Glycoside compound, amide compound and method for producing polynucleotide using the same

Abstract

The present invention provides an amide compound capable of improving the yield and purity of oligonucleotides, a glycoside compound as an intermediate thereof, and a method for producing a polynucleotide using the amide compound. The present invention also provides an amide compound of formula (1) and a glycoside compound of formula (10) capable of improving the yield and purity of oligonucleotides (in formulas (10) and (1), B a , R a , R b , R c , G 1 , G 2 and G 3 The same as defined in the specification, and R is represented by the following formula), and a method for producing a polynucleotide using the amide compound.

Description

Technical Field

[0001] This patent application claims patent priority and benefits under the Paris Convention based on Japanese Patent Application No. 2019-192899 (filed on October 8, 2019), and all the contents described in the above application are incorporated into this specification by reference.

[0002] The present invention relates to a glycoside compound, an amide compound thereof, and a method for producing a polynucleotide using the same. In addition, the present invention also relates to an intermediate compound of the above-mentioned glycoside compound and a method for producing the intermediate compound. Background Art

[0003] RNA is a useful material that can be used as an RNA probe, antisense RNA, ribozyme, siRNA, aptamer, and the like.

[0004] RNA can be synthesized by solid phase synthesis method, etc., in which phosphoramidite of nucleoside (hereinafter referred to as "amidite") is used as a raw material. As protecting groups for the hydroxyl group at the 2' position of such amidites, for example, TBDMS (tert-butyldimethylsilyl), TOM (triisopropylsilyloxymethyl), ACE (bis(2-acetoxyethoxy)methyl), etc. are known. In addition, as protecting groups for the hydroxyl group at the 2' position of amidites, protecting groups disclosed in Patent Documents 1 to 4 are reported, but the RNA synthesis method using amidites having these protecting groups is not necessarily satisfactory in terms of the yield and purity of the obtained RNA.

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent Document 1: Japanese Patent No. 5157168

[0008] Patent Document 2: Japanese Patent No. 5554881

[0009] Patent Document 3: International Publication No. 2007-064291

[0010] Patent Document 4: International Publication No. 2013-027843 Summary of the invention

[0011] Problems to be solved by the invention

[0012] The object of the present invention is to provide an amide compound capable of improving the yield and purity of a polynucleotide and a method for producing a polynucleotide using the amide compound. In addition, the object of the present invention is to provide a glycoside compound as an intermediate of the amide compound and a method for producing an ether compound as an intermediate of the glycoside compound.

[0013] Means for solving problems

[0014] The inventors of the present application have conducted intensive studies to achieve the above-mentioned object, and as a result, have found that RNA can be synthesized with high purity by using the following group as a protecting group for the 2′-hydroxyl group of an amidate.

[0015] [Chemical formula 1]

[0016]

[0017] (Where,

[0018] R a and R b are the same or different from each other, representing a methyl group, an ethyl group or a hydrogen atom. a and R b At the same time, it represents the situation of hydrogen atoms. And,

[0019] R c represents a phenyl group, a C1-C10 alkyl group or a benzyl group which may be substituted by a halogen atom, a methyl group, a nitro group, a methoxy group or a trifluoromethyl group.

[0020] The present invention has been completed after further studies based on the above findings, and provides the following glycoside compounds, amide compounds thereof, and methods for producing polynucleotides using the amide compounds, as well as ether compounds as intermediates of the glycoside compounds and methods for producing the ether compounds.

[0021] The present invention includes the embodiments described in the following items, but is not limited to these.

[0022] Item 1. An amide compound represented by formula (1) (hereinafter referred to as "the amide compound of the present invention").

[0023] [Chemical formula 2]

[0024]

[0025] In formula (1), R represents a group represented by the following formula:

[0026] [Chemical formula 3]

[0027]

[0028] (Where,

[0029] R a and R b are the same or different from each other, representing a methyl group, an ethyl group or a hydrogen atom. a and R b It also shows the situation of hydrogen atoms.

[0030] R c represents a phenyl group, a C1-C10 alkyl group or a benzyl group which may be substituted with a halogen atom, a methyl group, a nitro group, a methoxy group or a trifluoromethyl group. ),

[0031] B a represents a group having a protectable nucleic acid base backbone,

[0032] G 1 and G 2 are the same or different from each other, representing a protecting group for a hydroxyl group, and,

[0033] G 3 The same or different from each other represent an alkyl group.

[0034] Item 2. The amide compound according to Item 1, wherein R a is methyl, and R b A hydrogen atom.

[0035] Item 3. The amide compound according to Item 1, wherein R a , R b The same is methyl.

[0036] Item 4. The amide compound according to any one of Items 1 to 3, wherein G 1 The following groups.

[0037] [Chemical formula 4]

[0038]

[0039] (Where R 1 , R 2 and R 3 are the same or different from each other, and represent hydrogen or alkoxy. )

[0040] Item 5. The amide compound according to any one of Items 1 to 4, wherein G 2 The following groups.

[0041] [Chemical formula 5]

[0042]

[0043] Item 6. The amide compound according to any one of Items 1 to 5, wherein G 3 It is isopropyl.

[0044] Item 7. The amide compound according to any one of Items 1 to 6, wherein R c It is phenyl or tolyl.

[0045] Item 8. A method for producing a compound containing a polynucleotide backbone represented by formula (2), comprising the step of subjecting the amide compound described in any one of Items 1 to 7 to a solid phase synthesis reaction.

[0046] [Chemical formula 6]

[0047]

[0048] (In formula (2), B a are the same or different from each other, and represent a group having a nucleic acid base backbone that can be protected,

[0049] X represents an oxygen atom or a sulfur atom, and,

[0050] m represents a positive integer. ).

[0051] Item 9. The production method according to Item 8, wherein the compound containing the polynucleotide backbone of formula (2) is obtained by reacting tetraalkylammonium fluoride with a compound having an oligonucleotide backbone represented by formula (3) generated by a solid phase synthesis reaction using the amide compound,

[0052] [Chemical formula 7]

[0053]

[0054] (In formula (3), B a are the same or different from each other, and represent a group having a nucleic acid base backbone that can be protected,

[0055] X represents an oxygen atom or a sulfur atom, and,

[0056] R are the same or different from each other and represent a group represented by the following formula:

[0057] [Chemical formula 8]

[0058]

[0059] (Where,

[0060] R a and R b are the same or different from each other, representing a methyl group, an ethyl group or a hydrogen atom. a and R b At the same time, it represents the situation of hydrogen atoms. And,

[0061] R c represents a phenyl group, a C1-C10 alkyl group or a benzyl group which may be substituted with a halogen atom, a methyl group, a nitro group, a methoxy group or a trifluoromethyl group.

[0062] Item 10. The manufacturing method according to Item 9, wherein R ais methyl, and R b A hydrogen atom.

[0063] Item 11. An ether compound represented by the formula (4).

[0064] [Chemical formula 9]

[0065]

[0066] (In formula (4),

[0067] R a and R b are the same or different from each other, representing a methyl group, an ethyl group or a hydrogen atom. a and R b It also shows the situation of hydrogen atoms.

[0068] R c represents a phenyl group, a C1-C10 alkyl group or a benzyl group which may be substituted with a halogen atom, a methyl group, a nitro group, a methoxy group or a trifluoromethyl group, R d represents a C1-C10 alkyl group or a phenyl group. )

[0069] Item 12. The ether compound according to Item 11, wherein R a is methyl, R b is a hydrogen atom, and R c It is phenyl or tolyl.

[0070] Item 13. A method for producing an ether compound represented by formula (4), comprising step a: reacting a 2-hydroxyalkyl sulfone represented by formula (5) with a disulfide compound represented by formula (12) in a solvent in the presence of a halogenating agent and an acid;

[0071] [Chemical formula 10]

[0072]

[0073] (In formula (5),

[0074] R a and R b are the same or different from each other, representing a methyl group, an ethyl group or a hydrogen atom. a and R b At the same time, it represents the situation of hydrogen atoms. And,

[0075] R c represents a phenyl group, a C1-C10 alkyl group or a benzyl group which may be substituted with a halogen atom, a methyl group, a nitro group, a methoxy group or a trifluoromethyl group.

[0076] [Chemical formula 11]

[0077] (R d SCH2)2O (12)

[0078] (In formula (12), R d Same as defined in item 11 above.)

[0079] [Chemical formula 12]

[0080]

[0081] (In formula (4), R a , R b , R c and R d Same as the above definition. ).

[0082] Item 14. The manufacturing method according to Item 13, wherein R a is methyl or ethyl, R b is a hydrogen atom, and R c It is phenyl or tolyl.

[0083] Item 15. The manufacturing method according to Item 13, wherein R a is methyl, R b is a hydrogen atom, and R c It is phenyl or tolyl.

[0084] Item 16. A method for producing a compound represented by formula (8), characterized in that a compound represented by formula (7) is reacted with a compound represented by formula (4) in the presence of a halogenating agent.

[0085] [Chemical formula 13]

[0086]

[0087] (In formula (7), B a represents a compound having a protectable nucleic acid base backbone, and G 4 represents a protecting group for a hydroxyl group. )

[0088] [Chemical formula 14]

[0089]

[0090] (In formula (4), R a , R b , R c and R d Same as defined in item 11 above.)

[0091] [Chemical formula 15]

[0092]

[0093] (In formula (8), B a , R a , R b and R c Same as the above definition, and G 4 represents a protecting group for a hydroxyl group. ).

[0094] Item 17. A method for producing the compound of formula (1) according to Item 1, comprising the following steps:

[0095] A step of further deprotecting the compound of formula (8) to obtain a compound represented by formula (9);

[0096] A step of selectively protecting the 5' hydroxyl group of the compound of formula (9) to obtain a compound represented by formula (10); and

[0097] a step of reacting the compound of formula (10) with a phosphordiamidite represented by formula (11);

[0098] [Chemical formula 16]

[0099]

[0100] (In formula (9), B a , R a , R b and R c Same as defined in item 16 above.)

[0101] [Chemical formula 17]

[0102]

[0103] (In formula (10), B a , R a , R b , R c and G 1 Same as defined in item 17 above.)

[0104] [Chemical formula 18]

[0105]

[0106] (In formula (11), G 2 Represents a protective group for a hydroxyl group, G 3 are the same or different from each other, representing an alkyl group. ).

[0107] Item 18. The manufacturing method according to Item 17, wherein G 4 It has a G4-1 or G4-2 structure.

[0108] [Chemical formula 19]

[0109]

[0110] Item 19. A compound represented by formula (8).

[0111] [Chemical formula 20]

[0112]

[0113] (In formula (8), B a , R a , R b , R c , G 4 Same as defined in item 16 above.)

[0114] Item 20. A compound represented by formula (9).

[0115] [Chemical formula 21]

[0116]

[0117] (In formula (9), B a , R a , R b and R c Same as defined in item 16 above.)

[0118] Item 21. A compound represented by formula (10) (hereinafter referred to as "the glycoside compound of the present invention").

[0119] [Chemical formula 22]

[0120]

[0121] (In formula (10), B a , R a , R b , R c and G 1 Same as defined in item 17 above.)

[0122] Item 22. Use of the amide compound of the above formula (1) in the production of RNA.

[0123] Effects of the Invention

[0124] By using the glycoside compound of the present invention, an amide compound can be obtained, and as a result, the yield and purity of RNA in the solid phase synthesis method can be improved. DETAILED DESCRIPTION

[0125] Hereinafter, the present invention will be described in detail.

[0126] It should be noted that, in the present specification, “comprise” includes the meaning of “essentially consists of” and the meaning of “consist only of”.

[0127] The amide compound of the present invention is characterized in that it is represented by the formula (1).

[0128] [Chemical formula 23]

[0129]

[0130] (In formula (1), R represents the following formula:

[0131] [Chemical formula 24]

[0132]

[0133] (Where,

[0134] R a and R b are the same or different from each other, representing a methyl group, an ethyl group or a hydrogen atom. a and R b It also shows the situation of hydrogen atoms.

[0135] R c represents a phenyl group, a C1-C10 alkyl group or a benzyl group which may be substituted with a halogen atom, a methyl group, a nitro group, a methoxy group or a trifluoromethyl group. ),

[0136] B a represents a group having a protectable nucleic acid base backbone,

[0137] G 1 and G 2 are the same or different from each other, representing a protecting group for a hydroxyl group, and,

[0138] G 3 are the same or different from each other, representing an alkyl group. )

[0139] B a The nucleic acid base in is not particularly limited. As the nucleic acid base, adenine, cytosine, guanine, uracil, thymine, 5-methylcytosine, pseudouracil, 1-methylpseudouracil etc. can be enumerated. In addition, the nucleic acid base can be substituted with a substituent. As such a substituent, for example, a halogen atom, an acyl group, an alkyl group, an arylalkyl group, an alkoxy group, an alkoxyalkyl group, a cyanoalkyl group, a hydroxyl group, a hydroxymethyl group, an acyloxymethyl group, an amino group, a monoalkylamino group, a dialkylamino group, a carboxyl group, a cyano group, and a nitro group etc., and a combination of two or more substituents in the above-mentioned can be enumerated.

[0140] When the nucleic acid base has an amino group outside the ring, there is no particular limitation on the protecting group of the amino group, and known protecting groups used in nucleic acid chemistry can be used. Examples of such protecting groups include methyl, benzoyl, 4-methoxybenzoyl, acetyl, propionyl, butyryl, isobutyryl, phenylacetyl, phenoxyacetyl, 4-tert-butylphenoxyacetyl, 4-isopropylphenoxyacetyl, and (dimethylamino)methylene, as well as combinations of two or more of the above protecting groups.

[0141] B a More specifically, it represents a group represented by any of the following formulae.

[0142] [Chemical formula 25]

[0143]

[0144] (Where,

[0145] R 4 represents a hydrogen atom, a methyl group, a phenoxyacetyl group, a 4-tert-butylphenoxyacetyl group, a 4-isopropylphenoxyacetyl group, a phenylacetyl group, an acetyl group or a benzoyl group,

[0146] R 5 represents a hydrogen atom, an acetyl group, an isobutyryl group or a benzoyl group,

[0147] R 6 represents a hydrogen atom, a phenoxyacetyl group, a 4-tert-butylphenoxyacetyl group, a 4-isopropylphenoxyacetyl group, a phenylacetyl group, an acetyl group or an isobutyryl group,

[0148] R 7 represents 2-cyanoethyl,

[0149] R 8 represents a hydrogen atom, a methyl group, a benzoyl group, a 4-methoxybenzoyl group or a 4-methylbenzoyl group,

[0150] R 9 represents dimethylaminomethylene. )

[0151] As G 1 Any group that can function as a protecting group can be used without limitation, and a wide range of known protecting groups used in amide compounds can be used.

[0152] As G 1 , preferably the following groups.

[0153] [Chemical formula 26]

[0154]

[0155] (Where R1 , R 2 and R 3 are the same or different from each other, and represent hydrogen or alkoxy. )

[0156] R 1 , R 2 and R 3 It is preferred that one of them is hydrogen and the other two are alkoxy groups, and the alkoxy group is particularly preferably a methoxy group.

[0157] As G 2 As long as it can function as a protecting group, it can be used without limitation, and a wide range of known protecting groups used in amide compounds can be used. 2 , for example, a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, a haloalkyl group, an aryl group, a heteroaryl group, an arylalkyl group, a cycloalkenyl group, a cycloalkylalkyl group, a cyclylalkyl group, a hydroxyalkyl group, an aminoalkyl group, an alkoxyalkyl group, a heterocyclylalkenyl group, a heterocyclylalkyl group, a heteroarylalkyl group, a silyl group, a silyloxyalkyl group, a mono-, di- or tri-alkylsilyl group, a mono-, di- or tri-alkylsilyloxyalkyl group, etc., and they may also be substituted with one or more electron withdrawing groups.

[0158] As G 2 , preferably an alkyl group substituted with an electron withdrawing group. Examples of the electron withdrawing group include cyano, nitro, alkylsulfonyl, halogen, arylsulfonyl, trihalomethyl, and trialkylamino, and cyano is preferred.

[0159] As G 2 , the following groups are particularly preferred.

[0160] [Chemical formula 27]

[0161]

[0162] For G 3 For example, 2G 3 Bonded to each other to form a ring structure. 3 , preferably both parties are isopropyl.

[0163] The alkyl group may be straight chain or branched, and is preferably an alkyl group having 1 to 12 carbon atoms, and more preferably an alkyl group having 1 to 6 carbon atoms. Examples of the alkyl group include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, and hexyl. The alkyl group herein also includes the alkyl moiety of an alkoxy group or the like.

[0164] R aIt is preferably a methyl group. n is preferably an integer of 1 to 4, more preferably an integer of 1 to 3, further preferably 1 or 2, and particularly preferably 1.

[0165] In addition, the amide compound of the present invention can be used in a free state or in a salt state. The salt of the amide compound of the present invention is not particularly limited, and examples thereof include salts formed with inorganic bases such as sodium salts, magnesium salts, potassium salts, calcium salts, and aluminum salts; salts formed with organic bases such as methylamine, ethylamine, and ethanolamine; and salts and ammonium salts formed with basic amino acids such as lysine, ornithine, and arginine. The salt may be an acid addition salt, and as such a salt, specifically, examples thereof 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 acid addition salts formed with acidic amino acids such as aspartic acid and glutamic acid. The amide compound of the present invention also includes salts, hydrates, solvates, polymorphs, and the like.

[0166] The amide compound of the present invention can be produced by a known method described in Japanese Patent No. 5157168, Japanese Patent No. 5554881, etc., a method described in Examples described below, or a method in which these methods are appropriately modified as necessary.

[0167] In addition, specific examples of the amide compound of the present invention include the following compounds described in Examples.

[0168] [Chemical formula 28]

[0169]

[0170] The present invention includes a glycoside compound represented by the following formula (10).

[0171] [Chemical formula 29]

[0172]

[0173] (In formula (10), the definitions of the groups are the same as those mentioned above.)

[0174] In addition, specific examples of the glycoside compound of the present invention include the following compounds described in Examples.

[0175] [Chemical formula 30]

[0176]

[0177] The present invention also includes a production intermediate compound of the glycoside compound represented by formula (10). Examples of such an intermediate compound include an ether compound represented by formula (4).

[0178] [Chemical formula 31]

[0179]

[0180] (In formula (4),

[0181] R a and R b are the same or different from each other, representing a methyl group, an ethyl group or a hydrogen atom. a and R b It also shows the situation of hydrogen atoms.

[0182] R c represents a phenyl group, a C1-C10 alkyl group or a benzyl group which may be substituted with a hydrogen atom, a halogen atom, a methyl group, a nitro group, a methoxy group or a trifluoromethyl group, R d represents a C1-C10 alkyl group or a phenyl group. )

[0183] The ether compound represented by formula (4) can be produced by reacting a bissulfide compound represented by formula (12) (e.g., bis(alkylthiomethyl)ether or bis(phenylthiomethyl)ether) with 3-hydroxy-3-alkylpropanesulfone in the presence of a halogenating agent and an acid in a solvent, as shown in the following reaction formula.

[0184] [Chemical formula 32]

[0185]

[0186] The bissulfide compound of formula (12) can be obtained, for example, by reacting bis(chloromethyl)ether or bis(aryloxymethyl)ether with the corresponding alkylthiol or phenylthiol as shown in the following formula. Examples of the bis(aryloxymethyl)ether include bis(2,4,6-trichlorophenyloxymethyl)ether.

[0187] [Chemical formula 33]

[0188]

[0189] The disulfide compound of formula (12) can also be produced by a known production method (for example, the method described in Japanese Patent No. 6459852).

[0190] Step a for producing a compound of formula (6), which is one example of the ether compound of formula (4), will be described.

[0191] Next, step a will be described.

[0192] As the halogenating agent, N-halosuccinimides such as N-chlorosuccinimide, N-bromosuccinimide, and N-iodosuccinimide, N-halohydantoins such as 1,3-diiodo-5,5-dimethylhydantoin, and halogens such as chlorine, bromine, and iodine, and combinations of two or more thereof can be cited. In the present invention, N-halosuccinimide is preferably used, and N-iodosuccinimide is more preferably used.

[0193] The acid is not particularly limited, and examples thereof include perfluoroalkyl carboxylic acids and salts thereof, perfluoroalkyl sulfonic acids and salts thereof, and alkyl sulfonic acids and salts thereof, and combinations of two or more thereof. Examples of salts include copper salts and silver salts. Specifically, examples of acids include methanesulfonic acid, p-toluenesulfonic acid, camphorsulfonic acid, trifluoromethanesulfonic acid, silver trifluoromethanesulfonate, and combinations of two or more thereof. In the present invention, trifluoromethanesulfonic acid is preferably used.

[0194] Examples of the solvent include tetrahydrofuran, 2-methyltetrahydrofuran, cyclopentyl methyl ether, dioxane, dichloromethane, toluene, and combinations of two or more thereof. In the present invention, tetrahydrofuran is preferably used.

[0195] The amount of 2-hydroxyalkyl sulfone of formula (5) is usually 0.5 to 2.0 equivalents, preferably 0.8 to 1.5 equivalents, relative to the disulfide compound of formula (12). The amount of the halogenating agent is usually 0.5 to 2 equivalents, preferably 0.7 to 1.2 equivalents, relative to the compound of formula (12). The amount of the acid is usually 0.001 to 2.0 equivalents, preferably 0.01 to 0.1 equivalents, relative to the compound of formula (12).

[0196] The reaction temperature of the present reaction is usually -80°C to 0°C, preferably -50°C to -30°C. The reaction time of the present reaction is usually 1 to 24 hours, preferably 2 to 6 hours.

[0197] For the completion of the reaction, for example, a portion of the reaction mass can be sampled and confirmed by analytical methods such as GC, TLC, and LC. After the reaction is completed, a base such as triethylamine can be added to the reaction mass to stop the reaction. The reaction mass is added to water and subjected to conventional post-processing operations such as organic solvent extraction, washing, and concentration to obtain a residue containing the ether compound represented by formula (4). The residue is subjected to purification operations such as distillation and column chromatography to obtain a high-purity ether compound represented by formula (4).

[0198] In the aforementioned method, the preferred compound is formula (6):

[0199] [Chemical formula 34]

[0200]

[0201] (In formula (6), R a is methyl or ethyl, R b is a hydrogen atom, R c is phenyl or tolyl. More preferably, R a Methyl, R b is a hydrogen atom, R c is tolyl. ).

[0202] The amide compound of the present invention can be used as a material for producing RNA in a solid phase synthesis method. By using the amide compound of the present invention in a solid phase synthesis method, RNA can be produced with high purity.

[0203] The method for producing a compound containing a polynucleotide backbone represented by the following formula (2) of the present invention is characterized by comprising a step of performing a solid phase synthesis reaction using the above-mentioned amide compound.

[0204] [Chemical formula 35]

[0205]

[0206] (In formula (2), B a are the same or different from each other, and represent a group having a nucleic acid base backbone that can be protected,

[0207] X represents an oxygen atom or a sulfur atom, and,

[0208] m represents a positive integer. )

[0209] The production method of the present invention may also include a step of treating a compound having an oligonucleotide backbone represented by formula (3) with tetraalkylammonium fluoride to obtain a compound having an oligonucleotide backbone represented by formula (2).

[0210] [Chemical formula 36]

[0211]

[0212] (In formula (3), B a are the same or different from each other, and represent a group having a nucleic acid base backbone that can be protected,

[0213] X represents an oxygen atom or a sulfur atom, and,

[0214] R are the same or different from each other and represent the following formula:

[0215] [Chemical formula 37]

[0216]

[0217] (Where,

[0218] R a and R b are the same or different from each other, representing a methyl group, an ethyl group or a hydrogen atom. a and R b It also shows the situation of hydrogen atoms.

[0219] R c represents a phenyl group, a C1-C10 alkyl group or a benzyl group which may be substituted with a halogen atom, a methyl group, a nitro group, a methoxy group or a trifluoromethyl group.

[0220] B in formula (2) and (3) a and m are the same as in formula (1).

[0221] m is not particularly limited, but is preferably an integer of 2 to 300. )

[0222] The "compound containing a polynucleotide backbone" in the present invention refers to a compound containing at least one RNA, and preferably a compound consisting only of RNA.

[0223] The solid phase synthesis reaction can be carried out according to a known method such as the phosphoramidite method (for example, the method described in Japanese Patent Nos. 5157168 and 5554881). Alternatively, it can be carried out using a commercially available automatic nucleic acid synthesizer or the like.

[0224] The method for producing a compound containing a polynucleotide backbone represented by formula (2) specifically comprises the following steps: (A) treating the 5′-hydroxyl group of a first amide compound supported on a solid phase support (e.g., G of formula (1) 1 ) a step of deprotecting the amide compound after deprotection generated in step (A) with a second amide compound; (C) an optional step of capping the hydroxyl group at the 5' position of the unreacted compound in step (B); (D) a step of converting the phosphite group of the condensate generated in (B) or (C) into a phosphate group or a thiophosphate group; (E) a step of cutting the compound obtained in step (D) from the solid phase support and deprotecting the hydroxyl group at the 2' position and the nucleic acid base; (F) a step of deprotecting the hydroxyl group at the 5' position, etc. By repeating steps (A) to (D), a compound containing a polynucleotide backbone of a desired chain length (for example, a compound of formula (3)) can be produced.

[0225] The compound preferably having an oligonucleotide backbone represented by formula (3) is treated with tetraalkylammonium fluoride to remove the protecting group at the 2' position, thereby producing a compound having an oligonucleotide backbone represented by formula (2). The reaction conditions (reaction temperature, reaction time, amount of reagent, etc.) of the reaction can be based on known methods.

[0226] The compound with the oligonucleotide backbone represented by formula (2) obtained in the manufacturing method of the present invention can be separated and purified as needed. Generally, separation can be carried out by using a method for precipitation, extraction and purification of RNA. Specifically, a method for precipitating RNA by adding a solvent with low solubility for RNA such as ethanol and isopropyl alcohol to the solution after the reaction, or a method for adding a solution of phenol / chloroform / isoamyl alcohol (for example, phenol / chloroform / isoamyl alcohol=25 / 24 / 1) to the reaction solution and extracting RNA into the aqueous layer can be adopted. Then, separation and purification can be carried out by known high performance liquid chromatography (HPLC) methods such as reverse phase column chromatography, anion exchange column chromatography, affinity column chromatography, etc.

[0227] The production method of the present invention enables production of RNA at a higher purity than ever before.

[0228] The reaction conditions for producing the amide compound of the present invention represented by formula (1), the glycoside compound of the present invention represented by formula (10), and the intermediate ether compound of the present invention represented by formula (4) are not particularly limited. The intermediate ether compound represented by formula (4) can also be synthesized using a flow reactor.

[0229] In order to reduce impurities contained in the amide compound of the present invention, a reduction step using hydrogen in the presence of a transition metal catalyst such as palladium or a reduction step using magnesium or the like may be added.

[0230] The 2-hydroxyalkylsulfone represented by the formula (5) can be synthesized by the following production route with reference to the following documents 1 to 6.

[0231] Document 1 CHINESE JOURNAL OF CHEMISTRY 2003,21,917

[0232] Reference 2 SYNTHESIS 2012,44,3623

[0233] Reference 3 Chem. Commun., 2005, 5904

[0234] Reference 4 New J.Chem., 2009, 33, 972 (baker’s yeast)

[0235] Reference 4 Adv.Synth.Catal.2013,355,2860 (NaBH4 reduction)

[0236] Document 4 WO 2017 / 223414 Al (catalytic hydrogenation)

[0237] Reference 5 Tetrahedron Asymmetry 2005,16,2157

[0238] Document 6 Japanese Patent No. 6448867 (mCPBA)

[0239] Reference 6 Tetrahedron 2001,57,2469 (hydrogen peroxide, tungsten)

[0240] [Chemical formula 38]

[0241]

[0242] The compound of formula (1) can be produced from the compound of formula (7) according to steps 1, 2, 3 and 4 of the following scheme 1.

[0243] In the compound of formula (7), B a Means the same as above, G 4 Typically, the following G 4 -1 or G 4 -2 structure.

[0244] [Chemical formula 39]

[0245]

[0246] The above-mentioned compounds are commercially available or can be produced by the method described in, for example, Tetrahedron Letters, 2005, 46, 2961.

[0247] Step 1 (Etherification Step)

[0248] The etherification step can be carried out by reacting the compound of formula (7) with the compound of formula (4). The reaction is usually carried out by adding a halogenating agent. The halogenating agent used in this step is not particularly limited, and is preferably at least one selected from the group consisting of N-chlorosuccinimide, N-bromosuccinimide, N-iodosuccinimide, iodine, 1,3-diiodo-5,5'-dimethylhydantoin, bromine and chlorine.

[0249] In this step, an acid may be added. The acid to be used is not particularly limited, but is preferably at least one selected from the group consisting of perfluoroalkyl carboxylic acids, perfluoroalkyl sulfonic acids, alkyl sulfonic acids, and salts thereof.

[0250] The reaction solvent used in this step is not particularly limited, and examples thereof include ether, such as diethyl ether, THF (tetrahydrofuran), 2-methyltetrahydrofuran, tetrahydropyran, 4-methyltetrahydropyran, dimethoxyethane, diglyme, cyclopentyl methyl ether, dioxane, or nitriles such as acetonitrile, aromatic hydrocarbons such as toluene, chlorobenzene, dichlorobenzene, methylene chloride, and combinations of two or more of the above solvents. Preferred solvents include ether, such as diethyl ether, THF (tetrahydrofuran), 2-methyltetrahydrofuran, tetrahydropyran, 4-methyltetrahydropyran, dimethoxyethane, diglyme, cyclopentyl methyl ether, dioxane, and the like.

[0251] The reaction time in this step is not particularly limited, and is, for example, 10 minutes to 12 hours, and preferably 10 minutes to 6 hours.

[0252] The reaction temperature in this step is not particularly limited, and is, for example, -80 to 30°C, preferably -60 to 10°C.

[0253] The concentration of the ether compound represented by the above formula (4) in this step is not particularly limited and can be appropriately set.

[0254] In this step, the molar number of the ether compound represented by the above formula (4) is, for example, 0.5 to 2 times, preferably 0.8 to 1.5 times, the molar number of the compound represented by the formula (7).

[0255] In this step, the molar number of the halogenating agent is, for example, 0.5 to 10 times, preferably 0.8 to 6 times, the molar number of the compound represented by formula (7).

[0256] Step 2 (deprotection step)

[0257] The compound of formula (8) obtained in the above step 1 can be subjected to a deprotection reaction to be converted into a compound of formula (9). The deprotection step can be carried out by a known method, and typically, deprotection can be carried out by reacting hydrogen fluoride / triethylamine, hydrogen fluoride / pyridine, or triethylamine trihydrofluoride in a solvent.

[0258] Step 3 (5' hydroxyl group protection step)

[0259] The compound of formula (9) obtained in the above step is subjected to a protecting step. The introduction of a protecting group can be carried out by a known method. Typically, the protecting group can be introduced by reacting compound (9) with 4,4'-dimethoxytrityl chloride in pyridine to produce compound (10).

[0260] Step 4 (amidation step)

[0261] This step can be carried out by reacting the compound of formula (11) with the glycoside compound of formula (10) obtained in the above step. Typically, 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite as the compound of formula (11) is reacted in the presence of diisopropylammonium tetrazolide. Amidation can also be carried out based on the method described in Examples 2 to 5 of Japanese Patent No. 5554881.

[0262] [Chemical formula 40]

[0263]

[0264] As described above, the compounds of formula (7), (8), (9) and (10) can be used to produce the amide compound of formula (1).

[0265] Example

[0266] Hereinafter, examples will be given to explain the present invention in more detail, but the present invention is not limited to these examples and the like.

[0267] In this specification, the following abbreviations are used.

[0268] TPM = (1-(4-methylphenylsulfonyl)propan-2-yl)oxy)methoxy)methyl; A = adenine, G = guanine, C = cytosine, U = uracil.

[0269] Preparation of TPM Amidate U

[0270] Production Example 1

[0271] 1) Production of TPM agent (TPMR)

[0272] [Chemical formula 41]

[0273]

[0274] Bis(methylthiomethyl)ether (5.9 g, 0.043 mol) was dissolved in anhydrous tetrahydrofuran (THF) (60 mL), molecular sieves 4A (5.9 g) were added, and the mixture was stirred for 10 minutes. After the mixture was cooled to -50°C, N-iodosuccinimide (NIS) (11.5 g, 1.19 eq.) and trifluoromethanesulfonic acid (TfOH) (0.11 mL, 0.030 eq.) were added in sequence. A solution of 1-(4-methylbenzenesulfonyl)propane-2-ol (10 g, 1.09 eq.) (manufactured by ENAMINE Ltd.) in acetonitrile (20 mL) was added dropwise to the mixture, and the mixture was stirred at -50 to -45°C for 4 hours. After triethylamine (4.0 mL) was added dropwise to the reaction solution, the temperature was raised to -30 to -20°C, and then the reaction solution was added to a solution of sodium thiosulfate pentahydrate (17.1 g), sodium bicarbonate (6.0 g) and water (130 mL) previously cooled to 5 to 10°C in an ice-water bath. Ethyl acetate (42 mL) was added to the mixture, and after stirring at 10 to 15°C for 30 minutes, the mixed solution was filtered using diatomaceous earth (5.9 g). After the filtrate was separated, the organic layer was washed with 20% brine (24 mL). After the organic layer was dried with anhydrous magnesium sulfate (3 g), the solvent was distilled off under reduced pressure (bath temperature 40°C). The residue was purified by silica gel chromatography (hexane / ethyl acetate = 3 / 1, silica gel 207 mL) to obtain 3.1 g of a yellow oil.

[0275] Purity analysis by GC / FID revealed a purity of 91%.

[0276] The product was purified again by silica gel chromatography (hexane / ethyl acetate=8 / 1, silica gel 150 mL) to obtain 3.1 g of TPMR with a purity of 98.2% and 1.5 g of TPMR with a purity of 97.8%. The two products were mixed and used in the subsequent reaction.

[0277] 1 H-NMR(CDCl3): δ7.79(d,2H)7.36(d,2H)4.76(s,2H)4.63(d,2H)4.27(m,1H)3.44(dd,1H)3.14(dd,1H)2.45(s,3H)2.13(s,3H)1.32(d,3H)

[0278] Production Example 2

[0279] 2) Manufacturing of TPM-U-2

[0280] [Chemical formula 42]

[0281]

[0282] Anhydrous toluene (16.5 mL, 5 vol / wt) was added to U-1 (3.3 g, 6.78 mmol), and the mixture was concentrated to 3 vol / wt under reduced pressure. Anhydrous toluene (6.6 mL, 2.0 vol / wt) was further added, and the mixture was concentrated to 3 vol / wt under reduced pressure. Anhydrous tetrahydrofuran (6.6 mL, 2.0 vol / wt) was added, and the mixture was cooled to about -55°C, and PMMR (3.09 g, 10.17 mmol, 1.5 eq.) was dropped therein, and thoroughly washed with THF 2 mL. NIS (2.06 g, 9.15 mmol, 1.35 eq.) was added to the mixture, and TfOH (0.72 mL, 8.14 mmol, 1.2 eq.) was dropped at -55 to -45°C. After the mixture was stirred at -55 to -45°C for 1 hour, the reaction solution was added to a mixed solution consisting of sodium thiosulfate pentahydrate (3.3 g), sodium bicarbonate (1.12 g), water (22 mL) and toluene cooled in an ice-water bath. The mixture was stirred in an ice-water bath for 30 minutes and then separated. A solution consisting of sodium thiosulfate pentahydrate (1.65 g), sodium bicarbonate (0.6 g) and water (11 mL) was added to the organic layer, and the mixture was stirred at room temperature for 15 minutes before separation. The organic layer was dried over sodium sulfate (1 g) and concentrated to dryness under reduced pressure. The residue was purified by silica gel chromatography (hexane / ethyl acetate = 3 / 1, silica gel 250 mL) to obtain TPM-U-2 (5.3 g) as a colorless, clear, glassy solid.

[0283] Production Example 3

[0284] 3) Manufacturing of TPM-U-3

[0285] [Chemical formula 43]

[0286]

[0287] After TPM-U-2 (5.3 g, 7.13 mmol) was dissolved in acetone (10 mL), triethylamine trihydrofluoride (1.3 mL, 7.84 mmol) was added, and the mixture was stirred at room temperature for 2 hours. Methyl tert-butyl ether (MTBE) (53 mL) was added to the reaction solution, and the mixture was stirred for 30 minutes, and the MTBE layer was removed by decantation. After repeating this operation 3 times, the residue was dried under reduced pressure to obtain white amorphous TPM-U-3 (3.5 g).

[0288] Production Example 4

[0289] 4) Manufacturing of TPM-U-4

[0290] [Chemical formula 44]

[0291]

[0292] Pyridine (10.5 mL, 3 vol / wt) was added to TPM-U-3 (3.5 g, 7.00 mmol), and azeotropic dehydration was performed twice. Pyridine (7.0 mL, 2 vol / wt), toluene (17.5 mL, 5 vol / wt) and acetonitrile (7.0 mL, 2 vol / wt) were added, and the mixture was cooled to about 0°C. At this time, 4,4'-dimethoxytrityl chloride (2.85 g, 8.40 mmol, 1.2 eq.) was added. After the mixture was stirred at room temperature for 6 hours, methanol (1.75 mL. 0.5 vol / wt) was added, and the mixture was stirred for 10 minutes. A solution composed of sodium bicarbonate (0.53 g) and water (10.5 mL) was added, and the mixture was stirred for 15 minutes and separated (this operation was repeated once more). A solution composed of sodium chloride (1.05 g) and water (10.5 mL) was added to the organic layer, and the mixture was stirred for 15 minutes and separated. The organic layer was dried over sodium sulfate (1 g) and concentrated under reduced pressure, and the residue was purified by silica gel chromatography (hexane / ethyl acetate = 1 / 1 to ethyl acetate only, silica gel 280 mL) to obtain 3.8 g of white amorphous TPM-U-4 (yield: 67%).

[0293] Production Example 5

[0294] 5) Production of TPM-U-5 (TPM amidate U)

[0295] [Chemical formula 45]

[0296]

[0297] After TPM-U-4 (4.7 g, 5.85 mmol) was dissolved in anhydrous acetonitrile (47 mL, 10 vol / wt), diisopropylammonium tetrazolium salt (1.10 g, 6.56 mmol, 1.12 eq.) and molecular sieves 4A (0.94 g, 0.2 wt / wt) were added, and the mixture was stirred at room temperature for 30 minutes. 2-Cyanoethyl-N, N, N', N'-tetraisopropylphosphorodiamidite (Phos reagent) (2.65 g, 8.78 mmol, 1.5 eq.) was added to the solution, and the mixture was stirred at a bath temperature of 45°C for 1.5 hours. After the mixture was cooled to room temperature, it was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography (hexane / acetone = 2 / 1 + 5% pyridine, silica gel 380 mL) to obtain 4.75 g of TPM amidate U as a white solid.

[0298] 31 P-NMR(CDCl3): δ151.89,151.86,150.87

[0299] Example of nucleic acid production

[0300] Using the TPM amidate U prepared in the above Production Example 5, a uridine 50-mer represented by the sequence of SEQ ID NO: 1 below was synthesized.

[0301] 5'-UUUUUUUUU UUUUUUUUUU UUUUUUUUUU UUUUUUUUUU UUUUUUUUU-3'(serial number 1)

[0302] (wherein U represents uridine monophosphate sodium salt)

[0303] NTS M-4MX-E (manufactured by NIHON TECHNO SERVICE CO., LTD.) was used as a nucleic acid synthesizer, and solid phase synthesis was performed from the 3' side to the 5' side. In the synthesis, porous glass was used as a solid phase support, a high-purity trichloroacetic acid toluene solution was used as a deblocking solution, 5-benzylmercapto-1H-tetrazole was used as a condensing agent, an iodine solution was used as an oxidant, and a phenoxyacetic acid solution and an N-methylimidazole solution were used as a blocking solution.

[0304] The purity of the crude oligonucleotide product after solid phase synthesis was determined by HPLC. The crude product was separated into components by HPLC (wavelength 260 nm, chromatographic column ACQUITY UPLC Oligonucleotide BEH C18, 2.1 mm×100 mm), and the purity of the oligonucleotide was calculated from the area value of the main product in the total area value of the obtained chromatogram.

[0305] (Production Example of Nucleic Acid of the Present Invention)

[0306] Production Example 6

[0307] The TPM amidate U prepared in Example 5 was used to synthesize uridine 50-mer (molecular weight 15246.53). As a result, the OD 260 The OD was 37.21 and the purity was 53.5%. 260 The value of was calculated to be 8603 μg / μmol per 1 μmol.

[0308] The results are shown in Table 1 below.

[0309] (OD 260 It refers to the absorbance at UV260nm per 10mm pathlength in 1mL solution (pH=7.5). Generally speaking, 1OD=40μg is known for RNA, so the amount of RNA produced can be calculated using absorbance.

[0310] (Comparative Production Example of Nucleic Acid)

[0311] Comparative Example 1

[0312] Using the uridine EMM amidate described in Example 2 of Japanese Patent No. 5554881, a solid phase synthesis was performed in the same manner as described in Preparation Example 6 to produce a uridine 50-mer. As a result, 0.228 μmol of OD 260 The OD was 41.41, and the purity was 44.1%. 260 The value of was calculated to be 7264 μg / μmol per 1 μmol.

[0313] The results are shown in Table 1 below.

[0314] [Table 1]

[0315] Example 1 Comparative Example 1 Purity of uridine 50-mer (%) 53.5 44.1 Yield (μg / μmol) 8603 7264 Yield (%) 56.4 47.6

[0316] As shown in the above Table 1, when the amidated product produced by the present invention was used, good results were obtained in terms of the purity of the uridine 50-mer.

[0317] Industrial Applicability

[0318] The present invention provides a sulfo group-containing ether compound useful as a protecting group for a hydroxyl group at the 2' position of an amidated product, and an amide compound having the sulfo group-containing ether moiety. The amide compound of the present invention is suitable for the synthesis of a high-purity oligonucleic acid.

[0319] Matching table free text

[0320] SEQ ID NO: 1 in the sequence listing shows the base sequence of 50-mer of uridine.

Claims

1. An amide compound represented by formula (1), In formula (1), R represents a group represented by the following formula: In the formula, R a Represents methyl, R b represents a hydrogen atom, R c represents a phenyl group or a tolyl group, B a represents a group represented by any of the following formulae, In the formula, R 4 represents a hydrogen atom, a methyl group, a phenoxyacetyl group, a 4-tert-butylphenoxyacetyl group, a 4-isopropylphenoxyacetyl group, a phenylacetyl group, an acetyl group or a benzoyl group, R 5 represents a hydrogen atom, an acetyl group, an isobutyryl group or a benzoyl group, R 6 represents a hydrogen atom, a phenoxyacetyl group, a 4-tert-butylphenoxyacetyl group, a 4-isopropylphenoxyacetyl group, a phenylacetyl group, an acetyl group or an isobutyryl group, R 7 represents 2-cyanoethyl, R 8 represents a hydrogen atom, a methyl group, a benzoyl group, a 4-methoxybenzoyl group or a 4-methylbenzoyl group, R 9 represents dimethylaminomethylene, G 1 For the following groups, In the formula, R 1 , R 2 and R 3 are the same or different from each other, representing hydrogen or methoxy, G 2 For the following groups, And, G 3 The groups are the same or different and represent an alkyl group having 1 to 12 carbon atoms.

2. The amide compound according to claim 1, wherein G 3 It is isopropyl.

3. A method for producing a compound containing a polynucleotide backbone represented by formula (2), comprising the step of subjecting the amide compound according to claim 1 or 2 to a solid phase synthesis reaction, In formula (2), B a Same as defined in claim 1, X represents an oxygen atom or a sulfur atom, and, m represents an integer of 2 to 300.

4. A method for producing a compound of formula (1) according to claim 1, comprising the following steps: A step of deprotecting the compound of formula (8) to obtain a compound represented by formula (9); A step of selectively protecting the 5' hydroxyl group of the compound of formula (9) to obtain a compound represented by formula (10); and a step of reacting the compound of formula (10) with the phosphorodiamidite represented by formula (11), In formula (8), B a , R a , R b , and R c Same as defined in claim 1, and G 4 Having G4-1 or G4-2 structure, In formula (9), B a , R a , R b and R c Same as defined in claim 1, In formula (10), B a , R a , R b , R c and G 1 Same as defined in claim 1, In formula (11), G 2 Same as defined in claim 1, G 3 The groups are the same or different and represent an alkyl group having 1 to 12 carbon atoms.

5. A compound represented by formula (10), In formula (10), B a , R a , R b , R c and G 1 As defined in claim 1 above.

6. Use of the amide compound of formula (1) according to claim 1 or 2 in the production of RNA.

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