Novel artificial nucleic acids, methods of making and uses thereof

Abstract

The present application relates to a novel artificial nucleic acid, a method for producing the same, and a use thereof. The present application can be achieved by an artificial nucleic acid having a unit derived from a compound represented by the following formula (1) or a salt thereof: in the formula, Base is an optionally substituted aromatic heterocyclic group or an 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 alkenyl group, a cycloalkyl group, a cycloalkenyl group, or an aryl group, a protecting group for a hydroxyl group, a phosphino group having a substituent, or an optionally substituted dihydroxyphosphinyl group or a hydroxylmercapto phosphinyl group, or R 1 and R 2 together with the adjacent 2 oxygen atoms and the carbon atoms at positions 3 to 5 of the furanose form an optionally substituted ring, R 3 is a hydrogen atom, an optionally substituted alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, or an acyl group, a sulfonyl group having a substituent, or a silyl group, a functional molecular unit substituent, or a group represented by the formula: R 31 -X- (in the formula, R 31 is an optionally substituted amino group, X is an optionally substituted alkylene group, or at least one methylene group in the alkylene group is replaced with -N(R 32 ) - (in the formula, R 32 is a hydrogen atom or an alkyl group), -O-, or -S(=O) k - (in the formula, k is 0, 1, or 2), R 4 is a hydrogen atom, an optionally substituted alkyl group, or an aryl group, R 5 is a hydrogen atom or an optionally substituted alkyl group or an aryl group, R 4 and R 5 are not simultaneously a hydrogen atom, and n is 0 or 1.

Description

Technical Field

[0001] This invention relates to a novel artificial nucleic acid, its manufacturing method, and its uses. Background Technology

[0002] Oligonucleotides are short sequences of natural DNA, natural RNA, or artificial nucleic acids, and are known to be very useful for treating or diagnosing specific diseases by regulating gene expression at various gene transcription and translation levels or by examining the sequence status of genes.

[0003] Methods for regulating gene expression or examining / diagnosing gene information can be broadly divided into two types based on the target. The first type involves single-stranded RNA or single-stranded DNA, such as messenger RNA (mRNA) or microRNA (miRNA). The second type involves double-stranded genomic DNA as the target.

[0004] When the target is single-stranded RNA or single-stranded DNA, gene translation can be inhibited (or used for gene diagnosis) by using an antisense method where oligonucleotides bind complementary to single-stranded RNA or DNA to form a double strand. Alternatively, when the oligonucleotide is a double-stranded RNA molecule, the complementary binding of the oligonucleotide to the target mRNA causes the target mRNA to break down via a RISC complex cleavage enzyme (RNA interference). In the case of RNA interference, the oligonucleotide can be an endogenous microRNA equivalent to an endogenous microRNA that can bind to the 3'UTR region (3' untranslated region) of the target mRNA and inhibits target mRNA translation through incomplete complementarity (microRNA mimics).

[0005] Oligonucleotides can induce gene activation or increased transcription by, for example, by binding complementary to long antisense noncoding RNAs or by inhibiting complementary microRNAs, resulting in increased translation of the target mRNA of the microRNA (antimicroRNA).

[0006] Oligonucleotides, as functional materials used in methods for regulating gene expression or checking / diagnosing gene information, require excellent binding affinity to the target nucleic acid sequence, strong resistance to degrading enzymes, and safety in vivo. Natural materials such as DNA and RNA lack resistance to degrading enzymes and have insufficient binding affinity, making them unsuitable for use as functional materials. Therefore, to date, many artificial nucleic acids have been developed to achieve high functionalization of oligonucleotides.

[0007] Representative nucleic acids include: peptide nucleic acids (PNA), bridging structural nucleic acids, morpholino nucleic acids (PMO), and phosphothiophosphate-type nucleic acids (S oligonucleotides) where a non-bound oxygen atom in the phosphodiester portion of the nucleic acid is replaced by a sulfur atom. Representative examples of the aforementioned bridging structural nucleic acids include: LNA (structural formula 1 below), BNA... NC (Structure 2 below), ENA (Structure 3 below).

[0008] [Chemistry 1]

[0009]

[0010] The structure of the thiophosphate-type nucleic acid is represented by the following structural formula 4.

[0011] [Chemistry 2]

[0012]

[0013] These bridging structural nucleic acids have been shown to have excellent ability to selectively bind to single-stranded RNA sequences via Watson-Crick type hydrogen bonds (Patent Documents 1-3). Thus, conventional artificial nucleic acids have been utilized as functional materials for controlling the expression of specific genes and for detecting / diagnosing gene sequences with high sensitivity and accuracy.

[0014] Existing technical documents

[0015] Patent documents

[0016] Patent Document 1: U.S. Patent Application Publication No. 2003 / 105309

[0017] Patent Document 2: U.S. Patent Application Publication No. 2007 / 167387

[0018] Patent Document 3: U.S. Patent Application Publication No. 2003 / 207841 Summary of the Invention

[0019] The problem that the invention aims to solve

[0020] As the applications of oligonucleotides diversify, there is still room for improvement in the functionality of existing artificial nucleic acids, requiring the development of new artificial nucleic acids with the goal of further enhancing their functionality.

[0021] The purpose of this invention is to provide a novel artificial nucleic acid that is useful for a wide variety of genomic technologies, its manufacturing method, and its uses.

[0022] Technical solutions to the problem

[0023] In order to achieve the above-mentioned problem, the inventors conducted in-depth research and found that: in the BNA represented by the above structural formula 2 NC In this invention, an artificial nucleic acid with an optionally substituted alkyl or aryl group attached to the carbon atom between the carbon atom at position 4 of the furanose and the nitrogen atom bound to R exhibits both highly selective and strong binding ability to single-stranded DNA and single-stranded RNA sequences, as well as excellent resistance to degrading enzymes. Based on this discovery, the inventors conducted further and repeated research, thus completing this invention.

[0024] This invention includes the following methods:

[0025] Item 1.

[0026] A compound or a salt thereof represented by formula (1):

[0027] [Chemistry 3]

[0028]

[0029] In formula (1),

[0030] Base can be an aromatic heterocyclic group with optional substituents or an aromatic cyclic group with optional substituents.

[0031] A 1 It is a single bond or an alkylene group.

[0032] R 1 and R 2 Same or different, is a hydrogen atom, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted aryl, a protecting group of hydroxyl, a substituted phosphinyl, optionally substituted dihydroxyphosphonophosphonoyl or optionally substituted hydroxythiophosphonophosphonoyl, 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, it forms a ring with optional substituents.

[0033] R 3 It is a hydrogen atom, an alkyl group with optional substituents, an alkenyl group with optional substituents, a cycloalkyl group with optional substituents, an aryl group with optional substituents, an acyl group with optional substituents, a sulfonyl group with substituents, a silyl group with substituents, a functional molecular unit substituent, or the formula: R 31 -X-(where R is the formula) 31 X is an optional substituent amino group, X is an optional substituent alkylene group, or at least one methylene group in the alkylene group is replaced with -N(R) 32 )-(where R 32 It can be a hydrogen atom or an alkyl group, -O-, or -S (=O).k -(where k is 0, 1, or 2) represents the group.

[0034] R 4 It is a hydrogen atom, an alkyl group optionally substituted, or an aryl group optionally substituted.

[0035] R 5 It is a hydrogen atom, an alkyl group optionally substituted, or an aryl group optionally substituted.

[0036] R 4 and R 5 They are not both hydrogen atoms.

[0037] The following formula:

[0038] [Chemistry 4]

[0039]

[0040] The symbols represent single or double bonds.

[0041] When this notation represents a single bond, n is 1.

[0042] When this notation represents a double bond, n is 0.

[0043] Item 2.

[0044] According to the compound or salt thereof described in item 1, wherein A 1 It is a single key.

[0045] Item 3.

[0046] The compound or its salt according to item 1 or 2, wherein the following formula is used:

[0047] [Chemistry 5]

[0048]

[0049] The symbol represents a single bond, and n is 1.

[0050] Item 4.

[0051] The compound or salt thereof according to any one of items 1 to 3, wherein R 4 It is an alkyl group.

[0052] Item 5.

[0053] The compound or salt thereof according to any one of items 1 to 4, wherein R 5 It is a hydrogen atom or an alkyl group.

[0054] Item 6.

[0055] The compound or salt thereof according to any one of items 1 to 5, wherein R3 It is a hydrogen atom, alkyl, alkenyl, cycloalkyl, aryl, aralkyl, acyl, alkylsulfonyl, arylsulfonyl, formula: -Si(R 6 )3(where R is the value of each R) 6 The same or different (alkyl or aryl) group, labeled functional group, group with intercalation capability, group with nucleic acid binding capability, nucleic acid cleaving active functional group, group with intracellular or nuclear migration capability, or group with metal chelation capability.

[0056] Item 7.

[0057] The compound or salt thereof according to any one of items 1 to 5, wherein R 3 It is the formula: R 31 The group represented by -X-

[0058] R 31 It is either the group represented by formula (A) or the group represented by formula (B).

[0059] [Chemistry 6]

[0060]

[0061] (In formula (A), R) 3a and R 3b The same or different is 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 of an amino group, or R 3a and R 3b (Together with the adjacent nitrogen atom, it forms an optional ring with substituents)

[0062] [Chemistry 7]

[0063]

[0064] (In formula (B), R) 3c ~R 3f Whether they are the same or different, they are protecting groups of hydrogen atoms, alkyl groups, or amino groups.

[0065] X is -C m H 2m -(where m is an integer from 1 to 10).

[0066] Item 8.

[0067] The compound or salt thereof according to any one of items 1 to 7, wherein R 1 and R 2The same or different is a hydrogen atom, an alkyl group with optional substituents, an aryl group with optional substituents, an alkyl carbonyl group, an aryl carbonyl group, an alkyl sulfonyl group, an aryl sulfonyl group, or a group with the formula: -Si(R 6 )3(where R is the value of each R) 6 Whether the groups are the same or different (alkyl or aryl), the formula is: -P(R 7 (R) 8 (where R) 7 and R 8 The same or different is a group represented by hydroxyl, mercapto, amino, alkoxy, haloalkoxy, cyanoalkoxy, alkylthio, haloalkylthio, cyanoalkylthio, or alkylamino, dihydroxyphosphine, or hydroxythiolphosphine, or,

[0068] R 1 and R 2 Together with the two adjacent oxygen atoms and the carbon atoms at positions 3 to 5 of the furanose, it forms a ring with optional substituents.

[0069] Item 9.

[0070] The compound or salt thereof according to any one of claims 1 to 8, wherein Base is optionally substituted 2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl, optionally substituted 2-oxo-1,2-dihydropyrimidin-1-yl, optionally substituted purine-9-yl, or optionally substituted 6-oxo-1,6-dihydro-9H-purine-9-yl.

[0071] Item 10.

[0072] The compound or salt thereof according to item 1 is a compound or salt thereof represented by the following formula (1A).

[0073] [Chemistry 8]

[0074]

[0075] (In Equation (1A), Base and R) 1 ~R 5 Same as above)

[0076] Item 11.

[0077] The compound or salt thereof described in item 1 is a compound or salt thereof represented by the following formula (1B).

[0078] [Chemistry 9]

[0079]

[0080] (In Equation (1B), Base and R) 1 R 2 and R4 Same as above)

[0081] Item 12.

[0082] In a compound or salt thereof represented by formula (1) as described in item 1, n is 0 or n is 1 and R 5 A method for producing a compound containing hydrogen atoms or a salt thereof, the method comprising:

[0083] (I) Make the compound represented by the following formula (1E) and the formula: R 4 ·(where R) 4 The free radical or formula represented by (same as item 1): R 4 In the formula, M is a metal atom or a group of atoms containing a metal atom, R 4 The procedure for reacting with the organometallic reagents represented in item 1;

[0084] [Chemistry 10]

[0085]

[0086] (In equation (1E), Base and A) 1 R 1 and R 2 Same as item 1)

[0087] Further optional items include:

[0088] (II) A process of dehydrogenating the compound obtained in step (I);

[0089] (III) A process of dehydrogenating and then hydrogenating the compound obtained in step (I), or (IV) a process of dehydrogenating and then hydrogenating the compound obtained in step (I) and the compound of formula: R 3 -L(where L is the leaving group, R) 3 The process of reacting a compound represented by the same compound as in item 1 but not hydrogen.

[0090] Item 13.

[0091] In a compound or salt thereof represented by formula (1) as described in item 1, n is 1 and R. 3 It is a methyl group that optionally has one or two substituents and R 5 A method for producing a compound containing hydrogen atoms or a salt thereof, the method comprising:

[0092] (I) Make the compound represented by the following formula (1E) and the formula: R 4 ·(where R) 4 The free radical or formula represented by (same as item 1): R4 In the formula, M is a metal atom or a group of atoms containing a metal atom, R 4 The procedure for reacting with the organometallic reagents represented in item 1;

[0093] [Chemistry 11]

[0094]

[0095] (In equation (1E), Base and A) 1 R 1 and R 2 Same as item 1)

[0096] as well as,

[0097] (II) A process of reacting the compound obtained in step (I) with a carbonyl compound or of dehydrogenating the compound obtained in step (I) and then reacting the hydrogenated compound with a carbonyl compound.

[0098] Item 14.

[0099] An oligonucleotide or a salt thereof having a unit represented by the following formula (6):

[0100] [Chemistry 12]

[0101]

[0102] In formula (6),

[0103] Base can be an aromatic heterocyclic group with optional substituents or an aromatic cyclic group with optional substituents.

[0104] A 1 It is a single bond or an alkylene group.

[0105] R 3 It is a hydrogen atom, an alkyl group with optional substituents, an alkenyl group with optional substituents, a cycloalkyl group with optional substituents, an aryl group with optional substituents, an acyl group with optional substituents, a sulfonyl group with substituents, a silyl group with substituents, a functional molecular unit substituent, or the formula: R 31 -X-(where R is the formula) 31 X is an optional substituent amino group, X is an optional substituent alkylene group, or at least one methylene group in the alkylene group is replaced with -N(R) 32 )-(where R 32 It can be a hydrogen atom or an alkyl group, -O-, or -S (=O). k -(where k is 0, 1, or 2) represents the group.

[0106] R 4It is a hydrogen atom, an alkyl group optionally substituted, or an aryl group optionally substituted.

[0107] R 5 It is a hydrogen atom, an alkyl group optionally substituted, or an aryl group optionally substituted.

[0108] R 4 and R 5 They are not both hydrogen atoms.

[0109] The following formula:

[0110] [Chemistry 13]

[0111]

[0112] The symbols represent single or double bonds.

[0113] When this notation represents a single bond, n is 1.

[0114] When this notation represents a double bond, n is 0.

[0115] Item 15.

[0116] A method for detecting a target nucleic acid, comprising:

[0117] (I) The process of selectively amplifying the target nucleic acid using a nucleic acid amplification method; and,

[0118] (II) A process for detecting the target nucleic acid amplified in step (I);

[0119] The oligonucleotide used in the amplification or detection includes the oligonucleotide described in item 14 or a salt thereof.

[0120] Item 16.

[0121] A kit for detecting or selectively amplifying a target nucleic acid, wherein,

[0122] (a) The kit contains primers and probes, at least one of which contains the oligonucleotide described in item 14 or a salt thereof, or,

[0123] (b) The kit contains Clamp nucleic acid and primers, at least one of which contains the oligonucleotide or a salt thereof as described in item 14.

[0124] Item 17.

[0125] A pharmaceutical composition comprising any one of items 1 to 11, or a salt thereof, or an oligonucleotide or a salt thereof as described in item 14.

[0126] Invention Effects

[0127] According to the present invention, a novel artificial nucleic acid can be provided that is useful for a wide variety of genomic technologies. Attached Figure Description

[0128] Figure 1A This 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 residual rate of undigested oligonucleotides.

[0129] Figure 1B This is a graph showing the relationship between the reaction time of a digestive enzyme at a final concentration of 1.60 μg / mL and the residual rate of undigested oligonucleotides.

[0130] Figure 1C This is a graph showing the relationship between the reaction time of a digestive enzyme at a final concentration of 4.38 μg / mL and the residual rate of undigested oligonucleotides.

[0131] Figure 2A This is an example of a kit of the present invention, and is a schematic diagram of a kit comprising a container holding a composition containing primers and probes.

[0132] Figure 2B This is an example of a kit of the present invention, and is a schematic diagram of a kit comprising a container containing a composition containing primers and a container containing a composition containing probes.

[0133] Figure 2C This is an example of a kit of the present invention, and is a schematic diagram of a kit comprising 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. Detailed Implementation

[0134] <<Definition of Terminology>>

[0135] In this specification, "alkyl" refers to a monovalent group from which one hydrogen atom has been removed from a straight-chain or branched saturated hydrocarbon.

[0136] The number of carbon atoms in the alkyl group is not particularly limited, for example, 1 to 20, preferably 1 to 10, more preferably 1 to 6, and particularly preferably 1 to 4.

[0137] Examples of alkyl groups include: methyl, ethyl, propyl (e.g., n-propyl, isopropyl), butyl (e.g., n-butyl, isobutyl, sec-butyl, tert-butyl), pentyl (e.g., n-pentyl, isopentyl, neopentyl), hexyl, heptyl, octyl (e.g., n-octyl, 2-ethylhexyl), nonyl, decyl, etc.

[0138] In this specification, "alkylene" refers to a divalent group formed by removing two hydrogen atoms from a straight-chain or branched saturated hydrocarbon.

[0139] The number of carbon atoms in the alkylene group is not particularly limited, for example, it is 1 to 10, preferably 1 to 8, and more preferably 1 to 6.

[0140] Examples of alkylene groups include: C1 alkylene (e.g., methylene), C2 alkylene (e.g., methylmethylene, dimethylene), C3 alkylene (e.g., trimethylene, dimethylmethylene), C4 alkylene (e.g., tetramethylene), C5 alkylene (e.g., pentamethylene), and C6 alkylene (e.g., hexamethylene).

[0141] In this specification, "alkenyl" refers to a monovalent group that is straight-chain or branched and consists of an unsaturated hydrocarbon containing a carbon-carbon double bond with one hydrogen atom removed.

[0142] The number of carbon atoms in the alkenyl group is not particularly limited, for example, 2 to 20, preferably 2 to 10, and more preferably 2 to 6.

[0143] Examples of alkenyl groups include: vinyl (i.e., vinyl group), propenyl (e.g., 1-propenyl, allyl), butenyl, pentenyl, hexenyl, geranyl (geranyl group), farnesyl (farnesyl group), etc.

[0144] In this specification, "alkynyl" refers to a straight-chain or branched monovalent group that has one hydrogen atom removed from an unsaturated hydrocarbon containing a carbon-carbon triple bond.

[0145] The number of carbon atoms in the alkynyl group is not particularly limited, for example, 2 to 20, preferably 2 to 10, and more preferably 2 to 6.

[0146] Examples of alkynyl groups include ethynyl, propynyl, and 1-butynyl.

[0147] In this specification, "cycloalkyl" refers to a monovalent group derived from a saturated aliphatic hydrocarbon ring.

[0148] The number of carbon atoms in the cycloalkyl group is not particularly limited, for example, 3 to 20, preferably 5 to 12, and more preferably 5 to 10.

[0149] Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, norbornyl, and adamantyl.

[0150] In this specification, "cycloalkenyl" refers to a monovalent group derived from an unsaturated aliphatic hydrocarbon ring containing a carbon-carbon double bond.

[0151] The number of carbon atoms in the cycloalkenyl group is not particularly limited, for example, 3 to 20, preferably 5 to 12, and more preferably 5 to 10.

[0152] Examples of cycloalkenyl groups include: cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, norbornenyl, adamantenyl, etc.

[0153] In this specification, "aromatic hydrocarbon cyclogroup" refers to a monovalent group derived from an aromatic hydrocarbon ring, also known as "aryl".

[0154] The number of atoms constituting the aromatic hydrocarbon ring is not particularly limited, for example, 6 to 20, preferably 6 to 14, more preferably 6 to 12, and particularly preferably 6 to 10.

[0155] Aromatic hydrocarbon rings can be monocyclic or fused rings (e.g., bicyclic to tricyclic fused rings).

[0156] Examples of aromatic hydrocarbon cyclic groups include: phenyl, indene, naphthyl, fluorenyl, phenanthrene, anthracene, etc.

[0157] In this specification, the term "heterocyclic" is used to mean both "aliphatic heterocyclic" and "aromatic heterocyclic".

[0158] In this specification, "aliphatic heterocycle" refers to an aliphatic ring containing a carbon atom and at least one heteroatom selected from the group consisting of nitrogen, oxygen, sulfur, silicon, etc., as the constituent atoms of the ring.

[0159] The number of atoms constituting the aliphatic heterocycle is not particularly limited, for example, it is 5 to 20, preferably 5 to 12, and more preferably 6 to 10.

[0160] The number of heteroatoms in the constituent atoms of aliphatic heterocycles is not particularly limited, for example, it is 1 to 4.

[0161] Examples of aliphatic heterocycles include: oxygen-containing aliphatic heterocycles (e.g., tetrahydrofuran, dioxane, pyran, tetrahydropyran, dioxane), sulfur-containing aliphatic heterocycles (e.g., tetrahydrothiophene, thiopyran, tetrahydrothiopyran), nitrogen-containing aliphatic heterocycles (e.g., pyrrolidine, piperidine, azaheptan), nitrogen- and oxygen-containing aliphatic heterocycles (e.g., morpholine), nitrogen- and sulfur-containing aliphatic heterocycles (e.g., thiomorpholine), and aliphatic heterocycles containing siloxane bonds.

[0162] In this specification, "aromatic heterocycle" refers to an aromatic ring containing a carbon atom and at least one heteroatom selected from the group consisting of nitrogen, oxygen, sulfur, etc., as the constituent atoms of the ring.

[0163] The number of atoms constituting the aromatic heterocycle is not particularly limited, for example, it is 5 to 20, preferably 5 to 12, and more preferably 6 to 10.

[0164] The number of heteroatoms in the constituent atoms of aromatic heterocycles is not particularly limited, for example, it is 1 to 4.

[0165] Aromatic heterocycles can be monocyclic or fused-ring (e.g., bicyclic to tricyclic fused-ring).

[0166] Examples of aromatic heterocycles include: oxygen-containing aromatic heterocycles (e.g., furan, benzofuran, isobenzofuran, chromane, benzopyran, xanthan), sulfur-containing aromatic heterocycles (e.g., thiophene, thiathracene), nitrogen-containing aromatic heterocycles (e.g., pyrrole, imidazole, pyrazole, triazole, pyridine, pyrazine, pyrimidine, pyridazine, indole, isoindole, indoleazine, purine, quinoline, isoquinoline, 1,8-naphthidine, quinoxaline, quinazoline, cyclophosphine, phthalazine, pteridine, carbazole, phenanthridine, acridine, pyrimidine, phenazine), oxygen- and sulfur-containing aromatic heterocycles (e.g., phenoxazine), nitrogen- and oxygen-containing aromatic heterocycles (e.g., oxazole, isoxazole, furazine, phenoxazine), and nitrogen- and sulfur-containing aromatic heterocycles (e.g., thiazole, isothiazole, phenothiazine), etc.

[0167] In this specification, "heterocyclic group" refers to a monovalent group from which one hydrogen atom has been removed from the aforementioned heterocycle.

[0168] In this specification, "optionally substituented" or "optionally substituted" means both cases with no substituents and cases with one or more identical or different substituents replacing any hydrogen atom. It should be noted that, in the case of substituents, the number of substituents is not particularly limited, for example, 1 to 3, preferably 1 or 2.

[0169] In this specification, "substituent" refers to an atom or group of atoms that replaces a hydrogen atom. Examples of substituents include: halogen atoms (e.g., fluorine, chlorine, bromine, iodine), oxo groups (=O), thio groups (=S), hydroxyl groups, mercapto groups, amino groups, carboxyl groups, alkyl groups, alkenyl groups, cycloalkyl groups, cycloalkenyl groups, aryl groups, alkynyl groups, acyl groups, alkylsulfonyl groups, arylsulfonyl groups, cyano groups, heterocyclic groups, and combinations of two or more of them (e.g., haloalkyl, cyanalkyl, aralkyl, alkoxy, alkylamino groups), etc.

[0170] To clarify, "two or more combinations" includes any combination of groups exemplified as substituents of each group.

[0171] In this specification, "Cx-y" means that the number of carbon atoms in the subsequent group is x or more and y or less. x and y are positive integers, where x is a carbon atom or .... <y。

[0172] In this specification, "halogenated alkyl" means an alkyl group that is substituted by one or more identical or different halogen atoms.

[0173] A preferred example of a haloalkyl group is C. 1-6 Haloalkyl, more preferably C40, is a preferred example. 1-4The alkyl halogroup, and more preferred examples are trifluoromethyl, trichloromethyl or 2,2,2-trifluoroethyl.

[0174] In this specification, "cyanoalkyl" means an alkyl group substituted with one or more cyano groups.

[0175] A preferred example of a cyanoalkyl group is C. 1-6 Cyanoalkyl, more preferably C 1-4 Cyanoalkyl, with cyanomethyl or 2-cyanoethyl being further preferred examples.

[0176] In this specification, "aralkyl" refers to an alkyl group that is substituted by one or more homo- or hetero-aryl groups.

[0177] A preferred example of an aralkyl group is C 6-14 Aryl C 1-4 Alkyl groups, more preferably benzyl (i.e., benzyl), phenylethyl (i.e., phenylethyl), naphthylmethyl, naphthylethyl, triphenylmethyl (i.e., triphenylmethyl) or fluorenemethyl.

[0178] In this specification, "alkoxy" refers to a group represented by the formula: -O-alkyl. A preferred example of an alkoxy group is C... 1-6 Alkoxy, more preferably methoxy, ethoxy, propoxy (e.g., n-propoxy, isopropoxy) or butoxy (e.g., tert-butoxy).

[0179] It should be noted that haloalkoxy and cyanoalkoxy refer to the groups represented by the formula: -O-haloalkyl and -O-cyanoalkyl, respectively.

[0180] In this specification, "alkathiol" refers to a group represented by the formula: -S-alkyl. A preferred example of an alkathiol group is C... 1-6 Alkylthio, more preferably methylthio, ethylthio, propylthio (e.g., n-propylthio, isopropylthio) or butylthio.

[0181] It should be noted that haloalkylthio and cyanoalkylthio refer to the groups represented by the formula: -S-haloalkyl and -S-cyanoalkyl, respectively.

[0182] In this specification, "alkylamino" refers to an amino group substituted with one or two homo- or hetero-alkyl groups.

[0183] Alkylamino groups include monoalkylamino and dialkylamino groups.

[0184] A preferred example of a monoalkylamino group is a mono-C 1-6 Alkylamino, more preferably monomethylamino, monoethylamino, monopropylamino (e.g., mono(n-propyl)amino, mono(isopropyl)amino) or monobutylamino.

[0185] A preferred example of a dialkylamino group is a di-C 1-6 Alkylamino, more preferably dimethylamino, diethylamino, dipropylamino (e.g., di(n-propyl)amino, di(isopropyl)amino) or dibutylamino.

[0186] In this specification, "phosphine group with substituents" means a group in which at least one hydrogen atom of the phosphine group (-PH2) is replaced by other atoms or groups of atoms.

[0187] As an example of a phosphine group with substituents, one can cite the following formula: -P(R 7 (R) 8 (where R) 7 and R 8 The same or different groups are those represented by hydroxyl, mercapto, amino, alkoxy, haloalkoxy, cyanoalkoxy, alkylthio, haloalkylthio, cyanoalkylthio, or alkylamino.

[0188] In this specification, "optionally substituent dihydroxyphosphonoyl group" means dihydroxyphosphonoyl group (i.e., phosphonoyl group) (-P(=O)(OH)2) or dihydroxyphosphonoyl group in which at least one hydrogen atom is replaced by other atoms or groups (e.g., protecting group of hydroxyl group).

[0189] The latter group includes the following formula, which may optionally have substituents:

[0190]

[0191] The group represented (hereinafter referred to as "bisphosphonate") and the following formula with optional substituents:

[0192] [Chemistry 15]

[0193]

[0194] The group represented (hereinafter referred to as "triphosphate").

[0195] In this specification, "optionally substituted hydroxythiophosphines" refers to a hydroxythiophosphines (-P(=O)(OH)(SH)) or a hydroxythiophosphines in which at least one hydrogen atom is replaced by another atom or group (e.g., a protecting group of the hydroxyl group).

[0196] In this specification, "protecting group of hydroxyl group" refers to a monovalent group that prevents the hydroxyl group from participating in the reaction during the synthesis of a compound or its salt, or during the synthesis of an oligonucleotide or its salt.

[0197] Protecting groups for hydroxyl groups include, but are not limited to, groups that are stable under acidic or neutral conditions and can be cleaved by methods such as hydrogenation, hydrolysis, electrolysis, and photolysis.

[0198] Examples of protecting groups for hydroxyl groups include acyl groups with optional substituents, sulfonyl groups with substituents, and silyl groups with substituents.

[0199] In this specification, "acyl" refers to the group represented by the formula: -C(=O)-R (where R is a hydrocarbon group). The hydrocarbon group represented by R can be a straight-chain or branched hydrocarbon group (e.g., alkyl), a saturated or unsaturated cycloalkyl group (e.g., cycloalkyl, aryl), or a combination thereof (e.g., aralkyl).

[0200] Acyl groups include alkyl carbonyl, aryl carbonyl, and aralkyl carbonyl.

[0201] A preferred example of an alkyl carbonyl group is (C 1-10 Alkyl) carbonyl, more preferably acetyl, propionyl, butyryl, isobutyryl, valeryl, neovaleryl, valeryl, isovaleryl, octyl, nonanoyl or decyl.

[0202] A preferred example of an aryl carbonyl group is (C 6-14 Aryl)carbonyl, more preferably benzoyl or naphthyl (i.e., α-naphthyl, β-naphthyl).

[0203] A preferred example of an aralkyl carbonyl group is (C 6-14 Aryl C 1-4 Alkyl)carbonyl, more preferably benzylcarbonyl.

[0204] It should be noted that the acyl group in "acyloxy", "acylthio" and "acylamino" can also be the same group as described above.

[0205] In this specification, "sulfonyl group with substituent" refers to the group represented by the formula: -S(=O)2R (where R is the same as above).

[0206] The sulfonyl group having a substituent comprises a sulfonyl group having an alkyl group optionally substituented and a sulfonyl group having an aryl group optionally substituented. A preferred example of a sulfonyl group having an alkyl group is C. 1-6 Alkyl sulfonyl, more preferably methanesulfonyl or ethanesulfonyl.

[0207] A preferred example of a sulfonyl group having an aryl group is C. 6-14 Arylsulfonyl, more preferably benzenesulfonyl or p-toluenesulfonyl.

[0208] In this specification, "silyl group with substituents" refers to a silyl group in which at least one hydrogen atom of the silyl group (-SiH3) is replaced by other atoms or groups.

[0209] A typical example of a "substituent silyl group" is the formula: -Si(R 6 )3(where R is the value of each R)6 The same or different, whether it is an alkyl or aryl group. Examples of this group include: trialkylsilyl (e.g., trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, etc.). 1-6 Alkyl silyl), dialkylaryl silyl (e.g., dimethylphenylsilyl, etc., diC) 1-6 Alkyl C 6-14 Arylsilyl), alkyl diarylsilyl (e.g., tert-butyldiphenylsilyl, etc. C 1-6 Alkyl diC 6-14 Arylsilyl), triarylsilyl (e.g., triphenylsilyl, etc., tri-C) 6-14 Arylsilyl groups, etc.

[0210] In this specification, "protecting group of amino group" refers to a monovalent group that prevents the amino group from participating in the reaction during the synthesis of a compound or its salt, or during the synthesis of an oligonucleotide or its salt.

[0211] Protecting groups of amino groups include, but are not limited to, groups that are stable under acidic or neutral conditions and can be cleaved by methods such as hydrogenation, hydrolysis, electrolysis, and photolysis.

[0212] Examples of protecting groups for amino groups include acyl groups with optional substituents (e.g., alkyl carbonyl groups with optional substituents, aryl carbonyl groups with optional substituents, aralkyl carbonyl groups with optional substituents), N,N-dialkylaminomethyl methyl groups with optional substituents, alkoxy carbonyl groups with optional substituents, olefinic carbonyl groups with optional substituents, aryloxy carbonyl groups with optional substituents, arylalkoxy carbonyl groups with optional substituents, and sulfonyl groups with substituents.

[0213] In this specification, "formamidyl group" refers to the group represented by the formula: =CH-NH2. A preferred example of N,N-dialkylaminomethylene group is N,N-di(C 1-6 Alkyl)aminomethyl methyl group, more preferably N,N-di(C 1-4 Alkyl)aminomethyl subunit, a further preferred example being N,N-dimethylaminomethyl subunit or N,N-diethylaminomethyl subunit.

[0214] In this specification, "alkoxycarbonyl" refers to the group represented by the formula: -C(=O)-O-alkyl.

[0215] A preferred example of an alkoxycarbonyl group is (C 1-6 Alkoxy)carbonyl, more preferably methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl or butoxycarbonyl (e.g. tert-butoxycarbonyl).

[0216] In this specification, "olefinic carbonyl" refers to the group represented by the formula: -C(=O)-O-olefin.

[0217] A preferred example of an olefinic carbonyl group is (C 2-9 (Alkenyloxy)carbonyl, more preferably allyloxycarbonyl.

[0218] In this specification, "aryloxycarbonyl" refers to the group represented by the formula: -C(=O)-O-aryl.

[0219] A preferred example of an aryloxycarbonyl group is (C 6-14 Aryloxy)carbonyl, more preferably phenoxycarbonyl or naphthoxycarbonyl.

[0220] In this specification, "aranekoxycarbonyl" refers to the group represented by the formula: -C(=O)-O-aranealkyl.

[0221] A preferred example of an arylalkoxycarbonyl group is (C 6-14 Aryl C 1-4 Alkoxy)carbonyl, more preferably benzyloxycarbonyl or fluorenemethyloxycarbonyl.

[0222] In this specification, "functional molecular unit substituent" refers to the concept of groups including: labeled functional groups (e.g., fluorescently labeled functional groups, chemiluminescently labeled functional groups, radionuclide-containing functional groups), groups with intercalation capability, groups with nucleic acid binding capability, nucleic acid cleavage active functional groups, groups with intracellular or nuclear migration capability, and groups with metal chelation capability, etc.

[0223] Examples of fluorescently labeled functional groups include residues of fluorescent labeling reagents such as carboxyfluorescein (FAM), fluorescein isothiocyanate (FITC), carboxytetramethylrhodamine (TAMRA), and thiazolyl orange (1-methyl-4-[(3-methyl-2(3H)-benzothiazolyl)methyl]quinoline salt of p-toluenesulfonic acid). Examples of chemiluminescently labeled functional groups include residues of chemiluminescently labeled reagents such as tris(bipyridine)ruthenium(II) chloride. Examples of reagents containing radioactive nuclide groups include those containing... 11 CH3-、 14 CH3-、 18 F- or 32 P- groups, etc.

[0224] Examples of groups capable of intercalation include groups having an anthracene skeleton, groups having a pyrene skeleton, groups having an anthraquinone skeleton, groups having an acridine skeleton, and groups having a naphthamide skeleton. Other examples include residues of intercalating agents such as tamoxifen.

[0225] Examples of groups with nucleic acid binding capabilities include residues of nucleic acid binding agents such as fusiformin, pyromycin, and PI (pyrrolimidazole) polyamide.

[0226] Examples of functional groups that cleave nucleic acids include: endonucleases and bis(bipyridine). Residues of nucleic acid cleaving agents such as quinone diimine rhodium(II) complexes.

[0227] Examples of groups capable of intracellular or nuclear migration include residues of signal peptides such as TAT ​​(Twin-Arginine Translocation) signal peptide, polyarginine, GalNac (N-acetylgalactosamine), and signal peptides derived from the SV40T antigen.

[0228] Examples of groups with metal chelating ability include residues of metal chelating agents such as EDTA, crown ethers, and cavitary ligands.

[0229] In this specification, "hybridization" means the formation of a double helix of all or part of a specified polynucleotide or oligonucleotide by hydrogen bonds with all or part of another polynucleotide or oligonucleotide under stringent conditions. "Stringent conditions" are simply those conditions commonly used by those skilled in the art when performing hybridization of polynucleotides or oligonucleotides. For example, conditions under which one polynucleotide or oligonucleotide molecule can specifically hybridize with the other when there is at least 50%, preferably at least 75%, more preferably at least 90% sequence identity between the two molecules. The stringency of hybridization is known to be a function of temperature, salt concentration, base length and GC content of the polynucleotide or oligonucleotide, and the concentration of the chaotropic agent contained in the hybridization buffer. For example, conditions described in *Molecular Cloning: A Laboratory Manual (2nd Edition)*, edited by Sambrook, J. et al., published in Cold Spring Harbor Laboratory Press, New York, 1998, can be used as stringent conditions.

[0230] In this specification, "inspection" refers to examining the analytes such as nucleic acids in a sample for diagnostic, research, or other purposes. "Inspection sample" refers to a sample intended for inspection.

[0231] In this specification, the upper and lower limits of each numerical range can be combined arbitrarily.

[0232] <<Compounds or their salts>>

[0233] The compounds or salts thereof of the present invention are compounds or salts thereof represented by the following formula (1):

[0234] [Chemistry 16]

[0235]

[0236] (where Base and A are in the formula) 1 R 1 ~R 5 And n is the same as above. (Note: The carbon atoms of the furanose are labeled with position numbers.)

[0237] The compound represented by formula (N) or its salt will be referred to as "compound (N)" below.

[0238] Compound (1) in R 1 or R 2 When the substituent is a dihydroxyphosphonomethyl group or a hydroxythiophosphonomethyl group, it is called a "nucleotide"; when the substituent is another group, it is called a "nucleoside".

[0239] A preferred example of a base is an aromatic heterocyclic group with optional substituents.

[0240] The aforementioned aromatic heterocyclic group is preferably a nitrogen-containing aromatic heterocyclic group.

[0241] The aforementioned nitrogen-containing aromatic heterocyclic group is preferably a six- to ten-membered nitrogen-containing aromatic heterocyclic group. The aforementioned six- to ten-membered nitrogen-containing aromatic heterocyclic group is preferably 2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl, 2-oxo-1,2-dihydropyrimidin-1-yl, purine-9-yl, or 6-oxo-1,6-dihydro-9H-purine-9-yl.

[0242] Preferred examples of substituents replacing the aforementioned aromatic heterocyclic groups are at least one selected from the group consisting of alkyl, acyl, and amino groups optionally substituted with a protecting group of an amino group. The number of substituents is not particularly limited, and for example is 1 to 3.

[0243] More preferred examples of the base are optionally substituted thymine, optionally substituted cytosine, optionally substituted adenine, or optionally substituted guanine. The substituents are preferably at least one selected from the group consisting of alkyl, acyl, and N,N-dialkylaminomethyl subunits, more preferably C. 1-4 Alkyl, (C 1-4 alkyl)carbonyl, (C 6-14 aryl)carbonyl or N,N-di(C 1-4 Alkyl)aminomethyl subunit. The number of substituents is preferably 1 to 3.

[0244] Further preferred examples of Base are groups selected from the group consisting of:

[0245] 2,4-Dioxo-5-methyl-1,2,3,4-tetrahydropyrimidin-1-yl (e.g., thymine-1-yl),

[0246] 2-O-4-amino-1,2-dihydropyrimidin-1-yl (i.e., cytosine-1-yl),

[0247] 2-O-4-acylamino-1,2-dihydropyrimidin-1-yl (i.e., N-acyl-cytosine-1-yl),

[0248] 2-Oxo-4-amino-5-methyl-1,2-dihydropyrimidin-1-yl (i.e., 5-methylcytosine-1-yl),

[0249] 2-Oxo-4-acylamino-5-methyl-1,2-dihydropyrimidin-1-yl (i.e., N-acyl-5-methylcytosine-1-yl),

[0250] 6-Amino-9H-purine-9-yl (i.e., adenine-9-yl),

[0251] 6-Acylamino-9H-purine-9-yl (i.e., N-acyl-adenine-9-yl),

[0252] 2-Amino-6-oxo-1,6-dihydro-9H-purine-9-yl (e.g., guanine-9-yl)

[0253] 2-Acylamino-6-oxo-1,6-dihydro-9H-purine-9-yl (e.g., N-acyl-guanine-9-yl), and,

[0254] 2-(N,N-dialkylaminomethyl imide)amino-6-oxo-1,6-dihydro-9H-purine-9-yl (e.g., N-(N,N-dialkylaminomethyl imide)-guanine-9-yl).

[0255] The preferred example of Base is a group selected from the group consisting of the following:

[0256] Thymine-1-yl,

[0257] 5-Methylcytosine-1-yl,

[0258] N-acetyl-5-methylcytosine-1-yl,

[0259] N-Isobutyryl-5-methylcytosine-1-yl,

[0260] N-benzoyl-5-methylcytosine-1-yl,

[0261] adenine-9-yl

[0262] N-acetyl-adenine-9-yl

[0263] N-Isobutyryl-adenine-9-yl,

[0264] N-benzoyl-adenine-9-yl

[0265] Guanine-9-yl

[0266] N-acetyl-guanine-9-yl

[0267] N-Isobutyryl-guanine-9-yl,

[0268] N-benzoyl-guanine-9-yl, and,

[0269] N-(N,N-dimethylaminomethylsubyl)-guanine-9-yl.

[0270] A 1 Preferably a single bond or C 1-2 Alkylene (e.g., methylene, dimethylene), more preferably single bond.

[0271] R 1 Preferred examples are groups selected from the group consisting of:

[0272] hydrogen atom,

[0273] Optional alkyl groups,

[0274] Optional aryl groups,

[0275] Protecting group of hydroxyl group

[0276] Phosphine groups with substituents

[0277] Optional substituents, including dihydroxyphosphonoyl groups, and...

[0278] Optional hydroxythiophosphonomethyl group with substituents.

[0279] R 1 The term "optionally substituent alkyl" is preferably an alkyl group that optionally has at least one substituent selected from the group consisting of halogen atoms, alkoxy groups, and aryl groups, more preferably an alkyl group optionally substituted with an alkoxy group or an aralkyl group optionally substituted with an alkoxy group. The number of substituents is preferably 1 to 3.

[0280] R 1 The term "aryl group optionally substituted" is preferably an aryl group optionally having at least one substituent selected from the group consisting of halogen atoms, alkyl groups, and alkoxy groups, and more preferably an aryl group optionally substituted with an alkoxy group. The number of substituents is preferably 1 to 3.

[0281] R 1 The "protecting group of the hydroxyl group" is preferably an alkyl carbonyl group, an aryl carbonyl group, an alkyl sulfonyl group, an aryl sulfonyl group, or the formula: -Si(R 6 )3(where R is the value of each R) 6 The same or different groups are represented by alkyl or aryl groups.

[0282] R 1 The "phosphine group with substituents" represented is preferably of the formula: -P(R 7 (R) 8 (where R) 7 and R 8 Whether the group is the same or different, it is a group represented by hydroxyl, mercapto, amino, alkoxy, haloalkoxy, cyanoalkoxy, alkylthio, haloalkylthio, cyanoalkylthio, or alkylamino. A more preferred example of this group is the following formula:

[0283] [Chemistry 17]

[0284]

[0285] (where R is in the formula) 7a and R 7b Same or different, is a hydrogen atom or an alkyl group, R 8a Phosphine is represented by a hydrogen atom, alkyl group, haloalkyl group, or cyanoalkyl group.

[0286] R 1 The term "optionally substituent dihydroxyphosphonoyl group" is preferably dihydroxyphosphonoyl, diphosphate, or triphosphate, more preferably dihydroxyphosphonoyl. These groups may have a hydroxyl protecting group as a substituent, and all or part of the hydroxyl groups may be replaced by a hydroxyl protecting group.

[0287] R 1 The term "optionally substituted hydroxythiophosphonoamyl group" is preferably hydroxythiophosphonoamyl group.

[0288] R 1 The most preferred examples are hydrogen atom, methyl, ethyl, propyl, butyl, allyl, benzyl, triphenylmethyl, methoxymethyl, p-methoxybenzyl, monomethoxytriphenylmethyl, dimethoxytriphenylmethyl, acetyl, isobutyryl, benzoyl, methanesulfonyl, p-toluenesulfonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, phosphinyl, dihydroxyphosphineyl or hydroxythiophosphineyl represented by any of the following formulas.

[0289] [Chemistry 18]

[0290]

[0291] R 2 Preferred example and R 1 The preferred example is the same.

[0292] R 1 With R 2 The preferred example of the combination is R. 1 It is a hydrogen atom or dimethoxytriphenylmethyl (e.g., 4,4'-dimethoxytriphenylmethyl) with R 2 It is a combination of phosphine groups represented by the following formula.

[0293] [Chemistry 19]

[0294]

[0295] (where R is in the formula) 7a and R 7b Same or different, is a hydrogen atom or an alkyl group, R 8a (It is a hydrogen atom, alkyl, haloalkyl, or cyanoalkyl)

[0296] R 1 and R 2 When a ring is formed together with two adjacent oxygen atoms and carbon atoms at positions 3 to 5 of the furanose, a preferred example of the ring is an aliphatic heterocycle of a six- to ten-membered ring optionally with substituents, a preferred example of which is an alkyl group.

[0297] A more preferred example of this ring is an aliphatic heterocycle represented by any of the following formulas.

[0298] [Chemistry 20]

[0299]

[0300] (where R is in the formula) 9 and R 10 Same or different, is a hydrogen atom or an alkyl group, R 11 ~R 14 (Identical or different, alkyl)

[0301] A further preferred example of this ring is a ring represented by any of the following formulas.

[0302] [Chemistry 21]

[0303]

[0304] In one implementation, R 3 Preferably, the atom is hydrogen, alkyl, alkenyl, cycloalkyl, aryl, aralkyl, acyl, alkylsulfonyl, arylsulfonyl, or of the formula: -Si(R 6 )3(where R is the value of each R) 6The same or different (alkyl or aryl) group, labeled functional group, group with intercalation capability, group with nucleic acid binding capability, nucleic acid cleaving active functional group, group with intracellular or nuclear migration capability, or group with metal chelation capability.

[0305] In other embodiments, R 3 Preferred formula: R 31 -X- represents the group.

[0306] R 31 A preferred example is the group represented by the following formula (A).

[0307] [Chemistry 22]

[0308]

[0309] (where R is in the formula) 3a and R 3b The same or different is 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 of an amino group, or R 3a and R 3b (Together with the adjacent nitrogen atom, it forms an optional ring with substituents)

[0310] R 3a The protecting group is preferably a hydrogen atom, alkyl, aryl, aralkyl, or amino group, and more preferably a hydrogen atom, alkyl, or amino group.

[0311] R 3a The "protecting group of amino group" is preferably (C 1-4 alkyl) carbonyl or (C 1-4 (Halogenated alkyl) carbonyl, more preferably acetyl or trifluoromethyl carbonyl.

[0312] R 3b Preferred example and R 3a The preferred example is the same.

[0313] R 3a With R 3b The preferred example of the combination is R. 3a and R 3b It is also a combination of hydrogen atoms, R 3a It is a hydrogen atom and R 3b It is a combination of acetyl or trifluoromethyl carbonyl, R 3a and R 3b Simultaneously a combination of methyl groups, or R 3a and R 3b It is a combination of acetyl or trifluoromethyl carbonyl groups.

[0314] R3a and R 3b When a ring is formed together with an adjacent nitrogen atom, a preferred example of the ring is a nitrogen-containing aliphatic heterocycle of a five- to ten-membered ring optionally with substituents, preferably alkyl or acyl groups.

[0315] A more preferred example of this ring is a nitrogen-containing aliphatic heterocycle represented by the following formula.

[0316] [Chemistry 23]

[0317]

[0318] [In the formula, Z represents a single bond, an oxygen atom, and S (=O).] p (where p is 0, 1, or 2), C(R) 15 (R) 16 (where R) 15 and R 16 Same or different (is it hydrogen atom or alkyl group) or NR 17 (where R is in the formula) 17 [It is a hydrogen atom, alkyl group, or acyl group]

[0319] A further preferred example of this ring is a nitrogen-containing aliphatic heterocycle represented by the following formula.

[0320] [Chemistry 24]

[0321]

[0322] [In the formula, R] 17a It is a hydrogen atom, a straight-chain or branched carbon atom. 1-4 Alkyl (e.g., methyl, ethyl, propyl, butyl), or (straight-chain or branched C) 1-4 Alkyl) carbonyl (e.g., methyl carbonyl, ethyl carbonyl, propyl carbonyl, butyl carbonyl)

[0323] The most preferred example of this ring is 4-methylpiperazin-1-yl.

[0324] R 31 Another preferred example is the following formula (B):

[0325] [Chemistry 25]

[0326]

[0327] (In formula (B), R) 3c ~R 3f The same or different groups are represented by the protecting groups of hydrogen atoms, alkyl groups, or amino groups.

[0328] R 3c ~R 3f Preferred examples are hydrogen atoms or amino protecting groups.

[0329] R 3c Hydrogen atoms are preferred.

[0330] R 3d Preferably, it is a protecting group of amino, more preferably an alkoxycarbonyl, haloalkoxycarbonyl, or cyanoalkoxycarbonyl, and particularly preferably (C 1-4 alkoxy)carbonyl, (C 1-4 (haloalkoxy)carbonyl or (C) 1-4 (cyanoalkoxy)carbonyl.

[0331] R 3e Preferably, it is a protecting group of amino, more preferably an alkoxycarbonyl, haloalkoxycarbonyl, or cyanoalkoxycarbonyl, and particularly preferably (C 1-4 alkoxy)carbonyl, (C 1-4 (haloalkoxy)carbonyl or (C) 1-4 (cyanoalkoxy)carbonyl.

[0332] R 3f Hydrogen atoms are preferred.

[0333] R 31 The most preferred example is a group selected from the group below.

[0334] [Chemistry 26]

[0335]

[0336] A preferred example of X is an alkylene group, or a methylene group in the alkylene group bonded to a nitrogen atom and R. 31 At least one of the methylene groups other than the bound methylene group is replaced with -N(R) 32 )-(where R 32 It can be a hydrogen atom or an alkyl group, -O-, or -S (=O). k - (where k is 0, 1, or 2) groups. Alternatively, a preferred example of X is an alkylene group, or a group in which two adjacent carbon atoms have a -N(R) group between them. 32 )-(where R 32 Same as above), -O- or -S (=O) k - (where k is a group of 0, 1 or 2).

[0337] A further preferred example of X is a group selected from the group consisting of:

[0338] Formula: -C m H 2m -(where m is an integer from 1 to 10) represents the group,

[0339] Formula: -(CH2) m1 -(N(R321 )-(CH2) m2 ) m3 -(where R is in the formula) 321 It is a hydrogen atom or a carbon atom. 1-4 Alkyl groups, where m1 is an integer from 2 to 10, m2 is an integer from 2 to 4, m3 is an integer from 1 to 5, and when m3 is an integer greater than 2, each R 321 The groups represented by m2 (which can be the same or different from each other)

[0340] Formula: -(CH2) m4 -(O-(CH2) m5 ) m6 -(where m4 is an integer from 2 to 10, m5 is an integer from 2 to 4, m6 is an integer from 1 to 5, and when m6 is an integer greater than 2, each m5 can be the same or different) represents the group, and,

[0341] Formula: -(CH2) m7 -(S-(CH2) m8 ) m9 -(where m7 is an integer from 2 to 10, m8 is an integer from 2 to 4, m9 is an integer from 1 to 5, and when m9 is an integer greater than 2, each m8 can be the same or different) represents the group.

[0342] The preferred example of X is selected from the following groups:

[0343] Formula: -(CH2) m10 -(where m10 is an integer from 1 to 6) represents the group,

[0344] Formula: -C2H4-(N(R 322 )-C2H4) m11 -(where R is in the formula) 322 It is a hydrogen atom or a carbon atom. 1-4 Alkyl groups, where m11 is an integer from 1 to 5; when m11 is an integer greater than 2, each R... 322 The groups represented by (which may be the same or different from each other)

[0345] Formula: -C2H4-(O-C2H4) m12 -(where m12 is an integer from 1 to 5) represents the group, and,

[0346] Formula: -C2H4-(S-C2H4) m13 -(where m13 is an integer from 1 to 5) represents the group.

[0347] The substituents that can be arbitrarily substituted on the carbon atoms of the aforementioned alkylene group are preferably halogen atoms, hydroxyl groups, alkoxy groups, mercapto groups, alkylthio groups, amino groups, monoalkylamino groups, dialkylamino groups, acyloxy groups, acylamino groups, or acylthio groups. The number of substituents varies depending on the number of carbon atoms in the alkylene group, for example, from 1 to 3, preferably 2 or 3.

[0348] In one embodiment, R is preferred. 3 It is the formula: R 31 The group represented by -X-, R 31 X is the group represented by formula (A) or formula (B), where X is -C m H 2m -(where m is an integer from 1 to 10).

[0349] R 4 A preferred example is a hydrogen atom or an alkyl group optionally substituted. The substituent is preferably at least one selected from the group consisting of halogen atoms, oxo groups, hydroxyl groups, alkoxy groups, alkylthio groups, alkylamino groups, and cyano groups. The number of substituents is preferably 1 to 3.

[0350] R 4 A more preferred example is an alkyl group.

[0351] R 4 A further preferred example is C. 1-6 alkyl.

[0352] R 4 The optimal example is C. 1-4 alkyl.

[0353] R 5 A preferred example is a hydrogen atom or an alkyl group optionally substituted, wherein the substituent is preferably at least one selected from the group consisting of halogen atoms, oxo groups, hydroxyl groups, alkoxy groups, alkylthio groups, alkylamino groups, and cyano groups. The number of substituents is preferably 1 to 3.

[0354] R 5 A more preferred example is a hydrogen atom or an alkyl group.

[0355] R 5 Further preferred examples are hydrogen atoms or C atoms. 1-6 alkyl.

[0356] R 5 The optimal example is a hydrogen atom or a C atom. 1-4 alkyl.

[0357] Compound (1) is preferably one of the following compounds (1A) or (1B):

[0358] [Chemistry 27]

[0359]

[0360] (where Base and R are in the formula) 1 ~R 5 Same as above).

[0361] Compound (1) is further preferably compound (1A). In one embodiment, compound (1A) is preferably compound (1C) or (1D) of the following:

[0362] [Chemistry 28]

[0363]

[0364] (where R is in the formula) 33 ~R 35 Whether the groups are the same or different, they are protecting groups for hydrogen atoms, alkyl groups, or amino groups; Base, R 1 R 2 R 4 R 5 (and m are the same as above).

[0365] In other embodiments, compound (1A) is preferably compound (1A') or (1A''):

[0366] [Chemistry 29]

[0367]

[0368] (where Base and R are in the formula) 1 ~R 4 Same as above).

[0369] The salts mentioned above may or may not be pharmaceutically permissible. They may be inorganic or organic salts.

[0370] Examples of the aforementioned salts include: alkali metal salts (e.g., sodium, potassium, lithium), alkaline earth metal salts (e.g., calcium, magnesium), other metal salts (e.g., aluminum, iron, zinc, copper, nickel, cobalt), ammonium salts, tetramethylammonium salts, amine salts (e.g., tert-octylamine, dibenzylamine, morpholine, glucosamine, phenylglycine alkyl ester, ethylenediamine, N-methylglucosamine, guanidine, diethylamine, triethylamine, dicyclohexylamine, N,N'-dibenzylethylenediamine, chloroprocaine, procaine, diethanolamine). N-Benzyl-phenylethylamine salt, piperazine salt, tris(hydroxymethyl)aminomethane salt), inorganic acid salts (e.g., hydrofluoric acid, hydrochloride, hydrobromide, hydroiodide, nitrate, perchlorate, sulfate, phosphate), organic acid salts (e.g., methanesulfonate, trifluoromethanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, maleate), amino acid salts (e.g., glycine, lysine, arginine, ornithine, glutamate, aspartate).

[0371] <<Preparation method of compound (1)>>

[0372] 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.

[0373] [Chemistry 30]

[0374] Option 1

[0375]

[0376] (In the formula, L is the leaving group, Base, A) 1 and R 1 ~R 4 Same as above).

[0377] [Reaction Scheme 1]

[0378] <Process (I)>

[0379] Step (I) is the process of dehydrogenating compound (2A) (oxidizing -CH2-NH- to -CH=N-) to obtain compound (1E).

[0380] Compound (2A) can be obtained by known methods, such as those described in U.S. Patent Application Publication No. 2007 / 167387 (which is incorporated herein by reference).

[0381] Examples of reagents (oxidants) for dehydrogenating compound (2A) include high-valent iodine compounds. Examples of high-valent iodine compounds include (diacetoxyiodine)benzene, [bis(trifluoroacetoxy)iodine]benzene, 2-iodoacylbenzenesulfonic acid, 2-iodoacylbenzoic acid, and Dess-Martin reagent.

[0382] The amount of the above reagent is usually 0.5 to 6 moles relative to 1 mole of compound (2A), preferably 1 to 2 moles.

[0383] Dehydrogenation is preferably carried out in the presence of a solvent.

[0384] Examples of solvents include sulfoxide solvents (e.g., dimethyl sulfoxide), halogen solvents (e.g., dichloromethane), and mixtures of two or more of these solvents.

[0385] The temperature of dehydrogenation is not particularly limited as long as the reaction proceeds; for example, it is 0–100°C, preferably 20–60°C.

[0386] There is no particular limitation on the dehydrogenation time, for example, it is 1 to 12 hours, preferably 4 to 6 hours.

[0387] <Process (II)>

[0388] Step (II) involves reacting compound (1E) with formula (3A): R 4 ·(where R) 4 The free radical represented by formula (3B) (same as above): R 4 In the formula, M is a metal atom or a group of atoms containing a metal atom, R 4 The process of reacting an organometallic reagent (as described above) to obtain compound (1F).

[0389] Examples of compounds that generate free radicals (3A) include compounds that generate ethyl radicals in the presence of oxygen (e.g., triethylboron). 4 In the case of ethyl, triethylboron can be used alone, R 4 In cases other than ethyl, triethylboron can be reacted with the formula: R 4 -Halo (where Halo is a halogen atom) represents the combination of compounds used.

[0390] The amount of free radical (3A) generated in the above reaction system is typically 4 to 20 moles relative to 1 mole of compound (1E), preferably 8 to 12 moles.

[0391] In compound (3B), M can be, for example, Li, Na, K, Zn, Cu, Ce, MgCl, MgBr, MgI, etc. In one embodiment, compound (3B) is preferably a Grignard reagent.

[0392] The amount of compound (3B) used is usually 1 to 10 moles relative to 1 mole of compound (1E), preferably 2 to 6 moles.

[0393] The above reaction is preferably carried out in the presence of a Lewis acid.

[0394] Examples of Lewis acids include zinc chloride, tin tetrachloride, titanium tetrachloride, boron trifluoride, boron trifluoride diethyl ether (BF3·OEt2), boron trichloride, and trimethylsilyl trifluoromethanesulfonate ((CH3)3SiOSO2CF3).

[0395] The amount of Lewis acid used relative to 1 mole of compound (1E) is typically 4 to 20 moles, preferably 8 to 12 moles.

[0396] The above reaction is preferably carried out in the presence of a solvent.

[0397] Examples of solvents include aromatic hydrocarbon solvents (e.g., toluene, xylene), halogenated hydrocarbon solvents (e.g., dichloromethane), and mixtures of two or more of these solvents.

[0398] The reaction temperature of the above reaction is not particularly limited as long as the reaction proceeds, for example, it is -78 to 40°C, preferably 0 to 30°C.

[0399] The reaction time of the above reaction is not particularly limited, for example, it is 0.1 to 2 hours, preferably 0.5 to 1 hour.

[0400] <Process (IIIa)>

[0401] Step (IIIa) is to dehydrogenate compound (1F) (removing -C(H)(R) 4 -NH- is oxidized to -CR 4 =N-) to obtain compound (1G). This dehydrogenation can be carried out in the same way as step (I).

[0402] To illustrate, this can be achieved by reacting compound (1G) with formula (3C): R 5 ·(where R) 5 The free radical or formula (3D) represented by the same as above: R 5 In the formula, M is a metal atom or a group of atoms containing a metal atom, R 4 The compound represented by the organometallic reagent (as described above) reacts to introduce R. 5The reaction can be carried out by the methods described in Organic Letters 1999, 1, 4, 569-572 and Tetrahedron Letters 39 (1998) 3237-3240.

[0403] <Process (IIIb)>

[0404] Step (IIIb) is to hydrogenate compound (1G) (by removing -CR) 4 =N-restored to -C(H)(R) 4 The process of obtaining compound (1H) from )-NH-)

[0405] Examples of reagents (reducing agents) that hydrogenate compounds (1G) include: diisobutylaluminum hydride, lithium aluminum hydride, sodium borohydride, etc.

[0406] The amount of the above reagent is usually 1 to 10 moles relative to 1 mole of compound (1G), preferably 3 to 5 moles.

[0407] Hydrogenation is preferably carried out in the presence of a solvent.

[0408] Examples of solvents include aromatic hydrocarbon solvents (e.g., toluene, xylene), halogenated hydrocarbon solvents (e.g., dichloromethane), and mixtures of two or more of these solvents.

[0409] There are no particular restrictions on the hydrogenation temperature as long as the reaction proceeds; for example, it can be -20 to -78°C, preferably -60 to -78°C.

[0410] There is no particular limitation on the hydrogenation time, for example, it is 0.5 to 6 hours, preferably 1 to 3 hours.

[0411] <Process (IIIc)>

[0412] Step (IIIc) involves reacting compound (1H) with formula (4A): R 3 -L(where L is the leaving group, R) 3 The same process as above, but not the process of reacting the compound represented by hydrogen to obtain compound (1I).

[0413] Examples of leaving groups represented by L in compound (4A) include: halogen atoms (e.g., chlorine atoms, bromine atoms, iodine atoms), alkyl sulfonyloxy groups (e.g., methanesulfonyloxy groups), haloalkyl sulfonyloxy groups (e.g., trifluoromethylsulfonyloxy groups), or aryl sulfonyloxy groups (e.g., toluenesulfonyloxy groups).

[0414] The amount of compound (4A) used is usually 1 to 8 moles relative to 1 mole of compound (1H), preferably 1 to 4 moles.

[0415] The above reaction is preferably carried out in the presence of a solvent.

[0416] Examples of solvents include ether solvents (e.g., tetrahydrofuran), nitrile solvents (e.g., acetonitrile), aromatic hydrocarbon solvents (e.g., toluene, xylene), and mixtures of two or more of these solvents. Among these, aromatic hydrocarbon solvents are preferred, and at least one selected from the group consisting of toluene and xylene is more preferred.

[0417] The above reaction is preferably carried out in the presence of a base.

[0418] Examples of bases 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 trialkylamines are more preferred. 1-4 Alkylamines.

[0419] The amount of base used relative to 1 mole of compound (2A) is usually 2 to 10 moles, preferably 5 to 8 moles.

[0420] The reaction temperature of the above reaction is not particularly limited as long as the reaction proceeds, for example, it is 30 to 150°C, preferably 50 to 120°C.

[0421] The reaction time for the above reaction is not particularly limited, for example, it is 1 to 24 hours, preferably 1 to 12 hours.

[0422] To explain, R 3 A compound (1Q) is a methyl group that optionally has one or two substituents, which can be obtained, for example, by a method comprising the following steps (IIIc').

[0423] <Process (IIIc')>

[0424] Step (IIIc') involves reacting compound (1H) with formula (4B): R 3g -C(=O)-R 3h (where R is in the formula) 3g and R 3h It is R 3 The process of reacting a carbonyl compound represented by a residue to obtain compound (1Q).

[0425] The amount of compound (4B) used is usually 1 to 6 moles relative to 1 mole of compound (1H), preferably 1 to 3 moles.

[0426] The above reaction is preferably carried out in the presence of a reducing agent.

[0427] 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, and combinations thereof.

[0428] The amount of reducing agent used is usually 1 to 8 moles relative to 1 mole of compound (1H), preferably 1 to 4 moles.

[0429] The above reaction is preferably carried out in the presence of an acid catalyst. Examples of acid catalysts include pyridine p-toluenesulfonate (PPTS), acetic acid, and hydrochloric acid.

[0430] The above reaction is preferably carried out in the presence of a solvent. Examples of solvents include alcohol solvents (e.g., methanol), ether solvents (e.g., tetrahydrofuran), and mixtures of two or more of these solvents.

[0431] The reaction temperature of the above reaction is not particularly limited as long as the reaction proceeds; for example, it is 0 to 100°C, preferably 0 to 40°C.

[0432] The reaction time of the above reaction is not particularly limited, for example, it is 0.5 to 12 hours, preferably 1 to 4 hours.

[0433] To explain, R 3 It is the formula: R 31 Compounds (1I) with the group represented by -X- can be obtained, for example, by a method comprising the steps (IIId) to (IIIf) described below.

[0434] (IIId) The process of reacting compound (1H) with the compound represented by formula (4C) to obtain the compound represented by formula (1H');

[0435] [Chemistry 31]

[0436]

[0437] (In formula (4C), X and L are the same as above)

[0438] [Chemistry 32]

[0439]

[0440] (In formula (1H'), Base and A) 1R 1 R 2 R 4 (And X is the same as above)

[0441] (IIIe) The process of reacting compound (1H') with a hydrazine compound to obtain the compound represented by the following formula (1H”);

[0442] [Chemistry 33]

[0443]

[0444] as well as,

[0445] (IIIf) Optionally, the amino group of compound (1H”) is protected with an amino protecting group or the compound (1H”) is guanidineized.

[0446] <Process (IIId)>

[0447] Process (IIId) can be performed in the same way as process (IIIc).

[0448] <Process (IIIe)>

[0449] The amount of hydrazine compound used relative to 1 mole of compound (1H') is usually 1 to 10 moles, preferably 1.1 to 3.5 moles.

[0450] The reaction is preferably carried out in the presence of a solvent. Examples of solvents include water, alcohol-based solvents (e.g., methanol, ethanol), and mixtures of two or more such solvents.

[0451] <Process (IIIf)>

[0452] The method for protecting the amino group of compound (1H”) with an amino protecting group can be a known or conventional method. For example, the step of introducing a trifluoromethyl carbonyl group as a protecting group of 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 above reaction is preferably carried out in the presence of a solvent. A preferred example of a solvent is a cyclic amine (e.g., pyridine).

[0453] Guanidinization of compounds (1H”) is usually carried out by reaction with a guanidinizing agent. Examples of guanidinizing agents include nitrogen-based guanidinizing agents and sulfur-based guanidinizing agents.

[0454] Examples of nitrogen-based guanidine oxidizing agents include compounds represented by the following formula:

[0455] [Chemistry 34]

[0456]

[0457] (where L is in the formula) 3 It is a leaving group, R 3c ~R 3e Same as above).

[0458] L 3 The examples of leaving groups represented are the same as those for L.

[0459] The nitrogen-based guanidine oxidizing agent is preferably 1-aminopyrazole hydrochloride, 1-methamidinyl-1,2,4-triazole hydrochloride, 1-(N-tert-butoxy-amino)pyrazole, 1-(N-benzyloxy-amino)pyrazole, 1-[N,N'-(di-tert-butoxy)amino]pyrazole, 1-[N,N'-(di-benzyloxy)amino]pyrazole, 1,2,3-tris(tert-butoxycarbonyl)guanidine, or Goodman's reagent.

[0460] Examples of thioguanidine oxidizing agents include compounds represented by the following formula:

[0461] [Chemistry 35]

[0462]

[0463] (where R is in the formula) 3c ~R 3e Same as above).

[0464] The preferred thioguanidine oxidizing agent is N,N'-di-tert-butoxy-S-methylisothiourea or 1,3-di-tert-butoxythiourea.

[0465] The amount of guanidinizing agent used relative to 1 mole of compound (1H”) is usually 0.5 to 10 moles, preferably 0.8 to 2.0 moles.

[0466] The reaction of compound (1H”) with guanidinizing agent is preferably carried out in the presence of a solvent.

[0467] Examples of solvents include: halogenated hydrocarbon solvents (e.g., dichloromethane), amide solvents (e.g., N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone), and mixtures of two or more of these solvents. Among these, amide solvents are preferred, and N,N-dimethylformamide is more preferred.

[0468] There are no particular restrictions on the reaction temperature of the above reaction, as long as the reaction proceeds, for example, 15 to 30°C.

[0469] <Process (IV)>

[0470] Step (IV) is the step of reacting compound (1F) with compound (4A) to obtain compound (IJ). This reaction can be carried out in the same manner as step (IIIc). Additionally, R... 3 It is the formula: R 31Compounds (1J) with the group represented by -X- can be obtained, for example, by a method that includes the same steps as steps (IIId) to (IIIf).

[0471] <Process (IV')>

[0472] Step (IV') is the process of reacting compound (1F) with compound (4B) to obtain compound (1R). This step can be performed in the same manner as step (IIIc').

[0473] The bases of compounds (1), (1A) to (1J), (1Q), (1R) and (2A) can be transformed, for example, by the following reaction scheme 2.

[0474] [Chemistry 36]

[0475]

[0476] (where Q is in the formula) 1 It is a hydrogen atom or a substituent, Q 2 and Q 3 Whether they are the same or different, they are either protecting groups of hydrogen atoms or amino groups (but Q) 2 and Q 3 (not both hydrogen atoms), Q 4 ~Q 7 Whether the groups are the same or different, they are either protecting groups of hydrogen atoms or amino groups, and ring G is a five- or six-membered nitrogen-containing heterocycle.

[0477] [Reaction Scheme 2]

[0478] <Process (V)>

[0479] Step (V) is the process of converting the Base in compounds (1), (1A) to (1J), (1Q), (1R) and (2A) from “optionally substituted 2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl” to “optionally substituted 2-oxo-1,2-dihydropyrimidin-1-yl”, and includes steps (Va) to (Vc).

[0480] <Process (Va)>

[0481] Step (Va) is a step in which a compound (1K) whose Base is “optionally substituted 2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl” is reacted with the compound represented by formula (5A) and a phosphate halide to obtain compound (1L).

[0482] In compound (1K), Q 1 Preferably, hydrogen atoms or alkyl groups are used; more preferably, hydrogen atoms or C groups are used. 1-4 alkyl.

[0483] The compound (5A) is preferably a five-membered nitrogen-containing heterocyclic compound, more preferably a triazole.

[0484] The amount of compound (5A) used is typically 5 to 20 moles relative to 1 mole of compound (1K), preferably 7 to 9 moles.

[0485] Phosphoric acid halides are preferably phosphorus oxychloride.

[0486] The amount of phosphate halide used is usually 1 to 5 moles relative to 1 mole of compound (1K), preferably 1 to 3 moles.

[0487] The above reaction is preferably carried out in the presence of a solvent.

[0488] Examples of solvents include nitrile solvents (e.g., acetonitrile), ether solvents (e.g., tetrahydrofuran), halogen solvents (e.g., haloalkanes), and mixtures of two or more of these solvents. Among these, nitrile solvents (e.g., acetonitrile) are preferred.

[0489] The above reaction is preferably carried out in the presence of a base.

[0490] Examples of bases 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 trialkylamines are more preferred. 1-4 Alkylamines.

[0491] The amount of base used relative to 1 mole of compound (1K) is usually 5 to 20 moles, preferably 10 to 15 moles.

[0492] The reaction temperature for the above reaction is not particularly limited as long as the reaction proceeds; for example, it can be -5℃ to 10℃.

[0493] The above reaction can also be performed, for example, by the method described in U.S. Patent No. 5,359,067.

[0494] <Process (Vb)>

[0495] Step (Vb) is the step of reacting compound (1L) with ammonia to obtain compound (1M).

[0496] The amount of ammonia used is typically 5 to 100 moles relative to 1 mole of compound (1L), preferably 20 to 50 moles.

[0497] The above reaction is preferably carried out in the presence of a solvent.

[0498] Examples of solvents include amide solvents (e.g., N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone), ether solvents (e.g., tetrahydrofuran, dioxane, and other cyclic ethers), and mixtures of two or more of these solvents. Among these, ether solvents are preferred, cyclic ethers are more preferred, and at least one selected from tetrahydrofuran and dioxane is even more preferred.

[0499] The reaction temperature for the above reaction is not particularly limited as long as the reaction proceeds; for example, it can be 15–30°C.

[0500] <Process (Vc)>

[0501] Step (Vc) is the step of protecting the amino group of compound (1M) with a protecting group to obtain compound (1N). The method of protecting the amino group with a protecting group can be a known (e.g., U.S. Patent Application Publication No. 2007 / 167387) or conventional method.

[0502] <Process (VI)>

[0503] Step (VI) is the process of converting the Base in compounds (1), (1A) to (1J), (1Q), (1R) and (2A) from “optionally substituted 2,4-dioxo-1,2,3,4-tetrahydropyrimidine-1-yl” to “optionally substituted purine-9-yl”.

[0504] Step (VI) specifically involves reacting a compound (1K) whose Base is “optionally substituted 2,4-dioxo-1,2,3,4-tetrahydropyrimidine-1-yl” with the compound represented by formula (5B) to obtain compound (1O).

[0505] The amount of compound (5B) used is usually 1 to 5 moles relative to 1 mole of compound (1K), preferably 1 to 3 moles.

[0506] The above reaction is preferably carried out in the presence of a Lewis acid.

[0507] An example of a Lewis acid is trimethylsilyl trifluoromethanesulfonate.

[0508] The amount of Lewis acid used relative to 1 mole of compound (1K) is typically 0.5 to 5 moles, preferably 1 to 3 moles.

[0509] The above reaction is preferably carried out in the presence of a silylating agent.

[0510] 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-trimethylsilylheptafluorobutamide, N-methyl-N-trimethylsilyltrifluoroacetamide, N-trimethylsilylacetamide, and combinations of two or more of these. Among these, N,O-bis-trimethylsilylacetamide (BSA) is preferred.

[0511] The amount of silylating agent used relative to 1 mole of compound (1K) is usually 1 to 20 moles, preferably 3 to 8 moles.

[0512] The reaction temperature of the above reaction is not particularly limited as long as the reaction proceeds, for example, it is 30 to 150°C, preferably 50 to 120°C.

[0513] The above reaction can also be performed, for example, by the method described in U.S. Patent Application Publication No. 2012 / 071646 (which is incorporated herein by reference).

[0514] <Process (VII)>

[0515] Step (VII) is the process of converting the Base in compounds (1), (1A) to (1J), (1Q), (1R) and (2A) from “optionally substituted 2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl” to “optionally substituted 6-oxo-1,6-dihydro-9H-purine-9-yl”.

[0516] Step (VII) is specifically a step of reacting a compound (1K) whose Base is “optionally substituted 2,4-dioxo-1,2,3,4-tetrahydropyrimidine-1-yl” with the compound represented by formula (5C) to obtain compound (1P).

[0517] The reaction can be carried out under the same conditions as in process (VI).

[0518] As needed, the method for manufacturing compound (1) may further include a step of purifying the intermediate and final products by conventional methods, such as concentration, recrystallization, silica gel column chromatography, etc.

[0519] <<Compositions Containing Compound (1)>>

[0520] The composition of the present invention contains the compound (1) described above.

[0521] The composition may contain only one compound (1) or two or more compounds (1). For example, the composition may contain one, two, three or four compounds selected from the group consisting of:

[0522] Base is a compound of thymine-1-yl (1);

[0523] Base is a compound of 5-methylcytosine-1-yl, N-acetyl-5-methylcytosine-1-yl, N-isobutyryl-5-methylcytosine-1-yl or N-benzoyl-5-methylcytosine-1-yl (1);

[0524] Base is a compound of adenine-9-yl, N-acetyl-adenine-9-yl, N-isobutyryl-adenine-9-yl or N-benzoyl-adenine-9-yl (1); and,

[0525] Base is a compound of guanine-9-yl, N-acetyl-guanine-9-yl, N-isobutyryl-guanine-9-yl; N-benzoyl-guanine-9-yl or N-(N,N-dimethylaminomethylpyranyl)-guanine-9-yl (1).

[0526] Examples of forms of composition include: liquids.

[0527] When the composition is in liquid form, it typically contains a solvent. Known solvents can be used, and preferred examples include: halocarbon solvents (e.g., dichloromethane), nitrile solvents (e.g., acetonitrile), aromatic hydrocarbon solvents (e.g., toluene, xylene), water, TE buffer, etc. Among these, dichloromethane, toluene, and acetonitrile are preferred.

[0528] The composition is typically contained in a container and provided to the user.

[0529] The composition can be used in the synthesis of oligonucleotides or their salts described later. Additionally, the composition can be used as a pharmaceutical composition.

[0530] Oligonucleotides or their salts

[0531] The oligonucleotides or their salts of the present invention have units represented by the following formula (6).

[0532] [Chemistry 37]

[0533]

[0534] (where Base and A are in the formula) 1 R 3 ~R5 (and n is the same as above)

[0535] The aforementioned unit is preferably the unit represented by the following formula (6a).

[0536] [Chemistry 38]

[0537]

[0538] (where A is in the formula) 2 For OH, O - SH or S - Base, A 1 R 3 ~R 5 (and n is the same as above)

[0539] The unit represented by equation (6a) is preferably the unit represented by equation (6a-1), and more preferably the unit represented by equation (6a-2) or (6a-3):

[0540] [Chemistry 39]

[0541]

[0542] (where Base and A are in the formula) 1 A 2 R 3 ~R 5 R 33 ~R 35 (and m are the same as above).

[0543] Hereinafter, oligonucleotides or their salts having units represented by formula (6) or (6a) will be referred to as “oligonucleotide (6)”.

[0544] When an oligonucleotide (6) has two or more units represented by formula (6) or (6a), the structures of the units may be the same or different.

[0545] In addition to the unit represented by formula (6) or (6a), oligonucleotide (6) may contain other units. Examples of other units include at least one selected from the units represented by formulas (7) to (10).

[0546] [Chemistry 40]

[0547]

[0548] (where A is in the formula) 3 Identical or different, is a single bond or optionally substituted alkylene group, R a It is a hydrogen atom or a hydroxyl group, R b With R 3Same as above (Base is the same as above)

[0549] The other units mentioned above may preferably be selected from at least one of the units represented by formulas (7a) to (10a).

[0550] [Chemistry 41]

[0551]

[0552] (where A is in the formula) 4 ~A 7 The same or different, is OH or O - SH or S - Base, A 3 R a and R b Same as above)

[0553] It should be noted that other units may be, for example, units of nucleotides described in U.S. Patent Application Publication No. 2003 / 105309, U.S. Patent Application Publication No. 2017 / 044528, U.S. Patent Application Publication No. 2006 / 166908, U.S. Patent Application Publication No. 2012 / 208991, U.S. Patent Application Publication No. 2015 / 266917, and U.S. Patent Application Publication No. 2003 / 207841 (U.S. Patent Application Publication No. 2003 / 105309, U.S. Patent Application Publication No. 2017 / 044528, U.S. Patent Application Publication No. 2006 / 166908, U.S. Patent Application Publication No. 2012 / 208991, U.S. Patent Application Publication No. 2015 / 266917, and U.S. Patent Application Publication No. 2003 / 207841 are included in this specification by reference).

[0554] There are no particular restrictions on the base sequence of the oligonucleotide (6) as long as it is complementary to the base sequence (full length or part thereof) of the target DNA or target RNA.

[0555] The length of the oligonucleotide (6) is not particularly limited and can be selected according to the length of the target base sequence. The lower limit of the oligonucleotide (6) length is, for example, 5 mere, preferably 10 mere, more preferably 15 mere, and the upper limit of the oligonucleotide (6) length is, for example, 200 mere, preferably 100 mere, more preferably 50 mere, and more preferably 30 mere. The length of the oligonucleotide (6) is, for example, 5–200 mere, preferably 5–50 mere, more preferably 10–40 mere, and more preferably 15–30 mere. The longer the oligonucleotide (6), the stronger its binding affinity to the target base sequence.

[0556] In oligonucleotides (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 according to the purpose of use (primers, probes, Clamp nucleic acids, drugs, etc.).

[0557] Oligonucleotide (6) can be in the form of a salt. That is, at least one nucleotide unit of the nucleotide units constituting oligonucleotide (6) can be in the form of a salt. The salt can be a pharmaceutically permissible salt or a pharmaceutically unpermissible salt. The salt can be an inorganic salt or an organic salt. Examples of the salt, similar to those exemplified in compound (1), include: alkali metal salts, alkaline earth metal salts, other metal salts, ammonium salts, tetramethylammonium salts, amine salts, inorganic acid salts, organic acid salts, amino acid salts, etc.

[0558] Oligonucleotides (6) can be modified by labeling substances. There are no particular limitations on the labeling substances, and fluorescent substances, haptens (e.g., biotin, digoxigenin, DNP, etc.), radioactive isotopes, and other substances known in the art for labeling nucleic acids can be used.

[0559] Oligonucleotide (6) has high sequence specificity.

[0560] Oligonucleotide (6) has a high Tm value for both single-stranded DNA and single-stranded RNA. That is, oligonucleotide (6) binds firmly to both single-stranded DNA and single-stranded RNA and has a high double-strand forming ability. In particular, the Tm value of oligonucleotide (6) for single-stranded RNA is extremely high compared to that of DNA or RNA in formula (7).

[0561] Oligonucleotides (6) can be used as probes for the examination or highly selective detection of single-stranded RNA or single-stranded DNA base sequences.

[0562] Oligonucleotides (6) are not easily broken down by nucleases and can persist in organisms for a long time after being given to them. For example, oligonucleotides (6) can form double strands with positive RNA, inhibiting the transcription of mRNA, which is a component of pathogenic proteins in the organism. In addition, oligonucleotides (6) can also inhibit the proliferation of infectious viruses.

[0563] Oligonucleotides (6) are useful as drugs that inhibit gene activity to treat diseases, such as antitumor agents and antiviral agents.

[0564] In addition, oligonucleotides (6) have stable and excellent activity as antisense or antigenes or aptamers, or excellent activity as detection agents for specific genes or as primers for amplification initiation.

[0565] Oligonucleotides (6) are useful as materials for various physiological / biologically active substances, pharmaceutical materials, functional materials of double-stranded oligonucleotides used in RNA interference and decoy methods, DNA chips targeting single-stranded nucleic acids such as cDNA, functional materials such as molecular beacons, functional materials used in various antisense methods (including ribosomes and DNases), reverse gene methods, and gene homologous recombination methods, materials for highly sensitive analysis of trace components in organisms combined with fluorescent and luminescent substances, and materials for developing reagents for gene function elucidation and other research.

[0566] <<Methods for Manufacturing Oligonucleotides (6)>>

[0567] Oligonucleotides (6) can be synthesized using conventional methods, such as the phosphoramide scheme.

[0568] For example, the method for manufacturing oligonucleotides (6) includes the following steps.

[0569] (I) A step of reacting a compound represented by formula (6B) or a salt thereof with at least one selected from the compounds represented by formulas (6C) to (10C) below;

[0570] [Chemistry 42]

[0571]

[0572] (In formula (6B), R) 2a It can be an alkyl group with optional substituents, an alkenyl group with optional substituents, a cycloalkyl group with optional substituents, a cycloalkenyl group with optional substituents, an aryl group with optional substituents, a protecting group of a hydroxyl group, a phosphinyl group with optional substituents, a dihydroxyphosphonophosphonoyl group with optional substituents, or a hydroxythiophosphonophosphonoyl group with optional substituents, Base, A 1 R 3 ~R 5 (and n is the same as above)

[0573]

[0574] (where R is in the formula) 1a It is 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 of a hydroxyl group, a substituted phosphinyl group, an optionally substituted dihydroxyphosphonophosphonoyl group, or an optionally substituted hydroxythiophosphonophosphonoyl group, R A and R B Same or different, is a hydrogen atom or an alkyl group, R C It is a hydrogen atom, alkyl, haloalkyl, or cyanoalkyl, Base, A 1 A3 R 3 ~R 5 n, R a and R b Same as above)

[0575] And / or,

[0576] (II) A step of reacting a compound represented by formula (6C) or a salt thereof with at least one of the compounds represented by formulas (6B) to (10B) or a salt thereof; and (III) a step of oxidizing (in particular oxidizing phosphorus atoms) the compound obtained in step (I) and / or step (II).

[0577] [Chemistry 44]

[0578]

[0579] (In formula (6C), Base, A) 1 R 1a R 3 ~R 5 , n and R A ~R C Same as above)

[0580] [Chemistry 45]

[0581]

[0582] (where Base and A are in the formula) 1 A 3 R 2a R 3 ~R 5 n, R a and R b Same as above)

[0583] As needed, the method for manufacturing oligonucleotides (6) may further include the steps of purifying intermediates and final products by conventional methods, such as concentration, recrystallization, silica gel column chromatography, gel filtration, ethanol precipitation, preparative HPLC, etc.

[0584] <<Compositions Containing Oligonucleotides (6)>>

[0585] The compositions of the present invention contain oligonucleotides (6) as described above.

[0586] The composition may contain only one oligonucleotide (6) or two or more oligonucleotides (6).

[0587] Examples of the forms in which a composition can be formed include liquids and solids.

[0588] When the composition is in liquid form, it typically contains a solvent. Known solvents can be used, preferably including: 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 even more preferred. Examples of buffers include: Tris-HCl buffer (tris-hydroxymethylaminomethane), Tris-HCl buffer (tris-hydroxymethylaminomethane-hydrochloric acid), Tris-EDTA buffer (TE buffer), sodium phosphate, 2-morpholinoethanesulfonic acid (MES), N-(2-acetamide)iminodiacetic acid (ADA), piperazine-1,4-bis(2-ethanesulfonic acid), N-(2-acetamide)-2-aminoethanesulfonic acid (ACES), 2-amino-N,N,N-trimethylethaneammonium chloride (cholamine chloride), 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), acetamide glycine, tris(hydroxymethyl)methylglycine (Tricine), glycine, N,N-dihydroxyethylglycine (Bicine), etc.

[0589] The composition may further contain salts. Salts include metal chlorides, examples of which include NaCl, MgCl2, and KCl.

[0590] The composition may further contain additives and co-solvents. Examples include dimethyl sulfoxide (DMSO), glycerol, formamide, bovine serum albumin, ammonium sulfate, polyethylene glycol (PEG), gelatin, and nonionic surfactants. Examples of nonionic surfactants include Tween20 (registered trademark) and Triton X-100 (registered trademark).

[0591] When the composition is in solid form, it may be, for example, a composition formed by loading oligonucleotides (6) onto a solid support. Examples of solid supports include inorganic and organic materials capable of supporting biopolymers. Preferred supports include glass (e.g., porous glass (CPG), silica gel), and resins (e.g., cross-linked non-swellable polystyrene resin (HPS)). Among these, HPS or CPG are more preferred.

[0592] The composition is typically contained in a container and provided to the user.

[0593] The composition can be used for the amplification or detection of target nucleic acids, chain invasion, RNAi, etc., as described later. Additionally, the composition can be used as a pharmaceutical composition.

[0594] <<Detection Methods for Target Nucleic Acids>>

[0595] This invention includes a method for detecting target nucleic acids. The method comprises the following steps:

[0596] (I) The process of selectively amplifying the target nucleic acid using a nucleic acid amplification method; and,

[0597] (II) The procedure for detecting the target nucleic acid amplified in the above procedure (I).

[0598] The oligonucleotides used in the above amplification or detection preferably contain oligonucleotides (6).

[0599] In the nucleic acid amplification step (I), multiple primer molecules are typically used. At least some of these primer molecules may be oligonucleotides (6). For example, a forward primer containing oligonucleotides (6) and a reverse primer not containing oligonucleotides (6) may be used.

[0600] In the nucleic acid amplification process of step (I), multiple primer molecules and probe molecules are sometimes used. In this case, at least some of the primer molecules and probe molecules can be oligonucleotides (6). For example, a forward primer that does not contain oligonucleotides (6), a reverse primer that does not contain oligonucleotides (6), and a probe that contains oligonucleotides (6) can be used.

[0601] As a nucleic acid amplification method, there are no particular restrictions as long as the method can selectively amplify the 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 PCR, Clamp PCR, etc.), NASBA (refer to US Patent No. 5,130,238), TMA (refer to US Patent No. 5,399,491), TRC (refer to US Patent Application Publication No. 2001 / 0053518), LAMP (refer to US Patent No. 6,410,278), ICAN (refer to US Patent Application Publication No. 2003 / 073081), LCR (refer to European Patent Application No. 320,328), SDA (refer to US Patent No. 5,455,166), etc.

[0602] In one embodiment, the nucleic acid amplification method in step (I) is preferably Clamp PCR. In Clamp PCR, at least primers and Clamp nucleic acids are used. Either or both of the primers and Clamp nucleic acids may contain oligonucleotides (6).

[0603] Compared to target nucleic acids (e.g., variant genes), clamp nucleic acids typically clamp (or clamp) non-target nucleic acids (e.g., wild-type genes) more firmly in the test sample. Clamp nucleic acids bind tightly to non-target nucleic acids, inhibiting their amplification and thus selectively amplifying the target nucleic acid. For example, in detecting specific gene variants, nucleic acid amplification is performed in the presence of clamp nucleic acids with a base sequence completely complementary to the wild-type gene sequence; the amplification of the wild-type nucleic acid is inhibited, while the variant nucleic acid is selectively amplified.

[0604] Specifically, for example, when the target site of the target nucleic acid is taken as the variant site of the variant gene, and the base sequence containing the variant site (the target site of the target nucleic acid) is taken as "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.

[0605] Oligonucleotides (6) exhibit high sequence selectivity. Therefore, Clamp nucleic acids containing oligonucleotides (6) have very weak binding affinity to even base sequences differing by only one base, and can specifically bind to perfectly complementary base sequences. Furthermore, oligonucleotides (6) have high Tm values, forming stable double strands. That is, Clamp nucleic acids containing oligonucleotides (6) specifically and firmly bind to non-target nucleic acids, exhibiting high Clamping ability. The length of Clamp nucleic acids is not particularly limited, for example, 5–30 meters.

[0606] The nucleic acid amplification in step (I) can be a reverse transcription reaction. In the reverse transcription reaction, cDNA is synthesized using RNA as a template by RNA-dependent DNA polymerase. Since oligonucleotides (6) bind firmly to RNA, binding specific RNA in the test sample to oligonucleotides (6) can inhibit the reverse transcription reaction from that RNA. Thus, cDNA can be synthesized more specifically from the target RNA. The synthesized cDNA can be further amplified using methods such as PCR.

[0607] The target nucleic acid can also be the nucleic acid contained in the test sample. The test sample is typically a sample collected from a living organism (hereinafter also referred to as a "biological sample"). Samples obtained by pretreatment of samples collected from a living organism, such as removal of impurities, extraction / purification of nucleic acids, and pre-amplification, are commonly used. Specifically, blood, plasma, serum, pleural fluid, bronchial lavage fluid, bone marrow fluid, lymph, intestinal lavage fluid, excised tissue, nasopharyngeal swabs, saliva, nasal mucus, sputum, etc., can be used. Furthermore, these samples that have undergone the above pretreatment are also included in the "biological sample". Since the method of the present invention can detect nucleic acids with very high sensitivity, for example, excised tissue containing a mixture of normal and abnormal cells can be used to detect gene variations originating from abnormal cells. Furthermore, the method of the present invention is applicable even when the test sample is present in trace amounts or when the nucleic acid in the test sample is present in trace amounts. The test sample is preferably a solution containing the target nucleic acid. In the case of detecting intracellular target nucleic acids contained in blood or excised tissue, cell lysis can be performed using known methods. In addition to biological samples, excrement, drainage, river water, seawater, soil, and samples that have undergone the above pretreatment can also be used as test samples. Examples of excretions include urine and feces.

[0608] The target nucleic acid can be DNA or RNA, preferably DNA. DNA can also be cDNA synthesized from RNA via reverse transcription. The target nucleic acid can be a specific region on genomic DNA, such as a gene or a gene promoter region. The detection method of the present invention can be used to detect variations and polymorphisms in target sites of the target nucleic acid. Furthermore, the detection method of the present invention can be used to determine alleles. It can detect what type of allele is present in the sample. Additionally, the detection method of the present invention can be used to detect methylation. After treating the target nucleic acid with bisulfite, the presence of methylated cytosine in the target nucleic acid can be detected using the detection method of the present invention.

[0609] A gene containing a variation (hereinafter referred to as a variant gene) has a base sequence difference from that of the wild-type gene; that is, a variation. This difference arises from one or more variations selected from the group consisting of substitution, insertion, deletion, inversion, duplication, and transposition, or a combination thereof.

[0610] This difference is often related to the onset and / or treatment sensitivity of a specific disease. Here, "onset" includes not only the actual onset of the disease but also the risk of developing it. Furthermore, "treatment sensitivity" includes not only the effectiveness of treatment with drugs but also the strength of side effects. Examples of such diseases include, but are not limited to, cancer, myelodysplastic syndromes, and infectious diseases. Cancer is a preferred example of such a disease.

[0611] Preferred examples of the aforementioned genes include ABL / BCR fusion gene, HER2 gene, EGFR gene, c-KIT gene, KRAS gene, BRAF gene, PIK3CA gene, FLT3 gene, MYC gene, MYCN gene, MET gene, BCL2 gene, or EML4 / ALK fusion gene, etc.

[0612] In step (II), the amplified nucleic acid can be detected using known methods according to the nucleic acid amplification method described above. For example, the amplification can be detected qualitatively or quantitatively by detecting the fluorescence generated from the reaction solution or the turbidity of the reaction solution.

[0613] In addition, methods for detecting amplified target nucleic acids include base sequence analysis. The target nucleic acid can be detected by analyzing its base sequence using a known sequence analysis device (sequencing instrument).

[0614] It should be noted that the method for detecting the target nucleic acid in the test sample may, for example, be the method described in U.S. Patent Application Publication No. 2015 / 240299.

[0615] This invention includes a method for detecting a target nucleic acid in a test sample, the method comprising reacting a test sample containing an oligonucleotide (6) and a labeled probe with the target nucleic acid. Specific examples of this detection method include in situ hybridization, microarrays, etc.

[0616] When in situ hybridization is used as a detection method, the target nucleic acid in the test sample can be detected by hybridizing an oligonucleotide (6) labeled with a fluorescent dye or the like with the target nucleic acid in the test sample (e.g., cells) and measuring the label.

[0617] When using a microarray as a detection method, the target nucleic acid can be detected by reacting a microarray immobilized with a probe containing oligonucleotides (6) with a test sample containing the target nucleic acid and detecting the hybridization of the target nucleic acid with the probe.

[0618] Thus, using oligonucleotides (6), the target nucleic acid in the test sample can be detected with high sequence selectivity.

[0619] <<Reagent Kits for Detecting or Selectively Amplifying Target Nucleic Acids>>

[0620] Kits for detecting or selectively amplifying target nucleic acids contain oligonucleotides (6). It should be noted that the target nucleic acid may, for example, be a nucleic acid contained in the test sample.

[0621] In the kit, the oligonucleotide (6) can be used as a primer, probe, and / or Clamp nucleic acid for the amplification and / or detection of the target nucleic acid, as described above. The primers, probes, and / or Clamp nucleic acids can be appropriately designed based on the base sequence of the target or non-target nucleic acid.

[0622] One embodiment of the kit includes primers and / or probes. At least a portion of these primers and / or probes may be oligonucleotides (6).

[0623] Figure 2A This is a schematic diagram of an example kit, which includes a container holding a composition containing primers and probes. Kit 11 includes: an outer packaging box 12; a container support disposed within the outer packaging box 12 and having recesses formed on its surface; a container 13 mounted in the recesses and holding the composition containing primers and probes; and kit instruction manual 14. The kit instruction manual 14 may pre-describe the operating methods, storage conditions, and shelf life of kit 11.

[0624] Figure 2B This is a schematic diagram of an example kit, which includes a container containing a composition containing primers and a container containing a composition containing probes. Kit 21 includes: an outer packaging box 22; a container support disposed inside the outer packaging box 22 and having a first recess and a second recess spaced apart on its surface along its long side; a container 23a mounted in the first recess and containing the composition containing primers; a container 23b mounted in the second recess and containing the composition containing probes; and kit instruction manual 24.

[0625] Other embodiments of the kit include a forward primer, a reverse primer, and a probe. At least a portion of these may be an oligonucleotide (6). The composition containing the forward primer, the composition containing the reverse primer, and the composition containing the probe may all be contained in the same container (e.g.: Figure 2A The kit shown can be either a reagent kit or any two types can be contained in the same container (e.g.: Figure 2B The kit shown can be either a single reagent kit or all three types can be contained in their respective containers (e.g., [example of a kit]). Figure 2C (The kit shown).

[0626] Figure 2CThis is a schematic diagram of an example kit, which includes 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 packaging box 32; a container support disposed within the outer packaging box 32 and having first to third recesses spaced apart along its long side on its surface; a container 33a mounted in the first recess and containing a composition containing a forward primer; a container 33b mounted in the second recess and containing a composition containing a reverse primer; a container 33c mounted in the third recess and containing a composition containing a probe; and a kit instruction manual 34.

[0627] In addition, kits in other embodiments contain Clamp nucleic acids and primers. At least a portion of these may be oligonucleotides (6). The kit may be a kit for selectively amplifying target nucleic acids. The composition containing Clamp nucleic acids and the composition containing primers may be contained in the same container (e.g., Figure 2A The kit shown can also be contained in its own container (e.g.: Figure 2B (The kit shown).

[0628] Furthermore, kits in other embodiments comprise Clamp nucleic acid, primers, and probes. At least a portion of these may be oligonucleotides (6). The composition comprising Clamp nucleic acid, the composition comprising primers, and the composition comprising probes may all be contained in the same container (e.g.: Figure 2A The kit shown can be either a reagent kit or any two types can be contained in the same container (e.g.: Figure 2B The kit shown can be either a single reagent kit or all three types can be contained in their respective containers (e.g., [example of a kit]). Figure 2C (The kit shown).

[0629] The kit may contain DNA polymerase, deoxynucleoside triphosphates (dNTPs), reaction buffer, salt, restriction enzymes, etc.

[0630] This invention includes the use of oligonucleotides (6) for detecting or selectively amplifying target nucleic acids. The oligonucleotides (6) used herein, for example, have the same characteristics as those contained in the kit.

[0631] <Pharmaceutical Composition (or Formulation)>

[0632] The pharmaceutical composition (or formulation) of the present invention comprises a compound (1) or an oligonucleotide (6). Examples of pharmaceutical compositions (or formulations) comprising compound (1) include, for example, low-molecular-weight drugs such as AZT (azidothymidine), a nucleoside reverse transcriptase inhibitor (NRTI). Examples of pharmaceutical compositions (or formulations) comprising oligonucleotide (6) include, for example, medium- or high-molecular-weight nucleic acid drugs such as antisense, siRNA (small interfering RNA), aptamers, decoy nucleic acids, and CpG oligonucleotides.

[0633] The pharmaceutical composition may be 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 formulation known to those skilled in the art.

[0634] Preferred examples of pharmaceutical compositions are non-oral administration formulations (e.g., subcutaneous administration, intravenous administration, nasal administration, intramedullary administration, intraventricular administration, vitreous administration, etc.).

[0635] Other preferred examples of pharmaceutical compositions are topical formulations.

[0636] Pharmaceutical compositions typically further include pharmaceutically permissible carriers or additives.

[0637] Carriers can be categorized into solid and liquid carriers. Examples of solid carriers include starch, lactose, calcium sulfate dihydrate, sucrose, talc, gelatin, agar, pectin, gum arabic, magnesium stearate, stearic acid, and determinated collagen. Examples of liquid carriers include water (including physiological saline).

[0638] Additives include stabilizers, such as parabens (e.g., methylparaben, propylparaben); alcohols (e.g., benzyl alcohol); benzalkonium chloride; and phenols (e.g., phenol, cresol).

[0639] Oligonucleotides (6) are highly sequence-selective, have high Tm values, and are not easily broken down by nucleases. Therefore, pharmaceutical compositions (or formulations) containing compound (1) or oligonucleotides (6) can exert their effects by selectively targeting a target (e.g., a target gene) in vivo.

[0640] Example

[0641] The present invention will now be described in more detail with reference to embodiments, but the present invention is not limited to these embodiments.

[0642] <<Synthesis Example of Compound (1)>>

[0643] The notation and abbreviations in the synthesis example are as follows.

[0644] A BZ :N 6 -benzoyl adenine

[0645] Bz: Benzoyl group

[0646] DMTr: Dimethoxytriphenylmethyl

[0647] i-Pr: Isopropyl

[0648] BF3OEt2: Boron trifluoride-diethyl ether complex

[0649] BSA: N,O-bis(trimethylsilyl)acetamide

[0650] CIPS: 1,3-Dichloro-1,1,3,3-Tetraisopropyldisiloxane

[0651] DIBAL-H: Diisobutylaluminum Hydrogenation

[0652] DIPEA: N,N-Diisopropylethylamine

[0653] DMAP: 4-Dimethylaminopyridine

[0654] DMF: N,N-dimethylformamide

[0655] DMSO: Dimethyl sulfoxide

[0656] DMTrCl: 4,4'-Dimethoxytriphenylchloromethane

[0657] DBU: 1,8-diazabicyclo[5.4.0]-7-undecene

[0658] Et3B: Triethylboron

[0659] Et3N: Triethylamine

[0660] MeMgBr: Magnesium methyl bromide

[0661] MeOH: Methanol

[0662] NaBH3CN: Sodium cyanoborohydride

[0663] PPTS: Pyridine p-toluenesulfonate

[0664] TBAF: Tetra-n-Butylammonium Fluoride

[0665] Tf2O: Trifluoromethanesulfonic anhydride

[0666] THF: Tetrahydrofuran

[0667] TMSOTf: Trimethylsilyl trifluoromethanesulfonate

[0668] TsCl: p-Toluenesulfonyl chloride

[0669] rt: room temperature

[0670] h: hours

[0671] min: minutes

[0672] [Synthesis example 1]

[0673] Base is thymine-1-yl, A 1 It is a single key, R 1 It is DMTr, R 2 It is -P(N(i-Pr)2)(OC2H4CN), R 3 It is methyl, R 4 It is the methyl group configured with R, R 5 The compound (1) (hereinafter referred to as "compound (R)MT-4"), which is a hydrogen atom and n is 1, was synthesized according to the following reaction scheme.

[0674] [Chemistry 46]

[0675]

[0676] (Synthesis of compound T-1)

[0677] Compound 1 (20.0 g, 37.89 mmol) was dissolved in DMSO (500 mL) under a nitrogen stream. 2-iodobenzoic acid (27.89 g, 41.68 mmol) was added at room temperature, and the mixture was stirred for 4.5 hours at room temperature. The reaction mixture was cooled with water and the reaction was terminated with saturated sodium bicarbonate solution. The mixture was then diluted with ethyl acetate and water to separate the organic and aqueous layers, respectively. The aqueous layer was extracted with ethyl acetate in reverse. The organic layers obtained initially and in reverse extraction were combined, washed successively with saturated sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, and removed by vacuum distillation. The crude product was purified by silica gel column chromatography (hexane:ethyl acetate = 3:1–2:3) to give compound T-1 (17.48 g, 87% yield) as a white, foamy solid.

[0678] 1H NMR(CDCl3)δ1.01-1.12(28H,m),1.91(3H,d,J=1Hz),3.82,4.15(2H,ABq,J=14Hz),4.44 (1H,m),4.69(1H,d,J=4Hz),5.86(1H,s),7.09(1H,m),7.41(1H,d,J=1Hz),8.48(1H,s).

[0679] (Synthesis of compound (R)MT-1)

[0680] Compound T-1 (3.86 g, 7.33 mmol) was dissolved in toluene (55 mL). Boron trifluoride-diethyl ether complex (4.60 mL, 37.0 mmol) was added under dry ice / acetone cooling. Methyl magnesium bromide (12% tetrahydrofuran solution) was then added dropwise to the reaction mixture over 30 minutes, and the mixture was stirred under this cooling condition for 2 hours. The reaction was terminated with water, and the mixture was diluted with ethyl acetate and saturated brine to separate the organic layer. The obtained organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and removed by vacuum distillation. The crude product was purified by silica gel column chromatography (hexane:ethyl acetate = 2:1) to give compound (R)MT-1 (2.50 g, 63%) as a white, foamy solid.

[0681] 1 H NMR(CDCl3)δ0.88(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=2Hz),4.32(1H,d,J=3),6.12(1H,s),7.74(1H,s),8.55(1H,s).

[0682] As an alternative method, compound (R)MT-1 was synthesized according to the reaction scheme described below.

[0683] [Chemistry 47]

[0684]

[0685] (Synthesis of compound (R)MT-5)

[0686] Under a nitrogen stream, oxalyl chloride (5.6 mL, 64.92 mmol) was dissolved in dichloromethane (240 mL), and dimethyl sulfoxide (9.2 mL, 129.84 mmol) was added under a dry ice-acetone bath. After stirring at the same temperature for 30 minutes, a dichloromethane solution (40 mL) of compound S-1 (20.0 g, 49.94 mmol) was added to the reaction solution. After stirring at the same temperature for another 45 minutes, triethylamine (27.8 mL, 199.76 mmol) was added to the reaction solution. After stirring at the same temperature for another 15 minutes, the mixture was brought to room temperature and stirred for 1 hour. The reaction solution was diluted with dichloromethane and 1N hydrochloric acid, and the organic layer was separated. The organic layer was washed with saturated sodium bicarbonate solution and saturated brine, dried with anhydrous sodium sulfate, and removed by vacuum distillation to obtain the intermediate. Next, 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, methyl magnesium bromide (1 M in tetrahydrofuran, 100 mL, 100.0 mmol) was added in an ice bath. After stirring for another 1.5 hours at the same temperature, a tetrahydrofuran solution (120 mL) of the previously obtained intermediate was added in a dry ice-acetone bath. After stirring for 3 hours at the same temperature, the mixture was stirred for another hour at room temperature. The reaction solution was terminated with saturated ammonium chloride aqueous solution, diluted with ethyl acetate and water, and the organic and aqueous layers were separated. The obtained organic layer was washed successively with 1N hydrochloric acid, saturated sodium bicarbonate solution, and saturated brine, dried with anhydrous magnesium sulfate, and removed by vacuum distillation. The crude product was purified by silica gel column chromatography (hexane:ethyl acetate = 4:1 to 3:1) to give compound (R)MT-5 (19.0 g, 91%) as a colorless, transparent oil.

[0687] 1 H NMR(CDCl3)δ1.20(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=1 2),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).

[0688] (Synthesis of compound (R)MT-6)

[0689] Under a nitrogen stream, compound (R)MT-5 (9.06 g, 21.85 mmol) was dissolved in pyridine (143 mL), and p-toluenesulfonyl chloride (12.28 g, 64.43 mmol) was added. The mixture was stirred at 80 °C for 13 hours. The mixture was then cooled 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, then cooled 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 the reaction was terminated with water. The solution was then diluted with ethyl acetate and saturated brine to separate the organic layer. The obtained organic layer was washed successively with 1N hydrochloric acid, saturated sodium bicarbonate solution, and saturated brine, dried over anhydrous sodium sulfate, and removed by vacuum distillation. The crude product was purified by silica gel column chromatography (hexane:ethyl acetate = 3:1 to 3:1) to give compound (R)MT-6 (11.3 g, 91%) as a colorless, transparent oil.

[0690] 1 H NMR(CDCl3)δ1.32(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).

[0691] (Synthesis of compound (R)MT-7)

[0692] Under a nitrogen stream, (R)MT-6 (11.20 g, 19.69 mmol) was dissolved in acetic acid (111 mL), followed by the addition of acetic anhydride (18.4 mL, 194.76 mmol) and concentrated sulfuric acid (0.072 g, 0.73 mmol). The mixture was stirred at room temperature for 2.5 hours. The reaction solution was neutralized with saturated sodium bicarbonate solution, diluted with ethyl acetate and water, and the organic layer was separated. The obtained organic layer was washed successively with saturated sodium bicarbonate solution and saturated brine, dried with anhydrous magnesium sulfate, and removed by vacuum distillation. The obtained 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 cooling the reaction solution 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 solution was then cooled, and the reaction was terminated with saturated sodium bicarbonate solution. The mixture was then diluted with ethyl acetate and water to separate the organic layer. The obtained organic layer was washed with saturated brine, dried with anhydrous magnesium, and removed by vacuum distillation. 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.

[0693] 1 H NMR(CDCl3)δ1.29(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(5 H,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).

[0694] (Synthesis of compound (R)MT-8)

[0695] Under a nitrogen atmosphere, (R)MT-7 (11.72 g, 17.27 mmol) was dissolved in tetrahydrofuran (195 mL), and 40% methylamine aqueous solution (26 mL, 307.0 mmol) was added under ice-cold conditions. The mixture was stirred for 5 hours under the same ice-cold conditions. After removing the reaction solution by vacuum distillation, the solution was diluted with ethyl acetate and water to separate the organic layer. The obtained organic layer was washed with saturated brine, dried with anhydrous sodium sulfate, and removed by vacuum distillation. The obtained intermediate was dissolved in pyridine (110 mL) under a nitrogen stream, and methanesulfonyl chloride (1.9 mL, 24.95 mmol) was added under ice-cold conditions. The mixture was stirred at room temperature for 2 hours. The reaction solution was diluted with ethyl acetate, water, and saturated brine to separate the organic layer. The obtained organic layer was washed with saturated brine, dried with anhydrous sodium sulfate, and removed by vacuum distillation. The obtained intermediate was dissolved in ethanol (340 mL) and water (170 mL), and 1M sodium hydroxide aqueous solution (115 mL, 115.0 mmol) was added. The mixture was stirred at room temperature for 14 hours. The reaction solution was neutralized with 1N hydrochloric acid and then removed by vacuum distillation. The residue was diluted with ethyl acetate and water to separate the organic layer. The organic layer was washed with saturated brine, dried with anhydrous sodium sulfate, and removed by vacuum distillation. 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.

[0696] 1 H NMR(CDCl3)δ1.33(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(1 H,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).

[0697] (Synthesis of compound (R)MT-9)

[0698] Under a nitrogen atmosphere, compound (R)MT-8 (4.00 g, 6.03 mmol) was dissolved in ethanol (80 mL), followed by the addition of cyclohexene (8.9 mL, 87.76 mmol) and 20% palladium hydroxide powder on carbon (2.0 g), and the mixture was heated to reflux for 40 minutes. Then, cyclohexene (8.9 mL, 87.76 mmol) was added, and the mixture was heated to reflux for 55 minutes. Finally, cyclohexene (4.0 mL, 39.44 mmol) was added, and the mixture was heated to reflux for 25 minutes. The reaction mixture was filtered, and the filtrate was removed by vacuum distillation. The resulting intermediate was dissolved in N,N-dimethylformamide (56 mL) under a nitrogen atmosphere. Under ice-cold conditions, 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (2.4 mL, 7.54 mmol) and a 2.9 M imidazole solution in N,N-dimethylformamide (7.7 mL) were added sequentially, and the mixture was stirred at room temperature for 16 hours. After terminating the reaction with methanol, the solution was diluted with diethyl ether and water to separate the organic and aqueous layers. The aqueous layer was extracted with diethyl ether in reverse. The organic layers obtained initially and in the reverse extraction were combined, washed with saturated brine, dried with anhydrous sodium sulfate, and removed by vacuum distillation. 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, yield 65%) as a white, foamy solid.

[0699] 1 H NMR(CDCl3)δ0.91-1.16(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).

[0700] (Synthesis of compound (R)MT-10)

[0701] 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, and the mixture was stirred under ice-cold conditions for 3.5 hours. Then, 4-dimethylaminopyridine (0.13 g, 1.07 mmol) was added, and the mixture was stirred under ice-cold conditions for 3 hours. The reaction mixture was terminated with saturated brine, diluted with dichloromethane and water, and the organic and aqueous layers were separated. The aqueous layer was back-extracted with dichloromethane. The organic layers obtained initially and in the back-extraction were combined, washed sequentially with 1N hydrochloric acid, saturated sodium bicarbonate solution, and saturated brine, dried over anhydrous sodium sulfate, and removed by vacuum distillation. The obtained intermediate was dissolved in dimethyl sulfoxide (9.4 mL) under a nitrogen atmosphere, and 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. The mixture was stirred at room temperature for 86 hours. The reaction solution was diluted with diethyl ether and water, and the organic and aqueous layers were separated, respectively. The aqueous layer was extracted with diethyl ether in reverse. The organic layers obtained initially and in reverse extraction were combined, washed successively with water and saturated brine, and dried with anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (hexane:ethyl acetate = 2:1 to 1:1) to give compound (R)MT-10 (0.33 g, yield 36%) as a white foamy solid.

[0702] 1 H NMR(CDCl3)δ1.01-1.21(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).

[0703] <Synthesis of Compound (R)MT-1>

[0704] 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 solution 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 layers obtained initially and those obtained in the back-extraction were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and removed by vacuum distillation. The obtained 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 then at 70 °C for 23 hours. The reaction solution 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 layers obtained initially and in the back-extraction were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and removed by vacuum distillation. 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.

[0705] (Synthesis of compound (R)MT-2)

[0706] Under ice-cold conditions, 20% formaldehyde aqueous solution (0.31 mL, 2.0 mmol) was added to a 0.5 M methanol solution (7.2 mL) of pyridine p-toluenesulfonate containing compound (R)MT-1 (0.53 g, 0.98 mmol). Then, sodium cyanoborohydride (0.11 g, 1.80 mmol) was added under the same ice-cold conditions, and the mixture was stirred for 1 hour. The reaction solution was diluted with ethyl acetate, water, and saturated brine to separate the organic layer. The obtained organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and removed by vacuum distillation. 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.

[0707] 1H NMR(CDCl3)δ0.93(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=6Hz),3.6 2, 4.10 (2H, AB, J = 13), 4.02 (1H, d, J = 3Hz), 4.28 (1H, d, J = 3Hz), 6.23 (1H, s), 7.70 (1H, d, J = 1), 8.35 (1H, s).

[0708] (Synthesis of compound (R)MT-3)

[0709] 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 solution was removed by vacuum distillation, and the reaction residue was removed by silica gel column chromatography (ethyl acetate:methanol = 30:1–15:1) to obtain an intermediate. The intermediate was azeotropically dried with pyridine and dissolved in pyridine (4.5 mL) under a nitrogen stream. 4,4'-dimethoxytriphenylchloromethane (0.52 g, 1.52 mmol) was added, and the mixture was stirred at room temperature for 15 hours. After terminating the reaction with methanol, the mixture was diluted with ethyl acetate and water, and the organic layer was separated. The obtained organic layer was washed with saturated brine, dried with anhydrous sodium sulfate, and removed by vacuum distillation. The crude product was purified by silica gel column chromatography (hexane:ethyl acetate = 1:1) to give compound (R)MT-3 (0.58 g, 90%) as a white foamy solid.

[0710] 1 H NMR(CDCl3)δ0.77(3H,d,J=6Hz),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=3Hz),4.57(1H,dd,J=3,10Hz),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).

[0711] (Synthesis of compound (R)MT-4)

[0712] Compound (R)MT-3 (0.54 g, 1.57 mmol) was azeotropically dried in acetonitrile under a nitrogen stream, dissolving in acetonitrile (7 mL). 4,5-Dicyanoimidazole (0.22 g, 1.01 mmol) and 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphonic diamine (0.36 mL, 1.10 mmol) were added sequentially under ice-cold conditions, and the mixture was stirred at room temperature for 5 hours. The reaction solution was diluted with ethyl acetate and water, and the organic layer was separated. The obtained organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and removed by vacuum distillation. 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.

[0713] 31 P NMR (CDCl3) δ 148.7, 149.0.

[0714] HRMS (MALDI): calcd for C 43 H 55 N5O9P[M+H + ]816.3732,found 816.3746.

[0715] [Synthesis example 2]

[0716] Base is thymine-1-yl, A 1 It is a single key, R 1 It is DMTr, R 2 It is -P(N(i-Pr)2)(OC2H4CN), R 3 It is methyl, R 4 It is the ethyl compound of R, R 5 The compound (1) (hereinafter referred to as "compound (R)ET-4"), which is a hydrogen atom and n is 1, was synthesized according to the following reaction scheme.

[0717] [Chemistry 48]

[0718]

[0719] (Synthesis of compound (R)ET-1)

[0720] Under a nitrogen stream, compound T-1 (2.4 g, 4.63 mmol) was dissolved in dichloromethane (77 mL). At room temperature, a boron trifluoride-ethyl ether complex (1.5 mL, 24.39 mmol) and triethylboron (1 M in hexane, 12.2 mL, 24.39 mmol) were added. After stirring with air bubbled in at room temperature for 5 minutes, the boron trifluoride-ethyl ether complex (1.5 mL, 24.39 mmol) and triethylboron (1 M in hexane, 12.2 mL, 24.39 mmol) were added. After stirring with air bubbled in at room temperature for 10 minutes, a boron trifluoride-ethyl ether complex (0.2 mL, 1.59 mmol) and triethylboron (1 M in hexane, 2.0 mL, 2.00 mmol) were added, and the mixture was stirred with air bubbled in at room temperature for 5 minutes. The reaction solution was terminated with saturated sodium bicarbonate solution, diluted with dichloromethane and water, and the organic and aqueous layers were separated. The aqueous layer was extracted with dichloromethane by reverse extraction. The organic layers obtained in the initial separation and those obtained in the reverse extraction were combined, washed with saturated brine, dried with anhydrous sodium sulfate, and removed by vacuum distillation. 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, yield 79%) as a white foamy solid.

[0721] 1 H NMR(CDCl3)δ0.92-1.13(28H,m),1.21-1.46,(2H,m),1.92(3H,d,J=1Hz),3.58(1H,br),3.70,4.17(2H,ABq,J =13Hz), 4.13 (1H, d, J = 4Hz), 4.33 (1H, d, J = 3Hz), 5.51 (1H, br), 6.14 (1H, s), 7.73 (1H, d, J = 1Hz), 8.58 (1H, s).

[0722] (Synthesis of compound (R)ET-2)

[0723] Under a nitrogen stream, compound (R)ET-1 (0.97 g, 1.75 mmol) was dissolved in 0.5 M p-toluenesulfonic acid pyridine methanol solution (11.5 mL, 5.76 mmol). Then, under ice-cold conditions, 20% formaldehyde aqueous solution (0.49 mL, 3.24 mmol) and sodium cyanoborohydride (0.18 mg, 2.88 mmol) were added sequentially, and the mixture was stirred for 30 minutes under the same ice-cold conditions. The reaction solution was diluted with ethyl acetate, water, and saturated brine to separate the organic layer. The obtained organic layer was washed with saturated brine, dried with anhydrous sodium sulfate, and removed by vacuum distillation. The crude product was purified by silica gel column chromatography (hexane:ethyl acetate = 2:1–3:2) to give compound (R)ET-2 (0.92 g, 92%) as a white, foamy solid.

[0724] 1 H NMR(CDCl3)δ0.91-1.13(28H,m),1.29-1.36,(1H,m),1,57-1.68,(1H,m),1.91(3H,d,J=1Hz),2.74(3H,s),2.81(1H,m ), 3.73, 4.20 (2H, ABq, J = 13Hz), 3.98 (1H, d, J = 3Hz), 4.26 (1H, d, J = 3Hz), 6.26 (1H, s), 7.72 (1H, d, J = 1Hz), 8.34 (1H, s).

[0725] (Synthesis of compound (R)ET-3)

[0726] 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 solution was removed by vacuum distillation, and the reaction residue was removed by silica gel column chromatography (ethyl acetate:methanol = 40:1–20:1) to obtain an intermediate. The intermediate was dried azeotropically with pyridine and dissolved in pyridine (7 mL) under a nitrogen stream. 4,4'-dimethoxytriphenylchloromethane (0.77 g, 2.28 mmol) was added, and the mixture was stirred at room temperature for 15 hours. After terminating the reaction with methanol, the mixture was diluted with ethyl acetate and water, and the organic and aqueous layers were separated, respectively. The aqueous layer was extracted with ethyl acetate in reverse. The organic layers obtained initially and in reverse extraction were combined, washed with saturated brine, dried with anhydrous sodium sulfate, and removed by vacuum distillation. The crude product was purified by silica gel column chromatography (hexane:ethyl acetate = 2:1 to 1:4) to give compound (R)ET-3 (1.06 g, 91%) as a white foamy solid.

[0727] 1 H NMR(CDCl3)δ0.75(3H,t,J=8Hz),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=3Hz),4.51(1H,dd,J=3,9Hz),6.35(1H,s),6.84-6.87(4H,m),7.23-7.46(9H,m),7.89(1H,s),8.33(1H,s).

[0728] (Synthesis of compound (R)ET-4)

[0729] Compound (R)ET-3 (1.03 g, 1.64 mmol) was azeotropically dried in acetonitrile under a nitrogen stream, dissolving in acetonitrile (11 mL). 4,5-Dicyanoimidazole (0.22 g, 1.90 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphonic diamine (0.68 mL, 2.08 mmol) were added sequentially under ice-cold conditions, and the mixture was stirred at room temperature for 4 hours. The reaction solution was diluted with ethyl acetate and water, and the organic layer was separated. The obtained organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and removed by vacuum distillation. The crude product was purified by silica gel column chromatography (hexane:ethyl acetate = 1:1–2:3) to give compound (R)ET-4 (1.13 g, 82%) as a white, foamy solid.

[0730] 31 P NMR (CDCl3) δ 149.1, 150.5.

[0731] HRMS (MALDI): calcd for C 46 H 56 F3N6NaO 10 P[M+H + ]830.3888,found830.3883.

[0732] [Synthesis example 3]

[0733] Base is N-benzoyl-adenine-9-yl, A 1 It is a single key, R 1 It is DMTr, R 2 It is -P(N(i-Pr)2)(OC2H4CN), R 3 It is methyl, R 4 It is the ethyl compound of R, R 5The compound (1) (hereinafter referred to as "compound (R)EA-3"), which is a hydrogen atom and n is 1, was synthesized according to the following reaction scheme.

[0734] [Chemistry 49]

[0735]

[0736] (Synthesis of compound (R)EA-1)

[0737] Under a nitrogen stream, compound (R)ET-2 (0.33 g, 0.57 mmol) was dissolved in toluene (6.7 mL), and N2 was added sequentially. 6 -Benzyladenine (0.21 g, 0.88 mmol) and N,O-bis(trimethylsilyl)acetamide (0.87 mL, 3.50 mmol) were stirred at 90 °C for 0.5 h. Then, trimethylsilyl trifluoromethanesulfonate (0.15 mL, 0.82 mmol) was added, and the mixture was stirred at 90 °C for 1 h. The reaction mixture was chilled, and the reaction was terminated with saturated sodium bicarbonate solution. The mixture was then diluted with ethyl acetate and water, filtered through diatomaceous earth, and the filtrate was recovered to separate the organic layer. The obtained organic layer was washed with saturated brine, dried with anhydrous sodium sulfate, and removed by vacuum distillation. The crude product was purified by silica gel column chromatography (hexane:ethyl acetate = 2:1 to 1:1) to give compound (R)EA-1 (0.27 g, 71%) as a white, foamy solid.

[0738] 1 H NMR(CDCl3)δ0.98-1.15(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=13Hz),4.52(1H,d ,J=3Hz),4.64(1H,d,J=3Hz),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).

[0739] (Synthesis of compound (R)EA-2)

[0740] 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 solution was removed by vacuum distillation, and the reaction residue was removed by silica gel column chromatography (ethyl acetate:methanol = 30:1–10:1) to obtain an intermediate. The intermediate was azeotropically dried with pyridine and dissolved in pyridine (2 mL) under a nitrogen stream. 4,4'-dimethoxytriphenylchloromethane (0.18 g, 0.53 mmol) was added, and the mixture was stirred at room temperature for 16 hours. The reaction solution was cooled, the reaction was terminated with methanol, and the mixture was diluted with water and ethyl acetate to separate the organic layer. The obtained organic layer was washed with saturated brine, dried with anhydrous sodium sulfate, and removed by vacuum distillation. The crude product was purified by silica gel column chromatography (hexane:ethyl acetate = 1:2 to 0:1) to give compound (R)EA-2 (0.25 g, 75%) as a white solid.

[0741] 1 H NMR(DMSO-d6)δ0.69(3H,t,J=7Hz),1.05-1.13(1H,m),1.38-1.45(1H,m),2.69(3H,s),2.74(1H,d, J=3Hz),3.05,3.36(2H,ABq,J=10Hz),3.72(6H,d,J=2Hz),4.69(1H,d,J=3Hz),4.82(1H,dd,J=3,6Hz ),5.60(1H,d,J=6Hz),6.66(1H,s),6.86(4H,d,J=8Hz),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=7Hz),8.57(1H,s),8.80(1H,s),11.26(1H,s).

[0742] (Synthesis of compound (R)EA-3)

[0743] Compound (R)EA-2 (0.20 g, 0.27 mmol) was azeotropically dried with acetonitrile under a nitrogen stream, and dissolved by adding acetonitrile (3 mL) and tetrahydrofuran (2 mL). Then, 4,5-dicyanoimidazole (0.035 g, 0.30 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorous diamine (0.11 mL, 0.32 mmol) were added sequentially, and the mixture was stirred at room temperature for 6 hours. Next, 4,5-dicyanoimidazole (0.018 g, 0.15 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorous diamine (0.053 mL, 0.16 mmol) were added sequentially, and the mixture was stirred for another 16 hours. The reaction was terminated with water, and the mixture was diluted with ethyl acetate to separate the organic layer. The obtained organic layer was washed sequentially with water and saturated brine, dried over anhydrous sodium sulfate, and removed by vacuum distillation. 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.

[0744] 31 P NMR (CDCl3) δ 148.9, 149.1.

[0745] HRMS(MALDI):calcd for C51H59N8NaO8P[M+Na + ]965.4086,found965.4096.

[0746] [Synthesis example 4]

[0747] Base is thymine-1-yl, A 1 It is a single key, R 1 It is DMTr, R 2 It is -P(N(i-Pr)2)(OC2H4CN), R 4 The compound (1) (hereinafter referred to as "compound OM-T-3"), which is methyl and n is 0, was synthesized according to the following reaction scheme.

[0748] [Transformation 50]

[0749]

[0750] (Synthesis of compound OM-T-1)

[0751] Under a nitrogen stream, compound (R)MT-1 (2.88 g, 5.31 mmol) was dissolved in dimethyl sulfoxide (58 mL), and 2-iodobenzoic acid (3.89 g, 5.84 mmol) was added at room temperature. The mixture was stirred at 60 °C for 6 hours, and then stirred for another 9 hours while cooling to room temperature. The reaction mixture was cooled in water, and the reaction was terminated with saturated sodium bicarbonate solution. The solution was then diluted with ethyl acetate and water to separate the organic layer. The organic layer was washed with saturated brine, dried with anhydrous sodium sulfate, and removed by vacuum distillation. 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.

[0752] 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=1 3),4.45(1H,d,J=4Hz),4.61(1H,dd,J=4,1),5.86(1H,s),7.44(1H,s),8.40(1H,s).

[0753] (Synthesis of compound OM-T-2)

[0754] 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 solution was removed by vacuum distillation, and the reaction residue was removed by silica gel column chromatography (ethyl acetate:methanol = 30:1–10:1) to obtain an intermediate. The intermediate was azeotropically dried with pyridine and dissolved in pyridine (25 mL) under a nitrogen stream. 4,4'-dimethoxytriphenylchloromethane (1.62 g, 4.78 mmol) was added, and the mixture was stirred at room temperature for 15 hours. After terminating the reaction with methanol, the mixture was diluted with water and ethyl acetate, and the organic layer was separated. The obtained organic layer was washed with saturated brine, dried with anhydrous sodium sulfate, and removed by vacuum distillation. Dichloromethane was added to the crude product, and the precipitated solid was filtered out to obtain compound OM-T-2 (1.01 g, 69%) as a white solid.

[0755] 1H NMR(CDCl3)δ1.37(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).

[0756] (Synthesis of compound OM-T-3)

[0757] Compound OM-T-2 (0.40 g, 0.65 mmol) was azeotropically dried in acetonitrile under a nitrogen stream, dissolving in acetonitrile (4 mL). 4,5-Dicyanoimidazole (0.087 g, 0.73 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphonic diamine (0.26 mL, 0.80 mmol) were added sequentially under ice-cold conditions, and the mixture was stirred at room temperature for 5 hours. The reaction mixture was cooled, the reaction was terminated with water, and the mixture was diluted with ethyl acetate and saturated brine to separate the organic layer. The obtained organic layer was washed sequentially with water and saturated brine, dried over anhydrous sodium sulfate, and removed by vacuum distillation. 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.

[0758] 31 P NMR (CDCl3) δ 149.4, 149.7.

[0759] HRMS(MALDI):calcd for C42H50N5NaO9P[M+Na + ]822.3238,found822.3232.

[0760] [Synthesis example 5]

[0761] Base is thymine-1-yl, A 1 It is a single key, R 1 It is DMTr, R 2 It is -P(N(i-Pr)2)(OC2H4CN), R 3 It is methyl, R 4 It is the methyl group configured by S, R 5 The compound (1) (hereinafter referred to as "compound (S)MT-4"), which is a hydrogen atom and n is 1, was synthesized according to the following reaction scheme.

[0762] [Chemistry 51]

[0763]

[0764] (Synthesis of compound (S)MT-1)

[0765] Under a nitrogen stream, compound OM-T-1 (0.20 g, 0.37 mmol) was dissolved in toluene (4 mL). A 1.0 mol / L solution of diisobutylaluminum hydrogenation / hexane (1.5 mL, 1.5 mmol) was added under dry ice / acetone cooling, and the mixture was stirred for 2 hours under the same cooling condition. The reaction was terminated with a saturated Rochelle salt solution, and the mixture was diluted with ethyl acetate and water to separate the organic layer. The obtained organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and removed by vacuum distillation. The crude product was purified by silica gel column chromatography (hexane:ethyl acetate = 3:2–1:1) to give compound (S)MT-1 (0.046 g, 23%) as a white, foamy solid.

[0766] 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).

[0767] (Synthesis of compound (S)MT-2)

[0768] Under ice-cold conditions, 20% formaldehyde aqueous solution (0.16 mL, 1.0 mmol) was added to a 0.5 M methanol solution (3.6 mL) of pyridine p-toluenesulfonate containing compound (S)MT-1 (0.31 g, 0.57 mmol). Then, sodium cyanoborohydride (0.058 g, 0.92 mmol) was added under the same ice-cold conditions, and the mixture was stirred for 1 hour. The reaction solution was diluted with ethyl acetate, water, and saturated brine to separate the organic layer. The obtained organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and removed by vacuum distillation. The crude product was purified by silica gel column chromatography (hexane:ethyl acetate = 3:1–2:1) to give compound (S)MT-2 (0.29 g, 93%) as a white, foamy solid.

[0769] 1H NMR(CDCl3)δ0.94-1.15(28H,m),1.32(3H,d,J=7),1.91(3H,d,J=1),2.68(3H,s),2.87(1H,q,J=7Hz),3.9 7(1H,d,J=3),4.00,4.08(2H,ABq,J=13),4.34(1H,d,J=3Hz),6.24(1H,s),7.79(1H,d,J=1),8.40(1H,s).

[0770] (Synthesis of compound (S)MT-3)

[0771] 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 solution was removed by vacuum distillation, and the reaction residue was removed by silica gel column chromatography (ethyl acetate:methanol = 40:1–10:1) to obtain an intermediate. The obtained intermediate was azeotropically dried with pyridine and dissolved in pyridine (5.4 mL) under a nitrogen stream. 4,4'-dimethoxytriphenylchloromethane (0.32 g, 0.95 mmol) was added, and the mixture was stirred at room temperature for 15 hours. After terminating the reaction with methanol, the mixture was diluted with water and ethyl acetate, and the organic layer was separated. The crude product was purified by silica gel column chromatography (hexane:ethyl acetate = 1:2) to give compound (S)MT-3 (0.30 g, 88%) as a white, foamy solid.

[0772] 1 H NMR(CDCl3)δ1.04(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).

[0773] (Synthesis of compound (S)MT-4)

[0774] Compound (S)MT-3 (0.28 g, 0.46 mmol) was azeotropically dried in acetonitrile under a nitrogen stream, dissolving in acetonitrile (4 mL). 4,5-Dicyanoimidazole (0.064 g, 0.54 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphonic diamine (0.19 mL, 0.58 mmol) were added sequentially under ice-cold conditions, and the mixture was stirred at room temperature for 5 hours. The reaction solution was diluted with ethyl acetate and water, and the organic layer was separated. The obtained organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and removed by vacuum distillation. 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.

[0775] 31 P NMR (CDCl3) δ 149.1, 149.2.

[0776] HRMS(MALDI):calcd for C43H54N5NaO9P[M+Na + ]838.3551,found838.3564.

[0777] [Synthesis example 6]

[0778] Base is thymine-1-yl, A 1 It is a single key, R 1 It is DMTr, R 2 It is -P(N(i-Pr)2)(OC2H4CN), R 4 The compound (1) (hereinafter referred to as "compound OE-T-3"), which is ethyl and n is 0, was synthesized according to the following reaction scheme.

[0779] [Chemistry 52]

[0780]

[0781] (Synthesis of compound OE-T-1)

[0782] Under a nitrogen stream, compound (R)ET-1 (6.48 g, 11.66 mmol) was dissolved in dimethyl sulfoxide (102 mL), and 2-iodobenzoic acid (9.00 g, 13.50 mmol) was added at room temperature. The mixture was stirred at room temperature for 15 hours. The reaction solution was cooled with water, and the reaction was terminated with saturated sodium bisulfate aqueous solution. The solution was then diluted with ethyl acetate and water to separate the organic and aqueous layers, respectively. The aqueous layer was extracted with ethyl acetate in reverse. The organic layers obtained initially and in reverse extraction were combined, washed successively with saturated sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, and removed by vacuum distillation. 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.

[0783] 1 H NMR(CDCl3)δ0.92-1.14(28H,m),1.19(3H,t,J=7),1.91(3H,s),2.24(2H,q,J=7),3.94,4.27(2 H,ABq,J=13Hz),4.44(1H,d,J=4Hz),4.59(1H,d,J=4Hz),5.88(1H,s),7.45(1H,s),8.41(1H,s).

[0784] (Synthesis of compound OE-T-2)

[0785] 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 solution was removed by vacuum distillation, and the reaction residue was removed by silica gel column chromatography (ethyl acetate:methanol = 40:1–5:1) to obtain an intermediate. The obtained intermediate was azeotropically dried with pyridine and dissolved in pyridine (2 mL) under a nitrogen stream. 4,4'-dimethoxytriphenylchloromethane (0.18 g, 0.52 mmol) was added, and the mixture was stirred at room temperature for 19 hours. After terminating the reaction with methanol, the mixture was diluted with ethyl acetate and water, and the organic layer was separated. The obtained organic layer was washed with saturated brine, dried with anhydrous sodium sulfate, and removed by vacuum distillation. The crude product was purified by silica gel column chromatography (hexane:ethyl acetate = 1:2 to 1:3) to give compound OE-T-2 (0.11 g, 90%) as a white foamy solid.

[0786] 1H NMR(CDCl3)δ1.01(3H,t,J=7Hz),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=4Hz),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).

[0787] (Synthesis of compound OE-T-3)

[0788] Compound OE-T-2 (0.26 g, 0.42 mmol) was azeotropically dried in acetonitrile under a nitrogen stream, dissolving in acetonitrile (3 mL). 4,5-Dicyanoimidazole (0.062 g, 0.52 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphonic diamine (0.20 mL, 0.61 mmol) were added sequentially under ice-cold conditions, and the mixture was stirred at room temperature for 5 hours. The reaction mixture was cooled, the reaction was terminated with water, and the mixture was diluted with ethyl acetate to separate the organic layer. The obtained organic layer was washed sequentially with water and saturated brine, dried over anhydrous sodium sulfate, and removed by vacuum distillation. The crude product was purified by silica gel column chromatography (hexane:ethyl acetate = 1:1–1:2) to give compound OE-T-3 (0.30 g, 77%) as a white, foamy solid.

[0789] 31 P NMR (CDCl3) δ 149.1, 150.5.

[0790] HRMS(MALDI):calcd for C43H52N5NaO9P[M+Na + ]836.3395,found836.3398.

[0791] [Synthesis Example 7]

[0792] Base is thymine-1-yl, A 1 It is a single key, R 1 It is DMTr, R 2 It is -P(N(i-Pr)2)(OC2H4CN), R 3 It is 3-(N,N-dimethylamino)propyl, R 4 It is the ethyl compound of R, R 5 The compound (1) containing hydrogen atoms (hereinafter referred to as "compound (R)EDM-T-2") was synthesized according to the following reaction scheme.

[0793] [Chemistry 53]

[0794]

[0795] (Synthesis of compound (R)ET-5)

[0796] Under a nitrogen stream, compound (R)MT-1 (2.46 g, 4.42 mmol) was dissolved in tetrahydrofuran (30 mL), 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 solution was removed by vacuum distillation, and the reaction residue was removed by silica gel column chromatography (ethyl acetate:methanol = 10:1–8:1) to obtain an intermediate. The intermediate was azeotropically dried with pyridine and dissolved in pyridine (30 mL) under a nitrogen stream. 4,4'-dimethoxytriphenylchloromethane (2.59 g, 7.64 mmol) was added, and the mixture was stirred at room temperature for 15 hours. After terminating the reaction with methanol, the mixture was diluted with water and ethyl acetate to separate the organic and aqueous layers, respectively. The aqueous layer was extracted with ethyl acetate in reverse. The organic layers obtained initially and in reverse extraction were combined, washed with saturated brine, dried with anhydrous sodium sulfate, and removed by vacuum distillation. 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.

[0797] 1 H NMR(CDCl3)δ0.79(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(4 H,m),7.21-7.35(7H,m),7.42-7.45(2H,m),8.33(1H,d,J=1),8.82(1H,s).

[0798] (Synthesis of compound (R)EDM-T-1)

[0799] Under a nitrogen stream and ice-cold conditions, 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) and stirred for 20 minutes. Then, 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. After terminating the reaction with water, the mixture was diluted with saturated brine and toluene to separate the organic and aqueous layers. The aqueous layer was extracted with toluene in reverse. The organic layers obtained initially and in the reverse extraction were combined, washed with saturated brine, dried with anhydrous sodium sulfate, and distilled under reduced pressure until the residue showed a slight white turbidity. The resulting toluene solution of 3-(dimethylamino)-1-propane p-toluenesulfonic acid was used directly in subsequent reactions.

[0800] 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 a pre-prepared toluene solution of 3-(dimethylamino)-1-propyl p-toluenesulfonic acid was added dropwise over 20 minutes. The mixture was then stirred at 100 °C for 2 hours. The reaction mixture was brought back to room temperature, and the solvent was removed by vacuum distillation. The crude product was purified by silica gel column chromatography (ethyl acetate:triethylamine:methanol = 20:1:0 to 20:1:1.5) to give compound (R)EDM-T-1 (0.27 g, 14%) as a white, foamy solid.

[0801] 1 H NMR(CDCl3)δ0.73(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).

[0802] (Synthesis of compound (R)EDM-T-2)

[0803] Compound (R)EDM-T-1 (0.41 g, 0.59 mmol) was azeotropically dried in acetonitrile under a nitrogen stream, dissolving it in acetonitrile (5 mL). 4,5-Dicyanoimidazole (0.078 g, 0.66 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphonic diamine (0.24 mL, 0.72 mmol) were added sequentially under ice-cold conditions, and the mixture was stirred at room temperature for 6 hours. Next, 4,5-dicyanimidazole (0.039 g, 0.33 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorous diamine (0.12 mL, 0.36 mmol) were added at room temperature, and the mixture was stirred for 16 hours under these conditions. Then, 4,5-dicyanimidazole (0.039 g, 0.33 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorous diamine (0.12 mL, 0.36 mmol) were added again at room temperature, and the mixture was stirred for 1.5 hours under these conditions. After terminating the reaction with water, the mixture was diluted with ethyl acetate and saturated brine to separate the organic layer. The obtained organic layer was washed successively with water and saturated brine, dried with anhydrous sodium sulfate, and removed by vacuum distillation. The crude product was purified by silica gel column chromatography (ethyl acetate:triethylamine:methanol = 20:1:0 to 20:1:1) to give compound (R)EDM-T-2 (0.30 g, 55%) as a white foamy solid.

[0804] 31 P NMR (CDCl3) δ 148.9, 149.1.

[0805] HRMS(MALDI):calcd for C48H65N6NaO9P[M+Na + ]923.4443,found923.4420.

[0806] <<Examples of Oligonucleotide Synthesis>>

[0807] The oligonucleotides were synthesized using the compounds ((R)MT-4, (R)ET-4, (S)MT-4, OM-T-3, OE-T-3, (R)EDM-T-2) obtained in the above synthesis examples, according to the standard phosphoramide scheme, by an automated nucleic acid synthesizer (Expedite 8909 / ABI Corporation), to obtain the following oligonucleotides.

[0808] • In formula (6), Base is thymine-1-yl, A 1 It is a single bond, n is 1, R 3 It is methyl, R 4 It is an ethyl compound of R and R 5 Oligonucleotides consisting of hydrogen atoms (hereinafter referred to as (R)ET)

[0809] • In formula (6), Base is thymine-1-yl, A 1 It is a single bond, n is 1, R 3 It is methyl, R 4 It is a methyl group configured with R and R 5 Oligonucleotides consisting of hydrogen atoms (hereinafter referred to as (R)MT)

[0810] • In formula (6), Base is thymine-1-yl, A 1 It is a single bond, n is 0 and R 4 It is an oligonucleotide with an ethyl unit (hereinafter referred to as OE-T).

[0811] • In formula (6), Base is thymine-1-yl, A 1 It is a single bond, n is 1, R 3 It is methyl, R 4 It is a methyl group configured with S and R 5 Oligonucleotides consisting of hydrogen atoms (hereinafter referred to as (S)MT)

[0812] • In formula (6), Base is thymine-1-yl, A 1 It is a single bond, n is 0 and R 4 Oligonucleotides consisting of a methyl unit (hereinafter referred to as OM-T)

[0813] • In formula (6), Base is thymine-1-yl, A 1 It is a single bond, n is 1, R 3 It is 3-(N,N-dimethylamino)propyl, R 4 It is an ethyl compound of R and R 5 Oligonucleotides consisting of hydrogen atoms (hereinafter referred to as (R)EDM-T)

[0814] Oligonucleotides with 5'-terminus protected by dimethoxytriphenylmethyl and supported by a solid phase were cleaved from the chromatographic column with 28% ammonia (1.5 hours). The cleaved oligonucleotides were then reacted in 28% ammonia at 60°C for 16 hours to remove all protecting groups.

[0815] Simple purification was performed using a NAP-10 column, followed by reversed-phase HPLC [WakoPak WS-DNA column, 10.0 mm × 250 mm] [conditions: gradient elution of 8-16% acetonitrile at 3 ml / min for 30 min in 0.1 M triethylacetate buffer (pH 7.0), column temperature 50 °C].

[0816] The purity of the synthesized oligonucleotides was confirmed by reversed-phase HPLC [WakoPak WS-DNA column, 4.6 mm × 250 mm] [conditions: gradient elution of 8-16% acetonitrile at 1 ml / min for 30 min in 0.1 M triethylammonium acetate buffer (pH 7.0), column temperature 50 °C, detection wavelength 254 nm]. It should be noted that the purity of all synthesized oligonucleotides was above 90%.

[0817] In addition, the molecular weight of the synthesized oligonucleotides was determined by MALDI-TOF-MASS assay. The calculated and measured values ​​(results) of the molecular weights are shown in the table below. It should be noted that the unit represented by equation (6) is integrated into the n position of the antisense strand (SEQ ID NO:1~SEQ ID NO:5) in the table below, where Base is thymine-1-yl. The base sequence other than n is entirely composed of DNA (equation (7)R). a =H) constitutes. In addition, as a contrast, it is also given that the unit represented by equation (9) is integrated at the position of n, where Base is thymine-1-based and R b The values ​​are calculated and measured values ​​of the molecular weight of oligonucleotides with Me or H units (hereinafter referred to as NMe-T or NH-T, respectively, in the table).

[0818] [Table 1]

[0819]

[0820] <<Experimental Examples>>

[0821] [ Experimental Example 1 ] Determination of melting temperature (Tm) of oligonucleotides (evaluation of double-strand forming ability)

[0822] The double-stranding ability of the antisense strand was investigated by measuring the melting temperature (Tm) of the six oligonucleotides (6) ((R)ET, OE-T, (R)MT, (S)MT-4, OM-T-3, (R)EDM-T-2) obtained in the above synthetic examples, which were integrated into the n position of the sequence shown in SEQ ID NO:4. aThe oligonucleotide (DNA-T) synthesized by placing the unit (T) of H at the n position of SEQ ID NO:4, and the unit represented by formula (9) in which Base is a thymine 1-yl and R b The oligonucleotide (NMe-T) synthesized by integrating the Me unit at the n position of SEQ ID NO:4 serves as the antisense strand.

[0823] Sample solutions (120 μl) were prepared with final concentrations of 100 mM sodium chloride, 10 mM sodium phosphate buffer (pH 7.2), 4 μM antisense, and 4 μM sense. The temperature was increased from 15 °C to 110 °C at a rate of 0.5 °C / min. The absorbance at 260 nm was measured using a spectrophotometer (Shimadzu UV-1800, Ltd.). The Tm value was calculated from the measured values ​​using the differential method. The results are shown in the table below.

[0824] For the ability of single-stranded DNA to form double strands (Tm value)

[0825] [Table 2]

[0826]

[0827] For the ability of single-stranded RNA to form double strands (Tm value)

[0828] [Table 3]

[0829]

[0830] Therefore, the oligonucleotide (6) of the present invention exhibits excellent double-strand forming ability for either single-stranded DNA or single-stranded RNA, particularly for single-stranded RNA. Consequently, the oligonucleotide (6) of the present invention is suitable for use in nucleic acid drugs or gene diagnostics targeting DNA or RNA that require excellent double-strand forming ability.

[0831] [ Experimental Example 2 ] Enzyme resistance assay of oligonucleotides

[0832] 1) Preparation of oligonucleotides for enzyme resistance assay

[0833] Oligonucleotides having the sequence shown in SEQ ID NO:8 (TTTTTTTTTT) or a portion thereof modified with the sequence shown in SEQ ID NO:5, as described in the above examples of oligonucleotide synthesis, were prepared in the same manner.

[0834] [Table 4]

[0835]

[0836] In equation (7), T = Base is thymine-1-based and R aIt is a unit of H

[0837] ^=thiophosphate bond

[0838] t α = In equation (9), Base is thymine-1-based and R b It is a unit of H

[0839] t β = In equation (9), Base is thymine-1-based and R b It is the unit of Me

[0840] In equation (7), t = Base is a thymine-1-based unit.

[0841] t a = In equation (6), Base is thymine-1-yl, A 1 It is a single bond, n is 1, R 3 It is methyl, R 4 It is the methyl group configured by R

[0842] And R 5 It is a unit of hydrogen atom

[0843] t b = In equation (6), Base is thymine-1-yl, A 1 It is a single bond, n is 1, R 3 It is methyl, R 4 It is an ethyl compound of R configuration.

[0844] And R 5 It is a unit of hydrogen atom

[0845] t c = In equation (6), Base is thymine-1-yl, A 1 It is a single bond, n is 1, R 3 It is methyl, R 4 It is a methyl group configured with S.

[0846] And R 5 It is a unit of hydrogen atom

[0847] t d = In equation (6), Base is thymine-1-yl, A 1 It is a single bond, n is 0 and R 4 It is a methyl unit

[0848] t e = In equation (6), Base is thymine-1-yl, A 1 It is a single bond, n is 1, R 3 It is 3-(N,N-dimethylamino)propyl,

[0849] R4 It is an ethyl compound of R and R 5 It is a unit of hydrogen atom

[0850] 2) Preparation of sample solution

[0851] The sample solutions were prepared as shown in the table below.

[0852] [Table 5]

[0853] reagents Final concentration Tris HCl pH 8.0 50mM <![CDATA[MgCl2]]> 10mM Oligonucleotides 7.5μM

[0854] 3) Enzyme reaction

[0855] Using the apparatus (Major Science, MD-MINI), oligonucleotides numbered 1 to 7 were operated on (see (1) to (4) below) at a temperature of 37°C.

[0856] (1) Incubate the sample solution for 5 minutes.

[0857] (2) Add enzyme CAVP (Crotalus adamanteus venom phosphodiesterase I) to a final concentration of 1.60 μg / mL or 5.00 μg / mL to start the reaction.

[0858] (3) When the reaction time is over, add EDTA to make the concentration of the reaction solution 5.0mM and terminate the reaction.

[0859] (4) The reaction time is 0 minutes, 5 minutes, 10 minutes, 40 minutes and 80 minutes.

[0860] For oligonucleotides numbered 1, 2, 4, 6 and 8–10, except that in step (2) of step A, the reaction was initiated by adding the enzyme CAVP at a final concentration of 4.38 μg / mL, the same procedure as step A was performed in step B.

[0861] 4) Evaluation of enzyme resistance

[0862] The sample solution after the enzyme reaction in operation A was completed was analyzed by HPLC under the following conditions.

[0863] (condition)

[0864] Device: LC-2010A HT (manufactured by Shimadzu Corporation)

[0865] Chromatographic column: XBridge oligonucleotide (Oligonucleoties) BEH C18 column, 130, 2.5 μm, 4.6 mm × 50 mm.

[0866] mobile phase

[0867] Solution A: 0.1M triethylacetate ammonium buffer (pH 7.0)

[0868] Solution B: 0.1M triethylacetate ammonium buffer (pH 7.0): acetonitrile = 1:1 (v / v) gradient: 5–30% ((v / v) solution B), 15 minutes)

[0869] Flow rate: 0.8 mL / min

[0870] Column temperature: 50℃

[0871] Detection wavelength: 268nm

[0872] Injection volume: 15 μL (101.2 pmol)

[0873] The amount of undigested oligonucleotides was determined by HPLC analysis, and the residual rate (%) of undigested oligonucleotides at each reaction time was calculated using the following formula.

[0874] [Number 1]

[0875]

[0876] The sample solution after the enzyme reaction in Operation B was completed was analyzed by HPLC in the same manner as described above, except that the Alliance e2695 (manufactured by Waters Corporation) was used as the instrument.

[0877] 5) Results

[0878] The results for the enzyme CAVP at final concentrations of 5.00 μg / mL, 1.60 μg / mL, and 4.38 μg / mL are shown in Table 6A and 6B, respectively. Figure 1A Table 6B and Figure 1B and Table 6C and Figure 1C As shown.

[0879] [Table 6A]

[0880] Residual percentage of undigested oligonucleotides at each reaction time using CAVP 5.00 μg / mL (final concentration) (%)

[0881]

[0882] [Table 6B]

[0883] Residual percentage of undigested oligonucleotides at each reaction time using CAVP at a final concentration of 1.60 μg / mL (%)

[0884]

[0885] [Table 6C]

[0886] Residual percentage of undigested oligonucleotides at each reaction time using CAVP 4.38 μg / mL (final concentration) (%)

[0887]

[0888] The results show that the oligonucleotide (6) of the present invention has excellent enzyme resistance compared with natural and other non-natural oligonucleotides.

Claims

1. A compound represented by the following formula (1) or a salt thereof: ###0001### (1) wherein, Base is an aromatic heterocyclic group optionally having a substituent selected from the group consisting of an alkyl group, an acyl group and an amino group optionally substituted with a protecting group for an amino group; or an aromatic hydrocarbon ring group, ###0002### represented by the following formula: ###0003### the symbol represented by the formula is a single bond or a double bond, n is 1 when the symbol is a single bond, n is 0 when the symbol is a double bond, and Base is a 2,4-dioxo-l,2,3,4-tetrahydropyrimidin-l-yl group optionally having a substituent selected from the group consisting of an alkyl group, an acyl group and an amino group optionally substituted with a protecting group for an amino group, a 2-oxo-l,2-dihydropyrimidin-l-yl group optionally having a substituent selected from the group consisting of an alkyl group, an acyl group and an amino group optionally substituted with a protecting group for an amino group, a purin-9-yl group optionally having a substituent selected from the group consisting of an alkyl group, an acyl group and an amino group optionally substituted with a protecting group for an amino group, or a 6-oxo-l,6-dihydro-9H-purin-9-yl group.

8. The compound or a salt thereof according to claim 1, which is a compound represented by the following formula (1A) or a salt thereof, ###0006### (1A) 9. The compound or a salt thereof according to claim 1, which is a compound represented by the following formula (1B) or a salt thereof, ###0007### (1B) A 1 is a single bond or alkylene, R 1 and R 2 are the same or different, and are a hydrogen atom; an alkyl group optionally having a substituent selected from the group consisting of a halogen atom, an alkoxy group and an aryl group; an alkenyl group; a cycloalkyl group; a cycloalkenyl group; an aryl group optionally having a substituent selected from the group consisting of a halogen atom, an alkyl group and an alkoxy group; a protecting group for a hydroxyl group; a group represented by the formula: -P(R 7 )(R 8 ), wherein, in the formula -P(R 7 )(R 8 ), R 7 and R 8 are the same or different, and are a hydroxy 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 optionally having a substituent selected from a protecting group of a hydroxy group; or a hydroxymercapto phosphinyl group; or R 1 and R 2 together with the adjacent 2 oxygen atoms and the carbon atoms at positions 3 to 5 of the furanose form a ring optionally having a substituent selected from an alkyl group, 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 represented by the formula: -Si(R 6 )3, wherein, in the formula -Si(R 6 )3, each R 6 which are the same or different, is an alkyl group or an aryl group; a fluorescent labeling functional group; a chemiluminescent labeling functional group; a radionuclide-containing group; a residue of an intercalator; a residue of a nucleic acid binding agent; a residue of a nucleic acid cleaving agent; a residue of a cell-internal migration or nuclear-migration signal peptide; a residue of a metal chelating agent; or a group represented by the formula: R 31 -X-, wherein, in the formula R 31 -X-, R 31 is a group represented by the following formula (A) or is a group represented by the following formula (B), In formula (A), R 3a and R 3b are the same or different and are a hydrogen atom, an alkyl group, an alkenyl group, a cycloalkyl group, a cycloalkenyl group, an aryl group, an aralkyl group, or a protecting group for an amino group, or R 3a and R 3b together with the adjacent nitrogen atom form a ring, In formula (B), R 3c ~R 3f the same or different, is a hydrogen atom, an alkyl group, or a protecting group of an amino group, X is alkylene, or at least one methylene in the alkylene is replaced by a group -N(R 32 )-, -O- or -S(=O) k -, wherein, in the group -N(R 32 )-, R 32 is a hydrogen atom or an alkyl group, and in the group -S(=O) k -, k is 0, 1 or 2, R 4 is alkyl, R 5 is a hydrogen atom, 10. A method for producing a compound represented by the formula (1) or a salt thereof according to claim 1, the method comprising: (I) a step of reacting a compound represented by the following formula (2) or a salt thereof, ###0008### (2) with a compound represented by the following formula (3) or a salt thereof, ###0009### (3) in the presence of a base. further optionally comprising: (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 the compound, or 2. The compound according to claim 1, or a salt thereof, wherein, A 1 is a single bond.

3. The compound or salt thereof of claim 1 or 2, wherein, and, (II) a step of reacting the compound obtained in the step (I) with a carbonyl compound or a step of dehydrogenating the compound obtained in the step (I) and then hydrogenating the compound and then reacting the compound with a carbonyl compound.

4. The compound or salt thereof according to claim 1 or 2, wherein, 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 represented by the formula: -Si(R 6 )3, a fluorescent labeling functional group, a chemiluminescent labeling functional group, a radionuclide-containing group, a residue of an intercalator, a residue of a nucleic acid binding agent, a residue of a nucleic acid cleaving agent, a residue of a cell- or nucleus-migration signal peptide, or a residue of a metal chelating agent, wherein, in the formula -Si(R 6 )3, each R 6 is the same or different and is an alkyl group or an aryl group.

5. The compound according to claim 1 or 2, or a salt thereof, wherein, R 3 is a group of the formula: R 31 -X- represents a group, X is -C m H 2m - wherein, in the formula -C m H 2m - m is an integer from 1 to 10.

6. The compound according to claim 1 or 2, or a salt thereof, wherein, R 1 and R 2 are the same or different and are a hydrogen atom; an alkyl group optionally having a substituent selected from the group consisting of a halogen atom, an alkoxy group and an aryl group; an aryl group optionally having a substituent selected from the group consisting of a halogen atom, an alkyl group and an alkoxy group; an alkylcarbonyl group; an arylcarbonyl group; an alkylsulfonyl group; an arylsulfonyl group; a group represented by the formula: -Si(R 6 )3, wherein, in the formula -Si(R 6 )3, each R 6 is the same or different and is an alkyl group or an aryl group; a group represented by the formula: -P(R 7 )(R 8 ), wherein, in the formula -P(R 7 )(R 8 ), 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 hydroxymercapto phosphinyl group, or, R 1 and R 2 together with the adjacent 2 oxygen atoms and the carbon atoms at positions 3 to 5 of the furanose ring form a ring optionally having a substituent selected from an alkyl group.

7. The compound according to claim 1 or 2, or a salt thereof, wherein, 12. An oligonucleotide or a salt thereof having a unit represented by the following formula (6): ###0010### (6) wherein, Base is an aromatic heterocyclic group optionally having a substituent selected from the group consisting of an alkyl group, an acyl group and an amino group optionally substituted with a protecting group for an amino group; or an aromatic hydrocarbon ring group, ###0011### represented by the following formula: ###0012### the symbol represented by the formula is a single bond or a double bond, n is 1 when the symbol is a single bond, n is 0 when the symbol is a double bond, and Base is a 2,4-dioxo-l,2,3,4-tetrahydropyrimidin-l-yl group optionally having a substituent selected from the group consisting of an alkyl group, an acyl group and an amino group optionally substituted with a protecting group for an amino group, a 2-oxo-l,2-dihydropyrimidin-l-yl group optionally having a substituent selected from the group consisting of an alkyl group, an acyl group and an amino group optionally substituted with a protecting group for an amino group, a purin-9-yl group optionally having a substituent selected from the group consisting of an alkyl group, an acyl group and an amino group optionally substituted with a protecting group for an amino group, or a 6-oxo-l,6-dihydro-9H-purin-9-yl group.

13. A method for detecting a target nucleic acid, comprising: In formula (1A), Base and R 1 ~R 5 The same as claim 1. (I) a step of selectively amplifying a target nucleic acid by a nucleic acid amplification method; In formula (1B), Base and R 1 , R 2 and R 4 are the same as in claim 1. and, (I) reacting a compound represented by the following formula (1E) with an organic metal reagent represented by the formula: R 4 or the formula: R 4 M 1 , Base, A 1 , R 2 , and R 4 are the same as in claim 1, and the formula R 4 , R 4 M is a metal atom or a group containing a metal atom, and R 4 is the same as in claim 1; (II) a step of detecting the target nucleic acid amplified in the step (I), wherein the oligonucleotide used in the amplification or detection comprises the oligonucleotide or a salt thereof according to claim 12.

14. A kit for detecting a target nucleic acid or selectively amplifying a target nucleic acid, wherein, (IV) a process in which the compound obtained in the process (I) or the compound obtained in the process (I) after dehydrogenation followed by hydrogenation is reacted with a compound of the formula: R 3 -L, wherein L is a leaving group, R 3 -L, wherein L is a leaving group, R 3 is the same as in claim 1 but is not hydrogen.

11. A process for producing a compound represented by the formula (1) or a salt thereof in claim 1 wherein n is 1, R 3 is a methyl group, the process comprising: (I) reacting a compound represented by the following formula (1E) with an organic metal reagent represented by the formula: R 4 or the formula: R 4 M, wherein, in formula (1E), Base, A 1 , R 1 and R 2 are the same as in claim 1, and the formula R 4 , wherein R 4 is the same as in claim 1, and the formula R 4 M, wherein M is a metal atom or an atom group containing a metal atom, and R 4 is the same as in claim 1; (a) the kit comprises a primer and a probe, at least one of which comprises the oligonucleotide or a salt thereof according to claim 12, or (b) the kit comprises an oligonucleotide or a salt thereof according to claim 12. ​ ​ ​ A 1 is a single bond or alkylene, 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 represented by the formula: -Si(R 6 )3, wherein, in the formula -Si(R 6 )3, each R 6 is the same or different and is an alkyl group or an aryl group; a fluorescent label functional group; a chemiluminescent label functional group; a radionuclide-containing group; a residue of an intercalator; a residue of a nucleic acid binding agent; a residue of a nucleic acid cleaving agent; a residue of a cell internalization or nuclear internalization signal peptide; or a residue of a metal chelator; or a group represented by the formula: R 31 -X-, wherein, in the formula R 31 -X-, R 31 is a group represented by the formula (A) below or a group represented by the formula (B) below, In formula (A), R 3a and R 3b are the same or different and are a hydrogen atom, an alkyl group, an alkenyl group, a cycloalkyl group, a cycloalkenyl group, an aryl group, an aralkyl group, or a protecting group for an amino group, or R 3a and R 3b together with the adjacent nitrogen atom form a ring, In formula (B), R 3c ~R 3f the same or different, is a hydrogen atom, an alkyl group, or a protecting group of an amino group, X is alkylene, or at least one methylene in the alkylene is replaced by a group -N(R 32 )-, -O- or -S(=O) k - wherein in the group -N(R 32 )- R 32 is a hydrogen atom or an alkyl group, and in the group -S(=O) k k is 0, 1 or 2, R 4 is alkyl, R 5 is a hydrogen atom, ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ (b) the kit comprises a Clamp nucleic acid and a primer, at least one of which comprises the oligonucleotide or salt thereof of claim 12.

15. A pharmaceutical composition comprising the compound or salt thereof according to any one of claims 1 to 9, or the oligonucleotide or salt thereof according to claim 12.

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