JP6270742B2 - Method for producing cross-linked nucleic acid derivative - Google Patents

Description

The present invention relates to a method for producing an intermediate useful for the efficient production of amide-bridged nucleic acid derivatives.

    An antisense method is known as one of methods for treating diseases with nucleic acid drugs. Antisense method introduces oligonucleotide (antisense strand) complementary to disease-related mRNA from the outside to form a double strand, thereby inhibiting the translation process of pathogenic RNA and treating or preventing disease. It is a technique to do.

    When natural oligonucleotides are applied to the antisense method, there are problems such as being hydrolyzed by enzymes in the body and not having high cell membrane permeability. In order to solve this problem, many nucleic acid derivatives have been synthesized and studied. For example, phosphorothioate in which an oxygen atom on a phosphorus atom is substituted with a sulfur atom (Non-patent Document 1), methylphosphonate in which a methyl group is substituted (Non-Patent Document 2), BNA (bridged nucleic acid) and LNA (locked nucleic acid) ) And the like are synthesized (Patent Documents 1 to 3).

    Under these circumstances, Obiga et al. Are less susceptible to degradation by nucleases in vivo than conventional cross-linked types, have high binding affinity for target mRNA, and efficiently control the expression of specific genes. As a molecule applicable to the antisense method, a novel nucleic acid derivative in which an amide bond is introduced into a 2 ′, 4′-BNA cross-linked structure has been found (Patent Document 4).

    However, a multi-step process is required to produce this novel amide-bridged nucleic acid derivative, leaving room for improvement in terms of a production method for mass synthesis.

International Publication No. 2003/066875 International Publication No. 2005/021570 International Publication No. 2011/156202 International Publication No. 2011/052436

F. Eckstein et al. Biochem. , 18, 592 (1979) P. S. Miller et al. , Nucleic Acids Res. , 11, 5189 (1983)

An object of the present invention is to provide a method for efficiently producing an amide-bridged nucleic acid derivative useful as a molecule used in an antisense method.

The present inventors have found that an important intermediate for synthesizing an amide-bridged nucleic acid derivative can be efficiently synthesized by carrying out an aldol reaction under a specific condition, thereby completing the present invention. Further, the present invention was completed by finding a method for efficiently removing a ring by removing the protecting group of the intermediate.

（式中、Ｒ_１及びＲ_２は、同一または異なって、水素原子、核酸合成のアミノ基の保護基、アルキル基、アルケニル基、シクロアルキル基、アリール基、アラルキル基、アシル基、シリル基、リン酸基、核酸合成の保護基で保護されたリン酸基、−Ｐ（Ｒ₇）Ｒ₈［式中、Ｒ₇およびＲ₈は、同一または異なって、水酸基、核酸合成の保護基で保護された水酸基、メルカプト基、核酸合成の保護基で保護されたメルカプト基、アミノ基、炭素数１〜５のアルコキシ基、炭素数１〜５のアルキルチオ基、炭素数１〜６のシアノアルコキシ基、または、炭素数１〜５のアルキル基で置換されたアミノ基を示す］；
Ｒ_３は、水素原子、核酸合成の水酸基の保護基、アルキル基、アルケニル基、シクロアルキル基、アリール基、アラルキル基、アシル基、シリル基、リン酸基、核酸合成の保護基で保護されたリン酸基、−Ｐ（Ｒ_９）Ｒ_１０［式中、Ｒ_９およびＲ_１０は、同一または異なって、水酸基、核酸合成の保護基で保護された水酸基、メルカプト基、核酸合成の保護基で保護されたメルカプト基、アミノ基、炭素数１〜５のアルコキシ基、炭素数１〜５のアルキルチオ基、炭素数１〜６のシアノアルコキシ基、または、炭素数１〜５のアルキル基で置換されたアミノ基を示す］；
Ｒ_４は、水素原子、水酸基、または、炭素数１〜５のアルコキシ基；
Ｒ_５及びＲ_６は、同一または異なって、水素原子、炭素数１〜５のアルキル基、またはアリール基；
Ｂは、置換基を有していてもよい核酸塩基部分）
を含む、一般式（II）で表される化合物の製造方法、
（２）一般式（I）で表される化合物を塩基の存在下で式：Ｒ_５Ｃ（＝Ｏ）Ｒ_６で表される化合物と反応させる以下に示す工程：

（式中、各定義は前記（１）と同意義）を含む、一般式（II）で表される化合物の製造方法、
（３）前記塩基が、共役酸のｐＫａが６〜１０のものである前記（１）または（２）記載の製造方法、
（４）前記塩基が、トリエチルアミン、またはＮ−メチルモルホリンである前記（３）記載の製造方法、
（５）前記（１）〜（４）のいずれかに記載の方法により一般式（II）で表される化合物を得た後、Ｒ_４が、水素原子である場合、得られた一般式（II）で表される化合物を酸化反応に付する以下の工程：

（式中、Ｒ_４は水素原子、それ以外の定義は前記（１）と同意義）を含む、一般式（III）で表される化合物の製造方法、
（６）Ｒ₁およびＲ₂のいずれか一方が核酸合成のアミノ基の保護基であり、その他方が水素原子またはアルキル基である前記（１）〜（５）のいずれかに記載の製造方法、
（７）Ｒ₃が核酸合成の水酸基の保護基である前記（１）〜（６）のいずれかに記載の製造方法、
（８）Ｒ₄が水素原子である前記（１）〜（７）のいずれかに記載の製造方法、
（９）Ｒ₅およびＲ₆がいずれも水素原子である前記（１）〜（８）のいずれかに記載の製造方法、
（１０）Ｂが置換基を有していてもよい、ピリミジン核酸塩基もしくはプリン核酸塩基である前記（１）〜（９）のいずれかに記載の製造方法、
（１１）下記一般式（III）で表される化合物およびその塩

（式中、Ｒ_３は核酸合成の水酸基の保護基、それ以外の定義は前記（１）と同意義）、
（１２）一般式（III）で表される化合物を脱シリル化剤と反応させ、その反応液に縮合剤を反応させる以下に示す工程：

（式中、Ｒ_１は、炭素数１〜５のアルキル基、Ｒ_２は、核酸合成のアミノ基の保護基、Ｒ_３は核酸合成の水酸基の保護基であってシリル基を有する保護基、それ以外の定義は前記（１）と同意義）
を含む、一般式（ＩＶ）で表される化合物の製造方法、
（１３）前記（１２）に記載の方法により一般式（ＩＶ）で表される化合物を得た後、得られた一般式（ＩＶ）で表される化合物に、核酸合成の水酸基の保護に使用する試薬を反応させる以下に示す工程：

（式中、Ｒ_７は核酸合成の水酸基の保護基、それ以外の定義は前記（１２）と同意義）
を含む、一般式（Ｖ）で表される化合物の製造方法、
（１４）下記一般式（Ｖ）で表される化合物およびその塩、

（式中、各定義は、前記（１３）と同意義）
（１５）下記一般式（ＶＩ）で表される化合物をトランスグリコシル化反応に付する以下に示す工程：

（式中、Ｂ_１は置換基を有していてもよいピリミジン核酸塩基、Ｂ_２は置換基を有していてもよいチミン以外の核酸塩基、Ｘは水素原子、ナトリウム原子またはカリウム原子、それ以外の定義は前記（１）と同義）
を含む、一般式（ＶＩＩ）で表される化合物の製造方法、
（１６）下記一般式（ＶＩＩ）で表される化合物およびその塩、

（式中、各定義は、前記（１５）と同意義）
（１７）下記一般式（ＶＩＩＩ）で表される化合物をアルキルアミンと反応させる以下に示す工程：

（式中、Ｙ_１は置換基を有していてもよいアルキル基または置換基を有していてもよいフェニル基、Ｙ_２〜Ｙ_４は核酸合成の水酸基の保護基、Ｒ_１’およびＲ_２’は水素原子またはアルキル基、Ｂは置換基を有していてもよい芳香族複素環基）
を含む、一般式（ＩＸ）で表される化合物の製造方法、
（１８）下記一般式（Ｘ）で表される化合物をＮ−グリコシル化反応に付する以下に示す工程：

（式中、Ｙ_５は核酸合成の水酸基の保護基、Ｒ_４’はアルキル基、置換基を有していてもよい芳香環、Ｂは置換基を有していてもよい芳香族複素環基）
を含む、一般式（ＸＩ）で表される化合物の製造方法、および
（１９）下記一般式（ＸＩＩ）で表される化合物を分子内環化反応に付する以下に示す工程：

（式中、Ｙ_７およびＹ_８は核酸合成の水酸基の保護基、Ｒ_５’はアルキル基、水素原子、置換基を有していてもよいベンジル基、または置換基を有していてもよい芳香環、Ｒ_６’、Ｒ_７’、Ｒ_８’およびＲ_９’は水素原子またはアルキル基）を含む、一般式（ＸＩＩＩ）で表される化合物の製造方法、
に関する。That is, the present invention
(1) The following steps for subjecting a compound represented by the general formula (I) to an aldol reaction in the presence of a base:

(In the formula, R ₁ and R ₂ are the same or different and each represents a hydrogen atom, an amino group protecting group for nucleic acid synthesis, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a silyl group, Phosphate group, phosphate group protected with a protecting group for nucleic acid synthesis, -P (R ₇ ) R ₈ [wherein R ₇ and R ₈ are the same or different and protected with a hydroxyl group, a protecting group for nucleic acid synthesis Hydroxyl group, mercapto group, mercapto group protected with a protecting group for nucleic acid synthesis, amino group, alkoxy group having 1 to 5 carbon atoms, alkylthio group having 1 to 5 carbon atoms, cyanoalkoxy group having 1 to 6 carbon atoms, Or an amino group substituted with an alkyl group having 1 to 5 carbon atoms];
R ₃ was protected with a hydrogen atom, a hydroxyl protecting group for nucleic acid synthesis, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a silyl group, a phosphate group, or a protecting group for nucleic acid synthesis. Phosphoric acid group, —P (R ₉ ) R _{10 wherein} R ₉ and R ₁₀ are the same or different and are a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, a mercapto group, or a protecting group for nucleic acid synthesis. Substituted with a protected mercapto group, amino group, alkoxy group having 1 to 5 carbon atoms, alkylthio group having 1 to 5 carbon atoms, cyanoalkoxy group having 1 to 6 carbon atoms, or alkyl group having 1 to 5 carbon atoms Represents an amino group];
R ₄ represents a hydrogen atom, a hydroxyl group, or an alkoxy group having 1 to 5 carbon atoms;
R ₅ and R ₆ are the same or different and each represents a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, or an aryl group;
B is a nucleobase moiety optionally having a substituent)
A process for producing a compound represented by the general formula (II),
(2) reacting a compound represented by the general formula (I) with a compound represented by the formula: R ₅ C (═O) R ₆ in the presence of a base:

(Wherein each definition has the same meaning as in the above (1)), a method for producing a compound represented by the general formula (II),
(3) The production method according to (1) or (2), wherein the base is one having a pKa of a conjugate acid of 6 to 10,
(4) The production method according to (3), wherein the base is triethylamine or N-methylmorpholine,
(5) After obtaining the compound represented by the general formula (II) by the method according to any one of (1) to (4) above, when R ₄ is a hydrogen atom, the obtained general formula ( The following steps for subjecting the compound represented by II) to an oxidation reaction:

(Wherein, R ₄ is a hydrogen atom, and other definitions are as defined in the above (1)), a method for producing a compound represented by the general formula (III),
(6) The production method according to any one of (1) to (5), wherein either one of R ₁ and R ₂ is an amino-protecting group for nucleic acid synthesis, and the other is a hydrogen atom or an alkyl group. ,
(7) The production method according to any one of (1) to (6), wherein R ₃ is a hydroxyl-protecting group for nucleic acid synthesis,
(8) The production method according to any one of (1) to (7), wherein R ₄ is a hydrogen atom.
(9) The production method according to any one of (1) to (8), wherein both R ₅ and R ₆ are hydrogen atoms.
(10) The production method according to any one of (1) to (9), wherein B is a pyrimidine nucleobase or a purine nucleobase, which may have a substituent.
(11) A compound represented by the following general formula (III) and a salt thereof

(Wherein R ₃ is a protecting group for a hydroxyl group in nucleic acid synthesis, and other definitions are the same as defined in (1) above),
(12) reacting the compound represented by the general formula (III) with a desilylation agent and reacting the reaction solution with a condensing agent:

(Wherein R ₁ is an alkyl group having 1 to 5 carbon atoms, R ₂ is a protecting group for an amino group for nucleic acid synthesis, R ₃ is a protecting group for a hydroxyl group for nucleic acid synthesis and has a silyl group, Other definitions are the same as (1) above)
A process for producing a compound represented by the general formula (IV),
(13) After obtaining the compound represented by the general formula (IV) by the method described in the above (12), the obtained compound represented by the general formula (IV) is used for protecting hydroxyl groups in nucleic acid synthesis. The steps shown below for reacting the reagents to be reacted:

(Wherein R ₇ is a hydroxyl-protecting group for nucleic acid synthesis, and other definitions are the same as defined in (12) above)
A process for producing a compound represented by the general formula (V),
(14) A compound represented by the following general formula (V) and a salt thereof,

(Wherein each definition has the same meaning as (13) above)
(15) A step shown below for subjecting a compound represented by the following general formula (VI) to a transglycosylation reaction:

(Wherein B ₁ is a pyrimidine nucleobase which may have a substituent, B ₂ is a nucleobase other than thymine which may have a substituent, X is a hydrogen atom, sodium atom or potassium atom, Definitions other than are synonymous with (1) above)
A process for producing a compound represented by the general formula (VII),
(16) A compound represented by the following general formula (VII) and a salt thereof,

(Wherein each definition has the same meaning as in the above (15))
(17) The following process of reacting a compound represented by the following general formula (VIII) with an alkylamine:

(In the formula, Y ₁ is an optionally substituted alkyl group or an optionally substituted phenyl group, Y _{2 to} Y ₄ are hydroxyl protecting groups for nucleic acid synthesis, R _{1 ′} and R _{1) 2 ′} is a hydrogen atom or an alkyl group, B is an aromatic heterocyclic group which may have a substituent)
A process for producing a compound represented by the general formula (IX),
(18) Steps shown below for subjecting a compound represented by the following general formula (X) to N-glycosylation reaction:

(Wherein Y ₅ is a hydroxyl-protecting group for nucleic acid synthesis, R _{4 ′} is an alkyl group, an optionally substituted aromatic ring, and B is an optionally substituted aromatic heterocyclic group. )
A process for producing a compound represented by the general formula (XI), and (19) subjecting the compound represented by the following general formula (XII) to an intramolecular cyclization reaction:

Wherein Y ₇ and Y ₈ are hydroxyl-protecting groups for nucleic acid synthesis, R _{5 ′} is an alkyl group, a hydrogen atom, an optionally substituted benzyl group, or an optionally substituted group. An aromatic ring, R _{6 ′} , R _{7 ′} , R _{8 ′} and R _{9 ′} are a hydrogen atom or an alkyl group), and a method for producing a compound represented by the general formula (XIII),
About.

ADVANTAGE OF THE INVENTION According to this invention, the novel important intermediate required for manufacture of an amide bridge | crosslinking type nucleic acid derivative useful as a molecule | numerator used for an antisense method can be manufactured efficiently. More specifically, an important intermediate can be produced with a minimum of raw material residues and by-products. Further, by using this intermediate, it has become possible to produce an amide-bridged nucleic acid derivative useful as an antisense molecule more efficiently than the conventional method. Furthermore, it has become possible to obtain an intermediate in a high yield by finding a method for removing the protecting group of the intermediate and efficiently cyclizing the intermediate.

First, terms used in this specification are defined.

The “nucleobase moiety” of B is a structure in which a carbon atom which is a constituent atom of a hydrocarbon ring is replaced with one or more heteroatoms such as a nitrogen atom, a sulfur atom or an oxygen atom in addition to a hydrocarbon cyclic group And any heterocyclic group having a 5- to 20-membered ring exhibiting aromaticity and includes a single ring and a condensed ring. Specifically, examples of the hydrocarbon ring include benzene, naphthalene, anthracene, phenanthrene, indane, indene, tetrahydronaphthylene, biphenylene and the like. Examples of the heterocyclic ring include pyrimidine nucleobase or purine nucleobase. Any ring may have one or more substituents selected from the following α group. Here, the pyrimidine nucleobase or the purine nucleobase acts or substitutes as a base of a nucleic acid component generally known as a component of nucleic acid (for example, guanine, adenine, cytosine, thymine, uracil), and other similar nucleic acid components. Includes every chemical structure you get. Others, thiophene, thianthrene, furan, pyran, isobenzofuran, chromene, xanthene, phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridazine, indolizine, indole, isoindole, isoquinoline, quinoline, naphthyridine, quinoxaline, Also included are quinazoline, pteridine, carbazole, phenanthridine, acridine, perimidine, phenazine, phenalazine, phenothiazine, furazane, phenoxazine, pyrrolidine, pyrroline, imidazolidine, imidazoline, pyrazolidine and the like. Preferred are pyrimidine nucleobases or purine nucleobases, pyrimidine nucleobases or purine nucleobases which may have one or more substituents selected from the following α group, specifically, purine-9 A -yl group, a 2-oxo-pyrimidin-1-yl group, or a purin-9-yl group or a 2-oxo-pyrimidin-1-yl group having a substituent selected from the following α group is preferred.
α group: hydroxyl group, hydroxyl group protected with a protecting group for nucleic acid synthesis, alkoxy group having 1 to 5 carbon atoms, mercapto group, mercapto group protected with a protecting group for nucleic acid synthesis, alkylthio group having 1 to 5 carbon atoms, amino Group, an amino group protected with a protecting group for nucleic acid synthesis, an amino group substituted with an alkyl group having 1 to 5 carbon atoms, an alkyl group having 1 to 5 carbon atoms, and a halogen atom.

Here, a group suitable as “optionally substituted purine nucleobase” is 6-aminopurin-9-yl (ie, adeninyl), 6 in which the amino group is protected with a protecting group for nucleic acid synthesis. -Aminopurin-9-yl, 2,6-diaminopurin-9-yl, 2-amino-6-chloropurin-9-yl, 2-amino-6-amino group protected with a protecting group for nucleic acid synthesis Chloropurin-9-yl, 2-amino-6-fluoropurin-9-yl, 2-amino-6-fluoropurin-9-yl, 2-amino-6 in which the amino group is protected with a protecting group for nucleic acid synthesis -Bromopurin-9-yl, 2-amino-6-bromopurin-9-yl, amino-protected 2-amino-6-hydroxypurin-9-yl (ie, guanylyl) wherein the amino group is protected with a protecting group for nucleic acid synthesis The amino group is a protecting group for nucleic acid synthesis. Protected 2-amino-6-hydroxypurin-9-yl, 6-amino-2-methoxypurin-9-yl, 6-amino-2-chloropurin-9-yl, 6-amino-2-fluoropurine -9-yl, 2,6-dimethoxypurin-9-yl, 2,6-dichloroprolin-2-yl or 6-mercaptopurin-9-yl group, more preferably 6-benzoylaminopurine- 9-yl, adenynyl, 2-isobutyrylamino-6-hydroxypurin-9-yl or guaninyl group.

Further, a group suitable as “an optionally substituted pyrimidine nucleobase” is 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl (ie, cytosynyl), and an amino group is a nucleic acid. 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl protected with a synthetic protecting group, 2-oxo-4-amino-5-fluoro-1,2-dihydropyrimidin-1-yl, 2-oxo-4-amino-5-fluoro-1,2-dihydropyrimidin-1-yl, 4-amino-2-oxo-5-chloro-1, wherein the amino group is protected with a protecting group for nucleic acid synthesis -Dihydropyrimidin-1-yl, 2-oxo-4-methoxy-1,2-dihydropyrimidin-1-yl, 2-oxo-4-mercapto-1,2-dihydropyrimidin-1-yl, 2-oxo- 4-hide Xyl-1,2-dihydropyrimidin-1-yl (ie uracinyl), 2-oxo-4-hydroxy-5-methyl-1,2-dihydropyrimidin-1-yl (ie thyminyl) or 4-amino- 5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl (ie, 5-methylcytosinyl) group, more preferably 2-oxo-4-benzoylamino-1,2-dihydropyrimidine- 1-yl, cytosynyl, thyminyl, uracinyl, 2-oxo-4-benzoylamino-5-methyl-1,2-dihydropyrimidin-1-yl, or 5-methylcytosinyl group.

Among “optionally substituted purine nucleobases or pyrimidine nucleobases”, more preferably, 6-aminopurin-9-yl (ie, adeninyl), wherein the amino group is a protecting group for nucleic acid synthesis. Protected 6-aminopurin-9-yl, 2,6-diaminopurin-9-yl, 2-amino-6-chloropurin-9-yl, 2-amino group protected with a protecting group for nucleic acid synthesis Amino-6-chloropurin-9-yl, 2-amino-6-fluoropurin-9-yl, 2-amino-6-fluoropurin-9-yl in which the amino group is protected with a protecting group for nucleic acid synthesis, 2 -Amino-6-bromopurin-9-yl, 2-amino-6-bromopurin-9-yl, 2-amino-6-hydroxypurin-9-yl having an amino group protected with a protecting group for nucleic acid synthesis ( That is, Guaninyl), Ami 2-amino-6-hydroxypurin-9-yl, 6-amino-2-methoxypurin-9-yl, 6-amino-2-chloropurin-9-yl, wherein the group is protected with a protecting group for nucleic acid synthesis, 6-amino-2-fluoropurin-9-yl, 2,6-dimethoxypurin-9-yl, 2,6-dichloropurin-9-yl, 6-mercaptopurin-9-yl, 2-oxo-4- Amino-1,2-dihydropyrimidin-1-yl (ie, cytosynyl), 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl in which the amino group is protected with a protecting group for nucleic acid synthesis, 2 -Oxo-4-amino-5-fluoro-1,2-dihydropyrimidin-1-yl, 2-oxo-4-amino-5-fluoro-1,2-amino group protected with a protecting group for nucleic acid synthesis Dihydropyrimidine -Yl, 4-amino-2-oxo-5-chloro-1,2-dihydropyrimidin-1-yl, 2-oxo-4-methoxy-1,2-dihydropyrimidin-1-yl, 2-oxo-4 -Mercapto-1,2-dihydropyrimidin-1-yl, 2-oxo-4-hydroxy-1,2-dihydropyrimidin-1-yl (ie uracinyl), 2-oxo-4-hydroxy-5-methyl- 1,2-dihydropyrimidin-1-yl (ie, thyminyl), 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl (ie, 5-methylcytosinyl), or an amino group Is 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl protected with a protecting group for nucleic acid synthesis.

“Protecting group for amino group for nucleic acid synthesis” and “protecting group for hydroxyl group for nucleic acid synthesis” and “hydroxyl group protected by protecting group for nucleic acid synthesis” are an amino group or a stable group during nucleic acid synthesis. Although it is not particularly limited as long as it can protect a hydroxyl group, specifically, it is stable under acidic or neutral conditions, and is obtained by a chemical method such as hydrogenolysis, hydrolysis, electrolysis and photolysis. Examples of the protecting group that can be cleaved include, for example, formyl, acetyl, propionyl, butyryl, isobutyryl, pentanoyl, pivaloyl, valeryl, isovaleryl, octanoyl, nonanoyl, decanoyl, 3-methylnonanoyl, 8- Methylnonanoyl, 3-ethyloctanoyl, 3,7-dimethyloctanoyl, undecanoyl, dodecanoyl, tridecanoyl, te Radecanoyl, pentadecanoyl, hexadecanoyl, 1-methylpentadecanoyl, 14-methylpentadecanoyl, 13,13-dimethyltetradecanoyl, heptadecanoyl, 15-methylhexadecanoyl, octadecanoyl, 1-methylhepta Decanoyl, nonadecanoyl, 4-oxopentanoyl (levulinoyl), alkylcarbonyl groups such as eicosanoyl and henaicosanoyl, carboxylated alkylcarbonyl groups such as succinoyl, glutaroyl, adipoyl, chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl Such as halogeno lower alkylcarbonyl group, methoxyacetyl, phenoxyacetyl group, 2- (4-tert-butyl) phenoxyacetyl group An “aliphatic acyl group” such as an alkylcarbonyl group, an unsaturated alkylcarbonyl group such as (E) -2-methyl-2-butenoyl; methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s -Butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, “Lower” such as 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl Alkyl group "; ethenyl, 1-propenyl, 2-propenyl, 1-methyl-2-propenyl, 1- Methyl-1-propenyl, 2-methyl-1-propenyl, 2-methyl-2-propenyl, 2-ethyl-2-propenyl, 1-butenyl, 2-butenyl, 1-methyl-2-butenyl, 1-methyl- 1-butenyl, 3-methyl-2-butenyl, 1-ethyl-2-butenyl, 3-butenyl, 1-methyl-3-butenyl, 2-methyl-3-butenyl, 1-ethyl-3-butenyl, 1- Pentenyl, 2-pentenyl, 1-methyl-2-pentenyl, 2-methyl-2-pentenyl, 3-pentenyl, 1-methyl-3-pentenyl, 2-methyl-3-pentenyl, 4-pentenyl, 1-methyl- “Lower alkenyl such as 4-pentenyl, 2-methyl-4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl An arylcarbonyl group such as benzoyl, α-naphthoyl, β-naphthoyl, a halogenoarylcarbonyl group such as 2-bromobenzoyl, 4-chlorobenzoyl, 2,4,6-trimethylbenzoyl, 4-toluoyl, etc. Lower alkylated arylcarbonyl group, lower alkoxylated arylcarbonyl group such as 4-anisoyl, 2-carboxybenzoyl, 3-carboxybenzoyl, carboxylated arylcarbonyl group such as 4-carboxybenzoyl, 4-nitrobenzoyl, 2- Nitrated arylcarbonyl groups such as nitrobenzoyl; dialkylimino groups such as N, N-dimethylformimino group; lower alkoxycarbonylated arylcarbonyl groups such as 2- (methoxycarbonyl) benzoyl; 4-phenyl “Aromatic acyl groups” such as arylated arylcarbonyl groups such as benzoyl; tetrahydropyran-2-yl, 3-bromotetrahydropyran-2-yl, 4-methoxytetrahydropyran-4-yl, tetrahydrothiopyran- “Tetrahydropyranyl or tetrahydrothiopyranyl group” such as 4-yl, 4-methoxytetrahydrothiopyran-4-yl; “tetrahydrofuranyl or tetrahydro such as tetrahydrofuran-2-yl, tetrahydrothiofuran-2-yl” A thiofuranyl group ”; a tri-lower alkylsilyl group such as trimethylsilyl, triethylsilyl, isopropyldimethylsilyl, t-butyldimethylsilyl, methyldiisopropylsilyl, methyldi-t-butylsilyl, triisopropylsilyl; “Silyl groups” such as tri-lower alkylsilyl groups substituted with 1 to 2 aryl groups such as rumethylsilyl, diphenylbutylsilyl, diphenylisopropylsilyl, phenyldiisopropylsilyl; methoxymethyl, 1,1-dimethyl-1 “Lower alkoxymethyl groups” such as methoxymethyl, ethoxymethyl, propoxymethyl, isopropoxymethyl, butoxymethyl, t-butoxymethyl, 1-ethoxyethyl; “lower alkoxylated lower alkoxy” such as 2-methoxyethoxymethyl “Methyl group”; “halogeno lower alkoxymethyl group” such as 2,2,2-trichloroethoxymethyl and bis (2-chloroethoxy) methyl; “lower” such as 1-ethoxyethyl and 1- (isopropoxy) ethyl Alkoxylated ethyl group "; “Halogenated ethyl groups” such as 2,2-trichloroethyl; “benzyl, α-naphthylmethyl, β-naphthylmethyl, diphenylmethyl, triphenylmethyl, α-naphthyldiphenylmethyl, 9-anthrylmethyl” A methyl group substituted with 1 to 3 aryl groups "; 4-methylbenzyl, 2,4,6-trimethylbenzyl, 3,4,5-trimethylbenzyl, 4-methoxybenzyl, 4-methoxyphenyldiphenylmethyl, 4,4′-dimethoxytriphenylmethyl, 2-nitrobenzyl, 4-nitrobenzyl, 4-chlorobenzyl, 4-bromobenzyl, “lower alkyl, lower alkoxy, halogen, cyano group and aryl such as 4-cyanobenzyl A methyl group substituted with 1 to 3 aryl groups substituted on the ring; “Lower alkoxycarbonyl groups” such as nyl, ethoxycarbonyl, t-butoxycarbonyl, isobutoxycarbonyl; 4-chlorophenyl, 2-fluorophenyl, 4-methoxyphenyl, 4-nitrophenyl, 2,4-dinitrophenyl, etc. “Aryl group substituted with halogen atom, lower alkoxy group or nitro group”; “lower group substituted with halogen or tri-lower alkylsilyl group” such as 2,2,2-trichloroethoxycarbonyl, 2-trimethylsilylethoxycarbonyl “Alkoxycarbonyl group”; “alkenyloxycarbonyl group” such as vinyloxycarbonyl and aryloxycarbonyl; benzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 2 One or two “lower alkoxy or an aralkyloxycarbonyl group in which the aryl ring may be substituted with a nitro group” such as nitrobenzyloxycarbonyl and 4-nitrobenzyloxycarbonyl ”can be mentioned. In the “protecting group for hydroxyl group”, preferably, “aliphatic acyl group”, “aromatic acyl group”, “methyl group substituted with 1 to 3 aryl groups”, “lower alkyl, lower alkoxy, halogen” , A methyl group substituted with 1 to 3 aryl groups in which the aryl ring is substituted with a cyano group ”or“ silyl group ”, and more preferably an acetyl group, a benzoyl group, a benzyl group, p-methoxybenzoyl Group, dimethoxytrityl group, monomethoxytrityl group or tert-butyldiphenylsilyl group, tert-butyldimethylsilyl group In the protecting group of the group, 4-oxopentanoyl (levulinoyl) group, and “the hydroxyl group protected by the protecting group for nucleic acid synthesis”, preferably, “aliphatic acyl group”, “aromatic acyl group”, “Methyl group substituted with 1 to 3 aryl groups”, “aryl group substituted with halogen atom, lower alkoxy group or nitro group”, “lower alkyl group” or “lower alkenyl group”, more preferred Is a benzoyl group, a benzyl group, a 2-chlorophenyl group, a 4-chlorophenyl group, or a 2-propenyl group.

The “alkyl group” refers to a linear or branched alkyl group having 1 to 20 carbon atoms, and includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, tert-butyl, n -Pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2 Linear or branched having 1 to 6 carbon atoms such as 1,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl In addition to chain alkyl groups (in the present specification, these are also referred to as lower alkyl groups), heptyl, octyl, nonyl, deci Like include straight or branched chain alkyl group having 7 to 20 carbon atoms, preferably a straight or branched chain alkyl group having 1 to 6 carbon atoms described above.

The “alkenyl group” refers to a linear or branched alkenyl group having 2 to 20 carbon atoms, and includes ethenyl, 1-propenyl, 2-propenyl, 1-methyl-2-propenyl, 1-methyl-1-propenyl. 2-methyl-1-propenyl, 2-methyl-2-propenyl, 2-ethyl-2-propenyl, 1-butenyl, 2-butenyl, 1-methyl-2-butenyl, 1-methyl-1-butenyl, 3 -Methyl-2-butenyl, 1-ethyl-2-butenyl, 3-butenyl, 1-methyl-3-butenyl, 2-methyl-3-butenyl, 1-ethyl-3-butenyl, 1-pentenyl, 2-pentenyl 1-methyl-2-pentenyl, 2-methyl-2-pentenyl, 3-pentenyl, 1-methyl-3-pentenyl, 2-methyl-3-pentenyl, 4-pentenyl, 1-methyl C2-C6 linear or branched alkenyl such as ru-4-pentenyl, 2-methyl-4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl In addition to the groups (in the present specification, these are also referred to as lower alkenyl groups), geranyl, farnesyl, and the like are included, and preferably the above-described linear or branched alkenyl group having 2 to 6 carbon atoms. is there.

The “cycloalkyl group” refers to a cycloalkyl group having 3 to 10 carbon atoms, and examples thereof include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, norbornyl, adamantyl, and the like, preferably cyclopropyl, A cycloalkyl group having 3 to 8 carbon atoms such as cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and the like. In addition, the “cycloalkyl group” includes a heterocyclic group in which one or more methylenes on the ring of the cycloalkyl group are substituted with an oxygen atom, a sulfur atom, or a nitrogen atom substituted with an alkyl group, Examples thereof include a tetrahydropyranyl group.

The “aryl group” means a monovalent substituent having 6 to 14 carbon atoms obtained by removing one hydrogen atom from an aromatic hydrocarbon group, and examples thereof include phenyl, indenyl, naphthyl, phenanthrenyl, anthracenyl and the like. . Further, the aryl ring may be substituted with one or more groups such as a halogen atom, a lower alkyl group, a hydroxyl group, an alkoxy group, an aryloxy group, an amino group, a nitro group, trifluoromethyl, a phenyl group, Examples of the optionally substituted aryl group include 2-methylphenyl, 2,6-dimethylphenyl, 2-chlorophenyl, 4-chlorophenyl, 2,4-dichlorophenyl, 2,5-dichlorophenyl, and 2-bromo. Phenyl, 4-methoxyphenyl, 4-chloro-2-nitrophenyl, 4-nitrophenyl, 2,4-dinitrophenyl, biphenyl and the like can be mentioned. Preferable examples include a halogen atom, a phenyl group substituted with a lower alkoxy group nitro group, and a phenyl group.

The “aralkyl group” means an alkyl group having 1 to 6 carbon atoms substituted with an aryl group, and includes benzyl, α-naphthylmethyl, β-naphthylmethyl, indenylmethyl, phenanthrenylmethyl, anthracenyl “Methyl group substituted with 1 to 3 aryl groups” such as methyl, diphenylmethyl, triphenylmethyl, α-naphthyldiphenylmethyl, 9-anthrylmethyl, 4-methylbenzyl, 2,4,6 -Trimethylbenzyl, 3,4,5-trimethylbenzyl, 4-methoxybenzyl, 4-methoxyphenyldiphenylmethyl, 4,4'-dimethoxytriphenylmethyl, 2-nitrobenzyl, 4-nitrobenzyl, 4-chlorobenzyl, “Lower alkyl, lower alkoxy, halogen, such as 4-bromobenzyl, 4-cyanobenzyl, etc. 1-phenethyl, 2-phenethyl, 1-naphthylethyl, 2-naphthylethyl, 1-phenylpropyl, “methyl group substituted with 1 to 3 aryl groups in which the aryl ring is substituted with an ano group” -Phenylpropyl, 3-phenylpropyl, 1-naphthylpropyl, 2-naphthylpropyl, 3-naphthylpropyl, 1-phenylbutyl, 2-phenylbutyl, 3-phenylbutyl, 4-phenylbutyl, 1-naphthylbutyl, 2 -Naphthylbutyl, 3-naphthylbutyl, 4-naphthylbutyl, 1-phenylpentyl, 2-phenylpentyl, 3-phenylpentyl, 4-phenylpentyl, 5-phenylpentyl, 1-naphthylpentyl, 2-naphthylpentyl, 3 -Naphthylpentyl, 4-naphthylpentyl, 5-naphthylpentyl, 1 Phenylhexyl, 2-phenylhexyl, 3-phenylhexyl, 4-phenylhexyl, 5-phenylhexyl, 6-phenylhexyl, 1-naphthylpentyl, 2-naphthylpentyl, 3-naphthylpentyl, 4-naphthylpentyl, 5- “C3-C6 alkyl group substituted with aryl group” such as naphthylpentyl, 6-naphthylpentyl, and the like are included. Preferably, “methyl group substituted with 1 to 3 aryl groups”, “methyl substituted with 1 to 3 aryl groups with aryl ring substituted with lower alkyl, lower alkoxy, halogen, cyano group” Group ”, more preferably 4-methoxyphenyldiphenylmethyl, 4,4′-dimethoxytriphenylmethyl.

Examples of the “acyl group” include formyl, acetyl, propionyl, butyryl, isobutyryl, pentanoyl, pivaloyl, valeryl, isovaleryl, octanoyl, nonanoyl, decanoyl, 3-methylnonanoyl, 8-methylnonanoyl, 3-ethyloctanoyl, 3,7-dimethyl Octanoyl, undecanoyl, dodecanoyl, tridecanoyl, tetradecanoyl, pentadecanoyl, hexadecanoyl, 1-methylpentadecanoyl, 14-methylpentadecanoyl, 13,13-dimethyltetradecanoyl, heptadecanoyl, 15-methylhexayl Alkylcarbonyl groups such as decanoyl, octadecanoyl, 1-methylheptadecanoyl, nonadecanoyl, icosanoyl and heinacosanoyl, succinoyl, glutaroyl, Carboxylated alkylcarbonyl groups such as poyl, halogeno lower alkylcarbonyl groups such as chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl, lower alkoxy lower alkylcarbonyl groups such as methoxyacetyl, (E) -2-methyl “Aliphatic acyl groups” such as unsaturated alkylcarbonyl groups such as 2-butenoyl and arylcarbonyl groups such as benzoyl, α-naphthoyl, β-naphthoyl, 2-bromobenzoyl, 4-chlorobenzoyl, etc. Halogenoarylcarbonyl group, 2,4,6-trimethylbenzoyl, lower alkylated arylcarbonyl group such as 4-toluoyl, lower alkoxylated arylcarbonyl group such as 4-anisoyl, 2-carboxybenzoyl, 3-carb Carboxylated arylcarbonyl groups such as xoxybenzoyl, 4-carboxybenzoyl, nitrated arylcarbonyl groups such as 4-nitrobenzoyl, 2-nitrobenzoyl; lower alkoxycarbonylated arylcarbonyl groups such as 2- (methoxycarbonyl) benzoyl And an “aromatic acyl group” such as an arylated arylcarbonyl group such as 4-phenylbenzoyl, and preferably formyl, acetyl, propionyl, butyryl, isobutyryl, pentanoyl, pivaloyl, benzoyl group.

Examples of the “silyl group” include “tri-lower alkylsilyl group” such as trimethylsilyl, triethylsilyl, isopropyldimethylsilyl, t-butyldimethylsilyl, methyldiisopropylsilyl, methyldi-t-butylsilyl, triisopropylsilyl, diphenylmethylsilyl, Examples thereof include “tri-lower alkylsilyl groups substituted with 1 to 2 aryl groups” such as butyldiphenylbutylsilyl, diphenylisopropylsilyl, phenyldiisopropylsilyl, and preferably trimethylsilyl, triethylsilyl, triisopropylsilyl , T-butyldimethylsilyl, t-butyldiphenylsilyl, and more preferably trimethylsilyl.

The “protecting group” of the “phosphate group protected with a protecting group for nucleic acid synthesis” is not particularly limited as long as it can stably protect the phosphate group during nucleic acid synthesis, but is not specifically limited. Refers to a protecting group that is stable under acidic or neutral conditions and can be cleaved by chemical methods such as hydrogenolysis, hydrolysis, electrolysis and photolysis. For example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 4 -Methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2 “Lower alkyl groups” such as dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl and 2-ethylbutyl; “cyanated lower groups” such as 2-cyanoethyl and 2-cyano-1,1-dimethylethyl "Alkyl group"; "ethyl group substituted with silyl group" such as 2-methyldiphenylsilylethyl, 2-trimethylsilylethyl, 2-triphenylsilylethyl; 2,2,2-trichloroethyl, 2,2,2 “Halogenated lower alkyl groups” such as tribromoethyl, 2,2,2-trifluoroethyl, 2,2,2-trichloro-1,1-dimethylethyl; ethenyl, 1-propenyl, 2-propenyl, 1-methyl-2-propenyl, 1-methyl-1-propenyl, 2-methyl-2-propenyl, 2-ethyl-2-propenyl, 1-butyl Nyl, 2-butenyl, 1-methyl-2-butenyl, 1-methyl-1-butenyl, 3-methyl-2-butenyl, 1-ethyl-2-butenyl, 3-butenyl, 1-methyl-3-butenyl, 2-methyl-3-butenyl, 1-ethyl-3-butenyl, 1-pentenyl, 2-pentenyl, 1-methyl-2-pentenyl, 2-methyl-2-pentenyl, 3-pentenyl, 1-methyl-3- Of pentenyl, 2-methyl-3-pentenyl, 4-pentenyl, 1-methyl-4-pentenyl, 2-methyl-4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl Such as “lower alkenyl groups”; such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, norbornyl, adamantyl A “cycloalkylated lower alkenyl group” such as 2-cyanobutenyl; benzyl, α-naphthylmethyl, β-naphthylmethyl, indenylmethyl, phenanthrenylmethyl, anthracenylmethyl, diphenylmethyl , Triphenylmethyl, 1-phenethyl, 2-phenethyl, 1-naphthylethyl, 2-naphthylethyl, 1-phenylpropyl, 2-phenylpropyl, 3-phenylpropyl, 1-naphthylpropyl, 2-naphthylpropyl, 3- Naphthylpropyl, 1-phenylbutyl, 2-phenylbutyl, 3-phenylbutyl, 4-phenylbutyl, 1-naphthylbutyl, 2-naphthylbutyl, 3-naphthylbutyl, 4-naphthylbutyl, 1-phenylpentyl, 2- Phenylpentyl, 3-phenylpentyl, 4-phenyl Phenylpentyl, 5-phenylpentyl, 1-naphthylpentyl, 2-naphthylpentyl, 3-naphthylpentyl, 4-naphthylpentyl, 5-naphthylpentyl, 1-phenylhexyl, 2-phenylhexyl, 3-phenylhexyl, 4-phenyl “Aralkyl groups” such as hexyl, 5-phenylhexyl, 6-phenylhexyl, 1-naphthylpentyl, 2-naphthylpentyl, 3-naphthylpentyl, 4-naphthylpentyl, 5-naphthylpentyl, 6-naphthylpentyl; 4 "Nitro group, aryl at halogen atom, such as -chlorobenzyl, 2- (4-nitrophenyl) ethyl, o-nitrobenzyl, 4-nitrobenzyl, 2,4-dinitrobenzyl, 4-chloro-2-nitrobenzyl Aralkyl group with substituted ring ”; phenyl “Aryl groups” such as indenyl, naphthyl, phenanthrenyl, anthracenyl; 2-methylphenyl, 2,6-dimethylphenyl, 2-chlorophenyl, 4-chlorophenyl, 2,4-dichlorophenyl, 2,5-dichlorophenyl, 2-bromo Examples thereof include “lower alkyl group, halogen atom, aryl group substituted by nitro group” such as phenyl, 4-nitrophenyl and 4-chloro-2-nitrophenyl, preferably “lower alkyl group”, "Lower alkyl group substituted with a cyano group", "Aralkyl group", "Nitro group, an aralkyl group substituted with an aryl ring with a halogen atom" or "Aryl group substituted with a lower alkyl group, a halogen atom, or a nitro group" And more preferably a 2-cyanoethyl group, 2,2,2-trichloroethyl Group, a benzyl group, a 2-chlorophenyl group or 4-chlorophenyl group.

The protecting group of the “mercapto group protected with a protecting group for nucleic acid synthesis” is not particularly limited as long as it can stably protect the mercapto group during nucleic acid synthesis. Or a protecting group that is stable under neutral conditions and can be cleaved by a chemical method such as hydrogenolysis, hydrolysis, electrolysis and photolysis. , An alkylthio group such as ethylthio, tert-butylthio, an arylthio group such as benzylthio, and the like, and a “disulfide-forming group”, preferably an “aliphatic acyl group” or “aromatic acyl group” And more preferably a benzoyl group.

Examples of the “C 1-5 alkoxy group” include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, s-butoxy, tert-butoxy, and n-pentoxy. Is a methoxy or ethoxy group.

Examples of the “C1-C5 alkylthio group” include methylthio, ethylthio, propylthio, isopropylthio, butylthio, isobutylthio, s-butylthio, tert-butylthio, and n-pentylthio. A methylthio or ethylthio group;

The “C1-C6 cyanoalkoxy group” refers to a group substituted by the cyano group of the above “C1-C5 alkoxy group”, and examples of such groups include cyanomethoxy, 2-cyano. Examples include ethoxy, 3-cyanopropoxy, 4-cyanobutoxy, 3-cyano-2-methylpropoxy, or 1-cyanomethyl-1,1-dimethylmethoxy, and a 2-cyanoethoxy group is preferable.

Examples of the “amino group substituted with an alkyl group having 1 to 5 carbon atoms” include, for example, methylamino, ethylamino, propylamino, isopropylamino, butylamino, isobutylamino, s-butylamino, tert-butylamino, dimethyl Amino, diethylamino, dipropylamino, diisopropylamino, dibutylamino, diisobutylamino, di (s-butyl) amino, di (tert-butyl) amino can be mentioned, preferably methylamino, ethylamino, dimethylamino , Diethylamino or diisopropylamino group.

Examples of the “alkyl group having 1 to 5 carbon atoms” include methyl, ethyl, propyl, isopropyl, isopropyl, butyl, isobutyl, s-butyl, tert-butyl, n-pentyl, and the like. , Methyl or ethyl group.

Examples of the “halogen atom” include a fluorine atom, a chlorine atom, a bromine atom or an iodine atom, and preferably a fluorine atom or a chlorine atom.

The protecting group of the “amino group protected with a protecting group for nucleic acid synthesis” is not particularly limited as long as it can stably protect an amino group during nucleic acid synthesis. Or a protecting group that is stable under neutral conditions and can be cleaved by chemical methods such as hydrogenolysis, hydrolysis, electrolysis and photolysis, such as formyl, acetyl, propionyl, butyryl, isobutyryl, pentanoyl, Pivaloyl, valeryl, isovaleryl, octanoyl, nonanoyl, decanoyl, 3-methylnonanoyl, 8-methylnonanoyl, 3-ethyloctanoyl, 3,7-dimethyloctanoyl, undecanoyl, dodecanoyl, tridecanoyl, tetradecanoyl, pentadecanoyl, hexadecana Noyl, 1-methylpentadecanoyl, 14-methylpentadecano , 13,13-dimethyltetradecanoyl, heptadecanoyl, 15-methylhexadecanoyl, octadecanoyl, 1-methylheptadecanoyl, nonadecanoyl, nonadecanoyl, eicosanoyl and heinacosanoyl, succinoyl, glutaroyl, adipoyl Carboxylated alkylcarbonyl groups such as, chloroacetyl, dichloroacetyl, trichloroacetyl, halogeno lower alkylcarbonyl groups such as trifluoroacetyl, methoxyacetyl, phenoxyacetyl group, 2- (4-tert-butyl) phenoxy) acetyl An “aliphatic acyl group” such as an alkoxy lower alkylcarbonyl group such as a group, an unsaturated alkylcarbonyl group such as (E) -2-methyl-2-butenoyl; -Arylcarbonyl group such as naphthoyl, β-naphthoyl, halogenoarylcarbonyl group such as 2-bromobenzoyl, 4-chlorobenzoyl, lower alkylated arylcarbonyl such as 2,4,6-trimethylbenzoyl, 4-toluoyl Groups, lower alkoxylated arylcarbonyl groups such as 4-anisoyl, carboxylated arylcarbonyl groups such as 2-carboxybenzoyl, 3-carboxybenzoyl, 4-carboxybenzoyl, 4-nitrobenzoyl, 2-nitrobenzoyl, etc. Nitrated arylcarbonyl group; “aromatic acyl group” such as lower alkoxycarbonylated arylcarbonyl group such as 2- (methoxycarbonyl) benzoyl, arylated arylcarbonyl group such as 4-phenylbenzoyl; “Lower alkoxycarbonyl groups” such as xoxycarbonyl, ethoxycarbonyl, t-butoxycarbonyl, isobutoxycarbonyl; “halogen or tri-lower alkylsilyl groups such as 2,2,2-trichloroethoxycarbonyl, 2-trimethylsilylethoxycarbonyl” A lower alkoxycarbonyl group substituted with an “alkenyloxycarbonyl group” such as vinyloxycarbonyl or aryloxycarbonyl; benzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, 4-nitrobenzyloxy Examples thereof include 1 to 2 “aralkyloxycarbonyl groups in which the aryl ring may be substituted with a lower alkoxy or nitro group” such as carbonyl. A Le group "or" aromatic acyl group ", more preferably a benzoyl group. “Nucleoside analog” means a non-natural type of “nucleoside” in which a purine base or a pyrimidine base and a sugar are bonded, and a purine base or an aromatic heterocycle other than purine and pyrimidine and an aromatic hydrocarbon ring. A substance that can be substituted with a pyrimidine base is combined with a sugar.

The “salt” refers to a salt of the compound (III) of the present invention, and is preferably a salt such as sodium salt, potassium salt or lithium salt. Alkaline earth metal salts such as alkali metal salts, calcium salts and magnesium salts, metal salts such as aluminum salts, iron salts, zinc salts, copper salts, nickel salts and cobalt salts; inorganic salts such as ammonium salts, t- Octylamine salt, dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N, N′-di Benzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl -Amine salts such as organic salts such as phenethylamine salt, piperazine salt, tetramethylammonium salt, tris (hydroxymethyl) aminomethane salt; hydrofluoride, hydrochloride, hydrobromide, hydroiodide Inorganic acid salts such as halogen hydrides, nitrates, perchlorates, sulfates, phosphates, etc .; lower alkane sulfones such as methanesulfonates, trifluoromethanesulfonates, ethanesulfonates Acid salts, aryl sulfonates such as benzene sulfonate, p-toluene sulfonate, acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, maleic acid Organic acid salts such as salts; and amino acid salts such as glycine salts, lysine salts, arginine salts, ornithine salts, glutamate salts, aspartates Door can be.

The “reagent used for protecting a hydroxyl group for nucleic acid synthesis” refers to a reagent used for introducing the “protecting group for a hydroxyl group for nucleic acid synthesis” into a sugar hydroxyl group constituting a nucleic acid. For example, when the hydroxyl group at the 5 ′ position is 1) 4,4′-dimethoxytrityl group: in the presence of a base such as pyridine, DABCO (1,4-diazabicyclo [2.2.2] octane), triethylamine, diisopropylethylamine, 4,4′-dimethoxytrityl chloride or 4,4′-dimethoxytrityl trifluoromethanesulfonate,
2) In the case of a trityl group: in the presence of a base such as pyridine, DABCO (1,4-diazabicyclo [2.2.2] octane), triethylamine, diisopropylethylamine, trityl chloride or trityl trifluoromethanesulfonate,
3) In the case of tert-butyldimethylsilyl group: tert-butyldimethylsilyl chloride or tert-butyldimethylsilyl trifluoromethanesulfonate 4 in the presence of a base such as imidazole, N-methylimidazole, 4,4-dimethylaminopyridine, triethylamine, etc. ) In the case of tert-butyldiphenylsilyl group; tert-butyldimethylsilyl chloride or tert-butyldiphenylsilyl trifluoromethanesulfonate 5) in the presence of a base such as imidazole, N-methylimidazole, 4,4-dimethylaminopyridine, triethylamine In the case of a benzoyl group; i) in the presence of a base such as pyridine, triethylamine, potassium carbonate, benzoyl chloride, benzoic anhydride ii) a base such as triethylamine, N-methylmorpholine, and EDC (1-ethyl-3- (3 In the presence of a condensing agent such as dimethylaminopropyl) carbodiimide) hydrochloride or DCC (N, N′-dicyclohexylcarbodiimide), in the case of benzoic acid 6) levonoyl group; In the presence of a levulinic acid 7) 2-ethoxyethyl group in the presence of a condensing agent such as DCC, ethoxy vinyl ether is applicable in the presence of an acid catalyst such as pyridium paratoluenesulfonic acid or toluenesulfonic acid.

One aspect of the invention involves an aldol reaction represented by the following formula:

“Aldol reaction” is also referred to as aldol condensation or aldol addition. This is a reaction in which a carbonyl compound having a hydrogen atom at the α-position is reacted with another carbonyl compound to form a β-hydroxycarbonyl compound. An equilibrium reaction that occurs with either an acid or base catalyst.

Examples of the carbonyl compound to be reacted with the starting material in the present invention include ketone compounds such as acetone and aldehyde compounds such as formaldehyde, preferably formalin or paraformaldehyde.

Usually, it is carried out in the presence of a base. Examples of the base used at this time include inorganic bases such as sodium hydroxide and sodium carbonate, and organic bases such as N-methylmorpholine, DBU, triethylamine and pyridine. An organic base having a pKa of a conjugate acid of 6 to 10 is preferable, and examples thereof include triethylamine and N-methylmorpholine.

As the solvent to be used, a water-soluble solvent such as alcohol, acetonitrile or N, N′-dimethylformamide (DMF) is preferable.

A suitable combination is 1 to 3 volumes of an aqueous formalin solution, 1 to 3 volumes of N-methylmorpholine as a base, and 2 to 6 volumes of acetonitrile as a solvent. This is because as a result of carrying out the aldol reaction on the compound of the general formula (I) in this combination, it was possible to minimize the residual amount of the raw material (I) and the amount of by-products after the reaction.

The reaction temperature and reaction time are not particularly limited, but preferably the reaction temperature is 50 ° C to 100 ° C and the reaction time is 1 to 24 hours.

Further embodiments of the present invention also include a condensation reaction represented by the following formula: More specifically, the starting material compound and the desilylating agent are reacted, and the condensing agent is reacted with the reaction solution.

The “desilylating agent” is a reaction aid added to cause the elimination reaction of the silyl group. Generally, in addition to various acids such as hydrochloric acid, acetic acid, paratoluenesulfonic acid, fluorination Fluoride ions such as tetrabutylammonium, hydrofluoric acid and cesium fluoride can be mentioned.

A “condensation agent” is a reaction aid added to cause an amino group and a carboxyl group to undergo dehydration condensation. In general, EDC hydrochloride (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride), DCC (dicyclohexylcarbodiimide), DIC (diisopropylcarbodiimide), CDI (carbonyldiimidazole), PyBop [(benzotriazole- 1-yloxy) tripyrrolidinophosphonium hexafluorophosphate], HATU [O- (7-azabenzotriazol-1-yl) -N, N, N ′, N′-tetramethyluronium hexafluorophosphate], DMT- Reagents used for peptide synthesis such as MM [4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium chloride] and acidic such as aluminum chloride and titanium (IV) chloride A catalyst etc. are mentioned.

Furthermore, a further aspect of the invention also includes a transglycosylation reaction represented by the following formula:

Here, “transglycosylation reaction” refers to a reaction for substituting the base moiety of a nucleic acid derivative.

The general formula in the present invention includes both a cis form and a trans form. A trans form is preferable. Moreover, in the reaction process in this invention, the solid can be maintained and it is an industrially very useful method.

  The compound represented by the above general formula (I) (corresponding to A-5 below) can be obtained, for example, by the following method.

R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ and B in the formula are as defined above. P ₁ has the same meaning as the above-mentioned “protecting group for hydroxyl group in nucleic acid synthesis”. Preferred are a trityl group and a benzyl group which may be substituted with a methoxy group.

  The compound represented by the general formula (A-1) in the formula can be synthesized by the method described in Organic Letters, 7, 1569-1572 (2005).

(First step)
In this step, the compound represented by the general formula (A-1) is reacted with R ₃ -Hal (wherein Hal is halogen), (R ₃ ) ₂ O, etc., so that the 3′-position hydroxyl group is R ₃ —. This is a step of obtaining a compound represented by formula (A-2) protected by. This step can be performed according to the method described in Protective Groups in Organic Synthesis, Theodora W Green (John Wiley & Sons) and the like. For example, the compound represented by the general formula A-1 is converted into 1.0 equivalent to 10.0 equivalents, preferably 3.0 equivalents to 6.0 equivalents of R ₃ -Hal (in a solvent such as DMF, acetonitrile, or dichloromethane). In the formula, Hal is halogen), 1.0 equivalent to 10.0 equivalents, preferably 3.0 equivalents to 6.0 equivalents of an organic base (such as N-methylimidazole) or an inorganic base (such as sodium hydride). , Sodium carbonate, potassium carbonate, cesium carbonate, etc.) and 1.0 equivalent to 10.0 equivalents, preferably 2.0 equivalents to 4.0 equivalents of sodium iodide and the like, and 0 ° C. to 120 ° C., preferably Can react at 10-30 degreeC for 1 to 24 hours, and can obtain the compound represented by general formula (A-2).

(Second step)
In this step, the compound represented by formula (A-2) is reacted with a strong acid such as hydrochloric acid to obtain a compound A-3 in which the hydroxyl protecting group P ₁ bonded to the 4′-position is deprotected. It is a process. This step can be performed according to the method described in Protective Groups in Organic Synthesis, Theodora W Green (John Wiley & Sons) and the like. For example, the compound represented by the general formula A-2 is converted into a strong acid (1.0 equivalent to 10.0 equivalents, preferably 2.0 equivalents to 4.0 equivalents) in a solvent such as methanol, ethanol, ethyl acetate, or dioxane. An aqueous solution such as hydrochloric acid and sulfuric acid, an organic acid such as p-toluenesulfonic acid) and the like, by reacting at 0 ° C. to 100 ° C., preferably 20 ° C. to 30 ° C. for 0.5 to 24 hours. Can be obtained.

(Third step)
In this step, the amino group is protected with R ₂ -by reacting the compound represented by the general formula (A-3) with (R ₂ ) ₂ O, R ₂ -Hal (where Hal is halogen), or the like. This is a step of obtaining the prepared compound A-4. This step can be performed according to the method described in Protective Groups in Organic Synthesis, Theodora W Green (John Wiley & Sons) and the like. For example, the compound represented by the general formula A-3 is preferably used in a solvent such as tetrahydrofuran, dichloromethane, acetonitrile, or DMF, or in a mixed solvent such as water-tetrahydrofuran or water-toluene. 2.0 equivalents to 4.0 equivalents _{(R 2) 2} O and 1.0 equivalents of 5.0 equivalents, preferably 2.0 equivalents to 4.0 equivalents of an organic base (e.g., pyridine, etc.) or inorganic Reaction with a base (for example, sodium hydride, sodium hydroxide, sodium carbonate, potassium carbonate, cesium carbonate, sodium bicarbonate, etc.) at -20 ° C to 50 ° C, preferably 0 ° C to 30 ° C for 0.5 to 24 hours By doing so, a compound represented by the general formula (A-4) can be obtained.

(4th process)
This step is a step of obtaining a compound (A-5) having a carbonyl group by oxidizing the hydroxyl group bonded to the 4′-position of the compound represented by the general formula (A-4). This step can be performed according to the method described in Experimental Chemistry Course, 5th edition, volume 17 (Yamazen). For example, the compound represented by the general formula A-4 is added in an amount of 1.0 equivalent to 5.0 equivalent, preferably 2.0 equivalent to 4.0 equivalent of EDC and 1.0 equivalent to 3. equivalent in a solvent such as DMSO. Reaction with 0 equivalents, preferably 1.0 equivalents to 2.0 equivalents of an organic acid (eg, pyridinium paratoluenesulfonate) at 0 ° C. to 50 ° C., preferably 10 ° C. to 30 ° C. for 0.5 to 24 hours By doing so, a compound represented by the general formula (A-5) can be obtained.

(5th process)
About the compound represented by general formula (A-5) obtained by said method, the following aldol reaction is performed.

This step is a step of obtaining the compound represented by the general formula (A-6) by subjecting the compound represented by the general formula (A-5) to the aldol reaction shown in claim 1 of the present invention. For example, the compound represented by the general formula (A-5) is added in a solvent such as ethanol, butanol, acetonitrile, or DMF, preferably in acetonitrile, 1.0 to 20.0 times volume or 1.0 equivalent to 20. A carbonyl compound represented by 0 equivalent of R ₅ C (═O) R ₆ , preferably a 1.0 to 3.0-fold formalin aqueous solution, etc., and a 1.0 to 20.0-fold capacity or 1.0 Equivalent to 20.0 equivalents of organic base, preferably 1.0 to 3.0 times the volume of the conjugate acid pKa of 6 to 10 (for example, N-methylmorpholine, etc.) and 20 ° C to 100 ° C The compound represented by formula (A-6) can be obtained by reacting at 70 to 80 ° C. for 0.5 to 24 hours.

(Sixth step)
When R _{4 of the} compound represented by the general formula (A-6) obtained by the above method is a hydrogen atom, the following oxidation reaction is performed.

This step can be performed according to the method described in Experimental Chemistry Course, 5th edition, volume 17 (Yamazen). For example, the compound represented by the general formula (A-6) is mixed with a buffer solution such as an aqueous solution of sodium dihydrogen phosphate in a solvent such as ethyl acetate, acetonitrile, or tert-butanol, and 1.0 to 20.0 equivalents of 2 -In the presence of methyl-2-butene, squalene, etc., 1.0 equivalent to 10.0 equivalents, preferably 2.0 equivalents to 5.0 equivalents of sodium chlorite and 0 ° C to 60 ° C, preferably 10 The compound represented by the general formula (A-7) can be obtained by reacting at -40 ° C to 0.5-24 hours.

Further, when R _{4 of the} compound represented by the general formula (A-6) is an alkoxy group, for example, in a solvent such as methanol, ethanol, 1,4-dioxane, tetrahydrofuran, or a mixed solvent thereof, By reacting with 0.1 N to 10 N aqueous alkali solution (sodium hydroxide aqueous solution, potassium hydroxide aqueous solution, etc.) at 0 ° C. to 100 ° C., preferably 20 ° C. to 30 ° C. for 0.1 to 24 hours. A compound represented by A-7 can be obtained.

General production method of 4 types of amidites (1)
The amidite compound represented by the general formula (A-12) from the compound represented by the general formula (A-6) (provided that R ₄ is a hydroxyl group) or (A-7) obtained by the above method Can be obtained by the following production method.

R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ and B in the formula are as defined above. P ₂ has the same meaning as the above-mentioned “protecting group for hydroxyl group in nucleic acid synthesis”. A 4,4′-dimethoxytrityl group, a trityl group, a TBDPS group or a TBS group is preferable. P ₃ represents a “phosphoramidite group”. The “phosphoramidite group” is a group represented by the formula —P (OR ₁₁ ) (NR ₁₂ ) (wherein R ₁₁ is an alkyl group having 1 to 6 carbon atoms or a cyanoalkyl group having 1 to 7 carbon atoms). R ₁₂ represents an alkyl group having 1 to 6 carbon atoms.), And preferably a group or a formula represented by the formula —P (OC ₂ H ₄ CN) (N (iPr) ₂ ) It is a group represented by —P (OCH ₃ ) (N (iPr) ₂ ).

Step 7 This step is a step for obtaining compound A-8 by deprotecting the protecting group R ₂ substituted on the amino group of the compound represented by formula (A-7). This step can be performed according to the method described in Protective Groups in Organic Synthesis, Theodora W Green (John Wiley & Sons) and the like.

Step 8 This step is a step for obtaining a compound represented by the general formula (A-9) in which the 2′-position and the 4′-position are cyclized by an amide bond from the compound represented by the general formula (A-8). . 1.0 equivalent to 5 equivalents, preferably 1.0 equivalent to 1.5 equivalents of DCC, EDC, CDI (carbonyldiimidazole) in a solvent such as N, N′-dimethylformamide, acetonitrile, or a mixed solvent thereof. Etc.) (or HOBt, HOSu, etc. are added to these condensing agents) and reacted at −20 to 120 ° C., preferably 0 to 80 ° C. for 0.5 to 24 hours. A compound represented by the formula (A-9) can be obtained.

Ninth Step This step is a general formula (A-10) in which the 5′-position hydroxyl group of the compound represented by the general formula (A-9) is protected with a protecting group P ₂ (for example, 4,4′-dimethoxytrityl group). Is a step of obtaining a compound represented by the formula: This step can be performed according to the method described in Protective Groups in Organic Synthesis, Theodora W Green (John Wiley & Sons) and the like.

For example, the compound represented by the general formula (A-9) is dissolved in a solvent such as dichloromethane or toluene in the presence of a base such as pyridine, triethylamine, diisopropylethylamine, DABCO (1,4-diazabicyclo [2.2.2] octane). Or in a pyridine solvent, 1.0 equivalent to 5 equivalents, preferably 1.0 equivalent to 1.5 equivalents of 4,4′-dimethoxytrityl chloride is added, and 0 ° C. to 80 ° C., preferably 20 ° C. to 30 ° C. A compound represented by the general formula (A-10) in which P ₂ is a 4,4′-dimethoxytrityl group can be obtained by reacting at 0.5 ° C. for 24 hours.

Step 10 This step is a step of obtaining a compound represented by the general formula (A-11) in which the hydroxyl protecting group R ₃ bonded to the 3′-position of the compound represented by the general formula (A-10) is deprotected. It is. This step can be performed according to the method described in Protective Groups in Organic Synthesis, Theodora W Green (John Wiley & Sons) and the like.

In the case where R _{3 of the} compound represented by the general formula (A-10) is, for example, a silyl protecting group (TBS group, TBDPS group, TES group, etc.), in a solvent such as tetrahydrofuran, methanol, ethanol, dioxane, 1.0 Equivalent to 10.0 equivalents, preferably 1.1 equivalents to 3.0 equivalents of a desilylating agent (a fluorine-containing reagent such as tetrabutylammonium fluoride, ammonium fluoride, cesium fluoride, trifluoroacetic acid, hydrochloric acid, etc. The compound represented by the general formula (A-11) can be obtained by reacting with (acid) at 0 ° C to 100 ° C, preferably 20 ° C to 80 ° C for 0.1 hour to 24 hours.

In the case where R _{3 of the} compound represented by the general formula (A-10) is, for example, a benzyl protecting group (Bn group, p-methoxybenzyl group, etc.), in a solvent such as methanol, tetrahydrofuran, ethanol, dioxane, toluene and the like. Or in a mixed solvent thereof, palladium-carbon powder or palladium hydroxide-carbon powder is added, and the reaction is carried out at 0 ° C. to 40 ° C. for 1 hour to 24 hours in a hydrogen stream, thereby obtaining the general formula (A-11). Can be obtained.

Eleventh Step This step is represented by the general formula (A-12) in which the 3′-position hydroxyl group is amidated by reacting the compound represented by the general formula (A-11) with an amidite-forming reagent. This is a step of obtaining a compound. The compound represented by the general formula (A-11) is mixed with 2-cyanoethyl-N, N-diisopropyl in a solvent such as acetonitrile, dichloromethane, tetrahydrofuran or the like or in a mixed solvent thereof in the presence of a base such as diisopropylethylamine or triethylamine. 2-cyanoethyl-N, N, N ′, N′-tetraisopropyl phosphoramidite is added in the presence of an acid such as chlorophosphoramidite or tetrazole or dicyanoimidazole, and the reaction is carried out at 0 to 60 ° C. for 1 to 24 hours. By doing so, the general formula (A-12) can be obtained.

The order of the above steps may be changed, and it is sufficient that the compound represented by the general formula (A-7) can be converted into the compound represented by the general formula (A-12).

General production method of 4 amidites (2)
As an alternative to the general production method (1) of the four types of amidites, the general formula (A-11) can be obtained from the general formula (A-9) via the general formula (A-10-2).

Step 1 This step is a step of removing a compound represented by the general formula (A-10-2) in which the hydroxyl protecting group R ₃ bonded to the 3′-position of the compound represented by the general formula (A-9) is deprotected. It is a process to obtain. The compound represented by the general formula (A-10-2) can be obtained by the same method as the third step (steps A-10 to A-11) of the general production method (1) of the four amidites.
In the case where the nucleobase is adenine (A), guanine (G), or cytosine (C) and the amino group is unprotected, silylation with a silylating agent such as TMS chloride or TBS chloride in the presence of the base, An acylating agent such as benzoyl chloride, phenoxyacetyl chloride, various acid anhydrides or an iminating agent such as 1,1-dimethoxy-N, N′-dimethylmethanamine is −20 ° C. to 120 ° C., preferably 0 ° C. to 50 ° C. And then reacting with an acid treatment or a desilylating agent to obtain a compound represented by the general formula (A-10-2) in which the amino group of the nucleobase is protected. be able to.

Second Step In this step, the 5′-position hydroxyl group of the compound represented by the general formula (A-10-2) is protected with a protective group R ₇ (usually 4,4′-dimethoxytrityl group). This is a step of obtaining a compound represented by 11). The compound represented by the general formula (A-11) can be obtained by the same method as the second step (steps A-9 to A-10) of the general production method (1) of the four amidites.

The order of the steps described above may be changed as long as the compound represented by the general formula (A-9) can be converted into the compound represented by the general formula (A-11).

General production method of 4 kinds of amidites (3)
As an alternative to the general production method (1) of the four amidites, the general formula (A-7) to general formula (A-11) can also be obtained by the following steps.

R ₁ , R ₂ , R ₃ , R ₅ , R ₆ and B are as defined above. R ₇ is a hydroxyl-protecting group used for nucleic acid synthesis such as 4,4′-dimethoxytrityl group (or trityl group, TBDPS group, TBS group).

Step 1 This step is a step for obtaining a compound represented by the general formula (A-13) by removing the protecting groups at the 2′-position and the 3′-position from the compound represented by the general formula (A-7). . When the protecting group at the 2′-position is a trifluoroacetyl group, it is 1.0 equivalent to 5 equivalents, preferably 1.0 equivalent to 3 equivalents, in a solvent such as tetrahydrofuran, methanol, acetonitrile, pyridine, or a mixed solvent thereof. 0 equivalent of TBAF (tetrabutylammonium fluoride), hydrogen fluoride triethylamine complex, desilylating agent such as ammonium fluoride is added, and 0 to 100 ° C., preferably 25 to 80 ° C. for 0.5 hours to 24 A compound represented by the general formula (A-13) can be obtained by reacting for a period of time. The characteristic of this reaction is that the trifluoroacetyl group, which is not normally removed by the desilylating agent, is removed, but the nucleobase protecting group such as benzoyl group is not removed. It is a method that makes it possible to improve.
As another method, when R ₂ is an acyl protecting group or a carbamate protecting group containing a trifluoroacetyl group, it is 0 in a solvent such as methanol, ethanol, 1,4-dioxane, tetrahydrofuran, or a mixed solvent thereof. 2 by reacting with 1N to 10N alkaline solution (sodium hydroxide aqueous solution, potassium hydroxide-methanol solution, etc.) at 0 ° C to 100 ° C, preferably 20 ° C to 30 ° C for 0.1 to 24 hours. After removing the protecting group at the 'position, the compound represented by the general formula (A-13) can be obtained by desilylation with TBAF or the like.
When R ₂ is a tert-butyloxycarbonyl group (BOC group), an acid such as trifluoroacetic acid or hydrogen chloride and -20 ° C in a solvent such as methanol, ethanol, 1,4-dioxane, tetrahydrofuran, or without solvent After removing the protecting group at the 2 ′ position by reacting at ˜100 ° C., preferably 20 ° C. to 30 ° C. for 0.1 to 24 hours, desilylation with TBAF or the like is carried out to obtain the general formula (A-13) Can be obtained.
When R ₂ is a benzyl-based protecting group (CBz group, Bn group, MPM group, etc.), in a solvent such as methanol, tetrahydrofuran, ethanol, dioxane, toluene, or a mixed solvent thereof, palladium-carbon powder or water By adding palladium oxide-carbon powder and reacting at 0 ° C. to 40 ° C. for 1 hour to 24 hours under a hydrogen stream, the 2′-position protecting group is removed, and then desilylated with TBAF or the like, A compound represented by the general formula (A-13) can be obtained.

Second Step This step is a step of obtaining a compound represented by the general formula (A-14) in which the 2′-position and the 4′-position are cyclized by an amide bond from the compound represented by the general formula (A-13). . 1.0 equivalent to 10 equivalents, preferably 1.0 equivalent to 3 equivalents of DCC, EDC, CDI (carbonyldiimidazole) in a solvent such as N, N′-dimethylformamide, tetrahydrofuran, acetonitrile, or a mixed solvent thereof. Etc.) (or HOBt, HOSu, etc. are added to these condensing agents) and reacted at −20 to 120 ° C., preferably 25 to 80 ° C. for 0.5 to 24 hours. A compound represented by the formula (A-14) can be obtained.
In addition, when the nucleobase is adenine (A), guanine (G), or cytosine (C) and the amino group is unprotected, silylation with silylating agents such as TMS chloride, TBS chloride, etc. in the presence of the base results in benzoyl An acylating agent such as chloride, phenoxyacetyl chloride, various acid anhydrides, or an iminating agent such as 1,1-dimethoxy-N, N′-dimethylmethanamine is −20 ° C. to 120 ° C., preferably 0 ° C. to 50 ° C. After reacting for 0.5 to 24 hours, the compound represented by the general formula (A-14) in which the amino group of the nucleobase is protected can be obtained by reacting with an acid treatment or a desilylating agent. .

Third Step This step is a general formula (A-11) in which the 5′-position hydroxyl group of the compound represented by the general formula (A-14) is protected with a protecting group R ₇ (for example, 4,4′-dimethoxytrityl group). Is a step of obtaining a compound represented by This step can be performed according to the method described in Protective Groups in Organic Synthesis, Theodora W Green (John Wiley & Sons) and the like.
In the presence of a base such as pyridine, DABCO (1,4-diazabicyclo [2.2.2] octane), triethylamine, diisopropylethylamine, etc. in a solvent such as dichloromethane, toluene, tetrahydrofuran and the like. Alternatively, 1.0 equivalent to 5 equivalents, preferably 1.0 equivalent to 3 equivalents of 4,4′-dimethoxytrityl chloride is added in a pyridine solvent, and 0 ° C. to 80 ° C., preferably 20 ° C. to 30 ° C., By reacting for 0.5 to 24 hours, a compound represented by the general formula (A-11) in which R ₇ is a 4,4′-dimethoxytrityl group can be obtained.
In addition, it is also possible to implement 1st process-3rd process by 1 pot by selecting the reaction solvent appropriately.

General production method of 4 kinds of amidites (4)
In the general formula (A-7), when B is thymine (T) or a derivative thereof, it is converted into adenine (A), guanine (G), cytosine (C) or a derivative thereof by transglycosylation reaction. can do. Specifically, in a solvent such as 1,2-dichloroethane, toluene, acetonitrile, CPME (cyclopentylmethyl ether) or a mixed solvent thereof, BSA (N, O-bis (trimethylsilyl) acetamide), adenine, guanine, Alternatively, Lewis derivatives such as these derivatives, TMSOTf, BF3OEt2, etc. are added and reacted at 0 ° C. to 120 ° C., preferably 50 ° C. to 80 ° C. for 0.1 to 24 hours, whereby B becomes adenine (A), guanine (G ), Cytosine (C), or a compound represented by the general formula (A-7) of these derivatives.

General production method of amidite of cytidine type compound The compound represented by the general formula (A-11c) can be obtained from the general formula (A-10) by the following method.

Q ₁ , Q ₂ , and Q ₃ in the formula have the same meaning as the above-mentioned “alkyl group having 1 to 5 carbon atoms”.

First Step This step is a step represented by the step of obtaining a nucleobase (B-10) from a thymidine-type compound in which the nucleobase (B) is thymine or a thymine derivative (A-10t) in the compound represented by the general formula (A-10). ) Is a step of obtaining a cytidine type compound which is cytosine or a cytosine derivative (A-10c). A base such as triethylamine, imidazole, N, N-dimethylaminopyridine, and a sulfonyl chloride such as 2,4,6-triisopropylbenzenesulfonyl chloride in a solvent such as acetonitrile, dichloromethane, pyridine, or a mixed solvent thereof; After reacting at −20 ° C. to 80 ° C., preferably 0 ° C. to 30 ° C. for 0.1 to 24 hours, ammonia water is added, and 0 ° C. to 80 ° C., preferably 0 ° C. to 30 ° C., 0.5 to By reacting for 8 hours, the compound represented by the general formula (A-10c) can be obtained.

Step 2 This step is a step of obtaining a compound represented by the general formula (A-11c) by further modifying the amino group of cytosine or cytosine derivative from the compound represented by the general formula (A-10c). . Bases such as triethylamine, imidazole and N, N-dimethylaminopyridine, and acyl chlorides such as benzoyl chloride and acetyl chloride in solvents such as N, N′-dimethylformamide, pyridine, acetonitrile and dichloromethane, or mixed solvents thereof Alternatively, it is represented by the general formula (A-11c) by reacting with an acid anhydride such as benzoic anhydride and acetic anhydride at −20 ° C. to 80 ° C., preferably at 0 ° C. to 30 ° C. for 0.1 to 48 hours. Can be obtained.

Hereinafter, the synthesis of the nucleic acid derivative of the present invention will be described in more detail based on examples.
[Example 1]

  (2R, 3S, 4R, 5R) -3- (tert-butyldimethylalkyloxy) -2-hydroxymethyl-5- (5-methyl-2,4-dioxo-3,4-dihydropyrimidine-1 (2H ) -Yl) -4- (methylamino) tetrahydrofuran-2-carboxylic acid synthesis

Step 1 Synthesis of Compound 2 Suspension of Compound 1 (4.40 g, 8.05 mmol) in tetrahydrofuran (26 mL) under a nitrogen atmosphere was synthesized according to the method described in Organic Letters, 7, 1569-1572 (2005). To the mixture, saturated aqueous sodium hydrogen carbonate (13 mL) and Cbz chloride (1.26 mL, 8.86 mmol) were added under ice-cooling, and the mixture was stirred at room temperature for 1 hr. Water (13 mL) was added to the reaction mixture, and the mixture was extracted with dichloromethane (75 mL). The organic layer was washed with saturated brine (25 mL) and dried over sodium sulfate. The solvent was distilled off under reduced pressure, and ethyl acetate-isopropyl ether (2: 1, 66 mL) was added to the resulting residue. The precipitated solid was collected by filtration and washed with ethyl acetate-isopropyl ether (1: 1) to obtain compound 2 (4.87 g, yield 93%). The physical property data of the obtained compound 2 were as follows.
¹ H-NMR (CDCl ₃ ) δ: 8.04 (1H, s), 7.54 (1H, s), 7.44-7.27 (20H, m), 6.59 (1H, d, J = 7.3 Hz), 5.20 (1H, brs ), 4.63 (1H, brs), 4.17 (1H, s), 3.49 (1H, dd, J = 10.7, 2.4 Hz), 3.39 (1H, brs), 3.15 (3H, s), 1.36 (3H, s) .

Step 2 Synthesis of Compound 3 To a solution of compound 2 (4.35 g, 6.72 mmol) and imidazole (81.8 mg, 1.20 mmol) in DMF (9 mL) was added TBS chloride (140 mg, 0.929 mmol) under ice-cooling. The mixture was further stirred at room temperature for 19 hours. The reaction mixture was diluted with ethyl acetate (45 mL), and water (15 mL) and 2 mol / L hydrochloric acid (5 mL) were added under ice cooling. The organic layer was washed with water (20 mL) and saturated brine (20 mL), and dried over sodium sulfate. The solvent was distilled off under reduced pressure to obtain a crude product of compound 3.

Step 3 Synthesis of Compound 4 To a chloroform (40 mL) solution of the crude product of Compound 3 obtained in Step 2, 4 mol / L hydrogen chloride / ethyl acetate solution (2.00 mL, 8.00 mmol) was added under ice cooling, The mixture was stirred for 4.5 hours while warming to room temperature. Further, a 4 mol / L hydrogen chloride / ethyl acetate solution (1.34 mL, 5.36 mmol) was added under ice cooling, and the mixture was stirred for 30 minutes. Water (40 mL) was added to the reaction solution under ice-cooling, and the organic layer was washed with water (40 mL) and saturated brine (20 mL), and dried over sodium sulfate. The solvent was distilled off under reduced pressure, and the resulting residue was purified by silica gel column chromatography (n-hexane-ethyl acetate) to obtain compound 4 (2.20 g, yield 63%) and compound 3 (1.68 g, yield). 33%). The physical property data of the obtained compound 4 were as follows.
¹ H-NMR (CDCl ₃ ) δ: 8.24 (1H, s), 7.46 (0.8H, s), 7.33-7.24 (5.2H, m), 6.42 (0.8H, d, J = 9.3 Hz), 6.23 ( 0.2H, d, J = 8.3 Hz), 5.19-5.05 (2H, m), 4.73 (0.2H, t, J = 7.9 Hz), 4.66 (0.8H, t, J = 7.3 Hz), 4.49 (0.8H , d, J = 6.3 Hz), 4.42 (0.2H, d, J = 5.3 Hz), 4.02 (0.8H, s), 3.99 (0.2H, s), 3.88 (1H, d, J = 12.0 Hz), 3.78-3.71 (1H, m), 3.07 (0.7H, s), 3.06 (2.3H, s), 2.38 (1H, brs), 1.89 (2.3H, s), 1.84 (0.7H, s), 0.85 ( 9H, s), 0.01 (3H, s), -0.03 (3H, s).

Moreover, the physical property data of the obtained compound 3 were as follows.
¹ H-NMR (CDCl ₃ ) δ: 8.13 (0.4H, s), 8.08 (0.6H, s), 7.69 (0.4H, s), 7.56 (0.6H, s), 7.49-7.20 (20H, m) , 6.60-6.55 (1H, m), 5.47 (0.4H, d, J = 11.8 Hz), 5.19 (1.2H, s), 4.97 (0.6H, dd, J = 9.0, 6.4 Hz), 4.87 (0.4H , d, J = 12.2 Hz), 4.81 (0.4H, dd, J = 8.7, 5.8 Hz), 4.67 (0.6H, d, J = 5.9 Hz), 4.47 (0.4H, d, J = 5.3 Hz), 4.05 (0.6H, s), 3.98 (0.4H, s), 3.40-3.24 (2H, m), 3.16 (1.2H, s), 3.12 (1.8H, s), 1.41 (1.2H, s), 1.34 (1.8H, s), 0.82 (5.4H, s), 0.77 (3.6H, s), -0.03 (1.8H, s), -0.09 (1.8H, s), -0.21 (1.2H, s), -0.26 (1.2H, s).

Step 4 Synthesis of Compound 5 To a solution of Compound 4 (1.07 g, 2.06 mmol) in DMSO (10 mL) was added EDC hydrochloride (1.18 mg, 6.16 mmol) and pyridinium paratoluenesulfonate (518 mg, 2.06 mmol). Was added at room temperature and stirred for 3 hours. The reaction mixture was diluted with ethyl acetate (30 mL), washed with water (10 mL × 4 times) and saturated brine (10 mL), and dried over sodium sulfate. The solvent was distilled off under reduced pressure to obtain a crude product of compound 5.

Step 5 Synthesis of Compound 6 To a solution of the crude product of Compound 5 obtained in Step 4 in ethanol (4 mL), triethylamine (2.00 mL, 14.4 mmol) and 37% formalin aqueous solution (2.00 mL, 26.9 mmol) were added. It added at room temperature and stirred at 80 degreeC for 4 hours. 1 mol / L hydrochloric acid (20 mL) was added to the reaction mixture under ice cooling, and the mixture was extracted with ethyl acetate (30 mL and 20 mL). The organic layer was washed with water (20 mL) and saturated brine (20 mL), and dried over sodium sulfate. The solvent was distilled off under reduced pressure to obtain a crude product of compound 6.

Step 6 Synthesis of Compound 7 Crude product of Compound 6 obtained in Step 5, sodium dihydrogen phosphate dihydrate (551 mg, 3.53 mmol) and 2-methyl-2-butene (1.87 mL, 17.7 mmol) ) In tert-butanol (10 mL) and water (3 mL), a solution of sodium chlorite (800 mg, 8.85 mmol) in water (5 mL) was added dropwise under ice-cooling, and the reaction mixture was warmed to room temperature. The mixture was stirred for 2.5 hours. 0.2 mol / L sodium hydroxide aqueous solution (20 mL) was added to the reaction solution, and the mixture was washed with toluene (20 mL). The organic layer was extracted with water (5 mL), the aqueous layers were combined, 2 mol / L hydrochloric acid (4 mL) was added, and then extracted with ethyl acetate (20 mL × twice). The ethyl acetate extract was washed with saturated brine (20 mL) and dried over sodium sulfate. The solvent was distilled off under reduced pressure to obtain Compound 7 (816 mg, diastereomer ratio 93%, total yield of 70% for 3 steps). The physical property data of the obtained compound 7 were as follows.
¹ H-NMR (DMSO-d ₆ ) δ: 11.51 (1H, d, J = 4.6 Hz), 7.66 (0.8H, d, J = 4.5 Hz), 7.36-7.21 (5.2H, m), 6.59 (0.8 H, d, J = 8.8 Hz), 6.48 (0.2H, d, J = 9.5 Hz), 5.54-5.49 (0.8H, m), 5.22 (0.2H, d, J = 13.1 Hz), 5.11-4.94 ( 2H, m), 4.66-4.55 (1H, m), 4.49 (1H, dd, J = 6.4, 6.4 Hz), 4.23 (0.8H, s), 3.87 (0.8H, dd, J = 11.7, 3.6 Hz) , 3.78 (0.2H, d, J = 11.2 Hz), 3.72-3.54 (1H, m), 2.96 (2H, s), 2.91 (1H, s), 1.79 (2H, s), 1.76 (1H, s) , 0.89-0.82 (9H, m), 0.10-0.00 (3H, m), -0.10--0.18 (3H, m).

Step 7 (2R, 3S, 4R, 5R) -3- (tert-butyldimethylalkyloxy) -2-hydroxymethyl-5- (5-methyl-2,4-dioxo-3,4-dihydropyrimidine-1 Synthesis of (2H) -yl) -4- (methylamino) tetrahydrofuran-2-carboxylic acid (Compound 8) Compound 7 (816 mg, 1.45 mmol), 10% palladium on carbon (78.0 mg) in methanol (16 mL) The suspension was stirred for 5 hours under a hydrogen atmosphere at normal pressure. After filtering the reaction solution, the solvent of the filtrate was distilled off under reduced pressure to obtain a crude product of compound 8. The physical property data of the obtained Compound 8 were as follows.
¹ H-NMR (DMSO-d ₆ ) δ: 11.41 (1H, s), 7.83 (1H, s), 6.07 (1H, d, J = 5.5 Hz), 5.34 (1H, brs), 4.60 (1H, d , J = 6.0 Hz), 3.85-3.72 (3H, m), 2.47 (3H, s), 1.83 (4H, s), 0.91 (9H, s), 0.16 (3H, s), 0.15 (3H, s) .
[Example 2]

  (2R, 3S, 4R, 5R) -3- (tert-butyldimethylalkyloxy) -2-hydroxymethyl-5- (5-methyl-2,4-dioxo-3,4-dihydropyrimidine-1 (2H Synthesis of potassium) -yl) -4- (2,2,2-trifluoro-N-methylacetamide) tetrahydrofuran-2-carboxylate

Step 1 Synthesis of Compound 9 DMF (3.9 L) suspension of Compound 1 (1300 g, 2.53 mol) in accordance with the method described in Organic Letters, 7, 1569-1572 (2005) under nitrogen atmosphere To the solution, a solution of TBS chloride (1717 g, 11.39 mol) in DMF (2.6 L) was added dropwise at 5 to 15 ° C. Subsequently, sodium iodide (1138 g, 7.59 mol) was added at 10 to 20 ° C., and N-methylimidazole (999 mL, 12.7 mol) was added dropwise at the same temperature. After stirring at 20-30 ° C. for 6 hours, the mixture was allowed to stand overnight. The reaction mixture was poured into a mixture of ethyl acetate (3.9 L) and ice water (10.4 L), and ethyl acetate (2.6 L) and water (2.6 L) were further added. The organic layer was washed successively with 1 mol / L hydrochloric acid (6.5 L), 10% aqueous sodium carbonate solution (6.5 L), and 10% brine (6.5 L). The solvent was distilled off under reduced pressure, and methanol (2.6 L) was added to the obtained residue (2.68 kg). The solvent was distilled off again under reduced pressure to obtain a crude product of compound 9 (2.62 kg).

Step 2 Synthesis of Compound 10 To a solution of the crude product of Compound 9 obtained in Step 1 (2.62 kg) in methanol (6.5 L), 35% hydrochloric acid (633 mL, 7.59 mol) was added dropwise at 20-30 ° C. Stir for 4 hours. The reaction mixture was poured into a mixture of diisopropyl ether (6.5 L) and ice water (3.9 L), and diisopropyl ether (6.5 L) and water (2.6 L) were further added. The aqueous layer was washed with diisopropyl ether (3.25 L), then a solution of sodium carbonate (1073 g, 10.1 mol) in water (6.5 L) was added and extracted with ethyl acetate (19.5 L and 6.5 L), The organic layer was dried over anhydrous magnesium sulfate. The solvent was distilled off under reduced pressure, and tetrahydrofuran (2.6 L) was added to the obtained residue. The solvent was distilled off again under reduced pressure to obtain a crude product of compound 10 (1.08 kg). The physical property data of the obtained compound 10 were as follows.
¹ H-NMR (DMSO-d ₆ ) δ: 0.13 (s, 6H), 0.91 (s, 9H), 1.40-1.55 (br, 1H), 1.79 (s, 3H), 2.28 (s, 3H), 3.15 -3.20 (d, J = 4.1Hz, 1H), 3.52-3.65 (m, 2H), 3.85 (s, 1H), 4.30-4.35 (d, J = 4.1Hz, 1H), 5.18-5.24 (br, 1H ), 5.64-5.70 (d, J = 7.3Hz), 7.73 (s, 1H), 11.34 (s, 1H).

Step 3 Synthesis of Compound 11 To a solution of the crude product of compound 10 obtained in Step 2 (17.7 g) and pyridine (10.1 mL, 125 mmol) in tetrahydrofuran (80 mL) under a nitrogen atmosphere at 0 to 10 ° C. Acetic anhydride (17.8 mL, 125 mmol) was added dropwise and stirred for 1 hour. The reaction mixture was poured into a 20% aqueous sodium carbonate solution (112 mL) under ice-cooling, and tetrahydrofuran (24 mL) and water (48 mL) were added. After stirring for 20 minutes under ice cooling and 1 hour at room temperature, ethyl acetate (112 mL), n-hexane (80 mL), 10% brine (32 mL) and water (176 mL) were added. The organic layer was washed successively with 2 mol / L hydrochloric acid (80 mL), 0.4 mol / L hydrochloric acid (80 mL), 5% aqueous sodium hydrogen carbonate solution (80 mL), and 10% brine (80 mL). Each aqueous layer was extracted with ethyl acetate (80 mL). The organic layers were combined and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, ethyl acetate (24 mL) was added to the resulting residue, and the mixture was heated to 60 ° C. for dissolution, and then n-hexane (72 mL) was added. The resulting slurry was cooled to room temperature, and the solid obtained by filtration was washed with ethyl acetate / n-hexane (3.2 mL / 60.8 mL) and then air-dried for 1 day to give compound 11 (15. 8 g, the total yield from Step 1 was 79.1%). The physical property data of the obtained compound 11 were as follows.
¹ H-NMR (DMSO-d ₆ ) δ: -0.01 (2.1H, s), 0.02 (0.9H, s), 0.08 (2.1H, s), 0.09 (s, 0.9H, s), 0.86 (6.3 H, s), 0.87 (2.7H, s), 1.78 (0.9H, d, J = 0.8Hz), 1.79 (2.1H, d, J = 0.8Hz), 3.12 (0.9H, s), 3.20 (2.1 H, s), 3.61-3.57 (1.0H, m), 3.72-3.67 (1.0H, m), 3.99 (0.7H, q, J = 3.3Hz), 4.02 (0.3H, m), 4.45 (0.3H , m), 4.45 (0.3H, m), 4.52 (0.7H, dd, J = 6.8, 3.3Hz), 4.77 (0.7H, dd, J = 7.6, 6.8Hz), 5.30 (0.7H, t, J = 4.7Hz), 5.38 (0.3H, t, J = 4.7Hz), 6.37 (0.3H, m), 6.39 (0.7H, d, J = 7.6Hz), 7.73 (1.0H, s), 11.44 (0.7 H, brs), 11.5 (0.3H, brs).

Step 4 Synthesis of Compound 12 Under a nitrogen atmosphere, a solution of compound 11 (450 g, 935 mmol) in DMSO (1.35 L) was charged with EDC (538 g, 2800 mmol), DMSO (675 mL), pyridinium paratoluenesulfonate (235 g, 935 mmol) and DMSO (225 mL) was sequentially added, and the mixture was stirred at 20 to 30 ° C. for 2.5 hours. Subsequently, ethyl acetate (4.5 L), water (2.25 L) and ethyl acetate (2.25 L) were sequentially added at 10 to 20 ° C. The organic layer was washed twice with water (2.25 L), then the solvent was distilled off under reduced pressure, and acetonitrile (900 mL) was added to the resulting residue. The solvent was distilled off again under reduced pressure to obtain a crude product of compound 12 (540 g).

Step 5 Synthesis of Compound 13 First, conditions for the aldol reaction performed in Step 5 were examined. Specifically, the combination of the type / amount of the base used, the type / amount of the reagent, the type / amount of the solvent, the reaction temperature, and the reaction time were examined, and the target product, by-product formation rate, and raw material residual rate were determined by HPLC. Analyzed in The results are shown in the following table.

  Under the above condition 9, 37% formalin aqueous solution (900 mL) and N-methylmorpholine (900 mL) were sequentially added to a solution of the crude product of compound 12 obtained in step 4 (540 g) in acetonitrile (1.8 L), It stirred at 70-80 degreeC for 4 hours. After cooling the reaction solution to 20-30 ° C., ethyl acetate (2.25 L) was added, followed by 4 mol / L hydrochloric acid (2.25 L) at 10-20 ° C. The organic layer was washed twice with water (2.25 L) to obtain an ethyl acetate solution containing compound 13.

Step 6 (2R, 3S, 4R, 5R) -3- (tert-Butyldimethylalkyloxy) -2-hydroxymethyl-5- (5-methyl-2,4-dioxo-3,4-dihydropyrimidine-1 (2H) -yl) -4- (2,2,2-trifluoro-N-methylacetamide) tetrahydrofuran-2-carboxylate (compound 14) ethyl acetate solution containing compound 13 obtained in synthesis step 5 To the mixture, sodium dihydrogen phosphate dihydrate (292 g, 1870 mmol) in water (1.35 L), squalene (898 mL, 1870 mmol), and acetonitrile (1.35 L) were sequentially added. A solution of sodium chlorite (423 g, 4670 mmol) in water (2.25 L) was added dropwise at 20-30 ° C. and stirred for 3 hours. Subsequently, a 0.5 mol / L aqueous sodium hydroxide solution (3.6 L) and sodium thiosulfate pentahydrate (1160 g, 4670 mmol) were sequentially added at 10 to 15 ° C., and the mixture was stirred at 5 to 15 ° C. for 20 minutes. Ethyl acetate (900 mL) and water (900 mL) were added to the reaction solution, and the organic layer was washed with water (900 mL). The aqueous layers were combined and washed with ethyl acetate (900 mL), and then ethyl acetate (5.4 L) and a 2 mol / L aqueous hydrochloric acid solution (2.25 L) were added to the aqueous layer. The organic layer was washed with 10% brine (2.25 L) and dried over anhydrous sodium sulfate. The mixture was filtered, and the drying agent was washed with ethyl acetate (1.35 L). To the filtrate obtained, potassium acetate (82.5 g, 841 mmol) and ethanol (338 mL) were added. The mixture was stirred at 20-30 ° C. for 1 hour. The solid obtained by filtering the resulting slurry was washed with ethyl acetate / ethanol (3860 mL / 193 mL) and then dried by ventilation for 1 day to give compound 14 (283 g, total yield from step 4 of 53.8%). Got. The physical property data of the obtained compound 14 were as follows.
¹ H-NMR (DMSO-d ₆ ) δ: -0.11 (1.8H, s), -0.09 (1.2H, s), 0.09 (1.8H, s), 0.10 (1.2H, s), 0.82 (3.6H , s), 0.83 (5.4H, s), 1.77 (1.2H, s), 1.78 (1.8H, s), 3.18 (1.2H, s), 3.33 (1.8H, s), 3.54 (1.0H, d , J = 10.8Hz), 3.68 (0.6H, d, J = 10.8Hz), 3.69 (0.4H, d, J = 10.8Hz), 4.18 (0.4H, dd, J = 8.6, 7.8Hz), 4.49 ( 0.4H, d, J = 8.6Hz), 4.51 (0.6H, d, J = 8.6Hz), 4.73 (0.6H, dd, J = 8.6, 7.8Hz), 6.1 (1H, br), 6.90 (0.4H , d, J = 7.8Hz), 6.92 (0.6H, d, J = 7.8Hz), 7.61 (0.6H, s), 7.65 (0.4H, s).
[Example 3]

Synthesis of T amidite

Step 1 Synthesis of Compound 15 In a nitrogen atmosphere, a 1 mol / L TBAF-tetrahydrofuran solution (0.888 mL, 0.888 mmol) was added dropwise to a suspension of compound 14 (455 mg, 0.807 mmol) in pyridine (4 mL). The mixture was stirred at ˜70 ° C. for 9 hours and then allowed to stand overnight at room temperature. The next day, the mixture was further stirred at 60 to 70 ° C. for 9 hours, and allowed to stand at room temperature overnight. After adding EDC hydrochloride (310 mg, 1.62 mmol) and stirring at 60-70 ° C. for 9 hours, EDC hydrochloride (310 mg, 1.62 mmol) was added, and the mixture was allowed to stand overnight at room temperature. DMTr (4,4′-dimethoxytrityl) chloride (547 mg, 1.61 mmol) was added and stirred at room temperature for 5 hours, then DMTr chloride (547 mg, 1.61 mmol) was further added and stirred for 2 hours. 5% Aqueous sodium hydrogen carbonate solution (10 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (10 mL), and washed 3 times with water (5 mL). Each water wash was extracted with ethyl acetate (5 mL). The organic layers were combined, the solvent was distilled off under reduced pressure, and the resulting residue was purified by silica gel column chromatography (chloroform-methanol) to obtain Compound 15 (334 mg, yield 69.1%).
1H-NMR (CDCl3) δ: 1.70 (s, 3H), 1.97 (d, J = 5.1Hz, 1H), 3.02 (s, 3H), 3.61 (d, J = 12.3Hz, 1H), 3.79 (s, 6H), 3.95 (d, J = 12.3Hz, 1H), 4.11 (s, 1H), 4.37 (d, J = 4.9Hz, 1H), 5.41 (s, 1H), 6.85 (dd, J = 8.5, 2.5 Hz, 4H), 7.23-7.36 (m, 7H), 7.45 (d, J = 7.7Hz, 2H), 7.76 (s, 1H), 8.55 (s, 1H).

Step 2 Synthesis of Compound 16 (T Amidite) Compound 16 was synthesized according to a method according to the method described in WO2011 / 052436A1.

The route from compound 14 to compound 15 can also be synthesized by a method that does not go through a deTFA reaction as follows.

Step 1 Synthesis of Compound 30 Sodium borohydride (47 mg, 6 eq.) Was added to a methanol solution (1 ml) at 0 ° C., then Compound 14 (100 mg) was added, and the mixture was stirred at room temperature for 3.5 hours. Since the raw material did not disappear, sodium borohydride (32 mg) was further added, and the mixture was stirred at room temperature for 1 hour, adjusted to pH = 3 with hydrochloric acid-methanol solution, and further stirred for 1 hour. The reaction solution was concentrated as it was to obtain a crude product of compound 30 (218 mg). The obtained crude product of compound 30 was not purified, and the yield was quantitative, and it was advanced to the next step.

Step 2 Synthesis of Compound 31 The crude product of Compound 30 (196 mg) obtained in the previous step was suspended in N, N′-dimethylformamide (1.4 ml), EDC hydrochloride (76 mg) was added, and room temperature For 4 hours. The reaction mixture was filtered to remove inorganic substances (washed with N, N′-dimethylformamide (1.5 ml)), and then water (3.3 ml) was added to precipitate a slurry solid. . The solid was collected by filtration, washed with water, and dried to give Compound 31 (36.5 mg, yield from Compound 14 = 63%) as a colorless solid.

Step 3 Synthesis of Compound 32 Compound 32 was obtained by treating compound 31 with 4,4′-dimethoxytrityl chloride in pyridine.
Step 4 Conversion of Compound 32 to Compound 15 Compound 15 was obtained by treating Compound 32 with a tetrabutylammonium fluoride-tetrahydrofuran solution.
[Example 4]

Synthesis of A (Bz) amidite

Step 1 The conditions of the transglycosylation reaction performed in the synthesis step 1 of Compound 17 were examined. Specifically, the combination amount of the reagent and the type of the solvent was examined, and the target product, by-product formation rate, and the raw material residual rate were analyzed by HPLC. The results are shown in the following table.

According to the above condition 6, N, O-bis (trimethylsilyl) was added to a suspension of compound 14 (170 g, 271 mmol) and 6-N-benzoyladenine (64.9 g, 271 mmol) in cyclopentyl methyl ether (1700 mL) under a nitrogen atmosphere. ) Acetamide (466 mL, 1900 mmol) and TMSOTf (98 mL, 543 mmol) were added and stirred at 70-73 ° C. for 4 hours. Tetrahydrofuran (2920 mL), 2 mol / L hydrochloric acid (1700 mL) and water (1020 mL) were added to the reaction solution under ice cooling, and then the organic layer and aqueous layer were partitioned. The organic layer was 0.1 mol / L hydrochloric acid (1700 mL). And washed with water (1700 mL). Chloroform (340 mL) was added to the residue obtained by distilling off the solvent under reduced pressure, and the resulting slurry obtained by stirring for 20 minutes was filtered. The solid collected by filtration was washed with chloroform (510 mL) and then air-dried to obtain Compound 17 (165 g, yield 80.9%).
1H-NMR (DMSO-d6) δ: -0.06 (s, 3H), 0.13 (s, 2.1H), 0.14 (s, 0.9H), 0.89 (s, 9H), 3.22 (s, 0.9H), 3.31 (s, 2.1H), 3.82 (d, J = 12.0Hz, 1H), 3.89-3.95 (m, 1H), 4.70 (d, J = 7.3Hz, 0.3H), 4.75 (d, J = 7.3Hz, 0.7H), 5.04 (t, J = 7.5Hz, 0.3H), 5.41 (brs, 0.7H), 5.49 (t, J = 8.0Hz, 0.7H), 5.55 (brs, 0.3H), 7.02 (d, J = 8.5Hz, 0.3H), 7.04 (d, J = 8.5Hz, 0.7H), 7.56 (t, J = 7.4Hz, 2H), 7.66 (t, J = 7.4Hz, 1H), 8.04 (d, J = 7.8Hz, 2H), 8.78 (s, 1H), 8.80 (s, 1H), 11.31 (s, 1H), 13.34 (brs, 1H).

Step 2 Synthesis of Compound 18 Under a nitrogen atmosphere, 1 mol / L TBAF-tetrahydrofuran solution (14.9 mL, 14.9 mmol) was added dropwise to a solution of Compound 17 (4.75 g, 7.44 mmol) in tetrahydrofuran (25 mL). Stir at ~ 65 ° C for 3.5 hours. EDC hydrochloride (1.71 g, 8.92 mmol) was added and stirred at 60-65 ° C. for 1 hour. The solvent was distilled off under reduced pressure, acetonitrile (50 mL) was added to the resulting residue, and the solvent was distilled off again under reduced pressure three times to obtain a crude product of compound 18 (9.12 g).
1H-NMR (CDCl3) δ: 3.14 (s, 3H), 4.09 (dd, J = 13.8, 6.8Hz, 1H), 4.18 (dd, J = 14.0, 3.3Hz, 1H), 4.37 (s, 1H), 4.64 (s, 1H), 5.26 (m, 1H), 5.89 (s, 1H), 6.46 (brs, 1H), 7.52 (t, J = 7.5Hz, 2H), 7.60 (t, J = 7.3Hz, 1H ), 8.01 (d, J = 7.7Hz, 2H), 8.56 (s, 1H), 8.78 (s, 1H), 9.31 (s, 1H).

Step 3 Synthesis of Compound 19 To a solution of the crude product of Compound 18 (9.12 g) obtained in Step 2 (9.12 g) in dichloromethane (25 mL) under a nitrogen atmosphere was added DABCO (2.09 g, 18.6 mmol) and DMTr chloride (5. 04 g, 14.9 mmol) was added and stirred at room temperature for 2 hours. DMTr chloride (1.26 g, 3.72 mmol) was added, and the mixture was further stirred for 2 hours. A 5% aqueous sodium hydrogen carbonate solution (50 mL) and dichloromethane (50 mL) were added to the reaction solution, and the organic layer was washed with water (50 ml), and the aqueous washing solution was extracted with dichloromethane (50 mL). The organic layers were combined, methanol (50 mL) was added, the solvent was distilled off until the weight reached 60 g, methanol (50 mL) was added again, and the solvent was distilled off until the weight reached 67 g. Methanol (25 mL) was added again, and the resulting slurry was stirred for 30 minutes at room temperature and filtered. The solid collected by filtration was washed with methanol (35 mL) and then air-dried to obtain Compound 19 (4.31 g, total yield from Step 2 81.3%).
1H-NMR (DMSO-d6) δ: 2.98 (s, 3H), 3.26 (d, J = 11.0Hz, 1H), 3.42 (d, J = 11.7Hz, 1H), 3.73 (s, 6H), 4.67 ( s, 1H), 4.79 (brs, 1H), 6.14 (s, 1H), 6.19 (brs, 1H), 6.88 (d, J = 8.3Hz, 4H), 7.21-7.32 (m, 7H), 7.39 (d , J = 7.8Hz, 2H), 7.57 (t, J = 7.4 Hz, 2H), 7.67 (t, J = 7.4Hz, 1H), 8.06 (d, J = 7.4Hz, 2H), 8.53 (s, 1H ), 8.84 (s, 1H), 11.33 (brs, 1H). ¹ H-NMR (CDCl ₃ ) δ: 8.98 (1H, s), 8.81 (1H, s), 8.39 (1H, s), 8.03 (1H , d, J = 8.0 Hz), 7.63 (1H, t, J = 8.0 Hz), 7.55 (2H, t, J = 8.0 Hz), 7.46 (2H, d, J = 8.0 Hz), 7.38-7.24 (7H , m), 6.86 (4H, d, J = 8.0 Hz), 5.89 (1H, s), 4.51 (1H, s), 4.47 (1H, m), 3.91 (1H, d, J = 12.0 Hz), 3.79 (6H, s), 3.73 (1H, d, J = 12.0 Hz), 3.15 (3H, s), 2.07 (1H, br s, 4.0 Hz) .LC-MS: UPLC 4min base 2.28 min M + H = 713

Step 4 Synthesis of compound 20 (A amidite)

Under a nitrogen atmosphere, a suspension of compound 19 (107 g, 150 mmol) and DIEA (79 mL, 450 mmol, 3.0 eq.) In pyridine (428 mL, 4 V) under ice-cooling was cooled to 2-cyanoethyl-N, N-diisopropylchlorophosphoro. Amidite (53.6 mL, 240 mmol, 1.6 eq.) Was added dropwise and stirred at room temperature for 2 hours. Under ice-cooling, 5% aqueous sodium hydrogen carbonate solution (1.1 L) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (1.1 L). The organic layer was washed twice with 3% aqueous sodium chloride solution (1.1 L), and the washings were re-extracted with ethyl acetate (1.1 L), respectively, and the organic layers were combined and dried over sodium sulfate. The solvent was distilled off under reduced pressure, and the resulting residue was purified by silica gel column chromatography (ethyl acetate) to obtain compound 20 (120 g, containing 2.0 wt% pyridine and 5.0 wt% ethyl acetate, yield 81.5%). Got.
1H-NMR (DMSO-d6) δ: 0.87 (d, J = 6.8Hz, 2H), 0.92 (d, J = 6.5Hz, 4H), 1.06 (d, J = 6.8Hz, 2H), 1.08 (d, J = 6.8Hz, 4H), 2.55 (dd, J = 11.7, 5.4Hz, 1.3H), 2.70 (m, 0.7H), 2.99 (s, 2H), 3.01 (s, 1H), 3.18 (d, J = 11.5Hz, 0.7H), 3.22 (d, J = 11.5Hz, 0.3H), 3.43-3.57 (m, 4.3H), 3.65-3.70 (m, 0.7H), 3.72 (s, 2H), 3.73 ( s, 4H), 4.98 (s, 1H), 5.04 (d, J = 4.8Hz, 0.3H), 5.12 (d, J = 7.8Hz, 0.7H), 6.28 (s, 0.7H), 6.29 (s, 0.3H), 6.85-6.89 (m, 4H), 7.21-7.31 (m, 7H), 7.36-7.39 (m, 2H), 7.57 (t, J = 7.5Hz, 2H), 7.66 (t, J = 7.3 Hz, 1H), 8.06 (d, J = 7.5Hz, 2H), 8.50 (s, 0.3H), 8.52 (s, 0.7H), 8.83 (s, 1H), 11.31 (brs, 1H).
31P-NMR (DMSO-d6) δ: 150.13 (s, 0.3H), 150.42 (s, 0.7H).

About the path | route from the compound 14 to the compound 19, it can synthesize | combine also by the method which does not go through de-TFA reaction as follows.

Step 1 Synthesis of Compound 33
Under a nitrogen atmosphere, a suspension of compound 14 (5.0 g, 8.07 mmol) and adenine (1.091 g, 8.07 mmol) in cyclopentyl methyl ether (50 mL) was added to N, O-bis (trimethylsilyl) acetamide (13. 85 mL, 56.5 mmol) and TMSOTf (2.92 mL, 16.15 mmol) were added and stirred at 70 ° C. for 4 hours. Ethyl acetate (50 mL), 0.1 mol / L hydrochloric acid (40 mL), and tetrahydrofuran (70 mL) were added to the reaction solution under ice-cooling, and the organic layer and the aqueous layer were partitioned. Washed with L hydrochloric acid (110 mL) and water (110 mL). Ethyl acetate (50 mL) and n-hexane (50 ml) were added to the residue obtained by distilling off the solvent under reduced pressure, and the resulting slurry obtained by stirring was filtered. The solid collected by filtration was washed with a mixed solvent of ethyl acetate and n-hexane (1: 1, 20 ml) and then air-dried to obtain Compound 33 (3.58 g, yield 83%).

Step 2 Synthesis of Compound 34
To a solution of compound 33 (309 mg, 0.578 mmol) in ethanol (3 ml) was added 1 mol / L lithium borohydride tetrahydrofuran solution (1.156 ml) at 0 ° C., and the mixture was stirred at room temperature for 3 hours.
A 4 mol / L hydrogen chloride-dioxane solution (0.578 ml) was added to the reaction mixture, and the mixture was stirred at room temperature for 1 hr, EDC hydrochloride (222 mg, 1.156 mmol.) Was added, and the mixture was stirred at 65 ° C. for 2 hr. After allowing to cool, the obtained solid was collected by filtration, washed with methanol, and then air-dried to obtain Compound 34 (187 mg, yield 77%).
LC-MS: UPLC 4min base 1.57 min, M + H = 420

Step 3 Synthesis of Compound 35
Under a nitrogen stream, compound 34 (149 mg, 0.354 mmol) and methanol (4.5 mL) were added to a 20 ml Falcon tube, followed by addition of ammonium fluoride (52.5 mg, 1.417 mmol) at room temperature, and 60 ° C. And heated at reflux for 8 hours. After allowing to cool, methanol (1.5 ml) and tetrahydrofuran (2 ml) were added, and the resulting slurry was collected by filtration to give compound 35 (89.5 mg, yield 89.5%) as a white solid. .

Step 4 Synthesis of Compound 36
An azeotropic dehydration operation of compound 35 (8.00 g, 19.9 mmol) with pyridine (80 ml) was repeated three times with 500 ml of 1 L eggplant colben, and then suspended in pyridine (80 ml). N, N′-dimethylformamide-dimethylacetal (8.0 mL) was added and stirred at room temperature for 1 hour. The resulting orange solution was concentrated as it was, and the residue was subjected to a single azeotropic dehydration operation with pyridine (80 ml) and then dissolved in pyridine (80 ml). 4,4′-Dimethoxytrityl chloride (10.1 g, MR = 1.5) was added and stirred at room temperature for 3 hours. Methanol (5.0 ml) was added dropwise to the reaction solution, stirred for 40 minutes, and diluted with ethyl acetate. The organic layer was washed twice with saturated aqueous sodium hydrogen carbonate solution and saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated. Again, it was dissolved in pyridine (100 ml), 25% aqueous ammonia (30 ml) was added, and the mixture was stirred at room temperature for 20 hours. After concentrating the reaction solution as it was, the resulting residue was purified by silica gel column chromatography (chloroform / methanol, 10/0 → 9/1) to obtain compound 36 (8.83 g, purity 93%, yield). 73%) was obtained as a yellow powder.

Step 5 Conversion from Compound 36 to A (Bz) -DMTr Form (Compound 19) Under a nitrogen stream, HOBt (1.38 g, 10.19 mmol), dichloromethane (20 mL), N-methylmorpholine (2 .17 mL, 19.72 mmol) was added. Thereafter, BzCl (1.15 mL, 9.86 mmol) was added dropwise at room temperature to prepare Bz-OBt. Under a nitrogen stream, Compound 36 (2.00 g, 3.29 mmol), pyridine (20 mL), and N-methylmorpholine (2.17 mL, 19.72 mmol) were added to a 100 mL eggplant flask B. Subsequently, chlorotrimethylsilane (1.68 mL, 13.14 mmol) was added dropwise at room temperature to obtain a white suspension. The solution of Flask A was added dropwise to Flask B at room temperature and stirred overnight. Since the reaction proceeded only about 5%, the same amount of BzOt solution was prepared and added dropwise to the reaction solution at room temperature. Then, it stirred at 50 degreeC for 12 hours, and left still at room temperature on the weekend. After confirming the completion of the reaction, the reaction solution was poured into ethyl acetate, and the organic layer was washed with saturated multistory water and saturated brine by a liquid separation operation. Sodium sulfate was added to the obtained organic layer and stirred for 30 minutes, followed by filtration, concentration under reduced pressure, and drying to obtain a crude dibenzoyl compound. The residue was dissolved in methanol (20 mL), potassium carbonate (2.72 g, 19.72 mmol) was added at 0 ° C., and the mixture was stirred at 0 ° C. for 1 hr. Thereafter, filtration, concentration under reduced pressure, and drying were performed, and the resulting residue was purified by silica gel column chromatography (chloroform / methanol, 10/0 → 9/1) to obtain compound 19 (1.76 g, 75.1%). Was obtained as a white foamy solid.

(Comparative Example 1) Synthesis of A (Bz) amidite by conventional transglycosylation reaction

Step 1 Synthesis of Compound 37 Compound 37 was obtained by treating Compound 14 with trimethylsilyldiazomethane-n-hexane solution / methanol.

Step 2 Synthesis of Compound 38 Compound 38 was obtained by subjecting Compound 37 to the same conditions as in Step 2 of Example 2.

Step 3 Synthesis of Compound 39 Compound 37 (78.0 mg, 0.10 mmol), N-benzoyladenine (47.8 mg, 0.20 mmol), 1,2-dichloroethane (1 mL), BSA in a 10 mL eggplant flask under a nitrogen stream (0.15 mL, 0.60 mmol) was added. The reaction solution was heated to 60 ° C. and stirred for 40 minutes, and then allowed to cool to room temperature. After adding trimethylsilyl trifluoromethanesulfonate (0.020 mL, 0.11 mmol) at room temperature, the mixture was heated to reflux for 4 hours. After the disappearance of the raw materials, the mixture was allowed to cool to room temperature, and the reaction was stopped with saturated multistory water. Subsequently, the reaction solution was diluted with ethyl acetate, and then the organic layer was washed with water and saturated brine. Sodium sulfate was added to the obtained organic layer, and the mixture was stirred for 30 minutes, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / hexane: 1/9 → 2/3) to give compound 39 ( 86.4 mg, 97.0%) was obtained as a white foamy solid. The physical property data of the obtained compound were as follows.
¹ H-NMR (CDCl ₃ , 5: 1 mixture of rotamers) δ: 8.80 (1H, s), 8.19 (1H, s), 8.02 (2H, d, J = 4.0 Hz), 7.72-7.30 (14H, m ), 6.83 (1H, d, J = 12.0 Hz), 5.32 (1H, t, J = 8.0 Hz), 4.93 (1H, d, 8.0 Hz), 4.20 (1H, d, J = 12.0 Hz), 4.05 ( 1H, d, J = 12.0 Hz), 3.80 (3H, s), 3.39 (3H, s), 1.14 (9H, s), 1.00 (9H, s), 0.03 (3H, s), 0.00 (3H, s LC-MS: UPLC 4min base 3.60 min, M + H = 892

Step 4 Synthesis of Compound 40 Compound 38 was obtained by treating Compound 39 with NaOH / methanol.

Step 5 Conversion of Compound 40 to Compound 35 Compound 35 was obtained by carrying out under the same conditions as in Step 3 of Example 4.

Step 6 Conversion from Compound 35 to A (Bz) amidite (Compound 20) The reaction was carried out under the same conditions as in Step 5 of Example 4 and Step 4 of Example 4 without passing through the de-TFA reaction.
[Example 5]

Synthesis of A (Pac) amidite

Step 1 Synthesis of Compound 33 The reaction was performed under the same conditions as in Step 1 of the method of Example 4 without passing through the deTFA reaction.

Step 2 Conversion from Compound 33 to Compound 35 Synthesized by the same route as in Step 2 of Example 4.

Step 3 Conversion from Compound 35 to Compound 36 The step was carried out under the same conditions as in Step 4 of the method not passing through the deTFA reaction of Example 4.

Step 4 Synthesis of Compound 42 Under a nitrogen stream, Compound 40 (5.68 g, 9.33 mmol), pyridine (57 ml, 10 V), N-methylmorpholine (6.2 mL, MR = 6) were placed in a 100 mL eggplant flask. After stirring and dissolving, the mixture was cooled with an ice-cooled bath. Chlorotrimethylsilane (4.7 mL, MR = 4) was added dropwise for 5 minutes, and the mixture was further stirred for 20 minutes at room temperature for 1 hour.
Under a nitrogen stream, N-methylmorpholine (6.2 mL, MR = 6) was added to a suspension of HOBt (3.91 g, MR = 3.1) and dichloromethane (57 mL) in a 200 mL eggplant flask. PacCl (3.85 mL) was added dropwise at room temperature over 3 minutes and then stirred for 20 minutes to prepare Pac-OBt. The above-mentioned trimethylsilylation reaction solution was added to this reaction solution and stirred overnight, and the reaction was incomplete, so the same amount of Pac-Bt solution was added, and the mixture was further stirred for 23 hours. The reaction solution was poured into a saturated aqueous sodium hydrogen carbonate solution, the organic layer was separated, and washed successively with a saturated aqueous sodium hydrogen carbonate solution and saturated brine. Each aqueous layer was re-extracted with dichloromethane, and the organic layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was azeotroped with toluene to remove pyridine, dried, dissolved in tetrahydrofuran (50 ml), added with hydrogen fluoride / triethylamine complex (4 ml), and stirred at room temperature for 40 minutes. The reaction mixture was diluted with ethyl acetate, washed twice with saturated aqueous sodium hydrogen carbonate solution and then with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated. The obtained residue was purified by silica gel column chromatography (ethyl acetate / acetone, 100/0 → 80/20) to give compound 42 (9.91 g, purity 96%, yield 84%) as a beige powder. Got as.

Step 5 Synthesis of Compound 43 (A (Pac) amidite) Under a nitrogen atmosphere, a mixed solution of Compound 42 (5.54 g, 7.46 mmol) in acetonitrile (55 mL) -tetrahydrofuran (5.5 ml) was added diisopropylammonium tetrazolide ( After adding 1.34 g, MR = 1.05), 2-cyanoethyl-N, N, N ′, N′-tetraisopropyl phosphoramidite (3.37 g, MR = 1.5) was added in about 3 minutes. The solution was added dropwise and stirred at room temperature for 7 hours. The reaction mixture was diluted with ethyl acetate, washed successively with saturated aqueous sodium hydrogen carbonate solution and saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated. The solvent was distilled off under reduced pressure, and the resulting residue was purified by silica gel column chromatography (first time) hexane / ethyl acetate / acetone 2/1/1, (second time ethyl acetate) to obtain compound 43 (6 20 g, yield 71%, ratio of the two diastereomers (calculated from the integral value of ¹ H-NMR) = 60: 40)).
¹ H-NMR (CDCl3) δ: (major diastereomer) 9.54 (1H, br s), 8.82 (1H, s), 8.54 (1H, s), 7.45 (2H, d, J = 8.0 Hz), 7.40-7.25 (9H, m), 7.09 (2H, d, J = 8.0 Hz), 7.08 (1H, m), 6.89-6.83 (4H, m), 5.96 (1H, s, H-1 of sugar), 4.88 (2H , s), 4.68 (1H, dd, J = 8.5, 1.0 Hz), 4.64 (1H, s), 3.96 (1H, d, J = 12.8 Hz), 3.81 (3H, s), 3.80 (3H, s) , 3.57-3.39 (4H, m), 3.47 (1H, d, J = 12.8 Hz), 3.12 (3H, s, -NCH3), 2.27 (2H, t, J = 6.3 Hz), 1.09 (6H, d, J = 6.8 Hz), 0.90 (6H, d, J = 6.8 Hz). (Minor diastereomer) 9.54 (1H, br s), 8.84 (1H, s), 8.54 (1H, s), 7.45 (2H, d, J = 8.0 Hz), 7.40-7.25 (9H, m), 7.09 (2H, d, J = 8.0 Hz), 7.08 (1H, m), 6.89-6.83 (4H, m), 5.96 (1H, s, H -1 of sugar), 4.88 (2H, s), 4.76 (1H, s, H-2 of sugar), 4.65 (1H, dd, J = 5.0, 1.2 Hz), 3.89 (1H, d, J = 12.3 Hz ), 3.80 (6H, s), 3.70-3.55 (2H, m), 3.52 (1H, d, J = 12.3 Hz), 3.49-3.38 (2H, m), 3.14 (3H, s, -NCH3), 2.44 -2.39 (2H, m), 1.08 (6H, d, J = 6.8 Hz), 0.89 (6H, d, J = 6.8 Hz).

Furthermore, A (amidine) amidite can be synthesized from compound 40 by the following route.

Step 1 Synthesis of Compound 44 Compound 40 (1.82 g, 2.99 mmol) and pyridine (20 mL) were added to a 100 mL eggplant flask under a nitrogen stream. Thereafter, 1,1-dimethoxy-N, N-dimethylmethanamine (1.192 ml, 8.97 mmol) was added and stirred at room temperature for 30 minutes. The reaction solution was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (dichloromethane / methanol, 10/0 → 9/1) to obtain compound 44 (1.74 g, 87.7%) as white. Obtained as a foamy solid.
¹ H-NMR (CDCl ₃ ) δ: 8.99 (1H, s), 8.51 (1H, s), 8.25 (1H, s), 7.47-7.21 (9H, m), 6.86 (4H, d, J = 8.0 Hz ), 5.84 (1H, s), 4.50 (1H, s), 4.44 (1H, s), 3.91 (1H, d, J = 12.0 Hz), 3.78 (6H, s), 3.71 (1H, d, J = 12.0 Hz), 3.24 (3H, s), 3.21 (3H, s), 3.12 (3H, s) .LC-MS: UPLC 4min base 2.05 min, M + H = 664

Step 2 Synthesis of Compound 45
Under a nitrogen stream, Compound 44 (1.70 g, 2.56 mmol), N, N-diisopropylethylamine (2.68 mL, 15.37 mmol), and chloroform (17 mL) were added to a 100 mL eggplant flask. Thereafter, 2-cyanoethyl-N, N-diisopropylchlorophosphoramidite (1.714 ml, 7.68 mmol) was added at 0 ° C., and the mixture was warmed to room temperature and stirred for 4 hours. Saturated multistory water was added to the reaction solution to stop the reaction. The solution was poured into ethyl acetate, and the organic layer was washed with water and saturated brine. Sodium sulfate was added to the obtained organic layer, and the mixture was stirred for 30 minutes, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate) to give compound 45 (1.76 g, 79.5%). ) Was obtained as a white foamy solid.
¹ H-NMR (CDCl ₃ , 1: 1 mixture of diastereomers) δ: 9.04 (1H, s), 8.56 (1H, s), 8.42 (1H, s), 7.45 (2H, d, J = 8.0 Hz), 7.36-7.21 (9H, m), 6.84 (4H, m), 5.93 (1H, s), 4.72 (1H, s), 4.60 (1H, s), 4.14 (1H, d, J = 8.0Hz), 3.80 (6H, s), 3.52-3.40 (5H, m), 3.30 (3H, s), 3.24 (3H, s), 3.12 (3H, s), 2.38 (1H, m), 1.10 (6H, t, J = 8.0 Hz), 0.90 (6H, t, J = 8.0 Hz). ³¹ P-NMR (CDCl ₃ ) δ: 150.8 (s), 150.0 (s). LC-MS: UPLC 4min base 2.67 min, M + H = 864
[Example 6]

Synthesis of G (Pac) amidite

Step 1 Synthesis of Compound 21 In a nitrogen atmosphere, N, O-bis (trimethylsilyl) was added to a suspension of compound 14 (15.0 g, 26.6 mmol) and guanine (4.02 g, 26.6 mmol) in cyclopentyl methyl ether (135 mL). ) Acetamide (65.1 mL, 266 mmol) and cyclopentyl methyl ether (7.5 mL) were added and stirred at 60-70 ° C. for 10 minutes before TMSOTf (11.83 g, 53.20 mmol) and cyclopentyl methyl ether (7.5 mL). ) Was added and stirred for 2 hours. Ethyl acetate (150 mL) was added to the reaction solution at 20 to 30 ° C., and further 2 mol / L hydrochloric acid (75 mL), tetrahydrofuran (150 mL) and water (75 mL) were added at 15 to 30 ° C., and the organic layer was added with water (150 mL). Washed twice. The washings were combined and extracted with ethyl acetate (150 mL) and washed with water (75 mL). The organic layers were combined and the solvent was distilled off under reduced pressure. Ethyl acetate (37.5 mL) and n-hexane (112.5 mL) were added to the resulting residue, and the mixture was stirred at room temperature for 20 minutes. The obtained slurry was filtered, and the collected solid was washed with ethyl acetate-n-hexane (1: 9, 75 mL) and then air-dried to obtain Compound 21 (14.35 g, yield 91.8%). Obtained.
1H-NMR (DMSO-d6) δ: -0.09 (3H, s), 0.11 (3H, s), 0.85 (9H, s), 3.10-3.60 (4H, m), 3.74 (1H, dd, J = 11.8 , 6.8Hz), 3.90 (1H, dd, J = 11.9, 9.4Hz), 4.65 (1H, m), 5.09 (1H, t, J = 8.2Hz), 6.50-6.70 (3H, m), 8.00 (1H , d, J = 9.0Hz), 10.74 (1H, s), 13.24 (1H, brs).

Step 2 Synthesis of Compound 22 Under a nitrogen atmosphere, a solution of Compound 21 (14.0 g, 23.9 mmol) and N-methylimidazole (7.60 mL, 95.4 mmol) in DMA (70 mL) was added with a 1 mol / L TBAF-tetrahydrofuran solution. (28.6 mL, 28.6 mmol) and DMA (14 mL) were added and stirred at 70-80 ° C. for 8 hours. EDC hydrochloride (13.72 g, 71.55 mmol) and DMA (7 mL) were added at 20-30 ° C. and stirred for 4 hours. N-methylimidazole (7.60 mL, 95.4 mmol) was added to the reaction solution, TBS chloride (14.38 g, 95.40 mmol) was added at 20 ° C. or lower, and the mixture was stirred at room temperature for 2 hours. Ethyl acetate (280 mL) and 10% brine (140 mL) were added to the reaction solution, and the organic layer was washed twice with water (140 mL). Each washing solution was extracted with ethyl acetate (140 mL). The organic layers were combined, the solvent was distilled off under reduced pressure, and acetonitrile (56 mL), isopropyl alcohol (28 mL) and water (266 mL) were sequentially added to the obtained residue at room temperature, and the mixture was stirred at 0 to 10 ° C. for 1 hour. The obtained slurry was filtered, and the collected solid was washed with acetonitrile-water (1: 9, 70 mL), and then dried at 60 ° C. under reduced pressure to obtain Compound 22 (7.71 g, yield 74.1%). )
1H-NMR (DMSO-d6) δ: -0.09 (3H, s), 0.10 (3H, s), 0.81 (9H, s), 2.92 (3H, s), 3.89 (2H, dd, J = 33.0, 12.7 Hz), 4.33 (2H, s), 5.67 (1H, s), 5.93 (1H, brs), 6.54 (2H, brs), 7.69 (1H, s), 10.72 (1H, brs).

Step 3 Synthesis of Compound 23 Trimethylsilyl chloride (9.33 g, 85.9 mmol) was suspended in a pyridine (37.5 mL, 464 mmol) suspension of Compound 22 (7.50 g, 17.2 mmol) at 10-20 ° C. under a nitrogen atmosphere. ) Was added dropwise, followed by stirring at room temperature for 30 minutes. Next, phenoxyacetyl chloride (3.52 g, 20.6 mmol) was added dropwise at 0 to 5 ° C., followed by stirring at room temperature for 1 hour. The reaction solution was diluted with ethyl acetate (150 mL), and then a mixed solution of 10% brine (37.5 mL) and concentrated hydrochloric acid (30.0 mL) was added dropwise at 0 to 10 ° C. The organic layer was washed twice with 1 mol / L hydrochloric acid (75 mL) and water (75 mL), then ethyl acetate (37.5 mL) and 2 mol / L hydrochloric acid methanol solution (38.7 mL, 77.4 mmol) were added sequentially, Stir at room temperature for 1 hour. Methanol (15 mL) was added to the resulting slurry and stirred at room temperature for 30 minutes, followed by filtration. The solid collected by filtration was washed with ethyl acetate (45 mL) and then air-dried to obtain compound 23 (5.51 g, yield). Rate 70.3%).
1H-NMR (DMSO-d6) δ: 2.95 (3H, s), 3.68 (1H, d, J = 13.6Hz), 3.82 (1H, d, J = 13.3Hz), 4.35 (1H, s), 4.41 ( 1H, s), 4.92 (2H, s), 5.80 (1H, s), 6.99 (3H, m), 7.32 (2H, m), 8.19 (1H, s), 11.82 (2H, br).

Step 4 Synthesis of compound 24 (G amidite) Under a nitrogen atmosphere, a solution of compound 23 (500 mg, 1.10 mmol) in DMA (3.5 mL) was added DMTr chloride (1.11 g, 3.29 mmol) and DABCO (369 mg, 3 .21 mmol) was added and stirred at room temperature for 2.5 hours. DABCO (369 mg, 3.21 mmol) and 2-cyanoethyldiisopropylchlorophosphoramidite (0.587 mL, 2.63 mmol) were added to the reaction solution at 10 ° C. or lower, and the mixture was stirred at room temperature for 1 hour. Ethyl acetate (10 mL), 5% aqueous sodium hydrogen carbonate solution (2.5 mL) and 10% brine (2.5 mL) were added to the reaction mixture, and the organic layer was washed twice with 10% brine (5 mL). The washings were combined and extracted with ethyl acetate (2.5 mL). The organic layers were combined and dried over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure, and the resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate) to give compound 24 (720 mg, ethyl acetate 9.9 wt%). Content, yield 61.7%).
1H-NMR (DMSO-d6) δ: 11.87 (1.0H, br s), 11.77 (1.0H, br s), 8.07 (1.0H, s), 7.40 (1.9H, t, J = 7.4 Hz), 7.35 -7.23 (9.0H, m), 7.02-6.97 (3.0H, m), 6.93-6.88 (4.0H, m), 6.08 (1.0H, s), 4.91 (2.0H, s), 4.78 (1.0H, s), 4.64 (0.7H, d, J = 8.8 Hz), 4.59 (0.3H, d, J = 6.0 Hz), 3.75 (4.2H, s), 3.74 (1.8H, s), 3.72-3.60 (0.6 H, m), 3.60-3.49 (1.3H, m), 3.48-3.39 (1.4H, m), 3.21 (0.7H, dd, J = 15.4, 11.6 Hz), 2.98 (0.9H, s), 2.97 ( 2.1H, s), 2.70 (0.6H, dd, J = 10.1, 5.4 Hz), 2.58 (1.4H, t, J = 5.9 Hz), 1.09-1.05 (6.0H, m), 0.91-0.83 (6.0H , m).
31P-NMR (CDCl3) δ: 148.90, 149.98

The route from compound 14 to compound 21a can also be synthesized by a method that does not go through a deTFA reaction as follows.

Step 1 Synthesis of Compound 46 Under a nitrogen stream, cyclopentyl methyl ether of Compound 14 (5.56 g, 8.88 mmol) and 2-acetamido-9H-purin-6-yl-diphenylcarbamate (3.79 g, 9.77 mmol) (50 mL, 10 V) To the suspension, N, O-bis (trimethylsilyl) acetamide (BSA, 21.75 mL, 89 mmol) was added and stirred at 60 ° C. for 20 minutes, and then trimethylsilyl trifluoromethanesulfonate (3.95 mL, 17 .76 mmol) was added and heated at the same temperature for 4 hours. After cooling to room temperature, ethyl acetate (100 ml, 20 V) was added, followed by the dropwise addition of city water (7.5 ml, 1.5 V). The insoluble material was filtered off, and the filtrate was added to 10% brine (15 ml, 3V) and partitioned into an organic layer and an aqueous layer. The organic layer was washed successively with 2 mol / L hydrochloric acid (50 ml, 10 V) and city water (50 ml, 10 V), and then concentrated to obtain a crude product of compound 46 (9.52 g).
¹ H-NMR (DMSO-d ₆ ) δ: 8.65 (2H, d, J = 8.0 Hz), 7.91 (2H, d, J = 8.0 Hz), 7.51 (1H, t, J = 4.0 Hz), 7.43 ( 2H, m), 6.89 (1H, d, 12.0 Hz), 5.36 (1H, t, J = 8.0 Hz), 4.61 (1H, d, J = 8.0 Hz), 3.79 (1H, d, J = 12.0 Hz) , 3.70 (1H, d, J = 12.0Hz), 3.26 (1H, s), 2.38 (3H, s), 0.77 (9H, s), 0.06 (3H, s), -0.19 (3H, s) .LC -MS: UPLC 4min base 1.91 min, M + H = 789

Step 2 Synthesis of Compound 47 To a solution of crude compound 46 (9.52 g, corresponding to 8.88 mmol) in methanol (25 ml) was added 28% aqueous ammonia (25 ml), and the mixture was stirred at room temperature for 4 hours. After adding ethyl acetate (250 ml) and 10% brine (60 ml), the precipitated slurry-like solid was collected by filtration. The obtained solid was washed with ethyl acetate (25 ml) and then air-dried to obtain Compound 47 (3.89 g, purity = 91.4 wt%, yield 70.6% from Compound 14).

Step 3 A 1.8 mol / L lithium borohydride tetrahydrofuran solution (1.76 ml) was added dropwise to a tetrahydrofuran suspension (5 ml) of synthetic compound 46 (547 mg, purity = 91.4 wt%, corresponding to 0.881 mmol) of compound 48. And stirred at room temperature for 15 minutes. While continuing stirring, methanol (500 · L), acetone (500 · L), 2 mol / L hydrochloric acid-methanol solution (3.52 ml) were sequentially added, and then EDC hydrochloride (507 mg, 2.64 mmol, 3 eq.) Was added and stirred at 60-70 ° C. for 1.5 hours. After adding EDC hydrochloride (422 mg, 2.20 mmol, 2.5 eq.) And dimethylacetamide (1 ml), the mixture was stirred at the same temperature for 2.5 hours. After cooling to room temperature and adding 10% brine (10 ml), the solvent was distilled off until the reaction mixture became a slurry (residue weight: 12.95 g). After standing overnight, the resulting solid was collected by filtration, and the solid was washed with 5% aqueous methanol (5 ml) and air dried to obtain Compound 48 (273 mg, yield 70.9%).
LC-MS: UPLC 4min base 1.41 min, M + H = 437

Step 4 Conversion of Compound 48 to Compound 21a Methanol (1.5 mL) and ammonium fluoride (17 mg, 0.458 mmol) were added to compound 48 (100 mg, 0.229 mmol) at room temperature, and 4.5 hours at 70 ° C. Heated to reflux. After cooling to room temperature, the precipitate was collected by filtration, washed with methanol (0.5 ml), and air-dried to obtain Compound 21a (51 mg, yield 68.9%).

Comparative Example 2 Synthesis of G (Pac) amidite by conventional transglycosylation reaction

Process 1
It implemented on the same conditions as the process 1 of the comparative example 1.

Process 2
It implemented on the same conditions as the process 2 of the comparative example 1.

Process 3
Compound 38 (12 mg, 15 umol) and ON-N, N-diphenylcarbamoyl-N-acetylguanine (12 mg, 31 umol) were azeotroped with toluene in an eggplant flask (10 mL) (1 mL, once). After drying under a vacuum line, 1,2-dichloroethane (1 mL) was added under a nitrogen atmosphere. N, O-bis (trimethylsilyl) acetamide (BSA, 38 μL, 154 μmol) was added, heated to 60 ° C. and stirred for 10 minutes. After cooling to room temperature, trimethylsilyl trifluoromethanesulfonate (TMSOTf, 3 μL, 28 μmol) was added. Refluxed for 13 minutes and returned to room temperature. The reaction solution was extracted with a saturated aqueous solution of sodium bicarbonate (10 mL) and ethyl acetate (10 mL, twice), and the organic layers were combined and dried over anhydrous magnesium sulfate. The sample after filtration and concentration to dryness was purified by silica gel column chromatography (hexane / ethyl acetate = 2: 1 to 1: 1) to give colorless amorphous compound 49 (10 mg, 9.6 μmol, yield 62%). )

Process 4
Compound 49 (5 mg, 4.8 μmol) was dissolved in ethyl acetate (0.2 mL) in a tip vial and lithium iodide (1.2 mg, 9.6 umol) was added. After refluxing for 13.5 hours, the reaction solution was extracted with 10% aqueous sodium sulfate solution (5 mL) and chloroform (5 mL, twice). The organic layers were combined, washed sequentially with 0.1 mol / L hydrochloric acid, water, and saturated brine, and dried over anhydrous magnesium sulfate. After filtration, it was concentrated to dryness to obtain Compound 50 as a colorless amorphous sample (4 mg).

Process 5
The compound was synthesized by the same route as in Step 3 of the route not passing through the deTFAation reaction of Example 6.

Step 6
The compound was synthesized by the same route as in Step 3 of Example 6.

Step 7
It implemented on the same conditions as the process 4 of Example 6. FIG.
[Example 7]

G (Pac) amidite synthesis (part 2)

Step 1 Compound 21 was synthesized under the same conditions as in Step 1 of Example 6.

Step 2 Synthesis of Compound 21a The reaction was performed under the same conditions as in Step 2 of Example 6.

Step 3 Synthesis of Compound 23 Under a nitrogen stream, Compound 21a (19.5 g, 60.5 mmol) was made into a slurry solution with pyridine (200 ml) and dichloromethane (20 ml), and chlorotrimethylsilane (65.7 g, 605 mmol) was cooled with ice. Dropping was performed. After the dropwise addition, the internal temperature was raised to room temperature, the mixture was stirred for 1.5 hours, the reaction solution was cooled again to ice cooling, and PacCl (11.5 g, 67.4 mmol) was added dropwise. After the dropwise addition, the internal temperature was raised to room temperature, the mixture was stirred for 1.5 hours, the reaction solution was cooled again under ice cooling, and methanol (60 ml) was added dropwise. Then, the crude product of compound 23 was obtained by stirring at room temperature for 2 days and concentrating the reaction solution.

Step 4 Synthesis of Compound 23a Pyridine (200 g) and 4,4′-dimethoxytrityl chloride (21.5 g, 63.5 mmol) were added to the residue obtained by azeotropic distillation of the crude product of Compound 23 (60 ml × 3 times). The mixture was further stirred at room temperature for 2.5 hours. The reaction mixture was added to ethyl acetate (400 ml) and saturated aqueous sodium hydrogen carbonate solution (500 ml), and the layers were separated, the aqueous layer was re-extracted with ethyl acetate (100 ml), and the resulting organic layers were combined and saturated. The extract was washed with brine (100 ml), dried over anhydrous sodium sulfate and concentrated. The obtained residue was purified by silica gel column chromatography (hexane / ethyl acetate / acetonitrile), washed with slurry with acetonitrile (100 g) and ethyl acetate (145 g), then filtered and dried to give compound 23a (28 .3 g, 62% yield).

Step 5 Synthesis of compound 24 To compound 23a (28.3 g, 3 73 mmol), acetonitrile (220 ml) and tetrahydrofuran (30 ml) were added under a nitrogen stream, diisopropylammonium tetrazolide (7.34 g, 42.9 mmol), and then 2-cyanoethyl N, N, N ′, N′-tetraisopropyl phosphoramidite (16.9 g, 55.9 mmol) was added. After stirring at 30 ° C. for 4 hours, the reaction solution was added to ethyl acetate (200 ml) and saturated aqueous sodium hydrogen carbonate (20 ml) for liquid separation. The obtained aqueous layer was re-extracted with ethyl acetate (100 ml), and the organic layers were combined and washed with saturated brine (80 ml). The organic layer was dried over anhydrous sodium sulfate and concentrated, and then the resulting residue was purified by silica gel column chromatography (isopropyl ether / ethyl acetate / acetonitrile) to obtain compound 24 (17.0 g, yield 49%). It was.
[Example 8]

Synthesis of G (Ac) amidite body

Step 1 to Step 5 The steps can be performed under the same conditions as in Example 6.
[Example 9]

Synthesis of 5′-O-Lev-G (Pac) amidite

Step 1 Synthesis of Compound 58
Compound 58 can be obtained by adding levulinic acid, EDC hydrochloride and 4-dimethylaminopyridine to a dichloromethane solution of compound 23 (synthesized by the method described in Example 6) and stirring overnight at room temperature.

Step 2 Synthesis of Compound 59 Compound 59 can be synthesized in the same manner as in Step 4 of Example 6.
[Example 10]

Synthesis of 5'-O-TBDPS-G (Pac) amidite

Step 1 Synthesis of Compound 60
To a N, N-dimethylformamide solution of Compound 21a (synthesized by the method described in Example 6), tert-butyl-diphenylsilyl chloride and imidazole are added and reacted at room temperature to form a TBDPS at the 5′-position hydroxyl group. After the product is obtained, compound 60 can be obtained by adding trimethylsilyl chloride and phenoxyacetyl chloride to the pyridine solution and reacting at room temperature.

Step 2 Synthesis of Compound 61 Compound 61 can be synthesized in the same manner as in Step 4 of Example 6.
[Example 11]

Synthesis of MeC (Bz) amidite

Step 1 Synthesis of Compound 25 108.1 g of Compound 15 (Purity: 92.53%, Content: 100 g, 0.167 mol), DMF 300 ml, Imidazole 47.8 g (4.2 eq.), TBSCl 51.6 g (2.05 eq) .) Was added and stirred at around 25 ° C. for 23 hours. The solution was separated by adding 600 ml of AcOEt and 600 ml of water, and the aqueous layer was re-extracted with 300 ml of AcOEt. The organic layers were mixed, washed with 600 ml of 5% NaHCO _{3 and} 600 ml of 5% NaCl, and the solvent was recovered. After removing the solvent by toluene 500 ml × 3 for the purpose of removing TBSOH, the solidified amorphous was loosened to obtain the intended compound 25 (132.6 g, crude yield 111.2%).
1H-NMR (CDCl3) δ: 8.33 (1H, s), 7.89 (1H, s), 7.41 (2H, d, J = 7.0 Hz), 7.33-7.21 (7H, m), 6.82 (4H, dd, J = 8.8, 5.6 Hz), 5.43 (1H, s), 4.50 (1H, s), 4.04 (1H, s), 3.87 (1H, d, J = 11.8 Hz), 3.789 (3H, s), 3.787 (3H , s), 3.41 (1H, d, J = 11.8 Hz), 3.00 (3H, s), 1.56 (3H, s), 0.75 (9H, s), 0.04 (2H, s), -0.04 (2H, s ).

Step 2 Synthesis of Compound 26 Compound 25 132.6 g (corresponding to 0.167 mol), MeCN 800 ml, TEA 50.7 g (3 eq.), DMAP 2.0 g (0.1 eq.), TPBSO2Cl 65.8 g (1.3 eq. ) And stirred at around 25 ° C. for 22 hours. 25% NH ₃ aq. 800 ml was added and stirred at around 25 ° C. for 2 hours. MeCN was roughly recovered, and 400 ml of AcOEt and 400 ml of water were added for liquid separation, and the aqueous layer was re-extracted with 300 ml of AcOEt. The organic layers were mixed, washed with 400 ml of 5% NaCl × 2 times, and the solvent was recovered. The solidified amorphous was loosened to obtain the target compound 26 (192.8 g, crude yield 161.9% from compound 15).
1H-NMR (CDCl3) δ: 7.97 (1H, s), 7.53 (2H, d, J = 7.2 Hz), 7.35-7.25 (7H, m), 6.83 (4H, dd, J = 8.8, 6.8 Hz), 5.51 (1H, s), 4.46 (1H, s), 4.19 (1H, s), 3.88 (1H, d, J = 11.7 Hz), 3.79 (6H, s), 3.39 (1H, d, J = 11.7 Hz ), 3.02 (3H, s), 1.85 (3H, s), 1.66 (3H, s), 0.74 (9H, s), 0.04 (2H, s), -0.04 (2H, s).

Step 3 192.8 g (corresponding to 0.167 mol) of Compound 26 of Compound 27, 500 ml of DMF, and 75.6 g ( ₂ eq.) Of Bz ₂ O were added, and the mixture was stirred at around 25 ° C. for 22 hours. AcOEt 1000 ml, 5% NaHCO ₃ 1000 ml was added for liquid separation, and the aqueous layer was re-extracted with AcOEt 500 ml. The organic layers were mixed, washed with 500 ml of 5% NaHCO _{3 and} 500 ml of 5% NaCl, and the solvent was recovered to obtain crude compound 5. Purification is performed using 1.3 kg of silica gel, developing solvent Hex / AcOEt = 8/2 → 6/4, and the solidified amorphous is loosened to obtain the target compound 27 (124.1 g, yield 91% from compound 15). Obtained.
1H-NMR (CDCl3) δ: 8.33 (2H, d, J = 7.2 Hz), 8.05 (1H, s), 7.53 (1H, dd, J = 7.4, 7.4 Hz), 7.49-7.41 (5H, m), 7.35-7.28 (7H, m), 7.25 (1H, m), 6.84 (4H, dd, J = 8.8, 6.4 Hz), 5.49 (1H, s), 4.51 (1H, s), 4.10 (1H, s) , 3.90 (1H, d, J = 11.7 Hz), 3.803 (3H, s), 3.800 (3H, s), 3.42 (1H, d, J = 11.7 Hz), 3.11 (3H, s), 1.85 (3H, s), 0.74 (9H, s), 0.04 (2H, s), -0.04 (2H, s).

Step 4 Synthetic compound 27 124.1 g (0.152 mol), THF 869 ml, 1M-TBAF / THF 182 ml (1.2 eq.) Were added, and the mixture was stirred for 2 hours. The solvent was recovered, and liquid separation was performed by adding 620 ml of AcOEt and 620 ml of 5% NaHCO ₃ . The solvent was recovered by washing with 620 ml of 5% NaCl. After removing the solvent by toluene 620 ml × 3 for the purpose of removing TBSOH, the solidified amorphous was loosened to obtain the intended compound 28 (119.2 g, yield 119% from compound 27).
1H-NMR (CDCl3) δ: 13.44 (1H, br s), 8.32 (2H, d, J = 7.5 Hz), 7.92 (1H, s), 7.55 (1H, dd, J = 7.3, 7.3 Hz), 7.50 -7.43 (4H, m), 7.40-7.30 (7H, m), 7.26 (1H, m), 6.86 (4H, dd, J = 8.8, 3.6 Hz), 5.46 (1H, s), 4.37 (1H, br .s), 4.16 (1H, s), 3.92 (1H, d, J = 12.3 Hz), 3.803 (3H, s), 3.800 (3H, s), 3.57-3.42 (4H, m), 3.63 (1H, d, J = 12.3 Hz), 3.03 (3H, s), 1.90 (3H, s).

Step 5 Synthesis of Compound 29 (MeC (Bz) amidite) 108.2 g (equivalent to 0.138 mol) of Compound 28, 564 ml of dichloromethane and 35.7 g (2 eq.) Of DIPEA were charged and 2-cyanoethyldiisopropylchlorophosphoramidite at 5 ° C. or lower. 49.0 g (1.5 eq.) Was added dropwise. Then, it stirred at 25 degreeC vicinity for 2 hours. 564 ml of 5% NaHCO ₃ was added for liquid separation, and the solvent was recovered to obtain crude MeC (Bz). Purified with 2.3 kg of silica gel, developing solvent Hex / AcOEt = 7/3 → 5/5, loosened the solidified amorphous, and the target compound 29 (MeC (Bz) amidite, 105.2 g, yield 84) % From compound 27, the ratio of two diastereomers (calculated from the integrated value of HPLC) = 54: 46).
1H-NMR (CDCl3) δ: (major diastereomer) 13.47 (1H, br s), 8.33 (2H, d, J = 7.4 Hz), 7.98 (1H, s), 7.52 (1H, dd, J = 7.4, 7.4 Hz), 7.50-7.42 (4H, m), 7.37-7.24 (8H, m), 6.86 (4H, dd, J = 8.8, 6.6 Hz), 5.52 (1H, s), 4.64 (1H, d, J = 6.9 Hz), 4.37 (1H, s), 3.92 (1H, d, J = 11.7 Hz), 3.812 (3H, s), 3.807 (3H, s), 3.57-3.42 (4H, m), 3.51 (1H, d, J = 11.7 Hz), 3.02 (3H, s), 2.36 (2H, t, J = 5.9 Hz), 1.77 (3H, s), 1.14 (6H, d, J = 6.8 Hz), 1.05 (6H, d, J = 6.8 Hz). (minor diastereomer) 13.47 (1H, br s), 8.33 (2H, d, J = 7.3 Hz), 7.98 (1H, s), 7.56 (1H, dd, J = 7.3, 7.3 Hz), 7.47-7.43 (4H, m), 7.35-7.28 (7H, m), 7.22 (1H, m), 6.86 (4H, dd, J = 8.8, 5.5 Hz), 5.51 (1H, s), 4.60 (1H, d, J = 4.3 Hz), 4.43 (1H, s), 3.88 (1H, d, J = 11.7 Hz), 3.802 (3H, s), 3.798 (3H, s), 3.64 (1H, m) , 3.52 (1H, d, J = 11.7 Hz), 3.52-3.44 (3H, m), 3.04 (3H, s), 2.60-2.52 (2H, m), 1.79 (3H, s), 1.11 (6H, d , J = 6.8 Hz), 0.99 (6H, d, J = 6.8 Hz).
31P-NMR (CDCl3) δ: 151.24, 151.05.
[Example 12]

Synthesis of ^Me C (Bz) body

Process 1
Under a nitrogen atmosphere, a suspension of compound 14 (100 mg, 0.177 mmol) and 6-N-benzoylmethylcytosine (49 mg, 0.213 mmol) in cyclopentylmethyl ether (2 mL) was charged with N, O-bis (trimethylsilyl) acetamide ( 0.304 mL, 1.24 mmol) and TMSOTf (0.0641 mL, 0.355 mmol) were added and stirred at 70 ° C. for 4 hours. The reaction mixture was partitioned by adding tetrahydrofuran (20 mL) and 1 mol / L hydrochloric acid (20 mL) under ice-cooling, and the organic layer was washed with 0.1 mol / L hydrochloric acid (110 mL) and water (110 mL), and then added to anhydrous magnesium sulfate. After drying, the solvent was concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (chloroform / methanol) to obtain Compound 62 (84 mg, yield 75%). ¹ H-NMR was observed as a 71:29 rotamer mixture.
¹ H-NMR (CD3OD) δ: (Major) 8.30-8.17 (2H, m), 7.96 (1H, br.s), 7.56 (1H, dd, J = 7.3
7.3 Hz), 7.48 (2H, d, J = 7.5, 7.3 Hz), 6.95 (1H, J = 8.6 Hz, H-1 of sugar), 4.99 (1H, dd,
J = 8.6, 8.0 Hz), 4.79 (1H, d, J = 8.0 Hz), 4.10 (1H, d, J = 11.9 Hz), 3.90 (1H, d, J = 11.9 Hz), 2.11 (3H, s) , 0.93 (9H, s), 0.21 (3H, s), -0.01 (3H, s). (Minor) 8.30-8.17 (2H, m), 7.98 (1H, br.s), 7.56 (1H, dd, J = 7.3, 7.3 Hz), 7.48 (2H, d, J = 7.5, 7.3 Hz), 6.90 (1H, J = 7.8 Hz, H-1 of sugar), 4.73 (1H, d, J = 6.8 Hz), 4.51 (1H, dd, J = 7.8, 6.8 Hz), 4.12 (1H, d, J = 11.9 Hz), 3.86 (1H, d, J = 11.9 Hz), 2.11 (3H, s), 0.93 (9H, s ), 0.21 (3H, s), -0.01 (3H, s).

Process 2
The compound was synthesized under the same conditions as in Step 2 of Example 2.

Process 3
The compound was synthesized under the same conditions as in Step 3 of Example 2.

Process 4
The compound was synthesized under the same conditions as in Step 5 of Example 11.
[Example 13]

Synthesis of ^Me C (Ac) body

Step 1 Synthesis of Compound 25 Synthesis was performed under the same conditions as in Step 1 of Example 11.

Step 2 Synthesis of Compound 26 Synthesized under the same conditions as in Step 2 of Example 11.

Step 3 Synthesis of Compound 64 Under a nitrogen stream, Compound 26 (crude, 101.7 g) and pyridine (300 ml) were added to a 2 L four-necked flask and dissolved by stirring. Acetic anhydride (50 mL) was added, and the mixture was stirred at room temperature for 1 hour, diluted with ethyl acetate, washed successively with saturated aqueous sodium hydrogen carbonate solution (twice) and saturated brine. After drying with anhydrous sodium sulfate, filtration and concentration, the residue obtained was purified by silica gel column chromatography (toluene / ethyl acetate = 2/1 → 1/1 → 2/3 → 0/1). Compound 64 (49.1 g, 77% yield from compound 25) was obtained as a beige powder.

Step 4 Synthesis of Compound 65 Under a nitrogen stream, Compound 64 (61.7 g) and tetrahydrofuran (305 ml) were added to a 1 L four-necked flask and dissolved by stirring. A 1 mol / l tetrabutylammonium fluoride / tetrahydrofuran solution (98 ml, 1.2 eq.) Was added, and the mixture was stirred at room temperature for 1 hour, and then concentrated as it was. The organic layer obtained by partitioning the residue between ethyl acetate and water was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated. The obtained residue was purified by silica gel column chromatography (toluene / ethyl acetate = 1/0 → 1/1) to obtain Compound 65 (17.1 g).

Step 5 Synthesis of Compound 66 In a nitrogen atmosphere, Compound 65 (17.0 g, 26.5 mmol) and diisopropylammonium tetrazolide (3.41 g, MR = 0.75) were placed in a 500 mL four-necked flask and acetonitrile (170 mL). ), Tetrahydrofuran (57 ml) was added, and the mixture was stirred for 15 minutes, and then 2-cyanoethyl-N, N, N ′, N′-tetraisopropylphosphoramidite (9.60 g, MR = 1.2) was added and added. Stir at 0 ° C. for 3 hours. After partitioning with ethyl acetate-5% aqueous sodium hydrogen carbonate solution at room temperature, the organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated. The obtained residue was purified by silica gel column chromatography (ethyl acetate / acetone 10/1) and then washed with a mixed solvent of dichloromethane and hexane to give compound 66 (15.4 g, two diastereomers. Ratio (calculated from the integrated value of ¹ H-NMR) = 72: 28)).
¹ H-NMR (CDCl3) δ: (major diastereomer) 8.06 (1H, br s), 7.44 (2H, d, J = 7.3 Hz), 7.37-7.24 (8H, m), 6.88-6.80 (4H, m) , 5.56 (1H, br.s, H-1 of sugar), 4.59 (1H, d, J = 7.5 Hz), 4.47 (1H, br.s), 3.90 (1H, d, J = 11.8 Hz), 3.802 (3H, s), 3.798 (3H, s), 3.58-3.40 (5H, m), 3.03 (3H, s, -NCH3), 2.69 (2H, br.s), 1.58 (3H, s), 1.14 ( 6H, d, J = 6.8 Hz), 1.03 (6H, d, J = 6.8 Hz). (Minor diastereomer) 8.06 (1H, br s), 7.43 (2H, d, J = 7.3 Hz), 7.37-7.24 ( 8H, m), 6.88-6.80 (4H, m), 5.54 (1H, br.s, H-1 of sugar), 4.57 (1H, dd, J = 5.8, 1.5 Hz), 4.47 (1H, br.s ), 3.87 (1H, d, J = 11.8 Hz), 3.792 (3H, s), 3.789 (3H, s), 3.78 (1H, m), 3.61 (1H, m), 3.54 (1H, d, J = 11.8 Hz), 3.52-3.41 (2H, m), 3.05 (3H, s, -NCH3), 2.69 (2H, br.s), 1.58 (3H, s), 1.10 (6H, d, J = 6.8 Hz) , 0.98 (6H, d, J = 6.8 Hz).
[Example 14]

Synthesis of C (Bz) amidite

Process 1
Under a nitrogen atmosphere, a suspension of compound 14 (100 mg, 0.177 mmol) and 6-N-benzoyluridine (49 mg, 0.213 mmol) in cyclopentylmethyl ether (2 mL) was charged with N, O-bis (trimethylsilyl) acetamide (0 .304 mL, 1.24 mmol) and TMSOTf (0.0641 mL, 0.355 mmol) were added and stirred at 70 ° C. for 4 hours. The reaction mixture was partitioned by adding tetrahydrofuran (20 mL) and 1 mol / L hydrochloric acid (20 mL) under ice-cooling, and the organic layer was washed with 0.1 mol / L hydrochloric acid (110 mL) and water (110 mL), and then added to anhydrous magnesium sulfate. After drying, the solvent was concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (chloroform / methanol) to obtain Compound 67 (80 mg, yield 75%).

Process 2
The compound was synthesized under the same conditions as in Step 2 of Example 2.

Process 3
The compound was synthesized under the same conditions as in Step 3 of Example 2.

Process 4
The compound was synthesized under the same conditions as in Step 5 of Example 11.

The synthesis route of other bridged nucleic acid derivatives of amide bridged nucleic acid derivatives is shown below.

Reference example 1

Glucose method
2 '-(R)-(trifluoroacetyl) -N-methylamino-3', 5'-di-O-benzyl-4'-tert-butyldiphenylsilyloxymethylthymidine (2 '-(R)-( (trifluoroacetyl) -N-methylamino-3 ', 5'-di-O-benzyl-4'-tert-butyldiphenylsilyloxymethylthymidine) (Part 1)

Step 1 Synthesis of Compound 102 N, N′-dimethylformamide solution (10.5 L, 10.5 L) of Compound 101 (3.5 kg, 4.782 mol) derived from D-glucose by the method described in WO 2011/052436 3V) was added potassium carbonate (1652 g, 11.95 mol, 2.5 eq) and paramethoxybenzyl chloride (823.8 g, 5.260 mol, 1.1 eq), and the mixture was stirred at room temperature for 19 hours. Ethyl acetate 35.00L (10v / w) and city water 35.00L (10v / w) were added to this reaction liquid, and extraction and liquid separation were performed to obtain an organic layer I and an aqueous layer I. Then, the aqueous layer I was extracted with 10.50 L (3 v / w) of ethyl acetate to obtain an organic layer II. The obtained organic layers I and II were combined, washed with 24.50 L (7 v / w) of water and 24.50 L (7 v / w) of 10% brine, concentrated and dried under reduced pressure to give compound 102 (4470 g). The crude product was obtained as a brown oil. The obtained compound 102 was not purified, and the yield was quantitative, and it was advanced to the next step.

Step 2 Synthesis of Compound 103 Compound 102 (crude, 4470 g) was dissolved in tetrahydrofuran (16.30 L, 4 v / w), and city water (407.5 g, 0.1 w / w) was added. Next, n-tributylphosphine (1258 g, 6.218 mol, 1.3 eq) was added dropwise at a liquid temperature of 30 ° C. or lower, and the mixture was stirred at the same temperature for 5 hours. Dichloromethane (24.50 L, 6 v / w) and city water (4.100 L, 1 v / w) were added to this reaction solution, followed by extraction and separation, and the resulting organic layer was concentrated until almost no distillation occurred. went. After azeotroping with dichloromethane (5.000L, 1.2 v / w) × 2 times, drying under reduced pressure was performed to obtain a crude product of Compound 103 (5557 g) as a brown oil. As in the previous step, the obtained compound 103 was not purified, and the yield was quantitative and proceeded to the next step.

Step 3 Synthesis of Compound 104 Compound 103 (crude, 5557 g) was dissolved in dichloromethane (7.900 L (2 v / w)), and pyridine (945.6 g, 11.95 mol, 2.5 eq) was added. Next, TFAA (1507 g, 7.175 mol, 1.5 eq) was added dropwise at a liquid temperature of 35 ° C. or lower, and the mixture was stirred at the same temperature for 2 hours. The reaction was diluted with CH ₂ Cl ₂ (19.75 L, 5 v / w), cold 10% Na ₂ CO ₃ aq. (11.85 L, 3 v / w), extraction and liquid separation were performed, and the obtained organic layer was added with 5% NaHCO ₃ aq. (24.50 L, 7 v / w), concentrated and dried under reduced pressure to obtain Compound 104 (6550 g) as a reddish brown solid. Dichloromethane (2.000 L, 0.5 v / w) was added thereto, and the mixture was dissolved by heating at a liquid temperature of 40 ° C., isopropyl ether (39.50 L, 10 v / w) was gradually added, and the mixture was naturally cooled to room temperature. Thereafter, the mixture was stirred for 18 hours. And it stirred at the liquid temperature of 5 degrees C or less for 1 hour, and the precipitated crystal | crystallization was filtered. The obtained crystals were washed with isopropyl ether (10.00 L, 2.5 v / w) and dried under reduced pressure to obtain Compound 104 + n-Bu ₃ PO (4392 g) as a pale pink crystal compound. ¹ H-NMR of this crystal was measured, and it was converted that compound 104 contained 3544 g (3.844 mol). Yield from compound 101 = 80.4%.

Step 4 Synthesis of Compound 105 Compound 104 + n-Bu ₃ PO (4392 g) was dissolved in 10.60 L (3 v / w) of DMF at a liquid temperature of 50 ° C., and potassium carbonate (1062 g, 7.684 mol, 2 eq) was added. Subsequently, iodomethane (818.3 g, 5.765 mol, 1.5 eq) was added dropwise at a liquid temperature of 55 ° C. or lower, and the mixture was stirred at the same temperature for 7 hours and at room temperature for 15 hours. Ethyl acetate (35.50 L, 10 v / w) and 5% brine (24.80 L, 7 v / w) were added to this reaction solution for extraction and liquid separation to obtain an organic layer I and an aqueous layer I. Then, the aqueous layer I was extracted with ethyl acetate (10.00 L, 2.8 v / w) to obtain an organic layer II. The obtained organic layers I and II were combined, and 1908 g (7.688 mol, ₂ eq) of Na ₂ S ₂ O ₃ .5H ₂ O + 24.80 L (7 v / w) of city water (5% aqueous sodium hydrogen carbonate solution (24. 80 L, 7 v / w), 10% brine (24.80 L, 7 v / w), respectively, concentrated and dried under reduced pressure to obtain a crude product of compound 105 (4386 g) as a tan oil. The obtained crude product of compound 105 was not purified, and the yield was quantitative, and it was advanced to the next step.

Step 5 Synthesis of Compound 106 Crude product (4386 g) of Compound 105 was dissolved in acetonitrile (25.20 L, 7 v / w), and the solution temperature was 30 ° C. or less, and 7586 g (13.84 mol, 3.6 eq) + city of CAN A water (9.0000 L, 2.5 v / w) solution was added dropwise over 30 minutes, and the mixture was stirred at the same temperature for 2 hours. Ethyl acetate (36.00 L, 10 v / w) and city water (25.20 L, 7 v / w) were added to this reaction solution for extraction and liquid separation, and the resulting organic layer was washed with city water (25.20 L, 7 v / w). A 5% sodium hydrogen oxyacid aqueous solution (25.20 L, 7 v / w) was added thereto, and the resulting insoluble matter was filtered (AcOEt 8.000 L (2.2 v / w) was used for washing). The obtained organic layer was washed with 5% aqueous sodium hydrogen carbonate solution (25.20 L, 7 v / w) and 10% brine (25.20 L, 7 v / w), concentrated and dried under reduced pressure.
What dissolved the obtained residue 10% ethyl acetate-n-hexane solution (5.000 L) was used for silica gel column chromatography (silica gel: 43.00 kg, ethyl acetate / n-hexane = 0/1 to 1/4). (V / v)) and then recrystallized with toluene (4000 g) to obtain Compound 106 (2398 g, yield from Compound 104 = 76.5%) as slightly yellow crystals. It was observed as a 8: 2 rotamer mixture by ¹ H-NMR.
¹ H-NMR (CDCl ₃ ): (Major) 8.17 (1H, br.s), 7.66-7.05 (21H, m), 6.58 (1H, d, J = 9.7 Hz), 5.17 (1H, dd, J = 9.7, 5.7Hz), 4.69-4.48 (4H, m), 4.44 (1H, d, J = 11.3 Hz), 3.97 (1H, d, J = 10.2 Hz), 3.75 (1H, d, J = 10.2 Hz ), 3.65 (1H, d, J = 10.2 Hz), 3.60 (1H, d, J = 10.2 Hz), 3.20 (3H, s), 2.36 (3H, s), 1.03 (9H, s). (Minor) 8.10 (1H, br.s), 7.66-7.05 (21H, m), 6.53 (1H, d, J = 9.0 Hz), 3.69 (1H, d, J = 10.2 Hz), 3.55 (1H, d, J = 10.2 Hz), 3.15 (3H, s), 2.36 (3H, s), 1.03 (9H, s).

2 '-(R)-(trifluoroacetyl) -N-methylamino-3', 5'-di-O-benzyl-4'-tert-butyldiphenylsilyloxymethylthymidine (2 '-(R)-( (trifluoroacetyl) -N-methylamino-3 ', 5'-di-O-benzyl-4'-tert-butyldiphenylsilyloxymethylthymidine) (Part 2)

Step 1 Synthesis of Compound 107 Compound 100 (2.38 mmol) derived from D-glucose by the method described in WO 2011/052436 is dissolved in methylamine (20 mL, 2.0 mol / L THF solution, 40 mmol). And heated to 60 ° C. using a microwave and stirred for 8 hours. The reaction solution was concentrated to dryness to obtain Compound 107. The obtained compound was not purified and proceeded to the next reaction as it was.

Step 2 Conversion of Compound 107 to Compound 106 Compound 107 obtained in the previous step was dissolved in dichloromethane (10 mL), pyridine (481 μl, 5.96 mmol) was added, and TFAA (504 μL, 3.58 mmol) was added dropwise at room temperature. Stir for 2 hours. Saturated aqueous ammonium chloride solution was added and extracted. The obtained organic layer was washed with a saturated aqueous sodium hydrogen carbonate solution and saturated brine, and dried over magnesium sulfate. The solvent was distilled off under reduced pressure, and the resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate = 3/1) to obtain Compound 106 (1 g, yield 51%).
LC-MS: UPLC 4min acid 3.29 min, M + H = 816

Synthesis example of A (Bz) amidite

Step 1 Synthesis of Compound 107
Under a nitrogen stream, compound 106 (599.60 g, 734.85 mmol), Bz-adenine (351.82 g, 2 eq.), 1,2-DCE (3000 ml, 5 v / w) were added to a 20 L flask and dissolved by stirring. did. BSA (899.50 g, 6 eq.) Was added to this solution, the temperature was raised, and the mixture was stirred for 1 hour while maintaining at 49 ° C. After cooling to 24 ° C., TMSOTf (49.55 g, 0.3 eq.) Was added, the temperature was raised again, and stirring was continued for 5.5 hours while maintaining the temperature at 60 ° C. The reaction solution was cooled to 5 ° C., and 8% aqueous sodium hydrogen carbonate solution (900 g) was added dropwise. After completion of the dropwise addition, this was stirred at room temperature for 15 hours (beige slurry). DCM (7800 g) was added thereto, and the mixture was stirred at room temperature for 1 hour, and the solid was separated by filtration through celite. The filtered residue was slurried again with DCM (4800 g) and the solid was filtered off through celite filtration. Further, the residue was slurry washed with DCM (3000 g), and the solid was separated by suction filtration (this operation was repeated twice). All of the obtained mother liquor was transferred to a 20 L water washing kettle, 8% aqueous sodium hydrogen carbonate solution (900 g) was added to wash the organic layer, and the aqueous layer was re-extracted with DCM (900 g). Sodium sulfate (600 g) was added to the obtained organic layer and stirred for 30 minutes, followed by filtration, concentration under reduced pressure, and drying to obtain Compound 107 (645 g, yield 94.4%) as a tan amorphous solid.

Step 2 Synthesis of Compound 108
Under a nitrogen stream, 1.0 mol / L-TBAF / THF solution (766.44 g, 1.2 eq.) Was added to a THF (1290 ml, 2 v / w) solution of Compound 107 (645 g, 694.24 mmol) at room temperature. The mixture was stirred at 24 ° C. for 21 hours. The reaction solution was concentrated under reduced pressure, dissolved in ethyl acetate (1743 g), transferred to a 10 L water washing kettle, and washed with city water (2142 g). The organic layer was washed again with 5% brine (1290 g) and the brine was extracted again with ethyl acetate (322 g). The obtained organic layer was concentrated under reduced pressure, and then azeotroped with toluene (1000 g). Dichloromethane (712 g) was added to the crude product (405 g) obtained by drying this, and heated to 40 ° C. for dissolution. This was purified by silica gel (silica gel carrier amount: 3870 g, ethyl acetate / toluene = 1/2 → 1/1 → 2/1 → 1/0) to give compound 108 (405 g, yield 84.5%) as white. Obtained as a solid.

Step 3 Synthesis of Compound 109 Compound 108 (402 g, 582.04 mmol), acetonitrile (3216 ml, 8 v / w), 1 mol / l aqueous acetic acid solution (543 ml, 1.35 v / w) in a 10 L four-necked flask under a nitrogen stream 1 mol / l aqueous sodium acetate solution (1085 ml, 2.7 v / w), TEMPO (9.09 g, 0.1 eq.), 80% sodium chlorite (138.13 g, 2.1 eq.), 9% hypochlorous acid Sodium chlorate (6.0 ml, 0.015 v / w) was sequentially added, the internal temperature was raised to 36 ° C., and the mixture was stirred while keeping warm. After stirring for 4.5 hours, 9% sodium hypochlorite (2 ml, 0.005 v / w) was added, and the mixture was further stirred for 16 hours. After the reaction solution was cooled to 6 ° C., 20% aqueous sodium sulfite solution (804 g) was added dropwise, followed by acetic acid (177 g). The mixed solution was separated, the aqueous layer was re-extracted with ethyl acetate (1123 g), and the obtained organic layer was washed with 15% brine (1373 g). The organic layer was concentrated under reduced pressure, azeotroped with toluene (1200 ml × 2), and dried to obtain Compound 109 (468 g) as a white solid.

Step 4 Synthesis of Compound 110 Under a nitrogen stream, 25% aqueous ammonia (468 ml, 1 v / w) was added to a solution of compound 109 (468 g, content 410 g, 582.04 mmol) in methanol (2800 ml, 6 v / w) at room temperature. After that, the temperature was raised to 39 ° C., and the mixture was stirred while keeping warm. After stirring for 6 hours, 25% aqueous ammonia (140 ml, 0.3 v / w) was added. After further stirring for 16 hours, 25% aqueous ammonia (94 ml, 0.2 v / w) was added. After stirring while keeping the temperature for 8 hours, the reaction solution was allowed to cool and further stirred for 16 hours. The reaction solution was concentrated under reduced pressure, azeotroped with toluene (2500 ml × 2), and dried to obtain Compound 110 (470 g, purity calculated from HPLC: 80.8%) as an orange solid.

Step 5 Synthesis of Compound 111 Compound 110 (470 g, contents 294 g, 582.04 mmol) and DMF (1800 ml, 6.1 v / w) were added to a 10 L four-necked flask under a nitrogen stream, and the mixture was stirred while keeping at 24 ° C. . After confirmation of dissolution, EDC hydrochloride (169 g, 1.5 eq.) Was added and stirred while keeping warm. After stirring for 5 hours, EDC hydrochloride (23 g, 0.2 eq.) Was added, and the mixture was further stirred for 17.5 hours, and then city water (5124 g) was added dropwise over 15 minutes. After stirring this slurry solution for 1 hour, the crystals were filtered by suction filtration, washed with city water, the crystals were dried, and Compound 111 (226 g, yield of 79.9% in 3 steps from Compound 108) was cream crystals. Got as.

Step 6 Synthesis of Compound 112 Under a nitrogen stream, Compound 111 (225 g, 462.47 mmol) and acetic acid (1125 ml, 5 v / w) were added to a 3 L flask, and the mixture was heated to 50 ° C. and dissolved by stirring. 20% Pd (OH) ₂ -C (44.98 g, 0.2 w / w) was added thereto, and the inside of the system was replaced with hydrogen, followed by stirring while keeping hydrogen blown into the reaction solution. After stirring for 6 hours, 20% Pd (OH) ₂ -C (22.49 g, 0.1 w / w) was added. After stirring for 2.5 hours, 20% Pd (OH) ₂ -C (11.25 g, 0.05 w / w) was further added, and the mixture was further stirred for 3 hours. The reaction solution was then filtered with suction, washed with methanol (2030 g), and the mother liquor was concentrated under reduced pressure to about 1 L. Toluene (1300 ml × 2) was azeotroped (concentrated under reduced pressure to about 1 L), toluene (1000 ml) was added and stirred at room temperature, the solid was collected by suction filtration and dried, and compound 112 (139.7 g, 98 .7%) was obtained as a light gray solid.

The route from compound 106 to compound 110 can also be synthesized by the following method.

Step 1 Synthesis of Compound 113 The compound 113 can be synthesized under the same conditions as in Step 2 of the synthesis of A (Bz) amidite in Reference Example 1 described above.

Step 2 Synthesis of Compound 114 The compound 114 can be synthesized under the same conditions as in Step 3 of the synthesis of A (Bz) amidite in Reference Example 1 described above.

Step 3 Derivation of Compound 114 to Compound 110 Compound 114 (14 mg, 0.023 mmol) was dissolved in 1 mL of dichloroethane, and 16 mg (0.069 mmol) of N-benzoylguanine and 42 mg of (E) -trimethylsilyl N-trimethylsilylacetamide (0. 21 mmol) was added and heated to 60 ° C. and stirred for 20 minutes. After the reaction solution was lowered to room temperature, TMSOTf (4 μl, 0.023 mmol) was added, and the mixture was heated again to 60 ° C. and stirred for 6 hours. The reaction solution was purified by silica gel column chromatography (chloroform / methanol = 10/1) as it was to obtain compound 110 (5 mg, yield 30%). LC-MS: UPLC 4min acid 2.50, 2.59 (rotamers) min, M + H = 719

Synthesis example of G amidite

Step 1 Synthesis of Compound 115 Under a nitrogen stream, ON-N, N-diphenylcarbamoyl-N-isobutylguanine (93.0 g, 223 mmol), 1,2-dichloroethane (760 ml) was charged, and BSA (136. 2 g, 670 mmol) was added dropwise. The internal temperature was heated to 60 ° C. and stirred for 30 minutes. Thereafter, the internal temperature was lowered to ice-cooling, compound 106 (152 g, 186 mmol) was added, and after washing with 1,2-dichloroethane (150 ml), TMSOTf (l2.4 g, 55.8 mmol) was added dropwise. It was. The mixture was heated again to 60 ° C. and stirred for 2 hours. Saturated aqueous sodium bicarbonate (675 ml) was added dropwise to the reaction mixture under ice cooling, and then KC floc (45 g) was added as a filter aid. The reaction solution was filtered, washed with Nutsche with ethyl acetate, and the organic solvent was concentrated. The concentrated solution was extracted three times with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give compound 115 (92.9 g, 174 mmol, 94% yield).

Step 2 Synthesis of Compound 116 After adding ammonium fluoride (77.3 g, 2.09 mol) to a methanol (500 g) solution of Compound 115 (192.5 g, 1.74 mmol) at room temperature, the bath temperature was raised to 68 ° C. For 3.5 hours. After cooling to room temperature, the reaction solution was added to water (1 L) and ethyl acetate (1.5 L). Liquid separation was performed, and the aqueous layer was extracted with ethyl acetate (500 ml × 2). The organic layers were combined, washed with saturated brine (500 ml), and concentrated. The resulting crude product was purified by silica gel column chromatography (silica gel carrier amount: 1.1 kg, hexane / ethyl acetate / methanol) to obtain compound 116 (87.0 g, yield 68.5%). It was.

Step 3 Synthesis of Compound 117 Compound 116 (87 g) was dissolved in acetonitrile (440 g) at room temperature, and 1 mol / L acetic acid (87 ml) and 1 mol / L sodium acetate (174 ml) were added. Further TEMPO (1.8 g, 11.6 mmol) was added followed by 80% sodium chlorite (NaClO2, 27.5 g, 243 mmol) and 12% sodium hypochlorite (NaClO, 1.5 ml). The mixture was stirred at around 28 ° C. for 2.5 hours, and 12% sodium hypochlorite (NaClO, 1.5 ml) was added (the reaction solution changed from amber to brown). After stirring overnight, 2-methyl-2-butene (80 ml) was added and stirred for 40 minutes (the reaction solution changed from brown to amber). Acetic acid (35 ml) was added to this solution, and the mixture was extracted with ethyl acetate (200 ml), washed with saturated brine (50 ml), concentrated, and azeotroped with toluene (100 ml × 3 times) to give compound 117 (113 g). ) Was obtained.

Step 4 Synthesis of Compound 118 Crude compound 117 (113 g) was dissolved in methanol (400 g), 25% aqueous ammonia (200 ml) was added, and the mixture was stirred at 40 ° C. overnight. The reaction mixture was concentrated and azeotroped with toluene (200 ml × 3 times) to obtain a crude product of Compound 118 (112.8 g).

Step 5 Synthesis of Compound 119 Compound 118 (112.8 g, corresponding to 115.7 mmol) was dissolved in N, N′-dimethylformamide (440 g) at room temperature under a nitrogen stream, and EDC hydrochloride (33.3 g, 173) was dissolved. .6 mmol) was added. EDC hydrochloride (17.8 g, 92.8 mmol) was added while stirring as it was and confirming the progress of the reaction. After confirming the completion of the reaction, the reaction solution was dispersed under stirring with water (1760 g) and ethyl acetate (25 g). The resulting crystals were collected by filtration, washed with water (300 g) and ethyl acetate / hexane (1/4) (150 ml), and dried under reduced pressure to give compound 119 (40 g, 79.6 mmol, yield from compound 116). : 45.9%) as a colorless solid.

Step 6 Conversion from Compound 119 to Compound 21a Under a nitrogen stream, compound 119 (40 g, 79.6 mmol) was mixed with acetic acid (400 ml) at room temperature, palladium hydroxide (12.5 g) was added, and then hydrogen atmosphere was added. Stir for 24 hours at 30 ° C. Since crystals precipitated in the reaction system, water (80 g) was added, and the mixture was further stirred for 2 days. Then, the catalyst was filtered off, and the filtrate was concentrated. After azeotroping with toluene (80 g × 2 times), the obtained solid was slurry washed with methanol (50 g) and dried to obtain compound 21a (19.5 g, 60.5 mmol, yield 76.2%). It was.

Compound 21a to G (Pac) amidite (24) was induced by the method described in Example 6.

The route from compound 106 to compound 118 can also be synthesized by the method described in the following scheme.

Example of synthesis of T amidite Conversion of compound 106 to compound 14b can be carried out by the method described in the following scheme. The compound 14b can be synthesized by the method described in Example 3 or International Publication No. 2011/052436.

Reference example 2

Glucosamine root
Synthesis method using glucosamine as a starting material (part 1)

Step 1 Synthesis of Compound 124 Chemische Berichte, 101 (7), 2289-93 (1968). To obtain Compound 124 from D-glucosamine hydrochloride.

Step 2 Synthesis of Compound 125 In a nitrogen stream, sodium hydride (0.89 g, 22.2 mmol) was suspended in N, N′-dimethylformamide (30 mL) at 5 to 7 ° C. with Compound 124 (5.2 g, 17.1 mmol) in N, N′-dimethylformamide (30 mL) was added dropwise and stirred for 30 minutes. Subsequently, benzyl bromide (2.4 mL, 20.5 mmol) was added dropwise to the reaction solution at 7-8 ° C., and the mixture was stirred at room temperature for 90 minutes. A saturated aqueous ammonium chloride solution was added, and the mixture was extracted with tert-butyl methyl ether. The organic layer was washed with water and saturated brine. Thereafter, it was dried over sodium sulfate and distilled off under reduced pressure. The obtained crude product was purified by silica gel column chromatography (n-hexane / ethyl acetate) to obtain Compound 125 (5.1 g, 12.9 mmol) as a pale yellow solid.
¹ H-NMR (CDCl ₃ ) δ: 8.04 (2H, dt, J = 6.71, 1.60 Hz), 7.53-7.32 (10H, m), 6.25 (1H, d, J = 5.19 Hz), 5.02 (1H, d , J = 11.44 Hz), 4.84 (1H, dd, J = 6.71, 5.34 Hz), 4.71 (1H, d, J = 11.29 Hz), 4.42 (1H, td, J = 7.02, 2.90 Hz), 4.28 (1H , dd, J = 8.69, 6.86 Hz), 4.04-3.88 (3H, m), 1.40 (3H, s), 1.39 (4H, s).

Step 3 Synthesis of Compound 126 Under a stream of nitrogen, N, O-bistrimethylsilylacetamide (4.7 mL, 18.9 mmol) was added to a suspension of thymine (1.43 g, 11.4 mmol) in acetonitrile (15 mL) at 80 ° C. Stirred for 60 minutes under. Thereafter, a solution of compound 125 (3.0 g, 7.6 mmol) in acetonitrile (15 mL) and tin tetrachloride (0.89 mL, 7.6 mmol) were added, and the mixture was stirred at 80 ° C. for 6 hours. After cooling to room temperature, saturated multistory water was added, and the precipitated solid was removed by Celite filtration. The reaction mixture was concentrated under reduced pressure, extracted from the residue with ethyl acetate, and washed with water and saturated brine. The crude product 125a (4.1g) was obtained by drying with sodium sulfate and concentrating under reduced pressure. Subsequently, the crude product 126 (4.1 g) was dissolved in methanol (30 mL) and water (10 mL), camphorsulfonic acid (0.73 g, 3.1 mmol) was added at room temperature, and the mixture was stirred for 2 hours. The mixture was neutralized by adding a 1 mol / L aqueous sodium hydroxide solution and then concentrated under reduced pressure. The residue was extracted with ethyl acetate, washed with water and saturated brine, dried over sodium sulfate, and concentrated under reduced pressure. Recrystallization (n-hexane / ethyl acetate) gave Compound 126 (3.2 g, 6.7 mmol) as a white solid.
¹ H-NMR (CD ₃ OD) δ: 7.83 (1H, d, J = 1.18 Hz), 7.75-7.72 (2H, m), 7.55-7.52 (1H, m), 7.46-7.25 (7H, m), 6.19 (1H, d, J = 8.39 Hz), 4.91 (1H, dd, J = 8.48, 5.79 Hz), 4.75 (1H, d, J = 11.92 Hz), 4.55 (1H, d, J = 11.92 Hz), 4.36-4.32 (2H, m), 3.90 (1H, td, J = 5.75, 3.58 Hz), 3.61 (2H, d, J = 5.88 Hz), 1.91 (3H, d, J = 1.18 Hz).

Step 4 Synthesis of Compound 127 At room temperature, sodium periodate (2.3 g, 10.6 mmol) was added to a tetrahydron-water mixed solution of compound 126 (3.4 g, 7.1 mmol) (1: 1, 30 mL). And stirred for 1 hour. Ethylene glycol (0.4 mL, 7.1 mmol) was added and stirred for a while, and the resulting white solid was removed by filtration. The organic layer was extracted with ethyl acetate, washed with water and saturated brine, and dried over sodium sulfate. The organic layer was distilled off under reduced pressure to obtain a crude product 127 (3.6 g).

Step 5 A solution of the crude product 127 of compound 128 (3.4 g, 7.3 mmol) in tetrahydrofuran furan (20 mL) was cooled to −10 ° C., and then 37% formaldehyde (1.4 mL, 18.2 mmol) and 2 mol / L Aqueous sodium hydroxide solution (11 mL, 22 mmol) was added and stirred at 2 ° C. for 3 hours. The mixture was neutralized with 2 mol / L hydrochloric acid aqueous solution and extracted with ethyl acetate. The organic layer was washed with water and saturated brine, and dried over sodium sulfate. The organic layer was distilled off under reduced pressure to obtain a crude product 128 (3.6 g).

Step 6 Synthesis of Compound 129 To a mixed solution of crude product 128 (3.6 g) in tert-butanol-water-tetrahydrofuran (5: 5: 1, 33 mL) at room temperature, 2-methyl-2-butene (7.8 mL, 75 mmol), sodium dihydrogen phosphate (2.7 g, 22.5 mmol) and sodium chlorite (1.4 g, 15 mmol) were added and stirred for 2 hours 30 minutes. A 1 mol / L aqueous hydrochloric acid solution was added to adjust the reaction solution to pH 2 to 3, followed by extraction with chloroform. The organic layer was washed with water and saturated brine, and dried over sodium sulfate. The organic layer was distilled off under reduced pressure to obtain a crude product 129 (3.7 g).

Step 7 Synthesis of Compound 130 At room temperature, a 2 mol / L trimethylsilyldiazomethane solution (4.9 mL, 9.7 mmol) was added dropwise to a toluene-methanol solution (2: 1, 30 mL) of the crude product 129 (3.7 g). And stirred for 40 minutes. Acetic acid (0.2 mL) was added, and the reaction mixture was concentrated under reduced pressure. The obtained solid was recrystallized from ethyl acetate / methanol to obtain compound 130 (1.54 g, 3.1 mmol) as a white solid.
¹ H-NMR (CD ₃ OD) δ: 7.81 (1H, d, J = 1.22 Hz), 7.68-7.66 (2H, m), 7.53-7.51 (1H, m), 7.42-7.40 (3H, m), 7.26-7.21 (6H, m), 6.36 (1H, d, J = 8.69 Hz), 4.96-4.87 (28H, m), 4.66 (1H, d, J = 11.13 Hz), 4.60-4.51 (3H, m) , 4.12 (1H, dd, J = 13.12, 9.46 Hz), 3.88 (2H, t, J = 10.98 Hz), 3.72 (3H, s), 1.91 (3H, t, J = 3.13 Hz).

The conditions for the glycosylation reaction performed in step 3 were examined. Specifically, the combination of acid catalyst and solvent was examined, and the product and the remaining amount of raw material were analyzed by HPLC or the like. As a result, it has been found that the combination shown in Condition 7 as shown in the following table is the optimum condition that gives the highest product yield and the lowest raw material residual rate and byproduct production rate.

Synthesis method using glucosamine as a starting material (Part 2)
Compound 132 synthesized by the synthesis method (part 1) using glucosamine as a starting material can also be synthesized by the route described below.

Reference example 3

Route using cyclization reaction

Process 1
Compound 148 (2.0 g, 4.70 mmol) synthesized by a known method (Tetrahedron, 1998, 54, 3607., etc.) was dissolved in acetonitrile (20 ml) and acetate buffer (6 ml). 2,2,6,6-tetramethylpiperidinyloxy radical (74 mg, 0.47 mmol), sodium chlorite (894 mg, 9.88 mmol) and 5% aqueous sodium hypochlorite solution (1 ml) under ice cooling And stirred at room temperature overnight. 2-Methyl-2-butene (2 ml) was added to the reaction mixture, stirred at room temperature for 2 hours, and extracted with ethyl acetate. The obtained organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure to obtain a pale orange oil. Recrystallization (ethyl acetate / hexane) gave Compound 149 (1.58 g, 81% yield) as a white solid.
¹ H-NMR (DMSO-D ₆ ) δ: 12.68 (1H, s), 7.37-7.29 (10H, m), 5.78 (1H, d, J = 3.8 Hz), 4.76 (1H, t, J = 4.5 Hz ), 4.60 (2H, dd, J = 50.7, 11.9 Hz), 4.14 (1H, d, J = 5.3 Hz), 3.66 (2H, t, J = 10.7 Hz), 1.49 (3H, s), 1.29 (3H , s).

Process 2
Compound 149 (720 mg, 1.74 mmol) obtained in the first step was dissolved in N, N-dimethylformamide (5.5 ml). 2 mol / L methanamine in tetrahydrofuran (2.6 ml, 5.22 mmol), 1-hydroxybenzotriazole monohydrate (293 mg, 1.91 mmol) and 1-ethyl-3- (3-dimethylaminopropyl) at room temperature Carbodiimide hydrochloride (366 mg, 1.91 mmol) was added, and the mixture was stirred at room temperature for 8 hours. Saturated aqueous sodium hydrogen carbonate solution was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The obtained organic layer was washed with 1 mol / L hydrochloric acid aqueous solution, saturated aqueous sodium hydrogen carbonate solution and saturated brine, and dried over magnesium sulfate. The solvent was distilled off under reduced pressure to obtain Compound 150 (753 mg) as a pale orange solid.
¹ H-NMR (DMSO-D ₆ ) δ: 7.44 (1H, d, J = 4.6 Hz), 7.37-7.27 (10H, m), 5.86 (1H, d, J = 4.3 Hz), 4.82 (1H, t , J = 4.8 Hz), 4.60 (2H, dd, J = 40.0, 12.0 Hz), 4.48 (2H, dd, J = 14.2, 12.8 Hz), 4.12 (1H, d, J = 5.5 Hz), 3.63 (2H , dd, J = 56.9, 10.2 Hz), 2.63 (3H, d, J = 4.6 Hz), 1.41 (3H, s), 1.30 (3H, s).

Process 3
Compound 150 (500 mg, 1.11 mmol) obtained in the second step was dissolved in acetic acid (1.2 ml, 21.13 mmol). Acetic anhydride (0.58 ml, 6.12 mmol) was added, concentrated sulfuric acid (0.012 ml, 0.22 mmol) was added dropwise under ice cooling, and the mixture was stirred at room temperature for 7 hours. The reaction mixture was diluted with ethyl acetate (5 ml) and poured into ice water. Under ice cooling, the mixture was neutralized with an aqueous sodium hydroxide solution and extracted with ethyl acetate. The obtained organic layer was washed with 20% aqueous sodium carbonate solution and saturated brine, and dried over anhydrous magnesium sulfate. The solvent was distilled off under reduced pressure to obtain Compound 151 (543 mg) as a crude oil.

Process 4
Compound 151 (540 mg, 1.11 mmol) obtained in the third step was dissolved in acetonitrile (3 ml). 5-Methyluridine (188 mg, 1.49 mmol) and N, O-bistrimethylsilylacetamide (0.56 ml, 2.29 mmol) were added at room temperature, and the mixture was stirred at 55 ° C. for 15 minutes. Trimethylsilyl triflate (0.23 ml, 1.26 mmol) was added and stirred at 75 ° C. for 3.5 hours. Under ice-cooling, 3% saturated aqueous sodium hydrogen carbonate solution was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated aqueous sodium hydrogen carbonate solution and saturated brine, and dried over anhydrous magnesium sulfate. The solvent was distilled off under reduced pressure to obtain Compound 152 (549 mg, 93% yield) as amorphous.
¹ H-NMR (DMSO-D ₆ ) δ: 11.39 (1H, s), 8.26 (1H, d, J = 4.5 Hz), 7.61 (1H, s), 7.36 (10H, m), 6.31 (1H, d , J = 8.4 Hz), 5.39 (1H, dd, J = 8.3, 4.9 Hz), 4.72 (1H, d, J = 11.7 Hz), 4.62 (2H, dd, J = 11.2, 6.9 Hz), 4.49 (2H , dd, J = 16.1, 7.8 Hz), 4.12 (1H, d, J = 10.3 Hz), 3.73 (1H, d, J = 10.3 Hz), 2.61 (3H, d, J = 4.3 Hz), 2.02 (3H , s), 1.43 (3H, s).

Process 5
Compound 152 (549 mg, 1.03 mmol) obtained in the fourth step was dissolved in tetrahydrofuran (1.5 ml). A 40% aqueous methanamine solution (0.49 ml, 5.89 mmol) was added at room temperature, and the mixture was stirred at room temperature for 3 hours. A saturated aqueous ammonium chloride solution was added to the reaction mixture under ice cooling, and the mixture was extracted with ethyl acetate. The obtained organic layer was washed with saturated brine and dried over magnesium sulfate. The solvent was distilled off under reduced pressure to obtain Compound 153 (498 mg, 98% yield) as amorphous.
¹ H-NMR (DMSO-D ₆ ) δ: 11.36 (1H, s), 8.08 (1H, d, J = 4.6 Hz), 7.57 (1H, s), 7.38-7.29 (10H, m), 6.09 (1H , d, J = 8.4 Hz), 5.59 (1H, d, J = 6.3 Hz), 4.80 (1H, d, J = 11.2 Hz), 4.66 (1H, d, J = 11.7 Hz), 4.58 (2H, d , J = 10.7 Hz), 4.44 (1H, dd, J = 12.4, 6.8 Hz), 4.17 (1H, d, J = 4.3 Hz), 4.05 (1H, d, J = 10.7 Hz), 3.68 (1H, d , J = 10.2 Hz), 2.60 (3H, d, J = 4.5 Hz), 1.50 (3H, s).

Step 6
Compound 153 (480 mg, 0.97 mmol) obtained in the fifth step was dissolved in pyridine (1.5 ml). Under ice-cooling, mesyl chloride (0.18 ml, 2.28 mmol) was added, and the mixture was stirred at 0 ° C. for 30 minutes. The reaction solution was diluted with ethyl acetate, extracted with water. The obtained organic layer was washed 3 times with 2 mol / L aqueous hydrochloric acid, then washed with a saturated aqueous sodium hydrogen carbonate solution and saturated brine, and dried over magnesium sulfate. The solvent was distilled off under reduced pressure to obtain Compound 154 (520 mg, 93% yield) as amorphous.
¹ H-NMR (DMSO-D ₆ ) δ: 11.46 (1H, s), 8.28 (1H, d, J = 4.6 Hz), 7.55 (1H, s), 7.40-7.32 (10H, m), 6.31 (1H , d, J = 8.2 Hz), 5.36 (1H, dd, J = 8.0, 4.6 Hz), 4.73-4.60 (5H, m), 4.10 (1H, d, J = 10.3 Hz), 3.77 (1H, d, J = 10.3 Hz), 3.23 (3H, s), 2.61 (3H, d, J = 4.5 Hz), 1.43 (3H, s).

Step 7
Compound 154 (515 mg, 0.90 mmol) obtained in step 6 was dissolved in acetonitrile (5 ml). Diazabicycloundecene (0.18 ml, 1.20 mmol) was added at room temperature, and the mixture was stirred at room temperature for 7 hours. The reaction mixture was diluted with ethyl acetate under ice-cooling, and extracted with 5% aqueous citric acid solution. The obtained organic layer was washed with 5% aqueous citric acid solution, saturated aqueous sodium hydrogen carbonate solution and saturated brine, and dried over magnesium sulfate. The solvent was distilled off under reduced pressure to obtain a pale yellow oil. Recrystallization (ethyl acetate / hexane) gave Compound 155 (374 mg, 87% yield) as a white solid.
¹ H-NMR (DMSO-D ₆ ) δ: 7.96 (1H, d, J = 4.6 Hz), 7.75 (1H, s), 7.37-7.26 (8H, m), 7.08 (2H, d, J = 7.2 Hz ), 6.53 (1H, d, J = 5.9 Hz), 5.47 (1H, d, J = 5.9 Hz), 4.65 (2H, dd, J = 57.6, 11.5 Hz), 4.43 (1H, s), 4.24 (2H , dd, J = 32.7, 12.6 Hz), 3.69 (1H, d, J = 10.4 Hz), 3.32 (1H, d, J = 10.8 Hz), 2.61 (3H, d, J = 4.5 Hz), 1.84 (3H , s).

Process 8
To a solution of sodium tert-butoxide (18 mg, 0.19 mmol) in N, N-dimethylformamide (0.6 ml), the compound 155 (30 mg, 0.06 mmol) obtained in the seventh step was added, and 1. Stir for 5 hours. The mixture was diluted with ethyl acetate under ice-cooling, and a saturated aqueous ammonium chloride solution was added for extraction. The obtained organic layer was washed with a saturated aqueous sodium hydrogen carbonate solution and saturated brine, and dried over magnesium sulfate. The solvent was distilled off under reduced pressure, and the resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate) to obtain Compound 122 (19 mg, 63% yield) as amorphous.
¹ H-NMR (DMSO-D ₆ ) δ: 11.47 (1H, s), 7.38-7.28 (11H, m), 5.48 (1H, s), 4.62 (4H, dd, J = 20.7, 10.5 Hz), 4.40 (1H, s), 4.14 (1H, s), 3.92 (1H, d, J = 11.9 Hz), 3.79 (1H, d, J = 11.8 Hz), 2.87 (3H, s), 1.48 (3H, s) .

Step 9
Compound 122 (17 mg, 0.90 mmol) obtained in the eighth step was dissolved in a mixed solvent of tetrahydrofuran (1 ml) and methanol (0.2 ml). 20% Parazimed carbon (8 mg) was added and stirred for 2 hours under hydrogen atmosphere. Nitrogen substitution was performed, the catalyst was removed by filtration, and the solvent was distilled off from the filtrate under reduced pressure. Recrystallization of the obtained colorless oil (methanol / chloroform) gave Compound 14b (10 mg, 93% yield) as a white solid.
¹ H-NMR (MeOD) δ: 7.84 (1H, s), 5.47 (1H, s), 4.30 (1H, s), 4.15 (1H, s), 4.06 (1H, d, J = 13.3 Hz), 3.89 (1H, d, J = 13.6 Hz), 3.03 (3H, s), 1.91 (3H, s).

T amidite (compound 16) and MeC (Bz) amidite (compound 29) can be obtained from compound 14b by the method described in Example 3, Example 11 or International Publication No. 2011/052436.

Examination of conditions for cyclization reaction Conditions for the cyclization reaction performed in Step 9 were examined. Specifically, the types of base, solvent, and reaction temperature were examined, and the product (Compound 122) and the by-product (Compound 156) were analyzed by HPLC or the like. As a result, it has been found that the combination shown in Condition 8 as shown in the following table is the optimum condition that gives the highest product yield and the lowest raw material residual rate and by-product production rate.

Conversion to amidite Compound 122 was converted to 1) NaOH aq. Compound 156 was obtained in two steps by treatment with / THF 2) (CF ₃ CO) ₂ O / pyridine. Compound 156 can be derived into A (Bz) amidite (Compound 20) and G (Pac) amidite (Compound 24) by the method of Reference Example 1.

Claims (16)

The following steps for subjecting a compound represented by the general formula (I) to an aldol reaction in the presence of a base:

(In the formula, R ₁ and R ₂ are the same or different and each represents a hydrogen atom, an amino group protecting group for nucleic acid synthesis, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a silyl group, Phosphate group, phosphate group protected with a protecting group for nucleic acid synthesis, -P (R ₇ ) R ₈ [wherein R ₇ and R ₈ are the same or different and protected with a hydroxyl group, a protecting group for nucleic acid synthesis Hydroxyl group, mercapto group, mercapto group protected with a protecting group for nucleic acid synthesis, amino group, alkoxy group having 1 to 5 carbon atoms, alkylthio group having 1 to 5 carbon atoms, cyanoalkoxy group having 1 to 6 carbon atoms, Or an amino group substituted with an alkyl group having 1 to 5 carbon atoms];
R ₃ was protected with a hydrogen atom, a hydroxyl protecting group for nucleic acid synthesis, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a silyl group, a phosphate group, or a protecting group for nucleic acid synthesis. Phosphoric acid group, —P (R ₉ ) R _{10 wherein} R ₉ and R ₁₀ are the same or different and are a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, a mercapto group, or a protecting group for nucleic acid synthesis. Substituted with a protected mercapto group, amino group, alkoxy group having 1 to 5 carbon atoms, alkylthio group having 1 to 5 carbon atoms, cyanoalkoxy group having 1 to 6 carbon atoms, or alkyl group having 1 to 5 carbon atoms Represents an amino group];
R ₄ represents a hydrogen atom, a hydroxyl group, or an alkoxy group having 1 to 5 carbon atoms;
R ₅ and R ₆ are the same or different and each represents a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, or an aryl group;
B is a nucleobase moiety optionally having a substituent)
The manufacturing method of the compound represented by general formula (II) including this. The following steps of reacting a compound represented by the general formula (I) with a compound represented by the formula: R ₅ C (═O) R ₆ in the presence of a base:

(Wherein each definition has the same meaning as in claim 1), and a method for producing a compound represented by general formula (II). The production method according to claim 1 or 2, wherein the base has a pKa of a conjugate acid of 6 to 10. The method according to claim 3, wherein the base is triethylamine or N-methylmorpholine. After obtaining the compound represented by the general formula (II) by the method according to claim 1, when R ₄ is a hydrogen atom, the compound represented by the obtained general formula (II) is used. The following steps for subjecting the compound to an oxidation reaction:

(Wherein R ₄ is a hydrogen atom, and other definitions are as defined in claim 1), and a method for producing a compound represented by general formula (III). The production method according to any one of claims 1 to 5, wherein either one of R ₁ and R ₂ is an amino-protecting group for nucleic acid synthesis, and the other is a hydrogen atom or an alkyl group. The production method according to claim 1, wherein R ₃ is a hydroxyl-protecting group for nucleic acid synthesis. The process according to any one of claims 1 to 7 R ₄ is a hydrogen atom. R < ₅ > and R < ₆ > are both hydrogen atoms, The manufacturing method in any one of Claims 1-8. The production method according to any one of claims 1 to 9, wherein B is a pyrimidine nucleobase or a purine nucleobase which may have a substituent. Compounds represented by the following general formula (III) and salts thereof.

(Wherein R ₃ is a protecting group for a hydroxyl group in nucleic acid synthesis, and other definitions are the same as in claim 1) The following steps of reacting the compound represented by the general formula (III) with a desilylating agent and reacting the reaction solution with a condensing agent:

(Wherein R ₁ is an alkyl group having 1 to 5 carbon atoms, R ₂ is a trifluoroacetyl group , R ₃ is a protecting group for a hydroxyl group for nucleic acid synthesis and has a silyl group , and other definitions) Is equivalent to claim 1)
The manufacturing method of the compound represented by general formula (IV) including this. After obtaining the compound represented by the general formula (IV) by the method according to claim 12, the compound represented by the general formula (IV) is reacted with a reagent used for protecting the hydroxyl group in nucleic acid synthesis. The steps shown below:

(Wherein R ₇ is a hydroxyl-protecting group for nucleic acid synthesis, and other definitions are as defined in claim 12).
The manufacturing method of the compound represented by general formula (V) including this. Compounds represented by the following general formula (V) and salts thereof.

(Wherein each definition has the same meaning as in claim 13) The following steps for subjecting a compound represented by the following general formula (VI) to a transglycosylation reaction:

(Wherein B ₁ is an optionally substituted pyrimidine nucleobase, B ₂ is a nucleobase other than an optionally substituted pyrimidine , X is a hydrogen atom, sodium atom or potassium atom, Definitions other than are synonymous with claim 1)
The manufacturing method of the compound represented by general formula (VII) including this. The compound represented by the following general formula (VII) and its salt.

(Wherein each definition has the same meaning as claim 15)