Novel artificial nucleic acid, its production method and use

Abstract

The present invention addresses the problem of providing a novel artificial nucleic acid, a method for producing the same, and a use thereof. The problem can be solved by an artificial nucleic acid having a unit derived from a compound represented by formula (1) or a salt thereof: [in the formula, Base is an aromatic heterocyclic group or aromatic hydrocarbocyclic group optionally having a substituent; A1 is a single bond or an alkylene group; R1 and R2 are the same or different and are a hydrogen atom, an alkyl group, alkenyl group, cycloalkyl group, cycloalkenyl group, or aryl group optionally having a substituent, a hydroxyl-group protecting group, a phosphino group having a substituent, or a dihydroxyphosphinyl group or hydroxymercaptophosphinyl group optionally having a substituent, or R1 and R2 form a ring optionally having a substituent together with two adjacent oxygen atoms and the position 3-5 carbon atoms of furanose; R3 is a hydrogen atom, an alkyl group, alkenyl group, cycloalkyl group, aryl group, or acyl group optionally having a substituent, a sulfonyl group or silyl group having a substituent, a functional molecular unit substituent, or a group represented by formula: R31-X- (in the formula, R31 is an amino group optionally having a substituent, X is an alkylene group optionally having a substituent or a group in which at least one methylene group in this alkylene group is substituted by -N(R32)- (in the formula, R32 is a hydrogen atom or an alkyl group), -O-, or -S(=O)k- (in the formula, k is 0, 1, or 2)); R4 is a hydrogen atom, or an alkyl group or aryl group optionally having a substituent; R5 is a hydrogen atom, or an alkyl group or aryl group optionally having a substituent; R4 and R5 are not simultaneously hydrogen atoms; and n is 0 or 1].

Description

[Technical Field]

[0001] The present invention relates to a novel artificial nucleic acid, a method for producing the same, and uses thereof. [Background technology]

[0002] Oligonucleotides are short sequences of natural DNA or RNA or artificial nucleic acids, and have been shown to be very useful for regulating gene expression at the transcriptional and translational levels of various genes, and for examining the sequence status of genes, thereby treating and diagnosing specific diseases. Methods for regulating gene expression or testing / diagnosing genetic information can be broadly divided into two types depending on the target: the first type is when the target is single-stranded RNA or single-stranded DNA, such as messenger RNA (mRNA) or microRNA (miRNA), and the second type is when the target is double-stranded genomic DNA. When the target is single-stranded RNA or single-stranded DNA, the gene translation process can be inhibited (or genetic diagnosis) by antisense methods, in which oligonucleotides bind complementary to single-stranded RNA or single-stranded DNA to form duplexes. Alternatively, when the oligonucleotide is a double-stranded RNA molecule, complementary binding of the oligonucleotide to the target mRNA triggers degradation of the target mRNA by the "slicer" enzyme of the RISC complex (RNA interference method). In the case of RNA interference, the oligonucleotide can be an oligonucleotide equivalent to an endogenous microRNA (microRNA mimics) that binds to the 3' untranslated region (3' UTR) of the target mRNA and inhibits translation of the target mRNA due to incomplete complementarity. Oligonucleotides can induce gene activation or increased transcription, for example, by binding complementary to long antisense non-coding RNAs or by inhibiting complementary microRNAs, which can also result in increased translation of the microRNA's target mRNA (anti-microRNA). Oligonucleotides, which are functional materials used in methods for regulating gene expression and testing / diagnosing genetic information, are required to have characteristics such as excellent sequence-specific binding affinity with target nucleic acids, strong resistance to degradative enzymes, and safety in vivo. Naturally occurring DNA and RNA have poor resistance to degradative enzymes and insufficient binding affinity, making them unsuitable as functional materials. For this reason, numerous artificial nucleic acids have been developed to date with the aim of improving the functionality of oligonucleotides. Representative examples include peptide nucleic acids (PNA), bridged nucleic acids, morpholino nucleic acids (PMO), and phosphorothioate nucleic acids (S oligonucleotides) in which one non-bonded oxygen atom in the phosphate diester moiety of the nucleic acid is replaced with a sulfur atom. Representative examples of the bridged nucleic acids include LNA (Structural Formula 1 below), BNA, NC (Structural Formula 2 below) and ENA (Structural Formula 3 below). [ka] The structure of the phosphorothioate nucleic acid is represented by the following structural formula 4. [ka] These cross-linked nucleic acids have been demonstrated to have an excellent ability to bind to single-stranded RNA with high sequence selectivity via Watson-Crick hydrogen bonds (Patent Documents 1 to 3). Thus, conventional artificial nucleic acids are used as functional materials to control the expression of specific genes and to verify and diagnose gene sequences with high sensitivity and precision. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] US Patent Application Publication No. 2003 / 105309 [Patent Document 2] US Patent Application Publication No. 2007 / 167387 [Patent Document 3] US Patent Application Publication No. 2003 / 207841 Summary of the Invention [Problem to be solved by the invention]

[0004] As the uses of oligonucleotides become more diverse, there is still room for improvement in the functionality of existing artificial nucleic acids, and there is a need to develop new artificial nucleic acids with even higher functionality.

[0005] An object of the present invention is to provide novel artificial nucleic acids useful in various genome technologies, methods for producing them, and uses thereof. [Means for solving the problem]

[0006] As a result of extensive research to achieve the above object, the present inventors have discovered a BNA represented by the above structural formula 2. NC The inventors have found that an artificial nucleic acid in which an optionally substituted alkyl group or an optionally substituted aryl group is bound to the carbon atom between the carbon atom at position 4 of the furanose and the nitrogen atom to which R is bound has both highly sequence-selective and strong binding ability to single-stranded DNA and single-stranded RNA, and excellent resistance to degradative enzymes. Based on this finding, the inventors have conducted further studies and have completed the present invention.

[0007] The present invention includes the following aspects. Section 1. A compound represented by the following formula (1) or a salt thereof: [ka] [In the formula, Base is an aromatic heterocyclic group which may have a substituent, or an aromatic hydrocarbon ring group which may have a substituent, A 1 is a single bond or an alkylene group, R 1 and R 2are the same or different and are a hydrogen atom, an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted cycloalkyl group, an optionally substituted cycloalkenyl group, an optionally substituted aryl group, a protecting group for a hydroxyl group, a substituted phosphino group, an optionally substituted dihydroxyphosphinyl group, or an optionally substituted hydroxymercaptophosphinyl group, or R 1 and R 2 forms a ring which may have a substituent together with the two adjacent oxygen atoms and the carbon atoms at positions 3 to 5 of the furanose, R 3 represents a hydrogen atom, an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted cycloalkyl group, an optionally substituted aryl group, an optionally substituted acyl group, a substituted sulfonyl group, a substituted silyl group, a functional molecular unit substituent, or a group represented by the formula: R 31 -X-(wherein, R 31 is an amino group which may have a substituent, X is an alkylene group which may have a substituent, or at least one methylene group in this alkylene group is -N(R 32 )-(wherein, R 32 is a hydrogen atom or an alkyl group), -O-, or -S(=O) k - (wherein k is 0, 1, or 2), R 4 represents a hydrogen atom, an optionally substituted alkyl group, or an optionally substituted aryl group, R 5 represents a hydrogen atom, an optionally substituted alkyl group, or an optionally substituted aryl group, R 4 and R 5 is not a hydrogen atom at the same time, The following formula: [ka] represents a single or double bond, When the symbol is a single bond, n is 1; When the symbol is a double bond, n is 0. Section 2. A 1 Item 2. The compound or salt thereof according to Item 1, wherein is a single bond. Section 3. The following formula: [ka] Item 3. The compound or salt thereof according to Item 1 or 2, wherein the symbol represented by the formula: is a single bond and n is 1. Section 4. R 4 Item 4. The compound or salt thereof according to any one of Items 1 to 3, wherein is an alkyl group. Section 5. R 5 Item 5. The compound or salt thereof according to any one of Items 1 to 4, wherein is a hydrogen atom or an alkyl group. Section 6. R 3 is a hydrogen atom, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, an alkylsulfonyl group, an arylsulfonyl group, a group of the formula: -Si(R 6 )3(in the formula, each R 6 and are the same or different and are an alkyl group or an aryl group), a labeling functional group, a group having intercalating ability, a group having nucleic acid binding ability, a nucleic acid cleaving active functional group, a group having intracellular or nuclear transport ability, or a group having metal chelating ability. Section 7. R 3 However, the formula: R 31 is a group represented by -X-, R 31 But the following formula (A): [ka] (In the formula, R 3a and R 3bare the same or different and are a hydrogen atom, an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted cycloalkyl group, an optionally substituted cycloalkenyl group, an optionally substituted aryl group, or a protecting group for an amino group, or R 3a and R 3b forms, together with the adjacent nitrogen atom, a ring which may have a substituent. or a group represented by the following formula (B): [ka] (In the formula, R 3c ~R 3f are the same or different and are a protecting group for a hydrogen atom, an alkyl group, or an amino group. is a group represented by X is -C m H 2m - (wherein m is an integer of 1 to 10), Item 6. The compound or salt thereof according to any one of Items 1 to 5. Section 8. R 1 and R 2 are the same or different and are a hydrogen atom, an optionally substituted alkyl group, an optionally substituted aryl group, an alkylcarbonyl group, an arylcarbonyl group, an alkylsulfonyl group, an arylsulfonyl group, a group of the formula: -Si(R 6 )3(in the formula, each R 6 are the same or different and are an alkyl group or an aryl group), a group represented by the formula: -P(R 7 )(R 8 )(wherein, R 7 and R 8 are the same or different and are a hydroxyl group, a mercapto group, an amino group, an alkoxy group, a haloalkoxy group, a cyanoalkoxy group, an alkylthio group, a haloalkylthio group, a cyanoalkylthio group, or an alkylamino group), a dihydroxyphosphinyl group, or a hydroxymercaptophosphinyl group, or R 1 and R 2form a ring which may have a substituent together with the two adjacent oxygen atoms and the carbon atoms at positions 3 to 5 of the furanose. Item 8. The compound or salt thereof according to any one of Items 1 to 7. Section 9. Item 9. The compound or salt thereof according to any one of Items 1 to 8, wherein Base is an optionally substituted 2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl group, an optionally substituted 2-oxo-1,2-dihydropyrimidin-1-yl group, an optionally substituted purin-9-yl group, or an optionally substituted 6-oxo-1,6-dihydro-9H-purin-9-yl group. Section 10. Formula (1A): [ka] (Wherein, Base and R 1 ~R 5 is the same as above) Item 2. The compound or salt thereof according to Item 1, wherein the compound or salt thereof is represented by the formula: Section 11. Formula (1B): [ka] (Wherein, Base and R 1 , R 2 , and R 4 is the same as above) Item 2. The compound or salt thereof according to Item 1, wherein the compound or salt thereof is represented by the formula: Section 12. Item 1. The compound represented by formula (1) or a salt thereof, wherein n is 0 or n is 1 and R 5 is a hydrogen atom or a salt thereof, (I) Formula (1E): [ka] (In the formula, Base, A 1 , R 1 , and R 2 is the same as term 1) The compound represented by the formula: R 4 ·(In the formula, R 4 is the same as item 1), or a radical represented by the formula: R 4 M (wherein M is a metal atom or an atomic group containing a metal atom), R 4 is the same as in item 1), and (II) a step of dehydrogenating the compound obtained in the step (I); (III) a step of dehydrogenating the compound obtained in the step (I) and then hydrogenating it, or (IV) a step of treating the compound obtained in the step (I) or the compound obtained by dehydrogenating the compound obtained in the step (I) and then hydrogenating it with a compound of the formula: R 3 -L, where L is a leaving group and R 3 is the same as in item 1, but is not hydrogen) The method may include: Section 13. Item 1. The compound represented by formula (1) or a salt thereof, wherein n is 1 and R 3 is a methyl group which may have one or two substituents, and R 5 is a hydrogen atom or a salt thereof, (I) Formula (1E): [ka] (In the formula, Base, A 1 , R 1 , and R 2 is the same as term 1) The compound represented by the formula: R 4 ·(In the formula, R 4 is the same as item 1), or a radical represented by the formula: R 4 M (wherein M is a metal atom or an atomic group containing a metal atom), R 4 is the same as in item 1), and (II) A step of reacting the compound obtained in the step (I) or the compound obtained by dehydrogenating and then hydrogenating the compound obtained in the step (I) with a carbonyl compound. A method comprising: Section 14. The following formula (6): [ka] [In the formula, Base is an aromatic heterocyclic group which may have a substituent, or an aromatic hydrocarbon ring group which may have a substituent, A 1 is a single bond or an alkylene group, R 3 represents a hydrogen atom, an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted cycloalkyl group, an optionally substituted aryl group, an optionally substituted acyl group, a substituted sulfonyl group, a substituted silyl group, a functional molecular unit substituent, or a group represented by the formula: R 31 -X-(wherein, R 31 is an amino group which may have a substituent, X is an alkylene group which may have a substituent, or at least one methylene group in this alkylene group is -N(R 32 )-(wherein, R 32 is a hydrogen atom or an alkyl group), -O-, or -S(=O) k - (wherein k is 0, 1, or 2), R 4 represents a hydrogen atom, an optionally substituted alkyl group, or an optionally substituted aryl group, R 5 represents a hydrogen atom, an optionally substituted alkyl group, or an optionally substituted aryl group, R 4 and R 5 is not a hydrogen atom at the same time, The following formula: [ka] represents a single or double bond, When the symbol is a single bond, n is 1; When the symbol is a double bond, n is 0. An oligonucleotide or a salt thereof having a unit represented by the following formula: Section 15. A method for detecting a target nucleic acid, comprising: (I) selectively amplifying a target nucleic acid by a nucleic acid amplification method; and (II) detecting the target nucleic acid amplified in step (I); Item 15. A method for detecting a target nucleic acid, comprising: Section 16. A kit for detecting or selectively amplifying a target nucleic acid, comprising: (a) a kit comprising a primer and a probe, wherein at least one of the primer and the probe comprises the oligonucleotide or a salt thereof according to Item 14; or (b) A kit comprising a clamp nucleic acid and a primer, wherein at least one of the clamp nucleic acid and the primer comprises the oligonucleotide or a salt thereof according to Item 14. Section 17. A pharmaceutical composition comprising the compound or a salt thereof according to any one of items 1 to 11, or the oligonucleotide or a salt thereof according to item 14. [Effects of the Invention]

[0008] According to the present invention, it is possible to provide novel artificial nucleic acids that are useful in various genome technologies. [Brief explanation of the drawings]

[0009] [Figure 1A] FIG. 1A is a graph showing the relationship between the reaction time of a digestive enzyme at a final concentration of 5.00 μg / mL and the remaining rate of undigested oligonucleotide. [Figure 1B]FIG. 1B is a graph showing the relationship between the reaction time of the digestive enzyme at a final concentration of 1.60 μg / mL and the remaining rate of undigested oligonucleotide. [Figure 1C] FIG. 1C is a graph showing the relationship between the reaction time of the digestive enzyme at a final concentration of 4.38 μg / mL and the remaining rate of undigested oligonucleotide. [Figure 2A] FIG. 2A is a schematic diagram of an example of the kit of the present invention, which includes a container containing a composition including a primer and a probe. [Figure 2B] FIG. 2B is a schematic diagram of an example of the kit of the present invention, which includes a container containing a composition containing a primer and a container containing a composition containing a probe. [Figure 2C] Figure 2C is a schematic diagram of an example of a kit of the present invention, which includes a container containing a composition comprising a forward primer, a container containing a composition comprising a reverse primer, and a container containing a composition comprising a probe. DETAILED DESCRIPTION OF THE INVENTION

[0010] <<Terminology>> As used herein, the term "alkyl group" refers to a monovalent group obtained by removing one hydrogen atom from a linear or branched saturated hydrocarbon. The number of carbon atoms in the alkyl group is not particularly limited, but is, for example, 1 to 20, preferably 1 to 10, more preferably 1 to 6, and particularly preferably 1 to 4. Examples of alkyl groups include a methyl group, an ethyl group, a propyl group (e.g., n-propyl group, i-propyl group), a butyl group (e.g., n-butyl group, i-butyl group, s-butyl group, t-butyl group), a pentyl group (e.g., n-pentyl group, i-pentyl group, neopentyl group), a hexyl group, a heptyl group, an octyl group (e.g., n-octyl group, 2-ethylhexyl group), a nonyl group, and a decyl group.

[0011] As used herein, the term "alkylene group" refers to a divalent group formed by removing two hydrogen atoms from a linear or branched saturated hydrocarbon. The number of carbon atoms in the alkylene group is not particularly limited, but is, for example, 1 to 10, preferably 1 to 8, and more preferably 1 to 6. Examples of alkylene groups include C1 alkylene groups (e.g., methylene group), C2 alkylene groups (e.g., methylmethylene group, dimethylene group), C3 alkylene groups (e.g., trimethylene group, dimethylmethylene group), C4 alkylene groups (e.g., tetramethylene group), C5 alkylene groups (e.g., pentamethylene group), and C6 alkylene groups (e.g., hexamethylene group).

[0012] As used herein, the term "alkenyl group" refers to a monovalent group obtained by removing one hydrogen atom from a straight-chain or branched-chain unsaturated hydrocarbon containing a carbon-carbon double bond. The number of carbon atoms in the alkenyl group is not particularly limited, but is, for example, 2 to 20, preferably 2 to 10, and more preferably 2 to 6. Examples of alkenyl groups include ethenyl groups (ie, vinyl groups), propenyl groups (eg, 1-propenyl groups, allyl groups), butenyl groups, pentenyl groups, hexenyl groups, geranyl groups, and farnesyl groups.

[0013] As used herein, the term "alkynyl group" refers to a monovalent group obtained by removing one hydrogen atom from a straight-chain or branched-chain unsaturated hydrocarbon containing a carbon-carbon triple bond. The number of carbon atoms in the alkynyl group is not particularly limited, but is, for example, 2 to 20, preferably 2 to 10, and more preferably 2 to 6. Examples of the alkynyl group include an ethynyl group, a propargyl group, and a 1-butynyl group.

[0014] As used herein, the term "cycloalkyl group" refers to a monovalent group derived from a saturated aliphatic hydrocarbon ring. The number of carbon atoms in the cycloalkyl group is not particularly limited, but is, for example, 3 to 20, preferably 5 to 12, and more preferably 5 to 10. Examples of cycloalkyl groups include a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, a cycloheptyl group, a cyclooctyl group, a norbornyl group, and an adamantyl group.

[0015] As used herein, the term "cycloalkenyl group" refers to a monovalent group derived from an unsaturated aliphatic hydrocarbon ring containing a carbon-carbon double bond. The number of carbon atoms in the cycloalkenyl group is not particularly limited, but is, for example, 3 to 20, preferably 5 to 12, and more preferably 5 to 10. Examples of cycloalkenyl groups include a cyclopropenyl group, a cyclobutenyl group, a cyclopentenyl group, a cyclohexenyl group, a norbornenyl group, and an adamantenyl group.

[0016] In this specification, the term "aromatic hydrocarbon ring group" refers to a monovalent group derived from an aromatic hydrocarbon ring, and is also referred to as an "aryl group." The number of constituent atoms of the aromatic hydrocarbon ring is not particularly limited, but is, for example, 6 to 20, preferably 6 to 14, more preferably 6 to 12, and particularly preferably 6 to 10. The aromatic hydrocarbon ring may be a monocyclic ring or a fused ring (for example, a bicyclic or tricyclic fused ring). Examples of the aromatic hydrocarbon ring group include a phenyl group, an indenyl group, a naphthyl group, a fluorenyl group, a phenanthrenyl group, and an anthracenyl group.

[0017] In this specification, the term "heterocycle" is used to encompass "aliphatic heterocycle" and "aromatic heterocycle".

[0018] As used herein, the term "aliphatic heterocycle" refers to an aliphatic ring containing, as ring-constituting atoms, a carbon atom and at least one heteroatom selected from the group consisting of a nitrogen atom, an oxygen atom, a sulfur atom, a silicon atom, and the like. The number of constituent atoms of the aliphatic heterocycle is not particularly limited, but is, for example, 5 to 20, preferably 5 to 12, and more preferably 6 to 10. The number of heteroatoms among the constituent atoms of the aliphatic heterocycle is not particularly limited, but is, for example, 1 to 4. Examples of the aliphatic heterocycle include oxygen-containing aliphatic heterocycles (e.g., tetrahydrofuran, dioxolane, pyran, tetrahydropyran, dioxane), sulfur-containing aliphatic heterocycles (e.g., tetrahydrothiophene, thiopyran, tetrahydrothiopyran), nitrogen-containing aliphatic heterocycles (e.g., pyrrolidine, piperidine, azepane), nitrogen- and oxygen-containing aliphatic heterocycles (e.g., morpholine), nitrogen- and sulfur-containing aliphatic heterocycles (e.g., thiomorpholine), and aliphatic heterocycles containing a siloxane bond.

[0019] As used herein, the term "aromatic heterocycle" refers to an aromatic ring containing, as ring-constituting atoms, a carbon atom and at least one heteroatom selected from the group consisting of a nitrogen atom, an oxygen atom, a sulfur atom, and the like. The number of constituent atoms of the aromatic heterocycle is not particularly limited, but is, for example, 5 to 20, preferably 5 to 12, and more preferably 6 to 10. The number of heteroatoms among the constituent atoms of the aromatic heterocycle is not particularly limited, but is, for example, 1 to 4. The aromatic heterocycle may be a monocycle or a condensed ring (for example, a bicyclic to tricyclic condensed ring). Examples of aromatic heterocycles include oxygen-containing aromatic heterocycles (e.g., furan, benzofuran, isobenzofuran, chromene, benzopyran, xanthene), sulfur-containing aromatic heterocycles (e.g., thiophene, thianthrene), nitrogen-containing aromatic heterocycles (e.g., pyrrole, imidazole, pyrazole, triazole, pyridine, pyrazine, pyrimidine, pyridazine, indole, isoindole, indolizine, purine, quinoline, isoquinoline, 1,8-naphthyridine, quinoxaline, quinazoline, cinnoline, phthalazine, pteridine, carbazole, phenanthridine, acridine, perimidine, phenazine), oxygen- and sulfur-containing aromatic heterocycles (e.g., phenoxathiin), nitrogen- and oxygen-containing aromatic heterocycles (e.g., oxazole, isoxazole, furazan, phenoxazine), and nitrogen- and sulfur-containing aromatic heterocycles (e.g., thiazole, isothiazole, phenothiazine).

[0020] In this specification, the term "heterocyclic group" refers to a monovalent group obtained by removing one hydrogen atom from the above-mentioned heterocycle.

[0021] In this specification, the terms "optionally have a substituent" or "optionally substituted with a substituent" are used to mean both a case where no substituent is present and a case where any hydrogen atom is replaced with one substituent or two or more of the same or different substituents. When a substituent is present, the number of substituents is not particularly limited, but is, for example, 1 to 3, and preferably 1 or 2.

[0022] As used herein, the term "substituent" refers to an atom or atomic group replacing a hydrogen atom. Examples of the substituent include a halogen atom (e.g., a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom), an oxo group (=O), a thioxo group (=S), a hydroxyl group, a mercapto group, an amino group, a carboxy group, an alkyl group, an alkenyl group, a cycloalkyl group, a cycloalkenyl group, an aryl group, an alkynyl group, an acyl group, an alkylsulfonyl group, an arylsulfonyl group, a cyano group, a heterocyclic group, or a combination of two or more of these groups (e.g., a haloalkyl group, a cyanoalkyl group, an aralkyl group, an alkoxy group, or an alkylamino group). The term "combination of two or more types" includes any combination of the groups exemplified as the respective substituents.

[0023] In this specification, "Cx-y" means that the number of carbon atoms in the subsequent group is from x to y, where x and y are positive integers, and x <yである。

[0024] As used herein, the term "haloalkyl group" refers to an alkyl group substituted with one or more of the same or different halogen atoms. Suitable examples of haloalkyl groups include C 1-6 A more preferred example is a haloalkyl group. 1-4 A more preferred example is a haloalkyl group, such as a trifluoromethyl group, a trichloromethyl group, or a 2,2,2-trifluoroethyl group.

[0025] As used herein, the term "cyanoalkyl group" refers to an alkyl group substituted with one or more cyano groups. Suitable examples of cyanoalkyl groups include C 1-6 A more preferred example is a cyanoalkyl group, 1-4 A cyanoalkyl group is preferred, and more preferred examples include a cyanomethyl group or a 2-cyanoethyl group.

[0026] As used herein, the term "aralkyl group" refers to an alkyl group substituted with one or more of the same or different aryl groups. Suitable examples of aralkyl groups include C 6-14 Aryl C 1-4 More preferred examples of the alkyl group include a phenylmethyl group (i.e., a benzyl group), a phenylethyl group (i.e., a phenethyl group), a naphthylmethyl group, a naphthylethyl group, a triphenylmethyl group (i.e., a trityl group), and a fluorenylmethyl group.

[0027] As used herein, the term "alkoxy group" refers to a group represented by the formula: -O-alkyl. Suitable examples of the alkoxy group include C 1-6 It is an alkoxy group, and more preferred examples thereof include a methoxy group, an ethoxy group, a propoxy group (for example, an n-propoxy group, an i-propoxy group), and a butoxy group (for example, a t-butoxy group). The haloalkoxy group and the cyanoalkoxy group refer to a group represented by the formula: -O-haloalkyl and a group represented by the formula: -O-cyanoalkyl, respectively.

[0028] As used herein, the term "alkylthio group" refers to a group represented by the formula: -S-alkyl. Suitable examples of alkylthio groups include C 1-6 It is an alkylthio group, and more preferred examples thereof include a methylthio group, an ethylthio group, a propylthio group (e.g., an n-propylthio group, an i-propylthio group), or a butylthio group. The haloalkylthio group and the cyanoalkylthio group refer to a group represented by the formula: -S-haloalkyl and a group represented by the formula: -S-cyanoalkyl, respectively.

[0029] As used herein, the term "alkylamino group" refers to an amino group substituted with one or two alkyl groups of the same or different types. The alkylamino group includes a monoalkylamino group and a dialkylamino group. A suitable example of a monoalkylamino group is monoC 1-6 It is an alkylamino group, and more preferred examples thereof include a monomethylamino group, a monoethylamino group, a monopropylamino group (e.g., a mono(n-propyl)amino group, a mono(i-propyl)amino group), or a monobutylamino group. A suitable example of a dialkylamino group is diC 1-6 It is an alkylamino group, and more preferred examples thereof include a dimethylamino group, a diethylamino group, a dipropylamino group (e.g., a di(n-propyl)amino group, a di(i-propyl)amino group), or a dibutylamino group.

[0030] As used herein, the term "substituted phosphino group" refers to a phosphino group (-PH2) in which at least one hydrogen atom has been substituted with another atom or atomic group. Examples of substituted phosphino groups include those of the formula: -P(R 7 )(R 8 )(wherein, R 7 and R 8 are the same or different and are a hydroxyl group, a mercapto group, an amino group, an alkoxy group, a haloalkoxy group, a cyanoalkoxy group, an alkylthio group, a haloalkylthio group, a cyanoalkylthio group, or an alkylamino group).

[0031] As used herein, the term "optionally substituted dihydroxyphosphinyl group" refers to a dihydroxyphosphinyl group (i.e., a phosphono group) (-P(=O)(OH)2) or a dihydroxyphosphinyl group in which at least one hydrogen atom has been substituted with another atom or atomic group (e.g., a protecting group for a hydroxyl group). The latter group may have a substituent and is represented by the following formula: [ka] and a group represented by the following formula: [ka] (hereinafter referred to as "triphosphate group").

[0032] As used herein, the term "optionally substituted hydroxymercaptophosphinyl group" refers to a hydroxymercaptophosphinyl group (-P(=O)(OH)(SH)) or a hydroxymercaptophosphinyl group in which at least one hydrogen atom has been substituted with another atom or atomic group (e.g., a protecting group for a hydroxyl group).

[0033] As used herein, the term "hydroxyl-protecting group" refers to a monovalent group that prevents a hydroxyl group from participating in a reaction in the synthesis of a compound or a salt thereof, or in the synthesis of an oligonucleotide or a salt thereof. Examples of the hydroxyl protecting group include, but are not limited to, groups that are stable under acidic or neutral conditions and can be cleaved by methods such as hydrogenolysis, hydrolysis, electrolysis, and photolysis. Examples of the protecting group for a hydroxyl group include an acyl group which may have a substituent, a sulfonyl group which has a substituent, and a silyl group which has a substituent.

[0034] As used herein, the term "acyl group" refers to a group represented by the formula -C(=O)-R, where R is a hydrocarbon group. The hydrocarbon group represented by R may be a linear or branched hydrocarbon group (e.g., an alkyl group), a saturated or unsaturated hydrocarbon ring group (e.g., a cycloalkyl group, an aryl group), or a combination thereof (e.g., an aralkyl group). The acyl group includes an alkylcarbonyl group, an arylcarbonyl group, and an aralkylcarbonyl group. Suitable examples of alkylcarbonyl groups include (C 1-10 and more preferred examples thereof include an acetyl group, a propionyl group, a butyryl group, an isobutyryl group, a pentanoyl group, a pivaloyl group, a valeryl group, an isovaleryl group, an octanoyl group, a nonanoyl group, and a decanoyl group. Suitable examples of the arylcarbonyl group include (C 6-14 A more preferred example is a benzoyl group or a naphthoyl group (that is, an α-naphthoyl group, a β-naphthoyl group). Suitable examples of the aralkylcarbonyl group include (C 6-14 Aryl C 1-4 A more preferred example is a benzylcarbonyl group. The acyl group in the "acyloxy group," "acylthio group," and "acylamino group" can be exemplified by the same groups as those mentioned above.

[0035] As used herein, the term "substituted sulfonyl group" refers to a group represented by the formula: -S(=O)2R (wherein R is as defined above). The sulfonyl group having a substituent includes a sulfonyl group having an alkyl group which may have a substituent, and a sulfonyl group having an aryl group which may have a substituent. Preferred examples of the sulfonyl group having an alkyl group include C 1-6 The alkylsulfonyl group is more preferably a methanesulfonyl group or an ethanesulfonyl group. Suitable examples of sulfonyl groups having an aryl group include C 6-14An arylsulfonyl group is more preferred, and a more preferred example is a benzenesulfonyl group or a p-toluenesulfonyl group.

[0036] In this specification, the term "substituted silyl group" refers to a silyl group (-SiH3) in which at least one hydrogen atom has been substituted with another atom or atomic group. A typical example of a "substituted silyl group" is a silyl group of the formula: -Si(R 6 )3(in the formula, each R 6 are the same or different and are an alkyl group or an aryl group). Examples of such groups include trialkylsilyl groups (e.g., trimethylsilyl group, triethylsilyl group, triisopropylsilyl group, t-butyldimethylsilyl group, etc.) 1-6 alkylsilyl group), dialkylarylsilyl group (e.g., di-C such as dimethylphenylsilyl group) 1-6 Alkyl C 6-14 arylsilyl group), alkyldiarylsilyl group (e.g., t-butyldiphenylsilyl group, etc.) 1-6 Alkyldi-C 6-14 arylsilyl group), triarylsilyl group (e.g., tri-C such as triphenylsilyl group) 6-14 arylsilyl groups) .

[0037] As used herein, the term "amino-protecting group" refers to a monovalent group that prevents an amino group from participating in a reaction in the synthesis of a compound or a salt thereof, or in the synthesis of an oligonucleotide or a salt thereof. Examples of the amino-protecting group include, but are not limited to, groups that are stable under acidic or neutral conditions and can be cleaved by methods such as hydrogenolysis, hydrolysis, electrolysis, and photolysis. Examples of the protecting group for an amino group include an optionally substituted acyl group (e.g., an optionally substituted alkylcarbonyl group, an optionally substituted arylcarbonyl group, an optionally substituted aralkylcarbonyl group), an optionally substituted N,N-dialkylformamidyl group, an optionally substituted alkoxycarbonyl group, an optionally substituted alkenyloxycarbonyl group, an optionally substituted aryloxycarbonyl group, an optionally substituted aralkyloxycarbonyl group, and a substituted sulfonyl group.

[0038] As used herein, the term "formamidyl group" refers to a group represented by the formula: =CH-NH2. A suitable example of an N,N-dialkylformamidyl group is an N,N-di(C 1-6 A more preferred example is an N,N-di(C 1-4 A more preferred example is an N,N-dimethylformamidyl group or an N,N-diethylformamidyl group.

[0039] As used herein, the term "alkoxycarbonyl group" refers to a group represented by the formula: -C(=O)-O-alkyl. Suitable examples of the alkoxycarbonyl group include (C 1-6 and more preferred examples thereof include a methoxycarbonyl group, an ethoxycarbonyl group, a propoxycarbonyl group, and a butoxycarbonyl group (e.g., a t-butoxycarbonyl group).

[0040] As used herein, the term "alkenyloxycarbonyl group" refers to a group represented by the formula: -C(=O)-O-alkenyl. Suitable examples of the alkenyloxycarbonyl group include (C 2-9 A more preferred example is an allyloxycarbonyl group.

[0041] As used herein, the term "aryloxycarbonyl group" refers to a group represented by the formula: -C(=O)-O-aryl. Suitable examples of the aryloxycarbonyl group include (C 6-14 A more preferred example is a phenoxycarbonyl group or a naphthoxycarbonyl group.

[0042] As used herein, the term "aralkyloxycarbonyl group" refers to a group represented by the formula: -C(=O)-O-aralkyl. Suitable examples of the aralkyloxycarbonyl group include (C 6-14 Aryl C 1-4 A more preferred example is a benzyloxycarbonyl group or a fluorenylmethyloxycarbonyl group.

[0043] In this specification, the term "functional molecular unit substituent" is a concept that encompasses labeling functional groups (e.g., fluorescent labeling functional groups, chemiluminescent labeling functional groups, and radioactive nuclide-containing groups), groups with intercalating ability, groups with nucleic acid binding ability, nucleic acid cleavage active functional groups, groups with intracellular or nuclear transport ability, and groups with metal chelating ability. Examples of fluorescent labeling functional groups include residues of fluorescent labeling reagents such as carboxyfluorescein (FAM), fluorescein isothiocyanate (FITC), carboxytetramethylrhodamine (TAMRA), and thiazole orange (1-methyl-4-[(3-methyl-2(3H)-benzothiazolylidene)methyl]quinolinium p-tosylate). Examples of chemiluminescent labeling functional groups include residues of chemiluminescent labeling reagents such as tris(bipyridine)ruthenium(II) chloride. Examples of radionuclide-containing groups include: 11 CH3-, 14 CH3-, 18 F-, or 32 and groups containing P-. Examples of the group having intercalating ability include a group having an anthracene skeleton, a group having a pyrene skeleton, an anthraquinone skeleton, a group having an acridine skeleton, a group having a naphthalimide skeleton, etc. Further, other examples include residues of intercalating agents such as tamoxifen. Examples of groups capable of binding to nucleic acids include residues of nucleic acid binders such as netropsin, distamine, and PI (pyrrole imidazole) polyamide. Examples of nucleic acid cleaving active functional groups include residues of nucleic acid cleaving agents such as endonucleases and bis(bipyridine)chrysenequinonediiminerhodium(II) complexes. Examples of groups capable of intracellular or nuclear transport include residues of intracellular or nuclear transport signal peptides such as TAT (Twin-Arginine Translocation) signal peptide, polyarginine, GalNac (N-acetylgalactosamine), and signal peptides derived from SV40 T antigen. Examples of groups capable of chelating metals include residues of metal chelating agents such as EDTA, crown ethers, and cryptands.

[0044] As used herein, "hybridizing" refers to the formation of a duplex with all or a portion of a given polynucleotide or oligonucleotide through hydrogen bonding under stringent conditions. "Stringent conditions" may refer to conditions commonly used by those skilled in the art when hybridizing polynucleotides or oligonucleotides. For example, conditions under which one polynucleotide or oligonucleotide molecule can specifically hybridize to another polynucleotide or oligonucleotide molecule are present when there is at least 50%, preferably at least 75%, and more preferably at least 90% sequence identity between the two polynucleotides or oligonucleotide molecules. Hybridization stringency is known to be a function of temperature, salt concentration, base length and GC content of the polynucleotide or oligonucleotide, and the concentration of chaotropic agents contained in the hybridization buffer. Stringent conditions can be, for example, those described in Sambrook, J. et al., 1998, Molecular Cloning: A Laboratory Manual (Vol. 2), Cold Spring Harbor Laboratory Press, New York.

[0045] As used herein, "testing" refers to examining a test substance such as nucleic acid in a sample for the purpose of diagnosis, research, etc. "Test sample" refers to a sample that is subjected to testing.

[0046] In this specification, the upper and lower limits of each numerical range can be combined in any manner.

[0047] <<Compound or its salt>> The compound of the present invention or a salt thereof is a compound represented by the following formula (1) or a salt thereof: [ka] (In the formula, Base, A 1, R 1 ~R 5 and n are the same as above. The carbon atoms of the furanose are numbered.

[0048] Hereinafter, the compound represented by formula (N) or a salt thereof will be referred to as "compound (N)". Compound (1) is R 1 or R 2 When is an optionally substituted dihydroxyphosphinyl group or an optionally substituted hydroxymercaptophosphinyl group, it is called a "nucleotide," and when it is any other group, it is called a "nucleoside."

[0049] A suitable example of Base is an aromatic heterocyclic group which may have a substituent. The aromatic heterocyclic group is preferably a nitrogen-containing aromatic heterocyclic group. The nitrogen-containing aromatic heterocyclic group is preferably a 6- to 10-membered nitrogen-containing aromatic heterocyclic group, which is preferably a 2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl group, a 2-oxo-1,2-dihydropyrimidin-1-yl group, a purin-9-yl group, or a 6-oxo-1,6-dihydro-9H-purin-9-yl group. Suitable examples of the substituents on the aromatic heterocyclic group include at least one selected from the group consisting of alkyl groups, acyl groups, and amino groups optionally substituted with a protecting group for the amino group. The number of the substituents is not particularly limited, but is, for example, 1 to 3.

[0050] More preferred examples of Base include an optionally substituted thyminyl group, an optionally substituted cytosinyl group, an optionally substituted adenyl group, or an optionally substituted guanyl group. The substituent is preferably at least one selected from the group consisting of an alkyl group, an acyl group, and an N,N-dialkylformamidyl group, and more preferably C 1-4 Alkyl groups, (C 1-4 alkyl)carbonyl group, (C 6-14aryl)carbonyl group, or N,N-di(C 1-4 The number of the substituents is preferably 1 to 3.

[0051] Further preferred examples of Base include the following: 2,4-dioxo-5-methyl-1,2,3,4-tetrahydropyrimidin-1-yl group (e.g., thymin-1-yl group), 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl group (i.e., cytosin-1-yl group), 2-oxo-4-acylamino-1,2-dihydropyrimidin-1-yl group (i.e., N-acyl-cytosin-1-yl group), 2-oxo-4-amino-5-methyl-1,2-dihydropyrimidin-1-yl group (i.e., 5-methylcytosin-1-yl group), 2-oxo-4-acylamino-5-methyl-1,2-dihydropyrimidin-1-yl group (i.e., N-acyl-5-methylcytosin-1-yl group), 6-amino-9H-purin-9-yl group (i.e., adenin-9-yl group), 6-acylamino-9H-purin-9-yl group (i.e., N-acyl-adenin-9-yl group), 2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl group (e.g., guanin-9-yl group) 2-acylamino-6-oxo-1,6-dihydro-9H-purin-9-yl group (e.g., N-acyl-guanin-9-yl group), and 2-(N,N-dialkylformamidyl)amino-6-oxo-1,6-dihydro-9H-purin-9-yl group (e.g., N-(N,N-dialkylformamidyl)-guanin-9-yl group) is a group selected from the group consisting of:

[0052] The most preferred examples of Base are: thymin-1-yl group, 5-methylcytosin-1-yl group, N-acetyl-5-methylcytosin-1-yl group, N-isobutyryl-5-methylcytosin-1-yl group, N-benzoyl-5-methylcytosin-1-yl group, adenin-9-yl group, N-acetyl-adenine-9-yl group, N-isobutyryl-adenine-9-yl group, N-benzoyl-adenine-9-yl group, guanine-9-yl group, N-acetyl-guanine-9-yl group, N-isobutyryl-guanine-9-yl group, N-benzoyl-guanine-9-yl group, and N-(N,N-dimethylformamidyl)-guanine-9-yl group is a group selected from the group consisting of:

[0053] A 1 is preferably a single bond or C 1-2 It is preferably an alkylene group (eg, methylene group, dimethylene group), and more preferably a single bond.

[0054] R 1 Suitable examples of the following are: hydrogen atoms, an alkyl group which may have a substituent, an optionally substituted aryl group; hydroxyl protecting groups, a substituted phosphino group; an optionally substituted dihydroxyphosphinyl group, and Optionally substituted hydroxymercaptophosphinyl group is a group selected from the group consisting of:

[0055] R 1The "optionally substituted alkyl group" represented by the formula (I) is preferably an alkyl group which may have at least one substituent selected from the group consisting of a halogen atom, an alkoxy group, and an aryl group, and more preferably an alkyl group which may be substituted with an alkoxy group, or an aralkyl group which may be substituted with an alkoxy group. The number of the substituents is preferably 1 to 3.

[0056] R 1 The "aryl group which may have a substituent" represented by the formula (I) is preferably an aryl group which may have at least one substituent selected from the group consisting of a halogen atom, an alkyl group, and an alkoxy group, and more preferably an aryl group which may have an alkoxy group. The number of the substituents is preferably 1 to 3.

[0057] R 1 The "protective group for a hydroxyl group" represented by the formula: is preferably an alkylcarbonyl group, an arylcarbonyl group, an alkylsulfonyl group, an arylsulfonyl group, or a group represented by the formula: -Si(R 6 )3(in the formula, each R 6 are the same or different and are alkyl groups or aryl groups.

[0058] R 1 The "substituted phosphino group" represented by the formula: -P(R 7 )(R 8 )(wherein, R 7 and R 8 are the same or different and are a hydroxyl group, a mercapto group, an amino group, an alkoxy group, a haloalkoxy group, a cyanoalkoxy group, an alkylthio group, a haloalkylthio group, a cyanoalkylthio group, or an alkylamino group). A more preferred example of the group is a group represented by the following formula: [ka] (In the formula, R 7a and R 7b are the same or different and are a hydrogen atom or an alkyl group, and R 8ais a hydrogen atom, an alkyl group, a haloalkyl group, or a cyanoalkyl group. It is a phosphino group represented by the formula:

[0059] R 1 The "dihydroxyphosphinyl group which may have a substituent" represented by the formula (I) is preferably a dihydroxyphosphinyl group, a diphosphate group, or a triphosphate group, more preferably a dihydroxyphosphinyl group. These may have a hydroxyl-protecting group as a substituent, and all or part of the hydroxyl groups present may be substituted with a hydroxyl-protecting group.

[0060] R 1 The "hydroxymercaptophosphinyl group which may be substituted" represented by the following formula: is preferably a hydroxymercaptophosphinyl group.

[0061] R 1 Most preferred examples of the alkyl group include a hydrogen atom, a methyl group, an ethyl group, a propyl group, a butyl group, an allyl group, a benzyl group, a trityl group, a methoxymethyl group, a p-methoxybenzyl group, a monomethoxytrityl group, a dimethoxytrityl group, an acetyl group, an isobutyryl group, a benzoyl group, a methanesulfonyl group, a p-toluenesulfonyl group, a trimethylsilyl group, a triethylsilyl group, a triisopropylsilyl group, a t-butyldimethylsilyl group, a t-butyldiphenylsilyl group, and groups of the following formula: [ka] a phosphino group, a dihydroxyphosphinyl group, or a hydroxymercaptophosphinyl group represented by any one of the following formulae:

[0062] R 2 A suitable example of 1 This is the same as the preferred example of

[0063] R 1 and R 2 A suitable example of the combination of R 1is a hydrogen atom or a dimethoxytrityl group (e.g., 4,4'-dimethoxytrityl group), and R 2 But the following formula: [ka] (In the formula, R 7a and R 7b are the same or different and are a hydrogen atom or an alkyl group, and R 8a is a hydrogen atom, an alkyl group, a haloalkyl group, or a cyanoalkyl group. The combination is a phosphino group represented by the formula:

[0064] R 1 and R 2 When the ring is formed together with the two adjacent oxygen atoms and the carbon atoms at positions 3 to 5 of the furanose, a preferred example of the ring is a 6- to 10-membered aliphatic heterocycle which may have a substituent, and a preferred example of the substituent is an alkyl group.

[0065] More preferred examples of the ring include the following formula: [ka] (In the formula, R 9 and R 10 are the same or different and are a hydrogen atom or an alkyl group, and R 11 ~R 14 are the same or different and are alkyl groups. It is an aliphatic heterocycle represented by any one of the following:

[0066] Further preferred examples of the ring include the following formula: [ka] It is a ring represented by either

[0067] In one embodiment, R 3 represents a hydrogen atom, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, an alkylsulfonyl group, an arylsulfonyl group, a group of the formula: -Si(R 6)3(in the formula, each R 6 are the same or different and are an alkyl group or an aryl group), a labeling functional group, a group having intercalating ability, a group having nucleic acid binding ability, a nucleic acid cleaving active functional group, a group having intracellular or nuclear transport ability, or a group having metal chelating ability.

[0068] In another embodiment, R 3 is the formula: R 31 A group represented by -X- is preferred.

[0069] R 31 A suitable example is the following formula (A): [ka] (In the formula, R 3a and R 3b are the same or different and are a hydrogen atom, an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted cycloalkyl group, an optionally substituted cycloalkenyl group, an optionally substituted aryl group, or a protecting group for an amino group, or R 3a and R 3b forms, together with the adjacent nitrogen atom, a ring which may have a substituent. It is a group represented by the following formula:

[0070] R 3a is preferably a protecting group for a hydrogen atom, an alkyl group, an aryl group, an aralkyl group, or an amino group, and more preferably a protecting group for a hydrogen atom, an alkyl group, or an amino group.

[0071] R 3a The "amino protecting group" represented by the formula (C 1-4 alkyl)carbonyl group or (C 1-4 haloalkyl)carbonyl group, and more preferably an acetyl group or a trifluoromethylcarbonyl group.

[0072] R 3b A suitable example of3a This is the same as the preferred example of

[0073] R 3a and R 3b A suitable example of the combination of R 3a and R 3b are both hydrogen atoms, R 3a is a hydrogen atom, and R 3b is an acetyl group or a trifluoromethylcarbonyl group, 3a and R 3b are both methyl groups, or R 3a and R 3b and each represent an acetyl group or a trifluoromethylcarbonyl group.

[0074] R 3a and R 3b When the nitrogen atom forms a ring together with the adjacent nitrogen atom, a preferred example of the ring is a 5- to 10-membered nitrogen-containing aliphatic heterocycle which may have a substituent, and a preferred example of the substituent is an alkyl group or an acyl group.

[0075] More preferred examples of the ring include the following formula: [ka] [Wherein Z represents a single bond, an oxygen atom, or S(=O) p (wherein p is 0, 1, or 2), C(R 15 )(R 16 )(wherein, R 15 and R 16 are the same or different and are a hydrogen atom or an alkyl group), or NR 17 (In the formula, R 17 is a hydrogen atom, an alkyl group, or an acyl group). It is a nitrogen-containing aliphatic heterocycle represented by the formula:

[0076] Further preferred examples of the ring include the following formula: [ka] [In the formula, R 17ais a hydrogen atom, a straight-chain or branched C 1-4 Alkyl groups (e.g., methyl, ethyl, propyl, and butyl groups) or (linear or branched C 1-4 alkyl)carbonyl group (e.g., methylcarbonyl group, ethylcarbonyl group, propylcarbonyl group, butylcarbonyl group) It is a nitrogen-containing aliphatic heterocycle represented by the formula:

[0077] The most preferred example of the ring is a 4-methylpiperazin-1-yl group.

[0078] R 31 Another preferred example is the following formula (B): [ka] (In the formula, R 3c ~R 3f are the same or different and are a protecting group for a hydrogen atom, an alkyl group, or an amino group. It is a group represented by the following formula:

[0079] R 3c ~R 3f Suitable examples of are a protecting group for a hydrogen atom or an amino group. R 3c is preferably a hydrogen atom. R 3d is preferably a protecting group for an amino group, more preferably an alkoxycarbonyl group, a haloalkoxycarbonyl group, or a cyanoalkoxycarbonyl group, and particularly preferably (C 1-4 alkoxy)carbonyl group, (C 1-4 haloalkoxy)carbonyl group, or (C 1-4 cyanoalkoxy)carbonyl group. R 3e is preferably a protecting group for an amino group, more preferably an alkoxycarbonyl group, a haloalkoxycarbonyl group, or a cyanoalkoxycarbonyl group, and particularly preferably (C 1-4 alkoxy)carbonyl group, (C 1-4 haloalkoxy)carbonyl group, or (C 1-4cyanoalkoxy)carbonyl group. R 3f is preferably a hydrogen atom.

[0080] R 31 The most preferred examples are from the following group: [ka] is a group selected from

[0081] Suitable examples of X include an alkylene group, a methylene group bonded to a nitrogen atom in the alkylene group, and R 31 At least one of the methylene groups other than the methylene group bonded to -N(R 32 )-(wherein, R 32 is a hydrogen atom or an alkyl group), -O-, or -S(=O) k - (wherein k is 0, 1, or 2). Alternatively, a suitable example of X is an alkylene group, or an alkylene group having -N(R 32 )-(wherein, R 32 is the same as above), -O-, or -S(=O) k - (wherein k is 0, 1, or 2).

[0082] Further preferred examples of X include the following: Formula:-C m H 2m - (wherein m is an integer of 1 to 10), Formula:-(CH2) m1 -(N(R 321 )-(CH2) m2 ) m3 -(In the formula, R 321 is a hydrogen atom or C 1-4 m1 is an integer of 2 to 10, m2 is an integer of 2 to 4, and m3 is an integer of 1 to 5; when m3 is an integer of 2 or more, each R 321 may be the same or different from each other, and each m2 may be the same or different from each other), Formula:-(CH2)m4 -(O-(CH2) m5 ) m6 - (wherein m4 is an integer of 2 to 10, m5 is an integer of 2 to 4, and m6 is an integer of 1 to 5, and when m6 is an integer of 2 or greater, each m5 may be the same or different from each other), and Formula:-(CH2) m7 -(S-(CH2) m8 ) m9 - (wherein m7 is an integer of 2 to 10, m8 is an integer of 2 to 4, and m9 is an integer of 1 to 5, and when m9 is an integer of 2 or greater, each m8 may be the same or different from each other) is a group selected from the group consisting of:

[0083] The most preferred examples of X are: Formula:-(CH2) m10 - (wherein m10 is an integer of 1 to 6), Formula: -C2H4-(N(R 322 )-C2H4) m11 -(In the formula, R 322 is a hydrogen atom or C 1-4 alkyl group, m11 is an integer of 1 to 5, and when m11 is an integer of 2 or more, each R 322 may be the same or different), Formula:-C2H4-(O-C2H4) m12 - (wherein m12 is an integer of 1 to 5), and Formula: -C2H4-(S-C2H4) m13 - (wherein m13 is an integer of 1 to 5) is a group selected from the group consisting of:

[0084] The substituents that can be optionally substituted on the carbon atoms of the alkylene group are preferably halogen atoms, hydroxyl groups, alkoxy groups, mercapto groups, alkylthio groups, amino groups, monoalkylamino groups, dialkylamino groups, acyloxy groups, acylamino groups, and acylthio groups. The number of substituents varies depending on the number of carbon atoms of the alkylene group, but is, for example, 1 to 3, and preferably 2 or 3.

[0085] In one embodiment, R 3 However, the formula: R 31 -X-, and R 31 is a group represented by formula (A) or formula (B), and X is -C m H 2m - (wherein m is an integer of 1 to 10) is preferred.

[0086] R 4 Suitable examples of are a hydrogen atom or an alkyl group which may have a substituent. The substituent is preferably at least one selected from the group consisting of a halogen atom, an oxo group, a hydroxyl group, an alkoxy group, an alkylthio group, an alkylamino group, and a cyano group. The number of the substituents is preferably 1 to 3. R 4 A more preferred example is an alkyl group. R 4 A more preferred example of 1-6 It is an alkyl group. R 4 The most suitable example of 1-4 It is an alkyl group.

[0087] R 5 A suitable example of is a hydrogen atom or an alkyl group which may have a substituent, and the substituent is preferably at least one selected from the group consisting of a halogen atom, an oxo group, a hydroxyl group, an alkoxy group, an alkylthio group, an alkylamino group, and a cyano group. The number of the substituents is preferably 1 to 3. R 5 More preferred examples of are a hydrogen atom or an alkyl group. R 5More preferred examples of 1-6 It is an alkyl group. R 5 The most preferred examples of 1-4 It is an alkyl group.

[0088] The compound (1) is preferably the following compound (1A) or (1B): [ka] (Wherein, Base and R 1 ~R 5 is the same as above).

[0089] Compound (1) is more preferably compound (1A). In one embodiment, compound (1A) is preferably compound (1C) or (1D) shown below: [ka] (In the formula, R 33 ~R 35 are the same or different and are a protecting group for a hydrogen atom, an alkyl group, or an amino group; Base, R 1 , R 2 , R 4 , R 5 , and m are the same as above).

[0090] In another embodiment, compound (1A) is preferably compound (1A') or (1A'') below:

[0091] [ka] (Wherein, Base and R 1 ~R 4 is the same as above).

[0092] The salt may be a pharmaceutically acceptable salt or may not be a pharmaceutically acceptable salt, and may be an inorganic salt or an organic salt. Examples of such salts include alkali metal salts (e.g., sodium salts, potassium salts, lithium salts), alkaline earth metal salts (e.g., calcium salts, magnesium salts), other metal salts (e.g., aluminum salts, iron salts, zinc salts, copper salts, nickel salts, cobalt salts), ammonium salts, tetramethylammonium salts, amine salts (e.g., t-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucamine salts, guanidine salts, diethylamine salts, triethylamine salts, dicyclohexylamine salts, N,N'-dibenzylethylenediamine salts, chloroprocaine salts, procaine salts, salts of amino acids such as methyl acrylate, methyl meth ...

[0093] <<Method for producing compound (1)>> Compound (1) can be obtained, for example, by the following reaction scheme 1, but is not limited to this reaction scheme and can be synthesized by combining known reactions: [ka] (wherein L is a leaving group, Base, A 1 , and R 1 ~R 4 is the same as above).

[0094] [Reaction Scheme 1] <Process (I)> Step (I) is a step in which compound (1E) is obtained by dehydrogenating compound (2A) (oxidizing -CH2-NH- to -CH=N-).

[0095] Compound (2A) can be obtained by known methods, for example, the methods described in U.S. Patent Application Publication No. 2007 / 167387 (U.S. Patent Application Publication No. 2007 / 167387 is incorporated herein by reference).

[0096] Examples of reagents (oxidizing agents) for dehydrogenating compound (2A) include hypervalent iodine compounds, such as (diacetoxyiodo)benzene, [bis(trifluoroacetoxy)iodo]benzene, 2-iodoxybenzenesulfonic acid, 2-iodoxybenzoic acid, and Dess-Martin reagent. The amount of the reagent used is usually 0.5 to 6 mol, preferably 1 to 2 mol, per 1 mol of compound (2A).

[0097] The dehydrogenation is preferably carried out in the presence of a solvent. Examples of the solvent include sulfoxide solvents (eg, dimethyl sulfoxide), halogenated solvents (eg, dichloromethane), and mixed solvents of two or more of these.

[0098] The temperature for the dehydrogenation is not particularly limited as long as the reaction proceeds, but is, for example, 0 to 100°C, and preferably 20 to 60°C. The dehydrogenation time is not particularly limited, but is, for example, 1 to 12 hours, and preferably 4 to 6 hours.

[0099] <Process (II)> Step (II) is reacting compound (1E) with a compound of formula (3A): R 4 ·(In the formula, R 4 is the same as above), or a radical represented by formula (3B): R 4 M (wherein M is a metal atom or an atomic group containing a metal atom), R 4 is the same as above) to obtain compound (1F).

[0100] Examples of compounds that generate radicals (3A) include compounds that generate ethyl radicals in the presence of oxygen (e.g., triethylborane). 4 When R is an ethyl group, triethylborane can be used alone, and R 4 is other than an ethyl group, triethylborane and a compound of formula: R 4 -Halo (wherein Halo is a halogen atom) can be used in combination. The amount of the radical (3A) generated in the reaction system is usually 4 to 20 moles, preferably 8 to 12 moles, per mole of the compound (1E).

[0101] In compound (3B), examples of M include Li, Na, K, Zn, Cu, Ce, MgCl, MgBr, MgI, etc. In one embodiment, compound (3B) is preferably a Grignard reagent. The amount of compound (3B) used is usually 1 to 10 mol, preferably 2 to 6 mol, per 1 mol of compound (1E).

[0102] The reaction is preferably carried out in the presence of a Lewis acid. Examples of Lewis acids include zinc chloride, tin tetrachloride, titanium tetrachloride, boron trifluoride, boron trifluoride diethyl etherate (BF3·OEt2), boron trichloride, and trimethylsilyl triflate ((CH3)3SiOSO2CF3). The amount of the Lewis acid used is usually 4 to 20 mol, preferably 8 to 12 mol, per 1 mol of compound (1E).

[0103] The reaction is preferably carried out in the presence of a solvent. Examples of the solvent include aromatic hydrocarbon solvents (eg, toluene, xylene), halogenated hydrocarbon solvents (eg, dichloromethane), and mixed solvents of two or more of these.

[0104] The reaction temperature for the reaction is not particularly limited as long as the reaction proceeds, but is, for example, -78 to 40°C, and preferably 0 to 30°C. The reaction time for the reaction is not particularly limited, but is, for example, 0.1 to 2 hours, and preferably 0.5 to 1 hour.

[0105] <Process (IIIa)> In step (IIIa), compound (1F) is dehydrogenated (—C(H)(R 4 )-NH- to -CR 4 =N-) to obtain compound (1G). This dehydrogenation can be carried out in the same manner as in step (I). The compound (1G) can be prepared by the reaction of a compound of formula (3C): R 5 ·(In the formula, R 5 is the same as above), or a radical represented by formula (3D): R 5 M (wherein M is a metal atom or an atomic group containing a metal atom), R 4 is the same as above), to form a compound represented by an organometallic reagent represented by R 5 This reaction can be carried out by the method described in Organic Letters 1999, 1, 4, 569-572 and Tetrahedron Letters 39 (1998) 3237-3240.

[0106] <Process (IIIb)> In step (IIIb), compound (1G) is hydrogenated (-CR 4 =N- to -C(H)(R 4 )-NH-) to obtain compound (1H).

[0107] Examples of the reagent (reducing agent) for hydrogenating compound (1G) include diisobutylaluminum hydride, lithium aluminum hydride, sodium borohydride, and the like. The amount of the reagent used is usually 1 to 10 mol, preferably 3 to 5 mol, per 1 mol of compound (1G).

[0108] The hydrogenation is preferably carried out in the presence of a solvent. Examples of the solvent include aromatic hydrocarbon solvents (eg, toluene, xylene), halogenated hydrocarbon solvents (eg, dichloromethane), and mixed solvents of two or more of these.

[0109] The hydrogenation temperature is not particularly limited as long as the reaction proceeds, but is, for example, -20 to -78°C, and preferably -60 to -78°C. The hydrogenation time is not particularly limited, but is, for example, 0.5 to 6 hours, and preferably 1 to 3 hours.

[0110] <Process (IIIc)> In step (IIIc), compound (1H) is reacted with a compound of formula (4A): R 3 -L, where L is a leaving group and R 3 is the same as above, but is not hydrogen), to obtain compound (1I).

[0111] In compound (4A), examples of the leaving group represented by L include a halogen atom (e.g., a chlorine atom, a bromine atom, an iodine atom), an alkylsulfonyloxy group (e.g., a mesyloxy group), a haloalkylsulfonyloxy group (e.g., a trifluoromethylsulfonyloxy group), and an arylsulfonyloxy group (e.g., a tosyloxy group). The amount of compound (4A) used is usually 1 to 8 moles, preferably 1 to 4 moles, per mole of compound (1H).

[0112] The reaction is preferably carried out in the presence of a solvent. Examples of the solvent include ether solvents (e.g., tetrahydrofuran), nitrile solvents (e.g., acetonitrile), aromatic hydrocarbon solvents (e.g., toluene, xylene), mixed solvents of two or more of these, etc. Among these, aromatic hydrocarbon solvents are preferred, and at least one selected from the group consisting of toluene and xylene is more preferred.

[0113] The reaction is preferably carried out in the presence of a base. Examples of the base include inorganic bases [e.g., alkali metal carbonates (e.g., sodium carbonate, cesium carbonate), alkali metal bicarbonates (e.g., sodium bicarbonate), alkaline earth metal carbonates (e.g., calcium carbonate), alkali metal hydroxides (e.g., sodium hydroxide, potassium hydroxide), alkaline earth metal hydroxides (e.g., calcium hydroxide), metal alkoxides (e.g., sodium methoxide, sodium ethoxide)], organic bases [e.g., tertiary amines (e.g., trialkylamines), cyclic amines (e.g., 4-(dimethylamino)pyridine, diazabicycloundecene (DBU), diazabicyclononene (DBN))], and combinations thereof. Among these, tertiary amines are preferred, and triC 1-4 Alkylamines are more preferred. The amount of the base used is usually 2 to 10 mol, preferably 5 to 8 mol, per 1 mol of compound (2A).

[0114] The reaction temperature for the reaction is not particularly limited as long as the reaction proceeds, but is, for example, 30 to 150°C, and preferably 50 to 120°C. The reaction time for the reaction is not particularly limited, but is, for example, 1 to 24 hours, and preferably 1 to 12 hours.

[0115] In addition, R 3 Compound (1Q), in which is a methyl group optionally having one or two substituents, can be obtained, for example, by a method including the following step (IIIc'): <Process (IIIc')> In step (IIIc′), compound (1H) is reacted with a compound of formula (4B): R 3g -C(=O)-R 3h (In the formula, R 3g and R 3h is R 3 (which is a residue of the formula (1Q)) to obtain compound (1Q).

[0116] The amount of compound (4B) used is usually 1 to 6 moles, preferably 1 to 3 moles, per mole of compound (1H).

[0117] The reaction is preferably carried out in the presence of a reducing agent. Examples of reducing agents include sodium borohydride, sodium cyanoborohydride, lithium cyanoborohydride, lithium triethylborohydride, lithium tri(sec-butyl)borohydride, potassium tri(sec-butyl)borohydride, sodium triacetoxyborohydride, lithium aluminum hydride, sodium bis(2-methoxyethoxy)aluminum hydride, combinations thereof, and the like. The amount of the reducing agent used is usually 1 to 8 mol, preferably 1 to 4 mol, per 1 mol of compound (1H).

[0118] The reaction is preferably carried out in the presence of an acid catalyst, such as pyridinium p-toluenesulfonate (PPTS), acetic acid, or hydrochloric acid.

[0119] The reaction is preferably carried out in the presence of a solvent, such as an alcohol solvent (e.g., methanol), an ether solvent (e.g., tetrahydrofuran), or a mixed solvent of two or more of these.

[0120] The reaction temperature for the reaction is not particularly limited as long as the reaction proceeds, but is, for example, 0 to 100°C, and preferably 0 to 40°C. The reaction time for the reaction is not particularly limited, but is, for example, 0.5 to 12 hours, and preferably 1 to 4 hours.

[0121] In addition, R 3 is the formula:R 31 Compound (1I), which is a group represented by —X—, can be obtained, for example, by a method including the following steps (IIId) to (IIIf). (IIId) Compound (1H) is reacted with a compound of the following formula (4C): [ka] (wherein X and L are the same as above) and reacting the compound represented by the following formula (1H'): [ka] (In the formula, Base, A 1 , R 1 , R 2 , R 4 and X are the same as above. obtaining a compound represented by the formula: (IIIe) The compound (1H') is reacted with a hydrazine compound of the following formula (1H''): [ka] obtaining a compound represented by the formula: (IIIf) A step of optionally protecting the amino group of compound (1H″) with an amino-protecting group or guanidinating compound (1H″).

[0122] <Process (IIId)> Step (IIId) can be carried out in the same manner as step (IIIc).

[0123] <Process (IIIe)> The amount of the hydrazine compound used is usually 1 to 10 mol, preferably 1.1 to 3.5 mol, per 1 mol of compound (1H'). The reaction is preferably carried out in the presence of a solvent, examples of which include water, alcoholic solvents (e.g., methanol, ethanol), and mixed solvents of two or more of these.

[0124] <Process (IIIf)> A known or conventional method can be used to protect the amino group of compound (1H″) with an amino-protecting group. For example, the step of introducing a trifluoromethylcarbonyl group as a protecting group for the amino group of compound (1H″) is a step of reacting compound (1H″) with trifluoroacetic acid or a derivative thereof (e.g., trifluoroacetic anhydride). The reaction is preferably carried out in the presence of a solvent. A preferred example of the solvent is a cyclic amine (e.g., pyridine).

[0125] The guanidination of compound (1H″) is usually carried out by reaction with a guanidination agent. Examples of the guanidination agent include nitrogen-based guanidination agents and sulfur-based guanidination agents.

[0126] Examples of nitrogen-based guanidylating agents include compounds represented by the following formula: [ka] (In the formula, L 3 is a leaving group and R 3c ~R 3e is the same as above).

[0127] L 3 Examples of the leaving group represented by are the same as those of L.

[0128] The nitrogen-based guanidinating agent is preferably 1-amidinopyrazole hydrochloride, 1-carbamimidoyl-1,2,4-triazole hydrochloride, 1-(Nt-butoxy-amidino)pyrazole, 1-(N-benzyloxy-amidino)pyrazole, 1-[N,N'-(di-t-butoxy)amidino]pyrazole, 1-[N,N'-(di-benzyloxy)amidino]pyrazole, 1,2,3-tris(t-butoxycarbonyl)guanidine, or Goodman's reagent.

[0129] Examples of sulfur-based guanidination agents include compounds represented by the following formula: [ka] (In the formula, R 3c ~R 3e is the same as above). The sulfur-based guanidylating agent is preferably N,N'-di-t-butoxy-S-methylisothiourea or 1,3-di-t-butoxythiourea.

[0130] The amount of the guanidylating agent used is usually 0.5 to 10 mol, preferably 0.8 to 2.0 mol, per 1 mol of compound (1H″).

[0131] The reaction of compound (1H″) with a guanidylating agent is preferably carried out in the presence of a solvent. Examples of the solvent include halogenated hydrocarbon solvents (e.g., dichloromethane), amide solvents (e.g., N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone), mixed solvents of two or more of these, etc. Among these, amide solvents are preferred, and N,N-dimethylformamide is more preferred.

[0132] The reaction temperature for the reaction is not particularly limited as long as the reaction proceeds, but is, for example, 15 to 30°C.

[0133] <Process (IV)> Step (IV) is a step of reacting compound (1F) with compound (4A) to obtain compound (IJ). This reaction can be carried out in the same manner as in step (IIIc). 3 is the formula:R 31 Compound (1J), which is a group represented by —X—, can be obtained, for example, by a method including steps similar to steps (IIId) to (IIIf).

[0134] <Process (IV')> Step (IV') is a step of reacting compound (1F) with compound (4B) to obtain compound (1R). This step can be carried out in the same manner as step (IIIc').

[0135] The bases of the compounds (1), (1A) to (1J), (1Q), (1R), and (2A) can be converted, for example, by the following reaction scheme 2. [ka] (In the formula, Q 1 is a hydrogen atom or a substituent, and Q 2 and Q 3 are the same or different and are protecting groups for hydrogen atoms or amino groups (provided that Q 2 and Q 3 are not hydrogen atoms), Q 4 ~Q 7 are the same or different and are a protecting group for a hydrogen atom or an amino group, and ring G is a 5- or 6-membered nitrogen-containing heterocycle.

[0136] [Reaction Scheme 2] <Process (V)> Step (V) is a step of converting Base in compounds (1), (1A) to (1J), (1Q), (1R), and (2A) from an "optionally substituted 2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl group" to an "optionally substituted 2-oxo-1,2-dihydropyrimidin-1-yl group," and includes steps (Va) to (Vc).

[0137] <Process (Va)> Step (Va) is a step in which compound (1K), in which Base is an optionally substituted 2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl group, is reacted with a compound represented by formula (5A) and a phosphoric acid halide to obtain compound (1L).

[0138] In compound (1K), Q 1 is preferably a hydrogen atom or an alkyl group, more preferably a hydrogen atom or C 1-4 It is an alkyl group.

[0139] Compound (5A) is preferably a five-membered nitrogen-containing heterocyclic compound, more preferably a triazole. The amount of compound (5A) used is usually 5 to 20 mol, preferably 7 to 9 mol, per 1 mol of compound (1K).

[0140] The phosphoric acid halide is preferably phosphoric acid trichloride. The amount of phosphoric acid halide used is usually 1 to 5 mol, preferably 1 to 3 mol, per 1 mol of compound (1K).

[0141] The reaction is preferably carried out in the presence of a solvent. Examples of the solvent include nitrile solvents (e.g., acetonitrile), ether solvents (e.g., tetrahydrofuran), halogenated solvents (e.g., haloalkanes), and mixed solvents of two or more of these. Of these, nitrile solvents (e.g., acetonitrile) are preferred.

[0142] The reaction is preferably carried out in the presence of a base. Examples of the base include inorganic bases [e.g., alkali metal carbonates (e.g., sodium carbonate, cesium carbonate), alkali metal bicarbonates (e.g., sodium bicarbonate), alkaline earth metal carbonates (e.g., calcium carbonate), alkali metal hydroxides (e.g., sodium hydroxide, potassium hydroxide), alkaline earth metal hydroxides (e.g., calcium hydroxide), metal alkoxides (e.g., sodium methoxide, sodium ethoxide)], organic bases [e.g., tertiary amines (e.g., trialkylamines), cyclic amines (e.g., 4-(dimethylamino)pyridine, diazabicycloundecene (DBU), diazabicyclononene (DBN))], and combinations thereof. Among these, tertiary amines are preferred, and triC 1-4 Alkylamines are more preferred. The amount of the base used is usually 5 to 20 mol, preferably 10 to 15 mol, per 1 mol of compound (1K).

[0143] The reaction temperature for the reaction is not particularly limited as long as the reaction proceeds, but is, for example, -5°C to 10°C. For the reaction, for example, the method described in US Pat. No. 5,359,067 can be used.

[0144] <Process (Vb)> Step (Vb) is a step of reacting compound (1L) with ammonia to obtain compound (1M).

[0145] The amount of ammonia used is usually 5 to 100 mol, preferably 20 to 50 mol, per 1 mol of compound (1 L).

[0146] The reaction is preferably carried out in the presence of a solvent. Examples of the solvent include amide solvents (e.g., N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone), ether solvents (e.g., cyclic ethers such as tetrahydrofuran and dioxane), mixed solvents of two or more of these, etc. Among these, ether solvents are preferred, cyclic ethers are more preferred, and at least one selected from tetrahydrofuran and dioxane is even more preferred.

[0147] The reaction temperature for the reaction is not particularly limited as long as the reaction proceeds, but is, for example, 15 to 30°C.

[0148] <Process (Vc)> In step (Vc), the amino group of compound (1M) is protected with a protecting group to obtain compound (1N). The method for protecting the amino group with a protecting group can be a known method (for example, U.S. Patent Application Publication No. 2007 / 167387) or a conventional method.

[0149] <Process (VI)> Step (VI) is a step of converting Base from an "optionally substituted 2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl group" to an "optionally substituted purin-9-yl group" in compounds (1), (1A) to (1J), (1Q), (1R), and (2A).

[0150] Specifically, step (VI) is a step in which compound (1K), in which Base is an optionally substituted 2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl group, is reacted with a compound represented by formula (5B) to obtain compound (1O).

[0151] The amount of compound (5B) used is usually 1 to 5 mol, preferably 1 to 3 mol, per 1 mol of compound (1K).

[0152] The reaction is preferably carried out in the presence of a Lewis acid. An example of a Lewis acid is trimethylsilyl trifluoromethanesulfonate. The amount of the Lewis acid used is usually 0.5 to 5 mol, preferably 1 to 3 mol, per 1 mol of compound (1K).

[0153] The reaction is preferably carried out in the presence of a silylating agent. Examples of silylating agents include N,O-bis-trimethylsilylacetamide (BSA), N,O-bis-silyltrifluoroacetamide (BSTFA), hexamethyldisilazane (HMD), N,O-bis-tert-butyldimethylsilylacetamide, N-(trimethylsilyl)diethylamine, N-(trimethylsilyl)dimethylamine, N-methoxy-N,O-bis(trimethylsilyl)carbamate, N-methyl-N-trimethylsilylacetamide, N-methyl-N-trimethylsilylheptafluorobutyramide, N-methyl-N-trimethylsilyltrifluoroacetamide, N-trimethylsilylacetamide, and combinations of two or more thereof. Among these, N,O-bis-trimethylsilylacetamide (BSA) is preferred. The amount of the silylating agent used is usually 1 to 20 mol, preferably 3 to 8 mol, per 1 mol of compound (1K).

[0154] The reaction temperature for the reaction is not particularly limited as long as the reaction proceeds, but is, for example, 30 to 150°C, and preferably 50 to 120°C.

[0155] The reaction can also be carried out using, for example, the method described in U.S. Patent Application Publication No. 2012 / 071646 (the specification of U.S. Patent Application Publication No. 2012 / 071646 is incorporated herein by reference).

[0156] <Process (VII)> Step (VII) is a step of converting Base from an "optionally substituted 2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl group" to an "optionally substituted 6-oxo-1,6-dihydro-9H-purin-9-yl group" in compounds (1), (1A) to (1J), (1Q), (1R), and (2A). Specifically, step (VII) is a step in which compound (1K), in which Base is an optionally substituted 2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl group, is reacted with a compound represented by formula (5C) to obtain compound (1P). The reaction can be carried out under the same conditions as in step (VI).

[0157] The method for producing compound (1) may further include, if necessary, a step of purifying the intermediate product and the final product by a conventional method, such as concentration, recrystallization, silica gel column chromatography, or the like.

[0158] <<Composition containing compound (1)>> The composition of the present invention contains the compound (1) described above. The composition may contain only one type of compound (1), or may contain two or more types. For example, the composition may contain Compound (1) in which the base is a thymin-1-yl group; Compound (1), wherein the base is a 5-methylcytosin-1-yl group, an N-acetyl-5-methylcytosin-1-yl group, an N-isobutyryl-5-methylcytosin-1-yl group, or an N-benzoyl-5-methylcytosin-1-yl group; Compound (1) in which Base is an adenin-9-yl group, an N-acetyl-adenin-9-yl group, an N-isobutyryl-adenin-9-yl group, or an N-benzoyl-adenin-9-yl group; and Compound (1) in which the base is a guanine-9-yl group, an N-acetyl-guanine-9-yl group, an N-isobutyryl-guanine-9-yl group, an N-benzoyl-guanine-9-yl group, or an N-(N,N-dimethylformamidyl)-guanine-9-yl group. The composition may contain one, two, three, or four compounds selected from the group consisting of:

[0159] The composition may be in the form of a liquid. When the composition is in the form of a liquid, the composition usually contains a solvent. Known solvents can be used as the solvent, and preferred examples include halogenated hydrocarbon solvents (e.g., dichloromethane), nitrile solvents (e.g., acetonitrile), aromatic hydrocarbon solvents (e.g., toluene, xylene), water, and TE buffer. Among these, dichloromethane, toluene, and acetonitrile are preferably used.

[0160] The composition is typically provided to the user in a container.

[0161] The composition can be used for synthesizing the oligonucleotide or a salt thereof described below. The composition can also be used as a pharmaceutical composition.

[0162] <<Oligonucleotides or salts thereof>> The oligonucleotide or a salt thereof of the present invention has the following formula (6): [ka] (In the formula, Base, A 1 , R 3 ~R 5 , and n are the same as above) It has units expressed as:

[0163] The unit preferably has the following formula (6a): [ka] (In the formula, A 2 Oh, O - , SH, or S - and Base,A 1 , R 3 ~R 5 , and n are the same as above) It is a unit expressed as

[0164] The unit represented by formula (6a) is preferably a unit represented by the following formula (6a-1), more preferably a unit represented by the following formula (6a-2) or (6a-3): [ka] (In the formula, Base, A 1 , A 2 , R 3 ~R 5 , R 33 ~R 35 , and m are the same as above)

[0165] Hereinafter, an oligonucleotide having a unit represented by formula (6) or (6a) or a salt thereof will be referred to as "oligonucleotide (6)".

[0166] When oligonucleotide (6) has two or more units represented by formula (6) or (6a), the structures of the units may be the same or different.

[0167] The oligonucleotide (6) may contain other units in addition to the unit represented by formula (6) or (6a). Examples of the other units include the following formulae (7) to (10): [ka] (In the formula, A 3 are the same or different and are a single bond or an alkylene group which may have a substituent, and R a is a hydrogen atom or a hydroxyl group, and R bis R 3 and Base is the same as above) and at least one selected from units represented by the following formula:

[0168] The other units are preferably represented by the following formulae (7a) to (10a): [ka] (In the formula, A 4 ~A 7 are the same or different, OH, O - , SH, or S - and Base,A 3 , R a , and R b is the same as above.) At least one unit selected from the units represented by the following formulas can be given.

[0169] Other units are derived from nucleotides described in, for example, U.S. Patent Application Publication Nos. 2003 / 105309, 2017 / 044528, 2006 / 166908, 2012 / 208991, 2015 / 266917, and 2003 / 207841. (U.S. Patent Application Publication Nos. 2003 / 105309, 2017 / 044528, 2006 / 166908, 2012 / 208991, 2015 / 266917, and 2003 / 207841 are incorporated herein by reference).

[0170] The base sequence of oligonucleotide (6) is not particularly limited as long as it is complementary to the base sequence (full length or a part thereof) of the target DNA or target RNA.

[0171] The length of oligonucleotide (6) is not particularly limited and can be selected depending on the length of the target base sequence. The lower limit of the length of oligonucleotide (6) is, for example, a 5-mer, preferably a 10-mer, and more preferably a 15-mer, and the upper limit of the length of oligonucleotide (6) is, for example, a 200-mer, preferably a 100-mer, more preferably a 50-mer, and even more preferably a 30-mer. The length of oligonucleotide (6) is, for example, 5 to 200-mer, preferably a 5 to 50-mer, more preferably a 10 to 40-mer, and even more preferably a 15 to 30-mer. The longer the length of oligonucleotide (6), the stronger the binding strength to the target base sequence.

[0172] In oligonucleotide (6), the ratio of the number of units represented by formula (6) to the total number of nucleotide units is not particularly limited and can be appropriately designed depending on the intended use (primer, probe, clamp nucleic acid, pharmaceutical, etc.).

[0173] Oligonucleotide (6) may be in the form of a salt. That is, at least one of the nucleotide units constituting oligonucleotide (6) may be in the form of a salt. The salt may be a pharmaceutically acceptable salt or may not be a pharmaceutically acceptable salt. The salt may be an inorganic salt or an organic salt. Examples of the salt include alkali metal salts, alkaline earth metal salts, other metal salts, ammonium salts, tetramethylammonium salts, amine salts, inorganic acid salts, organic acid salts, and amino acid salts, as with the salts exemplified for compound (1).

[0174] The oligonucleotide (6) may be modified with a labeling substance, which may be, but is not limited to, a fluorescent substance, a hapten (e.g., biotin, digoxigenin, DNP, etc.), a radioisotope, or any other substance known in the art for labeling nucleic acids.

[0175] Oligonucleotide (6) has high sequence specificity. Oligonucleotide (6) has a high Tm value for single-stranded DNA and a high Tm value for single-stranded RNA. That is, oligonucleotide (6) binds tightly to single-stranded DNA and single-stranded RNA and has a high ability to form a double strand. In particular, the Tm value of oligonucleotide (6) for single-stranded RNA is extremely high compared to the DNA or RNA of formula (7). The oligonucleotide (6) can be suitably used as a probe for examining the base sequence of single-stranded RNA or single-stranded DNA, or for highly selectively detecting the sequence.

[0176] Oligonucleotide (6) is resistant to degradation by nucleases and can persist in vivo for a long time after administration. Oligonucleotide (6) can, for example, form a duplex with sense RNA to inhibit the transcription of mRNA for pathogenic biological components (proteins). Oligonucleotide (6) can also inhibit the proliferation of infecting viruses. Oligonucleotide (6) is useful as a drug for treating diseases by inhibiting the function of genes, such as antitumor and antiviral agents. Furthermore, the oligonucleotide (6) has stable and excellent activity as an antisense or antigene or aptamer, or as a primer for detecting or initiating amplification of a specific gene. Oligonucleotides (6) are useful as various physiologically and biologically active substances, pharmaceutical materials, functional materials for double-stranded oligonucleotides used in RNA interference and decoy methods, functional materials for DNA chips targeting single-stranded nucleic acids such as cDNA, functional materials for molecular beacons, functional materials for various antisense methods (including ribozymes and DNAzymes), antigene methods, and homologous recombination methods, materials for highly sensitive analysis of trace biological components in combination with fluorescent or luminescent substances, and materials for developing research reagents for elucidating gene function, etc.

[0177] <<Method for producing oligonucleotide (6)>> Oligonucleotide (6) can be synthesized according to conventional methods, such as phosphoramidite protocols. For example, the method for producing oligonucleotide (6) is as follows: (I) Formula (6B): [ka] (In the formula, R 2a represents an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted cycloalkyl group, an optionally substituted cycloalkenyl group, an optionally substituted aryl group, a protecting group for a hydroxyl group, a substituted phosphino group, an optionally substituted dihydroxyphosphinyl group, or an optionally substituted hydroxymercaptophosphinyl group; Base, A 1 , R 3 ~R 5 , and n are the same as above) or a salt thereof, [ka] (In the formula, R 1a represents a hydrogen atom, an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted cycloalkyl group, an optionally substituted cycloalkenyl group, an optionally substituted aryl group, a protecting group for a hydroxyl group, a substituted phosphino group, an optionally substituted dihydroxyphosphinyl group, or an optionally substituted hydroxymercaptophosphinyl group, R A and R B are the same or different and are a hydrogen atom or an alkyl group, R C is a hydrogen atom, an alkyl group, a haloalkyl group, or a cyanoalkyl group, Base, A 1 , A 3 , R 3 ~R 5 ,n,Ra , and R b is the same as above) and / or a step of reacting the compound represented by the formula (I) with at least one compound selected from the group consisting of a compound represented by the formula (I) and a salt thereof. (II) Formula (6C): [ka] (In the formula, Base, A 1 , R 1a , R 3 ~R 5 , n, and R A ~R C is the same as above) or a salt thereof, [ka] (In the formula, Base, A 1 , A 3 , R 2a , R 3 ~R 5 ,n,R a , and R b is the same as above) or a salt thereof, and (III) a step of oxidizing the compound obtained in step (I) and / or step (II) (particularly, oxidizing the phosphorus atom). Includes.

[0178] The method for producing oligonucleotide (6) may further include, if necessary, a step of purifying the intermediate and final products by conventional methods, such as concentration, recrystallization, silica gel column chromatography, gel filtration, ethanol precipitation, preparative HPLC, and the like.

[0179] <<Composition containing oligonucleotide (6)>> The composition of the present invention contains the oligonucleotide (6) described above. The composition may contain only one type of oligonucleotide (6), or may contain two or more types.

[0180] The composition may be in the form of a liquid, solid, or the like. When the composition is in the form of a liquid, it usually contains a solvent. Known solvents can be used as the solvent, and preferred examples include halogenated hydrocarbon solvents (e.g., dichloromethane), nitrile solvents (e.g., acetonitrile), aromatic hydrocarbon solvents (e.g., toluene, xylene), and water. Among these, water is more preferred, and water containing a buffer (buffer solution) is more preferred. Examples of buffers include trishydroxymethylaminomethane (Tris buffer), trishydroxymethylaminomethane-hydrochloric acid (Tris-HCl buffer), trishydroxymethylaminomethane-EDTA (TE buffer), sodium phosphate, 2-morpholinoethanesulfonic acid (MES), N-(2-acetamido)iminodiacetic acid (ADA), piperazine-1,4-bis(2-ethanesulfonic acid), N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), cholamine hydrochloride, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), N-tris(hydroxymethyl)methyl-2-methanesulfonic acid (TES), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), acetamidoglycine, tricine, glycinamide, and bicine.

[0181] The composition may further contain salts, including metal chlorides, examples of which include NaCl, MgCl, KCl, and the like.

[0182] The composition may further contain additives and cosolvents, such as dimethyl sulfoxide (DMSO), glycerol, formamide, bovine serum albumin, ammonium sulfate, polyethylene glycol (PEG), gelatin, and non-ionic surfactants, such as Tween 20® and Triton X-100®.

[0183] When the composition is in a solid form, the composition may be, for example, a composition in which the oligonucleotide (6) is supported on a solid support. Examples of solid supports include inorganic and organic materials capable of supporting biopolymers. Preferred examples include glass (e.g., porous glass (CPG)), silica gel, and resins (e.g., cross-linked non-swelling polystyrene resin (HPS)). Of these, HPS and CPG are more preferred.

[0184] The composition is typically provided to the user in a container.

[0185] The composition can be used for the amplification or detection of target nucleic acids, strand invasion, RNAi, etc., as described below. The composition can also be used as a pharmaceutical composition.

[0186] <<Method for detecting target nucleic acid>> The present invention includes a method for detecting a target nucleic acid, the method comprising: (I) selectively amplifying a target nucleic acid by a nucleic acid amplification method; and (II) detecting the target nucleic acid amplified in step (I); The oligonucleotide used for the amplification or detection preferably comprises oligonucleotide (6).

[0187] In the nucleic acid amplification of step (I), a plurality of primer molecules are typically used. At least some of the plurality of primer molecules may be oligonucleotide (6). For example, a forward primer containing oligonucleotide (6) and a reverse primer not containing oligonucleotide (6) are used. In the nucleic acid amplification in step (I), a plurality of primer molecules and probe molecules may be used. In this case, at least some of the primer molecules and probe molecules may be oligonucleotide (6). For example, a forward primer that does not contain oligonucleotide (6), a reverse primer that does not contain oligonucleotide (6), and a probe that contains oligonucleotide (6) may be used.

[0188] The nucleic acid amplification method is not particularly limited as long as it can selectively amplify target nucleic acid.Examples of nucleic acid amplification methods include PCR (including hot start PCR, multiplex PCR, nested PCR, RT-PCR, real-time PCR, digital PCR, TaqMan (registered trademark) PCR, clamp PCR, etc.), NASBA method (see US Patent No. 5,130,238), TMA method (see US Patent No. 5,399,491), TRC method (see US Patent Application Publication No. 2001 / 0053518), LAMP method (see US Patent No. 6,410,278), ICAN method (see US Patent Application Publication No. 2003 / 073081), LCR method (see European Patent Application No. 320,328), SDA method (see US Patent No. 5,455,166) etc.

[0189] In one embodiment, the nucleic acid amplification method in step (I) is preferably clamp PCR. In clamp PCR, at least a primer and a clamp nucleic acid are used. Either or both of the primer and the clamp nucleic acid may contain oligonucleotide (6). Clamp nucleic acids typically clamp non-target nucleic acids (e.g., wild-type genes) in a test sample more strongly than target nucleic acids (e.g., mutant genes). The clamp nucleic acids bind strongly to non-target nucleic acids, inhibiting their amplification, allowing for selective amplification of the target nucleic acid. For example, when detecting a specific gene mutation, nucleic acid amplification can be performed in the presence of a clamp nucleic acid with a base sequence completely complementary to the wild-type gene sequence, thereby inhibiting amplification of the wild-type nucleic acid and selectively amplifying the mutant nucleic acid. Specifically, for example, when the target site of the target nucleic acid is the mutation site of the mutant gene and the base sequence containing this mutation site (the target site of the target nucleic acid) is "sequence A," the clamp nucleic acid is completely complementary to the base sequence corresponding to sequence A in the wild-type gene, which is a non-target nucleic acid. Oligonucleotide (6) has high sequence selectivity. Therefore, clamp nucleic acids containing oligonucleotide (6) have extremely weak binding affinity to base sequences that differ by even a single base, but can specifically bind to perfectly complementary base sequences. Furthermore, oligonucleotide (6) has a high Tm value and forms a stable duplex. That is, clamp nucleic acids containing oligonucleotide (6) specifically and strongly bind to non-target nucleic acids, demonstrating high clamping ability. The length of the clamp nucleic acid is not particularly limited, but is, for example, 5 to 30 mers.

[0190] The nucleic acid amplification in step (I) may be a reverse transcription reaction. In the reverse transcription reaction, cDNA is synthesized by RNA-dependent DNA polymerase using RNA as a template. Because oligonucleotide (6) binds strongly to RNA, it can bind to specific RNA in the test sample and inhibit the reverse transcription reaction from that RNA. This allows cDNA to be synthesized more specifically from the target RNA. The synthesized cDNA may be further amplified by PCR or other methods.

[0191] The target nucleic acid may be contained in a test sample. A test sample is typically a sample collected from a living organism (hereinafter also referred to as a "biological sample"). Usually, a sample obtained by preprocessing, such as removing contaminants, extracting and purifying nucleic acids, and preamplification, from a sample collected from a living organism is used. Specifically, blood, plasma, serum, pleural effusion, bronchial lavage fluid, bone marrow fluid, lymphatic fluid, intestinal lavage fluid, resected tissue, nasopharyngeal swab, saliva, nasal secretion, and sputum can be used. Samples obtained by preprocessing these samples as described above are also included in the "biological sample." Because the method of the present invention can detect nucleic acids with extremely high sensitivity, it can detect genetic mutations originating from abnormal cells, for example, using resected tissue containing a mixture of normal and abnormal cells. Furthermore, the method of the present invention is suitable for use even when the test sample is small in volume or when the amount of nucleic acid in the test sample is small. The test sample is preferably a solution containing the target nucleic acid. When detecting target nucleic acids in cells contained in blood or resected tissue, the cells may be solubilized by a known method. In addition to biological samples, test samples that can be used include excrement, sewage, river water, seawater, soil, and samples of these that have been pretreated as described above. Examples of excrement include urine and feces.

[0192] The target nucleic acid may be DNA or RNA, but is preferably DNA. The DNA may be cDNA synthesized from RNA by reverse transcription. The target nucleic acid may be a specific region on genomic DNA, such as a gene or a promoter region of a gene. The detection method of the present invention can be used to detect mutations, polymorphisms, etc. at a target site of a target nucleic acid. The detection method of the present invention can also be used to determine alleles. It can detect the type of allele contained in a test sample. The detection method of the present invention can also be used to detect methylation. The presence or absence of methylated cytosine in a target nucleic acid can be detected by treating the target nucleic acid with bisulfite and then applying the detection method of the present invention. A gene containing a mutation (hereinafter referred to as a mutant gene) has a difference from the base sequence of a wild-type gene, i.e., a mutation. The difference is due to one or more mutations selected from the group consisting of substitution, insertion, deletion, inversion, duplication, and translocation, or a combination thereof. Such differences may usually be related to the onset of a particular disease and / or therapeutic sensitivity. Here, "onset" includes not only the actual onset of a disease but also the risk of onset. Furthermore, "therapeutic sensitivity" includes not only the response rate of treatment with a drug or the like but also the strength of side effects. Examples of such diseases include, but are not limited to, cancer, myelodysplastic syndrome, infectious diseases, etc. A preferred example of such a disease is cancer. Suitable examples of the gene include the ABL / BCR fusion gene, the HER2 gene, the EGFR gene, the c-KIT gene, the KRAS gene, the BRAF gene, the PIK3CA gene, the FLT3 gene, the MYC gene, the MYCN gene, the MET gene, the BCL2 gene, and the EML4 / ALK fusion gene.

[0193] In step (II), the amplified nucleic acid can be detected by a known method depending on the nucleic acid amplification method described above. For example, amplification can be detected qualitatively or quantitatively by detecting the fluorescence generated from the reaction solution or the turbidity of the reaction solution. Furthermore, an example of a method for detecting the amplified target nucleic acid is base sequence analysis, etc. The target nucleic acid can be detected by analyzing the base sequence of the amplified target nucleic acid using a known sequence analyzer (sequencer).

[0194] In addition, the method for detecting the target nucleic acid in the test sample can also be the method described in U.S. Patent Application Publication No. 2015 / 240299, for example.

[0195] The present invention encompasses a method for detecting a target nucleic acid in a test sample, which comprises reacting a probe comprising an oligonucleotide (6) and a label with the test sample containing the target nucleic acid. Specific examples of such detection methods include in situ hybridization and microarrays. When in situ hybridization is used as a detection method, an oligonucleotide (6) labeled with a fluorescent dye or the like is hybridized with a target nucleic acid in a test sample (e.g., a cell), and the target nucleic acid in the test sample can be detected by measuring the label. When a microarray is used as a detection method, the target nucleic acid can be detected by reacting a microarray on which a probe containing oligonucleotide (6) is immobilized with a test sample containing the target nucleic acid and detecting hybridization between the target nucleic acid and the probe.

[0196] Thus, by using oligonucleotide (6), it is possible to detect target nucleic acids in test samples with high sequence selectivity.

[0197] <<Kit for detecting or selectively amplifying a target nucleic acid>> A kit for detecting or selectively amplifying a target nucleic acid includes an oligonucleotide (6), which may be contained in, for example, a test sample.

[0198] In the kit, oligonucleotide (6) can be used as a primer, probe, and / or clamp nucleic acid in the amplification and / or detection of a target nucleic acid, as described above. The primer, probe, and / or clamp nucleic acid can be appropriately designed depending on the base sequences of the target nucleic acid and non-target nucleic acid.

[0199] In one embodiment, the kit includes primers and / or probes, at least some of which may be oligonucleotides (6).

[0200] 2A is a schematic diagram of an example of a kit including a container containing a composition including a primer and a probe. Kit 11 includes an outer box 12, a container support provided inside outer box 12 and having a recess formed on its surface, a container 13 attached to the recess and containing a composition including a primer and a probe, and an attached document 14. Instructions for handling kit 11, storage conditions, expiration date, etc., can be included in the attached document 14.

[0201] 2B is a schematic diagram of an example of a kit including a container containing a primer-containing composition and a container containing a probe-containing composition. Kit 21 includes an outer box 22, a container support provided in outer box 22 and having a first recess and a second recess formed on its surface at intervals along the longitudinal direction, container 23a mounted in the first recess and containing a primer-containing composition, container 23b mounted in the second recess and containing a probe-containing composition, and package insert 24.

[0202] In another embodiment, the kit includes a forward primer, a reverse primer, and a probe. At least some of these may be oligonucleotides (6). The forward primer-containing composition, the reverse primer-containing composition, and the probe-containing composition may all be contained in the same container (e.g., a kit as shown in FIG. 2A), any two of them may be contained in the same container (e.g., a kit as shown in FIG. 2B), or all three may be contained in separate containers.

[0203] 2C is a schematic diagram of an example of a kit including a container containing a composition containing a forward primer, a container containing a composition containing a reverse primer, and a container containing a composition containing a probe. Kit 31 includes an outer box 32, a container support provided within outer box 32 and having first to third recesses formed on its surface at intervals along the longitudinal direction, container 33a mounted in the first recess and containing a composition containing a forward primer, container 33b mounted in the second recess and containing a composition containing a reverse primer, container 33c mounted in the third recess and containing a composition containing a probe, and package insert 34.

[0204] In another embodiment, the kit comprises a clamp nucleic acid and a primer, at least some of which may be oligonucleotides (6). This kit may be for selectively amplifying a target nucleic acid. The clamp nucleic acid-containing composition and the primer-containing composition may be contained in the same container (e.g., a kit such as that shown in FIG. 2A) or in separate containers (e.g., a kit such as that shown in FIG. 2B).

[0205] In yet another embodiment, the kit includes a clamp nucleic acid, a primer, and a probe, at least some of which may be oligonucleotides (6). The clamp nucleic acid composition, the primer composition, and the probe composition may all be contained in the same container (e.g., a kit such as that shown in FIG. 2A), any two of the compositions may be contained in the same container (e.g., a kit such as that shown in FIG. 2B), or all three may be contained in separate containers (e.g., a kit such as that shown in FIG. 2C).

[0206] The kit may also contain DNA polymerase, deoxynucleoside triphosphates (dNTPs), a reaction buffer, salts, restriction enzymes, and the like.

[0207] The present invention includes the use of oligonucleotide (6) for detecting or selectively amplifying a target nucleic acid, where the oligonucleotide (6) used herein has the same characteristics as, for example, the oligonucleotide (6) contained in the kit.

[0208] <Pharmaceutical composition (or preparation)> The pharmaceutical composition (or formulation) of the present invention contains compound (1) or oligonucleotide (6). Examples of pharmaceutical compositions (or formulations) containing compound (1) include small molecule drugs such as AZT (azidothymidine), a nucleoside analogue reverse transcriptase inhibitor (NRTI). Examples of pharmaceutical compositions (or formulations) containing oligonucleotide (6) include medium-sized molecules such as antisense, siRNA (small interfering RNA), aptamer, decoy nucleic acid, and CpG oligonucleotide, as well as high molecular weight nucleic acid drugs.

[0209] The pharmaceutical composition may be in the form of a liquid preparation (e.g., injection, eye drops, nasal drops, suspension, etc.), a solid preparation (e.g., tablet, granule, powder, etc.), a semi-solid preparation (e.g., ointment, suppository, etc.), or any other preparation form known to those skilled in the art.

[0210] Suitable examples of pharmaceutical compositions include parenteral administration preparations (eg, subcutaneous administration preparations, intravenous administration preparations, intranasal administration preparations, intrathecal administration preparations, intracerebroventricular administration preparations, vitreous administration preparations, etc.). Another suitable example of a pharmaceutical composition is a topical formulation.

[0211] The pharmaceutical composition usually further comprises a pharmaceutically acceptable carrier or excipient. Carriers include solid carriers and liquid carriers. Examples of solid carriers include starch, lactose, calcium sulfate dihydrate, sucrose, talc, gelatin, agar, pectin, gum arabic, magnesium stearate, stearic acid, atelocollagen, etc. Examples of liquid carriers include water (including physiological saline), etc. The additives include stabilizers, examples of which include paraoxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as benzyl alcohol; benzalkonium chloride; phenols such as phenol and cresol; and the like. Oligonucleotide (6) has high sequence selectivity, a high Tm value, and is resistant to degradation by nucleases. Therefore, a pharmaceutical composition (or formulation) containing compound (1) or oligonucleotide (6) can bind to a target (e.g., a target gene) in the body with high sequence selectivity and act thereon. [Example]

[0212] The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples.

[0213] <<Synthesis example of compound (1)>> The symbols and abbreviations used in the synthesis examples are as follows. A BZ :N 6 -Benzoyladenine Bz: benzoyl DMTr: Dimethoxytrityl i-Pr: Isopropyl BF3OEt2: Boron trifluoride-ethyl ether complex BSA: N,O-bis(trimethylsilyl)acetamide CIPS: 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane DIBAL-H: Diisobutylaluminum hydride DIPEA: N,N-diisopropylethylamine DMAP: 4-dimethylaminopyridine DMF: N,N-dimethylformamide DMSO: dimethyl sulfoxide DMTrCl: 4,4'-dimethoxytrityl chloride DBU: 1,8-diazabicyclo[5.4.0]-7-undecene Et3B: Triethylborane Et3N: Triethylamine MeMgBr: methylmagnesium bromide MeOH: Methanol NaBH3CN: Sodium cyanoborohydride PPTS: Pyridinium p-toluenesulfonate TBAF: tetra-n-butylammonium fluoride TfO: Trifluoromethanesulfonic anhydride THF: tetrahydrofuran TMSOTf: Trimethylsilyl trifluoromethanesulfonate TsCl: p-toluenesulfonyl chloride rt: room temperature h: time min:minutes

[0214] [Synthesis Example 1] Base is a thymin-1-yl group, and A 1 is a single bond, and R 1 is DMTr and R 2 is -P(N(i-Pr)2)(OC2H4CN), and R 3 is a methyl group, and R 4 is a methyl group in the R configuration, and R 5 Compound (1) in which is a hydrogen atom and n is 1 (hereinafter referred to as "compound (R)MT-4") was synthesized according to the following reaction scheme.

[0215] [ka]

[0216] (Synthesis of Compound T-1) Compound 1 (20.0 g, 37.89 mmol) was dissolved in DMSO (500 mL) under a nitrogen atmosphere, and 2-iodoxybenzoic acid (27.89 g, 41.68 mmol) was added at room temperature. The mixture was stirred at room temperature for 4.5 hours. The reaction mixture was cooled with water and quenched with saturated aqueous sodium bicarbonate. The mixture was then diluted with ethyl acetate and water, and the organic and aqueous layers were separated. The aqueous layer was back-extracted with ethyl acetate. The organic layer from the first fraction and the organic layer from the back-extraction were combined and washed sequentially with saturated aqueous sodium bicarbonate and saturated brine, then dried over anhydrous sodium sulfate and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane:ethyl acetate = 3:1 to 2:3) to give compound T-1 (17.48 g, 87% yield) as a white foamy solid. 1 H NMR(CDCl3)δ 1.01-1.12 (28H, m), 1.91 (3H, d, J=1 Hz), 3.82, 4.15 (2H, ABq, J=14 Hz), 4.44 (1H, m), 4.69 (1H, d, J=4 Hz), 5.86 (1H, s), 7.09 (1H, m), 7.41 (1H, d, J=1 Hz), 8.48 (1H, s).

[0217] (Synthesis of Compound (R)MT-1) Compound T-1 (3.86 g, 7.33 mmol) was dissolved in toluene (55 mL). Boron trifluoride-ethyl ether complex (4.60 mL, 37.0 mmol) was added under dry ice / acetone cooling. Methylmagnesium bromide (12% tetrahydrofuran solution) was then added dropwise to the reaction mixture over 30 minutes, and the mixture was stirred for 2 hours while cooled. The reaction was quenched with water, and the mixture was diluted with ethyl acetate and saturated brine to separate the organic layer. The resulting organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane:ethyl acetate = 2:1) to obtain compound (R) MT-1 (2.50 g, 63%) as a white foamy solid. 1H NMR(CDCl3)δ 0.88 (3H, d, J=7), 0.95-1.14 (28H, m), 1.92 (3H, s), 3.67, 4.13 (2H, ABq, J=13), 3.76 (1H, m), 4.15 (1H, d, J=2 Hz), 4.32 (1H, d, J=3), 6.12 (1H, s), 7.74 (1H, s), 8.55 (1H, s).

[0218] Alternatively, compound (R)MT-1 was synthesized according to the following reaction scheme.

[0219] [ka]

[0220] (Synthesis of Compound (R)MT-5) Under a nitrogen stream, oxalyl chloride (5.6 mL, 64.92 mmol) was dissolved in methylene chloride (240 mL), and dimethyl sulfoxide (9.2 mL, 129.84 mmol) was added in a dry ice-acetone bath. After stirring at the same temperature for 30 minutes, a methylene chloride solution (40 mL) of compound S-1 (20.0 g, 49.94 mmol) was added to the reaction mixture. After stirring at the same temperature for an additional 45 minutes, triethylamine (27.8 mL, 199.76 mmol) was added to the reaction mixture. After stirring at the same temperature for an additional 15 minutes, the mixture was returned to room temperature and stirred for 1 hour. The reaction mixture was diluted with methylene chloride and 1N hydrochloric acid, and the organic layer was separated and washed with saturated aqueous sodium bicarbonate and saturated brine. It was then dried over anhydrous sodium sulfate and evaporated under reduced pressure to obtain the intermediate. Subsequently, cerium chloride (12.3 g, 49.9 mmol) was dissolved in tetrahydrofuran (240 mL) under a nitrogen stream. After stirring at room temperature for 30 minutes, methylmagnesium bromide (1 M in tetrahydrofuran, 100 mL, 100.0 mmol) was added in an ice bath. After stirring at the same temperature for an additional 1.5 hours, a tetrahydrofuran solution of the intermediate obtained earlier (120 mL) was added in a dry ice-acetone bath. After stirring at the same temperature for 3 hours, the mixture was stirred at room temperature for an additional 1 hour. The reaction mixture was quenched with saturated aqueous ammonium chloride, diluted with ethyl acetate and water, and separated into organic and aqueous layers. The resulting organic layer was washed sequentially with 1N hydrochloric acid, saturated aqueous sodium bicarbonate, and saturated brine, dried over anhydrous magnesium sulfate, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane:ethyl acetate = 4:1 to 3:1) to obtain compound (R)MT-5 (19.0 g, 91%) as a colorless, transparent oil. 1H NMR(CDCl3)δ 1.20 (3H, d, J=6), 1.35 (3H, s), 1.59 (3H, s), 3.29 (1H, d, J=2), 3.63, 3.79 (2H, ABq, J=10), 4.41, 4.54 (2H, ABq, J=12), 4.44 (1H, d, J=5), 4.48, 4.86 (2H, ABq, J=11), 4.57-4.62 (1H, m), 4.67 (1H, dd, J=4, 5), 5.79 (1H, d, J=4), 7.23-7.38 (10H, m).

[0221] (Synthesis of Compound (R)MT-6) Under a nitrogen stream, compound (R)MT-5 (9.06 g, 21.85 mmol) was dissolved in pyridine (143 mL), p-toluenesulfonyl chloride (12.28 g, 64.43 mmol) was added, and the mixture was stirred at 80 °C for 13 hours. The temperature was then lowered to 65 °C, and p-toluenesulfonyl chloride (3.07 g, 16.11 mmol) was added. The mixture was stirred at 80 °C for 7.5 hours. The temperature was then lowered again to 65 °C, and p-toluenesulfonyl chloride (3.07 g, 16.11 mmol) was added. The mixture was stirred at 80 °C for 2 hours. The reaction mixture was cooled and quenched with water. The mixture was then diluted with ethyl acetate and saturated brine, and the organic layer was separated. The resulting organic layer was washed sequentially with 1 N hydrochloric acid, saturated aqueous sodium bicarbonate, and saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The obtained crude product was purified by silica gel column chromatography (hexane:ethyl acetate=3:1 to 3:1) to obtain compound (R) MT-6 (11.3 g, 91%) as a colorless, transparent oil. 1H NMR(CDCl3)δ 1.32 (3H, s), 1.44 (3H, d, J=6), 1.53 (3H, s), 2.40 (3H, s), 3.48, 3.56 (2H, ABq, J=10), 4.22 (1H, d, J=5), 4.37-4.44 (3H, m), 4.59-4.62 (2H, m), 5.39 (2H, q, J=6), 5.75 (1H, d, J=4), 7.23-7.32 (12H, m), 7.72-7.75 (2H, m).

[0222] (Synthesis of Compound (R)MT-7) Under a nitrogen stream, (R)MT-6 (11.20 g, 19.69 mmol) was dissolved in acetic acid (111 mL), and acetic anhydride (18.4 mL, 194.76 mmol) and concentrated sulfuric acid (0.072 g, 0.73 mmol) were added sequentially. The mixture was stirred at room temperature for 2.5 hours. The reaction mixture was neutralized with saturated aqueous sodium bicarbonate, diluted with ethyl acetate, and water, and the organic layer was separated. The resulting organic layer was washed sequentially with saturated aqueous sodium bicarbonate and saturated brine, dried over anhydrous magnesium sulfate, and evaporated under reduced pressure. The resulting intermediate was azeotropically dried with acetonitrile and dissolved in acetonitrile (180 mL) under a nitrogen stream. Thymine (4.09 g, 32.46 mmol) and N,O-bis(trimethylsilyl)acetamide (21.4 mL, 86.56 mmol) were added sequentially, and the mixture was stirred at 80°C for 10 minutes. After the reaction mixture was cooled to 45°C, trimethylsilyl trifluoromethanesulfonate (4.6 mL, 25.97 mmol) was added and the mixture was stirred at 88°C for 1 hour. The reaction mixture was ice-cooled and quenched with saturated aqueous sodium bicarbonate. The mixture was then diluted with ethyl acetate and water, and the organic layer was separated. The resulting organic layer was washed with saturated brine, dried over anhydrous magnesium chloride, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane:ethyl acetate=3:2) to give compound (R)MT-7 (11.74 g, 87%) as a white foamy solid. 1H NMR(CDCl3)δ 1.29 (3H, d, J=7), 1.49 (3H, d, J=1), 2.02 (3H, s), 2.43 (3H, s), 3.56, 3.76 (2H, ABq, J=10), 4.46-4.63 (5H, m), 5.10 (1H, q, J=7), 5.43 (1H, dd, J=6, 7), 6.23 (1H, d, J=8), 7.26-7.42 (13H, m), 7.69-7.72 (2H, m), 8.05 (1H, s).

[0223] (Synthesis of Compound (R)MT-8) Under a nitrogen atmosphere, (R)MT-7 (11.72 g, 17.27 mmol) was dissolved in tetrahydrofuran (195 mL), and 40% aqueous methylamine solution (26 mL, 307.0 mmol) was added under ice-cooling. The mixture was stirred for 5 hours while still ice-cooled. The reaction mixture was evaporated under reduced pressure, then diluted with ethyl acetate and water, and the organic layer was separated. The resulting organic layer was washed with saturated saline, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The resulting intermediate was dissolved in pyridine (110 mL) under a nitrogen stream, and methanesulfonyl chloride (1.9 mL, 24.95 mmol) was added under ice-cooling. The mixture was stirred at room temperature for 2 hours. The reaction mixture was diluted with ethyl acetate, water, and saturated saline, and the organic layer was separated. The resulting organic layer was washed with saturated saline, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The resulting intermediate was dissolved in ethanol (340 mL) and water (170 mL), and 1M aqueous sodium hydroxide solution (115 mL, 115.0 mmol) was added. The mixture was stirred at room temperature for 14 hours. The reaction mixture was neutralized with 1N hydrochloric acid, and then evaporated under reduced pressure. The resulting residue was diluted with ethyl acetate and water, and the organic layer was separated. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane:ethyl acetate = 1:1 to 1:2) to give compound (R) MT-8 (9.86 g, 89%) as a white foamy solid. 1H NMR(CDCl3)δ 1.33 (3H, d, J=7), 1.62 (3H, s), 2.43 (3H, s), 3.58, 3.85 (2H, ABq, J=10), 4.11-4.15 (1H, m), 4.29 (1H, d, J=3), 4.44-4.45 (1H, m), 4.51 (2H, s), 4.57, 4.73 (2H, ABq, J=12), 5.06 (1H, q, J=6), 6.09 (1H, d, J=4), 7.23-7.41 (13H, m), 7.72-7.74 (2H, m), 8.77(1H, s).

[0224] (Synthesis of Compound (R)MT-9) Compound (R)MT-8 (4.00 g, 6.03 mmol) was dissolved in ethanol (80 mL) under a nitrogen atmosphere. Cyclohexene (8.9 mL, 87.76 mmol) and 20% palladium hydroxide-carbon powder (2.0 g) were added sequentially, and the mixture was heated to reflux for 40 minutes. Cyclohexene (8.9 mL, 87.76 mmol) was then added, and the mixture was heated to reflux for 55 minutes. Cyclohexene (4.0 mL, 39.44 mmol) was then added, and the mixture was heated to reflux for 25 minutes. The reaction mixture was filtered, and the filtrate was evaporated under reduced pressure. The resulting intermediate was dissolved in N,N-dimethylformamide (56 mL) under a nitrogen stream. 1,3-Dichloro-1,1,3,3-tetraisopropyldisiloxane (2.4 mL, 7.54 mmol) and 2.9 M imidazole in N,N-dimethylformamide (7.7 mL) were added sequentially under ice cooling, and the mixture was stirred at room temperature for 16 hours. The reaction mixture was quenched with methanol, diluted with diethyl ether and water, and separated into organic and aqueous layers. The aqueous layer was back-extracted with diethyl ether. The organic layer from the first fraction and the organic layer from the back-extraction were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane:ethyl acetate = 7:3 to 1:1) to give compound (R)MT-9 (2.76 g, 65% yield) as a white foamy solid. 1H NMR(CDCl3)δ 0.91-1.16 (28H, m), 1.47 (3H, d, J=6), 1.86 (3H, d, J=2), 2.45 (3H, s), 3.81-3.84 (2H,m), 3.94 (1H, ABq, J=12), 4.53 (1H, d, J=8), 4.66-4.72 (1H, m), 5.09 (1H, q, J=6), 6.05 (1H, d, J=7), 7.30-7.34 (3H, m), 7.80-7.82 (2H, m), 9.14 (1H, s).

[0225] (Synthesis of compound (R)MT-10) Under a nitrogen stream, (R)MT-9 (0.74 g, 1.06 mmol) was dissolved in dichloromethane (7.5 mL). Pyridine (0.41 mL, 5.07 mmol), 4-dimethylaminopyridine (0.39 g, 3.23 mmol), and trifluoromethanesulfonic anhydride (0.45 mL, 2.70 mmol) were added sequentially under ice-cooling, and the mixture was stirred for 3.5 hours. 4-Dimethylaminopyridine (0.13 g, 1.07 mmol) was then added, and the mixture was stirred for 3 hours under ice-cooling. The reaction mixture was quenched with saturated brine, diluted with dichloromethane and water, and separated into organic and aqueous layers. The aqueous layer was back-extracted with dichloromethane. The organic layer from the first fraction and the organic layer from the back-extraction were combined, washed sequentially with 1N hydrochloric acid, saturated aqueous sodium bicarbonate, and saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The resulting intermediate was dissolved in dimethyl sulfoxide (9.4 mL) under a nitrogen atmosphere. N-hydroxyphthalimide (0.48 g, 2.91 mmol) and 1,8-diazabicyclo[5.4.0]-7-undecene (0.42 mL, 2.80 mmol) were added and the mixture was stirred at room temperature for 86 hours. The reaction mixture was diluted with diethyl ether and water and fractionated into an organic layer and an aqueous layer. The aqueous layer was back-extracted with diethyl ether. The organic layer from the first fraction and the organic layer from the back-extraction were combined, washed sequentially with water and saturated brine, and then dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (hexane:ethyl acetate = 2:1 to 1:1) to obtain compound (R)MT-10 (0.33 g, 36% yield) as a white foamy solid. 1 H NMR(CDCl3)δ 1.01-1.21 (28H, m), 1.67 (3H, d, J=7), 1.85 (3H, d, J=1), 2.33 (3H, s), 3.92, 3.97 (2H, ABq, J=11), 4.90 (1H, dd, J=1, 8), 5.18 (1H, d, J=8), 5.43 (1H, q, J=7), 6.09 (1H, d, J=1), 7.20-7.23 (2H, m), 7.76-7.86 (7H, m).

[0226] <Synthesis of Compound (R)MT-1> Under a nitrogen stream, (R)MT-10 (0.26 g, 0.30 mmol) was dissolved in pyridine (3 mL), and hydrazine monohydrochloride (0.037 g, 0.54 mmol) and 1,8-diazabicyclo[5.4.0]-7-undecene (0.080 mL, 0.54 mmol) were added. The mixture was stirred at room temperature for 15 hours. The reaction mixture was diluted with ethyl acetate and water, and the organic and aqueous layers were separated. The aqueous layer was back-extracted with ethyl acetate. The organic layer from the first fraction and the organic layer from the back-extraction were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The resulting intermediate was azeotropically dried with toluene and dissolved in N,N-dimethylformamide (8.0 mL) under a nitrogen stream. 1,8-diazabicyclo[5.4.0]-7-undecene (0.32 mL, 2.14 mmol) was added and the mixture was stirred at 60°C for 1 hour and at 70°C for 23 hours. The reaction mixture was diluted with ethyl acetate and water and fractionated into organic and aqueous layers. The aqueous layer was back-extracted with ethyl acetate. The organic layer from the first fraction and the organic layer from the back-extraction were combined and washed with saturated brine, then dried over anhydrous sodium sulfate and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane:ethyl acetate = 1:1 to 1:2) to give compound (R)MT-1 (0.092 g, 55% yield) as a white foamy solid.

[0227] (Synthesis of Compound (R)MT-2) To a 0.5 M solution of compound (R)MT-1 (0.53 g, 0.98 mmol) in 0.5 M pyridinium p-toluenesulfonate in methanol (7.2 mL) was added 20% aqueous formaldehyde (0.31 mL, 2.0 mmol) under ice-cooling. Subsequently, sodium cyanoborohydride (0.11 g, 1.80 mmol) was added while still ice-cooling, and the mixture was stirred for 1 hour under ice-cooling. The reaction mixture was diluted with ethyl acetate, water, and saturated brine, and the organic layer was fractionated. The resulting organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane:ethyl acetate = 3:1 to 1:1) to give compound (R)MT-2 (0.48 g, 89%) as a white foamy solid. 1 H NMR (CDCl3) δ 0.93 (3H, d, J=6), 1.03-1.12 (28H, m), 1.90 (3H, d, J=1), 2.72 (3H, s), 2.94 (1H, q, J=6 Hz), 3.62, 4.10 (2H, AB, J=13), 4.02 (1H, d, J=3 Hz), 4.28 (1H, d, J=3 Hz), 6.23 (1H, s), 7.70 (1H, d, J=1), 8.35 (1H, s).

[0228] (Synthesis of Compound (R)MT-3) Compound (R)MT-2 (0.60 g, 1.02 mmol) was dissolved in tetrahydrofuran (6 mL), and tetra-n-butylammonium fluoride (1 M tetrahydrofuran solution, 2.3 mL, 2.30 mmol) was added. The mixture was stirred at room temperature for 15 minutes. The resulting reaction mixture was evaporated under reduced pressure, and the reaction residue was removed by silica gel column chromatography (ethyl acetate:methanol = 30:1 to 15:1) to obtain the intermediate. The resulting intermediate was azeotropically dried with pyridine and dissolved in pyridine (4.5 mL) under a nitrogen stream. 4,4'-Dimethoxytrityl chloride (0.52 g, 1.52 mmol) was added, and the mixture was stirred at room temperature for 15 hours. Methanol was added to quench the reaction, and the mixture was diluted with ethyl acetate and water. The organic layer was separated. The resulting organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The obtained crude product was purified by silica gel column chromatography (hexane:ethyl acetate=1:1) to obtain compound (R) MT-3 (0.58 g, 90%) as a white foamy solid. 1 H NMR(CDCl3)δ 0.77 (3H, d, J=6 Hz), 1.35 (3H, d, J=1), 2.69 (1H, d, J=12), 2.71 (3H, s), 2.80 (1H, q, J=6), 3.22, 3.41 (2H, ABq, J=10), 3.80 (6H, d, J=1), 4.35 (1H, d, J=3 Hz), 4.57 (1H, dd, J=3, 10 Hz), 6.33 (1H, s), 6.83-6.87 (4H, m), 7.22-7.46 (9H, m), 7.86 (1H, d, J=1), 8.39 (1H, s).

[0229] (Synthesis of Compound (R)MT-4) Compound (R)MT-3 (0.54 g, 1.57 mmol) was azeotropically dried with acetonitrile under a nitrogen stream and then dissolved in acetonitrile (7 mL). 4,5-Dicyanoimidazole (0.22 g, 1.01 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (0.36 mL, 1.10 mmol) were added sequentially under ice cooling, and the mixture was stirred at room temperature for 5 hours. The reaction mixture was diluted with ethyl acetate and water, and the organic layer was fractionated. The resulting organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane:ethyl acetate = 2:1 to 1:1) to give compound (R)MT-4 (0.50 g, 70%) as a white foamy solid. 31 P NMR(CDCl3) δ 148.7, 149.0. HRMS(MALDI): calculated for C 43 H 55 N5O9P [M+H + ] 816.3732, found 816.3746.

[0230] [Synthesis Example 2] Base is a thymin-1-yl group, and A 1 is a single bond, and R 1 is DMTr and R 2 is -P(N(i-Pr)2)(OC2H4CN), and R 3 is a methyl group, and R 4 is an ethyl group in the R configuration, and R 5 Compound (1) in which is a hydrogen atom and n is 1 (hereinafter referred to as "compound (R)ET-4") was synthesized according to the following reaction scheme. [ka]

[0231] (Synthesis of Compound (R)ET-1) Compound T-1 (2.4 g, 4.63 mmol) was dissolved in methylene chloride (77 mL) under a nitrogen stream. Boron trifluoride-ethyl ether complex (1.5 mL, 24.39 mmol) and triethylborane (1 M in hexane, 12.2 mL, 24.39 mmol) were added at room temperature. After stirring for 5 minutes while bubbling air at room temperature, boron trifluoride-ethyl ether complex (1.5 mL, 24.39 mmol) and triethylborane (1 M in hexane, 12.2 mL, 24.39 mmol) were added. After stirring for 10 minutes while bubbling air at room temperature, boron trifluoride-ethyl ether complex (0.2 mL, 1.59 mmol) and triethylborane (1 M in hexane, 2.0 mL, 2.00 mmol) were added. The mixture was stirred for 5 minutes while bubbling air at room temperature. The reaction mixture was quenched with saturated aqueous sodium bicarbonate, diluted with dichloromethane and water, and separated into an organic layer and an aqueous layer. The aqueous layer was back-extracted with dichloromethane. The organic layer from the first fraction and the organic layer from the back-extraction were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane:ethyl acetate = 3:2 to 1:1) to give compound (R)ET-1 (2.05 g, 79% yield) as a white foamy solid. 1 H NMR(CDCl3)δ 0.92-1.13 (28H, m), 1.21-1.46, (2H, m), 1.92 (3H, d, J=1 Hz), 3.58 (1H, br), 3.70, 4.17 (2H, ABq, J=13 Hz), 4.13 (1H, d, J=4 Hz), 4.33 (1H, d, J=3 Hz), 5.51 (1H, br), 6.14 (1H, s), 7.73 (1H, d, J=1 Hz), 8.58 (1H, s).

[0232] (Synthesis of Compound (R)ET-2) Under a nitrogen stream, compound (R)ET-1 (0.97 g, 1.75 mmol) was dissolved in 0.5 M pyridium p-toluenesulfonate methanol solution (11.5 mL, 5.76 mmol). 20% aqueous formaldehyde (0.49 mL, 3.24 mmol) and sodium cyanoborohydride (0.18 mg, 2.88 mmol) were added sequentially under ice-cooling, and the mixture was stirred for 30 min. The reaction mixture was diluted with ethyl acetate, water, and saturated brine, and the organic layer was fractionated. The resulting organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane:ethyl acetate = 2:1 to 3:2) to give compound (R)ET-2 (0.92 g, 92%) as a white foamy solid. 1 H NMR(CDCl3)δ 0.91-1.13 (28H, m), 1.29-1.36, (1H, m), 1,57-1.68, (1H, m), 1.91 (3H, d, J=1 Hz), 2.74 (3H, s), 2.81 (1H, m), 3.73, 4.20 (2H, ABq, J=13 Hz), 3.98 (1H, d, J=3 Hz), 4.26 (1H, d, J=3 Hz), 6.26 (1H, s), 7.72 (1H, d, J=1 Hz), 8.34 (1H, s).

[0233] (Synthesis of Compound (R)ET-3) Compound (R)ET-2 (0.98 g, 0.20 mmol) was dissolved in tetrahydrofuran (20 mL), and tetra-n-butylammonium fluoride (1 M tetrahydrofuran solution, 4.0 mL, 3.99 mmol) was added. The mixture was stirred at room temperature for 20 minutes. The resulting reaction mixture was evaporated under reduced pressure, and the reaction residue was removed by silica gel column chromatography (ethyl acetate:methanol = 40:1 to 20:1) to obtain the intermediate. The resulting intermediate was azeotropically dried with pyridine and dissolved in pyridine (7 mL) under a nitrogen stream. 4,4'-Dimethoxytrityl chloride (0.77 g, 2.28 mmol) was added, and the mixture was stirred at room temperature for 15 hours. After the reaction was quenched by the addition of methanol, the mixture was diluted with ethyl acetate and water, and the organic and aqueous layers were separated. The aqueous layer was back-extracted with ethyl acetate. The organic layer from the first fraction and the organic layer from the back-extraction were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The obtained crude product was purified by silica gel column chromatography (hexane:ethyl acetate=2:1 to 1:4) to obtain compound (R)ET-3 (1.06 g, 91%) as a white foamy solid. 1 H NMR(CDCl3)δ 0.75 (3H, t, J=8 Hz), 1.07-1.15, 1.42-1.51 (2H, m), 1.30 (3H, s), 2.59 (1H, m), 2.63 (1H, m), 2.71 (3H, s), 3.26, 3.54 (2H, ABq, J=11), 3.79 (6H, d, J=1), 4.32 (1H, d, J=3 Hz), 4.51 (1H, dd, J=3, 9 Hz), 6.35 (1H, s), 6.84-6.87 (4H, m), 7.23-7.46 (9H, m), 7.89 (1H, s), 8.33 (1H, s).

[0234] (Synthesis of Compound (R)ET-4) Compound (R)ET-3 (1.03 g, 1.64 mmol) was azeotropically dried with acetonitrile under a nitrogen stream and then dissolved in acetonitrile (11 mL). 4,5-Dicyanoimidazole (0.22 g, 1.90 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (0.68 mL, 2.08 mmol) were added sequentially under ice cooling and stirred at room temperature for 4 hours. The reaction mixture was diluted with ethyl acetate and water, and the organic layer was separated. The resulting organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane:ethyl acetate = 1:1 to 2:3) to give compound (R)ET-4 (1.13 g, 82%) as a white foamy solid. 31 P NMR(CDCl3) δ 149.1, 150.5. HRMS(MALDI): calculated for C 46 H 56 F3N6NaO 10 P [M+H + ] 830.3888, found 830.3883.

[0235] [Synthesis Example 3] Base is an N-benzoyl-adenine-9-yl group, and A 1 is a single bond, and R 1 is DMTr and R 2 is -P(N(i-Pr)2)(OC2H4CN), and R 3 is a methyl group, and R 4 is an ethyl group in the R configuration, and R 5 Compound (1) in which is a hydrogen atom and n is 1 (hereinafter referred to as "compound (R)EA-3") was synthesized according to the following reaction scheme. [ka]

[0236] (Synthesis of Compound (R)EA-1) Compound (R)ET-2 (0.33 g, 0.57 mmol) was dissolved in toluene (6.7 mL) under a nitrogen atmosphere. 6 N,O-benzoyladenine (0.21 g, 0.88 mmol) and N,O-bis(trimethylsilyl)acetamide (0.87 mL, 3.50 mmol) were added sequentially and stirred at 90°C for 0.5 h. Subsequently, trimethylsilyl trifluoromethanesulfonate (0.15 mL, 0.82 mmol) was added and stirred at 90°C for 1 h. The reaction mixture was cooled on ice and quenched with saturated aqueous sodium bicarbonate. The mixture was then diluted with ethyl acetate and water. The reaction mixture was filtered through Celite, and the filtrate was collected and the organic layer was fractionated. The resulting organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane:ethyl acetate = 2:1 to 1:1) to obtain compound (R)EA-1 (0.27 g, 71%) as a white foamy solid. 1 H NMR(CDCl3)δ 0.98-1.15 (31H, m), 1.33-1.44 (1H, m), 1.65-1.74 (1H, m), 2.80 (3H, s), 2.91 (1H, m), 3.81, 4.20 (2H, ABq, J=13 Hz), 4.52 (1H, d, J=3 Hz), 4.64 (1H, d, J=3 Hz), 6.78 (1H, s), 7.51-7.56 (2H, m), 7.59-7.64 (1H, m), 8.01-8.04 (2H, m), 8.36 (1H, s), 8.83 (1H, s), 9.01 (1H, s).

[0237] (Synthesis of Compound (R)EA-2) Compound (R)EA-1 (0.30 g, 0.44 mmol) was dissolved in tetrahydrofuran (3 mL), and tetra-n-butylammonium fluoride (1 M tetrahydrofuran solution, 0.92 mL, 0.92 mmol) was added. The mixture was stirred at room temperature for 30 minutes. The resulting reaction mixture was evaporated under reduced pressure, and the reaction residue was removed by silica gel column chromatography (ethyl acetate:methanol = 30:1 to 10:1) to obtain the intermediate. The resulting intermediate was azeotropically dried with pyridine and dissolved in pyridine (2 mL) under a nitrogen stream. 4,4'-Dimethoxytrityl chloride (0.18 g, 0.53 mmol) was added, and the mixture was stirred at room temperature for 16 hours. The reaction mixture was ice-cooled, the reaction was quenched with methanol, and then diluted with water and ethyl acetate. The organic layer was fractionated. The resulting organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The obtained crude product was purified by silica gel column chromatography (hexane:ethyl acetate=1:2 to 0:1) to obtain compound (R)EA-2 (0.25 g, 75%) as a white solid. 1 H NMR(DMSO-d6)δ0.69 (3H, t, J=7 Hz), 1.05-1.13 (1H, m), 1.38-1.45 (1H, m), 2.69 (3H, s), 2.74 (1H, d, J=3 Hz), 3.05, 3.36 (2H, ABq, J=10 Hz), 3.72 (6H, d, J=2 Hz), 4.69 (1H, d, J=3 Hz), 4.82 (1H, dd, J=3, 6 Hz), 5.60 (1H, d, J=6 Hz), 6.66 (1H, s), 6.86 (4H, d, J=8 Hz), 7.19-7.30 (7H, m), 7.37-7.40 (2H, m), 7.52-7.57 (2H, m), 7.62-7.67 (1H, m), 8.03-8.05 (2H, d, J=7 Hz), 8.57 (1H, s), 8.80 (1H, s), 11.26 (1H, s).

[0238] (Synthesis of Compound (R)EA-3) Compound (R)EA-2 (0.20 g, 0.27 mmol) was azeotropically dried with acetonitrile under a nitrogen stream and dissolved in acetonitrile (3 mL) and tetrahydrofuran (2 mL). 4,5-Dicyanoimidazole (0.035 g, 0.30 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (0.11 mL, 0.32 mmol) were added sequentially and stirred at room temperature for 6 hours. 4,5-Dicyanoimidazole (0.018 g, 0.15 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (0.053 mL, 0.16 mmol) were then added sequentially and stirred for an additional 16 hours. The reaction was quenched with water, diluted with ethyl acetate, and the organic layer was separated. The resulting organic layer was washed with water and saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane:ethyl acetate = 3:2 to 1:2) to give compound (R)EA-3 (0.15 g, 61%) as a white foamy solid. 31 P NMR(CDCl3) δ 148.9, 149.1. HRMS(MALDI): calcd for C51H59N8NaO8P [M+Na + ] 965.4086, found 965.4096.

[0239] [Synthesis Example 4] Base is a thymin-1-yl group, and A 1 is a single bond, and R 1 is DMTr and R 2 is -P(N(i-Pr)2)(OC2H4CN), and R 4 Compound (1) in which is a methyl group and n is 0 (hereinafter referred to as "compound OM-T-3") was synthesized according to the following reaction scheme. [ka]

[0240] (Synthesis of compound OM-T-1) Under a nitrogen stream, compound (R)MT-1 (2.88 g, 5.31 mmol) was dissolved in dimethyl sulfoxide (58 mL), and 2-iodoxybenzoic acid (3.89 g, 5.84 mmol) was added at room temperature. The mixture was stirred at 60°C for 6 hours and then allowed to cool to room temperature for an additional 9 hours. The reaction mixture was cooled with water and quenched with saturated aqueous sodium bicarbonate. The mixture was then diluted with ethyl acetate and water, and the organic layer was separated. The resulting organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane:ethyl acetate = 2:1) to give compound OM-T-1 (2.12 g, 73%) as a white foamy solid. 1 H NMR(CDCl3)δ1.00-1.14 (28H, m), 1.91 (3H, s), 1.94 (3H, s), 3.89, 4.22 (2H, ABq, J=13), 4.45 (1H, d, J=4 Hz), 4.61 (1H, dd, J=4, 1), 5.86 (1H, s), 7.44 (1H, s), 8.40 (1H, s).

[0241] (Synthesis of compound OM-T-2) Compound OM-T-1 (1.36 g, 2.51 mmol) was dissolved in tetrahydrofuran (14 mL), and tetra-n-butylammonium fluoride (1 M tetrahydrofuran solution, 5.4 mL, 5.40 mmol) was added. The mixture was stirred at room temperature for 15 minutes. The resulting reaction mixture was evaporated under reduced pressure, and the reaction residue was removed by silica gel column chromatography (ethyl acetate:methanol = 30:1 to 10:1) to obtain the intermediate. The resulting intermediate was azeotropically dried with pyridine and dissolved in pyridine (25 mL) under a nitrogen stream. 4,4'-Dimethoxytrityl chloride (1.62 g, 4.78 mmol) was added, and the mixture was stirred at room temperature for 15 hours. Methanol was added to quench the reaction, and the mixture was diluted with water and ethyl acetate to separate the organic layer. The resulting organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. Dichloromethane was added to the obtained crude product, and the precipitated solid was collected by filtration to obtain compound OM-T-2 (1.01 g, 69%) as a white solid. 1 H NMR (CDCl3) δ 1.37 (3H, d, J=1), 1.66 (3H, s), 3.37 (2H, brs), 3.75 (6H, s), 4.62-4.64 (2H, m), 5.81 (1H, s), 6.13 (1H, d, J=4), 6.90-6.93 (4H, m), 7.23-7.36 (7H, m), 7.42-7.45 (2H, m), 7.55 (1H, d, J=1), 11.48 (1H, brs).

[0242] (Synthesis of compound OM-T-3) Compound OM-T-2 (0.40 g, 0.65 mmol) was azeotropically dried with acetonitrile under a nitrogen stream and dissolved in acetonitrile (4 mL). 4,5-Dicyanoimidazole (0.087 g, 0.73 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (0.26 mL, 0.80 mmol) were added sequentially under ice cooling, and the mixture was stirred at room temperature for 5 hours. The reaction mixture was ice-cooled, quenched with water, diluted with ethyl acetate and saturated brine, and the organic layer was fractionated. The resulting organic layer was washed sequentially with water and saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane:ethyl acetate = 2:1 to 1:1) to give compound OM-T-3 (0.42 g, 80%) as a white foamy solid. 31 P NMR(CDCl3)δ 149.4, 149.7. HRMS(MALDI): calcd for C42H50N5NaO9P [M+Na + ] 822.3238, found 822.3232.

[0243] [Synthesis Example 5] Base is a thymin-1-yl group, and A 1 is a single bond, and R 1 is DMTr and R 2 is -P(N(i-Pr)2)(OC2H4CN), and R 3 is a methyl group, and R 4 is a methyl group in the S configuration, and R 5 Compound (1) in which is a hydrogen atom and n is 1 (hereinafter referred to as "compound (S)MT-4") was synthesized according to the following reaction scheme. [ka]

[0244] (Synthesis of Compound (S)MT-1) Under a nitrogen stream, compound OM-T-1 (0.20 g, 0.37 mmol) was dissolved in toluene (4 mL), and 1.0 mol / L diisobutylaluminum hydride hexane solution (1.5 mL, 1.5 mmol) was added under dry ice / acetone cooling. The mixture was stirred for 2 hours while cooled. The reaction was quenched with saturated Rochelle salt solution, followed by dilution with ethyl acetate and water, and the organic layer was separated. The resulting organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane:ethyl acetate = 3:2 to 1:1) to give compound (S)MT-1 (0.046 g, 23%) as a white foamy solid. 1 H NMR(CDCl3)δ0.97-1.15 (28H, m), 1.46 (3H, d, J=7), 1.92 (3H, d, J=1), 3.08 (1H, q, J=7), 4.03 (2H, s), 4.18 (1H, d, J=2), 4.41 (1H, d, J=2), 6.06 (1H, s), 7.75 (1H, d, J=1), 8.46 (1H, s).

[0245] (Synthesis of Compound (S)MT-2) To a 0.5 M solution of compound (S)MT-1 (0.31 g, 0.57 mmol) in 0.5 M pyridinium p-toluenesulfonate in methanol (3.6 mL) was added 20% aqueous formaldehyde (0.16 mL, 1.0 mmol) under ice-cooling. Subsequently, sodium cyanoborohydride (0.058 g, 0.92 mmol) was added while still ice-cooling, and the mixture was stirred for 1 hour under ice-cooling. The reaction mixture was diluted with ethyl acetate, water, and saturated brine, and the organic layer was separated. The resulting organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane:ethyl acetate = 3:1 to 2:1) to give compound (S)MT-2 (0.29 g, 93%) as a white foamy solid. 1H NMR (CDCl3) δ 0.94-1.15 (28H, m), 1.32 (3H, d, J=7), 1.91 (3H, d, J=1), 2.68 (3H, s), 2.87 (1H, q, J=7 Hz), 3.97 (1H, d, J=3), 4.00, 4.08 (2H, ABq, J=13), 4.34 (1H, d, J=3 Hz), 6.24 (1H, s), 7.79 (1H, d, J=1), 8.40 (1H, s).

[0246] (Synthesis of Compound (S)MT-3) Compound (S)MT-2 (0.31 g, 0.56 mmol) was dissolved in tetrahydrofuran (4 mL), and tetra-n-butylammonium fluoride (1 M tetrahydrofuran solution, 1.2 mL, 1.20 mmol) was added. The mixture was stirred at room temperature for 15 minutes. The resulting reaction mixture was evaporated under reduced pressure, and the reaction residue was removed by silica gel column chromatography (ethyl acetate:methanol = 40:1 to 10:1) to obtain the intermediate. The resulting intermediate was azeotropically dried with pyridine and dissolved in pyridine (5.4 mL) under a nitrogen stream. 4,4'-Dimethoxytrityl chloride (0.32 g, 0.95 mmol) was added, and the mixture was stirred at room temperature for 15 hours. Methanol was added to quench the reaction, and the mixture was diluted with water and ethyl acetate, and the organic layer was fractionated. The obtained crude product was purified by silica gel column chromatography (hexane:ethyl acetate=1:2) to obtain compound (S) MT-3 (0.30 g, 88%) as a white foamy solid. 1H NMR (CDCl3) δ 1.04 (3H, d, J=7), 1.43 (3H, s), 2.67 (3H, s), 3.26 (1H, q, J=7), 3.40, 3.58 (2H, ABq, J=11), 3.80 (7H, s), 4.13 (1H, dd, J=3, 10), 4.42 (1H, d, J=3), 6.12 (1H, s), 6.82-6.87 (4H, m), 7.22-7.40 (7H, m), 7.46-7.49 (2H, m), 7.65 (1H, d, J=1), 8.35 (1H, s).

[0247] (Synthesis of Compound (S)MT-4) Compound (S)MT-3 (0.28 g, 0.46 mmol) was azeotropically dried with acetonitrile under a nitrogen stream and then dissolved in acetonitrile (4 mL). 4,5-Dicyanoimidazole (0.064 g, 0.54 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (0.19 mL, 0.58 mmol) were added sequentially under ice cooling, and the mixture was stirred at room temperature for 5 hours. The reaction mixture was diluted with ethyl acetate and water, and the organic layer was separated. The resulting organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane:ethyl acetate = 1:1) to give compound (S)MT-4 (0.26 g, 70%) as a white foamy solid. 31 P NMR(CDCl3)δ 149.1, 149.2. HRMS(MALDI): calcd for C43H54N5NaO9P [M+Na + ] 838.3551, found 838.3564.

[0248] [Synthesis Example 6] Base is a thymin-1-yl group, and A 1 is a single bond, and R 1 is DMTr and R 2 is -P(N(i-Pr)2)(OC2H4CN), and R4 Compound (1) in which is an ethyl group and n is 0 (hereinafter referred to as "compound OE-T-3") was synthesized according to the following reaction scheme. [ka]

[0249] (Synthesis of Compound OE-T-1) Compound (R)ET-1 (6.48 g, 11.66 mmol) was dissolved in dimethyl sulfoxide (102 mL) under a nitrogen atmosphere, and 2-iodoxybenzoic acid (9.00 g, 13.50 mmol) was added at room temperature. The mixture was stirred for 15 hours. The reaction mixture was cooled with water and quenched with saturated aqueous sodium hydrosulfite. The mixture was then diluted with ethyl acetate and water and separated into organic and aqueous layers. The aqueous layer was back-extracted with ethyl acetate. The organic layer from the first fraction and the organic layer from the back-extraction were combined and washed sequentially with saturated aqueous sodium bicarbonate and saturated brine, then dried over anhydrous sodium sulfate and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane:ethyl acetate = 3:1 to 3:2) to give compound OE-T-1 (3.10 g, 48%) as a white foamy solid. 1 H NMR(CDCl3)δ 0.92-1.14 (28H, m), 1.19 (3H, t, J=7), 1.91 (3H, s), 2.24 (2H, q, J=7), 3.94, 4.27 (2H, ABq, J=13 Hz), 4.44 (1H, d, J=4 Hz), 4.59 (1H, d, J=4 Hz), 5.88 (1H, s), 7.45 (1H, s), 8.41 (1H, s).

[0250] (Synthesis of Compound OE-T-2) Compound OE-T-1 (0.11 g, 0.20 mmol) was dissolved in tetrahydrofuran (2 mL), and tetra-n-butylammonium fluoride (1 M tetrahydrofuran solution, 0.61 mL, 0.61 mmol) was added. The mixture was stirred at room temperature for 20 minutes. The resulting reaction mixture was evaporated under reduced pressure, and the reaction residue was removed by silica gel column chromatography (ethyl acetate:methanol = 40:1 to 5:1) to obtain the intermediate. The resulting intermediate was azeotropically dried with pyridine and dissolved in pyridine (2 mL) under a nitrogen stream. 4,4'-Dimethoxytrityl chloride (0.18 g, 0.52 mmol) was added, and the mixture was stirred at room temperature for 19 hours. Methanol was added to quench the reaction, and the mixture was diluted with ethyl acetate and water to separate the organic layer. The resulting organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The obtained crude product was purified by silica gel column chromatography (hexane:ethyl acetate=1:2 to 1:3) to obtain compound OE-T-2 (0.11 g, 90%) as a white foamy solid. 1 H NMR(CDCl3)δ 1.01 (3H, t, J=7 Hz), 1.47 (3H, d, J=1), 2.02-2.15 (2H, m), 2.65 (1H, br), 3.46, 3.66 (2H, ABq, J=11), 3.80 (6H, d, J=1), 4.65 (1H, d, J=4 Hz), 4.78 (1H, br), 6.00 (1H, s), 6.83-6.88 (4H, m), 7.24-7.45 (9H, m), 7.61 (1H, d, J=1), 8.62 (1H, s).

[0251] (Synthesis of Compound OE-T-3) Compound OE-T-2 (0.26 g, 0.42 mmol) was azeotropically dried with acetonitrile under a nitrogen stream and dissolved in acetonitrile (3 mL). 4,5-Dicyanoimidazole (0.062 g, 0.52 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (0.20 mL, 0.61 mmol) were added sequentially under ice cooling, and the mixture was stirred at room temperature for 5 hours. The reaction mixture was ice-cooled, quenched with water, diluted with ethyl acetate, and the organic layer was separated. The resulting organic layer was washed sequentially with water and saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane:ethyl acetate = 1:1 to 1:2) to give compound OE-T-3 (0.30 g, 77%) as a white foamy solid. 31 P NMR(CDCl3) δ 149.1, 150.5. HRMS(MALDI): calcd for C43H52N5NaO9P [M+Na + ] 836.3395, found 836.3398.

[0252] [Synthesis Example 7] Base is a thymin-1-yl group, and A 1 is a single bond, and R 1 is DMTr and R 2 is -P(N(i-Pr)2)(OC2H4CN), and R 3 is a 3-(N,N-dimethylamino)propyl group, and R 4 is an ethyl group in the R configuration, and R 5 Compound (1) in which is a hydrogen atom (hereinafter referred to as "compound (R)EDM-T-2") was synthesized according to the following reaction scheme. [ka]

[0253] (Synthesis of Compound (R)ET-5) Compound (R)MT-1 (2.46 g, 4.42 mmol) was dissolved in tetrahydrofuran (30 mL) under a nitrogen stream, and tetra-n-butylammonium fluoride (1 M tetrahydrofuran solution, 11.0 mL, 11.0 mmol) was added. The mixture was stirred at room temperature for 30 minutes. The resulting reaction mixture was evaporated under reduced pressure, and the residue was removed by silica gel column chromatography (ethyl acetate:methanol = 10:1 to 8:1) to obtain the intermediate. The resulting intermediate was azeotropically dried with pyridine and dissolved in pyridine (30 mL) under a nitrogen stream. 4,4'-Dimethoxytrityl chloride (2.59 g, 7.64 mmol) was added, and the mixture was stirred at room temperature for 15 hours. The reaction was quenched with methanol, and the mixture was diluted with water and ethyl acetate. The organic and aqueous layers were separated. The aqueous layer was back-extracted with ethyl acetate. The organic layer from the first fraction and the organic layer from the back-extraction were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane:ethyl acetate = 1:4 to 0:1) to give compound (R)ET-5 (2.63 g, 96%) as a white foamy solid. 1 H NMR(CDCl3)δ 0.79 (3H, t, J=7), 1.09-1.19 (2H, m), 1.27 (3H, d, J=1), 3.05-3.07 (1H, m), 3.28, 3.43 (2H, ABq, J=11), 3.41-3.44 (1H, m), 3.79 (6H, d, J=1), 4.40 (1H, d, J=3), 4.68 (1H, m), 5.60 (1H, brs), 6.25 (1H, s), 6.84-6.87 (4H, m), 7.21-7.35 (7H, m), 7.42-7.45 (2H, m), 8.33 (1H, d, J=1), 8.82 (1H, s).

[0254] (Synthesis of compound (R)EDM-T-1) Under a nitrogen stream, 3-(dimethylamino)-1-propanol (0.68 mL, 5.80 mmol) was slowly added dropwise to a toluene suspension (6.8 mL) of sodium hydride (60% in oil, 0.17 g, 4.14 mmol) under ice cooling, and the mixture was stirred for 20 minutes. Subsequently, p-toluenesulfonyl chloride (0.79 g, 4.14 mmol) was added in two portions, and the mixture was stirred at room temperature for 2 hours. The reaction was quenched with water, diluted with saturated brine and toluene, and separated into organic and aqueous layers. The aqueous layer was back-extracted with toluene. The organic layer from the first fraction and the organic layer from the back-extraction were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure until the residue became slightly cloudy. The resulting toluene solution of 3-(dimethylamino)-1-propyl p-toluenesulfonate was used directly in the next reaction. Under a nitrogen stream, (R)ET-5 (1.63 g, 2.65 mmol) was dissolved in toluene (14 mL) and N,N-diisopropylethylamine (1.1 mL, 6.35 mmol) was added at room temperature. The reaction mixture was heated to 100 °C, and the previously prepared toluene solution of 3-(dimethylamino)-1-propyl p-toluenesulfonate was added dropwise over 20 min. The mixture was stirred at 100 °C for an additional 2 h. The reaction mixture was then returned to room temperature, and the reaction solvent was removed under reduced pressure. The resulting crude product was purified by silica gel column chromatography (ethyl acetate:triethylamine:methanol = 20:1:0 to 20:1:1.5) to obtain compound (R)EDM-T-1 (0.27 g, 14%) as a white foamy solid. 1H NMR(CDCl3)δ 0.73 (3H, t, J=8), 1.01-1.13, 1.43-1.52 (2H, m), 1.28 (3H, s), 1.65-1.74, 1.83-1.92 (2H, m), 2.21 (6H, s), 2.27-2.36, 2.41-2.49 (2H, m), 2.73-2.80 (2H, m), 3.04-3.13 (1H, m), 3.27, 3.53 (2H, ABq, J=11), 3.80 (6H, s), 4.31 (1H, d, J=3), 4.50 (1H, d, J=3), 6.31 (1H, s), 6.83-6.87 (4H, m), 7.21-7.37 (7H, m), 7.44-7.47 (2H, m), 7.87 (1H, s).

[0255] (Synthesis of Compound (R)EDM-T-2) Compound (R)EDM-T-1 (0.41 g, 0.59 mmol) was dried azeotropically with acetonitrile under a nitrogen stream and then dissolved in acetonitrile (5 mL). 4,5-Dicyanoimidazole (0.078 g, 0.66 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (0.24 mL, 0.72 mmol) were added sequentially under ice cooling, and the mixture was stirred at room temperature for 6 hours. Next, 4,5-dicyanoimidazole (0.039 g, 0.33 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (0.12 mL, 0.36 mmol) were added at room temperature and stirred for 16 hours. 4,5-dicyanoimidazole (0.039 g, 0.33 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (0.12 mL, 0.36 mmol) were then added at room temperature and stirred for 1.5 hours. The reaction was quenched with water, diluted with ethyl acetate and saturated brine, and the organic layer was separated. The resulting organic layer was washed sequentially with water and saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The obtained crude product was purified by silica gel column chromatography (ethyl acetate:triethylamine:methanol=20:1:0 to 20:1:1) to obtain compound (R)EDM-T-2 (0.30 g, 55%) as a white foamy solid. 31 P NMR(CDCl3) δ 148.9, 149.1. HRMS(MALDI): calcd for C48H65N6NaO9P [M+Na + ] 923.4443, found 923.4420.

[0256] <<Oligonucleotide synthesis example>> The oligonucleotides were synthesized using the compounds obtained in the above synthesis examples ((R)MT-4, (R)ET-4, (S)MT-4, OM-T-3, OE-T-3, (R)EDM-T-2) according to the standard phosphoramidite protocol using an automated nucleic acid synthesizer (Expedite™ 8909 / ABI) to obtain the following oligonucleotides. In formula (6), Base is a thymin-1-yl group, and A 1 is a single bond, n is 1, and R 3 is a methyl group, and R 4 is an ethyl group in the R configuration, and R 5 is a hydrogen atom (hereinafter referred to as (R)ET). In formula (6), Base is a thymin-1-yl group, and A 1 is a single bond, n is 1, and R 3 is a methyl group, and R 4 is a methyl group in the R configuration, and R 5 is a hydrogen atom (hereinafter referred to as (R)MT). In formula (6), Base is a thymin-1-yl group, and A 1 is a single bond, n is 0, and R 4 An oligonucleotide having a unit in which is an ethyl group (hereinafter referred to as OE-T). In formula (6), Base is a thymin-1-yl group, and A 1 is a single bond, n is 1, and R 3 is a methyl group, and R 4 is a methyl group in the S configuration, and R 5 is a hydrogen atom (hereinafter referred to as (S)MT). In formula (6), Base is a thymin-1-yl group, and A 1 is a single bond, n is 0, and R 4 An oligonucleotide having a unit in which is a methyl group (hereinafter referred to as OM-T). In formula (6), Base is a thymin-1-yl group, and A 1 is a single bond, n is 1, and R 3 is a 3-(N,N-dimethylamino)propyl group, and R 4 is an ethyl group in the R configuration, and R 5 is a hydrogen atom (hereinafter referred to as (R)EDM-T). The oligonucleotides, each protected at the 5'-end with a dimethoxytrityl group and supported on a solid phase, were cleaved from the column with 28% aqueous ammonia (1.5 hours), and the cleaved oligonucleotides were reacted in 28% aqueous ammonia for 16 hours at 60°C to deprotect all protecting groups. A simple purification was performed using a NAP-10 column, followed by purification by reverse-phase HPLC (WakoPak™ WS-DNA column, 10.0 mm × 250 mm) [conditions: 30-minute 8-16% acetonitrile gradient at 3 ml / min in 0.1 M triethylammonium acetate buffer (pH 7.0), column temperature 50°C]. The purity of the synthesized oligonucleotides was confirmed by reverse-phase HPLC (WakoPak™ WS-DNA column, 4.6 mm × 250 mm) under the following conditions: 0.1 M triethylammonium acetate buffer (pH 7.0), 30 min gradient of 8-16% acetonitrile at 1 ml / min, column temperature 50°C, detection wavelength 254 nm. All synthesized oligonucleotides were 90% or higher in purity. The molecular weights of the synthesized oligonucleotides were determined by MALDI-TOF-MASS measurement. The calculated and measured molecular weights (measurement results) are shown in the table below. The unit represented by formula (6) is incorporated at position n of the antisense strand (SEQ ID NOs: 1 to 5) in the table below, and the base is a thymin-1-yl group. All base sequences other than n are DNA (formula (7)R a ═H). In addition, as a control, a unit represented by formula (9) is present at the position n, in which the base is a thymin-1-yl group and R b The calculated and measured molecular weights of oligonucleotides incorporating units where is Me or H (hereinafter referred to as NMe-T or NH-T, respectively, in the table) are also shown. [Table 1]

[0257] <<Test Example>> [Test Example 1] Measurement of melting temperature (Tm) of oligonucleotides (evaluation of duplex formation ability) Six types of oligonucleotides (6) of the present invention ((R)ET, OE-T, (R)MT, (S)MT, OM-T, (R)EDM-T) (antisense strands) were synthesized by incorporating the compounds obtained in the above synthesis examples ((R)ET-4, OE-T-3, (R)MT-4, (S)MT-4, OM-T-3, (R)EDM-T-2) into the n position of the sequence shown in SEQ ID NO: 4. The melting temperatures (Tm) of these oligonucleotides with single-stranded DNA (5'-d(agcaaaaaacgc)-3'; SEQ ID NO: 6 (sense strand)) or single-stranded RNA (5'-r(agcaaaaaacgc)-3'; SEQ ID NO: 7 (sense strand)) were measured to examine the duplex forming ability of the antisense strand. As a control, a unit represented by formula (7) in which the base is a thymin-1-yl group and R a is H) is located at the n position of SEQ ID NO: 4, and a unit represented by formula (9) in which Base is a thymine 1-yl group and R b An oligonucleotide (NMe-T) synthesized by incorporating a unit where n is Me at the n position of SEQ ID NO: 4 was prepared as the antisense strand. A sample solution (120 μl) was prepared with final concentrations of 100 mM sodium chloride, 10 mM sodium phosphate buffer (pH 7.2), 4 μM antisense strand, and 4 μM sense strand. The temperature was raised from 15°C to 110°C at a rate of 0.5°C / min, and the absorbance at 260 nm was measured at 0.5°C intervals using a spectrophotometer (Shimadzu UV-1800). The Tm values were calculated from the measured values using the differential method. The results are shown in the table below.

[0258] Ability to form a double strand with single-stranded DNA (Tm value) [Table 2]

[0259] Duplex formation ability with single-stranded RNA (Tm value) [Table 3]

[0260] It has been revealed that the oligonucleotide (6) of the present invention has excellent duplex formation ability with both single-stranded DNA and single-stranded RNA, particularly with single-stranded RNA. Therefore, the oligonucleotide (6) of the present invention is suitable for use in nucleic acid medicines and genetic diagnosis targeting DNA or RNA, which require excellent duplex formation ability.

[0261] [Test Example 2] Measurement of enzyme resistance of oligonucleotides 1) Preparation of oligonucleotides for enzyme resistance measurement In the same manner as in the above oligonucleotide synthesis example, the oligonucleotides in the table below having the sequence shown in SEQ ID NO: 8 (TTTTTTTTTT) or a partially modified version of the sequence shown in SEQ ID NO: 5 were prepared. [Table 4]

[0262] 2) Preparation of sample solution The sample solutions were prepared as shown in the table below. [Table 5]

[0263] 3) Enzyme reaction Oligonucleotides Nos. 1 to 7 were subjected to Procedure A ((1) to (4) below) at 37°C using an apparatus (Major Science, MD-MINI). (1) The sample solution was incubated (5 minutes). (2) The enzyme CAVP (Crotalus adamanteus Venom phosphodiesterase I) was added to a final concentration of 1.60 μg / mL or 5.00 μg / mL to initiate the reaction. (3) At the end of the reaction time, EDTA was added to the reaction solution to make the concentration 5.0 mM to stop the reaction. (4) The reaction times were 0, 5, 10, 40, and 80 minutes. For oligonucleotides Nos. 1, 2, 4, 6, and 8 to 10, procedure B was carried out in the same manner as procedure A above, except that in procedure A(2), the enzyme CAVP was added to a final concentration of 4.38 μg / mL to initiate the reaction.

[0264] 4) Evaluation of enzyme resistance The sample solution after the enzyme reaction in Procedure A was subjected to HPLC analysis under the following conditions. (conditions) Equipment: LC-2010A HT (Shimadzu Corporation) Column: XBridge Oligonucleoties BEH C18 Column, 130Å, 2.5μm, 4.6mm x 50mm. Mobile phase A: 0.1M triethylammonium acetate buffer (pH 7.0). Solution B: 0.1 M triethylammonium acetate buffer (pH 7.0):acetonitrile = 1:1 (v / v) Gradient: 5 to 30% ((v / v) Solution B), 15 min) Flow rate: 0.8mL / min Column temperature: 50℃ Detection wavelength: 268 nm Injection volume: 15μL (101.2pmol) The amount of enzyme-undigested oligonucleotide was measured from the HPLC analysis results, and the remaining rate (%) of undigested oligonucleotide at each reaction time was calculated using the following formula.

number

[0265] 5) Results The results when the final concentrations of the enzyme CAVP were 5.00 μg / mL, 1.60 μg / mL, and 4.38 μg / mL are shown in Table 6A and FIG. 1A, Table 6B and FIG. 1B, and Table 6C and FIG. 1C, respectively.

[0266] [Table 6A]

[0267] [Table 6B]

[0268] [Table 6C]

[0269] As is clear from the results, the oligonucleotide (6) of the present invention had superior enzyme resistance compared to natural and other non-natural oligonucleotides.

Claims

1. A compound represented by the following formula (1') or a salt thereof: 【Chemical 1】 [In the formula, Base represents an optionally substituted aromatic heterocyclic group or an optionally substituted aromatic hydrocarbon ring group, A １ is a single bond or an alkylene group, R １ and R ２ are the same or different and are a hydrogen atom, an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted cycloalkyl group, an optionally substituted cycloalkenyl group, an optionally substituted aryl group, an optionally substituted acyl group, a substituted sulfonyl group, a substituted silyl group, a substituted phosphino group, an optionally substituted dihydroxyphosphinyl group, or an optionally substituted hydroxymercaptophosphinyl group, or R １ and R ２ forms a ring, which may have a substituent, together with the two adjacent oxygen atoms and the carbon atoms at positions 3 to 5 of the furanose, R ３ is represented by the formula: R ３１ is a group represented by -X-, R ３１ is represented by the following formula (A): 【Chemistry 2】 (In the formula, R 3a and R 3b are the same or different and each represent a hydrogen atom, an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted cycloalkyl group, an optionally substituted cycloalkenyl group, an optionally substituted aryl group, an optionally substituted acyl group, an optionally substituted N,N-dialkylformamidyl group, an optionally substituted alkoxycarbonyl group, an optionally substituted alkenyloxycarbonyl group, an optionally substituted aryloxycarbonyl group, an optionally substituted aralkyloxycarbonyl group, or a substituted sulfonyl group, or R 3a and R 3b together with the adjacent nitrogen atom form a ring which may be substituted.) or a group represented by the following formula (B): 【Chemistry 3】 (In the formula, R 3c to R 3f are the same or different and each represent a hydrogen atom, an alkyl group, an acyl group which may have a substituent, an N,N-dialkylformamidyl group which may have a substituent, an alkoxycarbonyl group which may have a substituent, an alkenyloxycarbonyl group which may have a substituent, an aryloxycarbonyl group which may have a substituent, an aralkyloxycarbonyl group which may have a substituent, or a sulfonyl group which has a substituent.) is a group represented by X is —C m H 2m — (wherein m is an integer from 1 to 10); R ４ represents a hydrogen atom, an optionally substituted alkyl group, or an optionally substituted aryl group, R ５ represents a hydrogen atom, an optionally substituted alkyl group, or an optionally substituted aryl group, R ４ and R ５ is not a hydrogen atom at the same time.

2. A compound represented by the following formula (1J) or a salt thereof: 【Chemistry 4】 [In the formula, Base represents an optionally substituted aromatic heterocyclic group or an optionally substituted aromatic hydrocarbon ring group, A 1 is a single bond or an alkylene group; R 1 and R 2 are the same or different and each represent a hydrogen atom, an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted cycloalkyl group, an optionally substituted cycloalkenyl group, an optionally substituted aryl group, an optionally substituted acyl group, a substituted sulfonyl group, a substituted silyl group, a substituted phosphino group, an optionally substituted dihydroxyphosphinyl group, or an optionally substituted hydroxymercaptophosphinyl group, or R 1 and R 2 together with the two adjacent oxygen atoms and the carbon atoms at positions 3 to 5 of the furanose form a ring which may be substituted; R 3 is a hydrogen atom, an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted cycloalkyl group, an optionally substituted aryl group, an optionally substituted acyl group, a substituted sulfonyl group, a substituted silyl group, or a group represented by the formula: R 31 -X- (wherein R 31 is an optionally substituted amino group, and X is an optionally substituted alkylene group, or a group in which at least one methylene group in the alkylene group is replaced by -N(R 32 )- (wherein R 32 is a hydrogen atom or an alkyl group), -O-, or -S(═O) k - (wherein k is 0, 1, or 2)); R 4 is an alkyl group which may have a substituent, or an aryl group which may have a substituent.

3. A １ The compound or salt thereof according to claim 1 or 2, wherein is a single bond.

4. R ４ The compound or salt thereof according to any one of claims 1 to 3, wherein is an alkyl group.

5. R ５ The compound or salt thereof according to claim 1, wherein is a hydrogen atom or an alkyl group.

6. R ３ is a hydrogen atom, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, an alkylsulfonyl group, an arylsulfonyl group, or a group of the formula: —Si(R ６ ) ３ (In the formula, each R ６ and are the same or different and are an alkyl group or an aryl group, or a salt thereof.

7. R １ and R ２ are the same or different and are a hydrogen atom, an optionally substituted alkyl group, an optionally substituted aryl group, an alkylcarbonyl group, an arylcarbonyl group, an alkylsulfonyl group, an arylsulfonyl group, a group of the formula: —Si(R ６ ) ３ (In the formula, each R ６ are the same or different and are an alkyl group or an aryl group), a group represented by the formula: -P(R ７ )(R ８ ) (wherein, R ７ and R ８ are the same or different and are a hydroxyl group, a mercapto group, an amino group, an alkoxy group, a haloalkoxy group, a cyanoalkoxy group, an alkylthio group, a haloalkylthio group, a cyanoalkylthio group, or an alkylamino group), a dihydroxyphosphinyl group, or a hydroxymercaptophosphinyl group, or R １ and R ２ form a ring which may have a substituent together with the two adjacent oxygen atoms and the carbon atoms at positions 3 to 5 of the furanose. The compound or salt thereof according to any one of claims 1 to 6.

8. The compound or salt thereof according to any one of claims 1 to 7, wherein Base is an optionally substituted 2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl group, an optionally substituted 2-oxo-1,2-dihydropyrimidin-1-yl group, an optionally substituted purin-9-yl group, or an optionally substituted 6-oxo-1,6-dihydro-9H-purin-9-yl group.

9. In the compound represented by formula (1') or a salt thereof according to claim 1, R ５ is a hydrogen atom or a salt thereof, (I) The following formula (1E): 【Chemistry 5】 (In the formula, Base, A １ , R １ , and R ２ is the same as claim 1) The compound represented by the formula: R ４ ・(In the formula, R ４ is the same as in claim 1), or a radical represented by the formula: R ４ M (wherein M is a metal atom or an atomic group containing a metal atom, R ４ is the same as in claim 1), and (II) The compound obtained in the step (I) or the compound obtained by dehydrogenating and then hydrogenating the compound obtained in the step (I) is reacted with a compound of the formula: R ３ -L (wherein L is a leaving group and R ３ is the same as in claim 1) A method comprising:

10. Among the compounds represented by formula (1J) or salts thereof according to claim 2, R ３ is a methyl group optionally having one or two substituents, or a salt thereof, comprising the steps of: (I) The following formula (1E): 【Chemistry 6】 (In the formula, Base, A １ , R １ , and R ２ is the same as claim 2) The compound represented by the formula: R ４ ・(In the formula, R ４ is the same as in claim 2), or a radical represented by the formula: R ４ M (wherein M is a metal atom or an atomic group containing a metal atom, R ４ is the same as in claim 2), and (II) A step of reacting the compound obtained in the step (I) with a carbonyl compound. A method comprising:

11. The following formula (6'): 【Chemistry 7】 [In the formula, Base represents an optionally substituted aromatic heterocyclic group or an optionally substituted aromatic hydrocarbon ring group, A １ is a single bond or an alkylene group, R ３ is represented by the formula: R ３１ is a group represented by -X-, R ３１ is represented by the following formula (A): 【Chemistry 8】 (In the formula, R 3a and R 3b are the same or different and each represent a hydrogen atom, an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted cycloalkyl group, an optionally substituted cycloalkenyl group, an optionally substituted aryl group, an optionally substituted acyl group, an optionally substituted N,N-dialkylformamidyl group, an optionally substituted alkoxycarbonyl group, an optionally substituted alkenyloxycarbonyl group, an optionally substituted aryloxycarbonyl group, an optionally substituted aralkyloxycarbonyl group, or a substituted sulfonyl group, or R 3a and R 3b together with the adjacent nitrogen atom form a ring which may be substituted.) or a group represented by the following formula (B): 【Chemistry 9】 (In the formula, R 3c to R 3f are the same or different and each represent a hydrogen atom, an alkyl group, an acyl group which may have a substituent, an N,N-dialkylformamidyl group which may have a substituent, an alkoxycarbonyl group which may have a substituent, an alkenyloxycarbonyl group which may have a substituent, an aryloxycarbonyl group which may have a substituent, an aralkyloxycarbonyl group which may have a substituent, or a sulfonyl group which has a substituent.) is a group represented by X is —C m H 2m — (wherein m is an integer from 1 to 10); R ４ represents a hydrogen atom, an optionally substituted alkyl group, or an optionally substituted aryl group, R ５ represents a hydrogen atom, an optionally substituted alkyl group, or an optionally substituted aryl group, R ４ and R ５ is not a hydrogen atom at the same time. An oligonucleotide or a salt thereof having a unit represented by the following formula:

12. The following formula (6J): 【Chemistry 10】 [In the formula, Base represents an optionally substituted aromatic heterocyclic group or an optionally substituted aromatic hydrocarbon ring group, A 1 is a single bond or an alkylene group; R 3 is a hydrogen atom, an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted cycloalkyl group, an optionally substituted aryl group, an optionally substituted acyl group, a substituted sulfonyl group, a substituted silyl group, or a group represented by the formula: R 31 -X- (wherein R 31 is an optionally substituted amino group, and X is an optionally substituted alkylene group, or a group in which at least one methylene group in the alkylene group is replaced by -N(R 32 )- (wherein R 32 is a hydrogen atom or an alkyl group), -O-, or -S(═O) k - (wherein k is 0, 1, or 2)); R 4 is an alkyl group which may have a substituent, or an aryl group which may have a substituent. An oligonucleotide or a salt thereof having a unit represented by the following formula:

13. A method for detecting a target nucleic acid, comprising: (I) selectively amplifying a target nucleic acid by a nucleic acid amplification method; and (II) detecting the target nucleic acid amplified in the step (I); 13. A method for detecting a target nucleic acid, comprising:

14. A kit for detecting or selectively amplifying a target nucleic acid, comprising: (a) a kit comprising a primer and a probe, wherein at least one of the primer and the probe comprises the oligonucleotide or a salt thereof according to claim 11 or 12; or (b) A kit comprising a clamp nucleic acid and a primer, wherein at least one of the clamp nucleic acid and the primer comprises the oligonucleotide or a salt thereof according to claim 11 or 12.

15. A pharmaceutical composition containing the oligonucleotide or its salt described in claim 11 or 12.

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