5'-phosphonate modified nucleoside analogs and oligonucleotides produced thereby

Abstract

The present invention relates to 5'-modified nucleoside analogs and oligonucleotides produced therefrom, and more specifically, to modified nucleosides and their analogs that can be used to be incorporated into the oligonucleotide terminus, which can be bound to double-stranded oligonucleotides (short interfering RNA) or single-stranded oligonucleotides (e.g., antisense oligonucleotides). The oligonucleotides provided herein are expected to result in the loss of normal function of the target RNA by hybridizing to a portion of the target RNA.

Description

[Technical Field]

[0001] The present invention relates to 5'-phosphonate modified nucleoside analogs and oligonucleotides produced therefrom, and more specifically, to modified nucleosides and analogs useful for incorporation into oligonucleotide terminals that may be included in double-stranded compositions. [Background technology]

[0002] In recent years, oligonucleotides containing nucleotide sequences at least partially complementary to target RNA have been shown to alter the function and activity of the target both in vitro and in vivo. When delivered to cells containing target RNA (e.g., messenger RNA (mRNA)), such oligonucleotides can act as antisense compounds to regulate the expression of the target, leading to alterations in the transcription or translation of the target RNA. Such techniques are generally considered antisense techniques. The underlying principle of antisense techniques is that antisense compounds hybridize with target RNA, regulating gene expression activity or function, such as transcription or translation. Regulation of gene expression can be achieved, for example, by targeted degradation or occupational repression. An example of regulation of RNA target function by degradation is the degradation of target RNA by RNA enzyme H after hybridization with a DNA antisense compound. Another example of regulation of gene expression by targeted degradation is RNA interference (RNAi). RNAi is a biological process involving RNA or RNA-like molecules (e.g., chemically modified RNA molecules) that silences gene expression through degradation. The RNAi mechanism is initiated by the Dicer enzyme-mediated generation of longer non-coding RNA molecules from RNA molecules, which are then loaded into an RNA-inducible silencing complex (RISC), where the sense strand is discarded and the antisense or guide strand is hybridized to a fully or partially complementary mRNA sequence, subsequently inducing mRNA silencing via Ago2-mediated degradation or translational repression. Advances in RNAi technology and delivery methods have yielded increasingly positive results in RNAi-based therapies, which represent a promising treatment direction for diseases. However, such technologies are not widely applicable because natural oligonucleotides are easily degraded by cellular and extracellular nucleases in the body, causing intrinsic metabolic problems of natural RNA such as targetability and in vivo stability. Therefore, in conventional technologies, modifications around nucleotides, particularly these modifications, help improve nuclease resistance, binding affinity, targetability, and in vivo stability.

[0003] The development of various chemical modifications applicable to RNAi oligonucleotides has led to significant progress in overcoming the intrinsic metabolic problems of natural RNA. Such chemical modifications of RNAi oligonucleotides have a crucial role in fully utilizing the potential of such therapeutic schemes, as they can improve their pharmacokinetic and pharmacodynamic properties. For example, Choung et al. introduced 2'-OMe (2'-O-methyl), 2'F (2'-fluoro), and phosphorothioate modifications, as well as various combinations thereof, into nucleotides to achieve nucleotide stability in serum. Furthermore, the use of a class of 5'-modified phosphonate monomers in the manufacture of oligonucleotides has been described. Phosphonate-modified nucleotides located at the 5' end of the antisense strand can improve the likelihood of phosphorylation and maintenance of phosphorylation at the 5' end of the oligonucleotide, thereby improving the likelihood of loading specific strands into RISC, allowing RNAi drugs to enter the RNAi pathway, and improving and enhancing gene knockdown and gene silencing activity. For example, phosphonate furanose monomers with a vinyl group incorporated into the 5' end of the antisense chain of WO2011139702, or phosphonate furanose monomers with a cycloalkyl group of WO2017214112, are also recent research directions.

[0004] While chemically modified and improved delivery methods have made significant progress in overcoming the intrinsic metabolic problems of natural oligonucleotides, there remains a need in this field for oligonucleotides with improved duration of expression suppression and / or activity, suitable for therapeutic administration. [Overview of the project]

[0005] The present invention provides 5'-phosphonate modified nucleoside analogs and oligonucleotides produced therefrom, further providing 5'-phosphonate modified nucleoside analogs useful for incorporation into the terminals of oligonucleotides, particularly the 5'-terminus, to obtain double-stranded oligonucleotides (e.g., dsRNA) or single-stranded oligonucleotides (e.g., antisense oligonucleotides) with improved and enhanced gene silencing activity and / or duration. Oligonucleotides such as RNAi agents containing one or more 5'-phosphonate modified nucleoside analogs may be further ligated to a targeted ligand such as N-acetylgalactosamine or a peptide, or to a pharmacokinetic modifier such as a polyethylene glycol (PEG) moiety or a lipid.

[0006] According to one aspect of the present invention, compounds represented by formula (I), formula (II), and formula (III) or their stereoisomers are provided. [ka] Eventually, Each T1 is an independently and optionally protected phosphine moiety. Each T2 is an independently active phosphorus group. Each X1 is independently a chemical bond, O, S, NJ1, or CJ1J2, of which J1 and J2 are independently hydrogen, halogen, sulfonyl group, sulfinyl group, optionally substituted C1-C6 alkyl group, optionally substituted C3-C6 cycloalkyl group, optionally substituted C2-C6 alkenyl group, optionally substituted C2-C6 alkynyl group, or optionally substituted C5-C 12 These are aryl groups, optionally substituted 5-12 membered heteroaryl groups, and optionally substituted 5-12 membered heterocycles. Each X2 independently controls the CR 15 or N, Each X3 is independently a chemical bond, an optionally substituted C1-C3 alkylene group, SO, SO2, C(=O), P(=O)R, where R is OH, SH, a C1-C6 alkyl group, NH2, NHSO2CH3, Each Bx is independently a heterocyclic base moiety, Each R 1 and R 2 are each independently H, halogen, an optionally substituted C1-C6 alkyl group, an optionally substituted C1-C6 alkoxy group, an optionally substituted C2-C6 alkenyl group, an optionally substituted C2-C6 alkynyl group, a sulfinyl group, a sulfonyl group, an acetyl group, Each R 3 and R 15 are each independently H, halogen, an optionally substituted C1-C6 alkyl group, an optionally substituted C1-C6 alkoxy group, an optionally substituted C2-C6 alkenyl group, an optionally substituted C2-C6 alkynyl group, Each A independently has one of the following formulas,

Chemical formula

[0007] In one embodiment, the compound is such that each optionally substituted group independently comprises one or more substituents selected from halogen, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylthio, and CN.

[0008] In one embodiment, each T1 is an independently and optionally protected phosphine portion having the following formula: [ka] Eventually, Ra and Rc are each independently selected from a hydroxyl group or protected hydroxyl group, a mercapto group or protected mercapto group, an optionally substituted C1-C6 alkyl group, an optionally substituted C1-C6 alkoxy group, an amino group or protected / substituted amino group, a natural or modified nucleoside, and R b is O, S or NR 12 And R 12 These are hydrogen, a C1-C6 alkyl group, and an amino protecting group. The substituents on the substituted amino group are selected from optionally substituted C1-C6 alkyl groups, optionally substituted C2-C6 alkenyl groups, optionally substituted C2-C6 alkynyl groups, sulfinyl groups, sulfonyl groups, and acetyl groups.

[0009] In one embodiment, the sulfonyl group is preferably a methylsulfonyl group.

[0010] In one embodiment, each optionally substituted group independently comprises one or more substituents selected from halogens, hydroxyl groups, C1-C6 alkyl groups, C1-C6 alkoxy groups, C1-C6 alkylthio groups, and CN.

[0011] Examples of protecting groups commonly used to protect phosphate hydroxyl groups or phosphate mercapto groups include methyl group, ethyl group, benzyl group (Bn), phenyl group, isopropyl group, tert-butyl group, acetyl group, chloroacetyl group, trichloroacetyl group, trifluoroacetyl group, pivaloyl group, tert-butoxymethyl group, methoxymethyl group, 1-ethoxyethyl group, 1-(2-chloroethoxy)ethyl group, 2-trimethylsilylethyl group, allyl group, cyclohexyl group (cHex), 9-fluorenylmethoxycarbonyl group, and methanesulfonic acid. Group, toluenesulfonic acid group, trifluoromethanesulfonic acid group, benzoyl group, benzoyl formate, p-phenylbenzoyl group, 4-methoxybenzyl group, monomethoxytrityl group, dimethoxytrityl group, trimethoxytrityl group, 4-chlorobenzyl group, 4-nitrobenzyl group, 2,4-dinitrophenyl group, 4-acyloxybenzyl group, 2-methylphenyl group, 2,6-dimethylphenyl group, 2-chlorophenyl group, 2,6-dichlorobenzyl group, diphenylmethyl group, triphenylmethyl group, 4-methylthio-1-butyl group, 2-(S- Acetylthioethyl group (SATE), 2-cyanoethyl group, 2-cyano-1,1-dimethylethyl (CDM), 4-cyano-2-butenyl group, 2-(trimethylsilyl)ethyl group (TSE), 2-(phenylthio)ethyl group, 2-(triphenylsilyl)ethyl group, 2-(benzylsulfonyl)ethyl group, 2,2,2-trichloroethyl group, 2,2,2-tribromoethyl group, 2,3-dibromopropyl group, 2,2,2-trifluoroethyl group, phenylthio group, 2-chloro-4-tritylphenyl group, 2-bromophenyl group, 2-[N-I This includes, but is not limited to, sopropyl group-N-(4-methoxybenzoyl)aminoethyl group, 4-(N-trifluoroacetylamino)butyl group, 4-oxopentyl group, 4-tritylaminophenyl group, 4-benzylaminophenyl group, tetrahydropyranyl group, morpholino, trimethylsilyl group, triethylsilyl group, tert-butyldimethylsilyl group, tert-butyldiphenylsilyl group, triphenylsilyl group, triisopropylsilyl group, pivalate methyl ether group (POM), and 9-phenylxanthin-9-yl.

[0012] Common examples of amino protecting groups include, but are not limited to, 2-trimethylsilylethoxycarbonyl group (Teoc), 1-methyl-1-(4-biphenyl)ethoxycarbonyl group (Bpoc), tert-butoxycarbonyl group (BOC), allyloxycarbonyl group (Alloc), 9-fluorenylmethoxycarbonyl group (Fmoc), benzyloxycarbonyl group (Cbz), benzyl group, formyl group, acetyl group, pivaloyl group, trihaloacetyl group, benzoyl group, nitrophenyl group, acetyl group, 2-nitrobenzenesulfonyl group, phthalimide group (Pht), p-toluenesulfonyl group (Tos), trityl group (Trt), 2,4-dimethoxybenzyl group (PMB), and dithiosuccinyl group.

[0013] In one embodiment, each T1 is an independently and optionally protected phosphine portion having the following formula: [ka] Eventually, Ra and Rc are each independently selected from a protected hydroxyl group or a protected mercapto group, and R b It is either O or S.

[0014] In one embodiment, the hydroxy protecting groups are independently acetyl, tert-butyl, tert-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, trimethylsilyl, triethylsilyl, and tert-butyldimethyl The group is selected from the lusilyl group, tert-butyldiphenylsilyl group, triphenylsilyl group, triisopropylsilyl group, benzoyl formate, chloroacetyl group, trichloroacetyl group, trifluoroacetyl group, pivaloyl group, 9-fluorenylmethoxycarbonyl group, methanesulfonic acid ester, toluenesulfonic acid ester, trifluoromethanesulfonic acid ester, monomethoxytrityl group, dimethoxytrityl group, trimethoxytrityl group, pivalate methyl ether group (POM), or substituted 9-phenylxanthin-9-yl. In one embodiment, the preferred hydroxy protecting group is independently selected from the acetyl group, benzyl group, tert-butyldimethylsilyl group, pivalate methyl ether group (POM), tert-butyldiphenylsilyl group, and 4,4'-dimethoxytrityl group.

[0015] In one embodiment, the mercapto protecting groups are independently a methyl group, an ethyl group, an acetyl group, a tert-butyl group, a tert-butoxymethyl group, a methoxymethyl group, a tetrahydropyranyl group, a 1-ethoxyethyl group, a 1-(2-chloroethoxy)ethyl group, a 2-trimethylsilylethyl group, a p-chlorophenyl group, a 2,4-dinitrophenyl group, a benzyl group, a benzoyl group, a p-phenylbenzoyl group, a 2,6-dichlorobenzyl group, a diphenylmethyl group, a p-nitrobenzyl group, a trimethylsilyl group, and a triethylsilyl group. The group is selected from benzyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, triphenylsilyl, triisopropylsilyl, benzoyl formate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, 9-fluorenylmethoxycarbonyl, methanesulfonic acid ester, toluenesulfonic acid ester, trifluoromethanesulfonic acid ester, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, or substituted 9-phenylxanthin-9-yl. In one embodiment, preferred mercapto protecting groups are independently selected from benzyl, 4,4'-dimethoxytrityl, and trityl groups.

[0016] In one embodiment, R b is either O or S, and Ra and Rc are independently selected from a protected hydroxyl group, a C1-C6 alkyl group, and a C1-C6 alkoxy group, respectively.

[0017] In one embodiment, R b is O, and Ra and Rc are independently selected from OH, SH, OCH3, OCH2CH3, OCH(CH3)2, OCH2OC(=O)C(CH3)3, OCH2CH2CN, and NHSO2CH3, respectively.

[0018] In one embodiment, one of Ra and Rc is a hydroxyl group or a protected hydroxyl group, a mercapto group or a protected mercapto group, an optionally substituted C1-C6 alkyl group, an optionally substituted C1-C6 alkoxy group, an amino group or a protected / substituted amino group, the other is a natural or modified nucleoside, and Rb is O, S, or NR 12 And R 12 is hydrogen, a C1-C6 alkyl group, or an amino protecting group. More preferably, one of Ra and Rc is a hydroxyl group or a protected hydroxyl group, and the other is a natural nucleoside, and more preferably R b It is O.

[0019] In one embodiment, each T2 independently [ka] The active phosphorus group has the following structure, where M4 is H, an optionally substituted C1-C6 alkyl group, OH, OJ7, SH, SJ7, or NJ7J8, M5 is an optionally substituted C1-C6 alkyl group, OH, OJ7, SH, SJ7, or NJ7J8, each J7 or J8 is independently an optionally substituted C1-C6 alkyl group or sulfonyl group, and r is 0 or 1.

[0020] In one embodiment, each optionally substituted group of the active phosphorus group independently comprises one or more substituents selected from halogen, hydroxyl group, C1-C6 alkyl group, C1-C6 alkoxy group, C1-C6 alkylthio group, and CN.

[0021] In one embodiment, each J7 or J8 is an independently substituted C1-C6 alkyl group, and the substituent is selected from cyano groups and halogens.

[0022] In one embodiment, M4 is selected from a methyl group, an ethyl group, a propyl group, and an isopropyl group.

[0023] In one embodiment, M4 is selected from a methanesulfonamide group.

[0024] In one embodiment, M4 is OJ7, where J7 is a substituted C1-C6 alkyl group, and the substituent is selected from a cyano group and a halogen.

[0025] In one embodiment, M5 is selected from N(CH(CH3)2)2.

[0026] In one embodiment, M4 is O(CH2)2CN, M5 is N(CH(CH3)2)2, and r is 0.

[0027] In one embodiment, each T2 active phosphorus group is independently a phosphoramidite.

[0028] In one embodiment, each T2 active phosphorus group is independently selected from diisopropylcyanoethoxyphosphoramidite, diisopropylethylphosphoramidite, and H-phosphonate.

[0029] In one embodiment, each X1 is independently O.

[0030] In one embodiment, each B X The heterocyclic base moiety is independently selected from natural nucleic acid bases, modified nucleic acid bases, and universal bases.

[0031] In one embodiment, each B X The heterocyclic base moiety is independently a pyrimidine, a substituted pyrimidine, pseudouracil, a substituted pseudouracil, a purine, a hypoxanthine, or a substituted purine.

[0032] In one embodiment, each B XThe heterocyclic base moieties are independently uracil, 5-thiazolauracil, thymine, cytosine, pseudouracil, N1-methyl-psoiduracil, hypoxanthine, 5-methylcytosine, 5-methyluracil, 3-benzoyluracil, 2,6-diaminopurine, adenine, or guanine. In one embodiment, each B X The heterocyclic base moieties are independently 2-thiouracil, 5-fluorouracil, dihydrouridine (D), and 7-methylguanosine (m7G).

[0033] In one embodiment, each R 15 and R 3 These are H, independently of each other.

[0034] In one embodiment, each R 1 and R 2 These are, independently, H, a methylsulfonyl group, and an acetyl group.

[0035] In one embodiment, each X2 is independently N, and each R 1 and R 2 These are, independently, H, a methylsulfonyl group, and an acetyl group.

[0036] In one embodiment, A in formula (I), (II), or (III) has one of the following formulas: [ka] Of these, Q1 and Q2 are independently H, halogen, -CN, and optionally substituted C1-C6 alkyl groups, respectively, while Q8 is O, S, SO, SO2, PR 16 R 17 or NR 11 And R 16 and R 17 These are independently (=O), (=S), OH, SH, C1-C6 alkyl, and NR. 18 R 19 And, R 11 , R 18 and R 19Each of these is independently H, an optionally substituted C1-C6 alkyl group, an optionally substituted C1-C6 alkoxy group, a methanesulfonyl group, a sulfonic acid group, C(=O)J3, C(=O)OJ3, or C(=O)N(J3)(J4), where J3 and J4 are independently H or a C1-C6 alkyl group.

[0037] Each optionally substituted group independently comprises one or more substituents selected from halogen, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylthio, and CN.

[0038] In one embodiment, each Q1 and Q2 is independently H, F, -CN, or a methyl group, and more preferably H.

[0039] In one embodiment, Q8 is S, SO, or SO2.

[0040] In one embodiment, the conditions are that M1 is C(Rd)(Re), X2 is C, X3 is a chemical bond, and A is [ka] If so, Q8 is S, SO, SO2, PR 16 R 17 or NR 11 Q1, Q2, R 11 , R 16 and R 17 This is as defined by formula (I) herein.

[0041] In one embodiment, X2 is N, and X3 is a chemical bond, -CH2-, -CH2CH2-, SO, SO2.

[0042] In one embodiment, X2 is CH and X3 is a chemical bond.

[0043] In one embodiment, in compound formula (II) or (III) provided herein, n is 0 or 1.

[0044] In one embodiment, the compounds provided herein have the compound represented by formula (I-1) or a stereoisomer thereof. [ka] Of these, T1, T2, A, R3, and Bx are as defined in formula (I) and embodiments above, respectively, X3 is a chemical bond, C(=O), P(=O)R, SO, or SO2, M1 is C(Rd)(Re), C(Rd)(Re)C(Rg)(Rf), each Rd, Re, Rg, and Rf is independently a substituent selected from hydrogen, halogen, hydroxyl group, C1-C6 alkyl group, C1-C6 alkoxy group, C1-C6 alkylthio group, O(CH2)2-OCH3, NJ5, CN, OC(=O)J5, OC(=O)N(J5)(J6), J5 and J6 are independently H or C1-C6 alkyl group, and R is OH, SH, C1-C6 alkyl group, NH2, or NHSO2CH3.

[0045] In one embodiment, in compound formula (I-1) provided herein, each Rd, Re, Rg, and Rf is a substituent independently selected from hydrogen, fluorine, a hydroxyl group, a C1-C6 alkoxy group, and O(CH2)2-OCH3. More preferably, it is hydrogen.

[0046] In one embodiment, in compound formula (I-1) provided herein, R 3 It is hydrogen.

[0047] In one embodiment, in compound formula (I-1) provided herein, X3 is SO2 or a chemical bond.

[0048] In one embodiment, in compound formula (I-1) provided herein, A has one of the following formulas: [ka] Q1 and Q2 are independently H, a halogen, or an optionally substituted C1-C6 alkyl group. Q8 stands for O, S, SO, SO2, PR 16 R 17 or NR 11 And R 16 and R 17 These are independently (=O), (=S), OH, SH, C1-C6 alkyl, and NR. 18 R 19 And R 11 , R 18 and R 19 J3 is independently H, optionally substituted C1-C6 alkyl group, optionally substituted C1-C6 alkoxy group, methanesulfonyl group, sulfonic acid group, C(=O)J3, C(=O)OJ3, or C(=O)N(J3)(J4), where J3 and J4 are independently H or C1-C6 alkyl group.

[0049] Each optionally substituted group independently comprises one or more substituents selected from halogen, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylthio, and CN.

[0050] In one embodiment, the compounds provided herein have a compound represented by formula (I-2) or a stereoisomer thereof. [ka] Of these, T1, T2, A, R3, and Bx are as defined in formula (I) and embodiments above, respectively, M1 is C(Rd)(Re), C(Rd)(Re)C(Rg)(Rf), each Rd, Re, Rg, and Rf is independently a substituent selected from hydrogen, halogen, hydroxyl group, C1-C6 alkyl group, C1-C6 alkoxy group, C1-C6 alkylthio group, O(CH2)2-OCH3, NJ5, CN, OC(=O)J5, OC(=O)N(J5)(J6), and J5 and J6 are independently H or C1-C6 alkyl group.

[0051] In one embodiment, in compound formula (I-2) provided herein, each Rd, Re, Rg, and Rf is a substituent independently selected from hydrogen, fluorine, a hydroxyl group, a C1-C6 alkoxy group, and O(CH2)2-OCH3.

[0052] In one embodiment, in compound formula (I-2) provided herein, M1 is CH2 or CH2CH2.

[0053] In one embodiment, in compound formula (I-2) provided herein, R3 is hydrogen.

[0054] In one embodiment, in compound formula (I-2) provided herein, A has one of the following formulas: [ka] Q1 and Q2 are independently H, a halogen, or an optionally substituted C1-C6 alkyl group. Q8 stands for S, SO, SO2, PR 16 R 17 or NR 11 And R 16 and R 17 These are independently (=O), (=S), OH, SH, C1-C6 alkyl, and NR. 18 R 19 And R 11 , R 18 and R 19 J3 is independently H, optionally substituted C1-C6 alkyl group, optionally substituted C1-C6 alkoxy group, methanesulfonyl group, sulfonic acid group, C(=O)J3, C(=O)OJ3, or C(=O)N(J3)(J4), where J3 and J4 are independently H or C1-C6 alkyl group.

[0055] Each optionally substituted group independently comprises one or more substituents selected from halogen, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylthio, and CN.

[0056] In one embodiment, in a compound of formula (I-1) or formula (I-2) provided herein, T1 is an optionally protected phosphine moiety having the following formula: [ka] Eventually, Ra and Rc are each independently selected from a hydroxyl group or protected hydroxyl group, a mercapto group or protected mercapto group, an optionally substituted C1-C6 alkyl group, an optionally substituted C1-C6 alkoxy group, an amino group or protected / substituted amino group, a natural or modified nucleoside, and R b is O, S or NR 12 And R 12 These are hydrogen, a C1-C6 alkyl group, and an amino protecting group.

[0057] In one embodiment, the substituents on the substituted amino group are selected from optionally substituted C1-C6 alkyl groups, optionally substituted C2-C6 alkenyl groups, optionally substituted C2-C6 alkynyl groups, sulfinyl groups, sulfonyl groups, and acetyl groups.

[0058] In one embodiment, the sulfonyl group is preferably a methylsulfonyl group.

[0059] In one embodiment, R b is either O or S, and Ra and Rc are independently selected from a protected hydroxyl group, a C1-C6 alkyl group, and a C1-C6 alkoxy group, respectively.

[0060] In one embodiment, R bis O, and Ra and Rc are each independently selected from OH, OCH3, OCH2CH3, OCH(CH3)2, OCH2OC(=O)C(CH3)3, OCH2CH2CN, NHSO2CH3.

[0061] In certain embodiments, in the compound of formula (I-1) or formula (I-2) provided herein, T2 is an active phosphorus group, and the active phosphorus group is a phosphoramidite. In certain embodiments, the T2 active phosphorus group is selected from diisopropylcyanoethoxy phosphoramidite, diisopropylethyl phosphoramidite, and H-phosphonate.

[0062] In certain embodiments, in the compound of formula (I-1) or formula (I-2) provided herein, B X The heterocyclic base moiety is selected from natural nucleobases, modified nucleobases, and universal bases.

[0063] In certain embodiments, in the compound of formula (I-1) or formula (I-2) provided herein, B X The heterocyclic base moiety is pyrimidine, substituted pyrimidine, pseudouracil, substituted pseudouracil, purine, hypoxanthine, or substituted purine.

[0064] In certain embodiments, B X The heterocyclic base moiety is uracil, 5-thiazolyluracil, thymine, cytosine, pseudouracil, N1-methyl-pseudouracil, hypoxanthine, 5-methylcytosine, 5-methyluracil, 3-benzoyluracil, 2,6-diaminopurine, adenine, or guanine. In certain embodiments, each B X The heterocyclic base moiety is independently 2-thiouracil, 5-fluorouracil, dihydrouridine (D), 7-methylguanosine (m7G).

[0065] In certain embodiments, in the compound of formula (I-2) provided herein, it has the 1S,2S,4S or 1R,2R,4R configuration.

[0066] In one embodiment, the compounds provided herein have a compound represented by formula (II-1) or a stereoisomer thereof. [ka] Of these, T1, T2, X1, X3, A, R3, Bx, and n are as defined in the above formula (II) and embodiment, respectively, and R 1 and R 2 These are, independently, H, a methylsulfonyl group, and an acetyl group.

[0067] In one embodiment, in compound formula (II-1) provided herein, n is 0 or 1.

[0068] In one embodiment, in compound formula (II-1) provided herein, X3 is CH2 or CH2CH2.

[0069] In one embodiment, the compounds provided herein have a compound represented by formula (II-2) or formula (II-3) or a stereoisomer thereof. [ka] Of these, T1, T2, X1, A, and Bx are as defined in formula (II) and the embodiment above, respectively.

[0070] In one embodiment, the compounds provided herein have a compound represented by formula (III-1) or formula (III-2) or a stereoisomer thereof. [ka] Of these, T1, T2, X1, A, and Bx are as defined in formula (III) and the embodiment above, respectively.

[0071] In one embodiment, A in formula (II-1), formula (II-2), formula (II-3), formula (III-1) or formula (III-2) has one of the following formulas,

Chemical formula

[0072] Each optionally substituted group independently contains one or more substituents selected from halogen, hydroxy group, C1-C6 alkyl group, C1-C6 alkoxy group, C1-C6 alkylthio group, CN.

[0073] In one embodiment, the compounds provided herein have formula (II-1), formula (II-2), formula (II-3), formula (III-1) or formula (III-2), wherein each Q1 and Q2 is independently H, F, -CN, methyl group, more preferably H.

[0074] In one embodiment, the compounds provided herein have formula (II-1), formula (II-2), formula (II-3), formula (III-1) or formula (III-2), wherein T1 is an optionally protected phosphine moiety and has the following formula, [ka] Eventually, Ra and Rc are each independently selected from a hydroxyl group or protected hydroxyl group, a mercapto group or protected mercapto group, an optionally substituted C1-C6 alkyl group, an optionally substituted C1-C6 alkoxy group, an amino group or protected / substituted amino group, a natural or modified nucleoside, and R b is O, S or NR 12 And R 12 These are hydrogen, a C1-C6 alkyl group, and an amino protecting group.

[0075] In one embodiment, the substituents on the substituted amino group are selected from optionally substituted C1-C6 alkyl groups, optionally substituted C2-C6 alkenyl groups, optionally substituted C2-C6 alkynyl groups, sulfinyl groups, sulfonyl groups, and acetyl groups.

[0076] In one embodiment, the sulfonyl group is preferably a methylsulfonyl group.

[0077] In one embodiment, R b is either O or S, and Ra and Rc are independently selected from a protected hydroxyl group, a C1-C6 alkyl group, and a C1-C6 alkoxy group, respectively.

[0078] In one embodiment, R b is O, and Ra and Rc are independently selected from OH, OCH3, OCH2CH3, OCH(CH3)2, OCH2OC(=O)C(CH3)3, OCH2CH2CN, and NHSO2CH3, respectively.

[0079] In some embodiments, in the compound formulas (II-1), (II-2), (II-3), (III-1), or (III-2) provided herein, T2 is an active phosphorus group, and the active phosphorus group is a phosphoramidite. In some embodiments, the T2 active phosphorus group is selected from diisopropylcyanoethoxyphosphoramidite, diisopropylethylphosphoramidite, and H-phosphonate.

[0080] In one embodiment, in a compound formula (II-1), formula (II-2), formula (II-3), formula (III-1), or formula (III-2) provided herein, B X The heterocyclic base portion is selected from natural nucleic acid bases, modified nucleic acid bases, and universal bases.

[0081] In one embodiment, in a compound formula (II-1), formula (II-2), formula (II-3), formula (III-1), or formula (III-2) provided herein, B X The heterocyclic base moiety is a pyrimidine, a substituted pyrimidine, a pseudouracil, a substituted pseudouracil, a purine, a hypoxanthine, or a substituted purine. In one embodiment, B X The heterocyclic base moieties are uracil, 5-thiazolauracil, thymine, cytosine, pseudouracil, N1-methyl-psoiduracil, hypoxanthine, 5-methylcytosine, 5-methyluracil, 3-benzoyluracil, 2,6-diaminopurine, adenine, or guanine. In one embodiment, each B X The heterocyclic base moieties are independently 2-thiouracil, 5-fluorouracil, dihydrouridine (D), and 7-methylguanosine (m7G).

[0082] In another embodiment, the compounds provided herein have a compound represented by formula (IV) or a stereoisomer thereof. [ka] Of these, Q8 is S, SO, SO2, PR 16 R17 or NR 11 And R 16 and R 17 These are independently (=O), (=S), OH, SH, C1-C6 alkyl, and NR. 18 R 19 Ra and Rc are each independently selected from a hydroxyl group or a protected hydroxyl group, a mercapto group or a protected mercapto group, an optionally substituted C1-C6 alkyl group, an optionally substituted C1-C6 alkoxy group, a protected or optionally substituted amino group, a natural or modified nucleoside, and R b is O, S or NR 12 And R 12 These are hydrogen, a C1-C6 alkyl group, and an amino protecting group. The substituents on the substituted amino group are selected from optionally substituted C1-C6 alkyl groups, optionally substituted C2-C6 alkenyl groups, optionally substituted C2-C6 alkynyl groups, sulfinyl groups, sulfonyl groups, and acetyl groups. Q1 and Q2 are independently H, halogen, -CN, optionally substituted C1-C6 alkyl group, optionally substituted C1-C6 alkoxy group, optionally substituted C2-C6 alkenyl group, optionally substituted C2-C6 alkynyl group, or NR. 4 R 5 And, Each R 4 , R 5 , R 11 , R 18 and R 19 These are independently H, optionally substituted C1-C6 alkyl groups, optionally substituted C1-C6 alkoxy groups, methanesulfonyl groups, and sulfonic acid groups. Z is a phosphoramidite, a sugar, or a nucleoside containing a sugar substitution moiety.

[0083] In certain embodiments, in the compound of formula (IV) provided herein, each substituted group optionally and independently contains one or more substituents selected from halogen, hydroxy group, C1-C6 alkyl group, C1-C6 alkoxy group, C1-C6 alkylthio group, and CN.

[0084] Protecting groups for hydroxy, mercapto, and amino groups may optionally be derived from common protecting groups as described herein, for example, without limitation, those as described in formula (I).

[0085] In certain embodiments, in the compound of formula (IV) provided herein, among them, Q8 is SO or SO2.

[0086] In certain embodiments, in the compound of formula (IV) provided herein, among them, Q8 is S.

[0087] In certain embodiments, in the compound of formula (IV) provided herein, Q1 and Q2 are each independently H.

[0088] In certain embodiments, in the compound of formula (IV) provided herein, in the nucleoside containing the above sugar or sugar-substituted moiety, the sugar or sugar-substituted moiety contains a 5-membered furanose ring, a non-furanose ring, or a 5- to 6-membered carbocyclic ring system or an open-chain system.

[0089] In one embodiment, in a nucleoside comprising the above-mentioned sugar or sugar-substituted moiety of compound formula (IV) provided herein, the sugar-substituted moiety is a morpholinyl group, a cyclohexenyl group, a cyclohexyl group, a cyclopentyl group, a pyranyl group, or a cyclohexanehexaol group. In one embodiment, in a nucleoside comprising the above-mentioned sugar or sugar-substituted moiety, the sugar moiety is a furanose. In one embodiment, in a nucleoside comprising the above-mentioned sugar or sugar-substituted moiety, the nucleoside comprises an unlocked nucleic acid base analog (UNA) or a glycerol nucleic acid base analog (GNA). In one embodiment, in a nucleoside comprising the above-mentioned sugar or sugar-substituted moiety, the nucleoside comprises a locked nucleic acid (LNA) or a cross-linked nucleic acid (BNA).

[0090] In one embodiment, in compound formula (IV) provided herein, Q8 is bonded to the 4'-carbon or 5'-carbon of a sugar or sugar-substituted moiety.

[0091] In one embodiment, in compound formula (IV) provided herein, the sugar or sugar-substituted nucleoside has the following structural formula: [ka] Of these, M2 is independently C(q3)(q4), C(q3)(q4)C(q5)(q6), M3 independently controls O, S, and NR. 13 , C(q7)(q8), C(q7)(q8)C(q9)(q 10 ), C(q7)=C(q8), OC(q7)(q8), X1 is independently a chemical bond, O, S, NJ1 or CJ1J2, of which J1 and J2 are independently hydrogen, halogen, sulfonyl group, sulfinyl group, optionally substituted C1-C6 alkyl group, optionally substituted C3-C6 cycloalkyl group, optionally substituted C2-C6 alkenyl group, optionally substituted C2-C6 alkynyl group, or optionally substituted C5-C 12These are aryl groups, optionally substituted 5-12 membered heteroaryl groups, and optionally substituted 5-12 membered heterocycles. X2 each operates independently under CR 15 or N, X3 consists of independently chemically bonded, optionally substituted C1-C3 alkylene groups, SO, SO2, C(=O), and P(=O)R, while R consists of OH, SH, C1-C6 alkyl groups, NH2, and NHSO2CH3. Bx are each independently heterocyclic base parts, R 15 q1, q2, q3, q4, q5, q6, q7, q8, q9 and q 10 Each of these is independently hydrogen, halogen, hydroxyl group, C1-C6 alkyl group, C1-C6 alkoxy group, C1-C6 alkylthio group, O(CH2)2-OCH3, CN, OC(=O)J5, OC(=O)N(J5)(J6), or C(=O)N((J5)(J6), where J5 and J6 are independently H or C1-C6 alkyl group. R 13 These are, independently, hydrogen and a C1-C6 alkyl group.

[0092] In one embodiment, each R 15 q1, q2, q3, q4, q5, q6, q7, q8, q9 and q 10 These are groups that are independently selected from hydrogen, fluorine, a hydroxyl group, a C1-C6 alkyl group, a C1-C6 alkoxy group, and O(CH2)2-OCH3.

[0093] In one embodiment, each R 15 q1, q2, q3, q4, q5, q6, q7, q8, q9 and q 10 These are groups that are independently selected from hydrogen.

[0094] In one embodiment, in compound formula (IV) provided herein, the sugar or sugar-substituted nucleoside has the following structural formula: [ka] Among them, M2, M3, X1, X2, X3, q1, q2 and Bx are as defined in the above formula (IV) and the embodiments respectively.

[0095] In certain embodiments, in the compound formula (IV) provided herein, among them, the nucleoside of the sugar or sugar-substituted moiety has the following furanose structural formula,

Chemical formula

[0096] In certain embodiments, the compound provided herein has a compound represented by formula (IV-1) or a stereoisomer thereof,

Chemical formula

[0097] In certain embodiments, in the compound formula (IV-1) provided herein, R 15 , q1, q2, q3, q4, q5, q6, q7, q8, q9 and q 10 are each independently selected from hydrogen, fluorine, hydroxy group, methyl group, methoxy group, O(CH2)2-OCH3. In certain embodiments, in the compound formula (IV-1) provided herein, R 15 , q1, q2, q3, q4, q5, q6, q7, q8, q9 and q 10 are each independently selected from hydrogen.

[0098] In certain embodiments, M3 is O, S, C(q7)(q8), M2 is C(q3)(q4), X2 is CR15 Alternatively, N, X3 are chemical bonds, SO, SO2, and R 15 q1, q2, q3, q4, q7 and q8 are as defined herein, [ka] It has the following structural formula. In one preferred embodiment, M3 is O, S, CH2, M2 is CH2, X2 is CH, and X3 is a chemical bond. In one preferred embodiment, M3 is CH2, M2 is CH2, X2 is N, and X3 is a chemical bond, SO, or SO2.

[0099] In one embodiment, M3 is C(q7)(q8), M2 is C(q3)(q4)C(q5)(q6), and X2 is CR 15 Alternatively, N, X3 are chemical bonds, SO, SO2, and R 15 q1, q2, q3, q4, q5, q6, q7 and q8 are as defined herein, [ka] It has the following structural formula. In one preferred embodiment, M3 is CH2, M2 is CH2CH2, X2 is CH, and X3 is a chemical bond. In one preferred embodiment, M3 is CH2, M2 is CH2CH2, X2 is N, and X3 is a chemical bond, SO, or SO2.

[0100] In one embodiment, in compound formula (IV-1) provided herein, Q8 is SO or SO2.

[0101] In one embodiment, in compound formula (IV-1) provided herein, Q8 is S.

[0102] In one embodiment, in compound formula (IV-1) provided herein, R b is either O or S, and Ra and Rc are independently selected from a protected hydroxyl group, a C1-C6 alkyl group, and a C1-C6 alkoxy group, respectively.

[0103] In one embodiment, in compound formula (IV-1) provided herein, Ra and Rc are each independently selected from a hydroxyl group, OCH3, OCH2CH3, OCH(CH3)2OCH3, OCH2CH2CN, and NHSO2CH3.

[0104] In one embodiment, in a compound of formula (IV) or (IV-1) provided herein, B X The heterocyclic base portion is selected from natural nucleic acid bases, modified nucleic acid bases, and universal bases.

[0105] In one embodiment, in a compound of formula (IV) or (IV-1) provided herein, B X The heterocyclic base moieties are, independently, pyrimidine, substituted pyrimidine, pseudouracil, substituted pseudouracil, purine, hypoxanthine, or substituted purine. In one embodiment, B X The heterocyclic base moieties are, independently, uracil, 5-thiazolauracil, thymine, cytosine, pseudouracil, N1-methyl-psoiduracil, hypoxanthine, 5-methylcytosine, 5-methyluracil, 3-benzoyluracil, 2,6-diaminopurine, adenine, or guanine. In one embodiment, each B X The heterocyclic base moieties are, independently, 2-thiouracil, 5-fluorouracil, dihydrouridine (D), and 7-methylguanosine (m7G).

[0106] In one embodiment, in compound formula (IV) or (IV-1) provided herein, the phosphoramidite is selected from diisopropylcyanoethoxyphosphoramidite, diisopropylethylphosphoramidite, and H-phosphonate.

[0107] In one embodiment, the compounds provided herein have the following specific structures and stereoisomers thereof.

[0108] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8]

[0109] In one embodiment, the compounds provided herein have the following specific structures and stereoisomers thereof.

[0110] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7]

[0111] The present invention further defines the variables individually and in more detail, and it should be understood that the compounds described herein include all combinations of the embodiments and defined variables disclosed herein, for example, formula (I), formula (I-1), formula (I-2), formula (II), formula (II-1), (II-2), (II-3), formula (III), formula (III-1), formula (III-2), formula (IV), formula (IV-1) or their stereoisomers, and more technical features of such compounds can be made in any combination, and such compounds can be applied to oligonucleotides and further applied to the 5'-terminus to form oligonucleotides. The above oligonucleotides include, but are not limited to, single-stranded antisense oligonucleotides (ASOs), miRNAs, and double-stranded ribonucleic acid (dsRNAs). In some examples, it is applied to the 5'-terminus of the antisense strand in double-stranded ribonucleic acid (dsRNA).

[0112] According to another aspect of the present invention, an oligonucleotide comprising a 5'-terminal nucleotide represented by formula (V), formula (VI), or formula (VII) and one of their stereoisomers is provided. [ka] Of these, each T1 is an independently and selectively protected phosphine moiety. Each T3 is an internucleoside linking group that independently links the above 5'-terminal nucleotide to the oligonucleotide. Each X1 is independently a chemical bond, O, S, NJ1, or CJ1J2, of which J1 and J2 are independently hydrogen, halogen, sulfonyl group, sulfinyl group, optionally substituted C1-C6 alkyl group, optionally substituted C3-C6 cycloalkyl group, optionally substituted C2-C6 alkenyl group, optionally substituted C2-C6 alkynyl group, or optionally substituted C5-C 12 These are aryl groups, optionally substituted 5-12 membered heteroaryl groups, and optionally substituted 5-12 membered heterocycles. Each X2 independently controls the CR 15 or N, Each X3 is independently a chemically bonded, optionally substituted C1-C3 alkylene group, SO, SO2, C(=O), P(=O)R, where R is OH, SH, C1-C6 alkyl group, NH2, NHSO2CH3. Each Bx is independently a heterocyclic base moiety, Each R 1 and R 2 These are, independently, H, halogen, optionally substituted C1-C6 alkyl group, optionally substituted C1-C6 alkoxy group, optionally substituted C2-C6 alkenyl group, optionally substituted C2-C6 alkynyl group, sulfinyl group, sulfonyl group, and acetyl group. Each R 3 and R 15 These are, independently, H, halogen, optionally substituted C1-C6 alkyl group, optionally substituted C1-C6 alkoxy group, optionally substituted C2-C6 alkenyl group, and optionally substituted C2-C6 alkynyl group, Each A independently possesses one of the following equations: [ka] Q1 and Q2 are independently H, halogen, -CN, optionally substituted C1-C6 alkyl group, optionally substituted C1-C6 alkoxy group, optionally substituted C2-C6 alkenyl group, optionally substituted C2-C6 alkynyl group, or NR. 4 R 5 And, Each Q3 is independently O, S, NR 6 or CR 7 R 8 And, Each of Q4, Q5, Q6, Q7, Q9, Q 10 Q 11 and Q 12 Each of these is independently H, halogen, optionally protected hydroxyl group, acetoxy group, azide group, optionally substituted C1-C6 alkyl group, optionally substituted C1-C6 alkoxy group, optionally substituted C2-C6 alkenyl group, optionally substituted C2-C6 alkynyl group, NR 9 R 10 And, Each Q8 is independently O, S, SO, SO2, PR 16 R 17 or NR 11 And, Each R 16 and R 17 These are independently (=O), (=S), OH, SH, C1-C6 alkyl, and NR. 18 R 19 And, Each R4, R5, R6, R7, R8, R9, R 10 , R 11 , R 18 and R 19 These are independently H, optionally substituted C1-C6 alkyl groups, optionally substituted C1-C6 alkoxy groups, methanesulfonyl groups, sulfonic acid groups, C(=O)J3, C(=O)OJ3, or C(=O)N(J3)(J4), M1 is C(Rd)(Re), C(Rd)(Re)C(Rf)(Rg), and each Rd, Re, Rg and Rf is independently a substituent selected from hydrogen, halogen, hydroxyl group, C1-C6 alkyl group, C1-C6 alkoxy group, C1-C6 alkylthio group, O(CH2)2-OCH3, NJ5, CN, OC(=O)J5, OC(=O)N(J5)(J6), or C(=O)N((J5)(J6). Each of J3, J4, J5, and J6 is independently H or a C1-C6 alkyl group. n is 0, 1, or 2.

[0113] In one embodiment, in the above oligonucleotide, each optionally substituted group independently comprises one or more substituents selected from halogen, hydroxyl group, C1-C6 alkyl group, C1-C6 alkoxy group, C1-C6 alkylthio group, and CN.

[0114] In one embodiment, in a provided oligonucleotide, T1 is an optionally protected phosphine moiety having the following formula: [ka] Eventually, Ra and Rc are each independently selected from a hydroxyl group or protected hydroxyl group, a mercapto group or protected mercapto group, an optionally substituted C1-C6 alkyl group, an optionally substituted C1-C6 alkoxy group, an amino group or protected / substituted amino group, a natural or modified nucleoside, and R b is O, S or NR 12 And R 12 These are hydrogen, a C1-C6 alkyl group, and an amino protecting group. The substituents on the substituted amino group are selected from optionally substituted C1-C6 alkyl groups, optionally substituted C2-C6 alkenyl groups, optionally substituted C2-C6 alkynyl groups, sulfinyl groups, sulfonyl groups, and acetyl groups. Each optionally substituted group independently comprises one or more substituents selected from halogen, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylthio, and CN.

[0115] Examples of protecting groups commonly used to protect phosphate hydroxyl groups or phosphate mercapto groups include methyl group, ethyl group, benzyl group (Bn), phenyl group, isopropyl group, tert-butyl group, acetyl group, chloroacetyl group, trichloroacetyl group, trifluoroacetyl group, pivaloyl group, tert-butoxymethyl group, methoxymethyl group, 1-ethoxyethyl group, 1-(2-chloroethoxy)ethyl group, 2-trimethylsilylethyl group, allyl group, cyclohexyl group (cHex), 9-fluorenylmethoxycarbonyl group, and methanesulfonic acid. Group, toluenesulfonic acid group, trifluoromethanesulfonic acid group, benzoyl group, benzoyl formate, p-phenylbenzoyl group, 4-methoxybenzyl group, monomethoxytrityl group, dimethoxytrityl group, trimethoxytrityl group, 4-chlorobenzyl group, 4-nitrobenzyl group, 2,4-dinitrophenyl group, 4-acyloxybenzyl group, 2-methylphenyl group, 2,6-dimethylphenyl group, 2-chlorophenyl group, 2,6-dichlorobenzyl group, diphenylmethyl group, triphenylmethyl group, 4-methylthio-1-butyl group, 2-(S- Acetylthioethyl group (SATE), 2-cyanoethyl group, 2-cyano-1,1-dimethylethyl (CDM), 4-cyano-2-butenyl group, 2-(trimethylsilyl)ethyl group (TSE), 2-(phenylthio)ethyl group, 2-(triphenylsilyl)ethyl group, 2-(benzylsulfonyl)ethyl group, 2,2,2-trichloroethyl group, 2,2,2-tribromoethyl group, 2,3-dibromopropyl group, 2,2,2-trifluoroethyl group, phenylthio group, 2-chloro-4-tritylphenyl group, 2-bromophenyl group, 2-[N-I This includes, but is not limited to, sopropyl group-N-(4-methoxybenzoyl)aminoethyl group, 4-(N-trifluoroacetylamino)butyl group, 4-oxopentyl group, 4-tritylaminophenyl group, 4-benzylaminophenyl group, tetrahydropyranyl group, morpholino, trimethylsilyl group, triethylsilyl group, tert-butyldimethylsilyl group, tert-butyldiphenylsilyl group, triphenylsilyl group, triisopropylsilyl group, pivalate methyl ether group (POM), and 9-phenylxanthin-9-yl.

[0116] Common examples of amino protecting groups include, but are not limited to, 2-trimethylsilylethoxycarbonyl group (Teoc), 1-methyl-1-(4-biphenyl)ethoxycarbonyl group (Bpoc), tert-butoxycarbonyl group (BOC), allyloxycarbonyl group (Alloc), 9-fluorenylmethoxycarbonyl group (Fmoc), benzyloxycarbonyl group (Cbz), benzyl group, formyl group, acetyl group, pivaloyl group, trihaloacetyl group, benzoyl group, nitrophenyl group, acetyl group, 2-nitrobenzenesulfonyl group, phthalimide group (Pht), p-toluenesulfonyl group (Tos), trityl group (Trt), 2,4-dimethoxybenzyl group (PMB), and dithiosuccinyl group.

[0117] In one embodiment, in the provided oligonucleotide, R b is O or S, and Ra and Rc are independently selected from a protected hydroxyl group, a C1-C6 alkyl group, and a C1-C6 alkoxy group, respectively.

[0118] In one embodiment, in the provided nucleotide, Ra and Rc are OH, SH, OCH3, OCH2CH3, OCH(CH3)2, OCH2OC(=O)C(CH3)3, NH2, OCH2CH2CN, and NHSO2CH3, respectively.

[0119] In one embodiment, in the provided oligonucleotide, Ra and Rc are each OH, and R b It is O.

[0120] In one embodiment, one of Ra and Rc is an OH group, and the other is a natural nucleoside, R b It is O.

[0121] In one embodiment, in the provided oligonucleotide, T3 is an internucleoside linking group that links a 5'-nucleotide represented by formula (V), formula (VI), or formula (VII) and their stereoisomers to the 5'-terminus of the oligonucleotide, wherein the internucleoside linking group is selected from phosphorus-containing linking groups or phosphorus-free linking groups.

[0122] In one embodiment, in the provided oligonucleotide, the phosphorus-containing internucleoside linking group is independently a phosphodiester linking group, a phosphotriester linking group, a phosphorothioate linking group, a phosphorodithioate linking group, an alkylphosphonate linking group, an aminophosphonate linking group, a phosphonate linking group, a phosphinate linking group, a thiophosphoramidate linking group, or a phosphoramidate linking group.

[0123] In one embodiment, in the provided oligonucleotide, the nucleoside linking group is independently an alkylphosphonate linking group, a phosphodiester nucleoside linking group, or a phosphorothioate nucleoside linking group.

[0124] In one embodiment, in the provided oligonucleotide, B X The heterocyclic base portion is selected from natural nucleic acid bases, modified nucleic acid bases, and universal bases.

[0125] In one embodiment, in the provided oligonucleotide, B X The heterocyclic base portion is a pyrimidine, a substituted pyrimidine, pseudouracil, a substituted pseudouracil, a purine, hypoxanthine, or a substituted purine.

[0126] In one embodiment, in the provided oligonucleotide, B XThe heterocyclic base moieties are uracil, 5-thiazolauracil, thymine, cytosine, pseudouracil, N1-methyl-psoiduracil, hypoxanthine, 5-methylcytosine, 5-methyluracil, 3-benzoyluracil, 2,6-diaminopurine, adenine, or guanine. In one embodiment, each B X The heterocyclic base moieties are independently 2-thiouracil, 5-fluorouracil, dihydrouridine (D), and 7-methylguanosine (m7G).

[0127] In one embodiment, in the provided oligonucleotide, R 15 and R 3 H is H.

[0128] In one embodiment, in the provided oligonucleotide, X1 is O.

[0129] In one embodiment, in the provided oligonucleotide, each R 1 and R 2 These are, independently, H, a methylsulfonyl group, and an acetyl group.

[0130] In one embodiment, in the provided oligonucleotide, each X2 is independently N, and each R 1 and R 2 These are, independently, H, a methylsulfonyl group, and an acetyl group.

[0131] In one embodiment, in the provided oligonucleotide, A in formula (V), (VI), or (VII) has one of the following formulas: [ka] Of these, Q1 and Q2 are independently H, halogen, -CN, and optionally substituted C1-C6 alkyl groups, respectively, while Q8 is O, S, SO, SO2, PR 16 R 17 or NR 11 And R 16and R 17 These are independently (=O), (=S), OH, SH, C1-C6 alkyl, and NR. 18 R 19 And R 11 , R 18 and R 19 J3 is independently H, optionally substituted C1-C6 alkyl group, optionally substituted C1-C6 alkoxy group, methanesulfonyl group, sulfonic acid group, C(=O)J3, C(=O)OJ3, or C(=O)N(J3)(J4), where J3 and J4 are independently H or C1-C6 alkyl group.

[0132] Each optionally substituted group independently comprises one or more substituents selected from halogen, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylthio, and CN.

[0133] In one embodiment, in a provided oligonucleotide, Q 1 and Q 2 These are, independently, H, F, -CN, and a methyl group, and more preferably H.

[0134] In one embodiment, Q8 is S, SO, or SO2.

[0135] In one embodiment, the conditions are that M1 is C(Rd)(Re), X2 is C, X3 is a chemical bond, and A is [ka] If so, Q8 is S, SO, SO2, PR 16 R 17 or NR 11 Q1, Q2, R 11 , R 16 and R 17 This is as defined by formula (V) herein.

[0136] In one embodiment, in the provided oligonucleotide, X2 is N, and X3 is a chemical bond, -CH2-, -CH2CH2-, SO, SO2.

[0137] In one embodiment, in the provided oligonucleotide, X2 is CH and X3 is a chemical bond.

[0138] In one embodiment, the provided oligonucleotide is a 5'-terminal nucleotide represented by formula (VI) or (VII) provided herein, in which n is 0 or 1.

[0139] In one embodiment, the provided oligonucleotide comprises a 5'-terminal nucleotide represented by formula (V-1) or its stereoisomer, of which, [ka] Among them, T1, T3, A, R 3 Bx and Bx are as defined in formula (V) and embodiments above, respectively, X3 is a chemical bond, C(=O), P(=O)R, SO or SO2, M1 is C(Rd)(Re), C(Rd)(Re)C(Rg)(Rf), each Rd, Re, Rg and Rf is independently a substituent selected from hydrogen, halogen, hydroxyl group, C1-C6 alkyl group, C1-C6 alkoxy group, C1-C6 alkylthio group, O(CH2)2-OCH3, NJ5, CN, OC(=O)J5, OC(=O)N(J5)(J6) or C(=O)N((J5)(J6), J5 and J6 are independently H or C1-C6 alkyl group, and R is OH, SH, C1-C6 alkyl group, NH2, or NHSO2CH3.

[0140] In one embodiment, the provided oligonucleotide comprises a 5'-terminal nucleotide having formula (V-1) or a stereoisomer thereof, where X3 is SO2 or a chemical bond and R3 is hydrogen.

[0141] In one embodiment, the provided oligonucleotide comprises the 5'-terminal nucleotide of formula (V-1) or its stereoisomer, where Rd, Re, Rg, and Rf are substituents independently selected from hydrogen, fluorine, a hydroxyl group, a C1-C6 alkoxy group, and O(CH2)2-OCH3. More preferably, hydrogen.

[0142] In one embodiment, the provided oligonucleotide comprises the 5'-terminal nucleotide of formula (V-1) or its stereoisomer, where A has one of the following formulas: [ka] Q1 and Q2 are independently H, a halogen, or an optionally substituted C1-C6 alkyl group. Q8 stands for O, S, SO, SO2, PR 16 R 17 or NR 11 And R 16 and R 17 These are independently (=O), (=S), OH, SH, C1-C6 alkyl, and NR. 18 R 19 And, R 11 , R 18 and R 19 J3 is independently H, optionally substituted C1-C6 alkyl group, optionally substituted C1-C6 alkoxy group, methanesulfonyl group, sulfonic acid group, C(=O)J3, C(=O)OJ3, or C(=O)N(J3)(J4), where J3 and J4 are independently H or C1-C6 alkyl group.

[0143] Each optionally substituted group independently comprises one or more substituents selected from halogen, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylthio, and CN.

[0144] In one embodiment, the provided oligonucleotide comprises a 5'-terminal nucleotide represented by formula (V-2) or its stereoisomer, of which, [ka] Among them, T1, T3, A, R 3 Bx and Bx are as defined in formula (V) and embodiments above, respectively, M1 is C(Rd)(Re), C(Rd)(Re)C(Rg)(Rf), each Rd, Re, Rg and Rf is independently a substituent selected from hydrogen, halogen, hydroxyl group, C1-C6 alkyl group, C1-C6 alkoxy group, C1-C6 alkylthio group, O(CH2)2-OCH3, NJ5, CN, OC(=O)J5, OC(=O)N(J5)(J6) or C(=O)N((J5)(J6), and J5 and J6 are independently H or C1-C6 alkyl group.

[0145] In one embodiment, the oligonucleotide provided herein comprises a 5'-terminal nucleotide having formula (V-2) or a stereoisomer thereof, where each Rd, Re, Rg, and Rf is independently a substituent selected from hydrogen, fluorine, a hydroxyl group, a C1-C6 alkoxy group, and O(CH2)2-OCH3. More preferably, it is hydrogen.

[0146] In one embodiment, the oligonucleotide provided herein comprises a 5'-terminal nucleotide having formula (V-2) or a stereoisomer thereof, where M1 is CH2 or CH2CH2.

[0147] In one embodiment, the oligonucleotide provided herein comprises a 5'-terminal nucleotide having formula (V-2) or a stereoisomer thereof, of which R 3 It is hydrogen.

[0148] In one embodiment, the oligonucleotide provided herein comprises a 5'-terminal nucleotide having formula (V-2) or a stereoisomer thereof, where A has one of the following formulas: [ka] Q1 and Q2 are independently H, a halogen, or an optionally substituted C1-C6 alkyl group. Q8 stands for S, SO, SO2, PR 16 R 17 or NR 11 And R 16 and R 17 These are independently (=O), (=S), OH, SH, C1-C6 alkyl, and NR. 18 R 19 And R 11 , R 18 and R 19 J3 is independently H, optionally substituted C1-C6 alkyl group, optionally substituted C1-C6 alkoxy group, methanesulfonyl group, sulfonic acid group, C(=O)J3, C(=O)OJ3, or C(=O)N(J3)(J4), where J3 and J4 are independently H or C1-C6 alkyl group.

[0149] Each optionally substituted group independently comprises one or more substituents selected from halogen, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylthio, and CN.

[0150] In one embodiment, the oligonucleotides provided herein comprise a 5'-terminal nucleotide having formula (V-1), formula (V-2), or a stereoisomer thereof, where Q1 and Q2 are independently H, F, -CN, or a methyl group. More preferably, H.

[0151] In one embodiment, the provided oligonucleotide comprises a 5'-terminal nucleotide represented by formula (V-1) or formula (V-2), where T1 is an optionally protected phosphine moiety having the following formula: [ka] Eventually, Ra and Rc are each independently selected from a hydroxyl group or protected hydroxyl group, a mercapto group or protected mercapto group, an optionally substituted C1-C6 alkyl group, an optionally substituted C1-C6 alkoxy group, an amino group or protected / substituted amino group, a natural or modified nucleoside, and R b is O, S or NR 12 And R 12 These are hydrogen, a C1-C6 alkyl group, and an amino protecting group.

[0152] In one embodiment, the substituents on the substituted amino group are selected from optionally substituted C1-C6 alkyl groups, optionally substituted C2-C6 alkenyl groups, optionally substituted C2-C6 alkynyl groups, sulfinyl groups, sulfonyl groups, and acetyl groups.

[0153] In one embodiment, the sulfonyl group is preferably a methylsulfonyl group.

[0154] In one embodiment, in the provided oligonucleotide, R b is O. In one embodiment, in the provided oligonucleotide, R b S is.

[0155] In one embodiment, in the provided oligonucleotide, Ra and Rc are OH, SH, NH2, and NHSO2CH3, respectively.

[0156] In one embodiment, in the provided oligonucleotide, Ra and Rc are each OH, and R b It is O.

[0157] In one embodiment, the provided oligonucleotide comprises a 5'-terminal nucleotide represented by formula (V-1) or formula (V-2), where T3 is an internucleotide linking group.

[0158] In one embodiment, in the provided oligonucleotide, the internucleotide linking group is selected from alkylphosphonate linking groups, phosphodiester nucleoside linking groups, or phosphorothioate nucleoside linking groups.

[0159] In some preferred embodiments, the oligonucleotide provided comprises a 5'-terminal nucleotide represented by formula (V-1) or formula (V-2), where Q8 is SO2.

[0160] In some preferred embodiments, the oligonucleotide provided comprises a 5'-terminal nucleotide represented by formula (V-1) or formula (V-2), where Q8 is S.

[0161] In one embodiment, the provided oligonucleotide comprises a 5'-terminal nucleotide represented by formula (V-1) or formula (V-2), of which each B X The heterocyclic base portion is selected from natural nucleic acid bases, modified nucleic acid bases, and universal bases.

[0162] In one embodiment, the provided oligonucleotide comprises a 5'-terminal nucleotide represented by formula (V-1) or formula (V-2), of which each B X The heterocyclic base moiety is independently a pyrimidine, a substituted pyrimidine, pseudouracil, a substituted pseudouracil, a purine, a hypoxanthine, or a substituted purine.

[0163] In one embodiment, B X The heterocyclic base moieties are uracil, 5-thiazolauracil, thymine, cytosine, pseudouracil, N1-methyl-psoiduracil, hypoxanthine, 5-methylcytosine, 5-methyluracil, 3-benzoyluracil, 2,6-diaminopurine, adenine, or guanine. In one embodiment, each B X The heterocyclic base moieties are independently 2-thiouracil, 5-fluorouracil, dihydrouridine (D), and 7-methylguanosine (m7G).

[0164] In one embodiment, the 5'-terminal nucleotide in formula (V-2) provided herein has a 1S,2S,4S or 1R,2R,4R stereoconfiguration.

[0165] In one embodiment, the provided oligonucleotide comprises a 5'-terminal nucleotide represented by formula (VI-1) or its stereoisomer, of which, [ka] Of these, T1, T3, X1, X3, A, R3, Bx, and n are as defined in the above formula (VI) and embodiment, respectively, and R 1 and R 2 These are, independently, H, a methylsulfonyl group, and an acetyl group.

[0166] In one embodiment, in the provided oligonucleotide, n is 0 or 1.

[0167] In one embodiment, in the provided nucleotide, X3 is CH2 or CH2CH2.

[0168] In one embodiment, the provided oligonucleotide comprises a 5'-terminal nucleotide represented by formula (VI-2) or its stereoisomer, of which, [ka] Of these, T1, T3, X1, A, and Bx are as defined in formula (VI) and the embodiment above, respectively.

[0169] In one embodiment, the provided oligonucleotide comprises a 5'-terminal nucleotide represented by formula (VI-3) or its stereoisomer, of which, [ka] Of these, T1, T3, X1, A, and Bx are as defined in formula (VI) and the embodiment above, respectively.

[0170] In one embodiment, the provided oligonucleotide comprises a 5'-terminal nucleotide represented by formula (VII-1) or its stereoisomer, of which, [ka] Of these, T1, T3, X1, A, and Bx are as defined in formula (VII) and the embodiment above, respectively.

[0171] In one embodiment, the provided oligonucleotide comprises a 5'-terminal nucleotide represented by formula (VII-2) or its stereoisomer, of which, [ka] Of these, T1, T3, X1, A, and Bx are as defined in formula (VII) and the embodiment above, respectively.

[0172] In one embodiment, the provided oligonucleotide comprises a 5'-terminal nucleotide represented by formula (VI-1), formula (VI-2), formula (VI-3), (VII-1), (VII-2) or a stereoisomer thereof, where A independently has one of the following formulas: [ka] Of these, Q1 and Q2 are independently H, halogen, -CN, and optionally substituted C1-C6 alkyl groups, respectively, while Q8 is O, S, SO, SO2, PR 16 R 17 or NR 11 And R 16 and R 17 These are independently (=O), (=S), OH, SH, C1-C6 alkyl, and NR. 18 R 19 And R 11, R 18 and R 19 J3 is independently H, optionally substituted C1-C6 alkyl group, optionally substituted C1-C6 alkoxy group, methanesulfonyl group, sulfonic acid group, C(=O)J3, C(=O)OJ3, or C(=O)N(J3)(J4), where J3 and J4 are independently H or C1-C6 alkyl group.

[0173] Each optionally substituted group independently comprises one or more substituents selected from halogen, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylthio, and CN.

[0174] In some preferred embodiments, the oligonucleotides provided comprise a 5'-terminal nucleotide represented by formula (VI-1), formula (VI-2), formula (VI-3), (VII-1), (VII-2) or a stereoisomer thereof, where Q1 and Q2 are independently H, F, -CN, or a methyl group, respectively. More preferably, they are H.

[0175] In some preferred embodiments, the provided oligonucleotide comprises a 5'-terminal nucleotide represented by formula (VI-1), formula (VI-2), formula (VI-3), (VII-1), (VII-2) or a stereoisomer thereof, of which Q8 is independently NR 11 And R 11 These are independently a methyl group and a methylsulfonyl group.

[0176] In some preferred embodiments, the oligonucleotides provided comprise a 5'-terminal nucleotide represented by formula (VI-1), formula (VI-2), formula (VI-3), (VII-1), (VII-2) or a stereoisomer thereof, where Q8 is SO2.

[0177] In some preferred embodiments, the oligonucleotides provided comprise a 5'-terminal nucleotide represented by formula (VI-1), formula (VI-2), formula (VI-3), (VII-1), (VII-2) or a stereoisomer thereof, where Q8 is S.

[0178] In one embodiment, the provided oligonucleotide comprises a 5'-terminal nucleotide represented by formula (VI-1), formula (VI-2), formula (VI-3), (VII-1), (VII-2) or its stereoisomer, of which each B X The heterocyclic base portion is selected from natural nucleic acid bases, modified nucleic acid bases, and universal bases.

[0179] In one embodiment, the provided oligonucleotide comprises a 5'-terminal nucleotide represented by formula (VI-1), formula (VI-2), formula (VI-3), (VII-1), (VII-2) or its stereoisomer, of which each B X The heterocyclic base moiety is independently a pyrimidine, a substituted pyrimidine, pseudouracil, a substituted pseudouracil, a purine, a hypoxanthine, or a substituted purine.

[0180] In one embodiment, B X The heterocyclic base moieties are uracil, 5-thiazolauracil, thymine, cytosine, pseudouracil, N1-methyl-psoiduracil, hypoxanthine, 5-methylcytosine, 5-methyluracil, 3-benzoyluracil, 2,6-diaminopurine, adenine, or guanine. In one embodiment, each B X The heterocyclic base moieties are independently 2-thiouracil, 5-fluorouracil, dihydrouridine (D), and 7-methylguanosine (m7G).

[0181] In one embodiment, the provided oligonucleotide comprises a 5'-terminal nucleotide represented by formula (VI-1), formula (VI-2), formula (VI-3), (VII-1), (VII-2) or a stereoisomer thereof, where T1 is an optionally protected phosphine moiety having the following formula: [ka] Eventually, Ra and Rc are each independently selected from a hydroxyl group or protected hydroxyl group, a mercapto group or protected mercapto group, an optionally substituted C1-C6 alkyl group, an optionally substituted C1-C6 alkoxy group, an amino group or protected / substituted amino group, a natural or modified nucleoside, and R b is O, S or NR 12 And R 12 These are hydrogen, a C1-C6 alkyl group, and an amino protecting group.

[0182] In one embodiment, the substituents on the substituted amino group are selected from optionally substituted C1-C6 alkyl groups, optionally substituted C2-C6 alkenyl groups, optionally substituted C2-C6 alkynyl groups, sulfinyl groups, sulfonyl groups, and acetyl groups.

[0183] In one embodiment, the sulfonyl group is preferably a methylsulfonyl group.

[0184] In one embodiment, R b is O. In one embodiment, R b S is.

[0185] In one embodiment, in the provided oligonucleotide, Ra and Rc are OH, SH, NH2, and NHSO2CH3, respectively.

[0186] In one embodiment, in the provided oligonucleotide, Ra and Rc are each OH, and R b It is O.

[0187] In one embodiment, the provided oligonucleotide comprises a 5'-terminal nucleotide represented by formula (VI-1), formula (VI-2), formula (VI-3), (VII-1), (VII-2) or a stereoisomer thereof, where T3 is an internucleotide linking group.

[0188] In one embodiment, the provided oligonucleotide comprises a 5'-terminal nucleotide represented by formula (VI-1), formula (VI-2), formula (VI-3), (VII-1), (VII-2) or a stereoisomer thereof, wherein the internucleotide linking group is selected from alkylphosphonate linking groups, phosphodiester nucleoside linking groups, or phosphorothioate nucleoside linking groups.

[0189] In another embodiment, the provided oligonucleotide comprises a 5'-terminal nucleotide represented by formula (VIII) or its stereoisomer, of which, [ka] Of these, Q8 is S, SO, SO2, PR 16 R 17 or NR 11 And R 16 and R 17 These are independently (=O), (=S), OH, SH, C1-C6 alkyl, and NR. 18 R 19 Ra and Rc are each independently selected from a hydroxyl group or a protected hydroxyl group, a mercapto group or a protected mercapto group, an optionally substituted C1-C6 alkyl group, an optionally substituted C1-C6 alkoxy group, a protected or optionally substituted amino group, a natural or modified nucleoside, and R b is O, S, or NR 12 And R 12 These are hydrogen, a C1-C6 alkyl group, and an amino protecting group. Q1 and Q2 are independently H, halogen, -CN, or an optionally substituted C1-C6 alkyl group. The substituents on the substituted amino group are selected from optionally substituted C1-C6 alkyl groups, optionally substituted C2-C6 alkenyl groups, optionally substituted C2-C6 alkynyl groups, sulfinyl groups, sulfonyl groups, and acetyl groups. R 11 , R 18 and R 19 These are independently H, optionally substituted C1-C6 alkyl groups, optionally substituted C1-C6 alkoxy groups, methanesulfonyl groups, and sulfonic acid groups. Z is a nucleoside containing a sugar or a sugar substitution moiety. T3 is an internucleoside linking group that links the 5'-terminal nucleotide of formula (VIII) or its stereoisomer to an oligonucleotide. Each substituted group optionally and independently comprises one or more substituents selected from halogens, hydroxyl groups, C1-C6 alkyl groups, C1-C6 alkoxy groups, C1-C6 alkylthio groups, and CN.

[0190] In one embodiment, the provided oligonucleotide is such that Q8 is SO or SO2 in formula (VIII).

[0191] In one embodiment, the oligonucleotide provided is such that Q8 is S in formula (VIII).

[0192] In one embodiment, the provided oligonucleotide is such that Q1 and Q2 are independently H in formula (VIII).

[0193] In one embodiment, the provided oligonucleotide is a nucleoside in formula (VIII) comprising the above-mentioned sugar or sugar-substituted moiety, wherein the sugar or sugar-substituted moiety comprises a 5-membered furanose ring, a non-furanose ring, a 5-6 membered carbocyclic system, or a ring-opening system.

[0194] In one embodiment, the oligonucleotide provided is a nucleoside of formula (VIII) containing the above-mentioned sugar or sugar-substituted moiety, wherein the sugar-substituted moiety is a morpholinyl group, a cyclohexenyl group, a cyclohexyl group, a cyclopentyl group, a pyranyl group, or a cyclohexanehexaol group. In one embodiment, the nucleoside containing the above-mentioned sugar or sugar-substituted moiety is a furanose. In one embodiment, the nucleoside containing the above-mentioned sugar or sugar-substituted moiety contains an unlocked nucleic acid base analog (UNA) or a glycerol nucleic acid base analog (GNA). In one embodiment, the nucleoside containing the above-mentioned sugar or sugar-substituted moiety contains a locked nucleic acid (LNA) or a cross-linked nucleic acid (BNA).

[0195] In one embodiment, the provided oligonucleotide is of formula (VIII), wherein Q8 is bonded to the 4'-carbon or 5'-carbon of a sugar or sugar-substituted moiety.

[0196] In one embodiment, the provided oligonucleotide has formula (VIII), wherein the sugar or sugar-substituted nucleoside has the following structural formula: [ka] [ka] Of these, M2 is C(q3)(q4), C(q3)(q4)C(q5)(q6), M3 is O, S, NR 13 , C(q7)(q8), C(q7)(q8)C(q9)(q 10 ), C(q7)=C(q8), OC(q7)(q8), Each X1 is independently a chemical bond, O, S, NJ1, or CJ1J2, of which J1 and J2 are independently hydrogen, halogen, sulfonyl group, sulfinyl group, optionally substituted C1-C6 alkyl group, optionally substituted C3-C6 cycloalkyl group, optionally substituted C2-C6 alkenyl group, optionally substituted C2-C6 alkynyl group, or optionally substituted C5-C 12 These are aryl groups, optionally substituted 5-12 membered heteroaryl groups, and optionally substituted 5-12 membered heterocycles. Each X2 independently controls the CR 15 or N, Each X3 is independently a chemically bonded, optionally substituted C1-C3 alkylene group, SO, SO2, C(=O), P(=O)R, where R is OH, SH, C1-C6 alkyl group, NH2, NHSO2CH3. Each Bx is independently a heterocyclic base moiety, Each R 15 q1, q2, q3, q4, q5, q6, q7, q8, q9 and q 10 These are independently hydrogen, halogen, hydroxyl group, C1-C6 alkyl group, C1-C6 alkoxy group, C1-C6 alkylthio group, O(CH2)2-OCH3, CN, OC(=O)J5, C(=O)N(J5)(J6), and C(=O)N((J5)(J6), where J5 and J6 are independently H or C1-C6 alkyl group. Each R 13 These are independently hydrogen and a C1-C6 alkyl group.

[0197] In one embodiment, in the provided oligonucleotide, each R 15 q1, q2, q3, q4, q5, q6, q7, q8, and q9 are each independently selected from hydrogen, fluorine, a hydroxyl group, a C1-C6 alkyl group, a C1-C6 alkoxy group, and O(CH2)2-OCH3. More preferably, they are hydrogen.

[0198] In one embodiment, the provided oligonucleotide has formula (VIII), wherein the sugar or sugar-substituted nucleoside has the following structural formula: [ka] Of these, M2, M3, X1, X2, X3, q1, q2, and Bx are defined in formula (VIII) and the embodiment above, respectively.

[0199] In one mechanism, M3 is O, S, C(q7)(q8), C(q7)(q8)C(q9)(q 10 )

[0200] In one embodiment, q1, q2, q7, q8, q9, q 10 Each of these is independently selected from hydrogen, fluorine, hydroxyl group, methyl group, methoxy group, and O(CH2)2-OCH3.

[0201] In one embodiment, the provided oligonucleotide, in formula (VIII), wherein the sugar or sugar-substituted nucleoside has the following furanose structural formula: [ka] Of these, X1, q1, q2, q3, q4 and Bx are defined in formula (VIII) and the embodiment above, respectively.

[0202] In one embodiment, the provided oligonucleotide comprises a 5'-terminal nucleotide represented by formula (VIII-1) or its stereoisomer, of which, [ka] Among them, Q8, Ra, Rc, R b M2, M3, X1, X2, X3, q1, q2, T3, and Bx are as defined in formula (VIII) and the embodiment above, respectively.

[0203] In one embodiment, the oligonucleotide provided is R in formula (VIII-1). 15 q1, q2, q3, q4, q5, q6, q7, q8, q9 and q 10 Each is independently selected from hydrogen, fluorine, hydroxyl group, methyl group, methoxy group, and O(CH2)2-OCH3. In one embodiment, the oligonucleotide provided is R in formula (VIII-1). 15 q1, q2, q3, q4, q5, q6, q7, q8, q9 and q 10 Each of these is independently selected from hydrogen.

[0204] In one embodiment, in the provided oligonucleotide, M3 is O, S, C(q7)(q8), M2 is C(q3)(q4), and X2 is R 15 Or N, X3 is a chemical bond, SO, SO2, R 15 q1, q2, q3, q4, q7 and q8 are as defined herein, [ka] It has the following structural formula. In one preferred embodiment, M3 is O, S, CH2, M2 is CH2, X2 is CH, and X3 is a chemical bond. In one preferred embodiment, M3 is CH2, M2 is CH2, X2 is N, and X3 is a chemical bond, SO, or SO2.

[0205] In one embodiment, M3 is C(q7)(q8), M2 is C(q3)(q4)C(q5)(q6), and X2 is CR 15 Alternatively, N, X3 are chemical bonds, SO, SO2, and R 15 q1, q2, q3, q4, q5, q6, q7 and q8 are as defined herein, [ka] It has the following structural formula. In one preferred embodiment, M3 is CH2, M2 is CH2CH2, X2 is CH, and X3 is a chemical bond. In one preferred embodiment, M3 is CH2, M2 is CH2CH2, X2 is N, and X3 is a chemical bond, SO, or SO2.

[0206] In one embodiment, the provided oligonucleotide is of formula (VIII-1), where Q8 is SO or SO2.

[0207] In one embodiment, the provided oligonucleotide is of formula (VIII-1), where Q8 is S.

[0208] In one embodiment, the oligonucleotide provided is, in formula (VIII-1), of which R b That is oxygen.

[0209] In one embodiment, the provided oligonucleotide is of formula (VIII-1), where Q8 is SO or SO2, and Ra and Rc are independently selected from OH, SH, NH2, and NHSO2CH3, respectively.

[0210] In one embodiment, the provided oligonucleotide is in formula (VIII-1), where Q8 is SO or SO2, R b is oxygen, and Ra and Rc are each independently selected from OH.

[0211] In one embodiment, the provided oligonucleotide is of formula (VIII-1), where Q8 is SO or SO2 and X1 is oxygen.

[0212] In one embodiment, the provided oligonucleotide is of formula (VIII-1), where Q8 is S and X1 is oxygen.

[0213] In one embodiment, the provided oligonucleotide is in formula (VIII-1), where Q8 is S, Rb is oxygen, and Ra and Rc are each independently selected from OH.

[0214] In one embodiment, the oligonucleotide provided is B in formula (VIII) or formula (VIII-1). X The heterocyclic base portion is selected from natural nucleic acid bases, modified nucleic acid bases, and universal bases.

[0215] In one embodiment, the oligonucleotide provided is B in formula (VIII) or formula (VIII-1). X The heterocyclic base portion is a pyrimidine, a substituted pyrimidine, a pseudouracil, a substituted pseudouracil, a purine, a hypoxanthine, or a substituted purine.

[0216] In one embodiment, B X The heterocyclic base moieties are uracil, 5-thiazolauracil, thymine, cytosine, pseudouracil, N1-methyl-psoiduracil, hypoxanthine, 5-methylcytosine, 5-methyluracil, 3-benzoyluracil, 2,6-diaminopurine, adenine, or guanine. In one embodiment, each B X The heterocyclic base moieties are independently 2-thiouracil, 5-fluorouracil, dihydrouridine (D), and 7-methylguanosine (m7G).

[0217] In one embodiment, the provided oligonucleotide comprises linked monomer subunits in formula (VIII) or formula (VIII-1), where each internucleoside linking group is independently an alkylphosphonate linking group, a phosphodiester internucleoside linking group, or a phosphorothioate nucleoside linking group.

[0218] In one embodiment, an individually used oligonucleotide provided has a compound fragment of the following formula at its 5'-terminal nucleotide, [ka] Of these, Q8 is S, SO, SO2, PR 16 R 17 or NR 11 And R 16 and R 17 These are independently (=O), (=S), OH, SH, C1-C6 alkyl, and NR. 18 R 19 And R 16 and R 17 These are independently (=O), (=S), OH, SH, C1-C6 alkyl, and NR. 18 And, Ra and Rc are each independently selected from a hydroxyl group or a protected hydroxyl group, a mercapto group or a protected mercapto group, an optionally substituted C1-C6 alkyl group, an optionally substituted C1-C6 alkoxy group, a protected or optionally substituted amino group, a natural or modified nucleoside, and R b is O, S, or NR 12 And R 12 These are hydrogen, a C1-C6 alkyl group, and an amino protecting group. The substituents on the substituted amino group are selected from optionally substituted C1-C6 alkyl groups, optionally substituted C2-C6 alkenyl groups, optionally substituted C2-C6 alkynyl groups, sulfinyl groups, sulfonyl groups, and acetyl groups. R 11 , R 18 and R 19 These are independently H, optionally substituted C1-C6 alkyl groups, optionally substituted C1-C6 alkoxy groups, methanesulfonyl groups, and sulfonic acid groups. Each substituted group optionally and independently comprises one or more substituents selected from halogen, hydroxyl group, C1-C6 alkyl group, C1-C6 alkoxy group, C1-C6 alkylthio group, and CN. " [ka] The symbol '' represents the junction with the remaining portion of the 5'-terminal nucleotide.

[0219] In one embodiment, it should be understood that the 5'-terminal nucleotide has a compound fragment of the following formula, [ka] Among them, Q8, Ra, R b The definitions of and Rc are the same as those defined in the 5'-terminal nucleotides of formula (V), (VI), or formula (VII) of the oligonucleotides described herein and their embodiments.

[0220] In one embodiment, the provided oligonucleotide comprises a 5'-terminal nucleoside represented by one of the following specific structures or its stereoisomers, [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6]

[0221] [ka] This represents the linkage to the internucleoside linking group at the 5'-terminus of the oligonucleotide.

[0222] In one embodiment, the provided oligonucleotide comprises a 5'-terminal nucleoside represented by one of the following specific structures or its stereoisomers, [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5]

[0223] [ka] This represents the linkage to the internucleoside linking group at the 5'-terminus of the oligonucleotide.

[0224] The oligonucleotides described herein include, but are not limited to, single-stranded antisense oligonucleotides (ASOs), miRNAs, and double-stranded ribonucleic acid (dsRNAs). In one embodiment, the oligonucleotide provided is a single-stranded oligonucleotide. In one embodiment, the single-stranded oligonucleotide is a conventional antisense oligonucleotide (also called an ASO), a ribozyme, or an aptamer. In one embodiment, the oligonucleotide provided is a double-stranded ribonucleic acid (dsRNA) reagent, such a compound being a double-stranded nucleoside nucleic acid well known in the art, of which one or two strands are the oligonucleotides disclosed herein.

[0225] In one embodiment, the provided oligonucleotides are double-stranded ribonucleic acid (dsRNA) reagents, each comprising a sense strand and an antisense strand, wherein the sense strand and antisense strand are fully or partially complementary, and the antisense strand is partially or completely complementary to a nucleic acid target gene, and at least one of the sense strand and antisense strand is the oligonucleotide provided above, comprising at least one 5'-terminal nucleotide represented by formula (V), formula (V-1), formula (V-2), formula (VI), formula (VI-1), formula (VI-2), formula (VI-3), formula (VII), formula (VII-1), formula (VII-2), formula (VIII), formula (VIII-1) or a stereoisomer thereof, wherein the double-stranded ribonucleic acid (dsRNA) reagent further optionally comprises an independent target group.

[0226] In one embodiment, the provided oligonucleotide is a double-stranded ribonucleic acid (dsRNA) reagent, of which the antisense strand includes at least one 5'-terminal nucleotide represented by formula (V), formula (V-1), formula (V-2), formula (VI), formula (VI-1), formula (VI-2), formula (VI-3), formula (VII), formula (VII-1), formula (VII-2), formula (VIII), formula (VIII-1), or a stereoisomer thereof.

[0227] In one embodiment, the individually used oligonucleotides or double-stranded ribonucleic acid (dsRNA) provided each contain 8 to 40 nucleotides.

[0228] In one embodiment, a double-stranded ribonucleic acid (dsRNA) reagent is provided, optionally further comprising an independent target group, wherein the 5'-terminal and / or 3'-terminal nucleotide of any one strand further comprises one or more target groups. The target groups may include ligands conventionally used in the field of siRNA administration.

[0229] In some embodiments, in the provided double-stranded ribonucleic acid (dsRNA) reagent, the target group may be one or more ligands selected from target molecules or derivatives thereof, such as polymers, sugars, receptor ligands expressed by hepatocytes, antibodies, quantum dots, polypeptides, or small molecule ligands.

[0230] In some embodiments, in the provided double-stranded ribonucleic acid (dsRNA) reagent, at least one or each of the target groups is selected from ligands capable of binding to mammalian hepatocyte surface receptors.

[0231] In some embodiments, the provided double-stranded ribonucleic acid (dsRNA) reagents include, each of the target groups independently, a ligand having affinity for the asialoglycoprotein receptor (ASGPR) on the surface of mammalian hepatocytes.

[0232] In some embodiments, in the provided double-stranded ribonucleic acid (dsRNA) reagent, at least one or each of the target groups is a ligand comprising galactose or N-acetylgalactosamine.

[0233] In some embodiments, in a double-stranded ribonucleic acid (dsRNA) reagent provided, at least one or each of the target groups has a fragment of the following structure: [ka]

[0234] p is either 1 or 2.

[0235] In one embodiment, the use of oligonucleotides or double-stranded ribonucleic acid (dsRNA) reagents in the manufacture of drugs for suppressing gene expression is provided. In one embodiment, the suppression of gene expression includes contacting the oligonucleotides or double-stranded ribonucleic acid (dsRNA) with one or more types of cells, tissues, or animals.

[0236] In one embodiment, a method for suppressing gene expression is provided, comprising contacting cells with a reagent comprising an oligonucleotide or double-stranded ribonucleic acid (dsRNA) as described herein, wherein each strand of the oligonucleotide comprises 8 to 40 nucleotides, and the antisense strand of the oligonucleotide reagent is complementary to the target RNA.

[0237] In one embodiment, the cells are from an animal. In one embodiment, the cells are from a human. In one embodiment, the target RNA is selected from mRNA, pre-mRNA, and microRNA. In one embodiment, the target RNA is mRNA. In one embodiment, the target RNA is human mRNA. In one embodiment, the target RNA is cleaved to suppress its function. In one embodiment, the method further includes detecting the level of the target RNA. In one embodiment, a method for suppressing gene expression is provided, comprising contacting one or more types of cells or tissues with an oligonucleotide or double-stranded ribonucleic acid (dsRNA) reagent provided by a 5'-terminal nucleotide represented by formula (V), formula (V-1), formula (V-2), formula (VI), formula (VI-1), formula (VI-2), formula (VI-3), formula (VII), formula (VII-1), formula (VII-2), formula (VIII), formula (VIII-1) or a stereoisomer thereof.

[0238] Detailed description of the invention In one embodiment, a 5'-phosphonate modified nucleoside analog is provided, particularly as the 5'-terminus of an antisense strand, which is useful as the end of an oligonucleotide for incorporation into a nucleotide monomer compound. This specification further provides intermediates and methods for producing these oligonucleotides. The 5'-phosphonate modified nucleoside analogs provided herein can be used to improve the duration and / or activity of oligonucleotide expression repression. In one embodiment, the oligonucleotides and compositions provided herein are intended to hybridize with a portion of a target RNA to cause the target RNA to lose its normal function. These oligonucleotides are also intended to be used as primers and probes in diagnostic applications.

[0239] Unless otherwise specified, the terminology, procedures, and techniques used in conjunction with the analytical chemistry, organic synthesis, and pharmacochemistry described herein are all well-known and commonly used in the art. Standard techniques can be used in chemical synthesis and chemical analysis. They are incorporated by reference for any purpose. Where possible, all patents, patent applications, published patent applications, and other publications, as well as other data, referenced throughout this disclosure are incorporated in their entirety by reference.

[0240] As used herein, the terms “unsubstituted” and “substituted” mean that hydrogen atoms in the parent compound are not substituted by other substituents, or that one or more hydrogen atoms are substituted by substituents. As used herein, the terms “substituent” and “substituted” mean that they include other groups or groups that are commonly added to the parent compound to enhance a desired property or to provide other desired effects. Substituents may or may not be protected and may be added to one or more available sites in the parent compound. Substituents may be further substituted by other substituents and may be linked directly to the parent compound or to the parent compound via linking groups such as alkyl or hydrocarbon groups. Unless otherwise specified, substituents herein may be substituents that are well known in the art and conventionally preferred, and substituents preferred herein include halogens, hydroxyl groups, alkyl groups, alkenyl groups, alkynyl groups, and acyl groups (-C(=O)R aa ), carboxyl group (-C(=O)OR aa ), aliphatic group, alicyclic group, alkoxy group, substituted oxygen (-OR aa ), aryl group, aralkyl group, heterocyclyl group, heteroaryl group, heteroarylalkyl group, amino group (-N(R bb )(R cc )), imino group (=NR bb ), amide group (-C(=O)N(R bb )(R cc ) or N(R bb )C(=O)R aa , azide group (-N3), nitro group (-NO2), cyano group (-CN), ureido group (-N(R) bb )C(=O)N(R bb )(R cc )), thioureid group (-N(R bb )C(S)N(R bb )(R cc )), guanidino group (-N(R bb )C(=NR bb )N(R bb )(R cc )), amidine group (-C(=NR bb )N(Rbb )(R cc ) or N(R bb )C(=NR bb )(R aa )), thiol group or mercapto group (-SR bb ), sulfinyl group (-S(=O)R bb ), sulfonyl group (-S(=O)2R bb ) and sulfonamide group (sulfonamidyl)(-S(=O)2N(R bb )(R cc ) or -N(R bb )S(=O)2R bb This includes, but is not limited to, ) and each R aa , R bb and R cc The substituents are independently H, optionally linked to a chemically functional group, or another substituent, and preferably the list includes, but is not limited to, H, alkyl groups, alkenyl groups, alkynyl groups, aliphatic groups, alkoxy groups, acyl groups, aryl groups, aralkyl groups, heteroaryl groups, alicyclic groups, heterocyclic groups, and heteroarylalkyl groups. In some preferred embodiments of substituents not specifically described, the substituents are selected from halogens, hydroxyl groups, C1-C6 alkyl groups, C1-C6 alkoxy groups, C1-C6 alkylthio groups, O(CH2)2-OCH3, NJ5, CN, OC(=O)J5, OC(=O)N(J5)(J6), or C(=O)N((J5)(J6), where J5 and J6 are independently H or C1-C6 alkyl groups. As used herein, “substituent” is an intended aspect of the present invention, and the generality of such substituents should be consistent with the principle that, in the “substituent,” the total number thereof can form chemical bonds with the atom or group to which they are bonded, as will be understood to those skilled in the art in the fields of pharmaceuticals and organic chemistry. For example, Q8 is PR 16 R 17 And R 16 and R 17 These are independently (=O), (=S), OH, SH, C1-C6 alkyl, and NR. 18 R 19 If so, in order to satisfy the chemical form of pentavalent phosphorus, R 16 or R 17At least one of them is (=O) or (=S), and the other is OH, SH, C1-C6 alkyl, NR 18 R 19 That is the case.

[0241] As used herein, the terms “optionally protected,” “optionally substituted,” and “optionally substituted” mean substituted or protected to exist in an optional manner, and therefore include unsubstituted (unprotected) and substituted (protected) atoms and parts.

[0242] As used herein, the term “alkyl group” refers to a linear or branched saturated hydrocarbon group having 1 to about 24 carbon atoms. Where it appears herein, ranges of numbers such as “C1-C6 alkyl group” generally mean the number of carbon atoms in an alkyl group that includes only 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., and may contain up to 6 carbon atoms. Unless otherwise stated, generally the number of carbon atoms increased by further substitution of other atoms or groups in an alkyl group is not within the range of the count, and the same applies to other counts herein. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, isopropyl, n-hexyl, octyl, decyl, and dodecyl groups. The term “alkyl group” further includes cases where the range of the number of carbon atoms is not specified, for example, the term “alkyl group” means C1-C 10 The term may also refer to a subrange (e.g., C1-C6). "Substituted alkyl group" refers to an alkyl moiety having a substituent. As used herein, "lower alkyl group" refers to an alkyl moiety having 1 to about 6 carbon atoms. As used herein, the term "alkylene group" refers to a divalent radical induced by the substitution of another carbon atom of an alkyl group, which may be an independent group or part of another substituent, including, but not limited to, -CH2CH2-.

[0243] As used herein, the term “alkenyl group” refers to a linear or branched hydrocarbon group having at least one carbon-carbon double bond. Examples of alkenyl groups include, but are not limited to, vinyl groups, propenyl groups, butenyl groups, 1-methyl-2-buten-1-yl groups, and dienes such as 1,3-butadiene groups. Alkenyl groups generally contain 2 to about 24 carbon atoms, more generally 2 to about 12 carbon atoms, of which 2 to about 6 carbon atoms are more preferred. As used herein, alkenyl groups may optionally contain one or more further substituents.

[0244] As used herein, the term "alkynyl group" refers to a linear or branched hydrocarbon group having at least one carbon-carbon triple bond. Examples of alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, and 1-butynyl groups. Alkynyl groups generally contain 2 to about 24 carbon atoms, more generally 2 to about 12 carbon atoms, of which 2 to about 6 carbon atoms are more preferred. As used herein, alkynyl groups may optionally contain one or more further substituents.

[0245] As used herein, the term "acyl group" refers to a group having the general formula -C(O)-X, formed by removing a hydroxyl group from an organic acid, where X is generally aliphatic, alicyclic, or aromatic. Examples include aliphatic carbonyl groups, aromatic carbonyl groups, aliphatic sulfonyl groups, aromatic sulfinyl groups, aliphatic sulfinyl groups, aromatic phosphate esters, and the like. As used herein, the acyl group may optionally contain further substituents.

[0246] The term "cycloalkyl group" refers to a ring system in which the ring is aliphatic. This ring system may include one or more rings in which at least one ring is aliphatic. Preferred alicyclic compounds include rings having about 5 to about 9 carbon atoms within the ring. As used herein, cycloalkyl groups may optionally include further substituents.

[0247] As used herein, the term "alkoxy group" refers to a group formed between an alkyl group and an oxygen atom, the oxygen atom being used to bond the alkoxy group to the parent molecule. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentyloxy, neopentyloxy, and n-hexyloxy groups. As used herein, the alkoxy group may optionally contain further substituents.

[0248] As used herein, the term "aminoalkyl group" refers to a C1-C alkyl group substituted with an amino group. 12 This refers to an alkyl group. The alkyl portion of the group forms a covalent bond with the parent molecule. The amino group may be located at any position, and the aminoalkyl group may be substituted with further substituents at the alkyl and / or amino moieties.

[0249] As used herein, the terms “aryl group” and “aromatic” refer to monocyclic or polycyclic carbocyclic groups having one or more aromatic rings, and as used herein, the term “aromatic group” refers to a planar ring having a delocalized π-electron system containing 4n+2π electrons, where n is an integer. Aromatic rings may be formed from 5, 6, 7, 8, 9, or more than 9 atoms. The term “aromatic” is intended to include carbocyclic aryl groups (e.g., phenyl group) and heterocycloaryl groups (or “heteroaryl group” or “heteroaromatic” groups) (e.g., pyridine). The above terms include monocyclic or fused polycyclic rings, i.e., rings sharing adjacent pairs of carbon atoms. “Substituted aromatic group” refers to an aromatic group having one or more further substituents. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, and indenyl groups. A preferred aryl ring system has about 5 to about 20 carbon atoms in one or more rings. As used herein, the aryl group may optionally contain further substituents.

[0250] As used herein, the terms "aralkyl group" and "arylalkyl group" refer to C1-C 12 This refers to an aromatic group covalently bonded to an alkyl group. The alkyl portion of the resulting aralkyl group (or arylalkyl group) forms a covalent bond with the parent molecule. Examples include, but are not limited to, benzyl and phenylethyl groups. As used herein, the aralkyl group may optionally include further substituents bonded to the alkyl group, aryl group, or forming group.

[0251] As used herein, the terms “heteroaryl group” and “heteroaromatic” refer to groups comprising monocyclic or polycyclic aromatic rings, ring systems, or fused ring systems, of which at least one of the rings is aromatic and contains one or more heteroatoms. Heteroaryl groups are also intended to include fused ring systems, including systems in which one or more fused rings do not contain heteroatoms. Heteroaryl groups generally contain one ring atom selected from sulfur, nitrogen, or oxygen. Examples of heteroaryl groups include, but are not limited to, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isoxazolyl, thiadiazolyl, oxadiazolyl, phenylthio, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzoxazolyl, quinoxalinyl, and others. A heteroaryl group can be bonded to the parent molecule directly or via a bonding moiety such as an aliphatic group or heteroatom. As used herein, the heteroaryl group may optionally contain further substituents.

[0252] As used herein, the term "heteroarylalkyl group" refers to a covalently bonded C1-C 12This refers to the heteroaryl group defined above, further comprising an alkyl group. The alkyl portion of the resulting heteroarylalkyl group can form a covalent bond with the parent molecule. Examples include, but are not limited to, pyridylmethyl, pyridylethyl, and napthyridinylpropyl groups. As used herein, the heteroarylalkyl group may optionally contain further substituents on either or both of the heteroaryl group or the alkyl portion.

[0253] As used herein, the term "halo" or "halogen" refers to an atom selected from fluorine, chlorine, bromine, and iodine.

[0254] As used herein, the term “heterocyclyl group” refers to a monocyclic or polycyclic ring system that contains at least one heteroatom and is unsaturated, partially saturated, or fully saturated, and therefore includes heteroaryl groups. Heterocycles are also intended to include fused ring systems, of which one or more fused rings contain at least one heteroatom, and the other rings may contain one or more heteroatoms, or may not contain any heteroatoms. Heterocyclyl groups generally contain at least one atom selected from sulfur, nitrogen, or oxygen. Examples of heterocyclyl groups include the [1,3]dioxolanyl group, pyrrolidinyl group, pyrazolinyl group, pyrazolidinyl group, imidazolinyl group, imidazolidinyl group, piperidinyl group, pyrazinyl group, oxazolidinyl group, isoxazolidinyl group, morpholinyl group, thiazolidinyl group, isothiazolidinyl group, quinoxalinyl group, pyridadinyl group, tetrahydrofuran group, and the like. As used herein, an "n" membered heterocyclyl group means the total of n atoms in the ring system and does not include any atoms or groups outside the ring system. As used herein, a heterocyclyl group may optionally contain further substituents.

[0255] As used herein, the term “monocyclic or polycyclic structure” is intended to include all ring systems selected from monocyclic ring systems or polycyclic ring systems in which rings are fused or linked, and to include single and mixed ring systems independently selected from aliphatic groups, alicyclic groups, aryl groups, heteroaryl groups, aralkyl groups, arylalkyl groups, heterocyclic groups, heteroaryl groups, heteroaromatic groups, and heteroarylalkyl groups. Such monocyclic and polycyclic structures may include rings that each have the same degree of saturation, or each independently has different degrees of saturation (including fully saturated, partially saturated, or fully unsaturated). Each ring may include a heterocyclic ring and ring atoms selected from C, N, O, and S to obtain rings having only C ring atoms, and these rings may exist as a mixed motif such as benzimidazole, in which one ring has only a carbocyclic atom, while the fused ring has two nitrogen atoms. Monocyclic or polycyclic structures may be further substituted with substituents, such as phthalimides having two =O groups bonded to one of the rings. Monocyclic or polycyclic structures can be bonded to the parent molecule using various policies, including direct bonding by ring atoms, bonding by substituents, or bonding by bifunctional moieties.

[0256] The term "oxo" refers to the (=O) group.

[0257] As used herein, the term “sulfonyl group” (alone or as part of another group) refers to the group of formula RSO2-, where R is hydrogen, alkyl, substituted alkyl, aryl, or substituted aryl group, and CH3SO2- is called the methylsulfonyl group. As used herein, the term “sulfinyl group” (alone or as part of another group) refers to the group of formula RSO-, and as used herein, the term “sulfonamide group” (alone or as part of another group) refers to the group of formula RSO2-NH-, where R is hydrogen, alkyl, substituted alkyl, aryl, or substituted aryl group. Some non-limiting and exemplary sulfonamide groups include CH3-SO2-N(H)-.

[0258] The terms “alicyclic group” or “alicyclic group” refer to a cyclic ring system, of which the rings are aliphatic rings. The ring system association may include one or more rings, of which at least one ring is an aliphatic ring. Preferred aliphatic rings include rings having about 5 to about 9 carbon atoms. As used herein, aliphatic rings optionally include other substituents. As used herein, the term “aliphatic” refers to a linear or branched hydrocarbon group having up to 24 carbon atoms, of which the degree of saturation between any two carbon atoms is a single bond, a double bond, or a triple bond. The aliphatic group preferably has 1 to about 24 carbon atoms, more generally 1 to about 12 carbon atoms, and more preferably 1 to about 6 carbon atoms, and the linear or branched aliphatic group may be interrupted by one or more heteroatoms, including nitrogen, oxygen, sulfur, and phosphorus. The aliphatic group interrupted by the above heteroatoms includes, but is not limited to, polyalkoxy groups such as polyalkylene glycols, polyamines, and polyimines. As used herein, the aliphatic group optionally includes other substituents. Spacers are used to link oligonucleotides to position them away from the substrate or support during the synthesis process. This method allows for greater flexibility and more space for synthesis, and facilitates cleavage upon completion of synthesis.

[0259] As used herein, the term "carboxyl group" (either alone or as part of another group) refers to a group of the formula -COOH.

[0260] Linking groups or difunctional bonding moieties, such as those known in this field, can be used to bond chemical functional groups, conjugated groups, reporter groups, and other groups to selective sites in a parent compound, such as an oligonucleotide. Generally, a difunctional bonding moiety includes a hydrocarbon group moiety having two functional groups. One of the functional groups is selected to bond to the parent molecule or target compound, while the other is selected to bond to essentially any selected group, such as a chemical functional group or conjugated group. In some embodiments, the linking group includes polymers of chain structures or repeating units, such as ethylene glycol or amino acid units. Examples of functional groups commonly used in difunctional bonding moieties include, but are not limited to, electrophiles for reacting with nucleophiles and electrophiles for reacting with electrophiles. In some embodiments, the difunctional bonding moiety includes amino groups, hydroxyl groups, carboxylic acids, thiols, and degrees of unsaturation (e.g., double or triple bonds). Some non-limiting examples of the bifunctional bond include 8-amino-3,6-dioxaoctanoic acid (ADO), 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide (SMCC), and 6-aminocaproic acid (AHEX or AHA). Other linking groups include substituted C1-C 10 Alkyl alkyl groups, substituted or unsubstituted C2-C 10 Alkenyl group, or substituted or unsubstituted C2-C 10 The material includes, but is not limited to, alkynyl groups, and among them, a non-limiting list of preferred substituents includes hydroxyl groups, amino groups, alkoxy groups, carboxyl groups, benzyl groups, phenyl groups, nitro groups, thiol groups, thioalkoxy groups, halogens, alkyl groups, aryl groups, alkenyl groups, and alkynyl groups.

[0261] As used herein, the term "ether" refers to a product obtained by substituting a hydrogen atom in the hydroxyl group of an alcohol or phenol with a hydrocarbon group, with the general formula RO-R', where R and R' may be the same or different. Both identical ethers are called symmetric ethers, also known as simple ethers or monoethers, while ethers that are different from both are called asymmetric ethers, also known as mixed ethers.

[0262] When used in this specification, [ka] The term generally refers to a bond to an adjacent base or part. For example, any one of the structures of the fragment of formula A as herein, [ka] In, [ka] L1 represents the binding site to the phosphine moiety of T1, and L2 represents the binding site to the nucleoside sugar moiety or the substituted product.

[0263] As used herein, the term “protecting group” refers to an unstable chemical moiety known in the art used to protect a reactant group (including, but not limited to, hydroxyl, amino, and thiohydroxyl groups) from undesirable reactions in a synthetic procedure. Protecting groups are typically used selectively to protect other reaction sites during their reaction processes and are then removed to leave the unprotected group intact, or used in further reactions.

[0264] Common examples of protecting groups for hydroxyl or mercapto groups include methyl, ethyl, benzyl (Bn), phenyl, isopropyl, tert-butyl, acetyl, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, tert-butoxymethyl, methoxymethyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, allyl, cyclohexyl (cHex), 9-fluorenylmethoxycarbonyl, methanesulfonic acid, and toluenesulfonic acid. phenyl acid group, trifluoromethanesulfonic acid group, benzoyl group, benzoyl formate, p-phenylbenzoyl group, 4-methoxybenzyl group, monomethoxytrityl group, dimethoxytrityl group, trimethoxytrityl group, 4-chlorobenzyl group, 4-nitrobenzyl group, 2,4-dinitrophenyl group, 4-acyloxybenzyl group, 2-methylphenyl group, 2,6-dimethylphenyl group, 2-chlorophenyl group, 2,6-dichlorobenzyl group, diphenylmethyl group, triphenylmethyl group, 4-methylthio-1-butyl group, 2-(S-acetylthio O) Ethyl group (SATE), 2-cyanoethyl group, 2-cyano-1,1-dimethylethyl (CDM), 4-cyano-2-butenyl group, 2-(trimethylsilyl)ethyl group (TSE), 2-(phenylthio)ethyl group, 2-(triphenylsilyl)ethyl group, 2-(benzylsulfonyl)ethyl group, 2,2,2-trichloroethyl group, 2,2,2-tribromoethyl group, 2,3-dibromopropyl group, 2,2,2-trifluoroethyl group, phenylthio group, 2-chloro-4-tritylphenyl group, 2-bromophenyl group, 2-[N-isopropyl This includes, but is not limited to, pyryl groups -N-(4-methoxybenzoyl)aminoethyl group, 4-(N-trifluoroacetylamino)butyl group, 4-oxopentyl group, 4-tritylaminophenyl group, 4-benzylaminophenyl group, tetrahydropyranyl group, morpholino, trimethylsilyl group, triethylsilyl group, tert-butyldimethylsilyl group, tert-butyldiphenylsilyl group, triphenylsilyl group, triisopropylsilyl group, pivalate methyl ether group (POM), and 9-phenylxanthin-9-yl.

[0265] Commonly used amino protecting groups include, but are not limited to, 2-trimethylsilylethoxycarbonyl group (Teoc), 1-methyl-1-(4-biphenyl)ethoxycarbonyl group (Bpoc), tert-butoxycarbonyl group (BOC), allyloxycarbonyl group (Alloc), 9-fluorenylmethoxycarbonyl group (Fmoc), benzyloxycarbonyl group (Cbz), benzyl group, formyl group, acetyl group, pivaloyl group, trihaloacetyl group, benzoyl group, nitrophenyl group, acetyl group, 2-nitrobenzenesulfonyl group, phthalimide group (Pht), p-toluenesulfonyl group (Tos), trityl group (Trt), 2,4-dimethoxybenzyl group (PMB), and dithiosuccinyl group.

[0266] As used herein, the terms “phosphine moiety” and “phosphonate moiety” refer to a phosphorus-containing functional group or part thereof in a phosphate ester (-OP(=O)(OH)OH) molecule, wherein the phosphorus atom is directly bonded to carbon. As used herein, the term “protected phosphine moiety” means that a hydroxyl group, mercapto group, amino group, etc. in the phosphonate moiety and modified phosphonate moiety is protected by a protecting group.

[0267] As used herein, the term “phosphine moiety” refers to a monovalent p V This refers to a phosphorus group (pentavalent phosphorus). In one embodiment, the phosphine portion has the following formula: [ka] Eventually, Ra and Rc are each independently selected from a hydroxyl group or protected hydroxyl group, a mercapto group or protected mercapto group, an optionally substituted C1-C6 alkyl group, an optionally substituted C1-C6 alkoxy group, an amino group or protected / substituted amino group, a natural or modified nucleoside, and R b is O, S or NR 12 And R 12These are hydrogen, a C1-C6 alkyl group, and an amino protecting group. The substituents on the substituted amino group are selected from optionally substituted C1-C6 alkyl groups, optionally substituted C2-C6 alkenyl groups, optionally substituted C2-C6 alkynyl groups, sulfinyl groups, sulfonyl groups, and acetyl groups.

[0268] In one embodiment, the sulfonyl group is preferably a methylsulfonyl group.

[0269] In one embodiment, each optionally substituted group independently comprises one or more substituents selected from halogens, hydroxyl groups, C1-C6 alkyl groups, C1-C6 alkoxy groups, C1-C6 alkylthio groups, and CN.

[0270] As used herein, the term “phosphine moiety” may be located at any one end of the oligonucleotide, but preferably at the 5'-terminal nucleoside. In one embodiment, the phosphine moiety is located at the 5'-terminal nucleoside. [ka] It has the following formula.

[0271] In one embodiment, if one of Ra and Rc is a natural or modified nucleoside, the phosphine portion [ka] As shown, the natural bases are selected from C, U, G, A, and T. Modified nucleic acid bases may be those conventionally used in this article or in this art, such as nucleosides formed by substituting the 2'-hydroxyl group of a ribosyl group with a methoxy group or fluorine. If one of the Ra and Rc of the phosphine moiety is a natural or modified nucleoside, and is located at the 5' end of a double-stranded oligonucleotide, such as the 5' end of an antisense strand, then one protruding end is formed, and the base or modified base substituent does not pair with the sense strand's forming base, and even if the sense strand contains a 3' protruding end, it does not pair with the forming base of that protruding end. Methods for its synthesis can be found in the Chinese published patent CN115819484A, all of which are incorporated herein by reference.

[0272] As used herein, the term "active phosphorus" group refers to a group that can be used to form nucleoside bonds, for example, phosphodiester and phosphorothioate nucleoside bonds. These active phosphorus groups are known in the art and P III (Trivalent phosphorus) or p V The (pentavalent phosphorus) contains a phosphorus atom in a valence state, and the above-mentioned active phosphorus group includes, but is not limited to, phosphoramidites, H-phosphonic acid esters, phosphotryesters, and phosphorus-containing chiral additives. In one embodiment, it includes a reactive phosphorus group that provides a 5' or 3'-phosphinate nucleoside bond upon reaction with a free hydroxyl group. These reactive phosphorus groups are -(P=R e )(OR d )2, but not limited to this, and among them, each R d is independently a protecting group, a substituted or unsubstituted C1-C6 alkyl group, or a substituted or unsubstituted aryl group, and Re is O or S. Activated phosphorus group in one embodiment [ka] In this, M4 is H, an optionally substituted C1-C6 alkyl group, OH, OJ7, SH, SJ7, or NJ7J8, and M5 is an optionally substituted C1-C6 alkyl group, OH, OJ7, SH, SJ7, or NJ7J8, and each J7 or J8 is independently an optionally substituted C1-C6 alkyl group or sulfonyl group, and r is 0 or 1. Other active phosphates and phosphates are disclosed in Beaucage and Iyer, Tetrahedron, 1992, 48(12), 2223-2311.

[0273] As used herein, the term “phosphoramidite” refers to nitrogen-containing trivalent phosphorus derivatives, which are conventional active phosphorus. Examples of suitable phosphoramidites are provided herein.

[0274] As used herein, the terms “nucleoside bond” or “nucleoside linking group” are intended to include a variety of nucleoside linking groups known in the art, and in such embodiments, any nucleoside bond can be used to link nucleosides or analogs together. The two main internucleoside linking groups are defined by the presence or absence of a phosphorus atom and include, but are not limited to, phosphodiester linking groups, phosphotriester linking groups, phosphorothioate linking groups, phosphorodithioate linking groups, alkylphosphonate linking groups, aminophosphonate linking groups, phosphonate linking groups, phosphinate linking groups, thiophosphoramidate linking groups, or phosphoromidate linking groups, as well as internucleoside linking groups that do not contain phosphorus, such as thiodiesters (-OC(O)-S-), thiocarbonylcarbamates (-OC(O)(NH)-S-), siloxanes (-O-Si(H)2-O-), N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-), formyl groups, and methyleneimino groups (-CH2-N(CH3)-O-CH2-). Nucleoside interbonding further includes neutral nonionic nucleoside interbonding, and as used herein, the term “neutral nucleoside interbonding” is intended to include nonionic nucleoside interbonding. Neutral nucleoside interbonding includes, but is not limited to, phosphotriesters, methylphosphonates, MMI (3'-CH2-N(CH3)-O-5'), amide-3 (3'-CH2-C(=O)-N(H)-5'), amide-4 (3'-CH2-N(H)-C(=O)-5'), methylene acetal (formacetal) (3'-O-CH2-O-5'), and thiomethylene acetal (thioformacetal) (3'-S-CH2-O-5'). Further neutral nucleoside-to-nucleoside bonds include nonionic bonds, such as siloxanes (dialkylsiloxanes), carboxylic acid esters, formamides, sulfides, sulfonic acid esters, and amides (see, for example, "Carbohydrate Modifications in Antisense Research"; ACS Symposium Series 580, edited by YSSanghvi and PDCook; Chapters 3 and 4, pp. 40-65).Further neutral nucleoside bonds include nonionic bonds containing mixed N, O, S, and CH2 compositional moieties, of which phosphorus atoms are not always present. In some forms, alkylphosphonate linking groups are selected from C1-C6 alkylphosphonate linking groups.

[0275] In one embodiment, in the provided oligonucleotide, the phosphorus-containing nucleoside linking group is [ka] The structure has the structural fragment shown, where X is H, an optionally substituted C1-C6 alkyl group, OR 13 , SR 13 OH, SH, or NR 13 R 14 This represents, where Y represents O or S, and z may be 0 or 1, and each R 13 or R 14 These are independently hydrogen, optionally substituted C1-C6 alkyl groups, and sulfonyl groups. [ka] These terms independently represent the ligation site to the 5'-terminal nucleoside and the ligation site to the adjacent nucleotide as defined herein.

[0276] Modified bonds, compared to natural phosphodiester bonds, can be used to alter (generally enhance) the nuclease resistance of oligonucleotides. In some embodiments, nucleoside bonds containing chiral atoms can be produced as racemic mixtures or as individual enantiomers. Typical chiral bonds include, but are not limited to, alkylphosphonates and phosphorothioates. Methods for producing phosphorus-containing and phosphorus-free nucleoside bonds are well known to those skilled in the art.

[0277] As used herein, “nucleoside” refers to a naturally occurring or modified nucleoside compound, which includes a heterocyclic base moiety and a sugar moiety. Nucleosides include, but are not limited to, naturally occurring nucleosides (such as those found in DNA and RNA), baseless nucleosides, modified nucleosides, and nucleosides having a mimetic base and / or a glycosyl group. Nucleosides may be modified with any one of several substituents.

[0278] As used herein, “nucleotide” refers to a nucleoside further comprising a phosphate ester linking group. As used herein, “linked nucleoside” may or may not be linked by a phosphate ester bond, and thus includes “linked nucleotides.”

[0279] As used herein, the term "nucleotide position" refers to the position of a nucleotide in an oligonucleotide or within an oligonucleotide, counted from the 5' terminal nucleotide. For example, nucleotide position 1 refers to the 5'-terminal nucleotide of an oligonucleotide.

[0280] As used herein, “sugar moiety” means including natural or modified sugar rings or sugar substitutions. “Sugar substitution moiety” or “sugar substitution” refers to a five-membered furanose ring structure that can substitute a naturally occurring nucleoside. Such nucleosides having a sugar substitution group typically still retain a heterocyclic base moiety and maintain hybridizing ability. In some embodiments, the sugar substitution is a non-furanose (or 4'-substituted furanose) ring or ring system or ring-open system. While not limited to these, such structures include simple modifications compared to the natural furanose ring, such as six-membered rings, morpholinyl groups, tetrahydropyrans, and cyclohexanehexaol systems. Six-membered substitutions are further modified to replace tetrahydropyran with a naturally occurring nucleoside furanose, and modified tetrahydropyrans are called hexetol nucleic acids (HNA), anitol nucleic acids (ANA), mannitol nucleic acids (MNA) (see Leumann, CJ., Bioorg. & Med. Chem. (2002) 10:841-854), and fluoroHNA (F-HNA). The structure includes, but is not limited to, several nucleosides substituted with morpholinyl groups (see International Patent Publications WO200836127, WO2011150408), and since carbocyclic nucleosides are nucleoside analogs in which the oxygen atom of the furanose ring in the sugar portion is substituted with a carbon atom, carbocyclic nucleosides are also general sugar-substituted products, see, for example, US Patent No. 6,001,840, which has a cyclopentane ring substituted for the tetrahydrofuran ring of the nucleoside, and such are cyclohexyl groups, cyclopentyl groups, cyclohexenyl groups (PCT Application WO2010 / 036696, Robeyns et al., J.Am.Chem.Soc.,2008,130(6),1979-1984, Gu et al.,Nucleosides,Nucleotides&Nucleic Acids,2005,24(5-7),993-998, Nauwelaerts et al.,Nucleic AcidsResearch,2005,33(8),2452-2463, Gu et al.See Ligonucleotides, 2003, 13(6), 479-489. The "sugar substitution moiety" may be a bicyclic or tricyclic structure (see, for example, Leumann, Jc, Bioorganic & Medicinal Chemistry, 2002, 10, 841-854, US7399845, International Patent Publication WO2009006478, WO2008150150729, WO2011 / 139702), such as locked nucleic acids ("LNA") (see, for example, Koshkin et al. (1998), Tetrahedron, 54, 3607-3630), or cross-linked nucleic acids ("BNA") (see, for example, US Patent No. 7,427,672 and Mitsuoka et al. (2009), Nucleic Acids Res The “sugar-substituted moiety” may be acyclic, for example, an unlocked nucleic acid ("UNA") (see, e.g., U.S. Patent No. 8,314,227, Meghan A. et al., “Locked vs. unlocked nucleic acids (LNA vs. UNA): contrasting structures work towards common therapeutic goals.” Chem. Soc. Rev., 2011, 40, 5680-5689), the glycerol nucleic acid structure (GNA) may be more complex, as a sugar-substituted moiety (see, e.g., WO2016 / 028649), or as an acyclic system of peptide nucleic acids. Further novel “sugar-substituted moiety” nucleosides can be referenced from those described in International Patent Publication WO2011 / 139702. Each of these is incorporated herein by reference in its entirety.

[0281] With respect to furanose, the term "furanose" as used herein refers to a carbohydrate having a five-membered ring structure, of which the above ring structure having four carbon atoms and one oxygen atom is [ka] As shown, the numbers in the structure represent the positions of the four carbon atoms in the five-membered ring structure. The nucleoside of the sugar moiety substituted with a certain tetrahydropyranyl group is [ka] It has the following structural formula, where each variable can be the same as formula (I) herein, and the ring can be further substituted with any suitable group, and the nucleoside of the sugar moiety substituted with a certain morpholinyl group is [ka] It has the following structural formula, where the numbers represent the positions of the four carbon atoms in the five-membered ring structure, and each variable can correspond to formula (I) herein, and the ring can be understood to be further substituted with any suitable group, and the nucleoside of the sugar moiety substituted with a certain cyclohexenyl group is, [ka] It has the following structural formula, where each variable can be the same as formula (I) herein, and the ring can be further substituted with any suitable group.

[0282] Nucleosides of certain bicyclic substituted sugar moieties include, but are not limited to, nucleosides formed by bridges between 4' and 2' sugar ring atoms. [ka] It has the following structural formula, where Bx is the base portion, and R is independently H, a protecting group, or C1-C 12 It is an alkyl group.

[0283] The modified sugar (also referred to herein as the "sugar substitution moiety") comprises a modified deoxyribose or ribose moiety, where the modification occurs, for example, at the 2', 3', 4', or 5'-carbon position of the sugar.

[0284] As used herein, “nucleic acid base” or “heterocyclic base moiety” refers to the heterocyclic base moiety of a nucleoside, or the heterocyclic moiety located at the 1' position (or equivalent position in a nucleotide sugar moiety substitution that enables incorporation into a nucleic acid double helix) of a modified nucleotide that enables incorporation into a nucleic acid double helix. “Nucleic acid base” or “heterocyclic base moiety” may be a naturally occurring nucleic acid base, a modifiable nucleic acid base, or a universal nucleic acid base. In some embodiments, “nucleic acid base” or “heterocyclic base moiety” may include any atom or set of atoms capable of bonding to a base of another nucleic acid by hydrogen bond, and each heterocyclic base moiety of a nucleoside may be modified with one or more substituents to enhance one or more properties, such as affinity to a target chain, or to influence several other properties in a favorable manner. Modified nucleic acid bases include, but are not limited to, universal bases, hydrophobic bases, hybrid bases, amplified cyclic bases, and fluorinated bases as defined herein. These nucleic acid bases can be used to enhance the binding affinity of oligonucleotides provided herein. The two most common classes are purines and pyrimidines. For example, suitable natural nucleic acid bases include purine and pyrimidine bases such as adenine (A), thymine (T), cytosine (C), guanine (G), or uracil (U).Appropriately modified nucleic acid bases include diaminopurines and their derivatives, alkylated purines or pyrimidines, acylated purines or pyrimidines, thiolated purines or pyrimidines, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, adenine and guanine, 6-methyl and other alkyl derivatives, the 2-propyl group of adenine and guanine and other alkyl derivatives, 2-thiouracil, 2-thiothymine and 2-mercaptocytosine, 5-halouracil and 5-halocytosine, 5-propynyl(-C≡C-CH3)uracil and 5-propynylcytosine, and other pyrimidine bases. This includes rukinyl derivatives, 6-azouracil, 6-azocytosine and 6-azothimine, 5-uracil (pseudouracil), N1-methylpseudouracil, 4-thiouracil, 8-halogens, 8-amino groups, 8-thiols, 8-thioalkyl groups, 8-hydroxyl groups and other 8-substituted adenines and guanines, 5-halogens, especially 5-bromo, 5-trifluoromethyl groups and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, 3-deazaguanine and 3-deazaadenine, and the like. Further modified nucleic acid bases include tricyclic pyrimidines, such as phenoxazine cytosine nucleoside ([5,4-b][1,4]benzoxazine-2(3H)-one), phenothiadyl cytosine nucleoside (1H-pyrimido[5,4-b][1,4]benzothiadin-2(3H)-one), G-clamp-substituted phenoxazine cytosine nucleoside (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazine-2(3H)-one), carbazole cytosine nucleoside (2H-pyrimido[4,5-b]indole-2-one), and pyridoindole cytosine nucleoside (H-pyridine[3',2':4,5]pyrrolo[2,3-d]pyrimidine-2-one). The modified nucleic acid bases may further include those in which the purine or pyrimidine base is substituted with another heterocycle, such as 7-deazaadenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone.Representative examples of teaching the production of modified nucleic acid bases and other modified nucleic acid bases include WO2022007986 disclosed in the international application, and U.S. published patents 3,687,808, 4,845,205, 5,130,302, 5,134,066, 5,175,273, 5,367,066, 5,432,272, 5,457,187, 5,459,255, and 5,484. This includes, but is not limited to, those disclosed in 908, 5,502,177, 5,525,711, 5,552,540, 5,587,469, 5,594,121, 5,596,091, 5,614,617, 5,645,985, 5,681,941, 5,750,692, 5,763,588, 5,830,653 and 6,005,096. As used herein, “universal base” refers to a heterocyclic moiety having the same properties as a native base, located at the 1' position of a nucleotide sugar moiety in a modified nucleotide or at an equivalent position in a nucleotide sugar moiety substitution, i.e., when present in a nucleic acid double helix, it can be positioned opposite two or more bases without altering the double helix structure (e.g., the structure of a phosphate ester backbone). Some universal bases can base pair with all of the following bases: guanine (G), cytosine (C), adenine (A), thymine (T), and uracil (U), by forming hydrogen bonds under conditions that allow for base pairing. In a double helix, a universal base can form one or more hydrogen bonds with each of the opposing G, C, A, T, and U on the opposing strand of the double helix. In a double helix, base pairing between universal bases occurs, but the double helix structure of the phosphate ester backbone remains unchanged. Universal bases can also interact with bases in adjacent nucleotides on the same nucleic acid chain through stacking interactions. Such stacking interactions stabilize the double helix, especially when the universal base does not form hydrogen bonds with the base opposite it on the opposing strand of the double helix. In some cases, 2,4-difluorophenyl is also a common substitute for heterocyclic bases.Limited examples of common binding nucleotides include inosine, 1-OD-ribofuranosyl-5-nitroindole, and / or 1-β-D-ribofuranosyl-3-nitropyrrole (U.S. Patent Application No. 20070254362 by Quay et al., An acyclic 5-nitroindazole nucleoside analogue as ambiguous nucleoside, NUCLEIC ACIDS RES. November 11, 1995, 23(21)4363-70; Loakes et al., 3-Nitropyrrole and 5-nitroindole as universal bases in primers for DNA sequencing and PCR, NUCLEIC ACIDS RES. July 11, 1995, 23(13)2361-2366).

[0285] Some of these are owned together with the present application, and each of them, in whole, is incorporated herein by reference, but some non-exclusive structural examples are shown below.

[0286] [ka] That is the case.

[0287] As used herein, “modified nucleoside” means a nucleoside containing at least one modified nucleoside compared to a naturally occurring RNA or DNA nucleoside. Such modifications may be located at the sugar moiety and / or at the nucleic acid base. Modified nucleotides include those that do not contain a nucleic acid base (base-free).

[0288] As used herein, "2'-F" refers to a nucleoside containing a sugar further containing a fluorine group at the 2' position. As used herein, "2'-OMe" means that a sugar-containing nucleoside contains an -OCH3 group at the 2' position of the sugar ring. As used herein, "2'-MOE," "2'-OCH2CH2OCH3," or "2'-O-methoxyethyl group" means that a sugar-containing nucleoside further contains an -OCH2CH2OCH3 group at the 2' position of the sugar or sugar ring.

[0289] As used herein, the terms “5'-terminal nucleotide or 3'-terminal nucleotide” refer to an oligonucleotide or a nucleotide or nucleotide derivative located at the 5'-terminus or 3'-terminus of an oligonucleotide.

[0290] As used herein, “oligonucleotide” refers to a polymeric form of nucleotides consisting of 2 to 2500 nucleotides. Oligonucleotides may be single-stranded or double-stranded, and typically, for example, some of the oligonucleotides are used in gene therapy, and for example, some of the oligonucleotides are nucleic acid inhibitor molecules. In some embodiments, one or more of these many nucleotides are modified. In the context of the present invention, the term “oligonucleotide” refers to a polymer having at least one region that can hybridize with a nucleic acid molecule. Typically, an oligonucleotide comprises a main chain of bonded monomer subunits, and the bonding of the bonded monomer subunits, sugar moieties or substitutions and heterocyclic base moieties may be independently modified. The bonded sugar units may or may not contain heterocyclic bases and can be replaced with mimics such as peptide nucleic acid monomers. The ability to modify or substitute some or all of the monomers in each monomer of an oligonucleotide can result in a multitude of possible motifs. The term “oligonucleotide” includes oligonucleotide analogs and mixed polymers containing oligonucleotides and nucleotide mimics and / or nucleic acid and non-nucleic acid components. The term "oligonucleotide" further includes polymers comprising bonded monomer subunits, of which the monomer subunits include non-nucleic acid components such as nucleosides, modified nucleosides, nucleoside analogs, nucleoside mimetic compounds, and coupling groups. In some embodiments, mixtures of monomer subunits (exemplary, but not limited to these) provide oligonucleotides having enhanced properties for applications such as therapeutic and diagnostic use.

[0291] Oligonucleotides are typically manufactured in a conventional linear manner, cyclically by linking or other means, and may include branching. Oligonucleotides may form double-stranded constructs, for example, by hybridizing to form double strands of a double-stranded composition. The double-stranded composition may be linked or separated and may include overhangs at its ends.

[0292] As used herein, “expression” refers to the process by which a gene ultimately produces a protein. Expression includes, but is not limited to, transcription, splicing, post-transcriptional modification, and translation.

[0293] As used herein, “antisense oligonucleotide” refers to an antisense compound that is an oligonucleotide.

[0294] As used herein, “target nucleic acid” refers to any nucleic acid molecule whose expression level or activity can be regulated by an antisense compound. In some embodiments, the target nucleic acid is DNA or RNA. In some embodiments, the target RNA is mRNA, pre-mRNA, non-coding RNA, pri-microRNA, pre-microRNA, mature microRNA, promoter-regulating RNA, or native antisense transcript.

[0295] As used herein, “target mRNA” refers to a pre-selected RNA molecule that encodes a protein.

[0296] As used herein, “hybridization” refers to the pairing of complementary oligonucleotides (e.g., an antisense compound with its target nucleic acid). While not limited to specific mechanisms, the most common pairing mechanisms relate to hydrogen bonding between complementary nucleosides or nucleotide bases (nucleoside bases), which may be Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen bonds. For example, the natural nucleic acid base adenine is complementary to the natural nucleic acid bases thymidine and uracil, and they pair by forming hydrogen bonds. The natural nucleic acid base guanine is complementary to the natural nucleic acid bases cytosine and 5-methylcytosine.

[0297] In one embodiment, the oligonucleotide provided is a single-stranded oligonucleotide. In one embodiment, the single-stranded oligonucleotide is a conventional antisense oligonucleotide (also called an ASO), ribozyme, or aptamer. In one embodiment, the oligonucleotide provided is a double-stranded ribonucleic acid (dsRNA) reagent, such a compound being a double-stranded nucleoside nucleic acid well known in the art, of which one or two strands are the oligonucleotide disclosed herein.

[0298] As used herein, “antisense compound” and “antisense strand” refer to oligonucleotides whose at least portion is at least partially complementary to the target nucleic acid with which they hybridize. In some embodiments, an antisense compound modulates (increases or decreases) the expression or quantity of the target nucleic acid. In some embodiments, an antisense compound results in different splicing variants by altering the splicing of the target pre-mRNA. In some embodiments, an antisense compound modulates the expression of one or more different proteins. Antisense mechanisms envisioned herein include, but are not limited to, RNA enzyme H mechanisms, RNA mechanisms, splicing regulation, translation inhibition, alteration of RNA processing, suppression of microRNA function, or simulated microRNA function.

[0299] As used herein, the term "siRNA" refers to short interfering RNA or silencing RNA. siRNA is a class of double-stranded siRNA RNA molecules, which may be 20-25 (or even shorter) base pairs in length, similar to microRNAs (miRNAs) that function in the RNA interference (RNAi) pathway. siRNA interferes with the expression of specific genes having a nucleotide sequence complementary to the siRNA by degrading mRNA after transcription, thereby preventing translation. siRNA silences gene expression in cells by inducing the cleavage of messenger RNA (mRNA) by the RNA-induced silencing complex (RISC). As used herein, the term "single-stranded oligonucleotide" refers to a single-stranded oligomeric compound that has a sequence at least partially complementary to the target mRNA and can hybridize with the target mRNA by hydrogen bonding under mammalian physiological conditions (or similar in vitro conditions). In some embodiments, the single-stranded oligonucleotide is a single-stranded antisense oligonucleotide.

[0300] As used herein, unless otherwise indicated or modified, the term “double-stranded” refers to two individual oligonucleotides that hybridize with one another. Such double-stranded compounds may have one or more non-hybridized nucleosides (suspended) and / or one or more internal non-hybridized nucleosides (mispaired) at one or both ends of one or two strands, provided that sufficient complementarity exists to maintain hybridization under physiologically relevant conditions.

[0301] As used herein, the terms “silencing,” “reduction,” “suppression,” “downregulation,” or “knockdown” refer to a reduction in gene expression, as determined by the level of RNA transcribed from the gene or the level of polypeptides, proteins, or protein subunits translated from mRNA within the cells, cell aggregates, tissues, organs, or subjects from which the transcription of the gene occurred, when cells, cell aggregates, tissues, organs, or subjects are treated with an oligomeric compound such as an RNAi agent as described herein.

[0302] As used herein, the terms “self-complementary” or “hairpin” refer to a single oligonucleotide containing a double-stranded region formed by the self-hybridization of the oligonucleotide.

[0303] As used herein, the term "single-stranded" refers to oligonucleotides that do not hybridize with their complementary sequence and do not possess sufficient self-complementarity to form a hairpin structure under physiologically relevant conditions. Single-stranded compounds can bind to their complementary sequence to form double-stranded or partially double-stranded compounds.

[0304] As used herein, “target mRNA” or “target nucleic acid” refers to any nucleic acid molecule whose expression, quantity, or activity can be regulated by an antisense compound. In some embodiments, the target nucleic acid is DNA or RNA. In some embodiments, the target RNA is mRNA pre-mRNA, non-coding RNA, pri-microRNA, pre-microRNA, mature microRNA, promoter-regulating RNA, or a native antisense transcript. For example, the target nucleic acid may be a nucleic acid molecule expressing a cellular gene (or mRNA transcribed from a gene) associated with a particular disease or disease state, or a nucleic acid molecule derived from an infectious agent. In some embodiments, the target nucleic acid is viral nucleic acid or bacterial nucleic acid.

[0305] As used herein, “nucleic acid base complementarity,” “complementary,” or “complementary” refers to a nucleic acid base that can base-pair with another nucleic acid base, in the case of nucleic acid bases. For example, in DNA, adenine (A) is complementary to thymine (T). For example, in RNA, adenine (A) is complementary to uracil (U). In some embodiments, a complementary nucleic acid base refers to a nucleic acid base of an antisense compound that can base-pair with a nucleic acid base of its target nucleic acid. As used herein, “non-complementary” refers to a pair of nucleic acid bases that do not form hydrogen bonds with each other, or in other words, do not support hybridization, in the case of nucleic acid bases. In the case of a bound nucleoside oligonucleotide or nucleic acid, it refers to the ability of an oligonucleotide to hybridize with another oligonucleotide or nucleic acid by nucleic acid base complementarity. For example, when describing the relationship between the first nucleotide sequence of a dsRNA reagent (e.g., sense strand or target gene mRNA) and the second nucleotide sequence (e.g., dsRNA reagent antisense strand or single-stranded antisense polynucleotide), this refers to the ability of an oligonucleotide or polynucleotide containing the first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide containing the second nucleotide sequence [forming an interbase-pair hydrogen bond under mammalian physiological conditions (or similar in vitro conditions)] and to form a double helix or double helix structure under certain conditions. Other conditions, such as physiologically relevant conditions that may be encountered in vivo, can also be applied. Those skilled in the art can determine the optimal set of conditions for testing the two sequence complementarity from the final application of the hybridizing nucleotides. The complementary sequence includes Watson-Crick base pairs or non-Watson-Crick base pairs and includes natural or modified nucleotides or nucleotide mimeographs to the extent necessary for the above hybridization. Sequence identity or complementarity is independent of modification.

[0306] In some embodiments, at least a portion of the nucleic acid base sequence of the oligonucleotide is partially or completely complementary to the target nucleic acid. In some embodiments, it is 100% complementary to the target nucleic acid. In some embodiments, it is 90% complementary to the target nucleic acid. In some embodiments, it is 80% complementary to the target nucleic acid. In some embodiments, it is 90% complementary to the target nucleic acid. In some embodiments, it is 70% complementary to the target nucleic acid. In some embodiments, it is 90% complementary to the target nucleic acid. In some embodiments, it is 60% complementary to the target nucleic acid. For example, complementary sequences within a target gene dsRNA described herein include base pairings of one or two nucleotide sequences over the full length of an oligonucleotide or polynucleotide containing a first nucleotide sequence and an oligonucleotide or polynucleotide containing a second nucleotide sequence. Such sequences may be referred to herein as “completely complementary” to each other. In embodiments in which two oligonucleotides are designed to form one or more single-stranded overhangs during hybridization, it should be understood that such overhangs are not considered mispairs as determined herein based on complementarity. For example, a target gene dsRNA reagent includes an oligonucleotide having a length of 19 nucleotides and another oligonucleotide having a length of 20 nucleotides, of which the relatively longer oligonucleotide contains a sequence of 19 nucleotides that is completely complementary to the relatively shorter oligonucleotide, and for the purposes described herein, this can be referred to as "completely complementary." Thus, as used herein, "completely complementary" means that all (100%) of the bases in the contiguous sequence of the first polynucleotide hybridize with the same number of bases in the contiguous sequence of the second polynucleotide. The contiguous sequence may include all or part of the first or second nucleotide sequence.As used herein, the term “basically complementary” means that in a hybridization pair of nucleic acid sequences, at least about 85% (but not all) of the bases in the sequence of the first polynucleotide hybridize with the same number of bases in the sequence of the second polynucleotide. If the two sequences contain one or more mispaired base pairs during hybridization, e.g., at least 1, 2, 3, 4, or 5 mispaired base pairs, the term “basically complementary” can be used to mean that the first sequence forms a double helix of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs (bp) compared to the second sequence, while simultaneously retaining the ability to hybridize under the conditions most relevant to its ultimate application, such as the repression of target gene expression by the RISC pathway. The term “partially complementary” can be used herein to mean that, in a hybridization pair of nucleic acid base sequences, at least 75% (but not all) of the bases in the contiguous sequence of the first polynucleotide hybridize with the same number of bases in the contiguous sequence of the second polynucleotide. In some embodiments, “partially complementary” means that at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the bases in the contiguous sequence of the second polynucleotide hybridize with the same number of bases in the contiguous sequence of the second polynucleotide. The terms “complementary,” “fully complementary,” “basically complementary,” and “partially complementary,” as used herein, can be used to refer to base matching between the sense and antisense strands of a dsRNA reagent, base matching between the antisense strand of a dsRNA reagent and a target mRNA sequence, or base matching between a single-stranded antisense oligonucleotide and a target mRNA sequence. It should be understood that the term “antisense strand of a dsRNA reagent” can refer to the same sequence as “antisense polynucleotide reagent.”

[0307] As used herein, “mispairing” is acceptable to those skilled in the art for the effectiveness of dsRNA, particularly when the mispairing is located within the terminal region of the dsRNA. Some mispairs exhibit better resistance; for example, mispairs having fluctuating base pairs G:U and A:C exhibit better resistance (Du et el., A systematic analysis of the silencing effects of an active siRNA at all single-nucleotide mismatched target sites. Nucleic Acids Res. 2005 Mar 21;33(5):1671-7. Doi: 10.1093 / nar / gki312. Nucleic Acids Res. 2005;33(11):3698).

[0308] As used herein, “motif” and “sequence” refer to a sequence or order of nucleic acid bases or nucleotides, as expressed alphabetically using standard nucleotide nomenclature.

[0309] In one embodiment, the present invention provides oligonucleotides comprising various arbitrary length ranges. In one embodiment, in an oligonucleotide or double-stranded ribonucleic acid (dsRNA), any one of the single-stranded, sense, and / or antisense strands each comprises 8 to 40 nucleotides in length. In another embodiment, in an oligonucleotide or double-stranded ribonucleic acid (dsRNA), any one of the single-stranded, sense, and / or antisense strands each comprises 15 to 30 nucleotides in length. For example, in one embodiment, the nucleotide length of the oligonucleotide is selected from 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40. For example, in one embodiment, the present invention has a nucleotide length range of 8-9, 8-10, 8-11, 8-12, 8-13, 8-14, 8-15, 8-16, 8-17, 8-18, 8-19, 8-20, 8-21, 8-22, 8-23, 8-24, 8-25, 8-26, 8-27, 8-28, 8-29, 8-30, 9-10, 9-11, 9- 12, 9-13, 9-14, 9-15, 9-16, 9-17, 9-18, 9-19, 9-20, 9-21, 9-22, 9-23, 9-24, 9-25, 9-26, 9-27, 9-28, 9-29, 9-30, 10-11, 10-12, 10-13, 10-14, 10-15, 10-16, 10-17, 10-18, 10-19, 10-20, 10-2 1, 10-22, 10-23, 10-24, 10-25, 10-26, 10-27, 10-28, 10-29, 10-30, 11-12, 11-13, 11-14, 11-15, 11-16, 11-17, 11-18, 11-19, 11-20, 11-21, 11-22, 11-23, 11-24, 11-25, 11-26, 11-27, 11-28 , 11-29, 11-30, 12-13, 12-14, 12-15, 12-16, 12-17, 12-18, 12-19, 12-20, 12-21, 12-22, 12-23, 12-24, 12-25, 12-26, 12-27, 12-28, 12-29, 12-30, 13-14, 13-15, 13-16, 13-17, 13-18, 13-19,13-20, 13-21, 13-22, 13-23, 13-24, 13-25, 13-26, 13-27, 13-28, 13-29, 13-30, 14-15, 14-16, 14-17, 14-18, 14-19, 14-20, 14-21, 14-22, 14-23, 14-24, 14-25, 14-26, 14-27, 14-28, 14-29, 14-30, 15-16, 15-17, 15-18, 15-19, 15-20, 15-21, 15-22, 15-23, 15-24, 15-25, 15-26, 15 ~27, 15~28, 15~29, 15~30, 16~17, 16~18, 16~19, 16~20, 16~21, 16~22, 16~23, 16~24, 16~25, 16~26, 16~27, 16~28, 16~29, 16~30, 17~18, 17~19, 17~20, 17~21, 17~22, 17~23, 17~24, 17~25, 17~26, 17~27, 17~28, 17~29, 17~30, 18~19, 18~20, 18~21, 18~22, 18~23, 18~24, 18~25, 18~2 6, 18-27, 18-28, 18-29, 18-30, 19-20, 19-21, 19-22, 19-23, 19-24, 19-25, 19-26, 19-27, 19-28, 19-29, 19-30, 20-21, 20-22, 20-23, 20-24, 20-25, 20-26, 20-27, 20-28, 20-29, 20-30, 21-22, 21-23, 21-24, 21-25, 21-26, 21-27, 21-28, 21-29, 21-30, 22-23, 22-24, 22-25, 22-26, The present invention provides oligonucleotides comprising oligonucleotides consisting of 22-27, 22-28, 22-29, 22-30, 23-24, 23-25, 23-26, 23-27, 23-28, 23-29, 23-30, 24-25, 24-26, 24-27, 24-28, 24-29, 24-30, 25-26, 25-27, 25-28, 25-29, 25-30, 26-27, 26-28, 26-29, 26-30, 27-28, 27-29, 27-30, 28-29, 28-30, or 9-30 nucleosides. In embodiments in which the number of nucleosides in an oligonucleotide is limited, whether to a range or to a specific numerical value,Oligonucleotides or oligonucleotides may contain further other substituents. For example, an oligonucleotide containing 8 to 30 nucleosides does not include an oligonucleotide having 31 nucleosides, but unless otherwise indicated, such oligonucleotides may further contain, for example, one or more conjugates, terminal groups, or other substituents, and the monomers herein are generally considered to be terminal groups further contained in the oligonucleotide. In some embodiments, the terminal group includes, but is not limited to, a terminal group nucleoside. In such embodiments, the terminal group is modified to be different from the terminal nucleoside of the oligonucleotide, thereby distinguishing such a terminal group from the nucleoside of the oligonucleotide.

[0310] In some embodiments, at least one or each of the target groups is selected from ligands capable of binding to mammalian hepatocyte surface receptors. In some embodiments, each of the target groups is independently a ligand having affinity for asialoglycoprotein receptors (ASGPRs) on the surface of mammalian hepatocytes. In some embodiments, each of the target groups is independently an asialoglycoprotein or sugar-containing ligand. In some embodiments, each of the target groups is independently an asialoglycoprotein-containing ligand, such as asialoorosomucoid (ASOR) or asialofetuin (ASF).In some embodiments, each of the above target groups is independently D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, α-D-mannofuranose, β-D-mannofuranose, α-D-mannopyranose, β-D-mannopyranose, α-D-glucopyranose, β-D-glucopyranose, α-D-glu Cofuranose, β-D-glucofuranose, α-D-fructofuranose, α-D-fructopyranose, α-D-galactopyranose, β-D-galactopyranose, α-D-galactofuranose, β-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, Nn-butyrylgalactosamine, N-isobutylylgal Lactoseamine, 2-amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-carboxamide-2,3-di-O-methyl-D-mannopyranoose, 2-deoxy-2-sulfamino-D-glucopyranose, N-ethanolacyl-α-neuraminic acid, 5-thio-β-D-glucopyranose, 2,3,4- The ligand contains one selected from tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside methyl ester, 4-thio-β-D-galactopyranose, 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucopyranoside ethyl ester, 2,5-anhydro-D-alonitrile, ribose, D-ribose, D-4-thioribose, L-ribose, and L-4-thioribose.

[0311] In some embodiments, at least one or each of the target groups is a ligand comprising galactose or N-acetylgalactosamine.

[0312] In some embodiments, the target group comprises one or more "GalNAc" (N-acetylgalactosamine) derivatives bound via appropriate tethers by divalent or trivalent branched joints.

[0313] [ka] Eventually, q 2A , q 2B , q 3A , q 3B , q 4A , q 4B , q 5A , q 5B , q 5C , q 6A , q 6B and q 6C Each instance of a character independently represents a number between 0 and 20, and the overlapping units within that number may be homologous or distinct. P 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , P 5C , P 6A , P 6B , P 6C , T 2A , T 2B , T 3A , T 3B , T 4A , T 4B , T 4A , T 5B , T 5C , T 6A , T 6B , T 6C Each instance independently represents non-existence, CO, NH, O, S, OC(=O), NHC(=O), CH2, CH2NH, or CH2O. Q 2A Q 2B Q 3A Q 3B Q 4A Q 4B Q 5A Q5B Q 5C Q 6A Q 6B Q 6C Each instance of represents independently of absence, alkylene group, or substituted alkylene group, of which one or more methylene groups may be interrupted or terminated by one or more groups of O, S, S(=O), SO2, NH, C(R')=C(R''), C≡C, or C(=O). R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , R 5C , R 6A , R 6B , R 6C Each time it appears, it is independently non-existent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R a )C(O), -C(O)-CH(R a )-NH-, CO, CH=NO or heterocyclyl group, L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B , L 5C , L 6A , L 6B and L 6C Each instance of this symbol independently represents a monosaccharide (e.g., GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide.

[0314] In some non-limiting examples, the oligonucleotides described herein are selected from one of the following compound fragments as the target group.

[0315] [Table 5]

[0316] In some non-limiting examples, the oligonucleotides described herein include a 5'-terminal compound (or terminal nucleotide) in the antisense strand of a double-stranded RNA (dsRNA) represented by formula (IX-1) or (IX-2).

[0317] [ka] Rb represents the remaining portion of double-stranded RNA (dsRNA), and Rb is as described above.

[0318] When a 5'-phosphonate-modified nucleoside analog is the form of the phosphoramidite compound described herein, it can be used to ligate the 5'-phosphonate-modified nucleoside analog using nucleotide phosphoramidite synthesis methods known in the art. The 5'-phosphonate-modified nucleoside analog can be used to synthesize a phosphoramidite compound by linking the phosphorus atom of a phosphoramidite-forming agent through a coupling (e.g., phosphorylation) reaction, thereby forming the phosphoramidite compound. In some embodiments, the 5'-phosphonate-modified nucleoside analog-phosphoramidite compound is used to ligate the 5'-phosphonate-modified nucleoside analog to the 5' end of the antisense strand of double-stranded RNA.

[0319] The oligonucleotides described herein contain one or more chiral centers, resulting in enantiomers, diastereomers, and other stereoisomer configurations. In terms of absolute stereochemistry, these configurations can be defined as (R) or (S), α or β, with respect to sugar anomeric groups, or as (D) or (L) with respect to amino acids, etc. The oligonucleotides provided herein include all such possible isomers, as well as their racemic and optional pure forms.

[0320] In one embodiment, a method is provided for suppressing gene expression, comprising contacting cells with a reagent having an oligonucleotide or double-stranded ribonucleic acid (dsRNA) provided above, wherein the sense strand and antisense strand of the oligonucleotide or double-stranded ribonucleic acid (dsRNA) reagent each contain 8 to 40 nucleotides in length, and the antisense strand of the oligonucleotide or double-stranded ribonucleic acid (dsRNA) reagent is complementary to the target RNA. In one embodiment, the cells are in an animal. In one embodiment, the cells are in a human. In one embodiment, the target RNA is selected from mRNA, pre-mRNA, and microRNA. In one embodiment, the target RNA is mRNA. In one embodiment, the target RNA is human mRNA. In one embodiment, the target RNA is cleaved to suppress its function. In one embodiment, the method further comprises detecting the level of the target RNA. In one embodiment, a method for suppressing gene expression is provided, which comprises contacting one or more types of cells or tissues with an oligonucleotide or double-stranded ribonucleic acid (dsRNA) containing a 5'-terminal nucleotide represented by formula (V), formula (V-1), formula (V-2), formula (VI), formula (VI-1), formula (VI-2), formula (VI-3), formula (VII), formula (VII-1), formula (VII-2), formula (VIII), formula (VIII-1) or a stereoisomer thereof.

[0321] Therapeutic formulations of dsRNA reagents or targeted gene antisense polynucleotide reagents can be prepared for storage by mixing molecules or compounds of desired purity with optionally pharmaceutically acceptable carriers, excipients, or stabilizers [Remington's Pharmaceutical Sciences 21st edition, (2006)] in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to the recipient at the dosage and concentration used and include buffers such as phosphates, citrates, and other organic acids, antioxidants including ascorbic acid and methionine, preservatives (e.g., benzyldimethylstearylammonium chloride hydrate, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol, or benzyl alcohol, p-hydroxybenzoate esters such as methyl or propyl p-hydroxybenzoate, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), and low molecular weight (less than approximately 10 residues) polynucleotides. The product contains lipeptides, proteins such as serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose, or sorbitol, salt-forming counterions such as sodium, metal complexes (e.g., zinc-protein complexes), and / or nonionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0322] ● Administration method In some embodiments, the tissue to which the compound is administered is a tissue in which a disease or condition related to the target gene is present or likely to appear, non-limiting examples of which are the liver or kidney. Direct tissue administration can be achieved by direct injection or by other means. Many orally delivered compounds enter and pass through the liver and kidneys spontaneously, and some embodiments of the therapeutic methods of the present invention involve orally administering one or more target gene dsRNA reagents to a target. The dsRNA reagents or target gene antisense polynucleotides can be administered alone or in combination with other therapeutic agents, in a single dose, or they can be administered multiple times. In the case of multiple doses, the target gene dsRNA reagents or target gene antisense polynucleotides can be administered via different routes. For example, though not intended to be limiting, the first (or first few) doses may be administered subcutaneously, and one or more additional doses may be administered orally and / or systemically.

[0323] In embodiments of the present invention where systemic administration of a target gene dsRNA reagent or a target gene antisense polynucleotide reagent is desired, the target gene dsRNA reagent or the target gene antisense polynucleotide reagent can be prepared for parenteral administration by injection, such as bolus injection or continuous infusion. The injectable formulation can be in unit dosage forms such as ampoules or multi-dose containers with or without preservatives. The target gene dsRNA reagent formulation (also called a pharmaceutical composition) can take the form of a suspension, solution or emulsion in an oily or aqueous carrier and may contain preparing agents such as suspending agents, stabilizers and / or dispersants.

[0324] Parenteral formulations include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcohol / aqueous solutions, emulsions, or suspensions containing saline and a buffer medium. Parenteral carriers include sodium chloride solution, ringer's dextrose solution, glucose and sodium chloride solution, lactated ringer's solution, or fixative oils. Intravenous excipients include liquids and nutritional supplements, electrolyte supplements (e.g., those based on ringer's dextrose solution), etc. Preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, and inert gases, may also be present. Other forms of administration, such as intravenous administration, result in relatively low doses. If the response to the initial dose is insufficient, higher doses can be used within the patient's tolerance (or the dose can be effectively increased by a different, more localized delivery route). If necessary, the drug can be administered multiple times a day to achieve appropriate systemic or local levels of one or more target gene dsRNA reagents or target gene antisense polynucleotide reagents, thereby achieving an appropriate reduction in the activity of the target gene.

[0325] In other embodiments, the method of the present invention involves the use of a delivery carrier, such as a biocompatible microparticle, nanoparticle, or implant suitable for implantation of a target recipient. PCT Publication WO95 / 24929 (incorporated herein by reference) describes an exemplary biodegradable implant that can be used based on the method, which describes a biocompatible biodegradable polymer matrix containing a biopolymer.

[0326] Both non-biodegradable and biodegradable polymer matrices can be used in the methods of the present invention to deliver one or more target gene dsRNA reagents or target gene antisense polynucleotide reagents to a target. In some embodiments, the matrix may be biodegradable. The matrix polymer may be natural or synthetic. Based on the desired release period, polymers can be selected typically on the order of several hours to one year or more. Releases over periods of several hours to 3 to 12 months are typically available. The polymer is optionally in the form of a hydrogel capable of absorbing up to about 90% of its weight in water, and optionally crosslinked with polyvalent ions or other polymers.

[0327] Typically, target gene dsRNA reagents or target gene antisense polynucleotide reagents can be delivered in some embodiments of the present invention by diffusion or degradation of a polymer matrix using biodegradable implants. Exemplary synthetic polymers for such use are known in the art. Using methods known in the art, biodegradable and non-biodegradable polymers can be used to deliver target gene dsRNA reagents or target gene antisense polynucleotide reagents. Bioadhesive polymers such as bioerosive hydrogels (HSSawhney, CPPathak and JAHubell in Macromolecules, 1993, 26, 581-587) can also be used to deliver target gene dsRNA reagents or target gene antisense polynucleotide reagents to treat diseases or conditions related to the target gene. Other suitable delivery systems may include timed release, delayed release, or gradual delivery systems. Such systems can avoid repeated administration of target gene dsRNA reagents or target gene antisense polynucleotide agents, thereby increasing convenience for both the subject and medical professionals. Many types of release delivery systems are available and known to those skilled in the art. See, for example, U.S. Patents Nos. 5,075,109, 4,452,775, 4,675,189, 5,736,152, 3,854,480, 5,133,974 and 5,407,686. Furthermore, a pump-based hardware delivery system, some of which can also be applied to implantation, can be used.

[0328] The use of long-term sustained-release implants is applicable to the prophylactic treatment of subjects and to subjects at risk of developing diseases or conditions associated with recurrent target genes. As used herein, long-term release refers to constructing and positioning an implant to deliver therapeutic levels of target gene dsRNA reagents or target gene antisense polynucleotide reagents over periods of at least 10, 20, 30, 60, 90 days, 6 months, 1 year, or longer. Long-term sustained-release implants are known to those skilled in the art and include some of the release systems described above.

[0329] ● Effective amount In some embodiments, the methods of the present invention involve contacting cells with an effective amount of a dsRNA reagent or antisense polynucleotide reagent to reduce gene expression in the contacted cells. One embodiment of the methods of the present invention involves administering the dsRNA reagent or antisense polynucleotide agent to a subject in an amount that effectively reduces gene expression and treats the relevant disease or condition of the subject. For reduction of expression and / or treatment of the relevant disease or condition, the “effective amount” used is the amount necessary or sufficient to achieve the desired biological effect. For example, an effective amount of a dsRNA reagent or antisense polynucleotide reagent to treat a relevant disease or condition may be (i) the amount necessary to slow or stop the progression of the disease or condition, or (ii) the amount to reverse, reduce or eliminate one or more symptoms of the disease or condition. In some embodiments of the present invention, the effective amount is the amount of a dsRNA reagent or antisense polynucleotide agent that, when administered to a subject in need of treatment for the relevant disease or condition, results in a therapeutic response that prevents and / or treats the disease or condition. According to several embodiments of the present invention, the effective amount is the amount of the dsRNA reagent or antisense polynucleotide reagent of the present invention that, when combined with or used in combination with another therapeutic treatment for the associated disease or condition, results in a therapeutic response that prevents and / or treats the said disease or condition. In several embodiments of the present invention, the biological effect of treating a subject with the dsRNA reagent or antisense polynucleotide reagent of the present invention may be improvement and / or complete elimination of symptoms caused by the associated disease or condition. In several embodiments of the present invention, the biological effect is the complete elimination of the associated disease or condition, as demonstrated, for example, by a diagnostic test showing that the subject does not have the associated disease or condition. In several embodiments, the effective amount is the amount that results in a desired response, such as, for example, an amount that reduces the associated disease or condition in cells, tissues and / or subjects having the disease or condition.Some embodiments of the present invention include a method for determining the effectiveness of a target gene dsRNA reagent or antisense polynucleotide reagent of the present invention administered to a subject for treating a disease or condition related to a target gene, by evaluating and / or monitoring one or more "physiological features" of the disease or condition related to the target gene in the subject. Non-limiting examples of physiological features of a disease or condition related to a target gene include many patients. It should be recognized that gene silencing can be performed constitutively or by genome engineering in any cell of the expressed target gene and can be determined by any appropriate measurement. In some embodiments of the present invention, administration of the dsRNA reagent of the present invention reduces the expression of the target gene by at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments of the present invention, administration of the dsRNA reagent of the present invention reduces the expression of the target gene by 5% to 10%, 5% to 25%, 10% to 50%, 10% to 75%, 25% to 75%, 25% to 100%, or 50% to 100%.

[0330] The dsRNA reagent and antisense polynucleotide reagent in the present invention are delivered in a pharmaceutical composition in a dose sufficient for the expression of the target gene. In one embodiment of the present invention, the dose of the dsRNA reagent or antisense polynucleotide agent is 0.01 to 200.0 milligrams per kilogram of body weight per day of the recipient, and generally includes both extreme values, such as 1 to 50 mg / kg body weight, 5 to 40 mg / kg body weight, 10 to 30 mg / kg body weight, 1 to 20 mg / kg body weight, 1 to 10 mg / kg body weight, and 4 to 15 mg / kg body weight per day.

[0331] Various factors can be considered when determining the delivery dose and timing of the dsRNA reagent of the present invention. The absolute amount of the dsRNA reagent or antisense polynucleotide agent delivered depends on various factors, including concurrent treatment, dosage, and parameters of the individual subject, including age, physical condition, body size, and weight. These are factors known to those skilled in the art and can be resolved by conventional experiments. In some embodiments, the maximum dose, i.e., the safest dose based on reasonable medical judgment, can be used.

[0332] In some embodiments, the method of the present invention may include administering to a subject dose of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses of a reagent or antisense polynucleotide reagent. In some cases, doses of the pharmaceutical compound may be administered to a subject at least daily, every other day, weekly, bi-weekly, monthly, etc., and may be administered once a day or more times a day, for example, 2, 3, 4, 5 or more times in one 24-hour cycle. The pharmaceutical composition of the present invention may be administered once a day, or the dsRNA reagent or antisense polynucleotide reagent may be administered in two, three or more subdoses at appropriate intervals per day, or may be delivered using continuous infusion or through a sustained-release formulation. In some embodiments of the method of the present invention, the pharmaceutical composition of the present invention is administered to a subject once a day or more, once a week or more, once a month or more, or once a year or more.

[0333] One embodiment of the present invention involves the use of a pharmaceutical composition containing a dsRNA reagent or antisense polynucleotide reagent and a pharmaceutically acceptable carrier. A pharmaceutical composition containing a dsRNA reagent or antisense polynucleotide agent can be used in the method of the present invention to reduce gene expression and activity in cells and can be used to treat related diseases or conditions. Such pharmaceutical compositions can be prepared based on a delivery method. Non-limiting examples of formulations for delivery methods include compositions prepared for subcutaneous delivery, compositions prepared for systemic administration by parenteral delivery, compositions prepared for intravenous (IV) delivery, compositions prepared for intrathecal delivery, and compositions prepared for direct delivery into the brain. The compositions of the present invention can be administered by one or more methods to deliver the dsRNA reagent or antisense polynucleotide reagent to cells, including, for example, surface (e.g., by a transdermal patch), lung, by inhalation or blowing of a powder or aerosol, by a sprayer, intra-airway, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion, and intracranial, intrathecal, or intraventricular administration via implantation devices. dsRNA reagents or antisense polynucleotide agents can also be delivered directly to target tissues, such as directly to the liver or directly to the kidneys. It should be understood that "delivering" a "dsRNA reagent" or "antisense polynucleotide agent" to cells includes, respectively, delivering the dsRNA reagent or antisense polynucleotide agent, directly expressing the dsRNA reagent in cells, expressing the dsRNA reagent from a coding vector delivered intracellularly, or any appropriate method of making the dsRNA reagent or antisense polynucleotide agent appear in cells. The manufacture and use of the formulations and the means for delivering the suppressor RNA are known and conventionally used in this art.

[0334] In some embodiments, the composition further comprises one or more other therapeutic agents. The composition of the present invention may optionally comprise one or more dsRNA reagents and one or more pharmaceutically acceptable carriers, delivery agents, targeting agents, detectable labels, etc., and non-limiting examples of targeting agents available by some embodiments of the methods of the present invention are reagents for introducing and / or entering the dsRNA reagents of the present invention into the cells to be treated. The selection of targeting agents depends on factors such as the nature of the disease or condition in question and the type of target cells. In non-limiting examples, some embodiments of the present invention may require targeting and / or entry of the dsRNA reagent into hepatocytes. In some embodiments of the methods of the present invention, it should be understood that the therapeutic agent comprises a dsRNA reagent having only a delivery agent without any additional linking elements, for example, a delivery agent containing N-acetylgalactosamine (GalNAc). For example, in some embodiments of the present invention, the dsRNA reagent may be contained in a composition comprising a delivery compound containing GalNAc and a pharmaceutically acceptable carrier, and is administered to cells or subjects without any detectable label or targeting agent linked to the dsRNA reagent.

[0335] When the dsRNA reagent of the present invention is administered and / or linked to one or more delivery agents, targeting agents, labeling agents, etc., those skilled in the art will be able to recognize, select, and use appropriate reagents for use in the methods of the present invention. In some methods of the present invention, the labeling reagent can be used to determine the location of the dsRNA reagent in cells and tissues, and can be used in the methods of the present invention to determine the location of cells, tissues, or organs to which a therapeutic composition containing the dsRNA reagent has been administered. Means of attaching and using labeling reagents, such as enzyme labeling, dyes, and radiolabeling, are known in the art. In some embodiments of the compositions and methods of the present invention, it should be understood that the labeling reagent is linked to one or both of the sense polynucleotides and antisense polynucleotides contained in the dsRNA reagent.

[0336] In some embodiments, the compositions are packaged in reagent kits, containers, packaging, dispensers, pre-filled syringes, or vials. A reagent kit comprising one or more target gene dsRNA reagents and / or target gene antisense polynucleotide reagents, as well as instructions for their use in the methods of the present invention, is also within the scope of the present invention. The reagent kits of the present invention may contain one or more of the target gene dsRNA reagents, target gene sense polynucleotides, and target gene antisense polynucleotide reagents that can be used to treat diseases or conditions related to the target gene. Reagent kits comprising one or more target gene dsRNA reagents, target gene sense polynucleotides, and target gene antisense polynucleotide reagents can be manufactured for use in the therapeutic methods of the present invention. The components of the reagent kits of the present invention may be packaged in aqueous media or lyophilized form. The reagent kits of the present invention may include isolated carriers for the closed containment of one or more container devices or a series of container devices (e.g., test tubes, vials, flasks, bottles, syringes, etc.) therein. The first container device or a series of container devices may contain one or more compounds, such as a target gene dsRNA reagent and / or a target gene sense or antisense polynucleotide reagent. The second container device or a series of container devices may contain targeting agents, labeling agents, delivery agents, etc., which are included as part of the target gene dsRNA reagent and / or target gene antisense polynucleotide administered in embodiments of the therapeutic method of the present invention.

[0337] The reagent kit of the present invention may further include instructions. The instructions are usually in written form and provide instructions for carrying out treatment with the reagent kit and for making decisions based on such treatment.

[0338] ●Cells, subjects, and controls The methods of the present invention can be used in combination with cells, tissues, organs and / or subjects. In some embodiments of the present invention, the subjects are humans or vertebrate mammals, including, but not limited to, dogs, cats, horses, cattle, goats, mice, rats and monkeys. Accordingly, the present invention can be used to treat diseases or conditions related to target genes in human and non-human subjects.

[0339] In some embodiments of the present invention, the subject may be farm animals, zoo animals, livestock, or non-livestock animals, and the method of the present invention can be used in veterinary preventive and therapeutic schemes. In some embodiments of the present invention, the subject may be humans, and the method of the present invention can be used in preventive and therapeutic schemes for humans.

[0340] Non-limiting examples of subjects to whom the present invention can be applied are subjects diagnosed with, suspected of having, or at risk of having, a disease or condition related to the following diseases or conditions, also known as “elevated target gene expression levels,” which are higher than desired target gene expression and / or activity. Non-limiting examples of diseases and conditions related to higher-than-desired target gene expression and / or activity are described elsewhere in this specification. The methods of the present invention can be applied to subjects diagnosed with a disease or condition at the time of treatment, subjects related to higher-than-desired target gene expression and / or activity, or subjects considered to have or be at risk of developing a disease or condition related to higher-than-desired target gene expression and / or activity. In some embodiments of the present invention, the disease or condition related to higher-than-desired target gene expression and / or activity levels is an acute disease or condition, and in some embodiments of the present invention, the disease or condition related to higher-than-desired target gene expression and / or activity levels is a chronic disease or condition.

[0341] In another non-limiting example, the target gene dsRNA reagent of the present invention is administered to treat a disease or disorder caused by or related to the activation of the target gene, or a disease or disorder whose symptoms or progression are in response to the inactivation of the target gene. The term "disease related to the target gene" includes diseases, disorders, or conditions that benefit from reduced target gene expression.

[0342] Cells to which the methods of the present invention can be applied include in vitro, in vivo, and ex vivo cells. Cells may be in a subject, in a culture and / or suspension, or in any other suitable state or condition. Cells to which the methods of the present invention can be applied may also be liver cells, hepatocytes, cardiac cells, pancreatic cells, cardiovascular cells, kidney cells, or other types of vertebrate cells, including human and non-human mammalian cells. In some embodiments of the present invention, cells to which the methods of the present invention can be applied are healthy, normal cells not known as disease cells. In some embodiments of the present invention, the methods and compositions of the present invention are applied to liver cells, hepatocytes, cardiac cells, pancreatic cells, cardiovascular cells and / or kidney cells. In some embodiments of the present invention, while control cells are normal cells, it should be understood that in certain cases, cells with disease or disease may be used as control cells, for example, in the results of comparing treated cells with disease or disease with untreated cells with disease or disease.

[0343] According to the method of the present invention, the activity level of a target gene polypeptide can be determined and compared to a control level of the activity of the target gene polypeptide. The control may be a predetermined value in various forms. It may be a single cutoff value such as the median or mean. It can be established based on a comparison group, for example, in a group having normal levels of target gene polypeptide and / or target gene polypeptide activity, and in a group having increased levels of target gene polypeptide and / or target gene polypeptide activity. Another non-limiting example of a comparison group may be a population having one or more symptoms or diagnoses of a disease or condition related to the target gene and a population not having one or more symptoms or diagnoses of the disease or condition, a control group administered with the siRNA therapy of the present invention and a control group not administered with the siRNA therapy of the present invention. Typically, the control can be based on obviously healthy normal individuals or obviously healthy cells of an appropriate age group. In addition to predetermined values, it should be understood that the control according to the present invention may be a material sample tested in parallel with the experimental material. Examples include a sample from a control population, or a control sample produced by manufacturing for parallel testing with the experimental sample. In some embodiments of the present invention, the control may include cells or subjects that have not been contacted with or treated with the target gene dsRNA reagent of the present invention, in which case the control level of activity of the target gene polypeptide and / or target gene polypeptide can be compared with the level of activity of the target gene polypeptide and / or target gene polypeptide in cells or subjects that have been contacted with the target gene dsRNA reagent or target gene antisense polynucleotide reagent of the present invention.

[0344] In some embodiments of the present invention, the control level may be the target gene polypeptide level determined for a subject, where the target gene polypeptide level determined for the same subject at different time points is compared to the control level. In non-limiting examples, the target gene level is determined in a biological sample obtained from a subject that has not been treated with the target gene of the present invention. In some embodiments, the biological sample is a serum sample. The target gene polypeptide level measured from a sample obtained from a subject can be used as a baseline or control value for the subject. In the therapeutic method of the present invention, after one or more administrations of the target gene dsRNA reagent to a subject, one or more further serum samples may be obtained from the subject, and the target gene polypeptide level in one or more subsequent samples may be compared to the control / baseline level of the subject. Such comparisons can be used to assess the onset, progression, or regression of a disease or condition related to the target gene in the subject. For example, if the level of the target gene polypeptide in a baseline sample obtained from a subject is higher than the level obtained from the same subject after administering the target gene dsRNA reagent or target gene antisense polynucleotide reagent of the present invention to the subject, it indicates regression of the disease or condition related to the target gene and demonstrates the effectiveness of treating the disease or condition related to the target gene by administering the target gene dsRNA reagent of the present invention.

[0345] In one embodiment of the present invention, one or more values ​​in the activity level of the target gene polypeptide and / or target gene polypeptide determined for a subject can be used as a control value and subsequently used to compare the activity levels of the target gene polypeptide and / or target gene in the same subject, thereby enabling the evaluation of changes in the activity of the “baseline” target gene polypeptide in the subject. Accordingly, when the initial level is used as the control level for the subject, the initial target gene polypeptide level and / or initial target gene polypeptide activity level can be used as an indication and / or determination of the level in the subject of the method and compound of the present invention that can reduce the target gene polypeptide and / or target gene polypeptide activity in the subject.

[0346] The methods of the present invention can be used to administer the target gene dsRNA reagent and / or target gene antisense polynucleotide reagent of the present invention to a subject. The efficacy of administration and treatment with such dsRNAi reagents of the present invention can be evaluated as follows: after administration and treatment, the level of target gene polypeptide in a serum sample obtained from the subject is reduced by at least 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more, compared to the level of target gene polypeptide in a serum sample obtained from the subject at a prior time, or compared to a non-contact control level (e.g., the level of target gene polypeptide in a control serum sample). It should be understood that both the level of target gene polypeptide and the level of target gene polypeptide activity correlate with the level of target gene expression. One embodiment of the methods of the present invention involves administering the target gene dsRNA and / or target gene antisense reagent of the present invention to a subject in an amount that effectively suppresses the expression of the target gene, thereby reducing the level of target gene polypeptide and the level of target gene polypeptide activity in the subject.In some embodiments of the method of the present invention, the expression of a target gene in cells is suppressed by contact (also referred to herein as treatment) of cells with the siRNA reagent of the present invention by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, This results in 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or approximately 100%, for example, falling below the detection level of the test.

[0347] Some embodiments of the present invention involve determining the presence, absence, and / or amount (also referred to herein as level) of a target gene polypeptide from one or more biological samples obtained from one or more subjects. Such measurements can be used to evaluate the effectiveness of the therapeutic methods of the present invention. For example, the methods and compositions of the present invention can be used to determine the level of a target gene polypeptide in a biological sample obtained from a subject previously treated with the administration of the target gene dsNA reagent and / or target gene antisense agent of the present invention. If, after administration and treatment, the level of the target gene polypeptide in the serum sample obtained from the subject is reduced by at least 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more compared to the pre-administration level of the target gene polypeptide in a serum sample obtained from the subject at a previous point in time, or compared to a non-contact control level (e.g., the level of the target gene polypeptide in a control serum sample), it indicates the level of effectiveness of the treatment administered to the subject.

[0348] In some embodiments of the present invention, the physiological characteristics of a disease or condition associated with a target gene determined for a subject can be used as a control result, and the determination of the physiological characteristics of the same subject at different time points can be compared with the control result. In non-limiting examples, hemolysis of a pathological characteristic is measured from a subject that has not been treated with the target gene of the present invention, which can be used as a baseline or control value for the subject. In the treatment method of the present invention, after administering the target gene dsRNA reagent to a subject once or more times, blood cells are compared to the control / baseline level of the subject, respectively. Such comparisons can be used to assess the onset, progression, or regression of a disease or condition associated with the target gene in the subject. For example, if the baseline blood cell count obtained from a subject is higher than the thrombus measured from the same subject after administering the target gene dsRNA reagent or target gene antisense polynucleotide reagent of the present invention to the subject, it indicates regression of a disease or condition associated with the target gene and represents the effectiveness of treating the disease or condition associated with the target gene by administering the target gene dsRNA reagent of the present invention.

[0349] Some embodiments of the present invention include, but are not limited to, determining the presence, absence, and / or alteration of the physiological characteristics of a disease or disorder relating to a target gene using methods such as (1) measuring blood cells in a subject, (2) evaluating the physiological characteristics of one or more biological samples obtained from one or more subjects, and (3) performing a physical examination on a subject. Such measurements can be used to evaluate the effectiveness of the therapeutic method of the present invention.

[0350] ●Regarding modifications In some embodiments of the present invention, the RNA of the gene RNAi agent is chemically modified to obtain enhanced stability and / or one or more other beneficial properties. The nucleic acid in some embodiments of the present invention can be synthesized and / or modified by methods known in the art, see, for example, "Current protocols in Nucleic Acid Chemistry," Beaucage, S. Let al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. Modifications that may be present in some embodiments of the dsRNA reagent of the present invention include, for example, terminal modifications such as (a) 5'-end modifications (phosphorylation, conjugate, reverse bond, etc.) and 3'-end modifications (conjugate, DNA nucleotide, reverse bond, etc.), (b) base modifications such as stable bases, unstable bases or base substitutions that base-pair with an extended partner library, deletion bases (debased nucleotides) or conjugate bases, (c) sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions, and (d) skeletal modifications including modifications or substitutions of phosphodiester bonds. Specific examples of RNA compounds usable in certain embodiments of the dsRNA reagent, antisense polynucleotide, and sense polynucleotide of the present invention include, but are not limited to, RNA having a modified backbone or RNA lacking native internucleotide bonds. In non-limiting examples, RNA having a backbone modification may not have a phosphorus atom in its backbone. RNA without a phosphorus atom in its internucleoside backbone may be called an oligonucleoside. In a certain embodiment of the present invention, the modified RNA has a phosphorus atom in its internucleoside backbone.

[0351] The terms “RNA molecule” or “RNA” or “ribonucleic acid molecule” should be understood to include not only naturally expressed or found RNA molecules, but also RNA analogs and derivatives containing one or more ribonucleotide / ribonucleoside analogs or derivatives described herein or known in the art. The terms “ribonucleoside” and “ribonucleotide” can be used interchangeably herein. RNA molecules can be modified in their nucleic acid base structure or ribose-phosphate backbone structure (as described below, for example), and molecules containing ribonucleoside analogs or derivatives must retain the ability to form double-stranded structures.

[0352] The following examples are provided to illustrate specific examples of carrying out the present invention, and are not intended to limit the scope of the invention. It will be apparent to those skilled in the art that the present invention can be applied to a variety of compositions and methods.

[0353] About: As used herein, the terms “about” or “approximately” applied to one or more values ​​of interest refer to values ​​similar to the given reference values. In some embodiments, unless otherwise stated or made clear from the context, the terms “about” or “approximately” refer to a range of values ​​that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less than 25%, 20%, 1% of the above reference values ​​in any direction (greater than or less than) (unless such values ​​exceed 100% of the possible values).

[0354] Some abbreviations used herein: "Ac" refers to the acetyl group, "Bn" refers to the benzyl group, "Bz" refers to the benzoyl group, "DMTrCl" refers to 4,4-dimethoxytrityl chloride, "DMTr" refers to the 4,4-dimethoxytrityl group, "TIIP" refers to the tetrahydropyranyl group, "TBDMS" refers to the tert-butyldimethylsilyl group, "TIPDS" refers to the tetraisopropyldimethylsilyl group, and "DTBS" refers to the di(tert-butyl)silyl group. [Brief explanation of the drawing]

[0355] [Figure 1] This is the single-crystal X-ray diffraction pattern of Phos-15-1E-2(((((1R,2R,4R)-4-(2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl)-2-hydroxycyclopentyl)thio)methyl)diethylphosphonate). [Modes for carrying out the invention]

[0356] ●Specific examples The following examples are provided to illustrate specific examples of carrying out the present invention and are not intended to limit the scope of the invention. It will be apparent to those skilled in the art that the present invention is applicable to a variety of compositions and methods.

[0357] ●Example 1 ● Manufacturing of Phosphoramidite-01 [ka] ● Synthesis of Phos-01-1A To an anhydrous tetrahydrofuran solution of BWP5-B (3.71 g, 1.5 eq), PPh3 (8.87 g, 2.0 eq) was added, and then DEAD (6.19 g, 2.1 eq) was added to the mixture. After stirring for 30 minutes under nitrogen gas protection at 0°C, BOM-U (10.0 g, 1.0 eq) was added to the mixture and stirred for 16 hours. TLC (MeOH:DCM = 1:20, Rf = 0.6) showed that BOM-U was completely consumed. CaBr2 (13.6 g, 4.0 eq.) was added to the mixture and stirred at room temperature for 16 hours, then filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified and eluted by silica gel column chromatography (ethyl acetate:petroleum ether = 0:100~50:50) to obtain a grayish-white solid Phos-01-1A (2.3 g, yield 38%). MS(M+H) = 361.

[0358] ● Synthesis of Phos-01-1B Trifluoroacetic acid (10 mL) was added to an aqueous solution (10 mL) of Phos-01-1A (1.9 g, 1.0 eq). The reaction was stirred at room temperature for 3 hours, and TLC (MeOH:DCM = 1:20, Rf = 0.3) detection showed that Phos-01-1A was completely consumed. The mixture was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography and eluted with (MeOH:DCM = 0:100~1:10) to obtain colorless oily Phos-01-1B (0.97 g, yield 57%). MS(M+H)=321.

[0359] ● Synthesis of Phos-01-1C At 0°C in a nitrogen gas environment, DMTr-Cl (1.13 g, 1.1 eq.) was added to a pyridine solution of Phos-01-1B (0.97 g, 1.0 eq.), and the mixture was stirred at room temperature for 16 hours. TLC (MeOH:DCM=1:20, Rf=0.9) showed that Phos-01-1B had been completely consumed. The mixture was concentrated under reduced pressure until dry, and the crude product was purified by silica gel column chromatography and eluted with (MeOH:DCM=0:100~1:20) to obtain a brown solid Phos-01-1C (0.75 g, yield 40%). MS(M+H) = 623.

[0360] ● Synthesis of Phos-01-1D At 0°C in a nitrogen gas environment, NaH (139 mg, 1.1 eq) was added to a dry tetrahydrofuran solution of Phos-01-1C (0.75 g, 1.0 eq) and stirred at 0°C for 30 minutes. At 0°C, Phos-13-1A (1.3 g, 3.0 eq.) was added to the above mixture and stirred at room temperature for 5 hours. TLC (MeOH:DCM=1:20, Rf=0.7) showed that Phos-01-1C was completely consumed. The reaction mixture was injected into a saturated sodium bicarbonate aqueous solution and extracted with ethyl acetate. The ethyl acetate phase was washed with saline solution and dried over anhydrous sodium sulfate. After removing the drying agent by filtration, the filtrate was concentrated under reduced pressure until dry. The crude product was purified by silica gel column chromatography and eluted with (MeOH:DCM=0:100~1:25) to obtain yellow oily Phos-01-1D (0.75 g, yield 81%). MS(M+H) = 773.

[0361] ● Synthesis of Phos-01-1F Using nitrogen gas, Pd / C (300 mg) was added to a solution of Phos-01-1D (0.75 g, 1.0 eq.) in isopropanol-water (10:1, 0.5% formic acid), and the mixture was stirred overnight with hydrogen gas at atmospheric pressure. TLC (MeOH:DCM=1:20, Rf=0.3) showed that Phos-01-1D had been completely consumed. The mixture was filtered through a sand core funnel, and the filtrate was concentrated under reduced pressure until dry. The crude product was purified by silica gel column chromatography and eluted with (MeOH:DCM=0:100~1:20) to obtain a brown solid Phos-01-1F (200 mg, yield 59%). MS(M+H)=351.

[0362] ●Phosphoramidite-01 At room temperature, 20 mL of dichloromethane and Phos-01-1F (400 mg, 1.0 eq.) were added to a 100 mL flask using nitrogen gas. At room temperature, tetraazazole (132 mg, 1.65 eq., in a 0.45 M MeCN solution) was added dropwise, followed by bis(diisopropylamino)(2-cyanoethoxy)phosphine (775 mg, 2.25 eq.) in a 5 mL dichloromethane solution. The mixture was stirred continuously at room temperature for 3 hours. TLC (MeOH:DCM = 1:20, Rf = 0.8) showed that Phos-01-1D had been completely consumed. The reaction mixture was cooled to 0°C, injected into a saturated sodium bicarbonate aqueous solution (100 mL), extracted with dichloromethane (100 mL x 3), the combined organic layer was washed with saturated saline solution (100 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography and eluted with MeOH:DCM = 0:100~1:100 and 1% Et3N to obtain colorless oily phosphoramidite-01 (190 mg, yield 30%). 1 H NMR (400 MHz, CD3CN) δ 7.48 (d, J = 8.0 Hz, 1H), 5.54 (d, J = 8.0 Hz, 1H), 4.14-4.08 (m, 4H), 3.89-3.67 (m, 7H), 3.66-3.53 (m, 4H), 2.66-2.63 (m, 2H), 1.90-1.73 (m, 2H), 1.20-1.38 (m, 6H), 1.23-1.15 (m, 12H). MS(M+H)=551.

[0363] ● Manufacturing of Phosphoramidite-03 [ka] Under nitrogen gas protection, diethyl malonate (32 g, 0.2 mol, 1.0 eq.) and DMF (200 mL) were added to a 500 mL flask, and the solution was stirred and cooled to 0-5°C. Next, sodium hydrogen (60%, 8.4 g, 0.21 mol, 1.05 eq.) was added in several portions, and during the addition of sodium hydrogen, the temperature of the reaction mixture gradually rose from 5°C to 15°C. The reaction mixture was cooled to 5°C and stirred for 20 minutes, then potassium iodide (1.66 g, 0.01 mol, 0.05 eq.) was added to the mixture, followed by (2-bromoethoxy)(tert-butyl)dimethylsilane (57.41 g, 0.24 mol, 1.2 eq.). The reaction mixture was heated to 50°C and stirred overnight under nitrogen gas protection. TLC showed that the starting materials were converted to the new major product. The reaction mixture was quenched with saturated ammonium chloride solution (250 mL) and stirred at 0-25°C for 0.5 hours. The organic matter was extracted with ethyl acetate:petroleum ether (50:50, 500 mL x 2), and the crude product was obtained by vacuum concentration. The crude product was purified by flash column, and the product was eluted with an eluent (ethyl acetate:petroleum ether = 0%-20%). Vacuum concentration was performed to obtain 50.84 g of a colorless oily substance, phos-03-3A, with a yield of 79.8%. 1 H NMR:(400MHz,DMSO-d6),δppm4.13-4.07(q,4H),3.60(t,2H),3.50(t,1H),1.96-1.94(m,2H),1.20(t,6H),0.85(s,9H),0.00(s,6H).

[0364] 100 mL of tetrahydrofuran was added to a 500 mL flask and cooled to 0°C under nitrogen gas protection. Then, lithium tetrahydroaluminum (0.077 mol, 1.5 eq.) was added. At 0-5°C, 30 mL of a tetrahydrofuran solution of Phos-03-3A (16.4 g, 0.0515 mol, 1.0 eq.) was added to the reaction mixture, and the reaction mixture was stirred at 5°C for 1 hour. TLC showed that the starting materials had been converted to the new major product. The reaction mixture was gradually quenched with 100 mL of ethyl acetate and stirred at 5-25°C for 0.5 hours. The mixture was filtered through a thin layer of silica gel. The filtrate was concentrated to obtain the crude product, which was further purified by silica gel column chromatography, and the product was eluted with an eluent (0%-10% MeOH in DCM). Vacuum concentration was performed to obtain 4.8 g of a yellow oily substance, Phos-03-3A, with a yield of 39.8%. 1 H NMR:(400 MHz, DMSO-d6), δppm 4.27 (s, 2H), 3.62 (t,2H), 3.35-3.32 (m, 4H), 1.56-1.53 ​​(m,1H), 1.43-1.38 (m,2H), 0.84 (s,9H), 0.00 (s,6H).

[0365] Pyridine (50 mL) and Phos-03-3B (4.5 g, 0.019 mol, 1.0 eq.) were added to a 250 mL flask, stirred under nitrogen gas protection, and cooled to 0°C. Next, DMTrCl (7.2 g, 0.021 mol, 1.15 eq.) was added and stirred overnight at room temperature. TLC showed that the starting materials had been converted to the new major product. Volatile components were removed by vacuum distillation, and the residue was purified by silica gel column chromatography. The product was eluted with an eluent (EA:PE:Py = 1:8:0.2%). Vacuum concentration was performed to obtain 4.6 g of a yellow oily substance, Phos-03-3C, with a yield of 44.6%. 1H NMR: (400 MHz, DMSO-d6), δppm 7.45-7.39 (m, 5H), 7.359-7.24 (m, 8H), 4.48 (s, 1H), 3.78 (s,6H),3.57-3.40 (m, 4H), 3.04 (d, 2H), 1.89-1.83 (m,1H), 1.56-1.52 (m, 2H), 0.86 (s, 9H), 0.00 (s, 6H).

[0366] Dichloromethane (40 mL) and Phos-03-3C (4.6 g, 8.6 mmol, 1.0 eq.) were added to a 250 mL flask, followed by Dess-Martin reagent (18.3 g, 0.043 mol, 5.0 eq.). The reaction was stirred at room temperature under nitrogen gas protection for 2 hours. TLC showed that the starting materials were converted to a new major product. The solvent was filtered, and the filtrate was purified by silica gel column chromatography. The product was eluted with eluate (EA:PE:Py = 3:100:0.2%). The mixture was concentrated under vacuum to obtain 4.0 g of a yellow oily substance, Phos-03-3D, in yield 87.0%.

[0367] In a 250 mL flask at room temperature, acetonitrile (60 mL) and lithium chloride (523 mg, 1.2 mmol, 1.2 eq.) were added. Under nitrogen gas protection, tetraethylmethylene diphosphate (3.56 g, 1.2 mmol, 1.2 eq.), DBU (1.56 g, 1.0 mmol, 1.0 eq.), and Phos-03-3D (5.5 g, 1.0 mmol, 1.0 eq.) were added, and the mixture was stirred continuously at room temperature for 2 hours. TLC showed that the starting materials had been converted to the new major product. Volatile residues were removed by rotary evaporation, and the residue was purified by silica gel column chromatography. The product was eluted with an eluent (EA:PE:Py = 1:4:0.2%). The product was concentrated under vacuum to obtain 4.0 g of a pale yellow oily substance, Phos-03-3E, with a yield of 59.8%. 1H NMR (400 MHz, DMSO-d6) δ 7.42 (dd, 2H), 7.33 (t, 2H), 7.29-7.24 (m, 5H), 6.91 (d, 4H), 6.65 (ddd, 1H), 5.87 (s, 1H), 5.82 (d, 1H), 5.78 (s, 1H), 4.07 (q, 4H), 3.77 (s, 6H), 3.51 (dd, 2H), 3.12 (dd, 1H), 3.05-2.98 (m, 1H), 2.67 (d, 1H), 1.71-1.53 ​​(m, 2H), 1.27-1.23 (m, 6H), 0.86 (s, 9H), 0.00 (d, 6H).

[0368] In a 250 mL flask, ethanol (15 mL), pyridine (4.0 g, 51 mmol, 8.43 eq.), and Phos-03-3E (4.0 g, 6 mmol, 1.0 eq.) were added. The reaction mixture was stirred under nitrogen gas protection and cooled to 0°C. Pyridine hydrofluoride (3.0 g, 0.03 mol, 5.0 eq.) was then added to the flask. The reaction was stirred at room temperature for 2 hours. TLC showed that the starting materials were completely consumed. Volatiles were removed by rotary evaporation, and the residue was purified by silica gel column chromatography. By-products were eluted with an eluent (EA:PE = 0%~80%, +0.2% pyridine), followed by elution of the product with a 5%~10% MeOH DCM solution. The mixture was concentrated under vacuum to obtain 2.44 g of a colorless oily substance, Phos-03-3F, with a yield of 71.6%. 1 H NMR: (400 MHz, DMSO-d6), δ ppm 7.37-7.35 (m, 2 H), 7.32-7.28 (m, 2 H), 7.24-7.20 (m, 5H), 6.89-6.87 (m, 4H),6.64-6.52 (m, 1H), 5.85-5.75 (m, 1H),4.42 (s, 1H), 3.96-3.92 (m, 4H), 3.74 (s,6H),3.33-3.29 (m, 2H), 3.06-2.93(m,2H), 2.64 (s, 1H), 1.58-1.48 (m, 2 H), 1.23-1.18 (m, 6 H).

[0369] Under nitrogen gas protection, tetrahydrofuran (15 mL), triphenylphosphorus (1.36 g, 5.2 mmol, 1.2 eq.), 3-benzoyluracil (1.12 g, 5.2 mmol, 1.2 eq.), diethyl azodicarboxylate (0.91 g, 5.2 mmol, 1.2 eq.), and Phos-03-3F (2.4 g, 4.3 mmol, 1.0 eq.) were added sequentially to a 100 mL flask, and the reaction was stirred overnight at room temperature. LC-MS confirmed that the major product was the desired product. Volatiles were removed by rotary evaporation, and the residue was purified by silica gel column chromatography. By-products were eluted with an eluent (EA:PE = 0-80%, +0.2% Py). The product was eluted with a dichloromethane solution containing 5-10% MeOH. Vacuum concentration was performed to obtain 3.08 g of the yellow solid Phos-03-3G, with a yield of 95.1%. 1 H NMR: (400 MHz, DMSO-d6), δ ppm 8.59-8.57 (m, 1 H), 7.96 (dd, 2 H), 7.86 (d, 1 H), 7.79-7.75 (m,1 H), 7. 60 (t, 2 H), 7.35 (d, 2 H), 7.31 (t, 2 H), 7.24 (d, 4 H), 6.89 (d, 4 H), 6.64-6.52 (m, 1 H), 5.94-5.82 (m, 2 H), 3.96-3.88 (m, 4 H), 3.74 (s,6 H), 3.68-3.63 (m, 2 H), 3.07-2.97 (m, 2H), 2.56-2.53 (m,1 H), 1.81-1.77 (m, 2 H), 1.19 (t, 6 H).

[0370] Dichloromethane (50 mL) and Phos-03-3G (3.0 g, 4 mmol, 1.0 eq.) were added to a 250 mL flask. The reaction was stirred under nitrogen gas protection. Next, trichloroacetic acid (1.5 g, 13.2 mmol, 3.3 eq.) was added, and the reaction was stirred at room temperature for 2 hours. No residue of the starting materials was observed by TLC. The reaction mixture was then distilled, and the residue was purified by silica gel column chromatography. The product was eluted using eluate (0%-7% MeOH in DCM). The mixture was concentrated under vacuum to obtain 1.3 g of a colorless oily substance, Phos-03-3H, with a yield of 72.2%. HNMR showed residue of triethylamine, which was removed by purification using a reverse flash column (C18, 40 g, 40-63 μm, eluate, H2O / MeOH, 0-30% to elute the product). 1 H NMR: (400 MHz, DMSO-d6), δ ppm 7.96-7.94 (m, 2 H), 7.91 (d, 1 H), 7.81-7.76 (m, 1 H), 7.62-7.58 (m, 2 H), 6.60-6.48 (m, 1 H), 5.89-5.75 (m, 2 H),4.79 (t, 1 H), 3.97-3.89 (m, 4 H), 3.74-3.70 (m, 2 H),3.43 (t, 2 H), 2.40-2.35 (m,1 H), 1.88-1.69 (m, 2 H), 1.22-1.17 (m, 6 H).

[0371] Under nitrogen gas protection, 6 mL of dichloromethane, 0.061 g, 0.88 mmol, 1.1 eq. of tetraazazole, 0.31 g, 1.04 mmol, 1.3 eq. of bis(diisopropylamino)(2-cyanoethoxy)phosphine, and 0.36 g, 0.8 mmol, 1.0 eq. of Phos-O3-3H were added to a 100 mL flask and stirred at room temperature for 1.5 hours. LC-MS showed that the main peak was the desired product. The reaction mixture was quenched with 50 mL of saturated sodium bicarbonate aqueous solution and extracted twice with 50 mL of dichloromethane. The organic phases were combined and concentrated until dry after drying, and the residue was washed twice with 20 mL of petroleum ether. The residue was further purified by silica gel column chromatography, and the product was eluted with an eluent (MeOH:DCM = 0%~2%, +0.2% TEA). The solution was concentrated under vacuum at 35°C to obtain 0.32 g of a colorless oily substance, phosphoramidite-03, with a yield of 61.5%. 1 H NMR: (400 MHz, CD3CN), δ ppm 8.00-7.97 (m, 2 H), 7.80 -7.75 (m, 1 H), 7.62 -7.58 (m, 2 H), 7.53 -7.51 (m, 1 H), 6.66-6.55(m, 1 H),5.91-5.77 (m, 2 H), 4.02-3.97 (m, 4 H), 3.85-3.74 (m, 4 H), 3.73-3.59 (m, 3 H), 2.67-2.64 (m, 2 H), 2.63-2.57(m,1 H), 2.01-1.94 (m, 2 H), 1.87-1.82 (m, 1 H), 1.31-1.24 (m, 6 H), 1.20 (dd, 12 H).

[0372] ● Manufacturing of Phosphoramidite-13 [ka] At -50°C, trifluoroacetic anhydride (83.9 g, 297 mmol, 49.0 mL) was added dropwise to a 500 mL solution of compound Phos-13-SM1 (50 g, 297 mmol) and 2,6-dimethylpyridine (48.4 g, 452 mmol, 52.6 mL) in dichloromethane. The mixture was stirred for 10 minutes, then the reaction mixture was heated to 40°C and stirred for 3.0 hours. TLC showed that the starting materials were completely consumed and new spots had formed. The reaction mixture was ground with isopropyl ether to form a precipitate, which was then filtered through diatomaceous earth to remove the precipitate. The filtrate was washed with 1.0 M saturated HCl brine, and then vacuum concentrated to obtain a brown oily substance, Phos-13-1A (61 g, 203 mmol, yield 68.3%). 1 H NMR: EC10615-62-P1N (400 MHz, DMSO-d6) δ ppm 4.78 (d, J=7.63 Hz, 2H), 4.07 - 4.17 (m, 4 H), 1.25 - 1.30 (m, 6 H).

[0373] In a nitrogen gas environment, at 0°C, sodium hydrogen (60% purity, 787 mg, 19.7 mmol) was added in several portions to stirred tetrahydrofuran (50 mL), compound Phos-13-SM2 (5.0 g, 24.6 mmol) was added to the solution, and the mixture was stirred at 0°C for 45 minutes. Next, at 0°C, Phos-13-1A (3.54 g, 11.8 mmol) was added to the solution, and the mixture was stirred continuously for 2.0 hours while maintaining this temperature. LC-MS indicated that the reaction was complete. The reaction mixture was cooled to 0-5°C and carefully added dropwise to stirred saturated ammonium chloride solution (80 mL) (maintaining the temperature below 0-5°C). Then, the mixture was extracted with ethyl acetate (80 mL x 2), the organic phases were combined, washed with saline solution (80 mL), dried over anhydrous sodium bisulfate, and concentrated under reduced pressure to obtain the residue. The residue was purified by silica gel column chromatography (100-200 mesh silica gel) and eluted with methanol:dichloromethane (1:100:1:30) to obtain a brown oily substance Phos-13-1B (6.0 g, yield 76.7%).

[0374] At 0-5°C, HCl / dioxane (4M, 30.0 mL) was added to a solution of compound Phos-13-1B (6.0 g, 17.0 mmol) in dioxane (30 mL). The mixture was then stirred at 0-5°C for 1.0 hour, and LC-MS confirmed that the reaction was complete. The mixture was pulverized with isopropyl ether (300 mL x 2), filtered, and a solid was obtained. The solid was further subjected to preparative HPLC to obtain the brown oily compound Phos-13-1C (1.0 g, yield 23.2%). 1 H NMR: EC10615-84-all3 (400MHz,CHLOROFORM-d) δ ppm4.45 (d,J=3.25 Hz, 1H), 4.11 - 4.24 (m, 5H), 3.98 (d, J=9.13 Hz, 2H), 3.48 -3.56(m, 1H), 3.37 - 3.43 (m, 2H), 3.23 - 3.28 (m, 1H), 1.30-1.40 (m, 6H).

[0375] Uracil (3.0 g, 26.8 mmol) and SOCl2 (5.0 mL, 44.6 mmol) were added to a 100 mL reaction flask at 25°C. ClSO3H (8.0 g, 69.0 mmol) was then added, and the mixture was stirred at room temperature for 1.5 hours. The temperature was then raised to 60°C and the reaction was continued for 4.0 hours, followed by stirring at 75°C for 7.0 hours. TLC showed that the compound uracil was completely consumed and new spots were formed. The reaction mixture was added to a mixture of ice and acetic acid (1:1). The mixture was filtered to obtain a precipitate, and the resulting filtered cake was repeatedly washed with purified water and vacuum-dried to obtain a white powder Phos-13-1D (1.0 g, 4.75 mmol, yield 17.7%). 1 H NMR: EC4783-422-P1B1 (400 MHz, DMSO-d6) δ ppm 11.02 (br s, 1 H) 10.88 (br d, J=5.75 Hz, 1 H) 7.64 (d, J=6.00 Hz, 1 H).

[0376] At 0-5°C, a solution of compound Phos-13-1C (1.0 g, 3.95 mmol) and triethylamine (1.20 g, 11.9 mmol, 1.65 mL) in dichloromethane (10 mL) was added to a solution of compound Phos-13-1D (1.25 g, 5.92 mmol) in dichloromethane (5.0 mL), and the mixture was subsequently stirred at 25°C for 1.0 hour. LC-MS showed that compound Phos-13-1C was completely consumed, producing approximately 50.9% of the desired product. The resulting reaction mixture was diluted with water (20 mL), extracted with 30% CF3CH2OH / DCM (30 mL x 5), and the combined organic layers were dried over anhydrous sodium sulfate and concentrated under vacuum to obtain the residue. The residue was purified by silica gel column chromatography (100-200 mesh silica gel), eluted with methanol:dichloromethane (1:30-1:8), and a white solid Phos-13-1E (570 mg, yield 33.77%) was obtained. 1 H NMR: EC12346-4-P1B1-C (400 MHz, CHLOROFORM-d) δ ppm 8.09 (s, 1 H), 4.21 - 4.25 (m, 1 H), 4.10 - 4.19 (m, 4 H), 3.82 - 3.98 (m, 3 H), 3.66 - 3.70 (m, 2 H), 3.58 (dd, J=10.88, 4.25 Hz, 1 H), 3.36 (d, J=11.01 Hz, 1 H), 1.33 - 1.35 (m, 6 H).

[0377] Under a nitrogen gas atmosphere, a solution of compound Phos-13-1E (380 mg, 0.89 mmol) and diisopropylamine-tetraazazole salt (167 mg, 0.98 mmol) in dichloromethane (1.0 mL) was added to a solution of bis(diisopropylamino)(2-cyanoethoxy)phosphine (reagent P, 804 mg, 2.67 mmol, 0.85 mL) in dichloromethane (0.5 mL), and the mixture was stirred at 40°C for 1.0 hour. LC-MS showed that compound Phos-13-1E was completely consumed and the desired product was detected. The resulting reaction mixture was cooled to -20°C, injected into 10 mL of cold (0-5°C) saturated sodium bicarbonate aqueous solution, extracted with dichloromethane (10 mL x 2), washed with cold (0-5°C) saturated sodium bicarbonate aqueous solution / saline solution (5 mL / 5 mL), dried over Na2SO4, and concentrated under vacuum to obtain a residue (~2.0 mL). The residue was pulverized with isopropyl ether (20 mL x 2) to obtain the crude product, which was then purified by column chromatography (basic Al2O3, methanol:dichloromethane = 0 / 50~1 / 50, 0.1% TEA) to obtain white solid phosphoramidite-13 (95 mg, yield 17.0%). 1 H NMR: EC12346-6-P1Nre (400 MHz, DMSO-d6) δ ppm 9.75 (br d, J=8.13 Hz, 1 H), 7.97 (d, J=4.88 Hz, 1 H), 4.16 - 4.25 (m, 1 H), 4.01 (quin, J=7.29 Hz, 5 H), 3.82 (br dd, J=7.25, 2.00 Hz, 2 H), 3.63 - 3.75 (m, 2 H), 3.44 - 3.58 (m, 4 H), 3.42 (br s, 1 H), 3.39 (br d, J=2.00 Hz, 1 H), 3.24 (d, J=11.38 Hz, 1 H), 2.76 (q, J=5.46 Hz, 2 H), 1.21 (t, J=7.07 Hz, 6 H), 1.07 - 1.16 (m, 12 H).

[0378] ● Manufacturing of enantiomer phosphoramidite-15-1 and enantiomer phosphoramidite-15-2 [ka] At 0°C, benzoyl chloride (126 g, 893 mmol, 104 mL) was added to a solution of pyridine (735 g, 9.29 mol, 750 mL) containing uracil (50.0 g, 446 mmol) and acetonitrile (1.50 L). The reaction mixture was stirred at 20-25°C for 12.0 hours, and TLC showed that the compound uracil was completely consumed. The reaction mixture was concentrated under vacuum to obtain the residue, which was diluted with cold water (1.0 L) and extracted with ethyl acetate (1.0 L x 3). The combined organic layers were washed with saline solution (500 mL) and dried over anhydrous sodium sulfate to obtain the residue. The residue was purified by column chromatography (SiO2, ethyl acetate / petroleum ether = 1 / 10-1 / 1) to obtain a white solid Phos-15-1A (63 g, yield 65.3%). 1 H NMR: EC4783-420-P1N (400 MHz, DMSO-d6) δ ppm 7.96 (dd, J=8.4, 1.2 Hz, 2 H), 7.76 - 7.81 (m, 1 H), 7.67 (dd, J=7.6, 5.6 Hz, 1 H), 7.58 - 7.64 (m, 2 H), 5.75 (dd, J=7.6, 1.2 Hz, 1 H).

[0379] To a solution of Phos-15-SM2 (4.0 g, 47.6 mmol) and compound Phos-15-1A (7.91 g, 36.6 mmol) in tetrahydrofuran (80 mL), triphenylphosphorus (11.5 g, 43.9 mmol) and diethyl azodicarboxylate (7.64 g, 43.9 mmol, 7.98 mL) were added, and the mixture was stirred at 20-25°C for 16 hours. LC-MS showed that compound Phos-15-1A was completely consumed. The reaction mixture was concentrated under reduced pressure to remove the tetrahydrofuran. The residue was diluted with water (80 mL), then extracted with ethyl acetate (80 mL x 3), and the combined organic phase was washed with saline solution (80 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, MeOH / DCM = 0 / 10~1 / 10) to obtain the compound Phos-15-1B (14g, crude product), which is a white solid.

[0380] Under nitrogen gas protection, a mixture of compound Phos-15-1B (7.0 g, 9.30 mmol) and m-chloroperbenzoic acid (2.27 g, 11.1 mmol, 85% purity) in dichloromethane (70 mL) was reacted at 0-5°C for 16 hours. TLC showed that compound Phos-15-1B was completely consumed and a major new spot with relatively low polarity was detected. The pH of the reaction mixture was gradually adjusted to 7-8 with saturated NaHSO3 and NaHCO3 solution (1:1), then extracted with ethyl acetate (70 mL x 3), washed the combined organic phase with saline solution (700 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (100-200 mesh silica gel), eluted with ethyl acetate:petroleum ether (1:30-1:1) to obtain the white solid compound Phos-15-1C (1.2 g, crude product).

[0381] To a solution of compound Phos-15-SM3 (4.0 g, 14.4 mmol) in tetrahydrofuran (24.0 mL), KSAc (1.81 g, 15.8 mmol) and tetrabutylammonium iodide (TBAI, 531.4 mg, 1.44 mmol) were added, and the mixture was stirred at 70°C for 4.0 hours. LC-MS showed that the starting material Phos-15-SM3 was completely consumed and a main peak with the molecular weight of the desired target molecule was detected. The reaction mixture was cooled, concentrated under reduced pressure, filtered through a short silica gel pad to remove solid residue, rinsed with ethyl acetate, and the filtrate was concentrated under vacuum to obtain the brown oily compound Phos-15-1D (3.50 g, 98.5% yield). Compound Phos-15-1D can be used in the next step without requiring further purification. 1 H NMR: EC11950-13-P1B (400 MHz, DMSO-d6) δ ppm 3.96 - 4.07 (m, 4H) 3.27 (d, J=14.0 Hz, 2H) 2.40 (s, 3H) 1.22 (t, J=7.2 Hz, 6H).

[0382] To a solution of compound Phos-15-1C (1.20 g, 4.02 mmol) in ethanol (15.0 mL), potassium carbonate (1.11 g, 8.05 mmol) and compound Phos-15-1D (1.91 g, 8.45 mmol) were added, and the mixture was stirred at 20-25°C for 3.0 hours. TLC showed that compound Phos-15-1C was completely consumed, and a major new spot with relatively high polarity was detected. The resulting reaction mixture was filtered, diluted with water (20 mL), extracted with dichloromethane (20 mL x 3), washed with saline solution, dried over anhydrous sodium sulfate, and concentrated under vacuum to obtain the residue. The residue was purified by column chromatography (SiO2, MeOH / DCM = 1 / 100 to 10 / 100) to obtain the brown oily compound Phos-15-1E (1.00 g, yield 65.7%, a 1:1 mixture of enantiomer compound-1E-1 and compound-1E-2). 1H NMR: EC10615-82-P1N1 (400 MHz, DMSO-d6) δ ppm 11.23 (br s, 1 H), 7.69 (d, J=8.0 Hz, 1 H), 5.58 (dd, J=8.0, 1.6 Hz, 1 H), 4.93 (q, J=8.8 Hz, 1 H), 3.96 - 4.17 (m, 5 H), 3.08 - 3.17 (m, 1 H), 3.03 (dd, J=14.0, 2.0 Hz, 2 H), 2.38 - 2.47 (m, 1 H), 2.04-2.07 (m, 1 H), 1.82 - 1.90 (m, 1 H), 1.56-1.59 (m, 1 H), 1.25 (t, J=6.8 Hz, 6 H).

[0383] The compound Phos-15-1E can be resolved to obtain the enantiomers Phos-15-1E-1 and Phos-15-1E-2 under chiral resolution conditions: DAIELCHIRALPAK AD 40mm column, 140 mL / min, ethanol:carbon dioxide = 35:75. It should be understood that if it is necessary to obtain enantiomer phosphoramidite-15-1 or enantiomer phosphoramidite-15-2, it can be obtained simply by using the corresponding enantiomer Phos-15-1E-1 or Phos-15-1E-2 as a starting material and reacting it with a phosphorus reagent.

[0384] At room temperature under a nitrogen atmosphere, a solution of compound Phos-15-1E (400 mg, 1.06 mmol) and di-isopropylamine-tetraazazole salt (199 mg, 1.16 mmol) in dichloromethane (4.0 mL) was added to a solution of bis(diisopropylamino)(2-cyanoethoxy)phosphine (reagent P, 956 mg, 3.17 mmol, 1.01 mL) in dichloromethane (0.5 mL), and the mixture was then stirred at 40°C for 1.0 hour. LC-MS showed that compound Phos-15-1E was completely consumed, and several new peaks were observed on the LC-MS, with approximately 80% of the desired compound detected. The resulting reaction mixture was cooled to -20°C, injected into 10 mL of cold (0-5°C) saturated sodium bicarbonate aqueous solution, extracted with dichloromethane (10 mL x 2), washed with cold (0-5°C) saturated sodium bicarbonate aqueous solution / saline solution (5 mL / 5 mL), dried over anhydrous sodium sulfate, and concentrated under vacuum to obtain a residue (~2.0 mL). The residue was purified by column chromatography (basic Al2O3, MeOH / DCM = 1 / 80-1 / 40, 0.1% Et3N) to obtain phosphoramidite-15 (350 mg, 0.6 mmol, yield 57.2%, a 1:1 mixture of enantiomer phosphoramidite-15-1 and enantiomer phosphoramidite-15-2) as a colorless oil.

[0385] Enantiomerized phosphoramidite-15-1 or enantiomerized phosphoramidite-15-2 can be obtained using the corresponding Phos-15-1E-1 or Phos-15-1E-2 obtained by SFC separation and purification as a starting material, following the same process as described above. δ ppm 11.23 (br s, 1 H), 7.70 (d, J=8.0 Hz, 1 H), 5.55 - 5.60 (m, 1 H), 4.89 (q, J=8.4 Hz, 1H), 4.29 - 4.42 (m, 1H), 3.99 - 4.09 (m, 4H), 3.65 - 3.84 (m, 2H), 3.53 - 3.62 (m, 2H), 3.35 - 3.41 (m, 1H), 3.02 (dd, J=14.0, 8.0 Hz, 2H), 2.76-2.79 (m, 2H), 2.40 - 2.49 (m, 1H), 2.15 - 2.25 (m, 1H), 1.95 - 2.07 (m, 1H), 1.65 - 1.75 (m, 1H), 1.23-1.26 (m, 6H) 1.12 - 1.21 (m, 12H).

[0386] The specific manufacturing method for Phos-15-1E-1 or Phos-15-1E-2 chiral was as follows.

[0387] System: Waters SFC 150 Column name: DAISELCHIRALCEL(registered trademark)AD Column model number: 250 x 50 mm, 10 μm Mobile phase A: Supercritical CO2 Mobile phase B: EtOH Wavelength: 214nm Flow rate: 140mL / min Column temperature: RT Injection volume: 7.0mL Circulation time: 10.0min Solvent: Supercritical CO2: Food grade EtOH: Redistilled grade The single-crystal X-ray diffraction pattern of Phos-15-1E-2(((((1R,2R,4R)-4-(2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl)-2-hydroxycyclopentyl)thio)methyl)diethylphosphonate) is shown in Figure 1.

[0388] ● Manufacturing of enantiomer phosphoramidite-42-1 and enantiomer phosphoramidite-42-2 [ka] The method for producing enantiomer phosphoramidite-42-1 and enantiomer phosphoramidite-42-2 was the same as the method for producing enantiomer phosphoramidite-15-1 and enantiomer phosphoramidite-15-2, the only difference being the use of a different stereoisomer intermediate, phos12-2C.

[0389] Production of enantiomer phosphoramidite-11-1 and enantiomer phosphoramidite-11-2 [ka] At room temperature, 15 mL of ethanol and the above compound Phos-15-1E (0.53 g, 1.0 eq, a 1:1 mixture of enantiomers Phos-15-1E-1 and Phos-15-1E-2) were added to a 100 mL flask. Then, 10 mL of an aqueous solution of potassium peroxooxonate complex salt (1.72 g, 2.0 eq.) was added in several portions, and the mixture was stirred overnight at room temperature. TLC detection (MeOH:DCM=1:10, Rf=0.4) showed the formation of new spots, indicating that the compound Phos-15-1E had been completely consumed. The mixture was injected into a saturated sodium bicarbonate aqueous solution (20 mL), extracted with DCM:CF3CH2OH (30 mL x 5), washed with saturated saline solution (50 mL x 2), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a yellow oily Phos-11-1A (0.5 g, yield 87%, a 1:1 mixture of enantiomers Phos-11-1A-1 and Phos-11-1A-22).

[0390] At room temperature under nitrogen gas, 5 mL of dichloromethane, compound Phos-11-1A (0.3 g, 1.0 eq.), and tetraazazole (56 mg, 1.1 eq.) were added to a 100 mL flask. Then, a solution of bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.33 g, 1.5 eq.) in dichloromethane (1 mL) was added dropwise, and the reaction mixture was stirred at room temperature for 1 hour. TLC detection (MeOH:DCM:=1:10, Rf=0.6) showed the formation of new spots, indicating that compound Phos-11-1A had been completely consumed. The mixture was injected into saturated sodium bicarbonate aqueous solution (20 mL) and extracted with dichloromethane (30 mL x 3). The combined organic layer was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product of phosphoramidite-11 was purified by silica gel column chromatography and eluted with MeOH:DCM (=0:100~2:100, 1% Et3N) to obtain yellow, oily phosphoramidite-11 (0.2 g, yield 45%, a 1:1 mixture of enantiomer phosphoramidite-11-1 and phosphoramidite-11-2). 1 H NMR (400 MHz, CD3CN) δ 7.40 (d, J = 8.0, 1H), 5.58 (d, J = 8.0, 1H), 5.01-5.04 (m, 1H), 4.84-4.87 (m, 1H), 4.14-4.18 (m, 4H), 3.76-3.86 (m, 4H), 3.59-3.65 (m, 2H), 2.73-2.77 (m, 2H), 2.66-2.69 (m, 2H), 2.18-2.26 (m, 3H), 1.28-1.32 (m, 6H), 1.17-1.21 (m, 12H). 31 P NMR (400 MHz, CD3CN) δ 148.45, 149.21.

[0391] Enantiomer phosphoramidite-11-1 or enantiomer phosphoramidite-11-2 can be obtained by following the two steps described above, using the corresponding Phos-15-1E-1 or Phos-15-1E-2 obtained by SFC separation and purification as the starting material.

[0392] ● Manufacturing of Phosphoramidite-18 [ka] 1-Methyluracil (1.0 g, 7.9 mmol) and SOCl2 (2.36 g, 19.8 mmol) were added to a 100 mL reaction flask and stirred at 25°C for 1.5 hours. After adding ClSO3H (3.67 g, 31.7 mmol), the reaction mixture was stirred at 75°C for 16 hours. TLC showed that 1-methyluracil was completely consumed and new spots formed. The reaction mixture was added to a mixture of ice and glacial acetic acid (1:1), filtered, and the filtered cake was repeatedly washed with deionized water and vacuum dried to obtain 1.0 g of the gray powder intermediate Phos-18-1A (90% yield). 1 H NMR (400 MHz, DMSO): δ 14.25 (s, 1H), 11.26 (s, 1H), 7.99 (s, 1H), 3.27 (s, 3H).

[0393] A solution of Phos-18-1A (340 mg, 1.52 mmol) in tetrahydrofuran (10 mL) was added to a solution of Phos-13-1C (384 mg, 1.52 mmol) and triethylamine (460 mg, 4.56 mmol) in dichloromethane (30 mL) at 0-5°C, and the mixture was reacted at 25°C for 1.0 hour. LC-MS showed that Phos-13-1C was completely consumed. The resulting reaction mixture was diluted with water (20 mL), extracted with 30% CF3CH2OH / DCM (30 mL x 5), and the combined organic layer was dried over anhydrous sodium sulfate. The mixture was then vacuum concentrated to obtain the residue, which was purified by silica gel column chromatography and eluted with 0%-5% MeOH / DCM to obtain a colorless paste-like Phos-18-1B (400 mg, yield 60%). LC-MS: [M+H + ]=442

[0394] Under nitrogen gas protection, tetraazazole (51 mg, 0.726 mmol) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (reagent P, 327.8 mg, 1.09 mmol) were added to a solution of Phos-18-1B (400 mg, 0.907 mmol) in dried dichloromethane (10 mL), and the reaction was stirred at 25°C for 1.0 hour. LC-MS showed that the starting materials were completely consumed. The resulting reaction mixture was washed with saturated sodium bicarbonate aqueous solution (10 mL x 2) and saline solution (5 mL). The organic phase was dried and concentrated to obtain the crude product, which was purified by flash silica gel column chromatography and eluted with 0%-5% MeOH / DCM (containing 1% TEA) to obtain white solid phosphoramidite-18 (400 mg, yield 68.8%). 1 H NMR (400 MHz, CH3CN) δ 9.29 (s, 1H), 8.15 (d, J = 3.6 Hz, 1H), 4.44 - 4.31 (m, 1H), 4.09 (dd, J = 15.0, 7.4 Hz, 4H), 3.90 - 3.73 (m, 4H), 3.72 - 3.56 (m, 5H), 3.49 (dd, J = 31.5, 11.2 Hz, 1H), 3.36 (d, J = 2.3 Hz, 3H), 2.75 - 2.62 (m, 2H), 1.36 - 1.25 (m, 6H), 1.19 (dd, J = 9.1, 5.3 Hz, 12H). 31 P-NMR (162 MHz, DMSO-d6) δ ppm 146.99 - 147.13 (d, 1 P), 20.58 - 20.76 (s, 1 P).

[0395] Production of phosphoramidite-19 [ka] At room temperature, 60 mL of tetrahydrofuran, Phos-19-SM1 (10 g, 1.0 eq.), and CuBrSMe2 were added to a 250 mL flask. The mixture was cooled to -70°C, then vinyl magnesium bromide (216 mL, 4.0 eq.) was added dropwise, and the mixture was gradually heated to -10°C over 2 hours. TLC detection (petroleum ether:ethyl acetate = 2:1, Rf = 0.4) showed the formation of new spots, indicating that Phos-19-SM1 had been completely consumed. At -10°C, citric acid (150 mL, 10% aqueous solution) was added dropwise to the reaction mixture and extracted with ethyl acetate (100 mL x 3). The combined organic layer was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a brown oily Phos-19-1A (11 g, 95% yield).

[0396] Phos-19-1A contains a pair of enantiomers (3R,4S):(3S,4R)=1:1, [ka] It should be understood that, if necessary, Phos-19-1A racemate or the corresponding chiral pure enantiomer can be used as a starting material to obtain the following intermediates and the corresponding chiral pure compounds of phosphoramidite-19.

[0397] At room temperature and under nitrogen gas, 50 mL of dichloromethane, Phos-19-1A (5 g, 1.0 eq.), and Phos-19-SM2 (10.8 g, 3.0 eq.) were added to a 100 mL flask. Then, Grubbs catalyst (1 g, 0.05 eq.) was added with N2, and the mixture was heated and refluxed overnight. TLC detection of the mixture (ethyl acetate:petroleum ether = 1:1, Rf = 0.25) showed the formation of new spots, indicating that Phos-19-2 had been completely consumed. After removing the solvent, the residue was purified by silica gel column chromatography and eluted with MeOH:DCM (= 0:100~5:95) to obtain brownish oily Phos-19-1B (4.5 g, yield 55%).

[0398] At room temperature and under nitrogen gas, 15 mL of ethyl acetate and Phos-19-1B (1.5 g, 1.0 eq.) were added to a 100 mL flask, and then HCl / ₹ (15 mL, 4 M) was added dropwise. The mixture was stirred overnight at room temperature. LC-MS detection showed that Phos-19-1B had been completely consumed. The solvent was removed to obtain a brown solid, Phos-19-1C (1.5 g, crude product). MS (M+H) = 250.1

[0399] At room temperature and under nitrogen gas, 15 mL of tetrahydrofuran and Phos-19-1C (1.5 g, 1.0 eq.) were added to a 100 mL flask, followed by triethylamine (1.52 g, 3.5 eq.) and compound Phos-13-1D (0.72 g, 0.8 eq.). The mixture was stirred overnight at room temperature. LC-MS showed that Phos-19-1C was completely consumed. The reaction mixture was injected into citric acid (15 mL, 10% aqueous solution), extracted with dichloromethane (30 mL x 5), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with MeOH:DCM (=0:100~10:90) to obtain brownish oily Phos-19-1D (1 g, 39% yield). MS (M+H) = 424.1

[0400] At room temperature and under nitrogen gas, 5 mL of dichloromethane, Phos-19-1D (0.5 g, 1.0 eq.), and tetraazazole (83 mg, 1.1 eq.) were added to a 100 mL flask. Then, a solution of bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.46 g, 1.5 eq.) in dichloromethane (1 mL) was added dropwise, and the reaction mixture was stirred at room temperature for 1 hour. TLC (MeOH:DCM=1:10, Rf=0.65) showed the formation of new spots, indicating that Phos-19-1D had been completely consumed. The mixture was poured into saturated sodium bicarbonate aqueous solution (20 mL) and extracted with dichloromethane (20 mL x 3). The combined organic layer was washed with saturated saline solution (50 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography and eluted with MeOH:DCM (=0:100~5:95, 1% Et3N) to obtain brownish oily phosphoramidite-19 (0.27 g, yield 36%). 1 H NMR (400 MHz, DMSO) δ 11.21 (s,1H), 7.97 (s,1H), 6.45-6.57 (m, 1H), 5.88-5.98 (m, 1H), 4.18-4.23 (m, 1H), 3.91-3.96 (m, 4H), 3.71-3.74 (m, 3H), 3.57-3.62 (m, 1H), 3.49-3.53 (m, 2H), 3.15-3.24 (m, 1H), 2.91-3.01 (m, 1H), 2.83-2.85 (m, 2H), 2.74-2.78 (m, 2H), 1.18-1.23 (m, 6H), 1.06-1.12 (m, 12H). 31 P NMR (400 MHz, DMSO) δ 147.51, 17.01, 16.88.

[0401] Production of phosphoramidite-43 [ka] (3aR,6aR)-2,2-dimethyltetrahydro-3aH-cyclopentadienyl[d][1,3]dioxol-4(6aH)-one (16.2 g, 105 mmol, 1.0 eq), (mercaptomethyl)diethylphosphonate (19.3 g, 105 mmol, 1.0 eq), and dichloromethane (200 mL) were added to a 500 mL flask. The flask was stirred under nitrogen gas protection and cooled to 0-5°C. Then triethylamine (1.06 g, 10.5 mmol, 0.1 eq) was added dropwise. After the addition was complete, the temperature was returned to 25°C and the mixture was stirred overnight under nitrogen gas protection. When the completion of the reaction was detected by LC-MS, the reaction mixture was concentrated under vacuum to obtain the crude product. The crude product was purified by flash column chromatography, eluted with an eluent (EA:DCM = 0%~15%), and the product was concentrated under vacuum to obtain 25 g of Phos-43-1A, a pale yellow oily substance, with a yield of 70.3%. LCMS:M+H=339.5

[0402] Phos-43-1A (25 g, 73.9 mmol, 1.0 eq) and ethanol (250 mL) were added to a 500 mL flask. The flask was stirred under nitrogen gas protection and cooled to 0-5°C. Then sodium borohydride (3.1 g, 81.3 mmol, 1.1 eq) was added in several batches. After the addition was complete, the mixture was stirred for 0.5 hours while maintaining the temperature at 0-5°C. When the completion of the reaction was detected by LC-MS, ice water (200 mL) was added dropwise to the reaction mixture and stirred for 10 minutes. Then the mixture was extracted twice with dichloromethane (500 mL), and the combined organic phase was dried over anhydrous sodium sulfate and concentrated under vacuum to obtain the crude product. The crude product was purified by flash column chromatography, eluted with an eluent (MeOH:DCM = 0%~5%), and the product was concentrated under vacuum to obtain 24.5 g of Phos-43-1B, a pale yellow oily substance, with a yield of 97.4%. LCMS: M+H = 341.5. 1H NMR: (400 MHz, CD3CN), δ ppm 4.67-4.65 (d, J=8.0, 1 H), 4.47-4.42 (m, 2 H), 4.08-4.01 (m, 5 H), 3.27-3.25 (m, 1 H), 2.97-2.93 (d, J=16.0, 2 H), 2.03-1.99 (m, 1 H), 1.73-1.71 (m, 1 H), 1.38 (s, 3 H), 1.26-1.22 (m, 9 H). Phos-43-1B (10 g, 29.4 mmol, 1.0 eq), pyridine (7 g, 88.1 mmol, 3.0 eq), and dichloromethane (100 mL) were added to a 250 mL flask. The flask was stirred under nitrogen gas protection and cooled to -78 °C. Then, trifluoromethanesulfonic anhydride (12.4 g, 44.1 mmol, 1.5 eq) was added dropwise. After the addition was complete, the mixture was stirred for 3 hours under nitrogen gas protection while maintaining the temperature at -78 °C. When the completion of the reaction was detected by LC-MS, the reaction mixture was poured into 50 mL of ice water, then extracted twice with dichloromethane (100 mL). The combined organic phase was dried over anhydrous sodium sulfate and concentrated under vacuum to obtain the crude product Phos-43-1C, which was used directly in the next step. LCMS:M+H=473.4

[0403] Phos-43-1C (16 g, 33.8 mmol, 1.0 eq), 3-benzoyluracil (8.8 g, 40.6 mmol, 1.2 eq), cesium carbonate (22 g, 67.7 mmol), and acetonitrile (200 mL) were added to a 500 mL flask. The flask was stirred overnight at 25°C under nitrogen gas protection. Once the completion of the reaction was detected by LC-MS, the reaction mixture was filtered, and the filtrate was concentrated under vacuum to obtain the crude product. The crude product was purified by flash column chromatography, the product was eluted with an eluent (MeOH:DCM = 0%~5%), and the product was concentrated under vacuum to obtain 18 g of Phos-43-1D, a brown oily substance, with a yield of 98.7%. LCMS:M+H=539.4

[0404] Phos-43-1D (18 g, 33.4 mmol, 1.0 eq) and methanol (180 mL) were added to a 500 mL flask. Ammonia-methanol (180 mL) was added dropwise to the flask under nitrogen gas protection, and after the addition was complete, the mixture was stirred at 25°C under nitrogen gas protection for 5 hours. Once the completion of the reaction was detected by LC-MS, the reaction mixture was concentrated under vacuum to obtain the crude product Phos-43-1E, which was used directly in the next step. LCMS:M+H=435.4

[0405] Phos-43-1E (14.5 g, 33.4 mmol, 1.0 eq) and dioxane (180 mL) were added to a 500 mL flask. Dioxane hydrochloride (4 M, 180 mL) was added dropwise, and the flask was stirred overnight at 25°C under nitrogen gas protection. When the completion of the reaction was detected by LC-MS, the reaction mixture was concentrated under vacuum to obtain the crude product. The crude product was purified by flash column, the product was eluted with an eluent (MeOH:DCM = 0%~10%), and the product was concentrated under vacuum to obtain 5.2 g of Phos-43-1F, a white solid, with a yield of 39.5%. LCMS:M+H=395.4

[0406] Phos-43-1F (5.2 g, 13.2 mmol, 1.0 eq), toluene (100 mL), and acetonitrile (20 mL) were added to a 250 mL flask. Then, cyanomethylenetri-n-butylphosphine (6.4 g, 26.5 mmol, 2.0 eq) was added, and the flask was stirred at 90°C for 48 hours under nitrogen gas protection. When the completion of the reaction was detected by LC-MS, the reaction mixture was concentrated under vacuum to obtain the crude product. The crude product was purified by flash column, and the product was eluted with an eluent (MeOH:DCM = 0%~10%). The product was concentrated under vacuum to obtain 3.5 g of Phos-43-1G, a white solid, with a yield of 70.5%. LCMS:M+H=377.3

[0407] Phos-43-1G (2.0 g, 5.3 mmol, 1.0 eq), anhydrous methanol (20 mL), trimethyl borate (1.1 g, 10.6 mmol, 2.0 eq), methyl orthoformate (0.56 g, 5.3 mmol, 1.0 eq), and sodium bicarbonate (44.5 mg, 0.52 mmol, 0.2 eq) were added to a 250 mL steamer. The mixture was heated to 120 °C and stirred for 48 hours. The steamer was cooled to room temperature, and when the completion of the reaction was detected by LC-MS, the reaction mixture was concentrated under vacuum to obtain the crude product. The crude product was purified by flash column chromatography, and the product was eluted with an eluent (MeOH:DCM = 0%~10%). The product was concentrated under vacuum to obtain 1.3 g of Phos-43-1H, a white solid, with a yield of 60%. LCMS:M+H=409.4

[0408] Phos-43-1H (0.6 g, 1.47 mmol, 1.0 eq) and anhydrous dichloromethane (10 mL) were added to a 50 mL flask, followed by the sequential addition of tetraazazole (0.13 g, 1.76 mmol, 1.2 eq) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.66 g, 2.2 mmol, 1.5 eq). The mixture was stirred at 25°C for 1 hour under nitrogen gas protection. Once the completion of the reaction was detected by LC-MS, the reaction solution was injected into an aqueous sodium bicarbonate solution and extracted twice with dichloromethane (20 mL). The combined organic phase was dried over anhydrous sodium sulfate and then concentrated under vacuum to obtain the crude product. The crude product was purified by flash silica gel column chromatography, and the product was eluted with an eluent (DCM:MeOH:TEA = 0%~5% + 0.2%TEA). The mixture was concentrated under vacuum at 35°C to obtain phosphoramidite-43 (0.89 g, 100% yield), a colorless oily phosphoramidite. LCMS: M+H=609.6 1H NMR: (400 MHz, CD3CN), δ ppm 8.97 (s, 1 H), 7.43-7.41 (d, J=8.0, 1 H), 5.61-5.59 (d, J=8.0, 1 H), 4.76-4.69 (m, 1 H), 4.44-4.34(m, 1 H), 4.14-4.04 (m, 5 H), 3.90-3.81 (m, 2 H), 3.69-3.62 (m, 2 H), 3.46-3.44 (m, 1 H), 3.36-3.33 (d, J=12.0, 3 H), 2.97-2.87 (m, 2 H), 2.77-2.65(m,3H), 1.67-1.56 (m, 1 H), 1.32-1.27 (m, 6 H), 1.24-1.18 (m, 12 H). 31P NMR: (400 MHz, CD3CN), δ ppm 150.03, 148.62; 23.44, 23.24.

[0409] The method for producing phosphoramidite-47 was the same as that for phosphoramidite-43, the only difference being that the starting material 5-methyluracil was used as a nucleic acid base.

[0410] Production of Phosphoramidite-45 [ka] Magnesium waste (0.26 g, 10.6 mmol, 10.0 eq) and anhydrous ethanol (40 mL) were added to a 100 mL steamer, and the mixture was heated to 90°C and stirred for 18 hours. The steamer was cooled to room temperature, and Phos-43-1G (0.4 g, 1.06 mmol, 1.0 eq) was added, and the mixture was heated to 90°C and stirred for 18 hours. The steamer was cooled to room temperature, and LC-MS detected that the reaction was not complete and that approximately 50% had been converted to Phos-45-1A. The reaction mixture was concentrated under vacuum to obtain the crude product. The crude product was purified by flash column chromatography, and the product was eluted with an eluent (MeOH:DCM = 0%~8%). The product was concentrated under vacuum to obtain 0.13 g of Phos-45-1A, a pale yellow oily substance, with a yield of 29%. LCMS:M+H=423.4

[0411] Phos-45-1A (0.11 g, 0.26 mmol, 1.0 eq) and anhydrous dichloromethane (3 mL) were added to a 50 mL flask, followed by the sequential addition of tetraazazole (22 mg, 0.31 mmol, 1.2 eq) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.12 g, 0.4 mmol, 1.5 eq). The mixture was stirred at 25°C for 1 hour under nitrogen gas protection. Once the completion of the reaction was detected by LC-MS, the reaction solution was injected into an aqueous sodium bicarbonate solution and extracted twice with dichloromethane (10 mL). The combined organic phase was dried over anhydrous sodium sulfate and then concentrated under vacuum to obtain the crude product. The crude product was purified by flash silica gel column chromatography, and the product was eluted with an eluent (DCM:MeOH:TEA = 0%~3% + 0.2%TEA). The mixture was concentrated under vacuum at 35°C to obtain phosphoramidite Phos-45 (0.1 g, yield 61.7%), a yellow oily substance. LCMS: M+H=623.5, 1 H NMR: (400 MHz, CD3CN), δ ppm 7.42-7.40 (d, J=8.0, 1 H), 5.61-5.59 (d, J=8.0, 1 H), 4.73-4.68 (m, 1 H), 4.34-4.30(m, 1 H), 4.12-4.06 (m, 5 H), 3.88-3.83 (m, 2 H), 3.68-3.65 (m, 2 H), 3.58-3.42 (m, 2 H), 2.97-2.86 (m, 3 H), 2.72-2.62(m,3 H), 1.65-1.53 ​​(m, 1 H), 1.32-1.17 (m, 18H), 1.13-1.10 (t, J=6.8, 3 H). 31 P NMR: (400 MHz, CD3CN), δ ppm 149.85, 148.50; 23.46, 23.25.

[0412] ● Manufacturing of Phosphoramidite-46 [ka] Magnesium waste (0.48 g, 20.0 mmol, 15.0 eq) and anhydrous ethylene glycol monomethyl ether (50 mL) were added to a 100 mL steamer, and the mixture was heated to 90°C and stirred for 1 hour. The steamer was cooled to room temperature, Phos-43-1G (0.5 g, 1.33 mmol, 1.0 eq) was added, and the mixture was heated to 90°C and stirred for 18 hours. The steamer was cooled to room temperature, and LC-MS detected that the reaction materials had completely disappeared. The reaction mixture was transferred to a flask, 0.5 N dilute hydrochloric acid was added dropwise at 0°C to adjust the pH to 6, and the mixture was extracted five times with dichloromethane (50 mL). The combined organic phase was dried over anhydrous sodium sulfate and then concentrated under vacuum to obtain the crude product. The crude product was purified by flash column chromatography, and the product was eluted with an eluent (MeOH:DCM = 0%~10%). The product was concentrated under vacuum to obtain 0.13 g of Phos-46-1A, a pale yellow oily substance, with a yield of 20%. LCMS: M+H=513.4.

[0413] In a 50 mL flask, Phos-46-1A (0.1 g, 0.19 mmol, 1.0 eq) and anhydrous dichloromethane (3 mL) were added, followed by tetraazazole (16 mg, 0.23 mmol, 1.2 eq) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.09 g, 0.3 mmol, 1.5 eq). The mixture was stirred at 25°C for 1 hour under nitrogen gas protection. Once the completion of the reaction was detected by LC-MS, the reaction solution was injected into an aqueous sodium bicarbonate solution and extracted twice with dichloromethane (10 mL). The combined organic phase was dried over anhydrous sodium sulfate and then concentrated under vacuum to obtain the crude product. The crude product was purified by flash silica gel column chromatography, and the product was eluted with an eluent (DCM:MeOH:TEA = 0%~5% + 0.2%TEA). The mixture was concentrated under vacuum at 35°C to obtain phosphoramidite Phos-46 (93 mg, yield 66.9%), a yellow oily substance. LCMS: M+H = 713.6. 1H NMR: (400 MHz, CD3CN), δ ppm 8.92 (s, 1 H), 7.47-7.44 (dd, J=8.0, J=2.8, 1 H), 5.64-5.62 (d, J=8.0, 1 H), 4.78-4.68 (m, 1 H), 4.42-4.22 (m, 2H), 4.18-4.15 (m, 4 H), 3.92-3.85 (m, 2 H), 3.78-3.66 (m, 3 H), 3.60-3.52 (m, 5 H), 3.48-3.45 (m, 3 H), 3.36-3.35 (dd, J=2.4, J=0.8, 6 H), 3.28-3.27 (d, J=5.6, 3 H), 3.03-2.94 (m, 2 H), 2.75-2.63(m,3 H), 1.65-1.53 ​​(m, 1 H), 1.22-1.17 (m, 12 H). 31 P NMR: (400 MHz, CD3CN), δ ppm 149.85, 148.42; 24.43, 24.22.

[0414] Other 5'-phosphonate-modified nucleoside analogs described herein can be obtained by similar methods or by synthetic routes well known in the art.

[0415] ●Example 2. Synthesis of Alkyl Alkyls Oligonucleotides used in accordance with the present invention can be conveniently and conventionally produced by well-known solid-phase synthesis techniques. Apparatus for this synthesis, including, for example, Mermade12 (LGC), is available from several suppliers. Furthermore, as an alternative, any other apparatus known in the art can be used for such synthesis. Similar techniques are well known to be used to produce oligonucleotides such as alkylated derivatives and those having phosphorothioate bonds.

[0416] Oligonucleotides: Unsubstituted and substituted phosphodiester (O) oligonucleotides (including, but not limited to, oligonucleotides) can be synthesized using standard phosphoramidite chemistry and iodine oxidation in an automated DNA synthesizer, Mermade12 (LGC).

[0417] In one embodiment, the phosphorothioate nucleoside bond (S) was synthesized in a manner similar to that of the phosphodiester nucleoside bond, except that the phosphite ester bond was oxidized with a 10% w / v solution of 3,H-1,2-benzodithiol-3-one-1,1-dioxide in acetonitrile to achieve thiohybridization. The time of the thiohybridization reaction step was increased to 180 seconds and continued through a normal capping step. After treatment in concentrated ammonia water at 55°C (12-16 h), the oligonucleotide was recovered by cleavage and deblocking from the CPG column, and then precipitation in a 1 M NH4OAc solution with more than 3 times the volume of ethanol.

[0418] Phosphinate nucleoside bonds were prepared as described in US 5,508,270. Alkyl phosphate nucleoside bonds may be prepared as described in US 4,469,863, or by the phosphoramidite method described herein. 3'-Deoxy-3'-methylenephosphonate nucleoside bonds were prepared as described in US 5,610,289 or 5,625,050. Phosphoramidite nucleoside bonds were prepared as described in US 5,256,775 or US 5,366,878. Alkyl phosphorothioate nucleoside bonds were prepared as described in the published WO94 / 17093 and WO94 / 02499. The 3'-deoxy-3'-aminophosphoramidate nucleoside bond was prepared as described in US5,476,925. The phosphotriester nucleoside bond was prepared as described in US5,023,243, and several methods for preparing phosphate nucleoside bonds can be found in Beilstein J Org Chem. 2017, 13:1368-1387.

[0419] One or more phosphorus-free internucleoside bonds include, but are not limited to, oligonucleotides of methylenemethylimino-linked oligonucleotides, methylenedimethylhydrazine-linked oligonucleotides, methylenecarbonylamino-linked oligonucleotides, and methyleneaminocarbonyl-linked oligonucleotides, as well as mixed backbone oligonucleotides having, for example, alternating O or S bonds, prepared as described in US5,378,825, US5,386,023, US5,489,677, US5,602,240, and US5,610,289.

[0420] The double-stranded ribonucleic acid (dsRNA) reagent was obtained by forming a double-stranded molecule by mixing two complementary strands (sense strand and antisense strand) in a 1:1 molar ratio.

[0421] The double-stranded compounds in Table 2 were synthesized using an oligonucleotide synthesizer, typically employing a mature solid-phase synthesis method based on phosphoramidite chemistry, to obtain the sense and antisense strand sequences of dsRNA. Oligonucleotide chain extension was achieved through a four-step cycle consisting of deprotection, condensation, capping, and oxidation or sulfidation steps for the addition of each nucleotide. Synthesis was performed on a solid support made from porous control glass (CPG, 1000 angstroms). While common monomer phosphoramidites are commercially available, the 5'-phosphonate-modified nucleoside analog compounds described herein can be introduced into the oligonucleotide chain instead of monomer phosphoramidites. The 5'-phosphonate-modified nucleoside analog compounds described herein are used in the final coupling reaction if they can be ligated to the 5'-terminus instead of the monomer phosphoramidite. Trichloroacetic acid (TCA) in 3% dichloromethane was used to deprotect the 4,4'-dimethoxytrityl protecting group (DMT). 5-ethylthio-1H-tetrazolyl was used as an activator. I2 in THF / Py / H2O and phenylacetyl disulfide (PADS) in pyridine / MeCN were used for oxidation and sulfidation reactions, respectively. After the final solid-phase synthesis step, the oligomers bound to the solid support were cleaved and protecting groups removed by treatment with 1:1 volume 20 wt% aqueous methylamine solution and 28% ammonium hydroxide solution. The crude mixture was concentrated to synthesize oligonucleotides for use in in vitro screening. The remaining solid was dissolved in 1.0 M NaOAc and ice-cold EtOH was added to precipitate the single-chain product as a sodium salt, which could be used for annealing without further purification. To synthesize oligonucleotides for use in in vivo testing, the crude single-chain product was further purified by ion-pair reverse-phase HPLC (IP-RP-HPLC). The purified single-stranded oligonucleotide product obtained from IP-RP-HPLC was dissolved in 1.0 M NaOAc and precipitated by adding ice-cold EtOH, thereby converting it to a sodium salt.To form double-stranded oligonucleotide products, sense and antisense oligonucleotides were annealed in water using an equimolar complementation method.

[0422] The phosphoramidite compound described in Example 1 was coupled to the 5' end of an oligonucleotide by the method described in CN110072530A and CN103154014A to produce a 5'-terminal nucleotide. The atoms of each hydroxy protecting group of the phosphonate group, for example, the oxygen atom containing two methyl or ethyl protecting groups, were removed by removing one or two of the methyl or ethyl groups according to the deprotection step used. In some examples, the ethyl protecting group was removed using a carbonitride:trimethyliodosilane:pyridine e=50:2:2(v / v / v) deethyl solution.

[0423] The oligonucleotides containing the 5'-terminal nucleotide of the present invention were obtained by the methods described herein or by methods well known to those skilled in the art, and the dsRNA double-stranded bodies having an oligonucleotide sequence structure targeting FXII are shown in Table 2.

[0424] The 5'-terminal nucleoside structure of the double-stranded body in Table 2 is shown below. [Table 6-1] [Table 6-2] [Table 6-3]

[0425] As shown in Tables 2-3, the double-stranded nucleotide includes a sense strand and an antisense strand. Chemical modifications are represented by uppercase letters for 2'-fluoromodified nucleotides and lowercase letters for 2'-methoxy group modified nucleotides. The superscript "*" between two nucleosides represents a phosphorothioate internuclear bond, the absence of a superscript between two nucleosides represents a phosphodiester internuclear bond. Invab is reverse-baseless, and GLS-15 is the targeting group described herein.

[0426] Table 2 Targeting of FXII double-stranded bodies [Table 7] Table 3 Targeting of FXII double-stranded bodies [Table 8-1] [Table 8-2]

[0427] As shown in Tables 2 and 3 above, the distranded bodies are composed of the same sense chain, and the only difference between the distranded bodies is that the 5' end of the antisense chain contains a different uracil nucleoside derivative.

[0428] Example 3. In vivo evaluation of oligonucleotides containing the 5'-terminal nucleotide of the present invention. To evaluate the in vivo activity of FXII double-stranded nucleoside nucleic acid (also known as dsRNA), we used 6-week-old, pathogen-free female C57BL / 6 mice purchased from Shanghai Slack Laboratory Animals Co., Ltd.

[0429] ●a) Experimental plan In animal experiments, four female C57BL / 6 mice were administered to each group at a dose of 1 or 2 mg / kg, with a single administration frequency of S / C. Blood samples were collected on days 8, 22, and 29, and plasma FXII protein levels were detected.

[0430] b) Definition of experimental days: The day on which the first dose was administered to the mice was defined as day 1 of the experiment, the previous day as day -1, and the following day as day 2. The number of experimental days was then estimated by analogy.

[0431] c) All compounds awaiting measurement were prepared as a 5 mg / mL stock solution in PBS before administration. A small amount of the 5 mg / mL stock solution was taken, diluted 20-fold, and the OD value was detected using a Nanodrop machine. After converting based on the OD value to obtain the actual concentration of the stock solution, an appropriate amount was taken, diluted to the working fluid concentration (0.2 mg / mL), and administered.

[0432] d) All mice (C57BL / 6, female, 6 weeks old) underwent a 6-day adaptation period, after which they were administered by subcutaneous injection on day 1 as described above.

[0433] e) On days 8, 22, and 29, plasma was collected from all mice by submandibular vein sampling and used to detect FXII protein levels.

[0434] f) Sample detection analysis FXII protein levels in mouse plasma were detected using an ELISA reagent kit.

[0435] FXII protein levels in mouse plasma were detected using an ELISA reagent kit (Molecular Innovations, IMSFXIIKTT). Simply put, plasma samples collected from EDTA-K2 were diluted 30,000-fold, incubated on a detection plate coated with capture antibody, then the detection antibody and HRP-coupled secondary antibody were added sequentially. Finally, the samples were color-developed using TMB, and the absorbance at 450 nm was read. A standard curve was fitted using a four-parameter method, and the OD value of each detected sample was substituted to calculate the FXII protein content. Multiplying this by the dilution factor yielded the original plasma FXII protein concentration. The results are shown in Table 4.

[0436] Table 4. Relative expression levels of FXII protein using oligo compounds corresponding to the sequence, chemical modification, and delivery shown in Table 2. Table 4. Percentage of remaining FXII protein expression in mouse plasma [Table 9-1] [Table 9-2] .

[0437] Example 4. In vivo evaluation of oligonucleotides containing the 5'-terminal nucleotide of the present invention. To evaluate the in vivo activity of FXII double-stranded nucleoside nucleic acid (also known as dsRNA), we used 6-week-old, pathogen-free female C57BL / 6 mice purchased from Shanghai Slack Laboratory Animals Co., Ltd.

[0438] ●a) Experimental plan In animal experiments, four female C57BL / 6 mice were used in each group. The dosage was 0.5 or 1 mg / kg, administered once on day 1. Blood samples were collected on days 8, 22, and 29, and plasma FXII protein levels were detected.

[0439] b) Definition of experimental days: The day on which the first dose was administered to the mice was defined as day 1 of the experiment, the previous day as day -1, and the following day as day 2. The number of experimental days was then estimated by analogy.

[0440] c) All compounds awaiting measurement were prepared as a 5 mg / mL stock solution in PBS before administration. A small amount of the 5 mg / mL stock solution was taken, diluted 20-fold, and the OD value was detected using a Nanodrop machine. After converting based on the OD value to obtain the actual concentration of the stock solution, an appropriate amount was taken, diluted to the working fluid concentration (0.2 mg / mL), and administered.

[0441] d) All mice (C57BL / 6, female, 6 weeks old) underwent a 6-day adaptation period, after which they were administered by subcutaneous injection on day 1 as described above.

[0442] e) On days 8, 22, and 29, plasma was collected from all mice by submandibular vein sampling and used to detect FXII protein levels.

[0443] f) Sample detection analysis FXII protein levels in mouse plasma were detected using an ELISA reagent kit.

[0444] FXII protein levels in mouse plasma were detected using an ELISA reagent kit (Molecular Innovations, IMSFXIIKTT). Simply put, plasma samples collected from EDTA-K2 were diluted 30,000-fold, incubated on a detection plate coated with capture antibody, then the detection antibody and HRP-coupled secondary antibody were added sequentially. Finally, the samples were color-developed using TMB, and the absorbance at 450 nm was read. A standard curve was fitted using a four-parameter method, and the OD value of each detected sample was substituted to calculate the FXII protein content. Multiplying this by the dilution factor yielded the original plasma FXII protein concentration. The results are shown in Table 5.

[0445] Table 5. Relative expression levels of FXII protein using oligo compounds corresponding to the sequences, chemical modifications, and delivery shown in Tables 2 and 3. [Table 10]

[0446] Example 5. In vivo evaluation of oligonucleotides containing the 5'-terminal nucleotide of the present invention. The experimental method was the same as in Example 4, and the relative expression levels of the FXII protein were evaluated using oligo compounds corresponding to the sequences, chemical modifications, and delivery shown in Tables 2 and 3.

[0447] Table 6. Percentage of remaining FXII protein expression in mouse plasma [Table 11]

[0448] As can be seen from Table 6, the gene silencing effect using dsRNA containing a 5'-S phosphonate-modified nucleoside analog (e.g., AD01177) is superior to that using dsRNA containing a 5'-O phosphonate-modified nucleoside analog (AD01558).

[0449] Table 7. Percentage of remaining FXII protein expression in mouse plasma [Table 12] Table 8. Percentage of remaining FXII protein expression in mouse plasma [Table 13]

[0450] Example 6. In vivo evaluation of oligonucleotides containing the 5'-terminal nucleotide of the present invention. The experimental method was the same as in Example 4, and the relative expression levels of the FXII protein were evaluated using oligo compounds corresponding to the sequences, chemical modifications, and delivery shown in Tables 2 and 3.

[0451] Table 9. Percentage of FXII protein expression remaining in mouse plasma [Table 14]

[0452] ●Equivalent While several embodiments of the present invention have been described and explained herein, it will be readily apparent to those skilled in the art that various other means and / or structures for performing the functions described herein and / or obtaining the results and / or one or more advantages, as well as each of these variations and / or modifications, are considered to be within the scope of the present invention. More generally, those skilled in the art will readily apparent that all parameters, dimensions, materials and configurations described herein are illustrative, and that the actual parameters, dimensions, materials and / or configurations depend on the specific application taught by the present invention. Those skilled in the art will recognize many equivalents of the specific embodiments of the present invention described herein, or can determine them simply by using conventional experiments. Accordingly, it should be understood that the above embodiments are presented only as examples and are within the scope of the appended claims and their equivalents, and that the present invention can be implemented in ways different from those specifically described and claimed. The present invention relates to each of the individual features, systems, articles, materials and / or methods described herein. Furthermore, any combination of two or more such features, systems, articles, materials, and / or methods is also included within the scope of the present invention, provided that such features, systems, articles, materials, and / or methods are not contradictory to each other.

[0453] All definitions defined and used herein should be understood as the definitions in comparative dictionaries, the definitions in documents incorporated by citation, and / or the ordinary meanings of the defined terms.

[0454] Unless otherwise explicitly stated, if no numerical limitation is used in the specification or claims, it should be understood as "at least one".

[0455] The phrase "and / or" as used in the specification and claims should be understood to mean "one or two" of the elements thus combined, that is, such elements may appear together in some cases and separately in others. In addition to the elements specifically marked by "and / or," other elements may be optionally present, whether related to the specifically marked elements or not, unless the opposite is explicitly specified.

[0456] All references, patents and patent applications, and publications cited or referenced herein are incorporated herein by reference in their entirety.

Claims

1. An oligonucleotide comprising a 5'-terminal nucleotide represented by formula (V), formula (VI), or formula (VII) and one of their stereoisomers, 【Chemistry 1】 Among them, each T １ This is an independently and optionally protected phosphine moiety, Each T ３ This is an internucleoside linking group that independently links the 5'-terminal nucleotide to the oligonucleotide, Each X １ is independently a chemical bond, O, S, NJ １ or CJ １ J ２ wherein J １ and J ２ are each independently hydrogen, halogen, a sulfonyl group, a sulfinyl group, optionally substituted C １ -C ６ alkyl group, optionally substituted C ３ -C ６ cycloalkyl group, optionally substituted C ２ -C ６ alkenyl group, optionally substituted C ２ -C ６ alkynyl group, optionally substituted C ５ -C １２ aryl group, optionally substituted 5- to 12-membered heteroaryl group, optionally substituted 5- to 12-membered heterocyclic ring, Each X ２ CR independently １５ or N, Each X ３ These are C, which is independently chemically bonded and optionally substituted. １ -C ３ Alkylene group, SO, SO ２ C(=O), P(=O)R, where R is OH, SH, C １ -C ６ Alkyl, NH ２ NHSO ２ CH ３ And, Each Bx is independently a heterocyclic base moiety. Each R １ and R ２ These are H, halogen, and optionally substituted C, respectively, independently. １ -C ６ Alkyl alkyl groups, optionally substituted C １ -C ６ Alkoxy groups, optionally substituted C ２ -C ６ Alkenyl group, optionally substituted C ２ -C ６ These are alkynyl groups, sulfinyl groups, sulfonyl groups, and acetyl groups. Each R ３ and R １５ These are H, halogen, and optionally substituted C, respectively, independently. １ -C ６ Alkyl alkyl groups, optionally substituted C １ -C ６ Alkoxy groups, optionally substituted C ２ -C ６ Alkenyl group, optionally substituted C ２ -C ６ It is an alkynyl group, Each A independently possesses one of the following equations: 【Chemistry 2】 Q １ and Q ２ These are H, halogen, -CN, and optionally substituted C, respectively, independently. １ -C ６ Alkyl alkyl groups, optionally substituted C １ -C ６ Alkoxy groups, optionally substituted C ２ -C ６ Alkenyl group, optionally substituted C ２ -C ６ Alkynyl group or NR ４ R ５ And, Each Q ３ These are O, S, and NR independently. ６ or CR ７ R ８ And, Each Q ４ Q ５ Q ６ Q ７ Q ９ Q １０ Q １１ and Q １２ These are, independently, H, halogen, optionally protected hydroxyl group, acetoxy group, azide group, and optionally substituted C. １ -C ６ Alkyl alkyl groups, optionally substituted C １ -C ６ Alkoxy groups, optionally substituted C ２ -C ６ Alkenyl group, optionally substituted C ２ -C ６ Alkynyl group, NR ９ R １０ And, Each Q ８ These are O, S, SO, SO independently. ２ PR １６ R １７ Or NR １１ And, Each R １６ and R １７ is independently (=O), (=S), OH, SH, C １ -C ６ alkyl group, NR １８ R １９ wherein Each R ４ 、R ５ 、R ６ 、R ７ 、R ８ 、R ９ 、R １０ 、R １１ 、R １８ and R １９ is independently H, optionally substituted C １ -C ６ alkyl group, optionally substituted C １ -C ６ alkoxy group, methanesulfonyl group, sulfonic acid group, C(=O)J ３ 、C(=O)OJ ３ or C(=O)N(J ３ )(J ４ ) and M １ These are C(Rd)(Re), C(Rd)(Re)C(Rf)(Rg), where each Rd, Re, Rg and Rf is independently hydrogen, halogen, hydroxyl group, and C １ -C ６ alkyl group, C １ -C ６ Alkoxy group, C １ -C ６ Alkylthio group, O(CH ２ ) ２ - OCH ３ NJ ５ ,CN,OC(=O)J ５ ,OC(=O)N(J ５ ) (J ６ ) or C(=O)N((J ５ ) (J ６ A substituent selected from ) Each J ３ J ４ J ５ and J ６ H or C １ -C ６ It is an alkyl group, n is 0, 1, or 2. Each optionally substituted group can independently be a halogen, a hydroxyl group, or C １ -C ６ alkyl group, C １ -C ６ Alkoxy group, C １ -C ６ The alkylthio group comprises one or more substituents selected from CN. Oligonucleotides.

2. T １ This is an optionally protected phosphine moiety, having the following formula: 【Transformation 3】 Eventually, Ra and Rc are independently a hydroxyl group or a protected hydroxyl group, a mercapto group or a protected mercapto group, or optionally substituted with C １ -C ６ Alkyl alkyl groups, optionally substituted C １ -C ６ Selected from alkoxy groups, amino groups or protected / substituted amino groups, natural or modified nucleosides, and R ｂ is O, S or NR １２ And R １２ is hydrogen, C １ -C ６ Alkyl alkyl groups and amino protecting groups, The substituents in the substituted amino group are optionally substituted C １ -C ６ Alkyl alkyl groups, optionally substituted C ２ -C ６ Alkenyl group, optionally substituted C ２ -C ６ Selected from alkynyl group, sulfinyl group, sulfonyl group, and acetyl group, Each optionally substituted group can independently be a halogen, a hydroxyl group, or C １ -C ６ alkyl group, C １ -C ６ Alkoxy group, C １ -C ６ The alkylthio group comprises one or more substituents selected from CN. The oligonucleotide according to claim 1.

3. The protecting groups for the hydroxyl group or mercapto group are, independently, methyl group, ethyl group, benzyl group (Bn), phenyl group, isopropyl group, tert-butyl group, acetyl group, chloroacetyl group, trichloroacetyl group, trifluoroacetyl group, pivaloyl group, tert-butoxymethyl group, methoxymethyl group, 1-ethoxyethyl group, 1-(2-chloroethoxy)ethyl group, 2-trimethylsilylethyl group, allyl group, cyclohexyl group (cHex), 9-fluorenylmethoxycarbonyl group, methanesulfonic acid ester, and toluenesulfonic acid. phosphate ester, trifluoromethanesulfonic acid ester, benzoyl group, benzoyl formate, p-phenylbenzoyl group, 4-methoxybenzyl group, monomethoxytrityl group, dimethoxytrityl group, trimethoxytrityl group, 4-chlorobenzyl group, 4-nitrobenzyl group, 2,4-dinitrophenyl group, 4-acyloxybenzyl group, 2-methylphenyl group, 2,6-dimethylphenyl group, 2-chlorophenyl group, 2,6-dichlorobenzyl group, diphenylmethyl group, triphenylmethyl group, 4-methylthio-1-butyl group, 2-( S-acetylthio)ethyl group (SATE), 2-cyanoethyl group, 2-cyano-1,1-dimethylethyl (CDM), 4-cyano-2-butenyl group, 2-(trimethylsilyl)ethyl group (TSE), 2-(phenylthio)ethyl group, 2-(triphenylsilyl)ethyl group, 2-(benzylsulfonyl)ethyl group, 2,2,2-trichloroethyl group, 2,2,2-tribromoethyl group, 2,3-dibromopropyl group, 2,2,2-trifluoroethyl group, phenylthio group, 2-chloro-4-tritylphenyl group, 2-bromophenyl group, Selected from 2-[N-isopropyl group-N-(4-methoxybenzoyl)amino]ethyl group, 4-(N-trifluoroacetylamino)butyl group, 4-oxopentyl group, 4-tritylaminophenyl group, 4-benzylaminophenyl group, tetrahydropyranyl group, morpholino, trimethylsilyl group, triethylsilyl group, tert-butyldimethylsilyl group, tert-butyldiphenylsilyl group, triphenylsilyl group, triisopropylsilyl group, pivalate methyl ether group (POM), and 9-phenylxanthin-9-yl, The amino protecting group is independently selected from 2-trimethylsilylethoxycarbonyl group (Teoc), 1-methyl-1-(4-biphenyl)ethoxycarbonyl group (Bpoc), tert-butoxycarbonyl group (BOC), allyloxycarbonyl group (Alloc), 9-fluorenylmethoxycarbonyl group (Fmoc), benzyloxycarbonyl group (Cbz), benzyl group, formyl group, acetyl group, pivaloyl group, trihaloacetyl group, benzoyl group, nitrophenyl group, acetyl group, 2-nitrobenzenesulfonyl group, phthalimide group (Pht), p-toluenesulfonyl group (Tos), trityl group (Trt), 2,4-dimethoxybenzyl group (PMB), and dithiosuccinyl group. The oligonucleotide according to claim 2.

4. T １ This is an optionally protected phosphine moiety, having the following formula: 【Chemistry 4】 Eventually, R ｂ is O or S, and Ra and Rc are each independently protected hydroxyl groups, C １ -C ６ alkyl group, C １ -C ６ Selected from alkoxy groups, Oligonucleotides according to claims 2 to 3.

5. Ra and Rc are each OH, SH, OCH ３ , OCH ２ CH ３ , OCH(CH ３ ), ２ , OCH ２ OC(=O)C(CH ３ ), ３ , NH ２ , OCH ２ CH ２ CN, NHSO ２ CH ３ respectively. Oligonucleotides according to claims 2 to 3.

6. Ra and Rc are OH groups, and R ｂ is O, Oligonucleotides according to claims 2 to 3.

7. Said T ３ This is a nucleoside linking group selected from phosphorus-containing linking groups or phosphorus-free linking groups. The oligonucleotide according to claims 1 to 6.

8. The phosphorus-containing internucleoside linking groups are independently phosphodiester linking groups, phosphotriester linking groups, phosphorothioate linking groups, phosphorodithioate linking groups, alkylphosphonate linking groups, aminophosphonate linking groups, phosphonate linking groups, phosphinate linking groups, thiophosphoramidate linking groups, and phosphoramidate linking groups. The oligonucleotide according to claim 7.

9. The nucleoside linking group is independently an alkylphosphonate linking group, a phosphodiester nucleoside linking group, or a phosphorothioate nucleoside linking group. The oligonucleotide according to claim 7.

10. B Ｘ The heterocyclic base portion is selected from natural nucleic acid bases, modified nucleic acid bases, and universal bases. The oligonucleotide according to claims 1 to 9.

11. B Ｘ The heterocyclic base moiety is selected from pyrimidine, substituted pyrimidine, pseudouracil, substituted pseudouracil, purine, hypoxanthine, or substituted purine. The oligonucleotide according to claim 10.

12. B Ｘ The heterocyclic base moiety is selected from 2-thiouracil, 5-fluorouracil, dihydrouridine (D), 7-methylguanosine (m7G), uracil, 5-thiazolauracil, thymine, cytosine, pseudouracil, N1-methyl-psoidouracil, hypoxanthine, 5-methylcytosine, 5-methyluracil, 3-benzoyluracil, 2,6-diaminopurine, adenine, or guanine. The oligonucleotide according to claim 10.

13. The aforementioned R １５ and R ３ All of them are H. The oligonucleotide according to claims 1 to 12.

14. The aforementioned X １ is O, Oligonucleotides according to claims 1 to 13.

15. Each of the aforementioned R １ and R ２ These are, independently, H, a methylsulfonyl group, and an acetyl group. The oligonucleotide according to claims 1 to 14.

16. Each of the aforementioned X ２ Each of these is independently N, and each R １ and R ２ These are, independently, H, a methylsulfonyl group, and an acetyl group. The oligonucleotide according to claims 1 to 14.

17. In the above formulas (V), (VI), or (VII), A has one of the following formulas: 【Transformation 5】 Eventually, Q １ and Q ２ These are H, halogen, -CN, and optionally substituted C, respectively, independently. １ -C ６ It is an alkyl group, Q ８ is O, S, SO, SO ２ PR １６ R １７ Or NR １１ And R １６ and R １７ These are independently (=O), (=S), OH, SH, C １ -C ６ Alkyl, NR １８ R １９ And, R １１ , R １８ and R １９ H is independently substituted, and C is optionally substituted. １ -C ６ Alkyl alkyl groups, optionally substituted C １ -C ６ Alkoxy group, methanesulfonyl group, sulfonic acid group, C(=O)J ３ , C(=O)OJ ３ or C(=O)N(J ３ ) (J ４ ) and J ３ and J ４ H or C １ -C ６ It is an alkyl group, Each optionally substituted group can independently be a halogen, a hydroxyl group, or C １ -C ６ alkyl group, C １ -C ６ Alkoxy group, C １ -C ６ The alkylthio group comprises one or more substituents selected from CN. The oligonucleotide according to claims 1 to 16.

18. The aforementioned Q １ and Q ２ These are, independently, H, F, -CN, and a methyl group. The oligonucleotide according to claims 1 to 17.

19. The aforementioned Q １ and Q ２ Each of these is independently H. The oligonucleotide according to claim 18.

20. Condition M １ C(Rd)(Re), X ２ C, X ３ is a chemical bond, A is 【Transformation 6】 If that is the case, Q ８ is S, SO, SO ２ PR １６ R １７ Or NR １１ That is, The oligonucleotide according to claims 1 to 19.

21. Q ８ is S, SO, SO ２ PR １６ R １７ Or NR １１ That is, Oligonucleotides according to claims 1 to 20.

22. The aforementioned X ２ is N, X ３ This is a chemical bond, -CH ２ -ien-CH ２ CH ２ -, SO, SO ２ That is, The oligonucleotide according to claims 1 to 21.

23. The aforementioned X ２ C, X ３ It is a chemical bond. The oligonucleotide according to claims 1 to 21.

24. The aforementioned n is 0 or 1. Oligonucleotides according to claims 1 to 23.

25. It comprises a 5'-terminal nucleotide represented by formula (V-1) or its stereoisomer, 【Transformation 7】 Eventually, T １ , T ３ A, R ３ And Bx are as defined in claim 1, respectively, and X ３ These are chemical bonds, C(=O), P(=O)R, SO or SO ２ And M １ These are C(Rd)(Re), C(Rd)(Re)C(Rg)(Rf), where each Rd, Re, Rg and Rf is independently hydrogen, halogen, hydroxyl group, and C １ -C ６ alkyl group, C １ -C ６ Alkoxy group, C １ -C ６ Alkylthio group, O(CH ２ ) ２ - OCH ３ NJ ５ ,CN,OC(=O)J ５ ,OC(=O)N(J ５ ) (J ６ ) or C(=O)N((J ５ ) (J ６ A substituent selected from ) and J ５ J ６ H or C １ -C ６ It is an alkyl group, where R is OH, SH, or C. １ -C ６ Alkyl, NH ２ NHSO ２ CH ３ That is, The oligonucleotide according to claim 1.

26. The aforementioned X ３ SO ２ or chemical bond, R ３ It is hydrogen. The oligonucleotide according to claim 25.

27. Rd, Re, Rg, and Rf are each independently hydrogen, fluorine, a hydroxyl group, and C １ -C ６ Alkoxy group, O(CH ２ ) ２ - OCH ３ A substituent selected from, Oligonucleotides according to claims 25 to 26.

28. The Rd, Re, Rg, and Rf are substituents independently selected from hydrogen. The oligonucleotide according to claim 27.

29. A has one of the following equations: 【Transformation 8】 Q １ and Q ２ These are H, halogen, and optionally substituted C, respectively, independently. １ -C ６ It is an alkyl group, Q ８ is O, S, SO, SO ２ PR １６ R １７ Or NR １１ And R １６ and R １７ These are independently (=O), (=S), OH, SH, C １ -C ６ Alkyl, NR １８ R １９ And, R １１ , R １８ and R １９ H is independently substituted, and C is optionally substituted. １ -C ６ Alkyl alkyl groups, optionally substituted C １ -C ６ Alkoxy group, methanesulfonyl group, sulfonic acid group, C(=O)J ３ , C(=O)OJ ３ or C(=O)N(J ３ ) (J ４ ) and J ３ and J ４ H or C １ -C ６ It is an alkyl group, Each optionally substituted group can independently be a halogen, a hydroxyl group, or C １ -C ６ alkyl group, C １ -C ６ Alkoxy group, C １ -C ６ The alkylthio group comprises one or more substituents selected from CN. Oligonucleotides according to claims 25 to 28.

30. It comprises a 5'-terminal nucleotide represented by formula (V-2) or its stereoisomer, 【Chemistry 9】 Eventually, T １ , T ３ A, R ３ And Bx are as defined in claim 1, and M １ These are C(Rd)(Re), C(Rd)(Re)C(Rg)(Rf), where each Rd, Re, Rg and Rf is independently hydrogen, halogen, hydroxyl group, and C １ -C ６ alkyl group, C １ -C ６ Alkoxy group, C １ -C ６ Alkylthio group, O(CH ２ ) ２ - OCH ３ NJ ５ ,CN,OC(=O)J ５ ,OC(=O)N(J ５ ) (J ６ ) or C(=O)N((J ５ ) (J ６ A substituent selected from ) and J ５ J ６ H or C １ -C ６ It is an alkyl group. The oligonucleotide according to claim 1.

31. Each of the aforementioned Rd, Re, Rg, and Rf independently consists of hydrogen, fluorine, a hydroxyl group, and C. １ -C ６ Alkoxy group, O(CH ２ ) ２ - OCH ３ A substituent selected from, The oligonucleotide according to claim 30.

32. Said M １ CH ２ or CH ２ CH ２ That is, The oligonucleotide according to claim 30.

33. The aforementioned R ３ It is hydrogen. Oligonucleotides according to claims 30 to 32.

34. In the above formula (V-2) or its stereoisomer, A has one of the following formulas: 【Chemistry 10】 Q １ and Q ２ These are H, halogen, and optionally substituted C, respectively, independently. １ -C ６ It is an alkyl group, Q ８ is S, SO, SO ２ Or NR １１ PR １６ R １７ And R １６ and R １７ These are independently (=O), (=S), OH, SH, C １ -C ６ Alkyl, NR １８ R １９ And, R １１ , R １８ and R １９ H is independently substituted, and C is optionally substituted. １ -C ６ Alkyl alkyl groups, optionally substituted C １ -C ６ Alkoxy group, methanesulfonyl group, sulfonic acid group, C(=O)J ３ , C(=O)OJ ３ or C(=O)N(J ３ ) (J ４ ) and J ３ and J ４ H or C １ -C ６ It is an alkyl group, Each optionally substituted group can independently be a halogen, a hydroxyl group, or C １ -C ６ alkyl group, C １ -C ６ Alkoxy group, C １ -C ６ The alkylthio group comprises one or more substituents selected from CN. Oligonucleotides according to claims 30 to 33.

35. It comprises a 5'-terminal nucleotide represented by formula (VI-1) or its stereoisomer, 【Chemistry 11】 Eventually, T １ , T ３ , X １ , X ３ A, R ３ Bx and n are as defined in claim 1, respectively, and R １ and R ２ These are, independently, H, a methylsulfonyl group, and an acetyl group. The oligonucleotide according to claim 1.

36. The aforementioned n is 0 or 1. The oligonucleotide according to claim 35.

37. The aforementioned X ３ CH ２ or CH ２ CH ２ And R ３ H is Oligonucleotides according to claims 35 to 36.

38. It comprises a 5'-terminal nucleotide represented by formula (VI-2) or its stereoisomer, 【Chemistry 12】 Eventually, T １ , T ３ , X １ A and Bx are as defined in claim 1, The oligonucleotide according to claim 1.

39. It comprises a 5'-terminal nucleotide represented by formula (VI-3) or its stereoisomer, 【Chemistry 13】 Eventually, T １ , T ３ , X １ A and Bx are as defined in claim 1, The oligonucleotide according to claim 1.

40. It comprises a 5'-terminal nucleotide represented by formula (VII-1) or its stereoisomer, 【Chemistry 14】 Eventually, T １ , T ３ , X １ A and Bx are as defined in claim 1, The oligonucleotide according to claim 1.

41. It comprises a 5'-terminal nucleotide represented by formula (VII-2) or its stereoisomer, 【Chemistry 15】 Eventually, T １ , T ３ , X １ A and Bx are as defined in claim 1, The oligonucleotide according to claim 1.

42. In the 5'-terminal nucleotides represented by the aforementioned formulas (VI-1), (VI-2), (VI-3), (VII-1), (VII-2), or their stereoisomers, A independently has one of the following formulas: 【Chemistry 16】 Eventually, Q １ and Q ２ These are H, halogen, -CN, and optionally substituted C, respectively, independently. １ -C ６ It is an alkyl group, Q ８ is O, S, SO, SO ２ PR １６ R １７ Or NR １１ And R １６ and R １７ These are independently (=O), (=S), OH, SH, C １ -C ６ Alkyl, NR １８ R １９ And R １１ , R １８ and R １９ H is independently substituted, and C is optionally substituted. １ -C ６ Alkyl alkyl groups, optionally substituted C １ -C ６ Alkoxy group, methanesulfonyl group, sulfonic acid group, C(=O)J ３ , C(=O)OJ ３ or C(=O)N(J ３ ) (J ４ ) and J ３ and J ４ H or C １ -C ６ It is an alkyl group, Each optionally substituted group can independently be a halogen, a hydroxyl group, or C １ -C ６ alkyl group, C １ -C ６ Alkoxy group, C １ -C ６ The alkylthio group comprises one or more substituents selected from CN. Oligonucleotides according to claims 38 to 41.

43. Each of the aforementioned Q １ and Q ２ These are, independently, H, F, -CN, and a methyl group. Oligonucleotides according to claims 25 to 42.

44. Each of the aforementioned Q １ and Q ２ Each of these is independently H. The oligonucleotide according to claim 43.

45. The aforementioned Q ８ NR is independently １１ And R １１ These are independently a methyl group and a methylsulfonyl group. Oligonucleotides according to claims 25 to 42.

46. The aforementioned Q ８ is S or SO ２ That is, Oligonucleotides according to claims 25 to 42.

47. Said T １ This is an optionally protected phosphine moiety, having the following formula: 【Chemistry 17】 Eventually, Ra and Rc are independently a hydroxyl group or a protected hydroxyl group, a mercapto group or a protected mercapto group, or optionally substituted with C １ -C ６ Alkyl alkyl groups, optionally substituted C １ -C ６ Selected from alkoxy groups, amino groups or protected / substituted amino groups, natural or modified nucleosides, and R ｂ is O, S or NR １２ And R １２ is hydrogen, C １ -C ６ Alkyl alkyl groups are amino protecting groups. Oligonucleotides according to claims 25 to 46.

48. The substituted amino group is optionally substituted with C １ -C ６ Alkyl alkyl groups, optionally substituted C ２ -C ６ Alkenyl group, optionally substituted C ２ -C ６ Selected from alkynyl group, sulfinyl group, sulfonyl group, and acetyl group. The oligonucleotide according to claim 47.

49. The aforementioned R ｂ is either O or S. The oligonucleotide according to claim 47.

50. The aforementioned Ra and Rc are OH, SH, and NH, respectively. ２ , OCH ２ CH ２ CN, NHSO ２ CH ３ That is, The oligonucleotide according to claim 47.

51. The aforementioned Ra and Rc are OH, and R ｂ is O, The oligonucleotide according to claim 47.

52. Said T ３ This is an internucleotide linking group selected from alkylphosphonate linking groups, phosphodiester nucleoside linking groups, or phosphorothioate nucleoside linking groups. Oligonucleotides according to claims 25 to 51.

53. Each of the above B Ｘ The heterocyclic base portion is selected from natural nucleic acid bases, modified nucleic acid bases, and universal bases. Oligonucleotides according to claims 25 to 52.

54. Each of the above B Ｘ The heterocyclic base moiety is independently a pyrimidine, a substituted pyrimidine, pseudouracil, a substituted pseudouracil, a purine, hypoxanthine, or a substituted purine. The oligonucleotide according to claim 53.

55. Each of the BX heterocyclic base moieties is 2-thiouracil, 5-fluorouracil, dihydrouridine (D), 7-methylguanosine (m7G), uracil, 5-thiazolauracil, thymine, cytosine, pseudouracil, N1-methylpsoiduracil, hypoxanthine, 5-methylcytosine, 5-methyluracil, 3-benzoyluracil, 2,6-diaminopurine, adenine, or guanine. The oligonucleotide according to claim 53.

56. An oligonucleotide comprising a 5'-terminal nucleotide represented by formula (VIII) or its stereoisomer, [Chemistry 18] Eventually, Q ８ is S, SO, SO ２ PR １６ R １７ Or NR １１ And R １６ and R １７ These are independently (=O), (=S), OH, SH, C １ -C ６ Alkyl, NR １８ R １９ And, Ra and Rc are independently a hydroxyl group or a protected hydroxyl group, a mercapto group or a protected mercapto group, or optionally substituted with C １ -C ６ Alkyl alkyl groups, optionally substituted C １ -C ６ Selected from alkoxy groups, protected or optionally substituted amino groups, and natural or modified nucleosides, and R ｂ is O or S or NR １２ And R １２ is hydrogen, C １ -C ６ Alkyl alkyl groups and amino protecting groups, Q １ and Q ２ These are H, halogen, -CN, and optionally substituted C, respectively, independently. １ -C ６ It is an alkyl group, The substituents in the substituted amino group are optionally substituted C １ -C ６ Alkyl alkyl groups, optionally substituted C ２ -C ６ Alkenyl group, optionally substituted C ２ -C ６ Selected from alkynyl group, sulfinyl group, sulfonyl group, and acetyl group, R １１ , R １８ and R １９ H is independently substituted, and C is optionally substituted. １ -C ６ Alkyl alkyl groups, optionally substituted C １ -C ６ These are alkoxy groups, methanesulfonyl groups, and sulfonic acid groups. Z is a nucleoside containing a sugar or a sugar substitution moiety. T ３ This is an internucleoside linking group that links the 5'-terminal nucleotide of formula (VIII) or its stereoisomer to an oligonucleotide, Each substituted group can be optionally and independently a halogen, a hydroxyl group, or C １ -C ６ alkyl group, C １ -C ６ Alkoxy group, C １ -C ６ The alkylthio group comprises one or more substituents selected from CN. Oligonucleotides.

57. The aforementioned Q ８ is S, SO or SO ２ That is, The oligonucleotide according to claim 56.

58. The aforementioned Q １ and Q ２ Each of these is independently H. The oligonucleotide according to claim 57.

59. In the nucleoside containing the aforementioned sugar or sugar-substituted portion, the sugar or sugar-substituted portion includes a five-membered furanose ring, a non-furanose ring, or a five- to six-membered carbon ring system or a ring-opening system. Oligonucleotides according to claims 56 to 58.

60. The aforementioned sugar or sugar-substituted moiety is selected from morpholinyl group, cyclohexenyl group, cyclohexyl group, cyclopentyl group, pyranyl group, cyclohexanehexaol group, furanose, unlocked nucleic acid base analog (UNA), glycerol nucleic acid base analog (GNA), locked nucleic acid (LNA), or cross-linked nucleic acid (BNA). The oligonucleotide according to claim 59.

61. The aforementioned Q ８ It is bonded to the 4'-carbon or 5'-carbon of the sugar or sugar substitution moiety. Oligonucleotides according to claims 59 to 60.

62. The nucleoside containing the aforementioned sugar or sugar substitution has the following structural formula: 【Chemistry 19】 【Chemistry 20】 Eventually, M ２ is C(q ３ ) (q ４ ), C(q ３ ) (q ４ ) C(q ５ ) (q ６ ) and M ３ O, S, NR １３ , C(q ７ ) (q ８ ), C(q ７ ) (q ８ ) C(q ９ ) (q １０ ), C(q ７ ) = C(q ８ ), OC(q ７ ) (q ８ ) and Each X １ These are independently chemical bonds, O, S, NJ １ or CJ １ J ２ And among them, J １ and J ２ These are, independently, hydrogen, halogen, sulfonyl group, sulfinyl group, and optionally substituted C. １ -C ６ Alkyl alkyl groups, optionally substituted C ３ -C ６ Cycloalkyl groups, optionally substituted C ２ -C ６ Alkenyl group, optionally substituted C ２ -C ６ Alkynyl group, optionally substituted C ５ -C １２ These are aryl groups, optionally substituted 5- to 12-membered heteroaryl groups, and optionally substituted 5- to 12-membered heterocycles. X ２ CR １５ or N, X ３ This is a chemical bond, an optionally substituted C １ -C ３ Alkylene group, SO, SO ２ , C(=O), P(=O)R, R stands for OH, SH, C １ -C ６ Alkyl, NH ２ NHSO ２ CH ３ And, Bx is the heterocyclic base portion, R １５ , q １ , q ２ , q ３ , q ４ , q ５ , q ６ , q ７ , q ８ , q ９ and q １０ These are, independently, hydrogen, halogen, hydroxyl group, and C １ -C ６ alkyl group, C １ -C ６ Alkoxy group, C １ -C ６ Alkylthio group, O(CH ２ ) ２ - OCH ３ ,CN,OC(=O)J ５ ,OC(=O)N(J ５ ) (J ６ ) or C(=O)N((J ５ ) (J ６ ) and J ５ J ６ H or C １ -C ６ It is an alkyl group, R １３ is hydrogen, C １ -C ６ It is an alkyl group. Oligonucleotides according to claims 59 to 60.

63. The nucleoside containing the aforementioned sugar or sugar substitution has the following structural formula: 【Chemistry 21】 The oligonucleotide according to claim 62.

64. Said M ３ O, S, C(q ７ ) (q ８ ), C(q ７ ) (q ８ ) C(q ９ ) (q １０ ) The oligonucleotide according to claim 63.

65. It comprises a 5'-terminal nucleotide represented by formula (VIII-1) or its stereoisomer, 【Chemistry 22】 Eventually, Q ８ Ra, Rc, R ｂ M ２ M ３ , X １ , X ２ , X ３ , q １ , q ２ And Bx are as defined in claim 56, The oligonucleotide according to claim 56.

66. The aforementioned R １５ , q １ , q ２ , q ３ , q ４ , q ５ , q ６ , q ７ , q ８ , q ９ and q １０ These are, independently, hydrogen, fluorine, hydroxyl group, methyl group, methoxy group, and O(CH ２ ) ２ - OCH ３ Selected from, Oligonucleotides according to claims 62 to 65.

67. The aforementioned R １５ , q １ , q ２ , q ３ , q ４ , q ５ , q ６ , q ７ , q ８ , q ９ and q １０ These are each independently selected from hydrogen. The oligonucleotide according to claim 66.

68. Said M ３ O, S, C(q ７ ) (q ８ ) and M ２ is C(q ３ ) (q ４ ) and X ２ CR １５ or N, X ３ These are chemical bonds, SO, SO ２ That is, Oligonucleotides according to claims 65 to 67.

69. Said M ３ O, S, CH ２ And M ２ CH ２ X ２ CH is X ３ It is a chemical bond. The oligonucleotide according to claim 68.

70. Said M ３ CH ２ And M ２ CH ２ X ２ is N, X ３ is a chemical bond, SO or SO ２ That is, The oligonucleotide according to claim 68.

71. Said M ３ is C(q ７ ) (q ８ ) and M ２ is C(q ３ ) (q ４ ) C(q ５ ) (q ６ ) and X ２ CR １５ or N, X ３ These are chemical bonds, SO, SO ２ That is, Oligonucleotides according to claims 65 to 67.

72. Said M ３ CH ２ And M ２ CH ２ CH ２ X ２ CH is X ３ It is a chemical bond. The oligonucleotide according to claim 71.

73. Said M ３ CH ２ And M ２ CH ２ CH ２ X ２ is N, X ３ is a chemical bond, SO or SO ２ That is, The oligonucleotide according to claim 71.

74. The aforementioned Q ８ is S, SO or SO ２ That is, Oligonucleotides according to claims 56 to 73.

75. The aforementioned R ｂ It is oxygen. Oligonucleotides according to claims 56 to 73.

76. The aforementioned Q ８ is SO or SO ２ And Ra and Rc are independently OH, SH, and NH, respectively. ２ NHSO ２ CH ３ Selected from, Oligonucleotides according to claims 56 to 73.

77. The aforementioned Q ８ is S, SO or SO ２ And R ｂ is oxygen, and Ra and Rc are each independently selected from OH. Oligonucleotides according to claims 56 to 73.

78. The aforementioned X １ It is oxygen. Oligonucleotides according to claims 56 to 77.

79. B Ｘ The heterocyclic base portion is selected from natural nucleic acid bases, modified nucleic acid bases, and universal bases. Oligonucleotides according to claims 56 to 78.

80. B Ｘ The heterocyclic base portion is a pyrimidine, a substituted pyrimidine, pseudouracil, a substituted pseudouracil, a purine, hypoxanthine, or a substituted purine. The oligonucleotide according to claim 79.

81. B Ｘ The heterocyclic base moieties are 2-thiouracil, 5-fluorouracil, dihydrouridine (D), 7-methylguanosine (m7G), uracil, 5-thiazolauracil, thymine, cytosine, pseudouracil, N1-methyl-psoidouracil, hypoxanthine, 5-methylcytosine, 5-methyluracil, 3-benzoyluracil, 2,6-diaminopurine, adenine, or guanine. The oligonucleotide according to claim 79.

82. An oligonucleotide having a compound fragment of the following formula at its 5'-terminal nucleotide, 【Chemistry 23】 Eventually, Q ８ is S, SO, SO ２ PR １６ R １７ Or NR １１ And R １６ and R １７ These are independently (=O), (=S), OH, SH, C １ -C ６ Alkyl, NR １８ R １９ Ra and Rc are each independently a hydroxyl group or a protected hydroxyl group, a mercapto group or a protected mercapto group, or optionally substituted C １ -C ６ Alkyl alkyl groups, optionally substituted C １ -C ６ Selected from alkoxy groups, protected or optionally substituted amino groups, and natural or modified nucleosides, and R ｂ is O or S or NR １２ And R １２ is hydrogen, C １ -C ６ Alkyl alkyl groups and amino protecting groups, The substituents in the substituted amino group are optionally substituted C １ -C ６ Alkyl alkyl groups, optionally substituted C ２ -C ６ Alkenyl group, optionally substituted C ２ -C ６ Selected from alkynyl group, sulfinyl group, sulfonyl group, and acetyl group, R １１ , R １８ and R １９ H is independently substituted, and C is optionally substituted. １ -C ６ Alkyl alkyl groups, optionally substituted C １ -C ６ These are alkoxy groups, methanesulfonyl groups, and sulfonic acid groups. Each substituted group can be optionally and independently a halogen, a hydroxyl group, or C １ -C ６ alkyl group, C １ -C ６ Alkoxy group, C １ -C ６ It comprises one or more substituents selected from alkylthio groups and CNs, 【Chemistry 24】 This represents the junction with the remaining portion of the 5'-terminal nucleotide. Oligonucleotides.

83. The 5'-terminal nucleoside, which is represented by one of the following specific structures or its stereoisomer, Table 1-1 Table 1-2 Table 1-3 Table 1-4 Table 1-5 Table 1-6 or Table 2-1 Table 2-2 Table 2-3 Table 2-4 Table 2-5 Oligonucleotides according to claims 1 to 82.

84. The aforementioned nucleoside linking group is independently an alkylphosphonate linking group, a phosphodiester nucleoside linking group, or a phosphorothioate nucleoside linking group. Oligonucleotides according to claims 56 to 83.

85. The oligonucleotide is a double-stranded ribonucleic acid (dsRNA) reagent comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand are fully or partially complementary, and the antisense strand is partially or completely complementary to a nucleic acid target gene, and at least one of the sense strand and the antisense strand is an oligonucleotide with a 5'-terminal nucleotide represented as described in claims 1 to 84, wherein the double-stranded ribonucleic acid (dsRNA) reagent further optionally comprises an independent target group. Oligonucleotides according to claims 1 to 84.

86. The antisense strand in the double-stranded ribonucleic acid (dsRNA) reagent comprises an oligonucleotide of the 5'-terminal nucleotide as described in claims 1 to 84. Oligonucleotide according to claim 85.

87. In oligonucleotides or double-stranded ribonucleic acid (dsRNA), each strand contains 8 to 40 nucleotides. The oligonucleotide according to claims 1 to 86.

88. Use of oligonucleotide or double-stranded ribonucleic acid (dsRNA) reagents according to claims 1 to 87 in the manufacture of a drug, wherein the drug is used to suppress gene expression. use.

89. Compounds represented by formula (I), formula (II), and formula (III) or their stereoisomers, 【Chemistry 25】 Among them, each T １ This is an independently and optionally protected phosphine moiety, Each T ２ These are independently active phosphorus groups, Each X １ These are independently chemical bonds, O, S, NJ １ or CJ １ J ２ And among them, J １ and J ２ These are, independently, hydrogen, halogen, sulfonyl group, sulfinyl group, and optionally substituted C. １ -C ６ Alkyl alkyl groups, optionally substituted C ３ -C ６ Cycloalkyl groups, optionally substituted C ２ -C ６ Alkenyl group, optionally substituted C ２ -C ６ Alkynyl group, optionally substituted C ５ -C １２ These are aryl groups, optionally substituted 5- to 12-membered heteroaryl groups, and optionally substituted 5- to 12-membered heterocycles. Each X ２ CR independently １５ or N, Each X ３ These are C, which is independently chemically bonded and optionally substituted. １ -C ３ Alkylene group, SO, SO ２ C(=O), P(=O)R, where R is OH, SH, C １ -C ６ Alkyl, NH ２ NHSO ２ CH ３ And, Each Bx is independently a heterocyclic base moiety. Each R １ and R ２ These are H, halogen, and optionally substituted C, respectively, independently. １ -C ６ Alkyl alkyl groups, optionally substituted C １ -C ６ Alkoxy groups, optionally substituted C ２ -C ６ Alkenyl group, optionally substituted C ２ -C ６ These are alkynyl groups, sulfinyl groups, sulfonyl groups, and acetyl groups. Each R ３ and R １５ These are H, halogen, and optionally substituted C, respectively, independently. １ -C ６ Alkyl alkyl groups, optionally substituted C １ -C ６ Alkoxy groups, optionally substituted C ２ -C ６ Alkenyl group, optionally substituted C ２ -C ６ It is an alkynyl group, Each A independently possesses one of the following equations: 【Chemistry 26】 Each Q １ and Q ２ These are H, halogen, -CN, and optionally substituted C, respectively, independently. １ -C ６ Alkyl alkyl groups, optionally substituted C １ -C ６ Alkoxy groups, optionally substituted C ２ -C ６ Alkenyl group, optionally substituted C ２ -C ６ Alkynyl group or NR ４ R ５ And, Each Q ３ These are O, S, and NR independently. ６ or CR ７ R ８ And, Q ４ Q ５ Q ６ Q ７ Q ９ Q １０ Q １１ and Q １２ These are, independently, H, halogen, optionally protected hydroxyl group, acetoxy group, azide group, and optionally substituted C. １ -C ６ Alkyl alkyl groups, optionally substituted C １ -C ６ Alkoxy groups, optionally substituted C ２ -C ６ Alkenyl group, optionally substituted C ２ -C ６ Alkynyl group, NR ９ R １０ And, Q ８ is O, S, SO, SO ２ PR １６ R １７ Or NR １１ And, R １６ and R １７ These are independently (=O), (=S), OH, SH, C １ -C ６ Alkyl, NR １８ R １９ And, Each R ４ , R ５ , R ６ , R ７ , R ８ , R ９ , R １０ , R １１ , R １８ and R １９ H is independently substituted, and C is optionally substituted. １ -C ６ Alkyl alkyl groups, optionally substituted C １ -C ６ Alkoxy group, methanesulfonyl group, sulfonic acid group, C(=O)J ３ , C(=O)OJ ３ or C(=O)N(J ３ ) (J ４ ) and M １ These are C(Rd)(Re), C(Rd)(Re)C(Rf)(Rg), where each Rd, Re, Rg and Rf is independently hydrogen, halogen, hydroxyl group, and C １ -C ６ alkyl group, C １ -C ６ Alkoxy group, C １ -C ６ Alkylthio group, O(CH ２ ) ２ - OCH ３ NJ ５ ,CN,OC(=O)J ５ ,OC(=O)N(J ５ ) (J ６ ) or C(=O)N((J ５ ) (J ６ A substituent selected from ) Each J ３ J ４ J ５ and J ６ H or C １ -C ６ It is an alkyl group, Each n is independently 0, 1, or 2. Each optionally substituted group independently contains one or more substituents selected from halogen, a hydroxy group, C １ -C ６ an alkyl group, C １ -C ６ an alkoxy group, C １ -C ６ an alkylthio group, CN compound.

90. Each T １ This is an independently and optionally protected phosphine moiety, having the following formula: 【Chemistry 27】 Eventually, Ra and Rc are each independently a hydroxy group or a protected hydroxy group, a mercapto group or a protected mercapto group, an optionally substituted C １ -C ６ alkyl group, an optionally substituted C １ -C ６ alkoxy group, an amino group or a protected / substituted amino group, selected from natural or modified nucleosides, and R ｂ is O, S or NR １２ where R １２ is hydrogen, C １ -C ６ alkyl group, an amino protecting group, The substituents in the substituted amino group are optionally substituted C １ -C ６ Alkyl alkyl groups, optionally substituted C ２ -C ６ Alkenyl group, optionally substituted C ２ -C ６ Selected from alkynyl group, sulfinyl group, sulfonyl group, and acetyl group, Each optionally substituted group can independently be a halogen, a hydroxyl group, or C １ -C ６ alkyl group, C １ -C ６ Alkoxy group, C １ -C ６ The alkylthio group comprises one or more substituents selected from CN. The compound according to claim 89.

91. The protecting groups for the hydroxyl group or mercapto group are, independently, methyl group, ethyl group, benzyl group (Bn), phenyl group, isopropyl group, tert-butyl group, acetyl group, chloroacetyl group, trichloroacetyl group, trifluoroacetyl group, pivaloyl group, tert-butoxymethyl group, methoxymethyl group, 1-ethoxyethyl group, 1-(2-chloroethoxy)ethyl group, 2-trimethylsilylethyl group, allyl group, cyclohexyl group (cHex), 9-fluorenylmethoxycarbonyl group, methanesulfonic acid ester, and toluenesulfonic acid. phosphate ester, trifluoromethanesulfonic acid ester, benzoyl group, benzoyl formate, p-phenylbenzoyl group, 4-methoxybenzyl group, monomethoxytrityl group, dimethoxytrityl group, trimethoxytrityl group, 4-chlorobenzyl group, 4-nitrobenzyl group, 2,4-dinitrophenyl group, 4-acyloxybenzyl group, 2-methylphenyl group, 2,6-dimethylphenyl group, 2-chlorophenyl group, 2,6-dichlorobenzyl group, diphenylmethyl group, triphenylmethyl group, 4-methylthio-1-butyl group, 2-( S-acetylthio)ethyl group (SATE), 2-cyanoethyl group, 2-cyano-1,1-dimethylethyl (CDM), 4-cyano-2-butenyl group, 2-(trimethylsilyl)ethyl group (TSE), 2-(phenylthio)ethyl group, 2-(triphenylsilyl)ethyl group, 2-(benzylsulfonyl)ethyl group, 2,2,2-trichloroethyl group, 2,2,2-tribromoethyl group, 2,3-dibromopropyl group, 2,2,2-trifluoroethyl group, phenylthio group, 2-chloro-4-tritylphenyl group, 2-bromophenyl group, Selected from 2-[N-isopropyl group-N-(4-methoxybenzoyl)amino]ethyl group, 4-(N-trifluoroacetylamino)butyl group, 4-oxopentyl group, 4-tritylaminophenyl group, 4-benzylaminophenyl group, tetrahydropyranyl group, morpholino, trimethylsilyl group, triethylsilyl group, tert-butyldimethylsilyl group, tert-butyldiphenylsilyl group, triphenylsilyl group, triisopropylsilyl group, pivalate methyl ether group (POM), and 9-phenylxanthin-9-yl, The amino protecting group is independently selected from 2-trimethylsilylethoxycarbonyl group (Teoc), 1-methyl-1-(4-biphenyl)ethoxycarbonyl group (Bpoc), tert-butoxycarbonyl group (BOC), allyloxycarbonyl group (Alloc), 9-fluorenylmethoxycarbonyl group (Fmoc), benzyloxycarbonyl group (Cbz), benzyl group, formyl group, acetyl group, pivaloyl group, trihaloacetyl group, benzoyl group, nitrophenyl group, acetyl group, 2-nitrobenzenesulfonyl group, phthalimide group (Pht), p-toluenesulfonyl group (Tos), trityl group (Trt), 2,4-dimethoxybenzyl group (PMB), and dithiosuccinyl group. The compound according to claim 90.

92. Each T １ This is an independently and optionally protected phosphine moiety, having the following formula: 【Chemistry 28】 Eventually, Ra and Rc are each independently selected from a protected hydroxyl group or a protected mercapto group, and R ｂ is either O or S, The hydroxy protecting groups are, independently of each other, acetyl group, tert-butyl group, tert-butoxymethyl group, methoxymethyl group, tetrahydropyranyl group, 1-ethoxyethyl group, 1-(2-chloroethoxy)ethyl group, 2-trimethylsilylethyl group, p-chlorophenyl group, 2,4-dinitrophenyl group, benzyl group, benzoyl group, p-phenylbenzoyl group, 2,6-dichlorobenzyl group, diphenylmethyl group, p-nitrobenzyl group, trimethylsilyl group, triethylsilyl group, tert-butyldimethylsilyl group, te Selected from rt-butyldiphenylsilyl group, triphenylsilyl group, triisopropylsilyl group, benzoyl formate, chloroacetyl group, trichloroacetyl group, trifluoroacetyl group, pivaloyl group, 9-fluorenylmethoxycarbonyl group, methanesulfonic acid ester, toluenesulfonic acid ester, trifluoromethanesulfonic acid ester, trityl group, monomethoxytrityl group, dimethoxytrityl group, trimethoxytrityl group, pivalate methyl ether group (POM), or substituted 9-phenylxanthin-9-yl, The mercapto protecting groups are, independently of each other, a methyl group, an ethyl group, an acetyl group, a tert-butyl group, a tert-butoxymethyl group, a methoxymethyl group, a tetrahydropyranyl group, a 1-ethoxyethyl group, a 1-(2-chloroethoxy)ethyl group, a 2-trimethylsilylethyl group, a p-chlorophenyl group, a 2,4-dinitrophenyl group, a benzyl group, a benzoyl group, a p-phenylbenzoyl group, a 2,6-dichlorobenzyl group, a diphenylmethyl group, a p-nitrobenzyl group, a trimethylsilyl group, a triethylsilyl group, and te Selected from rt-butyldimethylsilyl group, tert-butyldiphenylsilyl group, triphenylsilyl group, triisopropylsilyl group, benzoyl formate, chloroacetyl group, trichloroacetyl group, trifluoroacetyl group, pivaloyl group, 9-fluorenylmethoxycarbonyl group, methanesulfonic acid ester, toluenesulfonic acid ester, trifluoromethanesulfonic acid ester, monomethoxytrityl group, dimethoxytrityl group, trimethoxytrityl group, or substituted 9-phenylxanthin-9-yl, The compounds according to claims 90 to 91.

93. The hydroxy protecting groups are independently selected from acetyl, benzyl, tert-butyldimethylsilyl, pivalate methyl ether (POM), tert-butyldiphenylsilyl, and 4,4'-dimethoxytrityl groups, and the mercapto protecting groups are independently selected from benzyl, 4,4'-dimethoxytrityl, and trityl groups. The compound according to claim 92.

94. R ｂ is O or S, and Ra and Rc are each independently protected hydroxyl groups, C １ -C ６ alkyl group, C １ -C ６ Selected from alkoxy groups, The compounds according to claims 90 to 93.

95. R ｂ is O, and Ra and Rc are independently OH, SH, and OCH, respectively. ３ , OCH ２ CH ３ , OCH(CH ３ ) ２ , OCH ２ OC(=O)C(CH ３ ) ３ , OCH ２ CH ２ CN, NHSO ２ CH ３ Selected from, The compounds according to claims 90 to 94.

96. Each T ２ Independently 【Chemistry 29】 This is an active phosphorus group having the following structure, and among them, M ４ H is replaced by C, which is optionally substituted. １ -C ６ Alkyl alkyl groups, OH, OJ ７ SH, SJ ７ Or NJ ７ J ８ And M ５ C is a C that has been optionally substituted. １ -C ６ Alkyl alkyl groups, OH, OJ ７ SH, SJ ７ Or NJ ７ J ８ And each J ７ or J ８ C is independently and arbitrarily substituted. １ -C ６ It is an alkyl group or a sulfonyl group, and r is 0 or 1. Each optionally substituted group independently contains one or more substituents selected from halogen, a hydroxy group, C １ -C ６ an alkyl group, C １ -C ６ an alkoxy group, C １ -C ６ an alkylthio group, CN The compounds according to claims 89 to 95.

97. Each J ７ or J ８ This is an independently substituted C １ -C ６ It is an alkyl group, and the substituent is selected from a cyano group and a halogen. The compound according to claim 96.

98. M ４ The group is selected from methyl, ethyl, propyl, and isopropyl groups. The compound according to claim 96.

99. M ４ It is selected from methanesulfonamide groups, The compound according to claim 96.

100. M ４ OJ ７ And among them, J ７ is a substituted C １ -C ６ It is an alkyl group, and the substituent is selected from a cyano group and a halogen. The compound according to claim 96.

101. M ４ is O(CH ２ ) ２ CN and M ５ is N(CH(CH ３ ) ２ ) ２ And r is 0. The compound according to claim 96.

102. Each T ２ The active phosphorus group is independently a phosphoramidite. The compound according to claim 96.

103. Each T ２ The active phosphorus group is independently selected from diisopropylcyanoethoxyphosphoramidite, diisopropylethylphosphoramidite, and H-phosphonate. The compound according to claim 96.

104. Each X １ These are independently O. The compounds according to claims 89 to 103.

105. Each B Ｘ The heterocyclic base moiety is independently selected from natural nucleic acid bases, modified nucleic acid bases, and universal bases. The compounds according to claims 89 to 104.

106. Each B Ｘ The heterocyclic base moiety is independently a pyrimidine, a substituted pyrimidine, pseudouracil, a substituted pseudouracil, a purine, hypoxanthine, or a substituted purine. The compound according to claim 105.

107. Each B Ｘ The heterocyclic base moieties are independently 2-thiouracil, 5-fluorouracil, dihydrouridine (D), 7-methylguanosine (m7G), uracil, 5-thiazolauracil, thymine, cytosine, pseudouracil, N1-methyl-psoidouracil, hypoxanthine, 5-methylcytosine, 5-methyluracil, 3-benzoyluracil, 2,6-diaminopurine, adenine, or guanine. The compound according to claim 105.

108. Each R １５ and R ３ Each of these is independently H. The compounds according to claims 89 to 106.

109. Each R １ and R ２ These are, independently, H, a methylsulfonyl group, and an acetyl group. The compounds according to claims 89 to 108.

110. Each X ２ Each of these is independently N, and each R １ and R ２ These are, independently, H, a methylsulfonyl group, and an acetyl group. The compounds according to claims 89 to 109.

111. A has one of the following equations: 【Transformation 30】 Eventually, Q １ and Q ２ These are H, halogen, -CN, and optionally substituted C, respectively, independently. １ -C ６ It is an alkyl group, Q ８ is O, S, SO, SO ２ PR １６ R １７ Or NR １１ And R １６ and R １７ These are independently (=O), (=S), OH, SH, C １ -C ６ Alkyl, NR １８ R １９ And R １１ , R １８ and R １９ H is independently substituted, and C is optionally substituted. １ -C ６ Alkyl alkyl groups, optionally substituted C １ -C ６ Alkoxy group, methanesulfonyl group, sulfonic acid group, C(=O)J ３ , C(=O)OJ ３ or C(=O)N(J ３ ) (J ４ ) and J ３ and J ４ H or C １ -C ６ It is an alkyl group, Each optionally substituted group can independently be a halogen, a hydroxyl group, or C １ -C ６ alkyl group, C １ -C ６ Alkoxy group, C １ -C ６ The alkylthio group comprises one or more substituents selected from CN. The compounds according to claims 89 to 110.

112. Each Q １ and Q ２ These are, independently, H, F, -CN, and a methyl group. The compounds according to claims 89 to 111.

113. Each Q １ and Q ２ Each of these is independently H. The compound according to claim 112.

114. Condition M １ C(Rd)(Re), X ２ C, X ３ is a chemical bond, A is 【Chemistry 31】 If that is the case, Q ８ is S, SO, SO ２ PR １６ R １７ Or NR １１ That is, The compounds according to claims 89 to 113.

115. Condition M １ C(Rd)(Re), X ２ C, X ３ is a chemical bond, A is 【Chemistry 32】 If that is the case, Q ８ is S, SO, SO ２ Or NR １１ That is, The compounds according to claims 89 to 114.

116. X ２ is N, X ３ This is a chemical bond, -CH ２ -ien-CH ２ CH ２ -, SO, SO ２ That is, The compounds according to claims 89 to 115.

117. X ２ CH is X ３ It is a chemical bond. The compounds according to claims 89 to 116.

118. n is either 0 or 1. The compounds according to claims 89 to 117.

119. Having the compound shown in formula (I-1) or its stereoisomer, 【Transformation 33】 Eventually, T １ , T ２ A, R ３ and Bx are as defined in claim 89, respectively, and X ３ These are chemical bonds, C(=O), P(=O)R, SO or SO ２ And M １ These are C(Rd)(Re), C(Rd)(Re)C(Rg)(Rf), where each Rd, Re, Rg and Rf is independently hydrogen, halogen, hydroxyl group, and C １ -C ６ alkyl group, C １ -C ６ Alkoxy group, C １ -C ６ Alkylthio group, O(CH ２ ) ２ - OCH ３ NJ ５ ,CN,OC(=O)J ５ ,OC(=O)N(J ５ ) (J ６ ) or C(=O)N((J ５ ) (J ６ A substituent selected from ) and J ５ J ６ H or C １ -C ６ It is an alkyl group, where R is OH, SH, or C. １ -C ６ Alkyl, NH ２ NHSO ２ CH ３ That is, The compound according to claim 89.

120. Each of Rd, Re, Rg, and Rf independently consists of hydrogen, fluorine, a hydroxyl group, and C. １ -C ６ Alkoxy group, O(CH ２ ) ２ - OCH ３ A substituent selected from, The compound according to claim 119.

121. Each of Rd, Re, Rg, and Rf is independently a hydrogen atom. The compound according to claim 120.

122. R ３ It is hydrogen. The compounds according to claims 89 to 121.

123. Eventually, X ３ SO ２ or chemical bond, The compounds according to claims 89 to 122.

124. A has one of the following equations: 【Transformation 34】 Q １ and Q ２ These are H, halogen, and optionally substituted C, respectively, independently. １ -C ６ It is an alkyl group, Q ８ is O, S, SO, SO ２ PR １６ R １７ Or NR １１ And R １６ and R １７ These are independently (=O), (=S), OH, SH, C １ -C ６ Alkyl, NR １８ R １９ And R １１ , R １８ and R １９ H is independently substituted, and C is optionally substituted. １ -C ６ Alkyl alkyl groups, optionally substituted C １ -C ６ Alkoxy group, methanesulfonyl group, sulfonic acid group, C(=O)J ３ , C(=O)OJ ３ or C(=O)N(J ３ ) (J ４ ) and J ３ and J ４ H or C １ -C ６ It is an alkyl group, Each optionally substituted group can independently be a halogen, a hydroxyl group, or C １ -C ６ alkyl group, C １ -C ６ Alkoxy group, C １ -C ６ The alkylthio group comprises one or more substituents selected from CN. The compounds according to claims 89 to 123.

125. Having the compound shown in formula (I-2) or its stereoisomer, 【Chemistry 35】 Eventually, T １ , T ２ A, R ３ And Bx are as defined in claim 89, respectively, and M １ These are C(Rd)(Re), C(Rd)(Re)C(Rg)(Rf), where each Rd, Re, Rg and Rf is independently hydrogen, halogen, hydroxyl group, and C １ -C ６ alkyl group, C １ -C ６ Alkoxy group, C １ -C ６ Alkylthio group, O(CH ２ ) ２ - OCH ３ NJ ５ ,CN,OC(=O)J ５ ,OC(=O)N(J ５ ) (J ６ ) or C(=O)N((J ５ ) (J ６ A substituent selected from ) and J ５ J ６ H or C １ -C ６ It is an alkyl group. Compound according to claim 89

126. Each of Rd, Re, Rg, and Rf independently consists of hydrogen, fluorine, a hydroxyl group, and C. １ -C ６ Alkoxy group, O(CH ２ ) ２ - OCH ３ A substituent selected from, The compound according to claim 125.

127. M １ CH ２ or CH ２ CH ２ That is, The compound according to claim 125.

128. R ３ It is hydrogen. The compounds according to claims 125 to 127.

129. A has one of the following equations: 【Transformation 36】 Q １ and Q ２ These are H, halogen, and optionally substituted C, respectively, independently. １ -C ６ It is an alkyl group, Q ８ is S, SO, SO ２ PR １６ R １７ Or NR １１ And R １６ and R １７ These are independently (=O), (=S), OH, SH, C １ -C ６ Alkyl, NR １８ R １９ And, R １１ , R １８ and R １９ H is independently substituted, and C is optionally substituted. １ -C ６ Alkyl alkyl groups, optionally substituted C １ -C ６ Alkoxy group, methanesulfonyl group, sulfonic acid group, C(=O)J ３ , C(=O)OJ ３ or C(=O)N(J ３ ) (J ４ ) and J ３ and J ４ H or C １ -C ６ It is an alkyl group, Each optionally substituted group can independently be a halogen, a hydroxyl group, or C １ -C ６ alkyl group, C １ -C ６ Alkoxy group, C １ -C ６ The alkylthio group comprises one or more substituents selected from CN. The compounds according to claims 125 to 128.

130. Having the compound shown in formula (II-1) or its stereoisomer, 【Chemistry 37】 Eventually, T １ , T ２ , X １ , X ３ A, R ３ Bx and n are as defined in claim 89, respectively, and R １ and R ２ These are, independently, H, a methylsulfonyl group, and an acetyl group. The compound according to claim 89.

131. n is either 0 or 1. The compound according to claim 130.

132. X ３ CH ２ or CH ２ CH ２ That is, The compounds according to claims 130 to 131.

133. Having a compound represented by formula (II-2) or formula (II-3) or a stereoisomer thereof, 【Transformation 38】 Eventually, T １ , T ２ , X １ A and Bx are as defined in claim 89, respectively. The compound according to claim 89.

134. Having a compound represented by formula (III-1) or formula (III-2) or a stereoisomer thereof, 【Chemistry 39】 Eventually, T １ , T ２ , X １ A and Bx are as defined in claim 89, respectively. The compound according to claim 89.

135. A has one of the following equations: 【Chemistry 40】 Eventually, Q １ and Q ２ These are H, halogen, -CN, and optionally substituted C, respectively, independently. １ -C ６ It is an alkyl group, Q ８ is O, S, SO, SO ２ PR １６ R １７ Or NR １１ And R １６ and R １７ These are independently (=O), (=S), OH, SH, C １ -C ６ Alkyl, NR １８ R １９ And R １１ , R １８ and R １９ H is independently substituted, and C is optionally substituted. １ -C ６ Alkyl alkyl groups, optionally substituted C １ -C ６ Alkoxy group, methanesulfonyl group, sulfonic acid group, C(=O)J ３ , C(=O)OJ ３ or C(=O)N(J ３ ) (J ４ ) and J ３ and J ４ H or C １ -C ６ It is an alkyl group, Each optionally substituted group can independently be a halogen, a hydroxyl group, or C １ -C ６ alkyl group, C １ -C ６ Alkoxy group, C １ -C ６ The alkylthio group comprises one or more substituents selected from CN. The compounds according to claims 130 to 134.

136. Each Q １ and Q ２ These are, independently, H, F, -CN, and a methyl group. The compounds according to claims 118 to 135.

137. Each Q １ and Q ２ Each of these is independently H. The compound according to claim 136.

138. T １ This is an optionally protected phosphine moiety, having the following formula: 【Chemistry 41】 Eventually, Ra and Rc are independently a hydroxyl group or a protected hydroxyl group, a mercapto group or a protected mercapto group, or optionally substituted with C １ -C ６ Alkyl alkyl groups, optionally substituted C １ -C ６ Selected from alkoxy groups, amino groups or protected / substituted amino groups, natural or modified nucleosides, and R ｂ is O, S or NR １２ And R １２ is hydrogen, C １ -C ６ Alkyl alkyl groups are amino protecting groups. The compounds according to claims 118 to 137.

139. The substituents in the substituted amino group are optionally substituted C １ -C ６ Alkyl alkyl groups, optionally substituted C ２ -C ６ Alkenyl group, optionally substituted C ２ -C ６ Selected from alkynyl group, sulfinyl group, sulfonyl group, and acetyl group. The compound according to claim 138.

140. R ｂ is O or S, and Ra and Rc are each independently protected hydroxyl groups, C １ -C ６ alkyl group, C １ -C ６ Selected from alkoxy groups, The compound according to claim 138.

141. R ｂ is O, and Ra and Rc are independently OH and OCH, respectively. ３ , OCH ２ CH ３ , OCH(CH ３ ) ２ , OCH ２ OC(=O)C(CH ３ ) ３ , OCH ２ CH ２ CN, NHSO ２ CH ３ Selected from, The compound according to claim 138.

142. T ２ This is an active phosphorus group, and the active phosphorus group is a phosphoramidite. The compounds according to claims 118 to 141.

143. T ２ The active phosphorus group is selected from diisopropylcyanoethoxyphosphoramidite, diisopropylethylphosphoramidite, and H-phosphonate. The compound according to claim 142.

144. B Ｘ The heterocyclic base portion is selected from natural nucleic acid bases, modified nucleic acid bases, and universal bases. The compounds according to claims 118 to 142.

145. B Ｘ The heterocyclic base portion is a pyrimidine, a substituted pyrimidine, pseudouracil, a substituted pseudouracil, a purine, hypoxanthine, or a substituted purine. The compound according to claim 144.

146. B Ｘ The heterocyclic base moieties are 2-thiouracil, 5-fluorouracil, dihydrouridine (D), 7-methylguanosine (m7G), uracil, 5-thiazolauracil, thymine, cytosine, pseudouracil, N1-methyl-psoidouracil, hypoxanthine, 5-methylcytosine, 5-methyluracil, 3-benzoyluracil, 2,6-diaminopurine, adenine, or guanine. The compound according to claim 144.

147. Compounds represented by formula (IV) or their stereoisomers, 【Chemistry 42】 Eventually, Q ８ is S, SO, SO ２ PR １６ R １７ Or NR １１ And R １６ and R １７ These are independently (=O), (=S), OH, SH, C １ -C ６ Alkyl, NR １８ R １９ Ra and Rc are each independently a hydroxyl group or a protected hydroxyl group, a mercapto group or a protected mercapto group, or optionally substituted C １ -C ６ Alkyl alkyl groups, optionally substituted C １ -C ６ Selected from alkoxy groups, protected or optionally substituted amino groups, and natural or modified nucleosides, and R ｂ is O, S or NR １２ And R １２ is hydrogen, C １ -C ６ Alkyl alkyl groups and amino protecting groups, The substituents in the substituted amino group are optionally substituted C １ -C ６ Alkyl alkyl groups, optionally substituted C ２ -C ６ Alkenyl group, optionally substituted C ２ -C ６ Selected from alkynyl group, sulfinyl group, sulfonyl group, and acetyl group, Q １ and Q ２ These are H, halogen, -CN, and optionally substituted C, respectively, independently. １ -C ６ Alkyl alkyl groups, optionally substituted C １ -C ６ Alkoxy groups, optionally substituted C ２ -C ６ Alkenyl group, optionally substituted C ２ -C ６ Alkynyl group or NR ４ R ５ And, Each R ４ , R ５ , R １１ , R １８ and R １９ H is independently substituted, and C is optionally substituted. １ -C ６ Alkyl alkyl groups, optionally substituted C １ -C ６ These are alkoxy groups, methanesulfonyl groups, and sulfonic acid groups. Z is a phosphoramidite, a sugar, or a nucleoside containing a sugar substitution moiety. Each substituted group can be optionally and independently a halogen, a hydroxyl group, or C １ -C ６ alkyl group, C １ -C ６ Alkoxy group, C １ -C ６ The alkylthio group comprises one or more substituents selected from CN. compound.

148. Q ８ is S, SO or SO ２ That is, The compound according to claim 147.

149. Q １ and Q ２ Each of these is independently H. The compounds according to claims 147 to 148.

150. In the nucleoside containing the aforementioned sugar or sugar-substituted portion, the sugar or sugar-substituted portion includes a 5-membered furanose ring, a non-furanose ring, or a 5-6 membered carbocyclic system or a ring-opening system. The compounds according to claims 147 to 149.

151. The sugar or sugar-substituted portion is selected from morpholinyl group, cyclohexenyl group, cyclohexyl group, cyclopentyl group, pyranyl group, cyclohexanehexaol group, furanose, unlocked nucleic acid base analog (UNA), glycerol nucleic acid base analog (GNA), locked nucleic acid (LNA), or cross-linked nucleic acid (BNA). The compound according to claim 150.

152. Q ８ It is bonded to the 4'-carbon or 5'-carbon of the sugar or sugar substitution moiety. The compound according to claim 151.

153. The sugar or the nucleoside of the sugar substitution has the following structural formula: 【Chemistry 43】 【Chemistry 44】 Eventually, M ２ is C(q ３ ) (q ４ ), C(q ３ ) (q ４ ) C(q ５ ) (q ６ ) and M ３ O, S, NR １３ , C(q ７ ) (q ８ ), C(q ７ ) (q ８ ) C(q ９ ) (q １０ ), C(q ７ ) = C(q ８ ), OC(q ７ ) (q ８ ) and X １ is a chemical bond, or O, S, NJ １ or CJ １ J ２ Selected from among them, J １ and J ２ These are, independently, hydrogen, halogen, sulfonyl group, sulfinyl group, and optionally substituted C. １ -C ６ Alkyl alkyl groups, optionally substituted C ３ -C ６ Cycloalkyl groups, optionally substituted C ２ -C ６ Alkenyl group, optionally substituted C ２ -C ６ Alkynyl group, optionally substituted C ５ -C １２ These are aryl groups, optionally substituted 5- to 12-membered heteroaryl groups, and optionally substituted 5- to 12-membered heterocycles. X ２ CR １５ or N, X ３ This is a chemical bond, an optionally substituted C １ -C ３ Alkylene group, SO, SO ２ , C(=O), P(=O)R, R stands for OH, SH, C １ -C ６ Alkyl, NH ２ NHSO ２ CH ３ And, Bx is the heterocyclic base portion, R １５ , q １ , q ２ , q ３ , q ４ , q ５ , q ６ , q ７ , q ８ , q ９ and q １０ These are, independently, hydrogen, halogen, hydroxyl group, and C １ -C ６ alkyl group, C １ -C ６ Alkoxy group, C １ -C ６ Alkylthio group, O(CH ２ ) ２ - OCH ３ ,CN,OC(=O)J ５ ,OC(=O)N(J ５ ) (J ６ ) or C(=O)N((J ５ ) (J ６ ) and J ５ J ６ H or C １ -C ６ It is an alkyl group, R １３ is hydrogen, C １ -C ６ It is an alkyl group. The compound according to claims 150 to 151.

154. Each R １５ , q １ , q ２ , q ３ , q ４ , q ５ , q ６ , q ７ , q ８ , q ９ and q １０ These are, independently, hydrogen, fluorine, hydroxyl group, and C １ -C ６ alkyl group, C １ -C ６ Alkoxy group, O(CH ２ ) ２ - OCH ３ The basis selected from, The compound according to claim 153.

155. Each R １５ , q １ , q ２ , q ３ , q ４ , q ５ , q ６ , q ７ , q ８ , q ９ and q １０ These are groups that are independently selected from hydrogen. The compound according to claim 154.

156. The sugar or the nucleoside of the sugar substitution has the following structural formula: 【Chemistry 45】 The compounds according to claims 153 to 155. 【Request Item 157】 【Chemistry 46】 Eventually, Q ８ Ra, Rc, R ｂ M ２ M ３ , X １ , X ２ , X ３ , q １ , q ２ and Bx are as shown in claim 147, The compounds according to claims 147 to 156.

158. R １５ , q １ , q ２ , q ３ , q ４ , q ５ , q ６ , q ７ , q ８ , q ９ and q １０ These are, independently, hydrogen, fluorine, hydroxyl group, methyl group, methoxy group, and O(CH ２ ) ２ - OCH ３ Selected from, The compound according to claim 157.

159. R １５ , q １ , q ２ , q ３ , q ４ , q ５ , q ６ , q ７ , q ８ , q ９ and q １０ These are each independently selected from hydrogen. The compound according to claim 158.

160. M ３ is O, S, C(q ７ ) (q ８ ) and M ２ is C(q ３ ) (q ４ ) and X ２ CR １５ or N, X ３ These are chemical bonds, SO, SO ２ That is, The compounds according to claims 157 to 159.

161. M ３ O, S, CH ２ And M ２ CH ２ X ２ CH is X ３ It is a chemical bond. The compound according to claim 160.

162. M ３ CH ２ And M ２ CH ２ X ２ is N, X ３ is a chemical bond, SO or SO ２ That is, The compound according to claim 160.

163. M ３ is C(q ７ ) (q ８ ) and M ２ is C(q ３ ) (q ４ ) C(q ５ ) (q ６ ) and X ２ CR １５ or N, X ３ These are chemical bonds, SO, SO ２ That is, The compounds according to claims 157 to 159.

164. M ３ CH ２ And M ２ CH ２ CH ２ X ２ CH is X ３ It is a chemical bond. The compound according to claim 163. 165 M ３ CH ２ And M ２ CH ２ CH ２ X ２ is N, X ３ is a chemical bond, SO or SO ２ That is, The compound according to claim 163.

165. Q ８ is S, SO or SO ２ That is, The compounds according to claims 147 to 164.

166. R ｂ is O or S, and Ra and Rc are each independently protected hydroxyl groups, C １ -C ６ alkyl group, C １ -C ６ Selected from alkoxy groups, The compounds according to claims 147 to 165.

167. Ra and Rc are each independently a hydroxyl group, OCH ３ , OCH ２ CH ３ , OCH(CH ３ ) ２ OCH ３ , OCH ２ CH ２ CN, NHSO ２ CH ３ Selected from, The compounds according to claims 147 to 165.

168. B Ｘ The heterocyclic base portion is selected from natural nucleic acid bases, modified nucleic acid bases, and universal bases. The compounds according to claims 147 to 167.

169. B Ｘ The heterocyclic base portion is a pyrimidine, a substituted pyrimidine, pseudouracil, a substituted pseudouracil, a purine, hypoxanthine, or a substituted purine. The compound according to claim 168.

170. B Ｘ The heterocyclic base moieties are 2-thiouracil, 5-fluorouracil, dihydrouridine (D), 7-methylguanosine (m7G), uracil, 5-thiazolauracil, thymine, cytosine, pseudouracil, N1-methyl-psoidouracil, hypoxanthine, 5-methylcytosine, 5-methyluracil, 3-benzoyluracil, 2,6-diaminopurine, adenine, or guanine. The compound according to claim 168.

171. The phosphoramidite is selected from diisopropylcyanoethoxyphosphoramidite, diisopropylethylphosphoramidite, and H-phosphonate. The compounds according to claims 147 to 170.

172. The following specific structure is present: Table 3-1 Table 3-2 Table 3-3 Table 3-4 Table 3-5 Table 3-6 Table 3-7 Table 3-8 or Table 4-1 Table 4-2 Table 4-3 Table 4-4 Table 4-5 Table 4-6 Table 4-7 The compounds according to claims 147 to 171.

Images