Process for the manufacture of glycoside compounds

Abstract

Provided is a method for synthesizing a target compound (phosphoramidite) with high purity, the method being a method for producing a glycoside compound represented by General Formula (3), the method including the following step: Step A: a step of producing a glycoside compound represented by General Formula (3) by reacting a glycoside compound represented by General Formula (1) with an ether compound represented by Formula (2) in the presence of an oxidizing agent and an acid (in the following formulas, B a represents a cytosine group which can be substituted with an acyl group, or a uracil group, R 1 represents a C1 to C6 alkyl group, or a phenyl group, and n represents 0 or 1. Here, the oxidizing agent is selected from the group consisting of N-halosuccinimide and N-halohydantoin, and the acid is selected from the group consisting of perfluoroalkyl carboxylic acid and a salt thereof, alkyl sulfonic acid and a salt thereof, aryl sulfonic acid and a salt thereof, perfluoroalkyl sulfonic acid and a salt thereof, and a combination of two or more of these.

Description

Technical Field

[0001] This patent application claims priority and interest under the Paris Treaty based on Japanese Patent Application No. 2018-168135 (filed on September 7, 2018), the entire contents of which are incorporated herein by reference.

[0002] This invention relates to methods for manufacturing glycoside compounds and phosphoramide compounds. Background Technology

[0003] RNA can be used as RNA probes, antisense RNA, ribozymes, siRNA, aptamers, etc., and is a useful raw material.

[0004] RNA can be synthesized using solid-phase synthesis methods, in which phosphoramidite (hereinafter referred to as "amidite") of nucleosides can be used as a starting material. Protecting groups for the 2'-hydroxyl group of such phosphoramidite include, for example, TBDMS (tert-butyldimethylsilyl), TOM (triisopropylsilyloxymethyl), and ACE (bis(2-acetoxyethoxy)methyl). Furthermore, protecting groups disclosed in Patent Documents 1, 2, and 3 have been reported as protecting groups for the 2'-hydroxyl group of phosphoramidite; however, the methods for synthesizing phosphoramidite with these protecting groups are not necessarily satisfactory in terms of the yield and purity of the obtained phosphoramidite.

[0005] The presence of known positional isomers of phosphoramidites can adversely affect the synthesis of the final product in RNA synthesis (see He, Kaizhang and Hasan, Ahmad Classification and Characterization of Impurities in Phosphoramidites Used in Making Therapeutic Oligonucleotides: Risk Mitigation Strategies for Entering Clinical Phases). <URL:https: / / assets.thermofisher.com / TFS-Assets / BID / Technical-Notes / amidit e-impurity-classification-technote.pdf> ).

[0006] Existing technical documents

[0007] Patent documents

[0008] Patent Document 1: Japanese Patent No. 5157168

[0009] Patent Document 2: Japanese Patent No. 5554881

[0010] Patent Document 3: WO2007 / 097447 Summary of the Invention

[0011] The problem that the invention aims to solve

[0012] The purpose of this invention is to provide a method for manufacturing glycoside compounds and phosphorus amide compounds that can synthesize target compounds (phosphoramide compounds) with high purity.

[0013] Methods for solving problems

[0014] In the synthesis of phosphoramide, as described in Patent Documents 1, 2, and 3, the following steps are performed when manufacturing the glycoside compound represented by the general formula (3): adding an acid such as trifluoromethanesulfonic acid or silver trifluoromethanesulfonic acid to the reaction system, followed by adding an oxidizing agent such as N-iodosuccinimide or N-bromosuccinimide. Thus, mass spectrometry analysis confirms the formation of an impurity with the same molecular weight as the target compound.

[0015] To achieve the aforementioned objectives, the inventors of this application conducted repeated and in-depth research, resulting in the following understanding: In the synthesis reaction of phosphoramidite compounds, when manufacturing glycoside compounds represented by general formula (3), by adding an oxidant (N-iodosuccinimide) to the reaction system, followed by the addition of an acid (trifluoromethanesulfonic acid), the generation of impurities with the same molecular weight as the target compound can be suppressed. As a result, the target compound (phosphoramidite compound) can be synthesized with high purity.

[0016] This invention was developed based on the above understanding and further repeated research, and provides the following methods for manufacturing glycoside compounds and phosphoramide compounds. This invention includes, but is not limited to, the embodiments described in the following items.

[0017] Item 1. A method for manufacturing a glycoside compound represented by general formula (3), said method comprising the following steps:

[0018] Step A: A step in which a glycoside compound represented by general formula (1) is reacted with an ether compound represented by formula (2) in the presence of an oxidizing agent and an acid to produce a glycoside compound represented by general formula (3). In this step, an oxidizing agent is added to the reaction system, followed by the addition of an acid.

[0019] [Chemical Formula 1]

[0020]

[0021] (in the formula,

[0022] B a This indicates a cytosine group or a uracil group that can be substituted with an acyl group.

[0023] R 1 It indicates C1-C6 alkyl or phenyl, and,

[0024] n represents 0 or 1.

[0025] Here,

[0026] The oxidizing agent is selected from the group consisting of N-halosuccinimide and N-halohydantoin, and,

[0027] The acid is selected from the group consisting of perfluoroalkyl carboxylic acids and their salts, alkyl sulfonic acids and their salts, aryl sulfonic acids and their salts, perfluoroalkyl sulfonic acids and their salts, and combinations of two or more of them.

[0028] Item 2. The manufacturing method as described in Item 1, wherein n is 1.

[0029] Item 3. The manufacturing method as described in Item 1, wherein n is 0.

[0030] Item 4. The manufacturing method as described in any one of items 1 to 3, wherein the oxidant is N-halosuccinimide.

[0031] Item 5. The manufacturing method as described in any one of items 1 to 4, wherein the oxidizing agent is N-iodosuccinimide.

[0032] Item 6. The manufacturing method according to any one of items 1 to 5, wherein the acid is at least one selected from the group consisting of trifluoromethanesulfonic acid, silver trifluoromethanesulfonate, and methanesulfonic acid.

[0033] Item 7. The manufacturing method as described in any one of items 1 to 6, wherein the acid is trifluoromethanesulfonic acid.

[0034] Item 8. The manufacturing method as described in any one of items 1 to 7, wherein tetrahydrofuran is used as a solvent.

[0035] Item 9. The manufacturing method as described in any one of items 1 to 8, wherein R 1 It is a methyl group.

[0036] Item 10. The manufacturing method as described in any one of items 1 to 9, wherein B a It can be a cytosine group substituted with an acetyl group or an unsubstituted uracil group.

[0037] Item 11. A method for manufacturing a phosphorus amide compound represented by general formula (I), said method comprising the following steps:

[0038] Step A: A step of manufacturing a glycoside compound represented by general formula (3) by reacting a glycoside compound represented by general formula (1) with an ether compound represented by formula (2) using any one of items 1 to 10.

[0039] [Chemical Formula 2]

[0040]

[0041] (where B is in the formula) a And n is the same as above.

[0042] G 1 and G 2 The same or different indicates the protecting group of the hydroxyl group, and,

[0043] G 3 (Identical or different, indicating alkyl groups.)

[0044] [Chemical Formula 3]

[0045]

[0046] (where B is in the formula) a R 1 And n is the same as above.

[0047] Item 12. The method as described in Item 11, further comprising the following steps:

[0048] Step B: A step of deprotecting the hydroxyl groups at the 3' and 5' positions of the glycoside compound represented by general formula (3) obtained in step A, thereby producing a glycoside compound represented by general formula (4).

[0049] [Chemical Formula 4]

[0050]

[0051] (where B is in the formula) a And n is the same as above.

[0052] Item 13. The method of Item 12, wherein triethylamine trihydrofluoride or pyridine hydrofluoride is used as a deprotecting agent.

[0053] Item 14. The method as described in Item 12 or 13, further comprising the following steps:

[0054] Step C: Introduce a protecting group G to the 5' hydroxyl group of the glycoside compound represented by general formula (4) obtained in step B. 1 The process of producing a phosphoramidite compound represented by general formula (I) by phosphoramidating the hydroxyl group at the 3' position.

[0055] [Chemical Formula 5]

[0056]

[0057] (where B is in the formula) a , n, G 1 G 2 and G 3 Same as above.

[0058] Item 15. The method of Item 14, wherein 4,4'-dimethoxytriphenylmethyl chloride is used as a protecting group introduction agent for the 5'-hydroxyl group.

[0059] Item 16. The method as described in Item 14 or 15, wherein 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphatidium diamide is used as the phosphoramidation agent for the 3'-hydroxyl group.

[0060] The effects of the invention

[0061] By means of the manufacturing method of the present invention, the generation of impurities in intermediate compounds can be suppressed in the synthesis reaction of phosphorus amide compounds, resulting in the production of the target compound (phosphorus amide compound) with high purity. Detailed Implementation

[0062] The present invention will now be described in detail.

[0063] It should be noted that, in this specification, the term "comprise" includes both the meaning of "essentially consist of" and the meaning of "consist of".

[0064] The present invention is a method for manufacturing a phosphorus amide compound represented by general formula (I), characterized by comprising the following steps A, steps A and B, or steps A to C.

[0065] [Chemical Formula 6]

[0066]

[0067] (in the formula,

[0068] B a This indicates a cytosine group or a uracil group that can be substituted with an acyl group.

[0069] n represents 0 or 1,

[0070] G 1 and G 2 The same or different indicates the protecting group of the hydroxyl group, and,

[0071] G 3 (Identical or different, indicating alkyl groups.)

[0072] Step A: A step in which a glycoside compound represented by general formula (1) is reacted with an ether compound represented by formula (2) in the presence of an oxidizing agent and an acid to produce a glycoside compound represented by general formula (3). In this step, an oxidizing agent is added to the reaction system, followed by the addition of an acid.

[0073] [Chemical Formula 7]

[0074]

[0075] (where B is in the formula) a And n are the same as above, and R 1 This indicates a C1-C6 alkyl group or a phenyl group.

[0076] Step B: The step of deprotecting the hydroxyl groups at the 3' and 5' positions of the glycoside compound represented by general formula (3) obtained in step A, thereby producing the glycoside compound represented by general formula (4).

[0077] [Chemical Formula 8]

[0078]

[0079] (where B is in the formula) a And n is the same as above.

[0080] Step C: Introduce a protecting group G to the 5' hydroxyl group of the glycoside compound represented by general formula (4) obtained in step B. 1 The process of phosphoramidizing the 3'-hydroxyl group to produce a phosphoramid compound represented by general formula (I) is described.

[0081] [Chemical Formula 9]

[0082]

[0083] (where B is in the formula) a , n, G 1 G 2 and G 3 Same as above.

[0084] As R 1 Preferably, it is methyl.

[0085] B a The nucleic acid bases in it are cytosine or uracil as shown below.

[0086] [Chemical Formula 10]

[0087]

[0088] (where R is in the formula) 2 (This represents a hydrogen atom, acetyl group, isobutyryl group, or benzoyl group.)

[0089] As G 1 Any group that can function as a protecting group can be used without particular restrictions, and known protecting groups used in phosphorusamide compounds can be used extensively.

[0090] As G 1 Preferably, the following groups are used.

[0091] [Chemical Formula 11]

[0092]

[0093] (where R is in the formula) 3 R 4 and R 5 "Same" or "different" indicates hydrogen or alkoxy groups.

[0094] For R 3 R 4 and R 5 In particular, one of them is hydrogen and the remaining two are alkoxy groups, and methoxy groups are especially preferred as alkoxy groups.

[0095] As G 2 Any group that can function as a protecting group can be used without particular restrictions; known protecting groups used in phosphorous amide compounds can be widely used. As G 2 Examples of such groups include hydrocarbon groups other than hydrogen atoms, alkyl, alkenyl, alkynyl, cycloalkyl, alkyl groups mentioned above, halogenated alkyl, aryl, heteroaryl, arylalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkyl, hydroxyalkyl, aminoalkyl, alkoxyalkyl, heterocyclic alkenyl, heterocyclic alkyl, heteroarylalkyl, silylalkyl, silylalkyloxyalkyl, mono-, di- or trialkylsilylalkyl, mono-, di- or trialkylsilyloxyalkyl, etc., which can be substituted with electron-withdrawing groups.

[0096] As G 2 Preferably, the alkyl group is substituted with an electron-withdrawing group. Examples of such electron-withdrawing groups include cyano, nitro, alkylsulfonyl, halogen, arylsulfonyl, trihalomethyl, and trialkylamino, with cyano being the most preferred.

[0097] As G 2 The following groups are particularly preferred.

[0098] [Chemical Formula 12]

[0099]

[0100] G 3Same or different, alkyl, 2 G 3 They can bond together to form a ring structure. As G 3 The preferred form is isopropyl.

[0101] The alkyl group can be either straight-chain or branched, preferably an alkyl group with 1 to 12 carbon atoms, and more preferably an alkyl group with 1 to 6 carbon atoms. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, and hexyl. The alkyl group here also includes the alkyl moiety of alkoxy groups, etc.

[0102] The acyl group represents a linear or branched aliphatic acyl group or an aromatic acyl group, with a total number of carbon atoms, including the carbonyl group, of 2 to 12, preferably 2 to 7. Examples of acyl groups include aliphatic acyl groups (e.g., acetyl, propionyl, butyryl (butyryl), isobutyryl (isobutyryl), pentyl, hexanoyl, heptanoyl, octanoyl, nonanoyl, decanoyl, undecanoyl, etc.) and aromatic acyl groups (e.g., benzoyl, 1-naphthoyl and 2-naphthoyl), preferably acetyl, isobutyryl or benzoyl.

[0103] Furthermore, the phosphorus amide compound of the present invention includes both a free state and a salt state. There are no particular limitations on the salt of the phosphorus amide compound of the present invention; it can be a base addition salt, such as salts of sodium, magnesium, potassium, calcium, and aluminum salts with inorganic bases; salts of methylamine, ethylamine, and ethanolamine salts with organic bases; salts of lysine, ornithine, and arginine salts with basic amino acids; and base addition salts such as ammonium salts. Additionally, the salt can be an acid addition salt. Specifically, examples of such salts include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, and phosphoric acid; organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, malic acid, tartaric acid, fumaric acid, succinic acid, lactic acid, maleic acid, citric acid, methanesulfonic acid, trifluoromethanesulfonic acid, and ethanesulfonic acid; and acidic amino acids such as aspartic acid and glutamic acid.

[0104] Process A:

[0105] In this reaction, a glycoside compound represented by general formula (1) is reacted with an ether compound represented by formula (2) in the presence of an oxidant and an acid (coupling reaction) to obtain a glycoside compound represented by general formula (3). In particular, it is characterized by adding an oxidant to the reaction system and then adding an acid.

[0106] Examples of oxidizing agents include N-halosuccinimides such as N-chlorosuccinimide, N-bromosuccinimide, and N-iodosuccinimide, and N-halohydantoins such as 1,3-diiodo-5,5-dimethylhydantoin. In this invention, N-halosuccinimides are preferred, and N-iodosuccinimide is even more preferred.

[0107] Examples of acids include perfluoroalkyl carboxylic acids and their salts, alkyl sulfonic acids and their salts, aryl sulfonic acids and their salts, perfluoroalkyl sulfonic acids and their salts, and combinations of two or more thereof. Examples of salts include metal salts (e.g., copper salts and silver salts). Specifically, examples of acids include methanesulfonic acid, p-toluenesulfonic acid, camphorsulfonic acid, trifluoromethanesulfonic acid, and silver trifluoromethanesulfonate, and combinations of two or more thereof. In this invention, trifluoromethanesulfonic acid is preferably used.

[0108] There are no particular limitations on the solvents used in this reaction. Examples include diethyl ether, tetrahydrofuran (THF), cyclopentylmethyl ether, 2-methyltetrahydrofuran, dimethoxyethane, dioxane, and other ethers; hydrocarbons such as toluene; nitriles such as acetonitrile; and halogenated hydrocarbons such as chlorobenzene and dichloromethane.

[0109] Regarding the amount of the ether compound represented by formula (2), it is generally 1 to 5 moles, preferably 1 to 3 moles, and more preferably 1 to 1.5 moles, relative to 1 mole of the glycoside compound represented by general formula (1). Regarding the amount of the oxidizing agent, it is generally 1 to 3 moles, preferably 1 to 2 moles, and more preferably 1 to 1.5 moles, relative to 1 mole of the glycoside compound represented by general formula (1). Regarding the amount of the acid, it is generally 0.1 to 5 moles, preferably 0.5 to 3 moles, and more preferably 0.5 to 1.5 moles, relative to 1 mole of the ether compound represented by general formula (1).

[0110] The reaction temperature is typically -80 to 0°C, preferably -60 to -10°C, and more preferably -50 to -40°C. The reaction time is typically 1 to 12 hours, preferably 1 to 8 hours, and more preferably 1 to 4 hours.

[0111] The glycosides represented by formula (1) can be manufactured using known methods or are available commercially available. The ether compounds represented by formula (2) can be manufactured using known methods (see the aforementioned patent documents 1, 2 and 3).

[0112] The reaction in step A can typically be carried out by adding an oxidant to a mixture of a glycoside compound represented by general formula (1) and an ether compound represented by formula (2), and then adding an acid to the resulting mixture. For example, it can be carried out by sequentially adding a glycoside compound represented by general formula (1), an ether compound of formula (2), N-iodosuccinimide, and trifluoromethanesulfonic acid.

[0113] Process B:

[0114] In this reaction, by deprotecting the hydroxyl groups at the 3' and 5' positions of the glycoside compound represented by general formula (3) obtained in step A, the glycoside compound represented by general formula (4) can be obtained.

[0115] For deprotection reactions, the protecting agent used can be appropriately changed depending on the protecting group. For example, it can be carried out by using a known deprotecting agent. There are no particular limitations on the deprotecting agent, and examples include pyridine hydrofluoride, triethylamine trihydrofluoride, pyridine hydrofluoride, ammonium fluoride, hydrofluoric acid, tetrabutylammonium fluoride, etc.

[0116] Regarding the amount of deprotectant, it is generally 0.1 to 20 moles, preferably 0.2 to 10 moles, and more preferably 1 to 5 moles, relative to 1 mole of the glycoside compound represented by general formula (3).

[0117] There are no particular limitations on the solvents used in this reaction; examples include ketones such as acetone, diethyl ether, ethers such as tetrahydrofuran (THF), hydrocarbons such as toluene, alcohols such as methanol and ethanol, and nitriles such as acetonitrile.

[0118] The reaction temperature is typically 0–100°C, preferably 10–60°C, and more preferably 10–30°C. The reaction time is typically 30 minutes to 72 hours, preferably 2–24 hours.

[0119] Process C:

[0120] In this reaction, a protecting group G is introduced at the 5' position of the glycoside compound represented by general formula (4) obtained in step B. 1 The hydroxyl group at the 3' position is phosphorylated to obtain a phosphorylated compound represented by general formula (I).

[0121] In this reaction, the protecting group G 1 The introduction and phosphorylation can be performed simultaneously (in one step), or after the protecting group G is introduced. 1 Then, phosphorous amidation is performed, or a protecting group G can be introduced after phosphorous amidation. 1 Among them, it is preferable to introduce the protecting group G. 1 Then, phosphorylation is performed.

[0122] Protective base G 1 In the introduction reaction, the protecting group introducer can be based on G 1Suitable choices include, for example, 4,4'-dimethoxytriphenylmethyl chloride. There are no particular limitations on the solvent; examples include aromatic solvents such as toluene and pyridine, nitriles such as acetonitrile, and ethers such as tetrahydrofuran. The reaction temperature is typically 0–100°C, preferably 10–60°C, and more preferably 20–30°C. The reaction time is typically 30 minutes to 24 hours, preferably 1–8 hours. Regarding the amount of the protecting group introducer, relative to 1 mole of the glycoside compound represented by general formula (4), it is typically 1–100 moles, preferably 1–20 moles, and more preferably 1–5 moles.

[0123] In the phosphorous amidation reaction, the phosphorous amidation reagent can be determined according to G. 2 and G 3 Appropriate choices include, for example, 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphite diamide. There are no particular limitations on the solvent; examples include nitrile solvents such as acetonitrile, ethers such as tetrahydrofuran, and halogenated solvents such as dichloromethane. The reaction temperature is typically 0–40°C, preferably 20–40°C. The reaction time is typically 30 minutes to 24 hours, preferably 1–6 hours. Regarding the phosphorylation reagent, it is typically 1–20 moles, preferably 1–5 moles, and more preferably 1–1.5 moles, relative to 1 mole of the glycoside compound represented by general formula (4).

[0124] The reaction products obtained through any of the steps A to C can be purified, washed, concentrated, etc., by known methods.

[0125] In the process of manufacturing the glycoside compound represented by general formula (3) using the manufacturing method of the present invention, the generation of impurities of the same molecular weight can be suppressed. As a result, the phosphoramidide compound represented by general formula (I) as the target compound can be manufactured with high purity.

[0126] Example

[0127] Hereinafter, embodiments are given to further illustrate the present invention in detail. However, the present invention is not limited to these embodiments, etc.

[0128] Example 1

[0129] [Chemical Formula 13]

[0130]

[0131] N 4Acetyl-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)cytidine (TIPS-C) (10.0 g, 19.0 mmol) and toluene (50 ml) were added to a flask, and the solution was concentrated to 30 ml. After adding tetrahydrofuran (22 ml), the reaction solution was cooled to -50 °C, and 2-cyanoethoxymethyl methyl thiomethyl ether (hereinafter also referred to as EMM agent) (4.58 g, 28.4 mmol), N-iodosuccinimide (5.76 g, 25.6 mmol) in tetrahydrofuran (13 ml) solution, and trifluoromethanesulfonic acid (4.27 g, 28.5 mmol) were added dropwise. After stirring at -50°C for 2 hours, the reaction mixture was added to an ice-cooled aqueous solution consisting of sodium bicarbonate (3.5 g), sodium thiosulfate pentahydrate (10.0 g), and water (65 ml). The mixture was separated at room temperature. The organic layer was further washed with a solution consisting of sodium bicarbonate (1.75 g), sodium thiosulfate pentahydrate (5.0 g), and water (32.5 ml). The organic layer was concentrated to obtain a crude product containing the target compound.

[0132] LC-MS analysis was performed using the target analyte in negative mode at an m / z of 639.3. The results showed that the target analyte peak was 99.1%, while the peak of the critical impurity with the same m / z was 0.9%.

[0133] Example 2

[0134] [Chemical Formula 14]

[0135]

[0136] Add 3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)uridine (TIPS-U) (5.0 g, 10.0 mmol) and toluene (25 ml) to a flask and concentrate to 15 ml. After adding tetrahydrofuran (3.5 ml), cool the solution to -50°C, and add dropwise an EMM agent (2.48 g, 15.4 mmol), a tetrahydrofuran (6.5 ml) solution of N-iodosuccinimide (3.18 g, 14.1 mmol), and trifluoromethanesulfonic acid (2.31 g, 15.4 mmol). After stirring at -50°C for 2 hours, add the reaction solution to an ice-cooled aqueous solution of sodium bicarbonate (1.75 g), sodium thiosulfate pentahydrate (5.0 g), and water (32.5 ml). Separate the mixture at room temperature. The organic layer was further washed with a solution consisting of sodium bicarbonate (0.85 g), sodium thiosulfate pentahydrate (2.5 g), and water (16 ml). The organic layer was concentrated to obtain a crude product containing the target compound.

[0137] [Chemical Formula 15]

[0138]

[0139] The aforementioned crude EMM U-1 was dissolved in tetrahydrofuran (15 ml) and acetone (15 ml), and hydrogen trifluoride / triethylamine (1.82 g, 11.3 mmol) was added. The mixture was stirred at 20 °C for 17 hours. The reaction solution was then injected into tert-butyl methyl ether (50 ml), and the mixture was stirred for 1 hour. The reaction solution was filtered, and the resulting solid was washed with tert-butyl methyl ether (10 ml). The solid was dried under reduced pressure to give the target compound (3.01 g, yield 84%).

[0140] LC-MS analysis was performed, and the target compound (chloride ion adduct from the device, glassware, etc.) was scanned in negative mode at an m / z of 392.1. The results showed that the target compound peak was 99.6%, while the peak of the main impurity with the same m / z as the target compound was 0.4%.

[0141] Example 3

[0142] [Chemical Formula 16]

[0143]

[0144] The aforementioned crude EMM C-1 was dissolved in tetrahydrofuran (40 ml), and hydrogen trifluoride / triethylamine (3.36 g, 20.8 mmol) was added. The mixture was stirred at 20 °C for 16 hours. The reaction solution was cooled to 0 °C, and tert-butyl methyl ether (60 ml) was added dropwise. The resulting solid was recovered by filtration and dried to obtain the target compound (7.95 g).

[0145] Example 4

[0146] [Chemical Formula 17]

[0147]

[0148] The aforementioned crude EMM C-2 (7.0 g, 17.6 mmol) was dissolved in pyridine (35 ml), acetonitrile (14 ml), and toluene (35 ml), and cooled to 0 °C. 4,4'-dimethoxytriphenylmethyl chloride (7.14 g, 21.1 mmol) was added, and the mixture was stirred at 20 °C for 4 hours. Methanol (3.5 ml) was added, and the mixture was stirred for 5 minutes. The reaction mixture was poured into a solution of sodium bicarbonate (1.75 g) and water (35 ml) and washed with toluene (7 ml). The mixture was separated at room temperature. Next, the organic layer was washed with a solution of sodium bicarbonate (1.75 g) and water (35 ml). The organic layer was further washed with a solution of sodium chloride (3.5 g) and water (35 ml), and the organic layer was concentrated to 21 ml. This process of adding toluene (28 ml) and concentrating to 21 ml was repeated three times to obtain a crude product containing the target compound. Purification was performed using silica gel column chromatography (1:1 solution of heptane / ethyl acetate-acetone = 40 / 60 to 10 / 90) to give the target compound (6.42 g, yield 61% based on TIPS-C).

[0149] Example 5

[0150] [Chemical Formula 18]

[0151]

[0152] Acetonitrile (18 ml) was added to the aforementioned EMM C-3 (6.0 g, 8.6 mmol), and diisopropylamine tetrazolide (1.68 g, 9.8 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphite diamide (3.09 g, 10.3 mmol) were added at 25 °C. The mixture was stirred at 35 °C for 2 hours. The reaction mixture was then injected into a solution of toluene (60 ml), water (30 ml), and sodium bicarbonate (1.5 g), and the mixture was separated at room temperature. The organic layer was washed four times with a solution of DMF (30 ml) and water (30 ml), twice with water (30 ml), and once with a solution of sodium chloride (3.0 g) and water (30 ml). Sodium sulfate (3.0 g) was added to the organic layer, and the mixture was filtered and concentrated to 18 ml to obtain a crude product containing the target compound. Purification was performed using silica gel column chromatography (heptane / acetone = 60 / 40 to 30 / 70) to give the target compound (6.45 g, 84% yield based on EMM C-3).

[0153] Example 6

[0154] [Chemical Formula 19]

[0155]

[0156] N 4 Acetyl-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)cytidine (TIPS-C) (1.0 g, 1.9 mmol) was dissolved in tetrahydrofuran (10 ml). Methylthiomethyl 2-cyanoethyl ether (hereinafter referred to as CEM agent) (0.37 g, 2.8 mmol) and molecular sieve 4A (0.8 g) were added. The mixture was stirred at -45°C for 30 minutes under a nitrogen atmosphere. N-iodosuccinimide (0.64 g, 2.8 mmol) was added, followed by the dropwise addition of trifluoromethanesulfonic acid (0.42 g, 2.8 mmol). The mixture was stirred at -45°C for 30 minutes. Triethylamine (1.6 ml) was added to the reaction mixture. After filtration, ethyl acetate was added, and the organic layer was washed twice with a mixed solution of water (10 ml), sodium thiosulfate pentahydrate (1.0 g), and sodium bicarbonate (0.35 g). The solvent was removed by distillation to obtain a crude product containing the target compound.

[0157] LC-MS analysis was performed, and the target analyte was scanned in negative mode at an m / z of 609.3. The results showed that the target analyte peak was 99.93%, while the peak of the main impurity, which has the same m / z as the target analyte, was 0.07%.

[0158] [Chemical Formula 20]

[0159]

[0160] The aforementioned crude CMM C-1 was dissolved in tetrahydrofuran (6 ml), and hydrogen trifluoride / triethylamine (0.37 g, 2.2 mmol) was added at 25 °C. The mixture was stirred at 45 °C for 2 hours. The precipitate was recovered by filtration, washed with tetrahydrofuran, and dried to obtain the target compound (0.68 g, 97% yield).

[0161] Example 7

[0162] [Chemical Formula 21]

[0163]

[0164] N4-acetyl-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)cytidine (TIPS-C) (3.0 g, 5.7 mmol) was azeotropically three times in toluene (15 ml), and the solvent was removed by vacuum distillation. The residue was dissolved in tetrahydrofuran (30 ml) under a nitrogen atmosphere, and the solution was cooled to -45°C. An EMM activator (2.8 g, 18 mmol), N-iodosuccinimide (2.0 g, 9.0 mmol), and trifluoromethanesulfonic acid (1.3 g, 8.8 mmol) were added dropwise to this solution. After stirring at -45°C for 5 hours, the reaction mixture was quenched by adding triethylamine. The reaction mixture was added to an ice-cooled solution of ethyl acetate (30 ml), sodium bicarbonate (1.5 g), sodium thiosulfate pentahydrate (3.0 g), and water (30 ml), and the mixture was separated at room temperature. The organic layer was concentrated to obtain a crude product containing the target compound.

[0165] LC-MS analysis was performed, and the target analyte was scanned in negative mode at an m / z of 639.3. The results showed that the target analyte peak was 99.87%, while the peak of the main impurity, which has the same m / z as the target analyte, was 0.13%.

[0166] Example 8

[0167] [Chemical Formula 22]

[0168]

[0169] Under a nitrogen atmosphere, 3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)uridine (TIPS-U) (3.0 g, 6.0 mmol) was dissolved in tetrahydrofuran (30 ml), and the solution was cooled to -45°C. An EMM quencher (1.56 g, 9.7 mmol), N-iodosuccinimide (2.16 g, 9.7 mmol), and trifluoromethanesulfonic acid (1.44 g, 9.7 mmol) were added dropwise to the solution. After stirring at -45°C for 5 hours, triethylamine was added to quench the reaction mixture. The reaction solution was then added to an ice-cooled aqueous solution consisting of sodium bicarbonate (1.5 g), sodium thiosulfate pentahydrate (3.0 g), water (30 ml), and ethyl acetate (15 ml). The mixture was separated at room temperature. The organic layer was further washed with a solution consisting of sodium bicarbonate (1.5 g), sodium thiosulfate pentahydrate (3.0 g), and water (30 ml). The organic layer was concentrated to obtain a crude product containing the target compound. The crude product was purified by silica gel column chromatography (ethyl acetate:heptane = 1:1) to give the target compound (3.0 g, yield 83%).

[0170] [Chemical Formula 23]

[0171]

[0172] The aforementioned EMM U-1 (2.0 g, 3.3 mmol) was dissolved in tetrahydrofuran (8 ml), and pyridine hydrogen fluoride (65.5%, 0.81 g) was added. The mixture was stirred at 25 °C for 17 hours. The precipitate was filtered to obtain the target compound (1.10 g, 92% yield).

[0173] LC-MS analysis was performed, and the target analyte was scanned in negative mode at m / z 356.1. The results showed that no peaks of substances with the same m / z as the target analyte, which are major impurities, were detected.

[0174] Comparative Example 1

[0175] [Chemical Formula 24]

[0176]

[0177] N4-acetyl-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)cytidine (TIPS-C) (10.0 g, 19.0 mmol) and toluene (50 ml) were added to a flask, and the solution was concentrated to 30 ml. Tetrahydrofuran (22 ml) was added, and the reaction solution was cooled to -50 °C. Then, a tetrahydrofuran (13 ml) solution of EMM activator (4.58 g, 28.4 mmol), trifluoromethanesulfonic acid (4.27 g, 28.5 mmol), and N-iodosuccinimide (5.76 g, 25.6 mmol) was added dropwise. After stirring at -50 °C for 1 hour, the reaction solution was added to an ice-cooled aqueous solution of sodium bicarbonate (3.5 g), sodium thiosulfate pentahydrate (10.0 g), and water (65 ml). The mixture was separated at room temperature. The organic layer was further washed with a solution consisting of sodium bicarbonate (1.75 g), sodium thiosulfate pentahydrate (5.0 g), and water (32.5 ml). The organic layer was concentrated to obtain a crude product containing the target compound.

[0178] LC-MS analysis was performed, and the target analyte was scanned in negative mode at an m / z of 639.3. The results showed that the target analyte peak was 96.4%, while the peak of the main impurity, which has the same m / z as the target analyte, was 3.6%.

[0179] Comparative Example 2

[0180] [Chemical Formula 25]

[0181]

[0182] Add 3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)uridine (TIPS-U) (5.0 g, 10.0 mmol) and toluene (25 ml) to a flask, and concentrate the solution to 15 ml. Add tetrahydrofuran (3.5 ml) to the solution, and cool the solution to -50°C. Then, add an EMM activator (2.48 g, 15.4 mmol), trifluoromethanesulfonic acid (2.31 g, 15.4 mmol), and N-iodosuccinimide (3.18 g, 14.1 mmol) in tetrahydrofuran (6.5 ml) dropwise. After stirring at -50°C for 2 hours, add the reaction solution to an ice-cooled aqueous solution of sodium bicarbonate (1.75 g), sodium thiosulfate pentahydrate (5.0 g), and water (32.5 ml). Separate the mixture at room temperature. The organic layer was further washed with a solution consisting of sodium bicarbonate (0.85 g), sodium thiosulfate pentahydrate (2.5 g), and water (16 ml). The organic layer was concentrated to obtain a crude product containing the target compound.

[0183] [Chemical Formula 26]

[0184]

[0185] The aforementioned crude EMM U-1 was dissolved in tetrahydrofuran (15 ml) and acetone (15 ml), and hydrogen trifluoride / triethylamine (1.82 g, 11.3 mmol) was added. The reaction mixture was stirred at 20 °C for 17 hours. The reaction mixture was then injected into tert-butyl methyl ether (50 ml), and the mixture was stirred for 1 hour. The reaction mixture was filtered, and the resulting solid was washed with tert-butyl methyl ether (10 ml). The solid was dried under reduced pressure to give the target compound (3.05 g, yield 85%).

[0186] LC-MS analysis was performed, and the target compound was scanned in negative mode at an m / z of 392.1 (chloride ion adducts from devices, glassware, etc.). The results showed that the peak of the target compound was 92.6%, while the peak of the main impurity with the same m / z as the target compound was 7.4%.

[0187] Comparative Example 3

[0188] [Chemical Formula 27]

[0189]

[0190] N4-acetyl-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)cytidine (TIPS-C) (1.0 g, 1.9 mmol) was dissolved in tetrahydrofuran (10 ml). CEM agent (0.37 g, 2.8 mmol) and molecular sieve 4A (0.8 g) were added. The mixture was stirred at -45°C for 30 minutes under a nitrogen atmosphere. Trifluoromethanesulfonic acid (0.42 g, 2.8 mmol) was added dropwise, followed by N-iodosuccinimide (0.64 g, 2.8 mmol). The mixture was stirred at -45°C for 30 minutes. Triethylamine (1.6 ml) was added to the reaction mixture. After filtration, ethyl acetate was added to the filtrate. The organic layer was washed twice with a mixed solution of water (10 ml), sodium thiosulfate pentahydrate (1.0 g), and sodium bicarbonate (0.35 g). The solvent was removed by distillation to obtain a crude product containing the target compound.

[0191] LC-MS analysis was performed, and the target analyte was scanned in negative mode at an m / z of 609.3. The results showed that the peak of the target analyte was 97.3%, while the peak of the main impurity, which has the same m / z as the target analyte, was 2.7%.

[0192] [Chemical Formula 28]

[0193]

[0194] The aforementioned crude CMM C-1 was dissolved in tetrahydrofuran (6 ml), and hydrogen trifluoride / triethylamine (0.37 g, 2.2 mmol) was added at 25 °C. The mixture was stirred at 45 °C for 2 hours. The precipitate was recovered by filtration, washed with tetrahydrofuran, and dried to obtain the target compound (0.63 g, 90% yield).

[0195] Comparative Example 4

[0196] [Chemical Formula 29]

[0197]

[0198] N4-acetyl-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)cytidine (TIPS-C) (3.0 g, 5.7 mmol) was azeotropically three times in toluene (15 ml), and the solvent was removed by distillation under vacuum. The solution was dissolved in tetrahydrofuran (30 ml) under a nitrogen atmosphere, and the solution was cooled to -45°C. An EMM activator (2.8 g, 18 mmol), trifluoromethanesulfonic acid (1.3 g, 8.8 mmol), and N-iodosuccinimide (2.0 g, 9.0 mmol) were added dropwise to this solution. After stirring at -45°C for 5 hours, triethylamine was added to quench the reaction. The reaction solution was added to an ice-cooled solution of ethyl acetate (30 ml), sodium bicarbonate (1.5 g), sodium thiosulfate pentahydrate (3.0 g), and water (30 ml), and the mixture was separated at room temperature. The organic layer was concentrated to obtain a crude product containing the target compound.

[0199] LC-MS analysis was performed, and the target analyte was scanned in negative mode at an m / z of 639.3. The results showed that the target analyte peak was 97.7%, while the peak of the main impurity, which has the same m / z as the target analyte, was 2.3%.

[0200] Comparative Example 5

[0201] [Chemical Formula 30]

[0202]

[0203] Under a nitrogen atmosphere, 3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)uridine (TIPS-U) (3.0 g, 6.0 mmol) was dissolved in tetrahydrofuran (30 ml), and the solution was cooled to -45°C. An EMM quencher (1.56 g, 9.7 mmol), trifluoromethanesulfonic acid (1.44 g, 9.7 mmol), and N-iodosuccinimide (2.16 g, 9.7 mmol) were added dropwise to the solution. After stirring at -45°C for 5 hours, triethylamine was added to quench the reaction. The reaction solution was added to an ice-cooled aqueous solution of sodium bicarbonate (1.5 g), sodium thiosulfate pentahydrate (3.0 g), water (30 ml), and ethyl acetate (15 ml), and the mixture was separated at room temperature. The organic layer was further washed with a solution consisting of sodium bicarbonate (1.5 g), sodium thiosulfate pentahydrate (3.0 g), and water (30 ml). The organic layer was concentrated to obtain a crude product containing the target compound. The crude product was purified by silica gel column chromatography (ethyl acetate:heptane = 1:1) to give the target compound (2.82 g, yield 78%).

[0204] [Chemical Formula 31]

[0205]

[0206] The aforementioned EMM U-1 (2.0 g, 3.3 mmol) was dissolved in THF (8 ml), and pyridine hydrogen fluoride (65.5%, 0.81 g) was added. The mixture was stirred at 25 °C for 17 hours. The precipitate was filtered off to obtain the target compound (1.08 g, 90% yield).

[0207] LC-MS analysis was performed, and the target analyte was scanned in negative mode at an m / z of 356.1. The results showed that the target analyte peak was 98.9%, while the peak of the main impurity, which has the same m / z as the target analyte, was 1.1%.

[0208] [Table 1]

[0209]

[0210] Reagent addition order 1:

[0211] The order of EMM or CEM oxidizing agent, N-iodosuccinimide, trifluoromethanesulfonic acid

[0212] Reagent addition order 2:

[0213] The order of EMM or CEM oxidizing agent, trifluoromethanesulfonic acid, and N-iodosuccinimide is shown in Table 1 above. Trifluoromethanesulfonic acid is added after N-iodosuccinimide, thereby inhibiting the formation of impurities and obtaining the target product with higher purity.

Claims

1. A method for manufacturing a glycoside compound represented by general formula (3), said method comprising the following steps: Step A: A step of producing a glycoside compound of general formula (3) by reacting a glycoside compound of general formula (1) with an ether compound of formula (2) in a solvent in the presence of an oxidizing agent and an acid, wherein an oxidizing agent is added to a mixture of the glycoside compound of general formula (1) and the ether compound of formula (2), and then an acid is added. In the formula, B a This indicates a cytosine group substituted with an acetyl group, or an unsubstituted uracil group. R 1 It represents C1 to C6 alkyl groups, and, n represents 0 or 1, Here, The solvent is a solvent containing tetrahydrofuran. The oxidizing agent is N-iodosuccinimide, and, The acid is selected from at least one of the groups consisting of trifluoromethanesulfonic acid and methanesulfonic acid.

2. The manufacturing method as described in claim 1, wherein, n is 1.

3. The manufacturing method as described in claim 1, wherein, n is 0.

4. The manufacturing method according to any one of claims 1 to 3, wherein, The acid is trifluoromethanesulfonic acid.

5. The manufacturing method according to any one of claims 1 to 3, wherein, The solvent is tetrahydrofuran.

6. The manufacturing method according to any one of claims 1 to 3, wherein, R 1 It is a methyl group.

7. A method for manufacturing a phosphorus amide compound represented by general formula (I), said method comprising the following steps: Step A: A step of manufacturing a glycoside compound represented by general formula (3) by reacting a glycoside compound represented by general formula (1) with an ether compound represented by formula (2) using the manufacturing method according to any one of claims 1 to 6. In the formula, B a And n is defined in the same way as in general formulas (1), (2) or (3). G 1 and G 2 The same or different indicates the protecting group of the hydroxyl group, and, G 3 Same or different, indicating alkyl groups, The method further includes the following steps: Step B: The step of deprotecting the hydroxyl groups at the 3' and 5' positions of the glycoside compound represented by general formula (3) obtained in step A, thereby producing the glycoside compound represented by general formula (4). ， In the formula, B a And n is the same as the definition in general formula (I), Step C: Introduce a protecting group G to the 5' hydroxyl group of the glycoside compound represented by general formula (4) obtained in step B. 1 The process of phosphoramidizing the 3'-position hydroxyl group to produce a phosphoramid compound represented by general formula (I) is described. In the formula, B a n, G 1 G 2 and G 3 Same as the definition in general formula (I).

8. The method of claim 7, wherein, Triethylamine trihydrofluoride or pyridine hydrofluoride was used as a deprotecting agent.

9. The method of claim 7 or 8, wherein, 4,4'-dimethoxytriphenylmethyl chloride was used as a protecting group introduction reagent for the 5'-hydroxyl group.

10. The method of claim 7 or 8, wherein, 2-Cyanoethyl-N,N,N',N'-Tetraisopropylphosphite was used as the phosphite amidation agent for the 3'-hydroxyl group.

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