Method for producing artificial nucleic acid monomers

Abstract

To provide a novel method for producing dDs amidite.SOLUTION: A method for producing 7-(2-thienyl)-3-[2'-deoxy-5'-O-(4,4'-dimethoxytrityl)-β-D-ribofuranosyl]-3H-imidazo[4,5-b]pyridine 2-cyanoethyl-N,N-diisopropylphosphoramidite includes (1) reacting 7-(2-thienyl)-3-[2'-deoxy-5'-O-(4,4'-dimethoxytrityl)-β-D-ribofuranosyl]-3H-imidazo[4,5-b]pyridine with 2-cyanoethyl tetraisopropylphosphorodiamidite in a solvent in the presence of an amine salt of trifluoroacetic acid.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing an artificial nucleic acid monomer. [Background technology]

[0002] Amidite monomers are used as raw materials for DNA oligomers, which are used in gene amplification analysis (PCR) and are also attracting attention as nucleic acid medicines.

[0003] In gene amplification diagnostic applications, very small amounts of DNA oligomers are used as templates.

[0004] On the other hand, for nucleic acid pharmaceutical applications, oligonucleotides are synthesized from amidite monomers by chemical synthesis. Oligonucleotide synthesis is typically performed by elongation reaction using solid-phase synthesis, which requires an excess of amidite monomer for each base elongation, necessitating the supply of amidite monomers in the gram to kilogram range. For this reason, a method for mass-supplying the raw material amidite monomers is needed.

[0005] Patent Document 1 discloses that first, 7-(2-thienyl)-3-[2'-deoxy-5'-O-(4,4'-dimethoxytrityl)-β-D-ribofuranosyl]-3H-imidazo[4,5-b]pyridine and 2-cyanoethyl tetraisopropylphosphorodiamidite are reacted at room temperature in an acetonitrile solution of 1H-tetrazole, and then the resulting mixture is purified using flash silica gel chromatography to obtain 7-(2-thienyl)-3-[2'-deoxy-5'-O-(4,4'-dimethoxytrityl)-β-D-ribofuranosyl]-3H-imidazo[4,5-b]pyridine 2-cyanoethyl-N,N-diisopropylphosphoramidite (dDs amidite).

[0006] Non-Patent Document 1 discloses a method for producing 2'-O-methoxyethyl (MOE) derivatives of nucleoside phosphoramidites by a precipitation method. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. WO2007 / 066737 [Non-patent literature]

[0008] [Non-Patent Document 1] Organic Process Research & Development 2005, 9, 730-737 Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to provide a novel method for producing dDs amidites. [Means for solving the problem]

[0010] The present inventors have conducted extensive research to solve the above problems and have found that dDs amidite can be easily mass-produced by solid separation and purification.

[0011] Based on this finding, the present inventors have conducted further research and have completed the present invention.

[0012] That is, the present invention includes the following method for producing a dDs amidite.

[0013] Section 1. 1. A method for preparing 7-(2-thienyl)-3-[2′-deoxy-5′-O-(4,4′-dimethoxytrityl)-β-D-ribofuranosyl]-3H-imidazo[4,5-b]pyridine 2-cyanoethyl-N,N-diisopropylphosphoramidite, comprising: (1) reacting 7-(2-thienyl)-3-[2'-deoxy-5'-O-(4,4'-dimethoxytrityl)-β-D-ribofuranosyl]-3H-imidazo[4,5-b]pyridine with 2-cyanoethyl tetraisopropylphosphorodiamidite in a solvent in the presence of an amine salt of trifluoroacetic acid; Including, The solvent is at least one solvent selected from the group consisting of dichloromethane, chloroform, tetrahydrofuran, dioxane, diethyl ether, and diisopropyl ether. Manufacturing method.

[0014] Section 2. Item 2. The production method according to Item 1, wherein the amine salt of trifluoroacetic acid is at least one amine salt of trifluoroacetic acid selected from the group consisting of alkyl-substituted amine salts of trifluoroacetic acid, substituted or unsubstituted pyridine salts, and imidazole salts.

[0015] Section 3. 1. A method for preparing 7-(2-thienyl)-3-[2′-deoxy-5′-O-(4,4′-dimethoxytrityl)-β-D-ribofuranosyl]-3H-imidazo[4,5-b]pyridine 2-cyanoethyl-N,N-diisopropylphosphoramidite, comprising: (2) dissolving a composition containing 7-(2-thienyl)-3-[2'-deoxy-5'-O-(4,4'-dimethoxytrityl)-β-D-ribofuranosyl]-3H-imidazo[4,5-b]pyridine 2-cyanoethyl-N,N-diisopropylphosphoramidite in alcohol, followed by mixing with water to precipitate 7-(2-thienyl)-3-[2'-deoxy-5'-O-(4,4'-dimethoxytrityl)-β-D-ribofuranosyl]-3H-imidazo[4,5-b]pyridine 2-cyanoethyl-N,N-diisopropylphosphoramidite; A manufacturing method comprising:

[0016] Section 4. Item 4. The production method according to Item 3, wherein the alcohol is at least one alcohol selected from the group consisting of methanol, ethanol, propanol, and isopropanol.

[0017] Section 5. 5. The method according to item 3 or 4, wherein an electrolyte is further added in the deposition step.

[0018] Section 6. Item 6. The manufacturing method according to item 5, wherein the electrolyte is at least one electrolyte selected from the group consisting of inorganic salts selected from the group consisting of lithium chloride, sodium chloride, potassium chloride, cesium chloride, calcium chloride, rubidium chloride, barium chloride, magnesium chloride, strontium chloride, sodium sulfate, potassium sulfate, magnesium sulfate, sodium nitrate, potassium nitrate, sodium bicarbonate, sodium carbonate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium borate, sodium bromide, and potassium bromide; organic salts selected from the group consisting of sodium acetate, sodium formate, ammonium acetate, and ammonium formate; and organic bases selected from the group consisting of trimethylamine and triethylamine.

[0019] Section 7. 1. A method for preparing 7-(2-thienyl)-3-[2′-deoxy-5′-O-(4,4′-dimethoxytrityl)-β-D-ribofuranosyl]-3H-imidazo[4,5-b]pyridine 2-cyanoethyl-N,N-diisopropylphosphoramidite, comprising: (1) reacting 7-(2-thienyl)-3-[2'-deoxy-5'-O-(4,4'-dimethoxytrityl)-β-D-ribofuranosyl]-3H-imidazo[4,5-b]pyridine with 2-cyanoethyl tetraisopropylphosphorodiamidite in a solvent in the presence of an amine salt of trifluoroacetic acid to obtain a composition containing 7-(2-thienyl)-3-[2'-deoxy-5'-O-(4,4'-dimethoxytrityl)-β-D-ribofuranosyl]-3H-imidazo[4,5-b]pyridine 2-cyanoethyl-N,N-diisopropylphosphoramidite; (2) dissolving the composition containing the 7-(2-thienyl)-3-[2'-deoxy-5'-O-(4,4'-dimethoxytrityl)-β-D-ribofuranosyl]-3H-imidazo[4,5-b]pyridine 2-cyanoethyl-N,N-diisopropylphosphoramidite in alcohol, followed by mixing with water to precipitate the 7-(2-thienyl)-3-[2'-deoxy-5'-O-(4,4'-dimethoxytrityl)-β-D-ribofuranosyl]-3H-imidazo[4,5-b]pyridine 2-cyanoethyl-N,N-diisopropylphosphoramidite; Including, The solvent in step (1) is at least one solvent selected from the group consisting of dichloromethane, chloroform, tetrahydrofuran, dioxane, diethyl ether, and diisopropyl ether. Manufacturing method. [Effects of the Invention]

[0020] The present invention can provide a new method for producing dDs amidites. [Brief explanation of the drawings]

[0021] [Figure 1] FIG. 1 is a diagram illustrating a crude product ( 1 H NMR) containing dDs amidite before solid separation and purification. [Figure 2] FIG. 1 is a diagram illustrating dDs amidite ( 1 H NMR) after solid separation and purification. [Figure 3] FIG. 1 is a diagram illustrating a crude product (31P NMR) containing dDs amidite before solid separation and purification. [Figure 4] FIG. 1 is a diagram illustrating dDs amidite (31P NMR) after solid separation and purification. [Figure 5] FIG. 1 is a diagram illustrating a crude product ( 1 H NMR) containing dDs amidite before column purification. [Figure 6] FIG. 1 is a diagram illustrating dDs amidite ( 1 H NMR) after silica gel column purification. [Figure 7]FIG. 1 is a diagram illustrating a crude product (31P NMR) containing dDs amidite before column purification. [Figure 8] FIG. 1 is a diagram illustrating dDs amidite (31P NMR) after silica gel column purification. [Figure 9] FIG. 1 is a diagram illustrating a stability test in a reaction solution of a dDs amidite produced by a conventional method. [Figure 10] FIG. 1 is a diagram illustrating a stability test of the dDs amidite produced by the method of the present invention in a reaction solution. DETAILED DESCRIPTION OF THE INVENTION

[0022] The present invention will be described in detail below.

[0023] In this specification, the terms "comprise" and "contain" are concepts that encompass all of "comprise," "consist essentially of," and "consist only of."

[0024] In this specification, when a numerical range is expressed as "A to B," it means A or more and B or less.

[0025] [1] Method for producing dDs amidite The method for producing 7-(2-thienyl)-3-[2'-deoxy-5'-O-(4,4'-dimethoxytrityl)-β-D-ribofuranosyl]-3H-imidazo[4,5-b]pyridine 2-cyanoethyl-N,N-diisopropylphosphoramidite (target compound (dDs amidite)) of the present invention (hereinafter also referred to as "the method for producing dDs amidite of the present invention") is as follows: (1) reacting 7-(2-thienyl)-3-[2'-deoxy-5'-O-(4,4'-dimethoxytrityl)-β-D-ribofuranosyl]-3H-imidazo[4,5-b]pyridine (a starting compound) with 2-cyanoethyl tetraisopropylphosphorodiamidite (an amiditizing reagent) in a solvent in the presence of an amine salt of trifluoroacetic acid; The solvent is at least one solvent selected from the group consisting of dichloromethane (methylene chloride, DCM), chloroform (trichloromethane, CHCl 3 ), tetrahydrofuran (THF), dioxane, diethyl ether (Et 2 O), and diisopropyl ether (IPE).

[0026] According to the method for producing a dDs amidite of the present invention, a raw material compound can be reacted with an amiditizing reagent (amiditizing reaction) to efficiently produce a dDs amidite (target compound).

[0027] Conventionally, 1H-tetrazole has been used as an activator for the amidite reagent under reaction conditions, and activation with 1H-tetrazole is the mainstream. However, 1H-tetrazole is classified as a Class 5 hazardous material under the Fire Service Act, so a hazardous material storage facility is required, and handling it in large-scale synthesis is dangerous.

[0028] Conventional reaction conditions include the use of acetonitrile as the solvent and 1H-tetrazole as the activator. However, when using acetonitrile and 1H-tetrazole, the reaction must be carried out at around room temperature, raising concerns about the stability of the resulting dDs amidite in solution for long periods of time. Solvent replacement is required when separating the organic solvent and aqueous layers.

[0029] In the method for producing dDs amidite of the present invention, the use of a pyridine salt of trifluoroacetic acid (TFA) (pyridine / TFA salt) or the like as an activator can avoid the use of 1H-tetrazole, which is a hazardous substance.

[0030] In the method for producing dDs amidites of the present invention, the amidite formation reaction proceeds efficiently by using a solvent such as dichloromethane (DCM) or chloroform and an amine salt of trifluoroacetic acid. The use of a solvent such as dichloromethane (DCM) and an amine salt of trifluoroacetic acid in the amidite formation reaction allows the reaction to proceed at low temperatures, improving the stability of the resulting dDs amidite. Furthermore, the use of dichloromethane as the reaction solvent avoids solvent replacement during separation, making production more efficient.

[0031] The method for producing dDs amidites of the present invention is suitable for mass production of dDs amidites.

[0032] [1-1] Raw material compound In the method for producing dDs amidite of the present invention, the starting compound is 7-(2-thienyl)-3-[2'-deoxy-5'-O-(4,4'-dimethoxytrityl)-β-D-ribofuranosyl]-3H-imidazo[4,5-b]pyridine, which is represented by the following chemical formula.

[0033] [ka]

[0034] In the above chemical formula, "DMTr" is a 4,4'-dimethoxytrityl group and is represented by the following chemical formula:

[0035] [ka]

[0036] [1-2] Amidite Reagents In the method for producing a dDs amidite of the present invention, the amiditizing reagent is 2-cyanoethyl tetraisopropylphosphorodiamidite, which is represented by the following chemical formula:

[0037] [ka]

[0038] The amount of the amiditizing reagent used is, in molar ratio relative to the raw material compound (substrate), preferably 1.0 to 3.0 equivalents, more preferably 1.1 to 2.0 equivalents, and even more preferably 1.3 to 1.7 equivalents.

[0039] As a preferred embodiment of the amount of the amiditizing reagent used, for example, about 1.3 equivalents in molar ratio relative to the starting compound (substrate) is first added, and if unreacted starting compound (substrate) remains, the amiditizing reagent and the amine salt of trifluoroacetic acid may then be added, preferably in amounts of 0.1 equivalent each, and this procedure may be repeated.

[0040] In the method for producing dDs amidite of the present invention, by adjusting the amount of amidite reagent used to fall within the above range when carrying out the amidite reaction, the amidite reaction can be carried out efficiently from the raw material compound and the amidite reagent, thereby producing dDs amidite.

[0041] [1-3] Solvent In the method for producing a dDs amidite of the present invention, the solvent is at least one solvent selected from the group consisting of dichloromethane, chloroform, tetrahydrofuran, dioxane, diethyl ether, and diisopropyl ether. Among these solvents, dichloromethane, chloroform, and tetrahydrofuran are preferred.

[0042] In the method for producing dDs amidite of the present invention, by using the above-mentioned solvent when proceeding with the amidite reaction, the amidite reaction can be efficiently carried out from the raw material compound and the amidite reagent, and dDs amidite can be produced.

[0043] In the method for producing dDs amidite of the present invention, the amidite formation reaction proceeds efficiently by using a solvent such as dichloromethane (DCM) or chloroform. The use of a solvent such as dichloromethane (DCM) in the amidite formation reaction allows the reaction to proceed at low temperatures and improves the stability of the resulting dDs amidite. Furthermore, the use of dichloromethane as the reaction solvent makes it possible to avoid solvent replacement during separation, thereby improving the efficiency of production.

[0044] The amount of solvent used is adjusted so that the concentration (molar concentration (M), mol / L) of the raw material compound (substrate) is preferably 0.01 M to 1 M, more preferably 0.05 M to 0.5 M, and even more preferably 0.1 M to 0.3 M.

[0045] The amount of solvent used is preferably 1.5 to 120 times, more preferably 3 to 24 times, and even more preferably 6 to 12 times, the amount of the raw material compound (substrate).

[0046] Amine salts of [1-4]trifluoroacetic acid The amine salt of trifluoroacetic acid used in the method for producing a dDs amidite of the present invention is preferably a complex of trifluoroacetic acid and an amine base, and is at least one amine salt of trifluoroacetic acid selected from the group consisting of alkyl-substituted amine salts of trifluoroacetic acid, substituted or unsubstituted pyridine salts, and imidazole salts. The amine salt of trifluoroacetic acid used in the method for producing a dDs amidite of the present invention is a complex of trifluoroacetic acid and an amine base, and examples thereof include alkyl-substituted amine salts of trifluoroacetic acid such as trimethylammonium, triethylammonium, diethylmethylammonium, tripropylammonium, and diisopropylethylammonium; aryl-substituted amine salts such as dimethylanilinium and triphenylammonium; aralkyl-substituted amine salts such as tribenzylammonium; polyamine salts such as tetramethylethylenediamine; substituted or unsubstituted pyridine salts such as pyridinium, 2-methylpyridinium, 3-methylpyridinium, 4-methylpyridinium, 4-dimethylaminopyridinium, 2,6-dimethylpyridinium, 2,4,6-trimethylpyridinium, 2,3,4,6-tetramethylpyridinium, and pentamethylpyridinium; saturated cyclic amine salts such as piperidinium, 2,2,6,6-tetramethylpiperidinium, and N-methylmorpholinium; and imidazole salts such as N-methylimidazolium. These amine salts may also be bound to a polymer. Among these, alkyl-substituted amine salts, substituted or unsubstituted pyridine salts, and imidazole salts of trifluoroacetic acid are more preferred, and pyridinium, N-methylimidazolium, and diisopropylethylammonium trifluoroacetic acid are particularly preferred.

[0047] In the method for producing dDs amidite of the present invention, the amine salt of trifluoroacetic acid is used when carrying out the amidite reaction, thereby enabling the amidite reaction to proceed efficiently from the raw material compound and the amidite reagent, thereby producing dDs amidite.

[0048] In the method for producing dDs amidite of the present invention, the use of a pyridine salt of trifluoroacetic acid (TFA) (pyridine / TFA salt) or the like can avoid the use of 1H-tetrazole, which is a dangerous substance.

[0049] The amount of the salt used is, in molar ratio relative to the raw material compound (substrate), preferably 1.0 to 3.0 equivalents, more preferably 1.1 to 2.0 equivalents, and even more preferably 1.2 to 1.7 equivalents.

[0050] As a preferred embodiment of the amount of the amine salt of trifluoroacetic acid used, for example, about 1.2 molar equivalents are first added to the starting compound (substrate), and if unreacted starting compound (substrate) remains, the amiditizing reagent and the amine salt of trifluoroacetic acid may be added, preferably in an amount of 0.1 equivalent each, and this procedure may be repeated.

[0051] [1-5] Target compound (dDs amidite) In the method for producing dDs amidite of the present invention, the target compound is 7-(2-thienyl)-3-[2'-deoxy-5'-O-(4,4'-dimethoxytrityl)-β-D-ribofuranosyl]-3H-imidazo[4,5-b]pyridine 2-cyanoethyl-N,N-diisopropylphosphoramidite (dDs amidite), which is represented by the following chemical formula:

[0052] [ka]

[0053] [1-6] Amidite formation process In the method for producing a dDs amidite of the present invention, the reaction temperature in the amidite formation reaction step is preferably -20°C to 30°C, preferably -15°C to 20°C, more preferably -10°C to 10°C, and particularly preferably -5°C to 5°C.

[0054] In the method for producing a dDs amidite of the present invention, the reaction time in the amidite-forming reaction step is preferably 0.5 to 4 hours, more preferably 0.5 to 3 hours, and further preferably 1 to 2 hours.

[0055] [2] Method for producing dDs amidite The method for producing 7-(2-thienyl)-3-[2'-deoxy-5'-O-(4,4'-dimethoxytrityl)-β-D-ribofuranosyl]-3H-imidazo[4,5-b]pyridine 2-cyanoethyl-N,N-diisopropylphosphoramidite (dDs amidite) of the present invention (hereinafter also referred to as "the method for producing dDs amidite of the present invention") comprises the steps of: (2) A step (crystallization step) is included in which a composition containing 7-(2-thienyl)-3-[2'-deoxy-5'-O-(4,4'-dimethoxytrityl)-β-D-ribofuranosyl]-3H-imidazo[4,5-b]pyridine 2-cyanoethyl-N,N-diisopropylphosphoramidite (dDs amidite) is first dissolved in alcohol, and then mixed with water (by adding water) to precipitate 7-(2-thienyl)-3-[2'-deoxy-5'-O-(4,4'-dimethoxytrityl)-β-D-ribofuranosyl]-3H-imidazo[4,5-b]pyridine 2-cyanoethyl-N,N-diisopropylphosphoramidite (dDs amidite).

[0056] The method for producing a dDs amidite of the present invention makes it possible to efficiently isolate a dDs amidite from a composition containing the dDs amidite.

[0057] In the method for producing a dDs amidite of the present invention, for example, an alcohol solution of a composition containing a dDs amidite can be dropped into water containing an electrolyte to precipitate the dDs amidite.

[0058] dDs amidite is a compound for which crystallinity has not been confirmed. When conventional purification methods using silica gel columns are used, the resulting product becomes oily after the solvent is removed. When the oily dDs amidite is then subjected to a drying process, it expands into a foam-like state and becomes very large in volume. For this reason, conventional methods for mass-producing dDs amidite require the use of specialized equipment such as large-capacity dryers or spray dryers, or additional procedures such as lyophilization after dissolving in an aqueous organic solvent. Furthermore, conventional methods for purifying dDs amidite using silica gel columns require precise separation to remove any residue of the amiditization reagent used in the reaction, which requires large amounts of solvent and silica gel.

[0059] In the method for producing a dDs amidite of the present invention, by applying a purification step in which the dDs amidite is precipitated (crystallized), it is possible to easily remove residues of the amidite-forming reagent used in the reaction.

[0060] According to the method for producing dDs amidite of the present invention, dDs amidite can be obtained with a purity equivalent to that obtained by the conventional purification method using a silica gel column.

[0061] In the method for producing dDs amidite of the present invention, a solid dDs amidite can be obtained in which the volume does not increase during the drying step, making it possible to produce it on a large scale without requiring special equipment for the drying process.

[0062] In the method for producing dDs amidite of the present invention, the precipitation step preferably further includes the addition of an electrolyte, which increases the coagulation of the precipitated dDs amidite solid, thereby improving the yield of dDs amidite and also increases the filtration rate during filtration of the dDs amidite, thereby improving workability. In the precipitation step, the dDs amidite is emulsified in a mixture of alcohol and water to form a very fine suspension. Adding an electrolyte such as sodium chloride to the suspension changes the dispersibility of the dDs amidite in the emulsion containing the dDs amidite, causing demulsification, and the dDs amidite can be obtained as aggregates of a size that can be separated into solids.

[0063] [2-1] Alcohol In the method for producing a dDs amidite of the present invention, the alcohol is preferably at least one alcohol selected from the group consisting of methanol, ethanol, propanol, and isopropanol.

[0064] In the method for producing dDs amidite of the present invention, in the precipitation step, the composition containing dDs amidite is dissolved in alcohol, thereby efficiently purifying the dDs amidite from the composition containing dDs amidite.

[0065] The amount of alcohol used is adjusted so that the concentration of the dDs amidite (molar concentration (M), mol / L) is preferably 0.001 M to 0.2 M, more preferably 0.003 M to 0.1 M, and even more preferably 0.005 M to 0.05 M.

[0066] [2-2]Water In the method for producing dDs amidite of the present invention, in the precipitation step, a composition containing dDs amidite is first dissolved in alcohol, and then water is added, thereby efficiently purifying the dDs amidite from the composition containing dDs amidite.

[0067] The amount of water used is adjusted so that the concentration of the dDs amidite (molar concentration (M), mol / L) is preferably 0.001 M to 0.1 M, more preferably 0.002 M to 0.05 M, and even more preferably 0.003 M to 0.03 M.

[0068] The blending ratio of the alcohol to water is preferably alcohol:water=3:7 to 9:1 by volume, more preferably alcohol:water=4:6 to 8:2, and even more preferably alcohol:water=5:5 to 7:3.

[0069] The blending ratio of the alcohol to water is, in volume ratio, 30% to 90% by volume of the alcohol relative to the total amount of the alcohol and water, more preferably 40% to 80% by volume, and even more preferably 50% to 70% by volume.

[0070] [2-3] Electrolyte In the method for producing a dDs amidite of the present invention, in the precipitation step, an electrolyte is preferably further added to carry out purification.

[0071] The electrolyte is preferably at least one electrolyte selected from the group consisting of inorganic salts selected from the group consisting of lithium chloride, sodium chloride, potassium chloride, cesium chloride, calcium chloride, rubidium chloride, barium chloride, magnesium chloride, strontium chloride, sodium sulfate, potassium sulfate, magnesium sulfate, sodium nitrate, potassium nitrate, sodium bicarbonate, sodium carbonate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium borate, sodium bromide, and potassium bromide; and organic salts selected from the group consisting of sodium acetate, sodium formate, ammonium acetate, and ammonium formate; and organic bases selected from the group consisting of trimethylamine and triethylamine.

[0072] The amount of the electrolyte and organic base used is preferably 1 mol to 300 mol, more preferably 1.5 mol to 100 mol, and even more preferably 2 mol to 20 mol, relative to 1 mol of the dDs amidite.

[0073] The amounts of the electrolyte and organic base used, in mass ratio relative to the water, are preferably 0.01 g / 100 mL to 36 g / 100 mL, more preferably 0.1 g / 100 mL to 10 g / 100 mL, and even more preferably 0.2 g / 100 mL to 4 g / 100 mL.

[0074] When sodium chloride, for example, is used as the electrolyte in the precipitation step, the upper limit of the amount used is preferably about 35.8 g / 100 ml water in saturated saline solution at 25°C.

[0075] In the method for producing a dDs amidite of the present invention, the dDs amidite can be efficiently purified from a composition containing the dDs amidite by adding an electrolyte in the precipitation step.

[0076] In the method for producing dDs amidite of the present invention, the precipitation step preferably further includes the addition of an electrolyte, which increases the coagulation of the precipitated dDs amidite solid, thereby improving the yield of dDs amidite and also increases the filtration rate during filtration of the dDs amidite, thereby improving workability. In the precipitation step, the dDs amidite is emulsified in a mixture of alcohol and water to form a very fine suspension. Adding an electrolyte such as sodium chloride to the suspension changes the dispersibility of the dDs amidite in the emulsion containing the dDs amidite, causing demulsification, and the dDs amidite can be obtained as aggregates of a size that can be separated into solids.

[0077] [2-4] Precipitation of dDs amidite In the method for producing a dDs amidite of the present invention, in the step of precipitating a dDs amidite, first, a composition containing the dDs amidite is dissolved in alcohol.

[0078] In the method for producing a dDs amidite of the present invention, in the step of precipitating a dDs amidite, a composition containing the dDs amidite dissolved in the alcohol is then mixed with water to precipitate (crystallize) the dDs amidite.

[0079] In the method for producing a dDs amidite of the present invention, the reaction temperature in the step of precipitating a dDs amidite is preferably -20°C to 25°C, preferably -10°C to 15°C, and more preferably -5°C to 10°C.

[0080] In a preferred embodiment of the precipitation step, for example, an alcohol (e.g., methanol) in which a composition containing a dDs amidite is dissolved is dropped into water cooled to 0°C. Because heat of dissolution of methanol / water is generated, the precipitation step is preferably carried out at 0°C to 10°C. When sodium chloride, for example, is used as the electrolyte, the lower limit of the reaction temperature in the precipitation step is preferably about -21.3°C, which is the freezing point of saturated saline.

[0081] In the method for producing a dDs amidite of the present invention, the reaction time in the step of precipitating a dDs amidite is preferably 0.1 hours to 24 hours, and more preferably 0.5 hours to 10 hours. More preferably, it is 1 hour to 6 hours.

[0082] In the precipitation step, the dropwise addition is usually carried out for about 1 hour, and then aging is carried out for about 5 hours.

[0083] [3] A method for producing dDs amidite, including purification of dDs amidite The method for producing 7-(2-thienyl)-3-[2'-deoxy-5'-O-(4,4'-dimethoxytrityl)-β-D-ribofuranosyl]-3H-imidazo[4,5-b]pyridine 2-cyanoethyl-N,N-diisopropylphosphoramidite (dDs amidite) of the present invention (the method for producing dDs amidite) is as follows: (1) reacting 7-(2-thienyl)-3-[2'-deoxy-5'-O-(4,4'-dimethoxytrityl)-β-D-ribofuranosyl]-3H-imidazo[4,5-b]pyridine (starting compound) with 2-cyanoethyl tetraisopropylphosphorodiamidite (amidite-forming reagent) in a solvent in the presence of an amine salt of trifluoroacetic acid to obtain a composition containing 7-(2-thienyl)-3-[2'-deoxy-5'-O-(4,4'-dimethoxytrityl)-β-D-ribofuranosyl]-3H-imidazo[4,5-b]pyridine 2-cyanoethyl-N,N-diisopropylphosphoramidite (target compound (dDs amidite)); (2) dissolving the composition containing the 7-(2-thienyl)-3-[2'-deoxy-5'-O-(4,4'-dimethoxytrityl)-β-D-ribofuranosyl]-3H-imidazo[4,5-b]pyridine 2-cyanoethyl-N,N-diisopropylphosphoramidite in alcohol, followed by mixing with water to precipitate 7-(2-thienyl)-3-[2'-deoxy-5'-O-(4,4'-dimethoxytrityl)-β-D-ribofuranosyl]-3H-imidazo[4,5-b]pyridine 2-cyanoethyl-N,N-diisopropylphosphoramidite (dDs amidite); Including, The solvent in step (1) is at least one solvent selected from the group consisting of dichloromethane, chloroform, tetrahydrofuran, dioxane, diethyl ether, and diisopropyl ether. The manufacturing method is included.

[0084] The step (1) of obtaining a composition containing a dDs amidite can employ the content explained in the above [1] Method for producing a dDs amidite.

[0085] The step (2) of precipitating a dDs amidite can be the same as that described in the above [2] Method for producing a dDs amidite.

[0086] Although the embodiments of the present invention have been described above, the present invention is not limited to these examples and can be embodied in various forms without departing from the spirit and scope of the present invention. [Example]

[0087] Hereinafter, the embodiments of the present invention will be described more specifically based on examples.

[0088] The present invention is not limited to these.

[0089] [1] Preparation of dDs amidite 1 Reagents and solvents used in preparing dDs amidites were purchased from standard suppliers and used without further purification.

[0090] The starting compound, 7-(2-thienyl)-3-[2′-deoxy-5′-O-(4,4′-dimethoxytrityl)-β-D-ribofuranosyl]-3H-imidazo[4,5-b]pyridine, was synthesized according to the method described in the prior art (International Publication No. WO2007 / 066737).

[0091] The instruments used to measure the physical properties in each example are as follows:

[0092] Nuclear magnetic resonance spectrometer: BRUKER AVANCE NEO console, Ascend TM 400 High performance liquid chromatography: Shimadzu Corporation SPD-20MA, LC-20AD, SIL-20ACHT, DGU-20A3R, CTO-20AC, CBM-20A (1) Preparation of dDs amidite Example 1 The starting compound was reacted with 2-cyanoethyl tetraisopropylphosphorodiamidite (amidite-forming reagent) in a solvent in the presence of an amine salt of trifluoroacetic acid to carry out an amidite formation reaction, thereby producing the target compound, 7-(2-thienyl)-3-[2'-deoxy-5'-O-(4,4'-dimethoxytrityl)-β-D-ribofuranosyl]-3H-imidazo[4,5-b]pyridine 2-cyanoethyl-N,N-diisopropylphosphoramidite (dDs amidite).

[0093] Dichloromethane (DCM) was used as the solvent.

[0094] As the amine salt of trifluoroacetic acid, a pyridine salt of trifluoroacetic acid (TFA) (pyridine / TFA salt) was used.

[0095] The method for producing dDs amidite is described in detail below.

[0096] A 200 mL four-neck flask was charged with 10.0 g of the starting compound, dissolved in 50 mL of dichloromethane, stirred under an argon gas atmosphere, and cooled to 0°C (reaction solution). 7.3 g of an amiditizing reagent was dissolved in 17 mL of dichloromethane and added to the reaction solution. A solution of 1.99 g of pyridine, 2.4 g of TFA, and 33 mL of dichloromethane was prepared separately and gradually added to the reaction solution. Consumption of the starting compound was confirmed by high-performance liquid chromatography (HPLC). 200 mL of 5% aqueous sodium bicarbonate was added to the reaction solution and stirred, and the organic layer was separated by a separation operation.

[0097] The organic solvent was removed from the organic layer by distillation under reduced pressure to obtain 16.85 g of a viscous oily composition containing 13.2 g of the target dDs amidite.

[0098] The 1H NMR and 31P NMR of the product are shown in Figures 1 and 3, respectively.

[0099] According to the method for producing dDs amidite of the example, the raw material compound was allowed to react with the amidite-forming reagent (amidite-forming reaction) efficiently to produce the target compound, dDs amidite.

[0100] (2) Purification of dDs amidite Examples 2 to 25 The composition containing the dDs amidite obtained in Example 1 was first dissolved in various alcohols, and then water was added to precipitate (crystallize) the dDs amidite of the target compound, thereby purifying the dDs amidite.

[0101] The alcohols used were methanol, ethanol, propanol (1-PrOH), and isopropanol (2-PrOH).

[0102] Additionally, electrolytes include sodium chloride (NaCl), lithium chloride (LiCl), potassium chloride (KCl), calcium chloride (CaCl2), magnesium chloride (hexahydrate, MgCl2·6H2O), rubidium chloride (RbCl), cesium chloride (CsCl), strontium chloride (hexahydrate, SrCl2·6H2O), barium chloride (dihydrate, BaCl2·2H2O), sodium sulfate (Na2SO4), potassium bromide (K Br), sodium bromide (NaBr), sodium nitrate (NaNO3), sodium bicarbonate (NaHCO3), disodium hydrogen phosphate (dodecahydrate, Na2HPO4·12H2O), sodium dihydrogen phosphate (dihydrate, NaH2PO4·2H2O), sodium borate (Na2B4O7 + boric acid (H3BO3)), sodium acetate (NaOAc), ammonium formate (HCO2NH4), and triethylamine were used.

[0103] The method for purifying dDs amidite is described in detail below.

[0104] The composition containing the dDs amidite obtained in Example 1 was dissolved in various alcohols under an argon atmosphere and cooled on ice. Water containing various electrolytes was cooled on ice, and the composition containing the dDs amidite dissolved in alcohol was added over 0.5 hours and aged for 1 hour. The precipitated solid was filtered using a Kiriyama funnel, and the resulting solid was dried under reduced pressure overnight to obtain the dDs amidite as a solid.

[0105] The amounts of the composition containing the dDs amidite obtained in Example 1, the type and amount of alcohol, the type and amount of electrolyte, and the amount of water were as shown in Tables 1 to 3 below.

[0106] The filtration rate of solids was evaluated by attaching filter paper No. 5A for Kiriyama Funnel (R) with a diameter of 2.1 cm, and filtering under reduced pressure, and measuring the time until no more droplets came out.

[0107] The HPLC purity was evaluated by area percentage (area%) of the obtained solid measured at 254 nm by HPLC.

[0108] The results are shown in Tables 1 to 3.

[0109] After applying the purification method of the present invention, the impurity peaks confirmed in the H NMR and P NMR of the crude product (FIGS. 1 and 3) were removed, and the H NMR and P NMR of the obtained product (e.g., FIG. 2 and FIG. 4 of Example 2) were consistent with the spectral values ​​described in the prior art (International Publication No. WO2007 / 066737).

[0110] Comparative Example 1 Based on the method described in the prior art (International Publication No. WO2007 / 066737), dDs amidite was synthesized by the following procedure. 7-(2-thienyl)-3-[2'-deoxy-5'-O-(4,4'-dimethoxytrityl)-β-D-ribofuranosyl]-3H-imidazo[4,5-b]pyridine (raw material compound) was dissolved in anhydrous acetonitrile, and an acetonitrile solution of 2-cyanoethyl tetraisopropylphosphorodiamidite (amidite reagent) and 1H-tetrazole was added, followed by stirring at room temperature (25°C) for 1 hour. A portion of this reaction solution was taken and subjected to the stability evaluation test described below. Anhydrous methanol was added to the remaining reaction mixture, which was then poured into water and extracted with dichloromethane containing 1% (v / v) triethylamine. The organic layer was dried and concentrated to give the crude dDs amidite. The H NMR and P NMR spectra of the crude product are shown in Figures 5 and 7, respectively.

[0111] The resulting crude dDs amidite product was subjected to column purification (hexane:ethyl acetate=4:1→3:2).

[0112] The resulting product formed a foamy solid (purity 97.5%) after drying. The H NMR and P NMR spectra (Figures 6 and 8) of the product were consistent with those reported in the prior art (International Publication No. WO2007 / 066737).

[0113] [Table 1]

[0114] [Table 2]

[0115] [Table 3]

[0116] [2] Evaluation of the stability of dDs amidite The stability of 7-(2-thienyl)-3-[2'-deoxy-5'-O-(4,4'-dimethoxytrityl)-β-D-ribofuranosyl]-3H-imidazo[4,5-b]pyridine 2-cyanoethyl-N,N-diisopropylphosphoramidite (dDs amidite) in the reaction mixture was evaluated.

[0117] (i) The reaction solution collected in Comparative Example 1 was used as the reaction solution for the conventional method. The reaction solution was stirred at 25°C, the same reaction temperature, and the reaction completion time was set as 0 hours, followed by HPLC measurements at 5 hours and 24 hours. As a result, impurities were gradually formed, and the purity of the dDs amidite decreased (Figure 9).

[0118] (ii) The reaction solution used in the method of the present invention was the same as in Example 1, and the reaction solution at the time when the consumption of the raw material compound was confirmed was used. Evaluation was performed by stirring at 0°C, the same reaction temperature, with the completion of the reaction defined as time 0, and by HPLC measurement at 5 hours and 24 hours. As a result, the increase in impurities was suppressed, and the purity of the dDs amidite did not decrease (Figure 10).

[0119] [3] Preparation of dDs amidite 2 Example 26 (1) Amidite reaction In a solvent, in the presence of an amine salt of trifluoroacetic acid, 7-(2-thienyl)-3-[2'-deoxy-5'-O-(4,4'-dimethoxytrityl)-β-D-ribofuranosyl]-3H-imidazo[4,5-b]pyridine (starting compound) was reacted with 2-cyanoethyl tetraisopropylphosphorodiamidite (amiditizing reagent) to obtain a composition containing 7-(2-thienyl)-3-[2'-deoxy-5'-O-(4,4'-dimethoxytrityl)-β-D-ribofuranosyl]-3H-imidazo[4,5-b]pyridine 2-cyanoethyl-N,N-diisopropylphosphoramidite (target compound (dDs amidite)).

[0120] In the amiditization reaction, dichloromethane (DCM) was used as a solvent.

[0121] In the amidite reaction, a pyridine salt of trifluoroacetic acid (TFA) (pyridine / TFA salt) was used as the amine salt of trifluoroacetic acid.

[0122] A 10 L, four-neck flask was charged with 232 g of the starting compound, dissolved in 1,500 mL of dichloromethane, stirred under an argon gas atmosphere, and cooled to 0°C (reaction solution). 169 g of an amiditizing reagent was added to the reaction solution while being washed down with 54 mL of dichloromethane. A solution of 46 g of pyridine, 55 g of TFA, and 765 mL of dichloromethane was prepared separately and gradually added to the reaction solution. Consumption of the starting compound was confirmed by high-performance liquid chromatography.

[0123] 4,800 mL of 5% aqueous sodium bicarbonate was added to the reaction mixture and stirred, and the organic layer was separated by a separation operation.

[0124] The organic solvent was removed from the organic layer by distillation under reduced pressure to obtain 401 g of a viscous oily composition containing the target compound, dDs amidite. (2) Precipitation treatment The obtained composition containing the dDs amidite was first dissolved in alcohol, and then water was added to precipitate the dDs amidite, thereby precipitating the dDs amidite.

[0125] In the precipitation step, methanol was used as the alcohol.

[0126] In the deposition step, sodium chloride was used as the electrolyte, and an aqueous sodium chloride solution was used.

[0127] Under an argon gas atmosphere, 401 g of a composition containing dDs amidite obtained by the above-mentioned method for producing dDs amidite and 18.5 L of methanol were added to a 20 L four-neck flask, and the mixture was stirred at room temperature to dissolve, and then cooled to 0° C. Under an argon gas atmosphere, 230 g of sodium chloride was added to a 30 L four-neck flask, and 12 L of water was added, stirred to dissolve, and cooled to 0° C.

[0128] The methanol solution was added over 1 hour so that the internal temperature was kept below 10°C, and the mixture was aged for 5 hours.

[0129] The precipitated solid was filtered using a Buchner funnel and washed with 1.1 L of water.

[0130] The solid was dried under reduced pressure to obtain 303 g of dDs amidite as a solid.

[0131] [ka]

[0132] In the precipitation step, 300 g of dDs amidite was synthesized by applying crystallization using methanol / salt water.

[0133] The method for producing dDs amidites, including the purification of dDs amidites in the Examples, can be evaluated as being suitable for mass production of dDs amidites. [Industrial Applicability]

[0134] According to the method for producing 7-(2-thienyl)-3-[2'-deoxy-5'-O-(4,4'-dimethoxytrityl)-β-D-ribofuranosyl]-3H-imidazo[4,5-b]pyridine 2-cyanoethyl-N,N-diisopropylphosphoramidite (dDs amidite) of the present invention, 7-(2-thienyl)-3-[2'-deoxy-5'-O-(4,4'-dimethoxytrityl)-β-D-ribofuranosyl]-3H-imidazo[4,5-b]pyridine (starting compound) and 2-cyanoethyl By reacting with tetraisopropyl phosphorodiamidite (amidite-forming reagent), 7-(2-thienyl)-3-[2'-deoxy-5'-O-(4,4'-dimethoxytrityl)-β-D-ribofuranosyl]-3H-imidazo[4,5-b]pyridine 2-cyanoethyl-N,N-diisopropylphosphoramidite (the target compound (dDs amidite)) can be produced.

Claims

1. 1. A method for preparing 7-(2-thienyl)-3-[2′-deoxy-5′-O-(4,4′-dimethoxytrityl)-β-D-ribofuranosyl]-3H-imidazo[4,5-b]pyridine 2-cyanoethyl-N,N-diisopropylphosphoramidite, comprising: (1) reacting 7-(2-thienyl)-3-[2'-deoxy-5'-O-(4,4'-dimethoxytrityl)-β-D-ribofuranosyl]-3H-imidazo[4,5-b]pyridine with 2-cyanoethyl tetraisopropylphosphorodiamidite in a solvent in the presence of an amine salt of trifluoroacetic acid to obtain a composition containing 7-(2-thienyl)-3-[2'-deoxy-5'-O-(4,4'-dimethoxytrityl)-β-D-ribofuranosyl]-3H-imidazo[4,5-b]pyridine 2-cyanoethyl-N,N-diisopropylphosphoramidite; the solvent is at least one solvent selected from the group consisting of dichloromethane, chloroform, tetrahydrofuran, dioxane, diethyl ether, and diisopropyl ether; The amine salt of trifluoroacetic acid is at least one amine salt of trifluoroacetic acid selected from the group consisting of alkyl-substituted amine salts, aryl-substituted amine salts, aralkyl-substituted amine salts, polyamine salts, substituted or unsubstituted pyridine salts, saturated cyclic amine salts, imidazole salts, and polymer-bound amine salts. Manufacturing method.

2. The amine salt of trifluoroacetic acid is of trifluoroacetic acid an alkyl-substituted amine salt selected from the group consisting of trimethylammonium, triethylammonium, diethylmethylammonium, tripropylammonium, and diisopropylethylammonium; an aryl-substituted amine salt selected from the group consisting of dimethylanilinium, and triphenylammonium; aralkyl-substituted amine salts selected from the group consisting of tribenzylammonium; a polyamine salt selected from the group consisting of tetramethylethylenediamine; a substituted or unsubstituted pyridine salt selected from the group consisting of pyridinium, 2-methylpyridinium, 3-methylpyridinium, 4-methylpyridinium, 4-dimethylaminopyridinium, 2,6-dimethylpyridinium, 2,4,6-trimethylpyridinium, 2,3,4,6-tetramethylpyridinium, and pentamethylpyridinium; saturated cyclic amine salts selected from the group consisting of piperidinium, 2,2,6,6-tetramethylpiperidinium, and N-methylmorpholinium; Imidazole salts selected from the group consisting of N-methylimidazolium; and polymer-bound amine salts; 2. The method of claim 1, wherein the amine salt of the trifluoroacetic acid is at least one selected from the group consisting of:

3. After the step (1), (2) dissolving the composition containing the 7-(2-thienyl)-3-[2'-deoxy-5'-O-(4,4'-dimethoxytrityl)-β-D-ribofuranosyl]-3H-imidazo[4,5-b]pyridine 2-cyanoethyl-N,N-diisopropylphosphoramidite in alcohol, followed by mixing with water to precipitate 7-(2-thienyl)-3-[2'-deoxy-5'-O-(4,4'-dimethoxytrityl)-β-D-ribofuranosyl]-3H-imidazo[4,5-b]pyridine 2-cyanoethyl-N,N-diisopropylphosphoramidite.

4. 4. The method according to claim 3, wherein the alcohol is at least one alcohol selected from the group consisting of methanol, ethanol, propanol, and isopropanol.

5. 5. The method according to claim 3, wherein an electrolyte is further added in the deposition step.

6. the electrolyte is an inorganic salt selected from the group consisting of lithium chloride, sodium chloride, potassium chloride, cesium chloride, calcium chloride, rubidium chloride, barium chloride, magnesium chloride, strontium chloride, sodium sulfate, potassium sulfate, magnesium sulfate, sodium nitrate, potassium nitrate, sodium bicarbonate, sodium carbonate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium borate, sodium bromide and potassium bromide; an organic salt selected from the group consisting of sodium acetate, sodium formate, ammonium acetate, and ammonium formate; and an organic base selected from the group consisting of trimethylamine and triethylamine; 6. The method according to claim 5, wherein the electrolyte is at least one selected from the group consisting of:

7. The reaction temperature in the step (1) is -20°C to 30°C, The reaction temperature in the step (2) is −20° C. to 25° C. The method of claim 3.

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