Methyl isobutyl ketone 8 in the residue
While adding 30 ml and stirring, 830 ml of water was added and stirred for 10 minutes. Next, the organic layer was collected, washed with saturated saline, dried over sodium sulfate, and then the solvent was distilled off under reduced pressure.
The residue was stirred vigorously with diisopropyl ether 800
The resulting precipitate was collected by filtration.

The precipitate was recrystallized from 750 ml of acetonitrile, and the crystalline product was collected by filtration. As a result of NMR analysis,
One molecule of the target compound was conjugated with one molecule of acetonitrile. The crystalline product was then dried under vacuum and the acetonitrile was lost. Its weight was 66.7 g. The product was eluted with a mixed solvent of acetonitrile / water (8/2) using an octadecyl silica gel column for reversed phase, and analyzed by high performance liquid chromatography detected with a UV detector (254 nm). As a result, the purity was 99.5%. . N6
The yield of -benzoyl-5'-O- (4,4'-dimethoxytrityl) -2'-deoxyadenosine was 79.4.
%Met. The largest impurity is N6-benzoyl-3 ′,
5'-O-bis (4,4'-dimethoxytrityl) -2 '
-Deoxyadenosine (0.15 area%).

Reference Example 1 N2-isobutyryl-3 ', 5'-O-bis (4,4'
-Dimethoxytrityl) -2'-deoxyguanosine
After concentration of the recrystallization filtrate obtained in Production Example 1, the concentrated residue was purified by column chromatography (ethyl acetate - hexane) was purified. The yellow oil obtained by concentrating the purified fraction was dropped into diisopropyl ether, and the precipitate was collected by filtration and dried to give the title compound as a pale-yellow powder. ^{1 H NMR (400MHz, DMSO-} d 6) δ: 12.1 (1H, s), 11.5 (1H, s), 7.9
(1H, s), 7.5-7.2 (18H, m), 6.8 (8H, m), 6.2 (1H, m), 4.4 (1H, m
m), 3.8 (1H, m), 3.7 (12H, s), 3.6 (1H, m), 3.0 (1H, m), 2.8 (1
H, m), 2.4 (1H, m), 1.8 (1H, m), 1.1 (6H, m), 1.0 (6H, m) Reference Example 2 N2-isobutyryl-5′-O- (t-butyldimethyl
Production of silyl) -2'-deoxyguanosine N2-isobutyryl-2'-deoxyguanosine 20.
5 g was dissolved in 200 mL of DMF. To this solution, 18.5 g of imidazole was added and dissolved. Thereafter, 21.4 g of t-butyldimethylsilyl chloride was added, and DM was added.
F100mL was added and stirred at room temperature. Eight hours later, the mixture was extracted with chloroform, washed with saturated saline, and then dried over anhydrous magnesium sulfate. After concentration, they were separated by column chromatography (methanol-chloroform). After fractionation,
The solution was concentrated to obtain 20.5 g of the title compound (yield 7).
4%). ¹ H NMR (400 MHz, CDCl ₃ ) δ: 12.4 (1 H, s), 10.9 (1 H, s), 8.0
(1H, s), 6.0 (1H, dd, J = 6.4,6.4Hz), 4.6 (2H, m), 4.1 (1H, d, J
= 2.0Hz), 3.8 (2H, m), 2.9 (1H, m), 2.4 (2H, m), 1.3 (6H, m), 0.
8 (9H, s), 0.02 (6H, s).

Reference Example 3 N2-isobutyryl-5'-O- (t-butyldimethyl
Silyl) -3'-O- (4,4'-dimethoxytriti
(1) Preparation of 2′-deoxyguanosine 19.8 g of N2-isobutyryl-5′-O- (t-butyldimethylsilyl) -2′-deoxyguanosine was dissolved in 100 mL of anhydrous pyridine. Add 4,4 'to this solution
-Dimethoxytrityl chloride (16.6 g) was added, anhydrous pyridine (140 mL) was added, and the mixture was stirred at 40 ° C. After completion of the reaction, pyridine was distilled off after neutralization with sodium bicarbonate. The mixture was extracted with chloroform, washed with saturated saline, and dried over anhydrous magnesium sulfate. After concentration, the residue was purified by column chromatography (ethyl acetate-hexane) to give the title compound (25.8).
g was obtained (78% yield). ¹ H NMR (400 MHz, CDCl ₃ ) δ: 11.9 (1 H, s), 8.1 (1 H, s), 7.8 (1
H, s), 7.5-7.2 (9H, m), 6.8 (4H, m), 6.2 (1H, dd, J = 6.0,8.4H
z), 4.4 (1H, m), 4.1 (1H, m), 3.8 (6H, s), 3.6 (1H, dd, J = 8.8,1
1.2Hz), 3.3 (1H, dd, J = 2.8,11.2Hz), 2.6 (1H, m), 2.0-1.6 (2
H, m), 1.3 (6H, m), 0.8 (9H, s), 0.02 (6H, s). Reference Example 4 N2-isobutyryl-3′-O- (4,4′-dimethoxy
Preparation of citrityl) -2'-deoxyguanosine N2-isobutyryl-5'-O- (t-butyldimethylsilyl) -3'-O- (4,4'-dimethoxytrityl) -2'-deoxyguanosine was dehydrated. THF2
Dissolved in 00 mL. To this solution was added 40 mL of a tetrabutylammonium fluoride / THF solution, and dehydrated T
HF (100 mL) was added and the mixture was stirred at room temperature. Eight hours later,
It was extracted with chloroform, washed with saturated saline, and dried over anhydrous magnesium sulfate. After concentration, it was purified by column chromatography (ethyl acetate-hexane-methanol),
12.9 g of the title compound were obtained as a white powder (yield 60
%). ¹ H NMR (400 MHz, CDCl ₃ ) δ: 12.0 (1 H, s), 8.3 (1 H, s), 7.7 (1
H, s), 7.5-7.2 (9H, m), 6.8 (4H, m), 6.2 (1H, dd, J = 5.2,10H
z), 4.5 (1H, m), 4.0 (1H, m), 3.8 (6H, s), 3.7 (1H, m), 3.3 (1H, m)
m), 2.6 (1H, m), 2.4 (1H, m), 1.7 (1H, m), 1.2 (6H, m).

Comparative Example 1 3'-substituted isomer [N2-isobutyryl-3'-O-
(4,4'-dimethoxytrityl) -2'-deoxyguanosine] or a polysubstituted 3 ', 5'-position [N2-isobutyryl-3', 5'-O-bis (4,4'-dimethoxytrityl)- The ability to remove impurities such as [2'-deoxyguanosine] was compared between a method of recrystallization with an acetonitrile solvent and a purification method of reprecipitation using dichloromethane as a soluble solvent and hexane or toluene as an insoluble solvent. In addition, the desired product [N2-isobutyryl-5'-O- (4,4'-dimethoxytrityl)-
2′-Deoxyguanosine] was also compared, and the obtained crystals were subjected to thermal analysis (DSC: endothermic peak, endothermic energy). Table 1 shows the results of the crude preparation (crystal) used, the content of the 3′-isomer and the polysubstituted product contained in the recrystallized crystal obtained for each solvent, and the like.

[0043]

[Table 1]

From the results of the above DSC (endothermic peak and endothermic energy), it was found that the different endothermic peaks resulted in different crystal forms. The purified product obtained from acetonitrile was found to be crystalline because of its high endothermic energy.

Reference Example 5 N6-benzoyl-3 ', 5'-O-bis (4,4'-
Preparation of (dimethoxytrityl) -2'-deoxyadenosine
After concentration of the recrystallization filtrate obtained in Concrete Example 2, the concentrated residue was purified by column chromatography (ethyl acetate - hexane) was purified. A yellow oil obtained by concentrating the purified fraction was dropped into diisopropyl ether, and the precipitate was collected by filtration and dried to obtain the title compound as a pale yellow powder. ^{1 H NMR (400MHz, DMSO-} d 6) δ: 11.2 (1H, br), 8.6 (1H, s), 8.
5 (1H, s), 8.0 (2H, m), 7.7-6.7 (29H, m), 6.4 (1H, m), 4.3 (1H, m
m), 4.1 (1H, m), 3.8 (14H, s), 3.6 (1H, m), 3.0 (1H, m), 1.0 (6
H, m). REFERENCE EXAMPLE 6 N6-benzoyl-5'-O- (t-butyldimethyloxy
Preparation of ril) -2'-deoxyadenosine N6-benzoyl-2'-deoxyadenosine 20.9
5 g was dissolved in 200 mL of DMF. To this solution, 15.7 g of imidazole was added and dissolved. Thereafter, 20.8 g of t-butyldimethylsilyl chloride was added, and DMF was added.
100 mL was added and stirred at room temperature. Eight hours later, the mixture was extracted with chloroform, washed with saturated saline, and dried over anhydrous magnesium sulfate. After concentration, the residue was purified by column chromatography (methanol-chloroform) to give the title compound 1
6.1 g was obtained (yield 61%). ¹ H NMR (400 MHz, CDCl ₃ ) δ: 9.1 (1 H, s), 8.8 (1 H, s), 8.3 (1
H, s), 8.0 (2H, d, J = 12.4Hz), 7.6-7.5 (3H, m), 6.6 (1H, dd, J =
6.8,6.8Hz), 4.7 (1H, m), 4.1 (1H, m), 3.9 (2H, m), 2.8 (1H, m)
m), 2.6 (1H, m), 0.9 (9H, s), 0.1 (6H, s).

Reference Example 7 N6-benzoyl-5'-O- (t-butyldimethylsi
Ryl) -3'-O- (4,4'-dimethoxytrityl)
Preparation of -2'-deoxyadenosine 16.1 g of N6-benzoyl-5'-O- (t-butyldimethylsilyl) -2'-deoxyadenosine was dissolved in 100 mL of anhydrous pyridine. 12.8 g of 4,4'-dimethoxytrityl chloride and 140 m of anhydrous pyridine
L was added and stirred at 45 ° C. After completion of the reaction, pyridine was distilled off after neutralization with sodium bicarbonate. Extract with chloroform,
After washing with saturated saline, it was dried over anhydrous magnesium sulfate. After concentration, the residue was purified by column chromatography (ethyl acetate-hexane) to obtain 16.7 g of the title compound (yield: 6).
3%). _{^{1 H NMR (400MHz, CDCl 3}} ) δ: 9.1 (1H, s), 8.9 (1H, s), 8.3 (1
H, s), 8.1 (2H, d, J = 7.2Hz), 7.7-7.3 (12H, m), 6.9 (4H, m), 6.
7 (1H, dd, J = 5.6,8.4Hz), 4.5 (1H, m), 4.2 (2H, m), 3.9 (6H,
s), 3.7 (1H, dd, J = 2.4,11.2Hz), 3.4 (1H, dd, J = 8.4,11.2H)
z), 2.2-2.1 (2H, m), 2.0-1.6 (2H, m), 1.3 (1.5H, m), 0.9 (9H,
s), 0.03 (6H, s). Reference Example 8 N6-benzoyl-3′-O- (4,4′-dimethoxy
Preparation of trityl) -2'-deoxyadenosine N6-benzoyl-5'-O- (t-butyldimethylsilyl) -3'-O- (4,4'-dimethoxytrityl)
16.2 g of -2'-deoxyadenosine is dehydrated in THF2
Dissolved in 00 mL. To this solution was added 31 mL of tetrabutylammonium fluoride / THF solution and dehydrated THF.
100 mL was added and stirred at room temperature. 8 hours later, TH
F was distilled off, extracted with ethyl acetate, washed with saturated saline, and dried over anhydrous magnesium sulfate. After concentration, the residue was purified by column chromatography (ethyl acetate-hexane-methanol) to obtain 13.8 g of the title compound as a white powder (yield 98%). ¹ H NMR (400 MHz, CDCl ₃ ) δ: 9.0 (1 H, br), 8.7 (1 H, s), 8.1 (1
H, s), 8.0 (2H, m), 7.7-7.2 (12H, m), 6.9 (4H, m), 6.4 (1H, m),
4.6 (1H, m), 4.1 (1.3H, m), 3.8 (6H, s), 3.7 (1H, m), 3.3 (1H, m)
m), 2.7 (1H, m), 2.0 (2H, m), 2.0-1.6 (2H, m), 1.7 (1H, m), 1.3
(2H, m).

Comparative Example 2 3'-substituted isomer [N6-benzoyl-3'-O-
(4,4'-dimethoxytrityl) -2'-deoxyadenosine] or a polysubstituted 3 ', 5'-position [N6-benzoyl-3', 5'-O-bis (4,4'-dimethoxytrityl)- [2'-deoxyadenosine], a method of recrystallization with an acetonitrile solvent and reprecipitation using a mixture of dichloromethane as a soluble solvent and a mixture of t-butyl methyl ether and hexane (1: 2) as an insoluble solvent. The purification method was compared. In addition, the desired product [N6-benzoyl-5'-O-
The recovery of (4,4'-dimethoxytrityl) -2'-deoxyadenosine] was also compared, and the obtained crystals were subjected to thermal analysis (DSC: endothermic peak, endothermic energy).

Table 2 shows the results of the crude preparation (crystals) used, the content of the 3'-isomer and the polysubstituted substance contained in the recrystallized crystals obtained for each solvent, and the like.

[0049]

[Table 2]

From the results of the above DSC (endothermic peak and endothermic energy), it was found that the different endothermic peaks resulted in different crystal forms. The purified product obtained from acetonitrile was found to be crystalline because of its high endothermic energy.

The conditions of each compound such as HPLC are shown below. (1) HPLC conditions of Comparative Examples 1 and 2 (analysis of polysubstituted amount): Column: Develosil TMS-UG-5, 150 mm × φ4.6 Flow rate: 1.0 mL / min Column temperature: 40 ° C. Detection wavelength: 254 nm Mobile phase: gradient conditions (shown in Table 3)

[0052]

[Table 3]

[Solution A] 100 mM triethylamine
Acetic acid (pH 7) 100 mL / water 880 mL / acetonitrile 20 mL [Solution B] 100 mM triethylamine-acetic acid (pH
7) 100 mL / acetonitrile 900 mL (2) HPLC conditions of Comparative Examples 1 and 2 (analysis of 3′-isomer amount): Column: Develosil TMS-UG-5, 150 mm × φ4.6 Moving layer: acetonitrile-water (55 : 45) Flow rate: 1.0 mL / min Column temperature: 40 ° C Detection wavelength: 254 nm (3) Thermal analysis conditions: Instrument: Perkin Elmer, DSC-7 Temperature rise rate: 10 ° C / min (4) XRD measurement: Equipment: RAD-RVC (Science) X-ray target: Cu 50 kV 200 mA

[0054]

According to the present invention, using a method capable of mass production, protected 2′-
Deoxypurine nucleosides can now be produced.

 ────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Katsutoshi Tsuchiya 1144 Togo, Mobara-shi, Chiba Mitsui Chemicals, Inc. F-term (reference) 4C057 AA04 BB02 DD03 LL29 LL40 LL42

Claims (8)

[Claims] [Claim 1] Formula [1] (Wherein R1 represents a 4-methoxytrityl group, a 4,4′-dimethoxytrityl group or a triphenylmethyl group,
B represents a purine group in which an amino group is protected. A) obtaining the compound represented by the formula (1) as a crystal in which the solvent for inclusion is immersed in a solvent containing the solvent for inclusion, and recovering the conjugated crystal from the solvent. For purifying 5′-protected 2′-deoxypurine nucleosides. 2. The 5′-protected 2 ′ according to claim 1, wherein the inclusion solvent is a nitrile compound represented by R3-CN (R3 represents a lower alkyl group or an aryl group).
-A method for purifying deoxypurine nucleosides. 3. The method for purifying 5′-protected 2′-deoxypurine nucleosides according to claim 2, wherein the conjugation solvent is acetonitrile. 4. A compound of the formula (1) and a compound of the following formula (2): (Wherein R2 is a hydrogen atom, a 4-methoxytrityl group,
4,4′-dimethoxytrityl group or triphenylmethyl group, R1 and B are as defined above. ) Is dissolved in the solvent, and the compound of the formula (1) is recovered from the solvent as crystals embracing the solvent, whereby the compound of the formula (2) is dissolved in the solvent. The method for purifying a 5'-protected 2'-deoxypurine nucleoside according to any one of claims 1 to 3, wherein the nucleoside is separated in a solvent. 5. The compound of the formula (1) is represented by the following formula (3): Or the following formula (4): The method for purifying a 5'-protected 2'-deoxypurine nucleoside according to any one of claims 1 to 4, wherein 6. The 5′-protected 2′-position according to claim 1, wherein the solvent-containing crystal of the compound of the formula (1) is recrystallized from a solvent comprising the solvent for the inclusion. A method for purifying deoxypurine nucleosides. 7. The method according to claim 1, wherein the solvent comprises a single solvent.
6. The method for purifying a 5′-protected 2′-deoxypurine nucleoside according to any one of 6. 8. The following formula (5): (Wherein m and n each independently represent any integer;
R1 represents a 4-methoxytrityl group, 4,4'-dimethoxytrityl group or triphenylmethyl group, R3 represents a lower alkyl group or an aryl group, and B represents a purine group in which an amino group is protected. Conjugated compound represented by).