JP4627625B2 - Method for producing N-acetylcytidines - Google Patents

Description

  The present invention relates to a method for producing N-acetylcytidines.

  N-acetylcytidines are used as synthesis intermediates in the synthesis of DNA oligomers, and demand is expected for DNA primers and DNA probes for gene research, or DNA drugs represented by antisense. Therefore, development of a practical synthesis method is demanded.

  N4-acetyl-5′-O- (4,4′-dimethoxytrityl) -2′-deoxycytidine, which is one of N-acetylcytidines, is used as an intermediate for oligonucleotide synthesis. It has been synthesized by a method of tritylating the 5 ′ position after acetylation (Non-patent Document 1 and Non-patent Document 2).

  Since the N4 position of cytidines is poor in nucleophilicity unlike a normal amino group, the reaction occurs in competition with acetylation to a hydroxyl group. In order to reduce by-products, it was necessary to selectively acetylate the hydroxyl group to the N4 position. Since it was difficult to control the selectivity between the N4 position and the hydroxyl group by the conventional method, only the acetyl group on the hydroxyl group was selectively deacetylated using an enzyme after acetylating all of the N4 position and the hydroxyl group. And a method of selectively acetylating to the N4 position using a microwave have been developed (Non-patent Documents 3 and 4).

Moreover, the method of heating in alcohol using excess acetic anhydride has been reported (nonpatent literature 5). Although the yield was stabilized by shortening the heating time to 1 hour, it was reported that the conversion rate was about 90% as a result of analysis by thin layer chromatography. Similarly, a method of carrying out a reaction in DMF using acetic anhydride has been reported, and DMF is concentrated and then added to diethyl ether for crystallization (Non-patent Document 6).
Tetrahedron Lett. 1994, 35, p4311; 1996, 37, p8691. Tetrahedron Lett. 1996, 37, p8691. Biosci. Biotechnol. Biochem. 2000, 64, p363. Tetrahedron Lett. 1997, 38, p7253. Tetrahedron, 1975, 31, p2423. Nucleosides & Nucleotides, 1989, 8, p179.

  However, in the method described in Non-Patent Document 3, it is necessary to acetylate all hydroxyl groups and amino groups in advance, and the number of steps is long. The method described in Non-Patent Document 4 requires a large apparatus that emits microwaves. Further, in the method described in Non-Patent Document 5, an excessive amount of acetic anhydride is required. Therefore, it is necessary to concentrate and remove the remaining high boiling point reagents such as acetic anhydride and acetic acid. Furthermore, in the method described in Non-Patent Document 6, there is a problem that the yield decreases due to low selectivity to the N4 position (see Comparative Examples 1 and 2 in the present specification). In view of these conventional problems, an object of the present invention is to provide a method for producing N4-acetylcytidines from cytidines with high yield and high selectivity.

As a result of intensive studies on the above problems, the present inventors have found that the acetylation can be selectively carried out at the N4 position by reacting cytidines with acetic anhydride in the presence of an acid, thereby completing the present invention.
That is, the present invention relates to the general formula (1)

(Wherein R1 represents a hydrogen atom or a hydroxyl protecting group, and R2 represents a hydrogen atom, a halogen atom, a hydroxyl group or a protected hydroxyl group) and a compound (A) represented by acetic anhydride (B) . At least selected from the group consisting of dichloroacetic acid, hydrochloric acid, sulfuric acid, trifluoroacetic acid, diisopropylamine hydrochloride, hydrochloride of the compound represented by the general formula (1), p-toluenesulfonic acid, and methanesulfonic acid Reacting in the presence of one acid (C) ,
General formula (2)

(Wherein R1 and R2 have the same meanings as R1 and R2 in the general formula (1)).

According to the present invention, N ⁴ -acetylcytidines can be produced from cytidines with high yield and high selectivity.

    Hereinafter, the present invention will be described in detail.

  In the compounds represented by the general formulas (1) and (2), R1 represents a hydrogen atom or a hydroxyl protecting group, and R2 represents a hydrogen atom, a halogen atom, a hydroxyl group or a protected hydroxyl group.

  The protecting group for the hydroxyl group in R1 in the compounds represented by the general formula (1) and the general formula (2) may be a protecting group that can be removed by a chemical method such as hydrogenolysis, hydrolysis, or photolysis. Examples of such protective groups include aliphatic acyl groups, aromatic acyl groups, trialkylsilyl groups, trialkoxysilyl groups, alkoxyalkyl groups, alkoxyalkyl groups substituted with silyl groups, and alkyl halides. Group, aralkyl group, alkoxycarbonyl group, aralkyloxycarbonyl group, orthoester group, trityl group and the like. A trialkylsilyl group, a trialkoxysilyl group, or a trityl group is preferable. Examples of the trialkylsilyl group include a triethylsilyl group, a triisopropylsilyl group, and a t-butyldimethylsilyl group. Examples of trialkoxysilyl groups include bis (trimethylsiloxy) diphenylmethoxysilyl group and bis (trimethylsiloxy) cyclododecyloxysilyl group. Examples of the trityl group include a trityl group, a 4-methoxytrityl group, and a 4,4'-dimethoxytrityl group.

  Among the hydroxyl protecting groups, bis (trimethylsiloxy) diphenylmethoxysilyl group, bis (trimethylsiloxy) cyclododecyloxysilyl group or 4,4'-dimethoxytrityl group is particularly preferable.

  The protecting group for the protected hydroxyl group in R2 in the compounds represented by the general formula (1) and the general formula (2) can be removed by a chemical method such as hydrogenolysis, hydrolysis, or photolysis. Any protecting group may be used, and examples of such protecting groups include formyl group, aliphatic acyl group, aromatic acyl group, silyl group, silyloxymethyl group, alkoxyalkyl group, and alkoxyalkyl substituted with acyloxy group. Groups, alkoxyalkyl groups substituted with silyl groups, halogenated alkyl groups, aralkyl groups, alkoxycarbonyl groups, aralkyloxycarbonyl groups, alkyl groups and the like. Preferred are a silyl group, a silyloxymethyl group, an alkoxyalkyl group substituted with a silyl group, an orthoester group, an alkyl group, and an alkoxyalkyl group. Examples of the silyl group include a triethylsilyl group, a triisopropylsilyl group, a t-butyldimethylsilyl group, and the like. Examples of the silyloxymethyl group include a trimethylsilyloxymethyl group, a triethylsilyloxymethyl group, a triisopropylsilyloxymethyl group, a t-butyldimethylsilyloxymethyl group, and the like. Examples of the alkoxyalkyl group substituted with a silyl group include a 2-trimethylsilylethyloxymethyl group. Examples of the alkoxyalkyl group substituted with an acyloxy group include bis (2-acetoxyethyloxy) methyl group, 2-acetoxyethyloxymethyl group, bis (2-benzoyloxyethyloxy) methyl group, and 2-benzoyloxyethyloxy. Examples thereof include a methyl group. Examples of the alkyl group include a methyl group, an ethyl group, an n-propyl group, and an allyl group. Examples of the alkoxyalkyl group include a methoxymethyl group, a methoxyethyl group, a methoxyethoxymethyl group, a dimethoxymethyl group, and a diethoxymethyl group.

  Among the hydroxyl groups substituted with a protecting group, an alkoxyalkyloxy group, an alkoxyalkyloxy group substituted with an acyloxy group, an alkoxyalkyloxy group substituted with a silyl group, and an alkyloxy group are preferred. The group and the bis (2-acetoxyethyloxy) methyloxy group are particularly preferred.

  In the compounds represented by the general formulas (1) and (2), R1 in the general formulas (1) and (2) is a hydrogen atom, a 4,4′-dimethoxytrityl group or a trialkoxysilyl group, and R2 A compound in which is a hydrogen atom, a halogen atom, a hydroxyl group, a methoxy group, a methoxyethoxy group or a bis (2-acetoxyethyl) methyloxy group is preferable for applying the production method of the present invention.

  The compound represented by the general formula (1) can be prepared by a known method such as J.I. Am. Chem. Soc. 1998, 120, p. 12395-12401. And J.A. Org. Chem. 1999, 64, p. 6087-6089. 2'-deoxycytidine can also be obtained commercially.

  The compound represented by the general formula (2) can be produced by reacting the compound represented by the general formula (1) with acetic anhydride in the presence of an acid.

  The amount of acetic anhydride used can be in the range of 0.8 to 3 molar equivalents, but is preferably in the range of 1 to 1.5 molar equivalents.

  In the reaction, the acid itself or a salt formed by the acid and the base can be used as the acid.

  As the acid, an acid whose pH of the 0.1 M aqueous solution is pH 2.9 or less of acetic acid, or a salt in which an aqueous solution of a salt formed by this acid and a base is acidic (hereinafter abbreviated as “acid salt”). ) Is not limited.

  Examples of the acid whose pH of the aqueous solution is lower than that of acetic acid include acetic acid, hydrochloric acid, sulfuric acid, trifluoroacetic acid, chloroacetic acid, dichloroacetic acid, 2-chlorobenzoic acid, 3-chlorobenzoic acid, phenoxyacetic acid, and fluoroacetic acid. , P-toluenesulfonic acid or methanesulfonic acid.

  These acids can be used alone or in combination of two or more.

  Examples of the acid salt include a salt formed by the acid and an organic base such as an alkylamine or a compound represented by the general formula (1).

  Examples of alkylamines that form acid salts include trialkylamines such as trimethylamine, triethylamine, ethyldiisopropylamine, and tributylamine, dialkylamines such as diethylamine, diisopropylamine, dibutylamine, and dicyclohexylamine, isopropylamine, and cyclohexylamine. And monoalkylamine.

  Among the acids whose pH of the aqueous solution is lower than that of acetic acid, acid dissociation constant (pKa value) in water is 4 or less, or acid dissociation in dimethylformamide (hereinafter abbreviated as “DMF”). An acid having a constant (pKa value) of 10 or less is preferred. Specifically, as the former, hydrochloric acid (-3.7), sulfuric acid (-3), trifluoroacetic acid (0.5), chloroacetic acid (2.87), dichloroacetic acid (1.26), 2-chlorobenzoic acid Acid (2.88), 3-chlorobenzoic acid (3.83), phenoxyacetic acid (3.17), and fluoroacetic acid (2.59). Specific examples of the latter include p-toluenesulfonic acid ( 2.6) and methanesulfonic acid (3.0).

  Among acids whose pH is lower than that of acetic acid, hydrochloric acid, sulfuric acid, trifluoroacetic acid, diisopropylamine hydrochloride, hydrochloride of the compound represented by the general formula (1), p-toluenesulfonic acid or methane Sulfonic acids are preferred.

  Among the above acid salts, diisopropylamine hydrochloride, dicyclohexylamine hydrochloride, diisopropylamine sulfate, dicyclohexylamine sulfate, and hydrochloride or sulfate of the compound represented by the general formula (1) are preferable.

  Among the acids, hydrochloric acid, sulfuric acid, trifluoroacetic acid, diisopropylamine hydrochloride, hydrochloride of the compound represented by the general formula (1), p-toluenesulfonic acid or methanesulfonic acid is particularly preferable.

  Although the said acid can also be used independently, 2 or more types can also be used together.

  The amount of the acid used is not particularly limited as long as it does not inhibit the reaction and does not decompose the compound, but it is preferably 0.01 mol% or more and 40 mol% or less, particularly preferably based on the compound represented by the general formula (1). Is 0.1 mol% or more and 20 mol% or less.

  A solvent is used for the reaction. The solvent is not limited as long as the reaction is not inhibited, but an aprotic solvent is preferable. Examples of the aprotic solvent include acetonitrile, dichloromethane, DMF, and 1,3-dimethyl-2-imidazolidinone. Among them, DMF is preferable.

  The reaction temperature can be selected from the range of −10 ° C. to the boiling point of the solvent to be used, but the range of 10 ° C. to 40 ° C. is particularly preferable.

  If the preferable implementation method of the manufacturing method of this invention is illustrated, the method of adding acetic anhydride to the mixture of the compound represented with General formula (1), an acid, and an aprotic solvent will be mention | raise | lifted.

  The compound represented by the general formula (2) obtained by the above reaction can be separated from the reaction mixture and recovered using a known method, for example, a method described in JP-A-6-189753. .

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples. In addition, 2'-deoxycytidine used for the following experiments was manufactured by Wako Pure Chemical Industries, Ltd.

Example 1
Synthesis of N ⁴ -acetyl-2′-deoxycytidine Acetic anhydride (0.94 g, 9.2 mmol) was added to a solution of 2′-deoxycytidine (2.0 g, 8.8 mmol) and trifluoroacetic acid (30 mg, 0.26 mmol) in DMF (20 g) at room temperature. ) Was added and stirred for 24 hours under the same temperature conditions. After completion of the reaction, the reaction solution was analyzed by liquid chromatography (hereinafter abbreviated as “HPLC”). As a result, the title compound was 96.7%, 2′-deoxycytidine (hereinafter abbreviated as “starting material”). 5%, N ⁴ , 5′-O-diacetyl-2′-deoxycytidine (hereinafter abbreviated as “By-product 1”) 0.3%, N ⁴ , 3′-O-diacetyl-2′- Deoxycytidine (hereinafter abbreviated as “By-product 2”) was 0%. N ⁴ -acetylcytosine was not observed.
HPLC analysis conditions: column, Develosil PRAQUEOUS 4.6 × 250 mm (Nomura Chemical); mobile phase, water-CH ₃ CN (9: 1); flow rate, 1 ml / min; column temperature, 40 ° C .; retention time (min), title compound 5.3, starting material 3.4, (byproduct 1) 21.8, (byproduct 2) 21.0.

Comparative Example 1
Synthesis of N ⁴ -acetyl-2′-deoxycytidine The reaction was performed in the same procedure as in Example 1 without adding trifluoroacetic acid. HPLC analysis of the reaction mixture after completion of the reaction revealed that the title compound was 94.7%, starting material 2.2%, (side product 1) 2.1%, and (side product 2) 0.6%.

Reference example 1
Synthesis of N ⁴ -acetyl-2′-deoxycytidine The reaction was carried out by adding 10 mol% of acetic acid instead of trifluoroacetic acid in the same procedure as in Example 1. HPLC analysis of the reaction mixture after completion of the reaction revealed that the title compound was 96.4%, starting material 1.7%, (side product 1) 1.3%, and (side product 2) 0.3%. N ⁴ -acetylcytosine was not observed.

Reference example 2
Synthesis of N ⁴ -acetyl-2′-deoxycytidine The reaction was performed by adding 100 mol% of acetic acid instead of trifluoroacetic acid in the same procedure as in Example 1. HPLC analysis of the reaction mixture after completion of the reaction showed that the title compound was 95.2%, starting material 0.9%, (Sub-product 1) 3.0%, and (Sub-product 2) 0.3%. N ⁴ -acetylcytosine was not observed.

Examples 2-8
Synthesis of N ⁴ -acetyl-2′-deoxycytidine In the same procedure as in Example 1, dichloroacetic acid, diisopropylamine hydrochloride and N ⁴ -acetyl-2′-deoxycytidine hydrochloride were added instead of trifluoroacetic acid. The results of the reaction are shown in Table 1.

Reference example 3
Synthesis of N ⁴ -acetyl-5′-O- (4,4′-dimethoxytrityl) -2′-deoxycytidine 11.7 g of DMF solution obtained in Example 8 (N ⁴ -acetyl-2′-deoxycytidine 1 Diisopropylamine (490 mg, 4.84 mmol) was added to room temperature at room temperature, and then 766 mg (9.68 mmol) of pyridine was added under ice cooling. Further, 1.64 g (4.84 mmol) of 4,4′-dimethoxytrityl chloride was added and stirred overnight. When the HPLC analysis was conducted after completion of the reaction, the title compound was abbreviated as 91.8%, 5′-O- (4,4′-dimethoxytrityl) -2′-deoxycytidine (hereinafter, “byproduct 3”). ) 0.17%, N ⁴ , 3′-O-diacetyl-5′-O- (4,4′-dimethoxytrityl) -2′-deoxycytidine (hereinafter abbreviated as “byproduct 4”) It was 0.56%.
HPLC analysis conditions: column, Develosil PRAQUEOUS 4.6 × 250 mm (Nomura Chemical); mobile phase, water—CH ₃ CN-10 mM NaH ₂ PO ₄ (pH 7) (65:35); flow rate, 1 ml / min (0-15 min), 1.5 ml / min (15.1-45 min); column temperature, 40 ° C .; retention time (min), title compound 5.4, (byproduct 3) 4.3, (byproduct 4) 38.

Reference example 4
Synthesis of N ⁴ -acetyl-5′-O- (4,4′-dimethoxytrityl) -2′-deoxycytidine The DMF solution obtained in Comparative Example 1 was treated in the same manner as in Reference Example 3 (in place of diisopropylamine). The reaction mixture obtained by using pyridine was analyzed by HPLC. As a result, the title compound was found to be 84.2%, (byproduct 3) 0.83%, and (byproduct 4) 2.6%.

Example 9
Synthesis of N ⁴ -acetyl-5′-O- (4,4′-dimethoxytrityl) -2′-deoxy-2′-fluorocytidine 5′-O- (4,4′-dimethoxytrityl) -2′- 0.23 g (2.3 mmol) of acetic anhydride was added to a 12 mL DMF solution of 1.2 g (2.2 mmol) of deoxy-2′-fluorocytidine and 7.4 mg (0.65 mmol) of trifluoroacetic acid at the same temperature. Stir below for 24 hours. HPLC analysis of the reaction mixture after completion of the reaction revealed that the title compound was 94.2%, 5′-O- (4,4′-dimethoxytrityl) -2′-deoxy-2′-fluorocytidine (starting material) 2.2. %, N ⁴ , 3′-O-diacetyl-5′-O- (4,4′-dimethoxytrityl) -2′-deoxy-2′-fluorocytidine (hereinafter abbreviated as “byproduct 5”). ) 2.7%. Water was added to the reaction mixture, and the mixture was extracted with ethyl acetate, and washed with water and saturated brine. The organic layer was dried over magnesium sulfate and concentrated under reduced pressure to give 1.3 g (yield 95%) of the title compound as a colorless powder.
HPLC analysis conditions: column, Develosil PRAQUEOUS 4.6 × 250 mm (Nomura Chemical); mobile phase, water-CH ₃ CN (9: 1); flow rate, 1 ml / min; column temperature, 40 ° C .; retention time (min), title compound 6.6, starting material 5.0, (byproduct 5) 10.5.

Examples 10 and 11 and Comparative Example 2
Synthesis of N ⁴ -acetyl-5′-O- (4,4′-dimethoxytrityl) -2′-deoxy-2′-fluorocytidine In the same procedure as in Example 9 , instead of trifluoroacetic acid (3 mol%) Table 2 [Table 2] shows the results of the reaction with addition of trifluoroacetic acid and diisopropylamine hydrochloride and the results of the comparison without addition of acid (Comparative Example 2).

Comparative Example 3
Synthesis of N ⁴ -acetyl-2′-deoxycytidine 6.26 g (61.3 mmol) of acetic anhydride was added over 30 minutes to a solution of 1.0 g (4.4 mmol) of 2′-deoxycytidine in 54 g of methanol under heating and reflux. The mixture was further heated to reflux for 30 minutes. After completion of the reaction, the reaction mixture was subjected to HPLC analysis. As a result, the title compound was 85.7%, 2′-deoxycytidine 2.4%, (by-products 1 and 2) 1.1%, N ⁴ -acetylcytosine (hereinafter referred to as “ Abbreviated as “by-product 6”.) 10.8%.

Comparative Example 4
Synthesis of N ⁴ -acetyl-2′-deoxycytidine The reaction was carried out using ethanol instead of methanol in the same procedure as in Comparative Example 3, and the title compound was 80.6%, 2′-deoxycytidine 0.73% (By-product 1 and 2) 1.3% and (by-product 6) 17.4%.

Comparative Example 5
Synthesis of N ⁴ -acetyl-2′-deoxycytidine The reaction was carried out using 2-propanol instead of methanol in the same procedure as in Comparative Example 3. As a result, 84.5% of the title compound, 2′-deoxycytidine 0. 35%, (by-products 1 and 2) 1.9%, and (by-product 6) 13.3%.

Claims (4)

General formula (1)

(Wherein R1 represents a hydrogen atom or a hydroxyl protecting group, and R2 represents a hydrogen atom, a halogen atom, a hydroxyl group or a protected hydroxyl group) and a compound (A) represented by acetic anhydride (B). At least selected from the group consisting of dichloroacetic acid, hydrochloric acid, sulfuric acid, trifluoroacetic acid, diisopropylamine hydrochloride, hydrochloride of the compound represented by the general formula (1), p-toluenesulfonic acid, and methanesulfonic acid The reaction is carried out in the presence of one kind of acid (C).

(Wherein R1 and R2 have the same meanings as R1 and R2 in the general formula (1)). The acid (C) according to claim 1 is hydrochloric acid, sulfuric acid, trifluoroacetic acid, diisopropylamine hydrochloride, general formula (1) (chemical formula 3)

(Wherein R1 represents a hydrogen atom or a protecting group for a hydroxyl group, and R2 represents a hydrogen atom, a halogen atom, a hydroxyl group or a protected hydroxyl group), a hydrochloride of the compound represented by the formula: The manufacturing method of Claim 1 which is methanesulfonic acid. Before the addition of hexane (C) is less than 20 mol%, the production method according to claim 1 or claim 2. R1 in the general formula (1) according to claim 1 is a hydrogen atom, a 4,4'-dimethoxytrityl group or a trialkoxysilyl group, R1 in the general formula (2) according to claim 1 is a hydrogen atom, It is a 4′-dimethoxytrityl group or a trialkoxysilyl group, and R2 in the general formula (1) according to claim 1 is a hydrogen atom, a halogen atom, a hydroxyl group, a methoxy group, a methoxyethoxy group or bis (2-acetoxyethyl) methyl. It is an oxy group, and R2 in the general formula (2) according to claim 1 is a hydrogen atom, a halogen atom, a hydroxyl group, a methoxy group, a methoxyethoxy group or a bis (2-acetoxyethyl) methyloxy group. The manufacturing method as described in any one of Claim 3.