Method for producing alkylated uracil derivative

Abstract

Provided is a method for synthesizing an N1-alkylated uracil derivative from a uracil derivative by one step, comprising a step for synthesizing, from a compound represented by general formula (1), a compound represented by general formula (2) by an alkylation reaction in the presence of a TMS group-containing compound. [In the formulas, is a hydrogen atom, an alkali metal atom, or -P(=O)(OR11)(OR12) (where R11 and R12 each independently represent a monovalent cation, an alkyl group which may have a substituent, or an acyl group); the ring A represents a 5- to 8-membered ring; when R1 is other than a hydrogen atom, the ring A is substituted with at least one hydroxy group; R2 is a group including a structure represented by formula (r2); and R3 represents a group including a structure represented by formula (r3) (where R31 is an alkyl group).]

Description

Method for producing alkylated uracil derivatives

[0001] The present invention relates to a method for synthesizing an N1-alkyluracil derivative from a uracil derivative in one step. This application claims priority to Japanese Patent Application No. 2024-111294, filed on July 10, 2024, the contents of which are incorporated herein by reference.

[0002] In recent years, research into nucleic acid medicines using oligonucleotides has been progressing. In particular, mRNA medicines, which use mRNA encoding a target protein as an active ingredient and achieve therapeutic or preventive effects by administering this to produce the target protein in the body, are expected to be therapeutic drugs for many diseases, including mRNA vaccines. However, nucleic acids have low in vivo stability due to the phosphodiester bonds that are susceptible to degradation by nucleases. If the stability of a nucleic acid medicine is low, it will be degraded in the body before reaching the target tissue, and the intended medicinal effect will not be achieved. Therefore, in order to increase stability in nucleic acid medicines, artificial nucleic acids are used in which some or all of the bases constituting the nucleic acid are replaced with bases that are more nuclease-resistant than natural bases (adenine, guanine, cytosine, thymine, and uracil).

[0003] Examples of bases that are more stable than natural bases include modified bases obtained by modifying natural bases with methylation, methoxylation, amination, fluorination, thiolation, etc., and isomers that can be translated in the same way as natural bases (Patent Document 1). By substituting all uridines (nucleosides consisting of uracil and ribose) in mRNA with pseudouridine (CAS No.: 1445-07-4), an isomer of uridine, or its methylated form, N1-methylpseudouridine (CAS No.: 13860-38-3), enzymatic degradation is suppressed, and as a result, the production efficiency of target proteins can be increased when administered to a living body.

[0004] N1-methylpseudouridine is synthesized by substituting a methyl group for the hydrogen atom bonded to the nitrogen atom at the N1 position of the pyrimidine base of pseudouridine using a methylating agent. Sites in pseudouridine that can be modified by a methylating agent include, in addition to the N1 position, the three hydroxy groups in ribose, the two carbonyl groups in the pyrimidine base, and the hydrogen atom bonded to the nitrogen atom at the N3 position of the pyrimidine base. In particular, the three hydroxy groups in ribose have a higher reactivity than the N1 position in the pyrimidine base. Therefore, when pseudouridine is subjected to a methylation reaction without modification, the hydroxy groups in ribose are also methylated (Patent Document 1). Therefore, in the past, when synthesizing N1-methylpseudouridine from pseudouridine, it was necessary to protect the three hydroxy groups in ribose with protecting groups before using a methylating agent, and then perform a deprotection reaction after methylation (Patent Document 2).

[0005] Furthermore, pseudouridine not only contains multiple hydroxy groups but also two modifiable nitrogen atoms in the nucleobase moiety, and therefore, when pseudouridine is methylated, both nitrogen atoms are methylated, making it difficult to selectively methylate only the nitrogen atom at the N1 position (Non-Patent Document 1).

[0006] U.S. Patent No. 5,646,265 International Publication No. 2015 / 196128

[0007] Chin et al., Biochemistry, 2000, col.39, p.12457-12464.

[0008] An object of the present invention is to provide a method for synthesizing an N1-alkyluracil derivative from a uracil derivative in one step.

[0009] The present inventors have discovered that the N1 position of the pyrimidine base of uracil can be selectively alkylated by carrying out an alkylation reaction in the presence of a trimethylsilyl (TMS) group-containing compound, and have completed the present invention.

[0010] That is, the present invention is as follows: [1] A compound represented by the following general formula (1): １is a hydrogen atom, an alkali metal atom, or -P(=O)(OR １１ )(OR １２ ) (R １１ and R １２ each independently represents a monovalent cation, an alkyl group having 1 to 6 carbon atoms which may have a substituent, or an acyl group having 2 to 7 carbon atoms; ring A is —OR １ and-R ２ represents a 5- to 8-membered ring which may further have a substituent in addition to the above; １ is other than a hydrogen atom, then ring A is substituted with at least one hydroxy group; ２ represents a group containing a structure represented by the following formula (r2): １ and ring A is the same as above; R ３ is expressed by the following formula (r3) (R ３１ is an alkyl group having 1 to 6 carbon atoms.) represents a group containing a structure represented by the following formula:

[0011]

[0012] [2] Furthermore, the compound represented by the general formula (2) is phosphorylated to obtain a compound represented by the following general formula (3):

[0013]

[0014] [In the formula, R ３ and ring A is the same as above; M ３ and M ４ are each independently a monovalent cation; n is an integer of 1 to 3; when n is 2 or 3, a plurality of M ３ and may be the same or different ions.] [3] The method for producing the alkylated uracil derivative according to [1] above, wherein the R ２ is represented by the following general formula (r2-1) or (r2-2): ２２represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom; ring B represents a 5- to 8-membered ring which may have a substituent; and the black circle represents the bonding site with ring A. ３ is represented by the following general formula (r3-1) or (r3-2): ２２ , ring B, and black circle are the same as above, and R ３１ is an alkyl group having 1 to 6 carbon atoms.] The method for producing an alkylated uracil derivative according to [1] or [2] above.

[0015]

[0016] [4] The method for producing an alkylated uracil derivative according to the above [3], wherein the ring B is a benzene ring, a pyridine ring, or a furan ring. [5] The method for producing an alkylated uracil derivative according to the above [3], wherein the ring A is a ring represented by the following general formula (a-1):

[0017]

[0018] [In the formula, R ４１ and R ４２ are each independently a hydrogen atom, a hydroxy group, an alkoxy group having 1 to 6 carbon atoms, a halogen atom, or —NHR′ (R′ is a protecting group); R ４３ is a hydrogen atom, or R ４１ is a group that bonds to form a ring structure; R ａ represents a methylene group (-CH) optionally substituted with a hydroxy group, an alkoxy group having 1 to 6 carbon atoms, a halogen atom, or -NHR' (R' is a protecting group). ２ -) or -NR'- (R' is a protecting group); a1 is 0 or 1; one black circle represents R ２ The two black circles represent the binding site for -OR １ where R １ is other than a hydrogen atom, R ４１ and R ４２ At least one of a1 is a hydroxy group, or a1 is 1 and R ａ[6] The method for producing an alkylated uracil derivative according to any one of the above [1] to [4], wherein the compound represented by the general formula (1) is a ring represented by the following general formulas (1-1) to (1-4): １ is a hydrogen atom, an alkali metal atom, or -P(=O)(OR １１ )(OR １２ ) (R １１ and R １２ each independently represents a monovalent cation, an alkyl group having 1 to 6 carbon atoms which may have a substituent, or an acyl group having 2 to 7 carbon atoms; R ２２ is a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom; R ４１ and R ４２ are each independently a hydrogen atom, a hydroxy group, an alkoxy group having 1 to 6 carbon atoms, a halogen atom, or —NHR′ (R′ is a protecting group); R ４３ is a hydrogen atom, or R ４１ is a group that bonds to form a ring structure; R ａ represents a methylene group (-CH) optionally substituted with a hydroxy group, an alkoxy group having 1 to 6 carbon atoms, a halogen atom, or -NHR' (R' is a protecting group). ２ -), or -NR'- (R' is a protecting group). １ is other than a hydrogen atom, R ４１ and R ４２ At least one of R is a hydroxy group, or ａ is a methylene group substituted with a hydroxy group], and the compound represented by the general formula (2) is a compound represented by any one of the following general formulas (2-1) to (2-4): １ , R ２２ , R ４１ , R ４２ , R ４３ , and R ａ is the same as above; R ３１ is an alkyl group having 1 to 6 carbon atoms.].

[0019]

[0020]

[0021] [7] The compound represented by the general formula (1) is represented by the following general formulas (1-1) to (1-4): １ is a hydrogen atom, an alkali metal atom, or -P(=O)(OR １１ )(OR １２ ) (R １１ and R １２ each independently represents a monovalent cation, an alkyl group having 1 to 6 carbon atoms which may have a substituent, or an acyl group having 2 to 7 carbon atoms; R ２２ is a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom; R ４１ and R ４２ are each independently a hydrogen atom, a hydroxy group, an alkoxy group having 1 to 6 carbon atoms, a halogen atom, or —NHR′ (R′ is a protecting group); R ４３ is a hydrogen atom, or R ４１ is a group that bonds to form a ring structure; R ａ represents a methylene group (-CH) optionally substituted with a hydroxy group, an alkoxy group having 1 to 6 carbon atoms, a halogen atom, or -NHR' (R' is a protecting group). ２ -), or -NR'- (R' is a protecting group). １ is other than a hydrogen atom, R ４１ and R ４２ At least one of R is a hydroxy group, or ａ is a methylene group substituted with a hydroxy group], and the compound represented by the general formula (2) is a compound represented by any one of the following general formulas (2-1) to (2-4): １ , R ２２ , R ４１ , R ４２ , R ４３ , and R ａ is the same as above; R ３１is an alkyl group having 1 to 6 carbon atoms.], and the compound represented by the general formula (3) is a compound represented by any one of the following general formulas (3-1) to (3-4): ３１ , R １ , R ２２ , R ４１ , R ４２ , R ４３ , and R ａ is the same as above; M ３ and M ４ are each independently a monovalent cation; n is an integer of 1 to 3; when n is 2 or 3, a plurality of M ３ may be the same type of ion or different types of ions.]

[0022]

[0023]

[0024]

[0025] [8] The compound represented by the general formula (1) is represented by the following general formula (1-1-1): １ is the same as the general formula (1).], and the compound represented by the general formula (2) is a compound represented by the following general formula (2-1-1): １ and R ３１ is the same as the general formula (2).]. The method for producing an alkylated uracil derivative according to any one of [1] to [7] above, which is a compound represented by the formula:

[0026]

[0027] [9] The trimethylsilyl group-containing compound is represented by the following general formula (e1), (e2), or (e3):

[0028]

[0029] [In the formula, R ５１ is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; R ５２ is -CO-CH ３ , —CO—CF３ , —CO—NH-TMS, or —TMS (TMS is a trimethylsilyl group); R ５３ is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; and ring E is a 5- or 6-membered ring.]

[10] A method for producing an alkylated uracil derivative according to any of the above [1] to [8], wherein ring E is a 5- or 6-membered aromatic heterocycle.

[11] A method for producing an alkylated uracil derivative according to any of the above [1] to

[10] , wherein the trimethylsilyl group-containing compound is one or more selected from the group consisting of N,O-bis(trimethylsilyl)acetamide, N-trimethylsilylimidazole, N-methyl-N-trimethylsilylacetamide, and bis(trimethylsilyl)amine.

[12] A method for producing an alkylated uracil derivative according to any of the above [1] to

[11] , wherein the alkylation reaction is carried out in an aprotic polar solvent.

[13] A method for producing an alkylated uracil derivative according to

[12] above, wherein the aprotic polar solvent is one or more selected from the group consisting of dichloromethane, dimethyl carbonate, and acetonitrile.

[14] A method for producing an alkylated uracil derivative according to any one of [1] to

[13] above, wherein the alkylation reaction is carried out at 10 to 50° C.

[15] A method for producing a nucleic acid, comprising synthesizing a compound represented by general formula (2-1-1) by the method for producing an alkylated uracil derivative according to any one of [8] to

[14] above, and synthesizing a nucleic acid using the obtained compound as a starting material.

[0030] According to the method of the present invention, an N1-alkylated uracil derivative can be synthesized from a uracil derivative in one step, without the need for a protection step with a protecting group and a deprotection step. That is, according to the present invention, an N1-alkylated uracil derivative can be obtained simply and in a short step.

[0031] In the present invention and the present specification, "C ｐ１－ｐ２ " (p1 and p2 are positive integers satisfying p1<p2) means that the group has p1 to p2 carbon atoms.

[0032] In the present invention and the present specification, "C １－６The "alkyl group" is an alkyl group having 1 to 6 carbon atoms, and may be a straight chain or a branched chain. １－６ Examples of the alkyl group include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, a pentyl group, an isopentyl group, a neopentyl group, a tert-pentyl group, and a hexyl group.

[0033] In the present invention and the present specification, "C １－６ The "alkoxy group" is an alkoxy group having 1 to 6 carbon atoms, and may be linear or branched. １－６ Examples of the alkoxy group include a methoxy group, an ethoxy group, a propyloxy group, an isopropyloxy group, a butoxy group, an isobutoxy group, a sec-butoxy group, a tert-butoxy group, a pentyloxy group, an isopentyloxy group, a neopentyloxy group, a tert-pentyloxy group, and a hexyloxy group.

[0034] In the present invention and the present specification, "C ２－７ An "acyl group" is an acyl group in which the hydrocarbon group moiety obtained by removing the carbonyl group is C １－６ Alkyl group, C ２－６ The term "acyl group" refers to a group that is an alkenyl group, a 5- or 6-membered aryl group, or a 5- or 6-membered heteroaryl group. The hydrocarbon group portion of the acyl group may be linear or branched. ２－７ Examples of the acyl group include an acetyl group, a propanoyl group, an isopropanoyl group, a butanoyl group, an isobutanoyl group, a sec-butanoyl group, a tert-butanoyl group, a pentanoyl group, an isopentanoyl group, a neopentanoyl group, a tert-pentanoyl group, a hexanoyl group, a heptanoyl group, and a benzoyl group.

[0035] In the present invention and this specification, the term "halogen atom" refers to a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom.

[0036] In the following description, "compound (n)" means a compound represented by formula (n).

[0037] In addition, the term "an alkyl group having 1 to 6 carbon atoms which may have a substituent" refers to an unsubstituted or substituted C １－６ "-OR alkyl group" means an "-OR alkyl group". １ and-R ２ The 5- to 8-membered ring may further have a substituent in addition to the group "-OR １ and-R ２ a 5- to 8-membered ring having no substituent other than -OR １ and-R ２ "A 5- to 8-membered ring which may have a substituent" means "an unsubstituted or substituted 5- to 8-membered ring". "A methylene group (-CH) which may be substituted with a hydroxy group, an alkoxy group having 1 to 6 carbon atoms, a halogen atom, or -NHR' (R' is a protecting group)" means "a 5- to 8-membered ring which may have a substituent" or "an unsubstituted or substituted 5- to 8-membered ring". ２ "-") means an unsubstituted methylene group, or a methylene group in which the hydrogen atom is substituted with a hydroxy group, an alkoxy group having 1 to 6 carbon atoms, a halogen atom, or -NHR'.

[0038] <Alkylation Reaction> The method for producing an alkylated uracil derivative according to the present invention is a method for synthesizing a compound represented by the following general formula (2) from a compound represented by the following general formula (1) by alkylation in the presence of a TMS group-containing compound. Compound (1) is a compound having a structure represented by formula (r2) and at least one hydroxy group on ring A. Compound (2) is a compound in which the structure represented by formula (r2) in compound (1) is replaced with a structure represented by formula (r3), i.e., only the hydrogen atom bonded to the nitrogen atom at the N1 position of the pyrimidine ring in the uracil structure is replaced with C １－６ In this production method, the alkylation is carried out in the presence of a TMS group-containing compound, thereby enabling selective alkylation of the nitrogen atom at the N1 position of the pyrimidine base without protecting other hydroxy groups in compound (1).

[0039]

[0040]

[0041] In general formulas (1) and (2), R １is a hydrogen atom, an alkali metal atom, or -P(=O)(OR １１ )(OR １２ ) is. R １１ and R １２ each independently represents a monovalent cation, a C １－６ Alkyl group, or C ２－７ It is an acyl group.

[0042] R １ When is an alkali metal atom, examples of the alkali metal atom include a sodium atom and a potassium atom.

[0043] R １ -P(=O)(OR １１ )(OR １２ ) among the cases where R １１ is a monovalent cation, R １ R in １１ and the oxygen atom are bonded by an ionic bond. １２ is a monovalent cation, R １ R in １２ and the oxygen atom are bonded by an ionic bond.

[0044] R １１ and R １２ When is a monovalent cation, the cation is not particularly limited, and may be a hydrogen ion, an alkali metal ion (Na ＋ , K. ＋ ammonium ions, quaternary ammonium ions (tetrabutylammonium ions, imidazolium ions, pyridinium ions, etc.), and the like.

[0045] R １１ and R １２ C which may have a substituent １－６ In the case of an alkyl group, the substituent may be a hydroxy group, a halogen atom, １－６Examples of the aryl group include an alkoxy group, an aryl group, a heteroaryl group, etc. Examples of the aryl group include a phenyl group, a naphthyl group, an anthryl group, a 9-fluorenyl group, etc., with a phenyl group being particularly preferred. Examples of the heteroaryl group include a pyridyl group, a pyrazinyl group, a pyrimidinyl group, a pyridazinyl group, a pyrazolyl group, a quinolyl group, an isoquinolyl group, a pyrrolyl group, an imidazolyl group, an indolyl group, a furyl group, a benzofuryl group, a thienyl group, a benzothienyl group, an oxazolyl group, a benzoxazolyl group, an isoxazolyl group, a benzoisoxazolyl group, a benzodioxazolyl group, a benzodioxolanyl group, a benzodiosolanyl group, a quinazolinyl group, a quinoxalinyl group, a thiazolyl group, a benzothiazolyl group, an isothiazolyl group, a benzoisothiazolyl group, a thiazinyl group, a benzothiazinyl group, a benzothiophenyl group, a chromenyl group, a dibenzofuranyl group, a carbazolyl group, a fluorenyl group, a phenazinyl group, and a phenoxazinyl group.

[0046] R １１ and R １２ C which may have a substituent １－６ In the case of an alkyl group, the group may be C １－６ Alkyl group or aryl group C １－６ Alkyl group (C １－６ A group in which one hydrogen atom of an alkyl group is substituted with an aryl group is preferred, a methyl group, an ethyl group, a propyl group, a benzyl group, a phenylethyl group, or a phenylpropyl group is more preferred, and a methyl group or a benzyl group is even more preferred.

[0047] R １１ and R １２ is C ２－７ In the case of an acyl group, the group is preferably an acetyl group or a propanoyl group, more preferably an acetyl group.

[0048] R １ -P(=O)(OR １１ )(OR １２ ), then R １ As for R １１ and R １２ are each independently a monovalent cation, C １－６ Alkyl group, aryl group １－６Preferred are alkyl, acetyl, or propanoyl groups; R １１ and R １２ are each independently a hydrogen ion, an alkali metal ion, an ammonium ion, a quaternary ammonium ion, a methyl group, an ethyl group, a propyl group, a benzyl group, a phenylethyl group, a phenylpropyl group, an acetyl group, or a propanoyl group; R １１ and R １２ are each independently a hydrogen ion, a sodium ion, a potassium ion, an ammonium ion, a tetrabutylammonium ion, an imidazolium ion, a pyridinium ion, a methyl group, an ethyl group, a propyl group, or a benzyl group, and R １１ and R １２ are each independently a hydrogen ion, a sodium ion, a potassium ion, an ammonium ion, a tetrabutylammonium ion, a methyl group, or a benzyl group, and R １１ and R １２ Even more preferably, is an ammonium ion.

[0049] R １ As the １１ and R １２ are each independently a monovalent cation, and R １１ and R １２ is the same type of monovalent cation group (-P(=O)(O － ２ )・2[M １ ] ＋ :[M １ ] ＋ is a monovalent cation. １ As -P(=O)(O － ２ )・2[M １ ] ＋ Among the groups represented by [M １ ] ＋ is preferably a hydrogen ion, a sodium ion, a potassium ion, an ammonium ion, a tetrabutylammonium ion, an imidazolium ion, or a pyridinium ion, and １ ]＋ is more preferably a group in which [M １ ] ＋ is most preferably a hydrogen ion. １ -P(=O)(OR １１ )(OR １２ ), acid treatment can be carried out to obtain [M １ ] ＋ It is preferable to replace with hydrogen ions.

[0050] R １ As for R １１ and R １２ In addition, a group in which at least one of the following is a methyl group or a benzyl group is also preferred. Specifically, —P(═O)(OH)(OCH ３ ), -P(=O)(OCH ３ ) ２ , -P(=O)(OH)(OBn), -P(=O)(OBn) ２ , -P(=O)(OCH ３ )(OBn) (Bn represents a benzyl group), etc.

[0051] In the general formulae (1) and (2), ring A is -OR １ and-R ２ represents a 5- to 8-membered ring which may further have a substituent in addition to the above. Ring A may be an aromatic ring, but is preferably an aliphatic ring. Ring A is a heterocycle having at least one oxygen atom, but may also be a ring having two or more heteroatoms. Examples of ring A include a tetrahydrofuran ring, a dioxolane ring, an oxazolidine ring, a tetrahydropyran ring, a morpholine ring, a 1,3-dioxane ring, a 1,4-dioxane ring, an oxepane ring, and an oxocane ring, with a tetrahydrofuran ring being preferred.

[0052] When Ring A is a heterocycle having a nitrogen atom, such as an oxazolidine ring or a morpholine ring, the hydrogen atoms bonded to the nitrogen atoms constituting the ring must be substituted with protecting groups. Examples of such protecting groups include various protecting groups used as protecting groups for amines in organic synthesis reactions. Specific examples of such protecting groups include tert-butoxycarbonyl (Boc), benzyloxycarbonyl, 9-fluorenylmethyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, allyloxycarbonyl, trifluoroacetyl, phthaloyl, p-toluenesulfonyl, and 2-nitrobenzenesulfonyl.

[0053] Ring A is -OR １ and-R ２ In addition, the alkyl group may further have a substituent. The substituent may be a hydroxy group, a halogen atom, a C １－６ Alkyl group, C １－６ Alkoxy groups and the like are particularly preferred. １ is other than a hydrogen atom, then ring A is substituted with at least one hydroxy group.

[0054] Specific examples of ring A include rings represented by the following general formula (a-1): In general formula (a-1), one black circle represents R ２ The two black circles represent the binding site with -OR １ represents the binding site with

[0055]

[0056] In general formula (a-1), R ４１ and R ４２ are each independently a hydrogen atom, a hydroxy group, or C １－６ An alkoxy group, a halogen atom, or —NHR′ (R′ is a protecting group). As the protecting group for R′, the same groups as those described above can be used.

[0057] R ４１ and R ４２ is C １－６ In the case of an alkoxy group, R ４１ and R ４２are each independently preferably a methoxy group, an ethoxy group, or a propoxy group, and more preferably a methoxy group.

[0058] R ４１ and R ４２ When is a halogen atom, R ４１ and R ４２ are each independently preferably a fluorine atom or a chlorine atom, more preferably a fluorine atom.

[0059] Among these, R ４１ and R ４２ is preferably a hydroxy group.

[0060] In the general formula (a-1), a1 is 0 or 1, and is preferably 0. When a1 is 0, R ４１ and the carbon atom to which R is bonded. ４２ The carbon atom to which is attached is connected by a single bond.

[0061] In general formula (a-1), R ａ is a hydroxy group, C １－６ a methylene group (-CH) optionally substituted with an alkoxy group, a halogen atom, or -NHR' (R' is a protecting group); ２ -), or -NR'- (R' is a protecting group). As the protecting group for R', the same as those mentioned above can be used.

[0062] R ａ is C １－６ In the case of a methylene group substituted with an alkoxy group, "C １－６ The "alkoxy group" is preferably a methoxy group, an ethoxy group, or a propoxy group, and more preferably a methoxy group.

[0063] R ａ When is a methylene group substituted with a halogen atom, the "halogen atom" is preferably a fluorine atom or a chlorine atom, more preferably a fluorine atom.

[0064] In general formula (a-1), R ４３ is a hydrogen atom, or R ４１ is a group that bonds with R to form a ring structure. ４１ is C １－６ In the case of an alkoxy group, the C１－６ The terminal carbon atom of the alkoxy group is R ４３ When a bridged structure is formed, ring A becomes a bicyclic bridging group. ４３ is preferably a hydrogen atom.

[0065] Compound (1) has at least one hydroxy group. Therefore, in general formula (1), R １ is other than a hydrogen atom, that is, -OR １ is not a hydroxy group, then ring A is substituted with at least one hydroxy group. ４１ and R ４２ At least one of a1 is a hydroxy group, or a1 is 1 and R ａ is a methylene group substituted with a hydroxy group. １ When is a hydrogen atom, R ４１ , R ４２ and R ａ may not have a hydroxy group, but R ４１ , R ４２ and R ａ It is preferable that at least one of them has a hydroxy group.

[0066] In general formula (1), R ２ represents a group containing a structure represented by formula (r2). ２ is not particularly limited as long as it is a group that maintains the structure of formula (r2), and may be a group in which one or both of the two carbon atoms constituting the unsaturated bond of the pyrimidine ring have a substituent, or may be a group having a structure in which the unsaturated bond is condensed with another ring.

[0067] In general formula (2), R ３ represents a group containing a structure represented by formula (r3). ３ is not particularly limited as long as it is a group that maintains the structure of formula (r3), and may be a group in which one or both of the two carbon atoms that constitute the unsaturated bond of the pyrimidine ring have a substituent, or may be a group having a structure in which the unsaturated bond is condensed with another ring.３１ is C １－６ is an alkyl group. ３１ "C" in １－６ The "alkyl group" is preferably a methyl group, an ethyl group, or a propyl group, and more preferably a methyl group.

[0068] R ２ Examples of R include groups represented by the following general formula (r2-1) or (r2-2). ３ Examples of R include groups represented by the following general formula (r3-1) or (r3-2). In the formula, the black circle represents the bonding site with ring A. In addition, in general formulas (r3-1) and (r3-2), R ３１ is the same as general formula (r3).

[0069]

[0070]

[0071] In general formulas (r2-1) and (r3-1), R ２２ is a hydrogen atom, C １－６ It is an alkyl group or a halogen atom.

[0072] R ２２ is C １－６ In the case of an alkyl group, R ２２ R is preferably a methyl group, an ethyl group, or a propyl group, and more preferably a methyl group. ２２ When is a halogen atom, R ２２ are each independently preferably a fluorine atom or a chlorine atom, more preferably a fluorine atom. ２２ is preferably a hydrogen atom.

[0073] In the general formulae (r2-2) and (r3-2), ring B represents a 5- to 8-membered ring which may have a substituent. Examples of ring B include a benzene ring, a pyridine ring, and a furan ring.

[0074] Compounds represented by the following general formulas (1-1) to (1-4) are preferred as compound (1). Compounds represented by the following general formulas (1-1) to (1-4) are alkylated in the presence of a TMS group-containing compound to synthesize compounds represented by the following general formulas (2-1) to (2-4).

[0075]

[0076]

[0077] In the general formulae (1-1) to (1-4) and the general formulae (2-1) to (2-4), R １ is the same as in the general formula (1), and R ２２ is the same as in general formula (r2-1), and R ４１ , R ４２ , R ４３ , and R ａ is the same as in general formula (a-1), and R ３１ is the same as the general formula (r3) above.

[0078] The compounds represented by the general formulas (1-1) to (1-4) include R １ is a hydrogen atom, an alkali metal atom, or -P(=O)(OR １１ )(OR １２ ) and R ４１ and R ４２ are each independently a hydrogen atom, a hydroxy group, a halogen atom, or C １－６ is an alkoxy group, and R ４３ is a hydrogen atom, and R ２２ is a hydrogen atom; １ is a hydrogen atom, an alkali metal atom, -P(=O)(OR １１ )(OR １２ ) and R ４１ and R ４２ are each independently a hydrogen atom, a hydroxy group, a halogen atom, or a methoxy group, and R ４３ is a hydrogen atom, and R ２２ is a hydrogen atom; １ is a hydrogen atom or an alkali metal atom, and R ４１ and R ４２ are each independently a hydrogen atom, a hydroxy group, a fluorine atom, or a methoxy group, and R ４３ is a hydrogen atom, and R ２２ is a hydrogen atom; or １ is a hydrogen atom, and R ４１ and R ４２is a hydroxy group, and R ４３ is a hydrogen atom, and R ２２ More preferred are compounds in which is a hydrogen atom.

[0079] The compounds represented by the general formulas (1-1) to (1-4) include R １ -P(=O)(OR １１ )(OR １２ ) among R １１ and R １２ are each independently a hydrogen ion, a sodium ion, a potassium ion, an ammonium ion, a tetrabutylammonium ion, an imidazolium ion, a pyridinium ion, a methyl group, an ethyl group, a propyl group, or a benzyl group, and R ４１ and R ４２ are each independently a hydrogen atom, a hydroxy group, a halogen atom, or C １－６ is an alkoxy group, and R ４３ is a hydrogen atom, and R ２２ is a hydrogen atom; １ -P(=O)(OR １１ )(OR １２ ) among R １１ and R １２ are each independently a hydrogen ion, a sodium ion, a potassium ion, an ammonium ion, a tetrabutylammonium ion, a methyl group, or a benzyl group, and R ４１ and R ４２ are each independently a hydrogen atom, a hydroxy group, a halogen atom, or a methoxy group, and R ４３ is a hydrogen atom, and R ２２ is a hydrogen atom; １ -P(=O)(OR １１ )(OR １２ ) among R １１ and R １２ are both groups that are hydrogen ions, sodium ions, potassium ions, ammonium ions, tetrabutylammonium ions, methyl groups, or benzyl groups, and R ４１ and R ４２ are each independently a hydrogen atom, a hydroxy group, a fluorine atom, or a methoxy group, and R ４３is a hydrogen atom, and R ２２ is a hydrogen atom; or １ -P(=O)(OR １１ )(OR １２ ) among R １１ and R １２ are both ammonium ions, and R ４１ and R ４２ is a hydroxy group, and R ４３ is a hydrogen atom, and R ２２ is a hydrogen atom, and R ３１ More preferred are compounds in which is a methyl group.

[0080] The compounds represented by the general formulas (2-1) to (2-4) include R １ is a hydrogen atom, an alkali metal atom, or -P(=O)(OR １１ )(OR １２ ) and R ４１ and R ４２ are each independently a hydrogen atom, a hydroxy group, a halogen atom, or C １－６ is an alkoxy group, and R ４３ is a hydrogen atom, and R ２２ is a hydrogen atom, and R ３１ is a methyl group; １ is a hydrogen atom, an alkali metal atom, or -P(=O)(OR １１ )(OR １２ ) and R ４１ and R ４２ are each independently a hydrogen atom, a hydroxy group, a halogen atom, or a methoxy group, and R ４３ is a hydrogen atom, and R ２２ is a hydrogen atom, and R ３１ is a methyl group; １ is a hydrogen atom or an alkali metal atom, and R ４１ and R ４２ are each independently a hydrogen atom, a hydroxy group, a fluorine atom, or a methoxy group, and R ４３ is a hydrogen atom, and R ２２ is a hydrogen atom, and R ３１ is a methyl group; or １is a hydrogen atom, and R ４１ and R ４２ is a hydroxy group, and R ４３ is a hydrogen atom, and R ２２ is a hydrogen atom, and R ３１ More preferred are compounds in which is a methyl group.

[0081] The compounds represented by the general formulas (2-1) to (2-4) include R １ -P(=O)(OR １１ )(OR １２ ) among R １１ and R １２ are each independently a hydrogen ion, a sodium ion, a potassium ion, an ammonium ion, a tetrabutylammonium ion, an imidazolium ion, a pyridinium ion, a methyl group, an ethyl group, a propyl group, or a benzyl group, and R ４１ and R ４２ are each independently a hydrogen atom, a hydroxy group, a halogen atom, or C １－６ is an alkoxy group, and R ４３ is a hydrogen atom, and R ２２ is a hydrogen atom, and R ３１ is a methyl group; １ -P(=O)(OR １１ )(OR １２ ) among R １１ and R １２ are each independently a hydrogen ion, a sodium ion, a potassium ion, an ammonium ion, a tetrabutylammonium ion, a methyl group, or a benzyl group, and R ４１ and R ４２ are each independently a hydrogen atom, a hydroxy group, a halogen atom, or a methoxy group, and R ４３ is a hydrogen atom, and R ２２ is a hydrogen atom, and R ３１ is a methyl group; １ -P(=O)(OR １１ )(OR １２ ) among R １１ and R １２are both groups that are hydrogen ions, sodium ions, potassium ions, ammonium ions, tetrabutylammonium ions, methyl groups, or benzyl groups, and R ４１ and R ４２ are each independently a hydrogen atom, a hydroxy group, a fluorine atom, or a methoxy group, and R ４３ is a hydrogen atom, and R ２２ is a hydrogen atom, and R ３１ More preferred are compounds in which R is a methyl group; １ -P(=O)(OR １１ )(OR １２ ) among R １１ and R １２ are both ammonium ions, and R ４１ and R ４２ is a hydroxy group, and R ４３ is a hydrogen atom, and R ２２ is a hydrogen atom, and R ３１ More preferred are compounds in which is a methyl group.

[0082] Among these, the compound (1) is preferably a compound represented by the general formula (1-1), and the compound (2) is preferably a compound represented by the general formula (2-1).

[0083] As the compound represented by general formula (1-1), a compound represented by the following general formula (1-1-1) is particularly preferred. The compound represented by general formula (1-1-1) is alkylated in the presence of a TMS group-containing compound to synthesize a compound represented by the following general formula (2-1-1). In general formulas (1-1-1) and (2-1-1), R １ is the same as in general formula (1), and R ３１ is the same as general formula (r3).

[0084]

[0085] In the general formulas (1-1-1) and (2-1-1), R １ Examples of the aryl group include a hydrogen atom, an alkali metal atom, and -P(=O)(OH)(OCH ３ ), -P(=O)(OCH ３ ) ２ , -P(=O)(OH)(OBn), -P(=O)(OBn)２ , -P(=O)(OCH ３ )(OBn), -P(=O)(O － ２ )・2[M １ ] ＋ ([M １ ] ＋ is preferably a monovalent cation), and is preferably a hydrogen atom, an alkali metal atom, or -P(=O)(O － ２ )・2[M １ ] ＋ is more preferred, and a hydrogen atom or an alkali metal atom is more preferred, or a hydrogen atom or —P(═O)(O － ２ )・2[M １ ] ＋ ([M １ ] ＋ is more preferably an ammonium ion). １ ] ＋ is most preferably a hydrogen ion. In particular, R １ -P(=O)(OR １１ )(OR １２ ), acid treatment can be carried out to obtain [M １ ] ＋ It is preferable that R is substituted with a hydrogen ion. ３１ As the alkyl group, a methyl group, an ethyl group, or a propyl group is preferred, and a methyl group is particularly preferred.

[0086] In the method for producing an alkylated uracil derivative according to the present invention, the N1 position of the pyrimidine base of compound (1) can be selectively alkylated by using a TMS group-containing compound. Therefore, a protection reaction or deprotection reaction of the hydroxy group in compound (1) is not required before the alkylation reaction, and compound (2) can be obtained from compound (1) in a single step of alkylation alone.

[0087] The TMS group-containing compound used in the method for producing an alkylated uracil derivative according to the present invention is not particularly limited, but is preferably a compound represented by the following general formula (e1), (e2), or (e3) in that it can be synthesized in a sufficient yield.

[0088]

[0089] In general formula (e1), R ５１ is a hydrogen atom or C １－６ R is an alkyl group, preferably a hydrogen atom or a methyl group. ５２ is -CO-CH ３ , —CO—CF ３ , —CO—NH-TMS, or —TMS, preferably —CO—CH ３ , —CO—CF ３ , or -TMS.

[0090] In general formula (e2), R ５３ is a hydrogen atom or C １－６ It is an alkyl group, preferably a methyl group.

[0091] Specific examples of the compound (e1) or the compound (e2) include the following compounds: In the following formula, "TMS" represents a trimethylsilyl group.

[0092]

[0093] In general formula (e3), ring E is a 5-membered or 6-membered ring. Ring E may be an aromatic ring or an aliphatic ring. Specific examples of ring E include 5-membered aliphatic heterocycles such as a pyrrolidine ring, an imidazolidine ring, a pyrazolidine ring, an oxazolidine ring, and an isoxazolidine ring; 6-membered aliphatic heterocycles such as a piperidine ring, a piperazine ring, and a morpholine ring; 5-membered aromatic heterocycles such as a pyrrole ring, an imidazole ring, a pyrazole ring, an oxazole ring, an isoxazole ring, a thiazole ring, and an isothiazole ring; and 6-membered aromatic heterocycles such as a pyridine ring, a pyrimidine ring, a pyrazine ring, a pyridazine ring, and an oxazine ring. As the TMS group-containing compound used in the method for producing an alkylated uracil derivative according to the present invention, among the compounds (e3), a 5- or 6-membered aromatic heterocycle is particularly preferred, a 5-membered aromatic heterocycle is more preferred, a pyrrole ring, an imidazole ring, or a pyrazole ring is more preferred, and an imidazole ring is even more preferred, from the viewpoint that a higher yield can be expected.

[0094] The TMS group-containing compound used in the method for producing an alkylated uracil derivative according to the present invention is preferably a silylating agent, since a higher yield can be expected. In particular, one or more methylating agents selected from the group consisting of N,O-bis(trimethylsilyl)acetamide (BSA) (CAS No.: 10416-59-8), N-trimethylsilylimidazole (CAS No.: 18156-74-6), N-methyl-N-trimethylsilylacetamide (CAS No.: 7449-74-3), N-methyl-N-trimethylsilyltrifluoroacetamide (CAS No.: 24589-78-4), and bis(trimethylsilyl)amine (CAS No.: 999-97-3) are preferred.

[0039] More preferred are one or more methylating agents selected from the group consisting of N-methyl-N-trimethylsilylacetamide (CAS No.: 18156-74-6), N-methyl-N-trimethylsilylacetamide (CAS No.: 7449-74-3), and bis(trimethylsilyl)amine (CAS No.: 999-97-3), and even more preferred are one or more methylating agents selected from the group consisting of BSA, N-trimethylsilylimidazole, and N-methyl-N-trimethylsilylacetamide.

[0095] The amount of the TMS group-containing compound to be added to the reaction system of the alkylation reaction is not particularly limited, but from the viewpoints of reaction efficiency and cost, it is preferably 0.1 to 20.0 equivalents of compound (1), more preferably 1.0 to 20.0 equivalents, even more preferably 1.0 to 10.0 equivalents, and particularly preferably 1.0 to 5.0 equivalents.

[0096] The alkyl group donor used in the alkylation reaction is not particularly limited and may be appropriately selected from compounds commonly used as alkyl group donors, such as methyl halides. Examples of the methyl halides used in the alkylation reaction include iodomethane, bromomethane, and methyl trifluoromethanesulfonate, with iodomethane being preferred.

[0097] The amount of the alkyl group donor added to the reaction system of the alkylation reaction may be at least a stoichiometric amount. From the viewpoints of reaction efficiency and cost, the amount of the alkyl group donor used in the alkylation reaction is preferably 1 to 20 equivalents, more preferably 1 to 10 equivalents, of compound (1).

[0098] The alkylation reaction can be carried out in a solvent inert to the reaction. The inert solvent is not particularly limited, but an aprotic polar solvent is preferred. Examples of aprotic polar solvents include dichloromethane (DCM), acetonitrile (MeCN), N,N'-dimethylformamide (DMF), N,N-dimethylacetamide, dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), diethyl ether, and dimethyl carbonate. The solvent used in the reaction may be a mixed solvent of two or more solvents. The reaction solvent used in the method for producing an alkylated uracil derivative according to the present invention is preferably at least one selected from the group consisting of dichloromethane, dimethyl carbonate, and acetonitrile, and more preferably at least one selected from the group consisting of dichloromethane and acetonitrile, since a higher yield can be expected.

[0099] The alkylation reaction is carried out by mixing compound (1), an alkyl group donor, and a TMS group-containing compound in a reaction solvent, and reacting the resulting mixture at an appropriate temperature for an appropriate time. The alkylation reaction proceeds under mild conditions. The reaction temperature is not particularly limited as long as the reaction solvent is in a liquid state, and the reaction can be carried out at a temperature of -40 to 130°C, preferably -40 to 80°C, and more preferably 10 to 50°C. R in general formula (2-1-1) １When synthesizing a compound in which R is a hydrogen atom, the target compound can be obtained in essentially quantitative yield by using one or more compounds selected from the group consisting of N,O-bis(trimethylsilyl)acetamide, N-trimethylsilylimidazole, N-methyl-N-trimethylsilylacetamide, and bis(trimethylsilyl)amine as the TMS group-containing compound and reacting at 10 to 50°C, preferably 20 to 40°C, for 12 to 24 hours. １ -P(=O)(OR １１ )(OR １２ When synthesizing a compound having the formula (I), the target compound can be obtained in essentially quantitative yield by using, as the TMS group-containing compound, one or more compounds selected from the group consisting of N,O-bis(trimethylsilyl)acetamide, N-trimethylsilylimidazole, N-methyl-N-trimethylsilylacetamide, N-methyl-N-trimethylsilyltrifluoroacetamide, and bis(trimethylsilyl)amine (more preferably N,O-bis(trimethylsilyl)acetamide or N-methyl-N-trimethylsilyltrifluoroacetamide), and reacting at −40 to 60° C., preferably −40 to 40° C., more preferably −30 to 10° C., for 12 to 24 hours.

[0100] <Phosphorylation Reaction> In the method for producing an alkylated uracil derivative according to the present invention, compound (2) is obtained from compound (1) by the alkylation reaction, and then compound (2) is phosphorylated, thereby synthesizing a compound represented by the following general formula (3):

[0101]

[0102] In general formula (3), R ３ and ring A are the same as in general formula (2).

[0103] In general formula (3), M ３ and M ４ are each independently a monovalent cation, and n is an integer of 1 to 3. When n is 2 or 3, a plurality of M ３ may be the same ion or different ions.３ and an oxygen atom, and M ４ and oxygen atoms are bonded by ionic bonds.

[0104] M ３ and M ４ The cation is not particularly limited as long as it is a monovalent cation, and examples thereof include hydrogen ions, alkali metal ions (Na ＋ , K. ＋ ammonium ions, quaternary ammonium ions (tetrabutylammonium ions, imidazolium ions, pyridinium ions, etc.), and the like.

[0105] The compound represented by the general formula (3) is preferably a compound represented by the following general formulas (3-1) to (3-4).

[0106]

[0107] In the general formulas (3-1) to (3-4), R １ is the same as in the general formula (1), and R ２２ is the same as in general formula (r2-1), and R ４１ , R ４２ , R ４３ , and R ａ is the same as in general formula (a-1), and R ３１ is the same as the general formula (r3), and M ３ , M ４ and n are the same as in the general formula (3).

[0108] Compound (3) is preferably a triphosphate compound in which n is 3, since it is suitable as a raw material for nucleic acid synthesis.

[0109] The phosphorylation reaction from compound (2) to compound (3) can be carried out by an organic synthesis reaction using a phosphorylating agent that is commonly used in the synthesis of phosphate esters. １ is a hydrogen atom or an alkali metal atom, phosphoryl chloride (POCl ３Compound (3) where n is 3 can be synthesized by a two-step phosphorylation reaction using bis(tetrabutylammonium)dihydrogen pyrophosphate (CAS RN: 857447-79-1) and then bis(tetrabutylammonium)dihydrogen pyrophosphate (CAS RN: 857447-79-1). １ -P(=O)(OR １１ )(OR １２ ), compound (3) in which n is 3 can be synthesized by phosphorylation using bis(tetrabutylammonium)dihydrogen pyrophosphate.

[0110] The phosphorylation reaction can be carried out in a variety of solvents inert to the reaction. ３ Phosphorylation using bis(tetrabutylammonium)dihydrogen pyrophosphate can be carried out in trimethyl phosphate (TMP) in the presence of a proton sponge (1,8-bis(dimethylamino)naphthalene) (CAS RN: 20734-58-1). Phosphorylation using bis(tetrabutylammonium)dihydrogen pyrophosphate can be carried out in N-methylimidazole and DMF.

[0111] The amount of the phosphorylating agent added to the reaction system of the phosphorylation reaction may be at least a stoichiometric amount. From the viewpoints of reaction efficiency and cost, the amount of the phosphorylating agent used in the phosphorylation reaction is preferably 1 to 20 equivalents, more preferably 1 to 10 equivalents, of compound (2).

[0112] The phosphorylation reaction is carried out by mixing compound (2) and a phosphorylating agent in a reaction solvent, and then reacting the resulting mixture at an appropriate temperature for an appropriate time. To monophosphorylate compound (2), the reaction is carried out at −20 to 20° C., preferably −10 to 10° C., and particularly preferably 0° C., for 3 to 24 hours, preferably 6 to 18 hours, and more preferably 9 to 15 hours. To triphosphorylate compound (2), the reaction is carried out at −20 to 20° C., preferably −10 to 10° C., and particularly preferably 0° C., for 10 minutes to 6 hours, preferably 10 minutes to 2 hours, and more preferably 15 minutes to 1 hour.

[0113] Compounds (2) and (3) synthesized by the method for producing alkylated uracil derivatives according to the present invention can be used as starting materials for nucleic acids, similar to known N1-methylpseudouridine. That is, compound (2) or compound (3) is synthesized by the method for producing alkylated uracil derivatives according to the present invention, and nucleic acids are synthesized using the resulting compound as a starting material. The triphosphate group of compound (3) is protected by a 4,4'-dimethoxytrityl (DMTr) group, and the hydrogen atom of the hydroxyl group in the ribose of compound (3) is substituted with a phosphorus atom bonded to a cyanoethoxy group and a diisopropylamino group to synthesize a phosphoramidite. The resulting phosphoramidite, along with phosphoramidites of other bases, can be used as starting materials to synthesize nucleic acids of the desired base sequence by the phosphoramidite method. Nucleic acid synthesis by the phosphoramidite method can be easily performed using commercially available automated synthesizers.

[0114] The synthesized target nucleic acid can be isolated and purified by various methods such as ion chromatography, gel filtration chromatography, reverse phase chromatography, and normal phase chromatography.

[0115] The nucleic acid synthesized using compound (2) or compound (3) synthesized by the method for producing an alkylated uracil derivative according to the present invention as a starting material is not particularly limited. The nucleic acid is preferably a functional nucleic acid that exhibits some physiological activity upon incorporation into target cells in vivo. Specific examples include nucleic acids for expressing proteins in cells, nucleic acids for suppressing gene expression in cells, nucleic acids for gene modification, nucleic acid aptamers that specifically bind to target biomolecules, and functional nucleic acids that affect the physiological functions of cells. Examples of nucleic acids for protein expression include mRNAs that encode proteins. Examples of nucleic acids for suppressing gene expression include functional nucleic acids used in RNA interference, such as siRNA, miRNA, shRNA, and antisense oligonucleotides.

[0116] The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[0117] Nuclear magnetic resonance (NMR) spectra were measured at ambient temperature using an NMR instrument (Bruker, AvanceNEO 400 MHz) unless otherwise specified. １ H-NMR was measured using tetramethylsilane as a standard. Mass spectrometry (LC-MS) was performed using a liquid chromatograph mass spectrum system (Agilent Technologies, LCMS6120B).

[0118] Example 1 N1-methylpseudouridine was synthesized in one step from pseudouridine using various TMS group-containing compounds as methylating agents.

[0119]

[0120] The alkylation reaction was carried out in a 25 cm ３ The reaction was carried out in a glass vessel. Pseudouridine (0.05 mmol) was dissolved in dichloromethane (DCM) (1 mL) in the glass vessel, and a TMS group-containing compound (TMS reagent) (2.5 equivalents) shown in Table 1 was added thereto, followed by stirring at room temperature for 1 hour. Subsequently, iodomethane (MeI) (1.5 equivalents, 0.005 mL) was added to the solution in the glass vessel, followed by stirring at 40°C for 16 hours. After cooling the reaction solution in the glass vessel to room temperature, the solvent was distilled off to obtain a crude product. The crude product was purified by silica column chromatography to obtain the target compound (N1-methylpseudouridine) as a colorless, transparent oil. The yield (%) of the target compound obtained is shown in Table 1.

[0121] When N,O-bis(trimethylsilyl)acetamide (0.07 mL) was used as the TMS group-containing compound (Test Group 1 in Table 1), 136.0 mg of the target compound (N1-methylpseudouridine) was obtained.

[0122]

[0123] As shown in Table 1, the target N1-methylpseudouridine was obtained in relatively high yields in all test plots in which a TMS group-containing compound was used as a methylating agent. Among them, the yield was high, at 70% or more, in test plots 1 to 4, 6, and 7, and particularly in test plots 1, 6, and 7, the yield was 98% or more, indicating that quantitative production was achieved.

[0124] Example 2 The effect of the solvent on the methylation reaction of pseudouridine using a TMS group-containing compound as a methylating agent was investigated.

[0125]

[0126] Specifically, the methylation reaction was carried out in the same manner as in Test Plot 1 of Example 1, except that the solvents shown in Table 2 were used. The yields (%) of the target compounds obtained are shown in Table 2. Test Plot 1 in Table 2 was carried out under the same conditions as Test Plot 1 in Table 1. In Table 2, "AcO ｎ "Bu" means butyl acetate, and "2-Me-THF" means 2-methyltetrahydrofuran. "NR" means that the raw material was returned without reacting at all.

[0127]

[0128] As shown in Table 2, the target N1-methylpseudouridine was obtained in all of the test plots 1 to 5 and 7, which used aprotic polar solvents. In particular, the test plots 1, 5, and 7, which used highly polar solvents, had high yields of 60% or more, and in particular, the yields in test plots 1 and 5 were 97% or more, meaning that the product was obtained almost quantitatively.

[0129] Example 3 N1-methylpseudouridine monophosphate ammonium salt was synthesized in one step from pseudouridine monophosphate ammonium salt using various TMS group-containing compounds as methylating agents.

[0130]

[0131] The alkylation reaction was carried out in a 25 cm ３The reaction was carried out in a glass vessel. Trifluoroacetic acid (TFA) (10.0 equivalents, 0.5 mmol) was added to pseudouridine monophosphate ammonium salt (0.05 mmol) in the glass vessel, and the mixture was stirred at room temperature for 10 minutes. The solution was then distilled off using an evaporator. The mixture was again dissolved in acetonitrile (MeCN) (1 mL), and a TMS group-containing compound (TMS reagent) (8.0 equivalents) listed in Table 3 was added, followed by stirring at 60°C for 1 hour. Iodomethane (MeI) (1.5 equivalents, 0.005 mL) was then added to the solution in the glass vessel, and the mixture was stirred at 60°C for 2 hours. The reaction solution in the glass vessel was cooled to room temperature, and the solvent was distilled off to obtain a crude product. The crude product was purified by silica column chromatography to obtain the target compound (N1-methylpseudouridine) as a colorless, transparent oil. The yield (%) of the target compound obtained is shown in Table 3.

[0132]

[0133] As shown in Table 3, in all test groups in which a TMS group-containing compound was used as a methylating agent, the target N1-methylpseudouridine monophosphate ammonium salt was obtained in high yield.

[0134] The present invention provides a production method that allows for the simple synthesis of an N1-alkylated uracil derivative from a uracil derivative in a single step. N1-alkylated uracil derivatives, including N1-methylpseudouridine, are more stable in vivo than uridine and are useful for improving the stability of nucleic acids administered to living organisms. Therefore, the present invention is particularly useful for synthesizing nucleic acids that are active ingredients in pharmaceuticals.

Claims

1. The following general formula (1) [In the formula, R １ is a hydrogen atom, an alkali metal atom, or -P(=O)(OR １１ )(OR １２ ) (R １１ and R １２ each independently represents a monovalent cation, an alkyl group having 1 to 6 carbon atoms which may have a substituent, or an acyl group having 2 to 7 carbon atoms; ring A is —OR １ and-R ２ represents a 5- to 8-membered ring which may further have a substituent in addition to the above; １ is other than a hydrogen atom, then ring A is substituted with at least one hydroxy group; ２ is expressed by the following formula (r2): is a group containing a structure represented by the following general formula (2): [In the formula, R １ and ring A is the same as above; R ３ is expressed by the following formula (r3): (R ３１ is an alkyl group having 1 to 6 carbon atoms.) represents a group containing a structure represented by the following formula:

2. Furthermore, the compound represented by the general formula (2) is phosphorylated to obtain a compound represented by the following general formula (3): [In the formula, R ３ and ring A is the same as above; M ３ and M ４ are each independently a monovalent cation; n is an integer of 1 to 3; when n is 2 or 3, a plurality of M ３ and may be the same ion or different ions.

3. The above R ２ is represented by the following general formula (r2-1) or (r2-2): [In the formula, R ２２ represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom; ring B represents a 5- to 8-membered ring which may have a substituent; and the black circle represents the bonding site with ring A. ３ is represented by the following general formula (r3-1) or (r3-2): [In the formula, R ２２ , ring B, and black circle are the same as above, and R ３１ is an alkyl group having 1 to 6 carbon atoms.] The method for producing an alkylated uracil derivative according to claim 1 or 2.

4. The method for producing an alkylated uracil derivative according to claim 3, wherein ring B is a benzene ring, a pyridine ring, or a furan ring.

5. The ring A is represented by the following general formula (a-1): [In the formula, R ４１ and R ４２ are each independently a hydrogen atom, a hydroxy group, an alkoxy group having 1 to 6 carbon atoms, a halogen atom, or —NHR′ (R′ is a protecting group); R ４３ is a hydrogen atom, or R ４１ is a group that bonds to form a ring structure; R ａ represents a hydroxy group, an alkoxy group having 1 to 6 carbon atoms, a halogen atom, or a methylene group optionally substituted with —NHR′ (R′ is a protecting group), or —NR′—; a1 is 0 or 1; one black circle represents R ２ The two black circles represent the binding site for -OR １ where R １ is other than a hydrogen atom, R ４１ and R ４２ At least one of a1 is a hydroxy group, or a1 is 1 and R ａ is a methylene group substituted with a hydroxy group.

6. The compound represented by the general formula (1) is represented by the following general formulas (1-1) to (1-4): [In the formula, R １ is a hydrogen atom, an alkali metal atom, or -P(=O)(OR １１ )(OR １２ ) (R １１ and R １２ each independently represents a monovalent cation, an alkyl group having 1 to 6 carbon atoms which may have a substituent, or an acyl group having 2 to 7 carbon atoms; R ２２ is a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom; R ４１ and R ４２ are each independently a hydrogen atom, a hydroxy group, an alkoxy group having 1 to 6 carbon atoms, a halogen atom, or —NHR′ (R′ is a protecting group); R ４３ is a hydrogen atom, or R ４１ is a group that bonds to form a ring structure; R ａ is a hydroxy group, an alkoxy group having 1 to 6 carbon atoms, a halogen atom, or a methylene group which may be substituted with —NHR′, or —NR′—. １ is other than a hydrogen atom, R ４１ and R ４２ At least one of R is a hydroxy group, or ａ is a methylene group substituted with a hydroxy group.], and the compound represented by the general formula (2) is a compound represented by any one of the following general formulas (2-1) to (2-4): [In the formula, R １ , R ２２ , R ４１ , R ４２ , R ４３ , and R ａ is the same as above; R ３１ and n is an alkyl group having 1 to 6 carbon atoms.] The method for producing an alkylated uracil derivative according to claim 1, 7. The compound represented by the general formula (1) is represented by the following general formulas (1-1) to (1-4): [In the formula, R １ is a hydrogen atom, an alkali metal atom, or -P(=O)(OR １１ )(OR １２ ) (R １１ and R １２ each independently represents a monovalent cation, an alkyl group having 1 to 6 carbon atoms which may have a substituent, or an acyl group having 2 to 7 carbon atoms; R ２２ is a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom; R ４１ and R ４２ are each independently a hydrogen atom, a hydroxy group, an alkoxy group having 1 to 6 carbon atoms, a halogen atom, or —NHR′ (R′ is a protecting group); R ４３ is a hydrogen atom, or R ４１ is a group that bonds to form a ring structure; R ａ is a hydroxy group, an alkoxy group having 1 to 6 carbon atoms, a halogen atom, or a methylene group which may be substituted with —NHR′, or —NR′. １ is other than a hydrogen atom, R ４１ and R ４２ At least one of R is a hydroxy group, or ａ is a methylene group substituted with a hydroxy group.], and the compound represented by the general formula (2) is a compound represented by any one of the following general formulas (2-1) to (2-4): [In the formula, R １ , R ２２ , R ４１ , R ４２ , R ４３ , and R ａ is the same as above; R ３１ is an alkyl group having 1 to 6 carbon atoms.], and the compound represented by the general formula (3) is a compound represented by any one of the following general formulas (3-1) to (3-4): [In the formula, R ３１ , R １ , R ２２ , R ４１ , R ４２ , R ４３ , and R ａ is the same as above; M ３ and M ４ are each independently a monovalent cation; n is an integer of 1 to 3; when n is 2 or 3, a plurality of M ３ and may be the same type of ion or different types of ions.] 8. The compound represented by the general formula (1) is represented by the following general formula (1-1-1): [In the formula, R １ is the same as the general formula (1).], and the compound represented by the general formula (2) is a compound represented by the following general formula (2-1-1): [In the formula, R １ and R ３１ is the same as the general formula (2).

9. The trimethylsilyl group-containing compound is represented by the following general formula (e1), (e2), or (e3): [In the formula, R ５１ is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; R ５２ is -CO-CH ３ , —CO—CF ３ , —CO—NH-TMS, or —TMS (TMS is a trimethylsilyl group); R ５３ is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; and ring E is a 5-membered or 6-membered ring.

10. The method for producing an alkylated uracil derivative according to claim 9, wherein ring E is a 5- or 6-membered aromatic heterocycle.

11. The method for producing an alkylated uracil derivative according to claim 1 or 2, wherein the trimethylsilyl group-containing compound is at least one selected from the group consisting of N,O-bis(trimethylsilyl)acetamide, N-trimethylsilylimidazole, N-methyl-N-trimethylsilylacetamide, and bis(trimethylsilyl)amine.

12. The method for producing an alkylated uracil derivative according to claim 1 or 2, wherein the alkylation reaction is carried out in an aprotic polar solvent.

13. The method for producing an alkylated uracil derivative according to claim 12, wherein the aprotic polar solvent is one or more selected from the group consisting of dichloromethane, dimethyl carbonate, and acetonitrile.

14. The method for producing an alkylated uracil derivative according to claim 1 or 2, wherein the alkylation reaction is carried out at 10 to 50°C.

15. A method for producing nucleic acid, comprising synthesizing a compound represented by the general formula (2-1-1) by the method for producing an alkylated uracil derivative according to claim 8, and synthesizing nucleic acid using the compound obtained as a starting material.

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