JPH0641129A - Purification of nucleoside derivative - Google Patents

Abstract

PURPOSE:To separate and purify the derivative useful as an anti-AIDS medicine or antiviral medicine from a crude 2',3'-dideoxynucleoside derivative accompanied by nucleic acid-related substances as impurities in a high purity and a high recovery rate. CONSTITUTION:The 2',3'-dideoxynucleoside derivative is (a) extracted with an organic solvent and/or (b) crystallized from the 12 or higher pH basic aqueous solution of the crude 2',3'-dideoxynucleoside derivative.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for purifying dideoxynucleosides, especially 2 ', 3'-dideoxynucleoside derivatives which have been approved or are being evaluated as anti-AIDS (AIDS) agents and antiviral agents. 2 ', 3'-dideoxyinosine (ddI), 2', 3'-dideoxycytidine (ddC) and 3'-deoxy-3'-azidothymidine (AZT) are already anti-AIDS drugs in the US Food and Drug Administration (FDA). Approved.

[0002]

2. Description of the Related Art Many methods for producing 2 ', 3'-dideoxynucleoside derivatives have been reported so far. In these reports, the method for isolating and purifying the target compound from the reaction crude product was 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 and synthetic adsorption resin.
(USP 3,817,982, and CK et al., J. Med Chem. (1990) 3
3,1553) is known. For example, when synthesizing 2 ', 3'-dideoxyadenosine (ddA) from adenosine as a raw material, adenine produced by cleavage of glycoside bond of adenosine as raw material, adenosine as an unreacted raw material, nucleobase such as deoxyadenosine, nucleoside ( Nucleic acid derivatives)
Is a byproduct. Separation and purification of ddA from these by-products are carried out by a method known per se such as a purification method by recrystallization, a silica gel chromatography or a purification method using a resin, but the target compound is similar to the by-products and physicochemical properties. From the sex, using any separation method known so far,
In order to obtain the target compound ddA with high purity, the recovery rate was very low, and its operation was complicated, so that it was not a purification method which can be industrially implemented.

Therefore, the present invention provides the above-mentioned ddA and adonin,
A mixture of adenosine, deoxyadenosine, etc., 2 ',
When ddA and ddI are isolated from a mixture of 3'-dideoxyinosine (ddI) and hypoxanthine, inosine, deoxyinosine, etc. by resin purification, a non-polar porous resin which is often used for purification of nucleic acid derivatives ( For example, "SP-207" manufactured by Mitsubishi Kasei Co., Ltd.) was used, and an ddA aqueous solution or a ddI aqueous solution (pH 7 to 10) was brought into contact therewith to selectively adsorb ddA or ddI, and an elution with alcohol was tried. It was found that purification was possible (Japanese Patent Laid-Open Nos. 1-98496, 1-175990, 1-165390, and 1-175991), but the purity and recovery of the target compound were not always satisfactory.

[0004]

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 earnest studies, the present inventor has found that a 2 ', 3'-dideoxynucleoside derivative containing impurities of the above-mentioned nucleic acid derivatives is made into a basic aqueous solution having a pH of 12 or more and then extracted with an organic solvent, or It was found that the 2 ', 3'-dideoxynucleoside derivative can be purified with high purity and high recovery rate by performing crystallization from a simple basic aqueous solution, and the present invention was completed based on such findings.

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

The present invention will be described in detail below.

FIG. 1 shows one column of the synthesis method of ddA and ddI. ddA and ddI can be synthesized by using adenosine and inosine as the raw materials, and reducing both hydroxyl groups at the 2'and 3'positions from them.
The final step of this reaction is the step of removing the protecting group of the 5'-hydroxyl group of the nucleic acid derivative, but after the reduction reaction one step before the step, 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 thereto to make it basic, and the elimination reaction of the protective group is performed.

The reaction-terminated solution contains adenine, adenosine, deoxyadenosine and the like in addition to the desired ddA. The reaction solution is kept basic, for example, 2
-Selectively extracting ddA into 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-completed solution of ddI synthesized by a similar method contains byproducts such as hypoxanthine, inosine, deoxyinosine, etc. in addition to the desired ddI, but alcohol is obtained by performing alcohol extraction under the same conditions. The ddI is selectively extracted therein, while the by-product remains in the basic aqueous solution, and as a result, the target substance and the impurities can be separated with high selectivity.

Further, a basic aqueous solution containing ddA, adenine, adenosine, deoxyadenosine, etc., having a pH of 12 or more,
Alternatively, a basic aqueous solution having a pH of 12 or more containing ddI, hypoxanthine, inosine, deoxyinosine, etc. is subjected to a crystallization treatment such as concentration and / or cooling to selectively give ddA or ddI as crystals with high purity. It can also be obtained with a high recovery rate. 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 target product can be obtained in high yield. In addition, the crystal grain size of the crystallized target substance is increased, and the crystal separability is improved. As described above, the crystallization method under the 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 made into a basic aqueous solution, and then re-purified by the above-mentioned crystallization method to obtain a desired product with a higher purity. Alternatively, the same purpose can be achieved by reversing the order and applying the organic solvent extraction method after crystallization. Other combinations of both methods are possible, as described below.

The 2 ', 3'-dideoxynucleoside derivative referred to in the present invention is, for example, guanosine, adenosine,
Purine nucleosides such as inosine and pyrimidine nucleosides such as uridine and cytidine, the 2'- and 3'-dideoxy forms thereof, their 2 ', 3'-didehydro forms, and their derivatives in the sugar moiety and the base moiety. Specifically,
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'-fluoronucleosides such as fluoroinosine; 2', 3'-
Examples thereof include dideoxy-3′-fluoronucleoside, and further, ribonucleosides having a purine base such as 2,6-diaminopurine, 6-chloropurine, and 2-aminopurine, and pyrimidines such as 5-methyluridine. The 2'- and 3'-position dideoxy forms of nucleosides and their 2 ', 3'-didehydro forms can be mentioned. Thus, it should be noted that the 2 ', 3'-dideoxynucleoside derivative of the present invention is broadly defined as including 2', 3'-dideoxynucleoside itself.

The basic conditions referred to in the present invention are pH 12 or higher, preferably pH 13 or higher. At pH 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, as the base used when preparing the basic aqueous solution, there are hydroxides of alkali metals such as sodium hydroxide and potassium hydroxide and hydroxides of alkaline earth metals such as calcium hydroxide. Used,
Sodium hydroxide is preferably used.

For example, when the pH of the 2 ', 3'-dideoxynucleoside derivative synthesis reaction completion liquid is 12 or more, such a synthesis reaction completion liquid 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 30% by weight of the 2', 3'-dideoxynucleoside derivative.
It is preferable 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. Carboxylic acid esters such as ethyl acetate and methyl acetate; hydrocarbons such as benzene, hexane, toluene; ethers such as diethyl ether, tetrahydrofuran, dioxane; halogenated hydrocarbons such as dichloromethane, chloroform, dichloroethane; methyl ethyl ketone, etc. Although it is an organic solvent such as ketones, the alcohols are preferable for the reason of the extraction rate. Usually, a 0.1 to 10-fold volume ratio of the crude 2 ', 3'-dideoxynucleoside derivative to the basic aqueous solution is used.

There is no particular limitation on the extraction temperature, and 0 to 100
It can be performed at ° C.

When performing extraction 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 the target substance is concentrated by removing the organic solvent layer by a method known per se, such as distilling off the solvent. The 2 ', 3'-dideoxynucleoside derivative, which is, can be precipitated and easily isolated. When removing the extraction solvent, add water to 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, a highly pure target product can be isolated by crystallization as a basic aqueous solution.

The crystallization referred to in the present invention is usually 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, if necessary 30 to
It is carried out by heating to 100 ° C to concentrate and then cooling. Although the crystallization concentration varies depending on the target compound, it is usually concentrated to a concentration of about 5 to 100 g / dl.

The concentrated solution thus obtained is forcibly cooled by leaving it at room temperature and, if desired, cooled to about 0 ° C., whereby crystals of the desired 2 ′, 3′-dideoxynucleoside derivative can be precipitated and filtered. It can be easily separated by another operation.

[0023]

EXAMPLES The present invention will be further described below with reference to examples.

Example 1 (Synthesis Example) Synthesis Example (a): Synthesis of 2 ', 3'-dideoxyadenosine (ddA) from adenosine (part 1) 20 g (74.9 mmol) of adenosine in 100 ml of acetic acid was added to trimethyl orthoacetate. Add 11.7 ml (1.3 times equivalent) and mix at 50 ° C for 3
Stir for hours. 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 times equivalent) of acetyl bromide was added dropwise thereto over 1 hour.
The reaction solution was further stirred for 2 hours at 15 ° C., neutralized with an aqueous sodium carbonate solution, and extracted with acetonitrile. In the extract
3 g (5 mol%) of a carbon catalyst supporting 10% palladium (10% Pd-C catalyst) was added, and sodium hydroxide (Na
The pH of the system was controlled to 9.5 with an aqueous solution (OH) and the system was placed in a hydrogen atmosphere, and the hydrogenation reaction was carried out at room temperature for 5 hours. After completion of the reaction, the reaction solution was filtered, the solvent was distilled off under reduced pressure, an aqueous NaOH solution was added, and the solution was kept at pH 12 and stirred for 5 hours.

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

[0026]

[Table 1]

Synthesis example (b): 2 ', 3'from adenosine
-Synthesis of dideoxyadenosine (ddA) (2) To a solution of adenosine 20 g (74.9 mmol) in acetic acid 100 ml, trimethyl orthoacetate 11.7 ml (1.3 times equivalent) was added and stirred at 50 ° C for 3 hours. 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 times equivalent) of acetyl bromide was added dropwise thereto over 1 hour. The reaction solution was further stirred for 2 hours at 15 ° C., neutralized with an aqueous sodium carbonate solution, and extracted with acetonitrile. Z in the extract
n powder 7.8 g (2 times equivalent) was added, and the mixture was stirred at room temperature for 2 hours. The reaction solution is ethylenediamine tetraacetic acid disodium salt 2
90 g (4 times equivalent) of the hydrate (EDTA / 2Na / 2H ₂ O) was added to a solution adjusted to pH 7 with an aqueous NaOH solution,
It was extracted with 200 ml of acetonitrile. 10% Pd- in the extract
3 g (5 mol%) of C catalyst was added, the system was made a hydrogen atmosphere, and hydrogenation reaction was carried out at room temperature for 5 hours. After completion of the reaction, the reaction solution was filtered, the solvent was distilled off under reduced pressure, an aqueous NaOH solution was added, and the solution was kept 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 ddA alkaline aqueous solution (pH 12) thus obtained.

[0029]

[Table 2]

Synthesis Example (c): 2 ', 3'from Adecinone
-Synthesis of dideoxyadenosine (ddA) (3) To a slurry solution of 20 g (74.9 mmol) of adenosine in 200 ml of acetonitrile, 0.67 ml (37.5 mmol) of water and 47.0 g (224.7 mmol, 3 times equivalent) of acetoxyisobutyryl bromide were added. ,
Reacted at room temperature for 2 hours. The mixture was neutralized with 10% aqueous sodium hydrogen carbonate, and the aqueous layer was separated. Zinc-copper complex (Zn-Cu complex) in the organic layer
19.3 g (2 times equivalent) was added, and the mixture was stirred at room temperature for 2 hours. 90 g of EDTA ・ 2Na ・ 2H ₂ O (4 times equivalent)
Was added to a solution adjusted to pH 7 with an aqueous NaOH solution, and extracted with 200 ml of acetonitrile. 3 g (5 mol%) of 10% Pd-C catalyst was added to the extract, the system was made a hydrogen atmosphere, and hydrogenation reaction was carried out at room temperature for 5 hours. After completion of the reaction, the reaction solution was filtered, the solvent was distilled off under reduced pressure, 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 ddA alkaline aqueous solution (pH 12) thus obtained.

[0032]

[Table 3]

Example 2 The saponification solution of composition A obtained in Synthesis Example (a) was adjusted to pH 7 with hydrochloric acid. From this pH-adjusted solution, 4 times 10 ml aliquots, keep the first of the 4 aliquots the same for the second, third and last ones of 1, 10 and 20 respectively.
Four kinds of test liquids were prepared in addition to the amount of the concentration by weight. The pH of these four test solutions is 7,>13,> 1 respectively.
3 and> 13.

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

The concentration of each nucleic acid derivative in the organic layer and the aqueous layer was analyzed by high performance liquid chromatography (HPLC),
The partition coefficient (organic layer / aqueous layer) and the 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, the distribution coefficient of ddA increases as the NaOH concentration increases, and the impurities 3 '
-Deoxyadenosine (3dA), adenosine (A
The partition coefficients of R) and adenine (Ad) were low, and as a result, ddA was extracted in the organic layer with high purity and high recovery rate.

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

[0039]

[Table 5]

[0040]

[Table 6]

From both tables, the saponification solutions obtained in Synthesis Examples (b) and (c) also had higher NaOH concentrations and ddA as in the saponification solutions obtained in Synthesis Example (a).
Has a higher distribution coefficient, and impurities 3dA, AR, and Ad
Has a low partition coefficient, and as a result, ddA was extracted in the organic layer with high purity and high recovery rate.

Example 3 ddA 1.0 g, 3dA 100 mg, AR 100 mg and Ad 100
The crystal mixture consisting of mg was added to 100 ml of water (solvent 1), and 60
It melt | dissolved by heating at ℃. The solution was concentrated at room temperature under reduced pressure (10 mmHg) until crystallization occurred and then cooled to 5 ° C.

The precipitated crystals were filtered and dried, and the dried crystals were washed with H
Analysis by PLC was performed to determine the purity of this crystal and the recovery rate of each nucleic acid derivative.

Other solvents shown in Table 7 below (2) to (6)
The same experiment was also 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 cooled to 5 ° C.

[0045]

[Table 7]

From the above table, when the solvent is an aqueous solution having a high concentration of NaOH, the ddA crystal obtained by recrystallization has a high recovery rate, and does not contain 3dA, AR and Ad, or contains only a very small amount. It can be seen that it is of high purity.

Example 4 1 L of saponification liquid obtained by the same reaction procedure as in Synthesis Example (a)
(115.05 g of ddA, 13.10 g of 3dA, 2.04 of 2dA
g, AR 4.07g, and Ad 3.67g), 25% N
10 L of an aOH aqueous solution was added, and extraction was performed 3 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 reconcentrate. 25% NaO on the way
200 ml of H aqueous solution was added and stirred, cooled to 20 ° C. and crystallized. The precipitated crystal was collected by filtration to obtain a purified ddA crystal.

A high-purity ddA crystal was obtained by a combination of the above extraction operation and crystallization operation under basic conditions. The content composition of the nucleic acid derivative at each stage of the saponification 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, in purifying a 2 ', 3'-dideoxynucleoside derivative, it is possible to industrially and simply remove impurities and purify, and obtain the target compound with high purity and high recovery rate. became.

[Brief description of drawings]

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

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Satoshi Takahashi 1730 Niigata, Yokkaichi, Mie Prefecture Ajinomoto Co., Ltd. Tokai Plant

Claims (6)

[Claims] 1. A crude aqueous solution of a 2 ', 3'-dideoxynucleoside derivative from a basic aqueous solution having a pH of 12 or more is used to obtain the 2', 3'-
A method for purifying a 2 ', 3'-dideoxynucleoside derivative, which comprises extracting the dideoxynucleoside derivative with an organic solvent. 2. A crude 2 ', 3'-dideoxynucleoside derivative prepared from a basic aqueous solution having a pH of 12 or more is used to prepare the 2', 3'-
A method for purifying a 2 ', 3'-dideoxynucleoside derivative, which comprises crystallizing a dideoxynucleoside derivative. 3. The 2 ', 3'-dideoxynucleoside derivative is 2', 3'-dideoxynucleoside, 2 ',
3'-dideoxy-2 ', 3'-didehydronucleoside, 2', 3'-dideoxy-3'-azidonucleoside, 2 ', 3'-dideoxy-2'-fluoronucleoside and 2', 3'-dideoxy The purification method according to claim 1 or 2, which is one kind selected from -3'-fluoronucleosides. 4. The purification method according to claim 1, wherein the 2 ', 3'-dideoxynucleoside derivative is 2', 3'-dideoxyadenosine. 5. The purification method according to claim 1, 3 or 4, wherein the organic solvent is alcohol. 6. The purification method according to claim 1, wherein the basic aqueous solution has a pH of 13 or more.