JP3123238B2 - Purification method of nucleoside derivative - Google Patents

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 dideoxynucleosides, particularly 2 ', 3'-dideoxynucleoside derivatives which have been approved or are being evaluated as anti-AIDS (AIDS) or antiviral agents. 2 ', 3'-Dideoxyinosine (ddI), 2', 3'-dideoxycytidine (ddC) and 3'-deoxy-3'-azidothymidine (AZT) have already been identified as anti-AIDS drugs by the US Food and Drug Administration (FDA). Approved.

[0002]

2. Description of the Related Art Many processes for producing 2 ', 3'-dideoxynucleoside derivatives have been reported. In these reports, the method of isolating and purifying the target compound from the reaction crude product includes a purification method by recrystallization using an organic solvent (McCarthy et al., J. Am. Chem. Soc., (1966) 88, 1549,
Mansuri et al., J. Org.Chem., (1989) 43,4780, and Robi
ns et al., T etrahedron Lett., (1984) 25, 367), purification method using silica gel chromatography or synthetic adsorption resin.
(USP 3,817,982, and CK et al., J. Med Chem. (1990) 3
3,1553) are known. For example, when 2 ', 3'-dideoxyadenosine (ddA) is synthesized from adenosine as a raw material, adenine generated by cleavage of a glycoside bond of the raw material adenosine, unreacted raw material adenosine, nucleic acid bases such as deoxyadenosine, nucleosides and the like ( Nucleic acid derivatives)
Is by-produced. The separation and purification of ddA from these by-products is performed by a method known per se, such as a purification method by recrystallization, a silica gel chromatography or a purification method using a resin. From the nature, using any of the separation methods known so far,
In order to obtain the target compound ddA with high purity, the recovery rate was extremely low, and the operation was complicated, and the purification method was not an industrially feasible purification method.

[0003] Accordingly, the present invention provides the above-mentioned ddA and adonin,
A mixture with adenosine, deoxyadenosine, etc. or 2 ',
When ddA or ddI is isolated from a mixture of 3′-dideoxyinosine (ddI) and hypoxanthine, inosine, deoxyinosine, or the like by resin purification, a non-polar porous resin commonly used for purification of nucleic acid derivatives ( For example, using "SP-207" manufactured by Mitsubishi Kasei Co., Ltd.), a ddA aqueous solution or a ddI aqueous solution (pH 7 to 10) is brought into contact with this to selectively adsorb ddA or ddI, and an elution method with alcohol is attempted. It has been found that the compound can be purified (JP-A-1-98496, 1-175990, 1-165390, and 1-175991), but the purity and recovery of the target compound are not always satisfactory.

[0004]

SUMMARY OF THE INVENTION High purity 2 ', 3'-
It is an object of the present invention to develop an industrially advantageous method capable of separating and purifying a dideoxynucleoside derivative from impurities at a high recovery rate.

[0005]

Means for Solving the Problems In order to solve this problem,
As a result of intensive studies, the present inventors have found that a 2 ', 3'-dideoxynucleoside derivative containing impurities of the above-mentioned nucleic acid derivatives is converted into a basic aqueous solution having a pH of 12 or more, and then subjected to extraction with an organic solvent. It has been found that crystallization from a basic aqueous solution can purify a 2 ', 3'-dideoxynucleoside derivative with high purity and high recovery, and based on such findings, the present invention has been completed.

That is, the present invention provides a method for purifying a crude 2 ', 3'-dideoxynucleoside derivative, wherein the crude 2', 3'-dideoxynucleoside derivative has a pH of 12 or less.
The present invention relates to a purification method characterized by extracting or crystallizing the 2 ', 3'-dideoxynucleoside derivative with an organic solvent from the above basic aqueous solution.

Hereinafter, the present invention will be described in detail.

FIG. 1 shows one sequence of a method for synthesizing ddA and ddI. ddA and ddI can be synthesized by starting from adenosine and inosine, respectively, and reducing both 2 ′ and 3 ′ hydroxyl groups thereof.
In this reaction, the final step is a step of removing the protecting group for the 5'-hydroxyl group of the nucleic acid derivative. After the reduction reaction one step before, the reaction solution is concentrated to remove the organic solvent, and then water is removed. In addition, for example, an aqueous solution of sodium hydroxide is added to make the mixture basic, and the elimination reaction of the protecting group is performed.

The reaction termination solution contains adenine, adenosine, deoxyadenosine and the like in addition to the desired ddA. While maintaining the basicity of the reaction solution, for example, 2
-Selectively extracting ddA in 2-propanol by performing extraction with propanol, while adenine,
Adenosine and deoxyadenosine remain in the basic aqueous solution, and as a result, the target substance and impurities can be separated with high selectivity. The reaction ending solution of ddI synthesized by the same method contains by-products such as hypoxanthine, inosine, and deoxyinosine in addition to the target ddI. DdI is selectively extracted therein, while by-products remain in the basic aqueous solution. As a result, the target substance and impurities can be separated with high selectivity.

A basic aqueous solution containing ddA, adenine, adenosine, deoxyadenosine or the like and having a pH of 12 or more;
Alternatively, by subjecting a basic aqueous solution having a pH of 12 or more containing ddI, hypoxanthine, inosine, deoxyinosine and the like to crystallization treatment such as concentration and / or cooling, ddA and ddI are selectively purified to high purity and crystalline. It can also be obtained with high recovery. This utilizes the advantage that the solubility of the 2 ', 3'-dideoxynucleoside derivative is reduced, while the solubility of the related substance is not significantly reduced, and the desired product can be obtained in high yield. In addition, the crystal size of the crystallized target product increases, and the separability of the crystal improves. As described above, the crystallization method under basic conditions of the present invention provides an industrially superior purification method.

Of course, the desired 2 ', 3'-dideoxynucleoside derivative once purified by the above-mentioned organic solvent extraction method is converted into a basic aqueous solution, which is then re-purified by the above-mentioned crystallization method to obtain a higher-purity target compound. It is also possible to achieve the same object by reversing the order and applying crystallization followed by organic solvent extraction. As described below, other combinations of both methods are possible.

The 2 ', 3'-dideoxynucleoside derivative according to the present invention includes, for example, guanosine, adenosine,
Purine nucleosides such as inosine, and pyrimidine nucleosides such as uridine and cytidine, 2'- and 3'-dideoxy forms, 2 ', 3'-didehydro forms thereof, and derivatives in the sugar portion and base portion thereof. In general,
2 ', 3'-dideoxyadenosine (ddA) and 2',
2 ', 3' such as 3'-dideoxyinosine (ddI)
-Dideoxynucleoside; 2 ', 3'-dideoxy-
2 ', 3'-didehydronucleoside;2',3'-dideoxy-3'-azidonucleoside; 2 ', 3'-dideoxy-2'-fluoroadenosine, 2', 3'-dideoxy-2'- 2 ', 3'-dideoxy-2'-fluoronucleoside such as fluoroinosine; 2', 3'-
Dideoxy-3'-fluoronucleoside and the like, and further, ribonucleosides having a purine base such as 2,6-diaminopurine, 6-chloropurine and 2-aminopurine, and pyrimidines such as 5-methyluridine Examples include the 2'-position and 3'-position dideoxy forms of nucleosides and their 2 ', 3'-didehydro forms. Thus, it should be noted that the 2 ', 3'-dideoxynucleoside derivative of the present invention is broadly defined to include 2', 3'-dideoxynucleoside itself.

The basic condition in the present invention refers to pH 12 or more, preferably pH 13 or more. If the pH is 11 or less, the separability from impurities is not sufficient. Specifically, 2 ',
A basic aqueous solution obtained by dissolving 3'-dideoxynucleoside in an aqueous solution of an inorganic or organic base having a concentration of 0.1 to 50% by weight, preferably 1 to 25% by weight can be used.

In the present invention, the base used in preparing the basic aqueous solution includes alkali metal hydroxides such as sodium hydroxide and potassium hydroxide and alkaline earth metal hydroxides such as calcium hydroxide. Used
Preferably, sodium hydroxide is used.

[0015] For example, when the pH of the solution for terminating the synthesis reaction of the 2 ', 3'-dideoxynucleoside derivative is 12 or more, such a solution for terminating the synthesis reaction may be directly subjected to the purification treatment of the present invention. Of course.

The basic aqueous solution of the crude 2 ', 3'-dideoxynucleoside derivative of the present invention contains 0.1 to 30% by weight, preferably 1 to 3% by weight of the 2', 3'-dideoxynucleoside derivative.
It is preferred to contain 20% by weight for productivity reasons.

The extraction solvent used in the present invention includes alcohols such as 2-propanol, 1-butanol, 2-butanol, 2-methyl-1-propanol, 2-methyl-2-propanol and 1-pentanol; acetonitrile Carboxylates such as ethyl acetate and methyl acetate; hydrocarbons such as benzene, hexane and toluene; ethers such as diethyl ether, tetrahydrofuran and dioxane; halogenated hydrocarbons such as dichloromethane, chloroform and dichloroethane; methyl ethyl ketone and the like Organic solvents such as ketones; alcohols are preferred for reasons of extraction rate. Usually, 0.1 to 10 times the volume of the basic aqueous solution of the crude 2 ', 3'-dideoxynucleoside derivative is used.

There is no particular limitation on the extraction temperature.
C. can be performed.

When extracting under such conditions, shaking or stirring can shorten the extraction time. Usually 1 minute ~
Extraction is completed in about 24 hours.

After completion of the extraction, the organic solvent layer containing the target substance is separated and concentrated by removing the organic solvent layer by a method known per se, that is, by distilling off the solvent. 2 ′, 3′-dideoxynucleoside derivative can be precipitated and easily isolated. When removing the extraction solvent, add water and concentrate on the way,
When the aqueous solution is used, the target substance can be recovered more efficiently. At this time, according to the present invention, it is possible to isolate a high-purity target product by crystallization as a basic aqueous solution.

The crystallization referred to in the present invention is usually carried out by a method known per se, for example, a basic aqueous solution of a 2 ', 3'-dideoxynucleoside derivative, preferably under reduced pressure, more preferably 1 to 200 mmHg. Under reduced pressure, 30 ~
It is performed by heating to 100 ° C., concentrating, and then cooling. The crystallization concentration varies depending on the target compound, but it is usually concentrated to a concentration of about 5 to 100 g / dl.

The resulting concentrated solution is left to stand at room temperature, forcibly cooled and, if desired, cooled to about 0 ° C., whereby the desired crystal of the 2 ′, 3′-dideoxynucleoside derivative can be precipitated. It can be easily separated by another operation.

[0023]

The present invention will be further described with reference to the following examples.

Example 1 (Synthesis Example) Synthesis Example (a): Synthesis of 2 ', 3'-dideoxyadenosine (ddA) from adenosine (Part 1) Trimethyl orthoacetate was added to a solution of 20 g (74.9 mmol) of adenosine in 100 ml of acetic acid. Add 11.7 ml (1.3 equivalents) and add 3 at 50 ° C.
Stirred for hours. After the reaction solution was concentrated under reduced pressure, 100 ml of acetonitrile was added, the reaction solution was cooled to 10 ° C., and 22 ml (4 equivalents) of acetyl bromide was added dropwise thereto over 1 hour.
The reaction solution was further stirred at 15 ° C. for 2 hours, neutralized with an aqueous solution of sodium carbonate, and extracted with acetonitrile. To extract
3 g (5 mol%) of a carbon catalyst (10% Pd-C catalyst) supporting 10% palladium was added, and sodium hydroxide (Na
While controlling the pH of the system to 9.5 with an aqueous solution of OH), the inside of the system was set to a hydrogen atmosphere, and a hydrogenation reaction was carried out at room temperature for 5 hours. After completion of the reaction, the reaction solution was filtered, and the solvent was distilled off under reduced pressure. Then, an aqueous NaOH solution was added, and the solution was maintained at pH 12 and stirred for 5 hours.

Each nucleic acid derivative having the composition shown in Table 1 was contained in 1000 ml of the thus obtained ddA alkaline aqueous solution (pH 12).

[0026]

[Table 1]

Synthesis example (b): 2 ', 3' from adenosine
Synthesis of dideoxyadenosine (ddA) (part 2) To a solution of 20 g (74.9 mmol) of adenosine in 100 ml of acetic acid was added 11.7 ml (1.3 equivalents) of trimethyl orthoacetate, and the mixture was stirred at 50 ° C for 3 hours. After the reaction solution was concentrated under reduced pressure, 100 ml of acetonitrile was added, the reaction solution was cooled to 10 ° C., and 22 ml (4 equivalents) of acetyl bromide was added dropwise thereto over 1 hour. The reaction solution was further stirred at 15 ° C. for 2 hours, neutralized with an aqueous solution of sodium carbonate, and extracted with acetonitrile. Z in the extract
7.8 g (2 equivalents) of n powder was added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was treated with ethylenediaminetetraacetic acid disodium salt 2
Hydrate _{(EDTA · 2Na · 2H 2 O} ) 90g (4 equivalents) was charged to a solution adjusted to pH7 with aqueous NaOH,
Extracted with 200 ml of acetonitrile. 10% Pd- in extract
3 g (5 mol%) of C catalyst was added, and the system was set to a hydrogen atmosphere, and a hydrogenation reaction was performed at room temperature for 5 hours. After completion of the reaction, the reaction solution was filtered, and the solvent was distilled off under reduced pressure. Then, an aqueous NaOH solution was added, and the solution was maintained at pH 12 and stirred for 5 hours.

Each of the nucleic acid derivatives having the composition shown in Table 2 was contained in 1000 ml of the thus obtained ddA alkaline aqueous solution (pH 12).

[0029]

[Table 2]

Synthesis example (c): 2 ′, 3 ′ from adesinone
-Synthesis of dideoxyadenosine (ddA) (part 3) To a slurry solution of adenosine (20 g, 74.9 mmol) in acetonitrile (200 ml) was added water (0.67 ml, 37.5 mmol) and acetoxyisobutyryl bromide (47.0 g, 224.7 mmol, 3 equivalents). ,
The reaction was performed at room temperature for 2 hours. Neutralized with 10% aqueous sodium bicarbonate, the aqueous layer was separated and removed. Zinc-copper complex (Zn-Cu complex) for organic layer
19.3 g (2 equivalents) was added, and the mixture was stirred at room temperature for 2 hours. The reaction mixture _{EDTA · 2Na · 2H 2 O 90g} (4 equivalents)
Was poured into a solution adjusted to pH 7 with an aqueous NaOH solution, and extracted with 200 ml of acetonitrile. 3 g (5 mol%) of a 10% Pd-C catalyst was added to the extract, and the inside of the system was set to a hydrogen atmosphere, and a hydrogenation reaction was performed at room temperature for 5 hours. After completion of the reaction, the reaction solution was filtered, and the solvent was distilled off under reduced pressure. Then, an aqueous NaOH solution was added, and the solution was kept at pH 12 and stirred for 5 hours.

Each of the acid derivatives having the composition shown in Table 3 was contained in 1000 ml of the thus obtained ddA alkaline aqueous solution (pH 12).

[0032]

[Table 3]

Example 2 The pH of the saponified solution of composition A obtained in Synthesis Example (a) was adjusted to 7 using hydrochloric acid. Four 10 ml aliquots were taken from this pH-adjusted solution, and the first of the four aliquots was left as it was, and the second, third and last were NaOH 1, 10, and 20, respectively.
Four kinds of test liquids were prepared in addition to the amount to give the concentration by weight. The pHs of these four test solutions were 7,> 13 and> 1 respectively.
3, and> 13.

To each test solution, 10 ml of 2-methyl-1-propanol was added, mixed well, and allowed to stand to separate layers.

The concentration of each nucleic acid derivative in the organic layer and the aqueous layer is analyzed by high performance liquid chromatography (HPLC).
The partition coefficient (organic layer / water layer) and ddA purity in the organic layer were calculated. The results are shown in Table 4.

[0036]

[Table 4]

As is clear from the above table, as the NaOH concentration increases, the partition coefficient of ddA increases, and the impurity 3 '
-Deoxyadenosine (3dA), adenosine (A
The partition coefficients of R) and adenine (Ad) became low, and as a result, ddA was extracted into the organic layer with high purity and high recovery.

The saponification solutions of the compositions B and C obtained in the synthesis examples (b) and (c) were also used for the composition A obtained in the synthesis example (a).
And the same treatment as the saponification solution of
The results shown in Table 6 and Table 6 were obtained.

[0039]

[Table 5]

[0040]

[Table 6]

As can be seen from both tables, the saponified solutions obtained in Synthesis Examples (b) and (c) also have a higher NaOH concentration and ddA as in the case of the saponified solution obtained in Synthesis Example (a).
Is higher, and impurities 3dA, AR, and Ad
The ddA was extracted with high purity and high recovery rate in the organic layer as a result.

Example 3 1.0 g of ddA, 100 mg of 3dA, 100 mg of AR and 100 mg of Ad 100
mg of the crystal mixture is added to 100 ml of water (solvent 1),
The mixture was dissolved by heating to ° C. The solution was concentrated at room temperature under reduced pressure (10 mmHg) until crystals formed, then cooled to 5 ° C.

The precipitated crystals were filtered and dried.
Analysis by PLC was performed to determine the purity of the crystals and the recovery of each nucleic acid derivative.

Other solvents (2) to (6) shown in Table 7 below
A similar experiment was performed for However, in the case of the solvents (5) and (6), the heating dissolution temperature was 50 ° C., the pressure in the vacuum concentration was 10 mmHg, and then the temperature was cooled to 5 ° C.

[0045]

[Table 7]

From the above table, it can be seen that when the solvent is an aqueous solution having a high NaOH concentration, the ddA crystals obtained by recrystallization have a high recovery, and do not contain 3dA, AR and Ad, or contain only a small amount. It turns out that it is a thing of high purity.

Example 4 1 L of saponified solution obtained by the same reaction procedure as in Synthesis Example (a)
(115.05 g of ddA, 13.10 g of 3dA, 2.04 g of 2dA
g, 4.07 g of AR and 3.67 g of Ad) and 25% N
10 L of an aOH aqueous solution was added, and extraction was performed three times with 10 L of 2-propanol. The obtained 2-propanol extract layer was concentrated, and 250 ml of water was added on the way to re-concentrate. 25% NaO on the way
H 2 aqueous solution (200 ml) was added, stirred, cooled to 20 ° C., and crystallized. The precipitated crystals were collected by filtration to obtain purified ddA crystals.

By the combination of the extraction operation and the crystallization operation under the above basic conditions, high-purity ddA crystals were obtained. The content composition of the nucleic acid derivative at each stage of the saponified solution, the 2-propanol extract, and the crystal after recrystallization was measured using HPLC. The results are shown in Table 8.

[0049]

[Table 8]

[0050]

Industrial Applicability According to the present invention, when purifying a 2 ', 3'-dideoxynucleoside derivative, it is possible to industrially and easily remove and purify impurities, and to obtain a target compound with high purity and high recovery. became.

[Brief description of the drawings]

FIG. 1. 2 ′, 3′-dideoxyadenosine (ddA)
And a method for synthesizing 2 ′, 3′-dideoxyinosine (ddI).

──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Satoshi Takahashi 1730 Hinaga, Yokkaichi, Mie Pref. Ajinomoto Co., Inc. Tokai Plant (56) References JP-A-1-98496 (JP, A) JP Hei 1-175991 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C07D 473/00-473/40 C07B 63/00 CA (STN) CAOLD (STN) REGISTRY (STN)

Claims (4)

(57) [Claims] 1. A crude 2 ', the two from pH12 or more basic aqueous solution of 3'-deoxyadenosine', 3'-dideoxy
A method for purifying 2 ', 3'- dideoxyadenosine , comprising extracting siadenosine with an organic solvent. 2. A crude 2 ', the two from pH12 or more basic aqueous solution of 3'-deoxyadenosine', 3'-dideoxy
2, characterized in that crystallization shea adenosine ', 3'
A method for purifying dideoxyadenosine . 3. The method according to claim 1, wherein the organic solvent is an alcohol.
Purification method. 4. The purification method according to claim 1 or 2 pH of said basic aqueous solution is 13 or more.