5'-modified nucleoside derivative and oligonucleotide comprising same

Abstract

The present disclosure relates to a 5'-modified nucleoside derivative and an oligonucleotide comprising same. Specifically, the oligonucleotide comprises at least one structure represented by formula (I), wherein each functional group is defined in the context of the present disclosure.

Description

5'-Modified Nucleoside Derivatives and Oligonucleotides Containing Them

[0001] Both applications claim priority to CN202410720451.6 filed on June 5, 2024 and CN202410767353.8 filed on June 14, 2024. Technical Field

[0002] This disclosure relates to a 5'-modified nucleoside derivative and an oligonucleotide containing the same, and also to a method for preparing and using the aforementioned oligonucleotide. Background Technology

[0003] RNA interference (RNAi) is an effective way to silence gene expression. Statistics show that over 80% of disease-related proteins in the human body cannot be targeted by conventional small-molecule drugs and large-molecule biological agents, thus becoming undrugable proteins. Using RNA interference technology, suitable siRNAs can be designed based on the mRNA encoding these proteins, specifically targeting and degrading the target mRNA, thereby inhibiting the production of the related proteins. Therefore, siRNAs hold significant promise for drug development. However, unmodified siRNAs suffer from in vivo instability and non-specific distribution, necessitating the development of specific chemical modifications to further enhance their drug potential. Summary of the Invention

[0004] This disclosure provides an oligonucleotide comprising at least one structure as shown in formula (I):

[0005] in,

[0006] X is selected from O or C(R) X )2;

[0007] Each R X Each is independently selected from hydrogen, halogens (e.g., fluorine, chlorine, bromine, iodine), and C. 1-6 Alkyl groups (e.g., methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, 2-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl, and 3-methylpentyl), wherein the C 1-6 Alkyl groups are optionally surrounded by one or more components selected from R A The substituents replaced by R A Selected from halogens (e.g., fluorine, chlorine, bromine, iodine), hydroxyl, cyano, and oxo groups;

[0008] R 1A R 1B Each is independently selected from hydrogen, hydroxyl, and C. 1-6alkyl (e.g., methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, 2-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl, and 3-methylpentyl), C 1-6 alkoxy (e.g., methoxy, ethoxy, propoxy, butoxy), amino, or halogen (e.g., fluorine, chlorine, bromine, iodine), said C 1-6 alkyl, C 1-6 alkoxy, amino is optionally substituted with one or more substituents selected from R A , said R A is selected from halogen, hydroxyl, cyano, oxo;

[0009] or R 1A , R 1B forms a 3- to 6-membered heterocyclyl (e.g., 3-membered, 4-membered, 5-membered, 6-membered) with the carbon atom to which R 1A , R 1B is attached, said 3- to 6-membered heterocyclyl is optionally substituted with one or more substituents selected from R A , said R A is selected from halogen, hydroxyl, cyano, oxo;

[0010] R2, R3are each independently selected from hydrogen or C 1-6 alkyl (e.g., methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, 2-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl, and 3-methylpentyl), said C 1-6 alkyl is optionally substituted with one or more substituents selected from R A , said R A is selected from halogen, hydroxyl, cyano, oxo;

[0011] m is selected from 0, 1, 2, or 3;

[0012] said ring A is selected from 5- to 12-membered heteroaryl (e.g., 5-membered, 6-membered, 7-membered, 8-membered, 9-membered, 10-membered, 11-membered, 12-membered) or 3- to 12-membered heterocyclyl (e.g., 3-membered, 4-membered, 5-membered, 6-membered, 7-membered, 8-membered, 9-membered, 10-membered, 11-membered, 12-membered);

[0013] R4is each independently selected from hydrogen, cyano, halogen (e.g., fluorine, chlorine, bromine, iodine), hydroxyl, amino, C 1-6 alkyl (e.g., methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, 2-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl, and 3-methylpentyl), C 2-6 alkynyl (e.g., ethynyl, prop-1-ynyl, but-1-ynyl, pent-1-ynyl, pent-4-ynyl, and pent-1,4-diynyl), C2-6 alkenyl (e.g., ethenyl, 1-propenyl, 2-propenyl, 2-methyl-l- propenyl, 1-butenyl, 2-butenyl), -S-C 1-6 alkyl (e.g., methylthio, ethylthio, propylthio, butylthio), C 1-6 alkoxy (e.g., methoxy, ethoxy, propoxy, butoxy), -N(C 1-6 alkyl)2(e.g., -NMe2, -NEt2, -N(nPr)2, -N(iPr)2), oxo, 3- to 6-membered cycloalkyl (e.g., cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl, cyclohexenyl, cyclohexadienyl), 6- to 10-membered aryl (e.g., phenyl, naphthyl), 5- to 12-membered heteroaryl (e.g., 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-membered), 3- to 12-membered heterocyclyl (e.g., 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-membered), -NH-(C=0)-C 1-6 alkyl, -NH-(C=0)-C 3-6 cycloalkyl, -NH(C=0)-OC 1-6 alkyl, -NH(C=0)-OC 3-6 cycloalkyl, -O(C=0)NHC 1-6 alkyl, -O(C=0)NH-C 3-6 cycloalkyl, -(C=0)NH-C 1-6 alkyl, -(C=0)-NH-C 3-6 cycloalkyl, -(C=0)-C 1-6 alkyl, -(C=0)-C 3-6 cycloalkyl, -SO2-C 1- 6alkyl, -SO2-C 3-6 cycloalkyl, -SO2-NH2, -SO2-NH-C 1-6 alkyl, -SO2-NH-C 3-6 cycloalkyl, -SO2-N(C 1-6 alkyl)2, -SO2-NH(C 3-6 cycloalkyl)2, -S(O)(NH)-C 1-6 alkyl, -S(O)(NH)-C 3-6 cycloalkyl;

[0014] n is selected from 0, 1, 2, 3, 4, or 5;

[0015] * indicates the direction towards the 5' end of the sequence, # indicates the direction towards the 3' end of the sequence;

[0016] Base represents a base.

[0017] In some embodiments, n is selected from 0 or 1.

[0018] In some embodiments, the structure shown in formula (I) is the structure shown in formula (I-1), and the oligonucleotide contains at least one structure shown in formula (I-1):

[0019] Among them, R 1A R2, R3, R4, m, n, ring A, and Base are defined as in equation (I), where * indicates the direction toward the end of sequence 5' and # indicates the direction toward the end of sequence 3'.

[0020] In some embodiments, the structure of formula (I) is the structure shown in formula (I-2), and the oligonucleotide contains at least one structure shown in formula (I-2):

[0021] Among them, R 1A R4, m, n, ring A, and Base are defined as in equation (I), where * indicates the direction toward the end of sequence 5' and # indicates the direction toward the end of sequence 3'.

[0022] In some embodiments, the structure shown in formula (I) is a structure shown in formula (I-3), and the oligonucleotide contains at least one structure shown in formula (I-3):

[0023] Among them, R 1A R2, R3, R4, n, ring A, and Base are defined as in equation (I), where * indicates the direction toward the end of sequence 5' and # indicates the direction toward the end of sequence 3'.

[0024] In some embodiments, the structure shown in formula (I) is a structure shown in formula (I-4), and the oligonucleotide contains at least one structure shown in formula (I-4):

[0025] Among them, R 1A R4, n, ring A, and Base are defined as in equation (I), where * indicates the direction toward the end of sequence 5' and # indicates the direction toward the end of sequence 3'.

[0026] In some implementation schemes, R 1A Selected from hydroxyl, C 1-6 Alkyl groups (e.g., methoxy, ethoxy, propoxy, butoxy), the C 1-6 Alkyl groups are optionally surrounded by one or more R groups. A The substituents replaced by R A Selected from halogens, hydroxyl groups, cyano groups, and oxo groups.

[0027] In some embodiments, R 1A is selected from C 1-6 alkyl, said C 1-6 alkyl is optionally substituted with one or more substituents selected from R A , said R A is selected from halogen, hydroxyl, cyano, oxo.

[0028] In alternative embodiments, R 1A is methoxy.

[0029] In some embodiments, said R 1A is halogen (e.g., fluorine, chlorine, bromine, iodine).

[0030] In alternative embodiments, R 1A is fluorine.

[0031] In some embodiments, said ring A is a 5- to 12-membered heteroaryl (e.g., 5-membered, 6-membered, 7-membered, 8-membered, 9-membered, 10-membered, 11-membered, 12-membered).

[0032] In some embodiments, ring A is a 5- to 6-membered heteroaryl.

[0033] In some embodiments, ring A is a 1,2,4-triazole ring.

[0034] In some specific embodiments, ring A is

[0035] In some embodiments, said ring A is a 3- to 12-membered heterocyclyl (e.g., 3-membered, 4-membered, 5-membered, 6-membered, 7-membered, 8-membered, 9-membered, 10-membered, 11-membered, 12-membered).

[0036] In some embodiments, ring A is a 3- to 6-membered heterocyclyl.

[0037] In some embodiments, ring A is a morpholine ring or azetidine.

[0038] In some specific embodiments, ring A is selected from

[0039] In some embodiments, said R4is each independently selected from hydrogen, cyano, halogen (e.g., fluorine, chlorine, bromine, iodine), hydroxyl, amino, C 1-6 alkyl (e.g., methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, t-butyl, pentyl, 2-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl, and 3-methylpentyl), C 2-6 alkynyl (e.g., ethynyl, prop-1-ynyl, but-1-ynyl, pent-1-ynyl, pent-4-ynyl, and pent-1,4-diynyl), C 2-6alkenyl (e.g., ethenyl, 1-propenyl, 2-propenyl, 2-methyl-l-propenyl, 1-butenyl, 2-butenyl), -S-C 1-6 alkyl (e.g., methylthio, ethylthio, propylthio, butylthio), C 1-6 alkoxy (e.g., methoxy, ethoxy, propoxy, butoxy), -N(C 1-6 alkyl)2(e.g., -NMe2, -NEt2, -N(nPr)2, -N(iPr)2).

[0040] In some embodiments, each R4is independently selected from hydrogen, C 1-6 alkyl, -S-C 1-6 alkyl, C 1-6 alkoxy, -N(C 1-6 alkyl)2.

[0041] In some specific embodiments, each R4is independently selected from hydrogen or -N(C 1-6 alkyl)2.

[0042] In some embodiments, the structure according to Formula (I) is a structure according to any one of Formulas (I-5) - (I-12), and the oligonucleotide contains at least one structure according to any one of Formulas (I-5) - (I-12):

[0043] wherein Base represents a base, * represents a direction toward the 5' end of the sequence, and # represents a direction toward the 3' end of the sequence, and the structure according to any one of Formulas (I-5) - (I-12) is linked to the nucleoside adjacent thereto by a phosphodiester group or a phosphodiester group having a modifying group.

[0044] In some embodiments, the modified phosphodiester group is a phosphorothioate group.

[0045] In some specific embodiments, the structure according to Formula (I) is a structure according to any one of Formulas (I-5A) - (I-12B). The oligonucleotide contains at least one structure according to any one of Formulas (I-5A) - (I-12B):

[0046] wherein Base represents a base, * represents a direction toward the 5' end of the sequence, and # represents a direction toward the 3' end of the sequence, and the structure according to any one of Formulas (I-5A) - (I-12B) is linked to the nucleoside adjacent thereto by a phosphodiester group or a phosphodiester group having a modifying group.

[0047] In some embodiments, the structure according to Formula (I) is a structure according to any one of Formulas (I-11) or (I-12):

[0048] * indicates the direction towards the 5' end of the sequence, and # indicates the direction towards the 3' end of the sequence.

[0049] In some specific embodiments, the structure according to Formula (I) is a structure according to any one of Formulas (I-5A), (I-5B), (I-6A), or (I-6B):

[0050] * indicates the direction towards the 5' end of the sequence, and # indicates the direction towards the 3' end of the sequence.

[0051] In some embodiments, the modified phosphodiester group is a phosphorothioate diester group.

[0052] In some embodiments, the oligonucleotide is a single-stranded oligonucleotide, a double-stranded oligonucleotide, an antisense oligonucleotide (ASO), a small interfering RNA (siRNA), a double-stranded RNAi agent (dsRNA), a microRNA (miRNA), a short hairpin RNA (shRNA), a ribozyme, an RNAi inhibitor molecule, and a Dicer enzyme substrate.

[0053] In some embodiments, the oligonucleotide is a single-stranded oligonucleotide.

[0054] In some embodiments, the single-stranded oligonucleotide is an antisense oligonucleotide (ASO) or a single-stranded RNAi molecule (ssRNAi), the single-stranded oligonucleotide consisting of 15-50 nucleotides.

[0055] In some embodiments, the single-stranded oligonucleotide molecule has 16 to 50, 16 to 45, 17 to 45, 17 to 40, 18 to 40, 18 to 35, 18 to 32, 18 to 31, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 19 to 25, 19 to 24, or 19 to 23 nucleotides.

[0056] In some embodiments, the oligonucleotide is a double-stranded RNAi agent, the RNAi agent comprising a sense strand and an antisense strand that are at least partially complementary to form a double-stranded region.

[0057] In some embodiments, only 1 nucleotide in the oligonucleotide contains a structure according to Formula (I).

[0058] In some embodiments, at least one of the 2ndto 10th(eg., 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th) nucleotides from the 5' terminus of the antisense strand comprises a structure according to Formula (I).

[0059] In some embodiments, the 6th, 7th, 8th, or 9thnucleotide from the 5' terminus of the antisense strand comprises a structure according to Formula (I). In some particular embodiments, the 7thor 8thnucleotide from the 5' terminus of the antisense strand comprises a structure according to Formula (I).

[0060] In some embodiments, the sense strand is at least partially reverse complementary to the antisense strand to form a double stranded region.

[0061] In some embodiments, there are no more than 5, no more than 4, no more than 3, no more than 2, no more than 1 mismatches between the sense strand and the antisense strand.

[0062] In some embodiments, the sense strand is fully reverse complementary to the antisense strand.

[0063] In some embodiments, the double stranded RNAi agent molecule comprises one or two blunt ends.

[0064] In some embodiments, the double stranded RNAi agent molecule comprises overhangs having from 1 to 4 unpaired nucleotides, for example 1, 2, 3, or 4 unpaired nucleotides.

[0065] In some embodiments, the 3' end of the antisense strand comprises an overhang.

[0066] In some embodiments, the sense strand is 15-35 nucleotides in length and the antisense strand is 15-35 nucleotides in length.

[0067] In some embodiments, the sense strand and antisense strand each independently have from 16 to 35, from 16 to 34, from 17 to 34, from 17 to 33, from 18 to 33, from 18 to 32, from 18 to 31, from 18 to 30, from 18 to 29, from 18 to 28, from 18 to 27, from 18 to 26, from 18 to 25, from 18 to 24, from 18 to 23, from 19 to 25, from 19 to 24, or from 19 to 23 nucleotides.

[0068] In some embodiments, the sense strand and antisense strand are the same or different in length, the sense strand is 19-23 nucleotides in length and the antisense strand is 19-26 nucleotides in length.

[0069] The length ratio of the sense strand and the antisense strand in the RNAi agent provided by the present disclosure can be 19 / 20, 19 / 21, 19 / 22, 19 / 23, 19 / 24, 19 / 25, 19 / 26, 20 / 20, 20 / 21, 20 / 22, 20 / 23, 20 / 24, 20 / 25, 20 / 26, 21 / 20, 21 / 21, 21 / 22, 21 / 23, 21 / 24, 21 / 25, 21 / 26, 22 / 20, 22 / 21, 22 / 22, 22 / 23, 22 / 24, 22 / 25, 22 / 26, 23 / 20, 23 / 21, 23 / 22, 23 / 23, 23 / 24, 23 / 25, or 23 / 26.

[0070] In some embodiments, the length of the sense strand and the antisense strand is 19 / 21, 21 / 21, 21 / 23, or 23 / 25.

[0071] In some embodiments, the length ratio of the sense strand and the antisense strand is 19 / 21 or 21 / 21.

[0072] In some embodiments, the oligonucleotide of the present disclosure further comprises one or more delivery groups. Each of the delivery groups is attached to any one of the nucleotides in the oligonucleotide. In some embodiments, the oligonucleotide is a double-stranded RNAi agent, and the delivery groups are attached to the sense strand and / or the antisense strand. In the context of the present disclosure, "attached" includes covalent and non-covalent attachment. The delivery groups are capable of delivering the oligonucleotide of the present disclosure to a location where the target gene expression exists.

[0073] In some embodiments, the oligonucleotide is a double-stranded RNAi agent, and the one or more delivery groups are attached to any one or more of the nucleotides in the sense strand or the antisense strand of the oligonucleotide. In some embodiments, the one or more delivery groups are attached to the sense strand of the RNAi agent. In some embodiments, the one or more delivery groups are attached to the antisense strand of the RNAi agent. In some embodiments, at least one of the delivery groups is attached to the sense strand of the RNAi agent, and at least one of the delivery groups is attached to the antisense strand of the RNAi agent. In some embodiments, the delivery groups are attached to the 3' end of the sense strand of the RNAi agent.

[0074] In some embodiments, the delivery groups are attached to the base of the nucleotide.

[0075] In some embodiments, the delivery groups are attached to the sugar ring of the nucleotide.

[0076] In some embodiments, the delivery groups are attached to the internucleoside linking group between two adjacent nucleotides.

[0077] In some embodiments, the delivery group is a lipophilic group.

[0078] In some embodiments, the lipophilic group comprises a saturated or unsaturated C 4-30 hydrocarbon chain, and optionally a functional group selected from halogen, alkoxy, hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.

[0079] In some embodiments, the lipophilic group comprises a saturated or unsaturated C 6-18 hydrocarbon chain.

[0080] In some embodiments, the lipophilic group comprises a saturated or unsaturated C 16 hydrocarbon chain.

[0081] In some embodiments, the delivery group comprises a targeting ligand that targets the liver.

[0082] In some embodiments, the targeting ligand binds to the Asialoglycoprotein Receptor (ASGPR).

[0083] In some embodiments, the targeting ligand comprises a cluster of galactose or a cluster of galactose derivatives selected from N-acetyl-galactosamine, N- trifluoroacetyl galactosamine, N-propionyl galactosamine, N-n-butyryl galactosamine, or N- isobutyryl galactosamine.

[0084] In some embodiments, the delivery group is a delivery group as disclosed in PCT application WO2025067317A1 or WO2023274395A. In some embodiments, the delivery group has a structure as shown in formula (V):

[0085] In some embodiments, the delivery group is directly linked to the end of the RNAi agent by a phosphodiester group, a thiophosphodiester group, or a phosphonate group. In some embodiments, the delivery group is directly linked to the end of the RNAi agent by a phosphodiester group. In some embodiments, the delivery group is indirectly linked to the end of the RNAi agent by a phosphodiester group, a thiophosphodiester group, or a phosphonate group.

[0086] In some embodiments, the delivery group is directly linked to the end of the RNAi agent by a phosphodiester group, a thiophosphodiester group, or a phosphonate group. In some embodiments, the delivery group is directly linked to the end of the RNAi agent by a phosphodiester group. In some embodiments, the delivery group is directly linked to the 3’ end of the sense strand of the RNAi agent by a phosphodiester group or a thiophosphodiester group. In some embodiments, the delivery group is directly linked to the 3’ end of the sense strand of the RNAi agent by a phosphodiester group.

[0087] In some embodiments, at least one additional nucleotide in the oligonucleotide described in the present disclosure is also a modified nucleotide. In some embodiments, at least one modified nucleotide is comprised in the sense strand and the antisense strand, respectively. In some embodiments, all nucleotides in the oligonucleotide are modified nucleotides.

[0088] In some embodiments, the modified nucleotide is selected from the group consisting of: 2'-methoxy modified nucleotide, 2'-substituted alkoxy modified nucleotide, 2'-alkyl modified nucleotide, 2'-substituted alkyl modified nucleotide, 2'-amino modified nucleotide, 2'-substituted amino modified nucleotide, 2'-fluoro modified nucleotide, 2'-deoxynucleotide, 2'-deoxy-2'-fluoro modified nucleotide, 3'-deoxy-thymine (dT) nucleotide, iso-nucleotides, LNA, ENA, cET, UNA, GNA.

[0089] In some embodiments, the 5' position of the sugar ring of the first nucleotide at the 5' end of one strand in the oligonucleotide is a 5'-vinyl phosphodiester group (VP). In some embodiments, the 5'-vinyl phosphodiester group is a 5'-trans vinyl phosphodiester group (E-VP). In some embodiments, the oligonucleotide is a double stranded RNAi agent, the 5' position of the sugar ring of the first nucleotide at the 5' end of the sense strand or the antisense strand is a 5'-vinyl phosphodiester group (VP).

[0090] In some embodiments, the modified nucleotide is independently selected from the group consisting of: 2'-methoxy modified nucleotide, 2'-fluoro modified nucleotide.

[0091] In some embodiments, at least one phosphodiester group in the sense strand and / or the antisense strand is a phosphodiester group with a modification group.

[0092] In some embodiments, a plurality of phosphodiester groups with a modification group is comprised in the sense strand and / or the antisense strand.

[0093] In some embodiments, a plurality of phosphodiester groups with a modification group is comprised in both the sense strand and the antisense strand.

[0094] In some embodiments, four phosphodiester groups with a modification group are comprised in the sense strand and four phosphodiester groups with a modification group are comprised in the antisense strand.

[0095] In some embodiments, the phosphodiester group with a modifying group is present at one or more of the positions selected from the group consisting of: between any two adjacent nucleotides from the 1stto the 4thnucleotide of the 5' terminal end and / or the 3' terminal end of the sense strand, and / or between any two adjacent nucleotides from the 1stto the 4thnucleotide of the 5' terminal end and / or the 3' terminal end of the antisense strand, for example, can be located at:

[0096] between the 1stand 2ndnucleotides of the 5' terminal end of the sense strand;

[0097] between the 2ndand 3rdnucleotides of the 5' terminal end of the sense strand;

[0098] between the 1stand 2ndnucleotides of the 3' terminal end of the sense strand;

[0099] between the 2ndand 3rdnucleotides of the 3' terminal end of the sense strand;

[0100] between the 1stand 2ndnucleotides of the 5' terminal end of the antisense strand;

[0101] between the 2ndand 3rdnucleotides of the 5' terminal end of the antisense strand;

[0102] between the 1stand 2ndnucleotides of the 3' terminal end of the antisense strand; and

[0103] between the 2ndand 3rdnucleotides of the 3' terminal end of the antisense strand.

[0104] In some embodiments, the phosphodiester group with a modifying group is a phosphorothioate group.

[0105] In some embodiments, the oligonucleotide of the present disclosure targets the Inhibin beta E gene (INHBE) or the Sterile Alpha and TIR Containing 1 (SARM1) gene.

[0106] In some embodiments, the oligonucleotide is any one of the oligonucleotides set forth in Table 2. In some embodiments, the oligonucleotide is TJR102840, TJR103687, or TJR103688.

[0107] The present disclosure also provides a compound of Formula (II) or a tautomer thereof:

[0108] wherein R 1A , R 1B , R2, R3, R4, m, n, ring A, Base, X are as defined in Formula (I).

[0109] W is selected from H or a hydroxyl protecting group;

[0110] Q is a phosphorus-containing reactive group.

[0111] In some embodiments, the compound of Formula (II) or a tautomer thereof, W is selected from MMTr, DMTr, or trityl.

[0112] In some embodiments, the compound of Formula (II) or a tautomer thereof, Q is

[0113] In some embodiments, the compound of Formula (II) or a tautomer thereof, is a compound of Formula (II-1) or a tautomer thereof:

[0114] wherein R 1A , R2, R3, R4, m, n, ring A, Base are as defined in Formula (I);

[0115] W, Q are as defined in Formula (II).

[0116] In some embodiments, the compound of Formula (II) or a tautomer thereof, is a compound of Formula (II-2) or a tautomer thereof:

[0117] wherein R 1A , R4, m, n, ring A, Base are as defined in Formula (I);

[0118] W, Q are as defined in Formula (II).

[0119] In some embodiments, the compound of Formula (II) or a tautomer thereof, is a compound of Formula (II-3) or a tautomer thereof:

[0120] wherein R 1A , R2, R3, R4, n, ring A, Base are as defined in Formula (I);

[0121] W, Q are as defined in Formula (II).

[0122] In some embodiments, the compound of Formula (II) or a tautomer thereof, is a compound of Formula (II-4) or a tautomer thereof:

[0123] wherein R 1A , R4, n, ring A, Base are as defined in Formula (I);

[0124] W and Q are defined as in equation (II).

[0125] In some embodiments, the compound represented by formula (II) or its tautomer, wherein R 1A Selected from hydroxyl, C 1-6 Alkyl groups (e.g., methoxy, ethoxy, propoxy, butoxy), the C 1-6 Alkyl groups are optionally surrounded by one or more components selected from R A The substituents replaced by R A Selected from halogens, hydroxyl groups, cyano groups, and oxo groups.

[0126] In some embodiments, the compound represented by formula (II) or its tautomer, wherein R 1A Selected from C 1-6 Alkoxy, the C 1-6 Alkyl groups are optionally surrounded by one or more R groups. A The substituents replaced by R A Selected from halogens, hydroxyl groups, cyano groups, and oxo groups.

[0127] In an optional embodiment, in the compound represented by formula (II) or its tautomer, R 1A Selected from methoxy groups.

[0128] In some embodiments, in the compound represented by formula (II) or its tautomers, wherein R 1A It is a halogen (e.g., fluorine, chlorine, bromine, iodine).

[0129] In an optional embodiment, in the compound represented by formula (II) or its tautomer, R 1A It's fluorine.

[0130] In some embodiments, in the compound represented by formula (II) or its tautomers, ring A is a 5- to 12-membered heteroaryl group (e.g., 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-membered).

[0131] In some embodiments, in the compound of formula (II) or its tautomers, ring A is a 5- to 6-membered heteroaryl group.

[0132] In some embodiments, in the compound of formula (II) or its tautomers, ring A is a 1,2,4-triazole ring.

[0133] In some specific embodiments, in the compound represented by formula (II) or its tautomers, ring A is

[0134] In some embodiments, the compound of Formula (II) or a tautomer thereof, ring A is a 3- to 12-membered heterocyclyl (e.g., 3-membered, 4-membered, 5-membered, 6-membered, 7-membered, 8-membered, 9-membered, 10-membered, 11-membered, 12-membered).

[0135] In some embodiments, the compound of Formula (II) or a tautomer thereof, ring A is a 3- to 6-membered heterocyclyl.

[0136] In some embodiments, the compound of Formula (II) or a tautomer thereof, ring A is a morpholine ring or azetidine.

[0137] In some embodiments, the compound of Formula (II) or a tautomer thereof, ring A is a morpholine ring or azetidine.

[0138] In some embodiments, the compound of Formula (II) or a tautomer thereof, wherein each R4is independently selected from hydrogen, cyano, halogen (e.g., fluorine, chlorine, bromine, iodine), hydroxyl, amino, C 1-6 alkyl (e.g., methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, t-butyl, pentyl, 2-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl, and 3-methylpentyl), C 2-6 alkynyl (e.g., ethynyl, prop-1-ynyl, but-1-ynyl, pent-1-ynyl, pent-4-ynyl, and pent-1,4-diynyl), C 2-6 alkenyl (e.g., ethenyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl), -S-C 1-6 alkyl (e.g., methylthio, ethylthio, propylthio, butylthio), C 1-6 alkoxy (e.g., methoxy, ethoxy, propoxy, butoxy), -N(C 1-6 alkyl)2(e.g., -NMe2, -NEt2, -N(nPr)2, -N(iPr)2).

[0139] In some embodiments, the compound of Formula (II) or a tautomer thereof, wherein each R4is independently selected from hydrogen, C 1-6 alkyl, -S-C 1-6 alkyl, C 1-6 alkoxy, -N(C 1-6 alkyl)2.

[0140] In some embodiments, the compound of Formula (II) or a tautomer thereof, wherein each R4is independently selected from hydrogen, C 1-6 alkyl, -S-C

[0141] In some embodiments, the compound of formula (II) or a tautomer thereof is selected from:

[0142] wherein Base is as defined in formula (I).

[0143] In some embodiments, the compound of formula (II) or a tautomer thereof is selected from:

[0144] wherein Base is as defined in formula (I).

[0145] The present disclosure also provides a method for preparing the oligonucleotide of the present disclosure, comprising the steps of:

[0146] (1) synthesizing the compound of formula (II) or a tautomer thereof according to the present disclosure;

[0147] (2) synthesizing the oligonucleotide according to the present disclosure using the compound of formula (II) or a tautomer thereof obtained in step (1).

[0148] The present disclosure also provides a pharmaceutical composition comprising the oligonucleotide of the present disclosure.

[0149] In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

[0150] In some embodiments, the pharmaceutically acceptable excipient can be, for example, a carrier, a vehicle, a diluent, and / or a delivery polymer, various drug delivery systems are known and can be used for the RNAi agents of the present disclosure, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the RNAi agent, receptor-mediated endocytosis, construction of the nucleic acid as part of a retroviral or other vector.

[0151] In some embodiments, the pharmaceutical composition can further comprise a pharmaceutically acceptable adjuvant and / or an adjuvant. The adjuvant can be one or more of various preparations or compounds conventionally used in the art, for example, the pharmaceutically acceptable adjuvant can include at least one of a pH buffering agent, a protective agent, and an osmotic pressure adjusting agent.

[0152] In some embodiments, the unit dose of the pharmaceutical composition is 0.001 mg-1000 mg.

[0153] In some embodiments, the pharmaceutical composition contains 0.01-99.99% of the oligonucleotide of the disclosure, or a pharmaceutically acceptable salt thereof, or an isotopically substituted version thereof, based on the total weight of the composition.

[0154] In some embodiments, the pharmaceutical composition contains 0.1-99.9% of the oligonucleotide of the disclosure, or a pharmaceutically acceptable salt thereof, or an isotopically substituted version thereof.

[0155] In some embodiments, the pharmaceutical composition contains 0.5-99.5% of the oligonucleotide of the disclosure, or a pharmaceutically acceptable salt thereof, or an isotopically substituted version thereof.

[0156] In some embodiments, the pharmaceutical composition contains 1-99% of the oligonucleotide of the disclosure, or a pharmaceutically acceptable salt thereof, or an isotopically substituted version thereof.

[0157] In some embodiments, the pharmaceutical composition contains 2-98% of the oligonucleotide of the disclosure, or a pharmaceutically acceptable salt thereof, or an isotopically substituted version thereof.

[0158] In some embodiments, the pharmaceutical composition contains 0.01-99.99% of a pharmaceutically acceptable excipient, based on the total weight of the composition. In some embodiments, the pharmaceutical composition contains 0.1-99.9% of a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition contains 0.5-99.5% of a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition contains 1-99% of a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition contains 2-98% of a pharmaceutically acceptable excipient.

[0159] In some embodiments, the oligonucleotide or pharmaceutical composition of the disclosure, when contacted to a cell expressing a target gene, inhibits the expression of the target gene by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, as determined by, for example: psiCHECK activity screening and luciferase reporter assay, other methods such as PCR or branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence assays, for example Western Blot or flow cytometry.

[0160] In some embodiments, the oligonucleotide or pharmaceutical composition of the present disclosure, when contacted to a cell expressing a target gene, causes the percentage of residual expression of the mRNA of the target gene to be no more than 99%, no more than 95%, no more than 90%, no more than 85%, no more than 80%, no more than 75%, no more than 70%, no more than 65%, no more than 60%, no more than 55%, no more than 50%, no more than 45%, no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, or no more than 10% as determined by, for example, psiCHECK activity screening and luciferase reporter assay, other methods such as PCR or branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence assays, for example, Western Blot or flow cytometry.

[0161] The oligonucleotide and / or pharmaceutical composition of the present disclosure can be delivered by local administration (e.g., direct injection, implantation), systemic administration, or any suitable mode of administration commonly known in the art, including subcutaneous, intravenous, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal and intrathecal), intramuscular, transdermal, airway (aerosol), nasal, oral, rectal, or buccal and sublingual administration.

[0162] In alternative embodiments, the pharmaceutical composition provided by the present disclosure can be administered by injection, for example, intravenous, intramuscular, intradermal, subcutaneous, intraduodenal or intraperitoneal injection.

[0163] In another aspect, the present disclosure provides a cell comprising the oligonucleotide of the present disclosure.

[0164] In another aspect, the present disclosure provides a kit comprising the oligonucleotide and / or pharmaceutical composition of the present disclosure.

[0165] In alternative embodiments, the conjugate can be packaged in a kit when the delivery group is linked to the oligonucleotide.

[0166] The present disclosure also provides a method for silencing the mRNA of a target gene in a cell, the method comprising the step of introducing the oligonucleotide and / or pharmaceutical composition of the present disclosure into the cell.

[0167] The present disclosure also provides a method for silencing a target gene or the mRNA of a target gene in a cell in vivo or in vitro, the method comprising the step of introducing the oligonucleotide and / or pharmaceutical composition according to the present disclosure into the cell.

[0168] The present disclosure also provides a method for inhibiting a target gene or mRNA expression of a target gene, the method comprising administering to a subject in need thereof an effective amount or effective dose of the oligonucleotide and / or pharmaceutical composition according to the present disclosure.

[0169] In some embodiments, the effective amount or effective dose of the oligonucleotide and / or pharmaceutical composition is about 0.001 mg / kg body weight to about 200 mg / kg body weight, about 0.01 mg / kg body weight to about 100 mg / kg body weight, or about 0.5 mg / kg body weight to about 50 mg / kg body weight.

[0170] In another aspect, the present disclosure also provides use of the oligonucleotide and / or pharmaceutical composition of the present disclosure in the manufacture of a medicament for preventing and / or treating a disease associated with the expression of a target gene.

[0171] In another aspect, the present disclosure provides a method for preventing and / or treating a disease, comprising administering to a subject an effective amount or effective dose of the oligonucleotide and / or pharmaceutical composition of the present disclosure described above, the disease being a disease associated with the expression of a target gene.

[0172] In some embodiments, the target gene is an INHBE gene, and the disease is a disease associated with the expression of the INHBE gene, including but not limited to a metabolic disorder.

[0173] In some embodiments, the metabolic disorder is selected from one or more of metabolic syndrome (MS), cardiovascular disease, obesity, hepatitis, and kidney disease.

[0174] In some embodiments, the target gene is a SARM1 gene, and the disease is an axonal degenerative disease. In some embodiments, the axonal degenerative disease is a central nervous system degenerative disease and an ocular disease associated with axonal degeneration. In some embodiments, the central nervous system degenerative disease is selected from one or more of diabetic induced neuropathic pain, optic nerve injury, multiple sclerosis (MS), spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD), Alzheimer’s disease (AD), neuropathic pain (e.g., post-chemotherapy neuropathic pain), chemotherapy induced peripheral neuropathy (CIPN), traumatic brain injury, spinal cord injury, Charcot-Marie-Tooth disease, and / or peripheral neuropathy. In some embodiments, the ocular disease is selected from glaucoma, optic nerve injury.

[0175] In some embodiments, the subject has been previously identified as having a pathological upregulation of the target gene or mRNA thereof in the targeted cell, cell population, tissue, or subject.

[0176] Unless otherwise specified, the "RNAi agent," "nucleic acid," "oligonucleotide," "single-stranded oligonucleotide," "double-stranded oligonucleotide," "antisense oligonucleotide," "small interfering RNA (siRNA)," "double-stranded RNA (dsRNA)," "micro RNA (miRNA)," "short hairpin RNA (shRNA)," "ribozyme," "RNAi inhibitor molecule," "double-stranded RNAi inhibitor molecule," and "Dicer substrate," "compound" of the disclosure can independently exist in a salt, a mixed salt, or a non-salt (e.g., free acid or free base) form, and the "compound" of the disclosure includes the oligonucleotides of the disclosure. When in a salt or mixed salt form, it can be a pharmaceutically acceptable salt.

[0177] In the absence of specified configurations, the compounds of the disclosure (e.g., oligonucleotides, RNAi agents, siRNAs, chemical structures represented by Formula (I), targeting ligands, compounds represented by Formula (II), etc.) can exist in particular geometric or stereoisomeric forms. The disclosure contemplates all such compounds, including cis- and trans-isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)- isomers, (L)-isomers, as well as the racemic mixtures and other mixtures thereof, such as those that are enantiomeric or diastereomeric mixtures. Additional asymmetric carbon atoms can be present in a substituent group. All such isomers, as well as mixtures thereof, are included within the scope of the disclosure. The compounds of the disclosure containing an asymmetric carbon atom can be isolated in optically active or racemic forms. This can be achieved by

[0178] Optically active (R)- and (S)-isomers, and D and L isomers, can be prepared by chiral synthesis or by chiral reagents or other conventional techniques. If desired, one enantiomer of a compound of the disclosure can be obtained by asymmetric synthesis or derivatization with a chiral auxiliary, separation of the resulting diastereomeric mixture, and cleavage of the auxiliary to provide the pure desired enantiomer. Alternatively, when a basic functional group (e.g., amino) or an acidic functional group (e.g., carboxyl) is present in the molecule, diastereomeric salts can be formed with an appropriate optically active acid or base, and the diastereomeric salt separated by conventional means, and the pure enantiomer recovered by treating with base or acid, as appropriate. Additionally, separation of the enantiomers and diastereomers is typically accomplished by chromatography using a chiral stationary phase, optionally in combination with chemical derivatization (e.g., formation of carbamates from amines).

[0179] In the chemical structures of the compounds described herein (including oligonucleotides, RNAi agents, siRNAs, chemical structures depicted in Formula (I), targeting ligands, compounds depicted in Formula (II), etc.) where a bond is drawn without indicating a configuration, the bond represents no specified configuration, i.e., if a chiral isomer is present in the chemical structure, the bond may be or or both configurations are included. and The bond in the chemical structure of the compounds described herein is not specified as to configuration, i.e., the configuration of the bond may be E or Z, or both configurations are included.

[0180] The present disclosure also includes some isotopically-labeled compounds of the present disclosure which are identical to those recited herein, but for the inclusion of one or more atoms of an element naturally found in the atom, but present at an abnormal proportion or an abnormal number. Examples of isotopes that can be incorporated into a compound of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, sulfur, fluorine, iodine, and chlorine, such as 2 H, 3 H, 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F, 123 I, 125 I, and 36 Cl.

[0181] Deuterated drugs have advantages of reducing side effects, increasing drug stability, enhancing efficacy, prolonging the biological half-life of drugs, etc. compared to non-deuterated drugs. All isotopic compositions of the compounds of the present disclosure, whether radioactive or not, are included within the scope of the present disclosure. Each of the available hydrogen atoms attached to a carbon atom can be independently replaced with a deuterium atom, wherein the replacement of deuterium can be partial or complete, partial replacement of deuterium means that at least one hydrogen is replaced with at least one deuterium. Those skilled in the art can refer to the relevant literature to synthesize deuterated forms of compounds. Commercially available deuterated starting materials can be used when preparing deuterated forms of compounds, or they can be synthesized using conventional techniques using deuterated reagents, including but not limited to deuterated borane, tritetradeuterated borane tetrahydrofuran solution, deuterated lithium aluminum hydride, deuterated iodoethane, and deuterated iodomethane, etc.

[0182] Additionally, unless otherwise indicated, the compounds and intermediates of the present disclosure (including oligonucleotides, RNAi agents, siRNAs, chemical structures depicted in Formula (I), targeting ligands, compounds depicted in Formula (II), etc.) can exist in different tautomeric forms, and all such forms are encompassed by the scope of the present disclosure. The term “tautomer” or “tautomeric form” refers to different energy structures that can interconvert via a low energy barrier.

[0183] Terminology

[0184] For the purposes of the present disclosure, certain technical and scientific terms are specifically defined below. Unless otherwise clearly indicated, all other technical and scientific terms used herein have the meanings that would be given to them by one of ordinary skill in the art to which the present disclosure pertains.

[0185] As used herein, “oligonucleotide” refers to a single- or double-stranded oligonucleotide molecule composed of a plurality of linked nucleotides, including single-stranded oligonucleotides, double-stranded oligonucleotides, antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), double-stranded RNAi agents (dsRNAs), microRNAs (miRNAs), short hairpin RNAs (shRNAs), ribozymes, RNAi inhibitor molecules, and Dicer enzyme substrates.

[0186] As used herein, “RNAi agent” (also referred to as “RNAi trigger”) means an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule that contains a sequence capable of degrading or inhibiting (e.g., under appropriate conditions) the translation of a messenger RNA (mRNA) transcript of a target mRNA in a sequence-specific manner. As used herein, an RNAi agent can act through an RNA interference mechanism (i.e., by interacting with the machinery of the RNA interference pathway of a mammalian cell (the RNA-induced silencing complex or RISC) to induce RNA interference) or through any alternative mechanism or pathway. While the term RNAi agent as used herein is believed to act primarily through an RNA interference mechanism, the disclosed RNAi agents are not bound or limited to any particular mechanism or pathway of action. The RNAi agents disclosed herein are composed of a sense strand and an antisense strand, and include, but are not limited to, short (or small) interfering RNAs (siRNAs). The antisense strand of the RNAi agent described herein is at least partially complementary to the targeted mRNA. The RNAi agent can comprise one or more modified nucleotides and / or one or more non-phosphodiester linkages.

[0187] The term "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of a target gene, including mRNA that is a product of RNA processing of the primary transcript. The portion of the target sequence that is targeted should be long enough to serve as a substrate for iRNA-directed cleavage. In one embodiment, the target sequence is within the protein coding region of the target gene. As used herein, in the context of RNA-mediated gene silencing, the sense strand (also referred to as SS, SS strand, or sense strand) refers to the strand comprising a sequence identical or substantially identical to the sequence of the target mRNA; the antisense strand (also referred to as AS or AS strand) refers to the strand having a sequence complementary to the sequence of the target mRNA.

[0188] The terms "Sterile Alpha and TIR Motif 1", "SARM1", are used interchangeably in the present disclosure, unless otherwise specified. SARM1 includes, but is not limited to, human SARM1, cynomolgus monkey SARM1, rabbit SARM1, mouse SARM1, rat SARM1, the amino acid and complete coding sequences, mRNA sequences of which are readily available using publicly available databases, e.g., GenBank, UniProt, OMIM, and the Macaca Genome Project website.

[0189] The term "SARM1" also refers to naturally occurring DNA sequence variations of the SARM1 gene, such as single nucleotide polymorphisms (SNPs) in the SARM1 gene. Exemplary SNPs can be found in the dbSNP database.

[0190] The terms "Inhibin Beta E", "INHBE", are used interchangeably in the present disclosure, unless otherwise specified. INHBE includes, but is not limited to, human INHBE, cynomolgus monkey INHBE, mouse INHBE, rat INHBE, the amino acid and complete coding sequences, mRNA sequences of which are readily available using publicly available databases, e.g., GenBank, UniProt, OMIM, and the Macaca Genome Project website.

[0191] The term "INHBE" also refers to naturally occurring DNA sequence variations of the INHBE gene, such as single nucleotide polymorphisms (SNPs) in the INHBE gene. Exemplary SNPs can be found in the dbSNP database.

[0192] In the present disclosure, the "5' region" or "5' end" or "5' terminal" of the sense strand or antisense strand can be used interchangeably. For example, the 2nd to 10th nucleotides of the 5' region of the antisense strand can be used interchangeably with the 2nd to 10th nucleotides of the 5' end of the antisense strand. Similarly, the "3' region", "3' end" and "3' terminal" of the sense strand or antisense strand can be used interchangeably.

[0193] In the context of the present disclosure, "G", "C", "A", "T" and "U" represent nucleotides comprising the bases guanine, cytosine, adenine, thymine and uracil, respectively, unless otherwise specified. It is well known to those skilled in the art that the replacement of bases T and U does not significantly affect the properties of the RNAi agent sequence, and that U in the sequences of the present disclosure can be replaced by T at will, and the resulting sequence is also within the scope of the present disclosure. In the sequences of the present disclosure, for the same nucleic acid strand, in the direction from 5' end to 3' end, the direction from left to right, the lower case letter m indicates that the nucleotide adjacent to the left of the letter m is a 2'-methoxy-modified nucleotide; the lower case letter f indicates that the nucleotide adjacent to the left of the letter f is a 2'-fluoro-modified nucleotide; the lower case letter s indicates that the two nucleotides adjacent to the letter s are connected by a phosphorothioate diester group, and unless otherwise specified, the two nucleotides are connected by a phosphodiester group. Unless otherwise specified, "RNAi agent", "nucleotide", "compound", "chemical modification", "oligonucleotide", "double-stranded RNAi inhibitor molecule", "siRNA", "dsRNA", "nucleic acid" and "RNAi" in the present disclosure can independently exist in the form of a salt, a mixed salt or a non-salt (e.g. free acid or free base). When it exists in the form of a salt or a mixed salt, it can be a pharmaceutically acceptable salt.

[0194] The term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts. When it exists in the form of a salt, some groups can be ionized to form anions / cations, for example, phosphodiester groups, thiophosphodiester groups can exist in the form of anions, and unless otherwise specified, the structures of the following salts are also within the scope of the present disclosure. Unless otherwise specified, the 3' position of the first nucleotide at the 3' end of each strand is a hydroxyl group; the 5' position of the first nucleotide at the 5' end of each strand is a hydroxyl group. The above-mentioned modification and linker groups have the structures shown in Table 1 below, respectively, wherein Base represents the base at the corresponding position:

[0195] Table 1

[0196] A "nucleotide analogue" refers to a group that can replace a nucleotide in a nucleic acid, but is structurally different from an adenine ribonucleotide, a guanine ribonucleotide, a cytosine ribonucleotide, a uracil ribonucleotide, or a thymine deoxyribonucleotide. Such as an iso-nucleotide, a bridged nucleic acid (BNA) or an acyclic nucleotide. The methoxy modified nucleotide refers to a nucleotide in which the 2'-hydroxyl group of the ribosyl group is replaced by a methoxy group. The iso-nucleotide refers to a compound in which the base in the nucleotide is changed in position on the ribose ring. In some embodiments, the iso-nucleotide can be a compound in which the base is moved from the 1'-position to the 2'-position or the 3'-position on the ribose ring. The BNA refers to a constrained or inaccessible nucleotide. The BNA can contain a five-membered ring, a six-membered ring, or a seven-membered ring with a "fixed" C3'-endo sugar contraction bridged structure. The bridge is usually incorporated at the 2'-, 4'-position of the ribose to provide a 2', 4'-BNA nucleotide. In some embodiments, the BNA can be LNA, ENA, cET BNA, etc. The acyclic nucleotide is a class of nucleotides in which the sugar ring of the nucleotide is opened. In some embodiments, the acyclic nucleotide can be an unlocked nucleic acid (UNA) or a glycerol nucleic acid (GNA).

[0197] In the context of the present disclosure, one nucleotide sequence has "nucleotide difference" or "differs by no more than n nucleotides" from another nucleotide sequence, means that the base type of the nucleotide at the same position is changed in the former compared to the latter, for example, when the nucleotide base in the latter is A, and the corresponding nucleotide base at the same position in the former is U, C, G or T, it is considered that there is a nucleotide difference between the two nucleotide sequences at that position, or differs by one nucleotide. In some embodiments, when a nucleotide at a position that is not a chemical structure represented by formula (I) or a tautomer thereof is replaced by an abasic nucleotide or its equivalent, it is also considered that a nucleotide difference is generated at that position.

[0198] The term "lipophilic group" or "lipophilic moiety" broadly refers to any chemical compound or chemical moiety that has an affinity for lipids. One way to characterize the lipophilicity of a lipophilic moiety is by the octanol-water partition coefficient logK ow , where K ow is the ratio of the concentration of a chemical in the octanol phase to its concentration in the water phase of a two-phase system at equilibrium. In principle, a chemical has lipophilicity when logK ow exceeds 0. Typically, a lipophilic moiety has a logK ow exceeding 1, exceeding 1.5, exceeding 2, exceeding 3, exceeding 4, exceeding 5, or exceeding 10, such as the logK owlogK of cholesteryl N-(hexan-6-ol)carbamate is about 0.7 ow is 10.7.

[0199] The lipophilicity of a molecule can vary relative to the functional groups it carries. For example, the addition of a hydroxyl or amine group at the end of a lipophilic moiety can increase or decrease the partition coefficient (e.g., logK ow ) value of the lipophilic moiety. For example, the lipophilic moiety can be an aliphatic, cyclic such as alicyclic, or polycyclic such as a polyalicyclic compound, such as a steroid (e.g., a sterol) or a straight chain or branched aliphatic hydrocarbon. The lipophilic moiety can generally comprise a hydrocarbon chain, which can be cyclic or acyclic. The hydrocarbon chain can comprise various substituents and / or one or more heteroatoms, such as oxygen or sulfur atoms. Such lipophilic aliphatic moieties include, but are not limited to, saturated or unsaturated C4-C 30 hydrocarbons (e.g., C 10 -C 30 hydrocarbons), saturated or unsaturated fatty acids, waxes (e.g., monoalcohol esters of fatty acids and fatty diamides), terpenes (e.g., C 10 terpenes, C 15 sesquiterpenes, C 20 diterpenes, C 30 triterpenes, and C 40 tetra terpenes), and other polyalicyclic hydrocarbons; for example, the lipophilic moiety can be an optionally substituted straight chain alkyl of C 10-30 ; for example, the lipophilic moiety can be an optionally substituted straight chain alkyl of C 14-24 .

[0200] As used herein, the terms "complementary" or "reverse complementary" are used interchangeably and have the meaning well known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one strand pair with the bases on the other strand in a complementary manner. In DNA, the purine base adenine always pairs with the pyrimidine base thymine (or, in RNA, uracil); the purine base guanine always pairs with the pyrimidine base cytosine. Each base pair includes one purine and one pyrimidine. When the adenine on one strand always pairs with the thymine (or uracil) on the other strand, and the guanine always pairs with the cytosine, the two strands are considered to be complementary to each other, and the sequence of one strand can be inferred from the sequence of its complementary strand. Correspondingly, "mismatch" means in the art that the bases at the corresponding positions in a double-stranded nucleic acid are not paired in a complementary manner.

[0201] As used herein, the term "inhibit," can be used interchangeably with "reduce," "silence," "down-regulate," "repress," and other similar terms, and includes inhibition at any level. Inhibition can be assessed by a decrease in absolute or relative levels of one or more of these variables compared to a control level. The control level can be any type of control level used in the art, such as a pre-dosing baseline level or a level determined from a subject, cell, or sample that has not been treated or has been treated with a control (e.g., a buffer control or an inert agent control). For example, the degree of inhibition of target gene expression by an RNAi agent can be characterized by the amount of residual mRNA expression, such as no more than 99%, no more than 95%, no more than 90%, no more than 85%, no more than 80%, no more than 75%, no more than 70%, no more than 65%, no more than 60%, no more than 55%, no more than 50%, no more than 45%, no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, or no more than 10%. The rate of inhibition of target gene expression can be characterized by the fold decrease in mRNA expression, such as a decrease of no more than 2-fold, no more than 3-fold, no more than 4-fold, no more than 5-fold, no more than 6-fold, no more than 7-fold, no more than 8-fold, no more than 9-fold, no more than 10-fold, no more than 15-fold, no more than 20-fold, no more than 25-fold, no more than 30-fold, no more than 35-fold, no more than 40-fold, no more than 45-fold, no more than 50-fold, no more than 55-fold, no more than 60-fold, no more than 65-fold, no more than 70-fold, no more than 75-fold, no more than 80-fold, no more than 85-fold, no more than 90-fold, no more than 95-fold, or no more than 100-fold. The rate of inhibition of target gene expression can also be characterized by the fold increase in the level of a reporter gene, such as a luciferase gene, in the presence of an RNAi agent, such as no more than 2-fold, no more than 3-fold, no more than 4-fold, no more than 5-fold, no more than 6-fold, no more than 7-fold, no more than 8-fold, no more than 9-fold, no more than 10-fold, no more than 15-fold, no more than 20-fold, no more than 25-fold, no more than 30-fold, no more than 35-fold, no more than 40-fold, no more than 45-fold, no more than 50-fold, no more than 55-fold, no more than 60-fold, no more than 65-fold, no more than 70-fold, no more than 75-fold, no more than 80-fold, no more than 85-fold, no more than 90-fold, no more than 95-fold, or no more than 100-fold. Luciferase Assay System, reading the firefly chemiluminescence value (Fir) and the Renilla chemiluminescence value (Ren), respectively, and calculating the relative value Ratio = Ren / Fir; in the present disclosure, the residual mRNA expression ratio (or the residual activity %) = Ratio (RNAi agent treatment group) / Ratio (no RNAi agent control group), and the inhibition rate (%) = 100% - residual mRNA expression (%).

[0202] The term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0203] A "pharmaceutically acceptable acid addition salt" means a salt formed by combination of an inorganic or organic acid with the free base which retains the biological effectiveness of the free base and has no other undesirable side effects. Inorganic acid salts include, but are not limited to, hydrochloride, hydrobromide, sulfate, nitrate, phosphate, and the like; and organic acid salts include, but are not limited to, formate, acetate, 2,2-dichloroacetate, trifluoroacetate, propionate, hexanoate, octanoate, decanoate, undecylenate, glycolate, gluconate, lactate, sebacate, adipate, glutarate, malonate, oxalate, maleate, succinate, fumarate, tartrate, citrate, palmitate, stearate, oleate, cinnamate, laurate, malate, glutamate, pyroglutamate, aspartate, benzoate, methanesulfonate, benzenesulfonate, p-toluenesulfonate, alginate, ascorbate, salicylate, 4-aminosalicylate, naphthalene-2-disulfonate, and the like. These salts can be prepared by methods known in the art.

[0204] "Pharmaceutically acceptable base addition salt" refers to those salts which retain the biological effectiveness and non-toxicity of the free acids and are formed with inorganic or organic bases. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, and the like. In some embodiments, the inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines, including naturally occurring substituted amines, cyclic amines, and basic ion-exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethyl ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like. In some embodiments, the organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. These salts can be prepared by methods known in the art.

[0205] "Effective amount" or "effective dose" means the amount of a drug, compound, or pharmaceutical composition necessary to effect any one or more of the beneficial or desired results. For prophylactic use, beneficial or desired results include eliminating or reducing risk, lessening severity, or delaying onset of a disease or disorder, including biochemical, histological and / or behavioral symptoms of the disorder, its complications, and intermediate pathological phenotypes presenting during development of the disorder. For therapeutic use, beneficial or desired results include clinical results such as decreasing the incidence of various target gene, target mRNA, or target protein related disorders of the disclosure, or ameliorating one or more symptoms of such disorders, reducing the dose of other medications required to treat the disorders, enhancing effect of another medication, and / or delaying development of a target gene, target mRNA, or target protein related disorder of the disclosure in a patient.

[0206] As used herein, "patient," "subject," or "individual" are used interchangeably and include a human or non-human animal, such as a mammal, for example, a human or a monkey.

[0207] The RNAi agents provided by the present disclosure can be obtained by methods of preparation conventional in the art, such as methods of solid phase synthesis and liquid phase synthesis. Among them, solid phase synthesis has been commercialized as a subscription service. Methods of preparing nucleoside monomers with corresponding modifications and methods of introducing modified nucleotide groups into the RNAi agents described in the present disclosure by using nucleoside monomers with corresponding modifications are also well known to those skilled in the art.

[0208] The term “chemical modification” or “modification” includes all alterations to nucleotides by chemical means, such as the addition or removal of a chemical moiety, or the substitution of one chemical moiety for another.

[0209] The term "base" includes any known DNA and RNA base, base analogues such as purines or pyrimidines, and also includes natural compounds such as adenine, thymine, guanine, cytosine, uracil, hypoxanthoside, and natural analogues.

[0210] The terms "blunt-ended" or "knuckle-ended" are used interchangeably and refer to the absence of unpaired nucleotides or nucleotide analogues at a given end of an RNAi agent; that is, no nucleotide protrusions. In most cases, RNAi agents with blunt ends will be double-stranded throughout their entire length.

[0211] The terms “about” and “approximately” mean that a numerical value is within an acceptable margin of error for a specific value as determined by a person skilled in the art, the numerical value depending in part on how it is measured or measured (i.e., the limits of the measurement system). For example, “about” may mean within or above 1 standard deviation. Alternatively, “about” or “substantially includes” may mean a range of up to 20%, such as between 1% and 15%, between 1% and 10%, between 1% and 5%, between 0.5% and 5%, or between 0.5% and 1%. In this disclosure, each instance of a number or range of values ​​preceded by the term “about” also includes embodiments of a given number. Unless otherwise stated, when a specific value appears in this application and claims, the meaning of “about” or “substantially includes” should be assumed to be within an acceptable margin of error for that specific value.

[0212] Unless otherwise stated, "optionally," "optionally," "optional," or "optional" means that the event or situation described below may but does not have to occur, and this description includes the possibility that the event or situation may or may not occur. For example, "optionally, R1 and R2 are directly connected to form a loop" means that R1 and R2 being directly connected to form a loop may occur but is not required to exist, and this description includes both the case where R1 and R2 are directly connected to form a loop and the case where R1 and R2 are not connected to form a loop.

[0213] In the chemical structural formula disclosed herein, It can be connected with one or more groups according to the scope of the invention described herein.

[0214] The term "connection" refers to the link between two molecules, either through a covalent bond or through a non-covalent bond (e.g., hydrogen bond or ionic bond), including direct and indirect connections.

[0215] The term "direct link" refers to the connection between a first compound or group and a second compound or group without any intercalating atoms or atomic groups.

[0216] The term "indirectly connected" means that a first compound or group is connected to a second compound or group through an intervening group, compound or molecule (e.g., a linker).

[0217] "Substituted" means that one or more hydrogen atoms, preferably up to 5, more preferably 1 to 3, of a group are independently of each other replaced with a corresponding number of substituents. When the substituent is a ketone or oxo (i.e., =0), then two (2) hydrogens on the atom are replaced.

[0218] "Substituted with one or more" means that a single or multiple substituents can be present. When multiple substituents are present, these can be multiple identical substituents or a combination of one or multiple different substituents.

[0219] The prefix "C u-v " indicates that the following group has from u to v carbon atoms. For example, "C 1-6 alkyl" indicates that the alkyl group has 1 to 6 carbon atoms, in particular it can be an alkyl group having 1, 2, 3, 4, 5 or 6 carbon atoms.

[0220] "Alkyl" refers to saturated aliphatic hydrocarbon groups, both straight-chain and branched-chain, comprising 1 to 20 carbon atoms. Alkyl groups comprising 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, and various branched isomers thereof, and the like. Alkyl groups can be substituted or unsubstituted, and when substituted, the substituents can be substituted at any available point of attachment, preferably with one or more groups independently selected from halogen, hydroxyl, oxo, cyano, amino, C 1-6 alkyl, C 1-6 alkoxy, 3- to 6-membered cycloalkyl, or 3- to 6-membered heterocycloalkyl, said alkyl, alkoxy, cycloalkyl, or heterocycloalkyl being optionally substituted with halogen, hydroxyl, nitro, cyano, or amino.

[0221] The term "alkenyl" refers to unsaturated aliphatic straight-chain or branched hydrocarbon groups and contain one or more carbon-carbon double bonds. Exemplary alkenyl groups include C2-C8, C2-C7, C2-C6, C2-C4, C3-C12, and C3-C6 alkenyl groups. These include, but are not limited to, ethenyl (i.e., vinyl), 1-propenyl, 2-propenyl (i.e., allyl), 2-methyl-1-propenyl, 1-butenyl, 2-butenyl (i.e., crotyl), and the like. Alkenyl groups used in any context herein are optionally substituted in the same manner as alkyl groups.

[0222] The term "alkynyl" refers to unsaturated aliphatic straight chain or branched chain hydrocarbon groups and contains one or more carbon-carbon triple bonds. Exemplary alkynyl groups include C2-C8, C2-C7, C2-C6, C2-C4, C3-C12, and C3-C6 alkynyl groups. Included are, but are not limited to, ethynyl, prop-1-ynyl, but-1-ynyl, pent-1-ynyl, pent-4-ynyl, and pent-1,4-diynyl. An alkynyl group used in any context herein is optionally substituted in the same manner as an alkyl group.

[0223] The term "cycloalkyl" refers to saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituents, the cycloalkyl ring containing 3 to 20 carbon atoms, preferably containing 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, and the like; polycyclic cycloalkyl groups include spiro, fused, and bridged cycloalkyl groups. Cycloalkyl groups can be substituted or unsubstituted, and when substituted, the substituents can be substituted at any available point of attachment, preferably one or more groups independently selected from halogen, hydroxyl, oxo, cyano, amino, C1-C6 alkyl, C1-C6 alkoxy, 3- to 6-membered cycloalkyl, or 3- to 6-membered heterocycloalkyl, said alkyl, alkoxy, cycloalkyl, or heterocycloalkyl optionally substituted with halogen, hydroxyl, nitro, cyano, or amino. 1-6 alkyl, C 1-6 alkoxy, 3- to 6-membered cycloalkyl, or 3- to 6-membered heterocycloalkyl, said alkyl, alkoxy, cycloalkyl, or heterocycloalkyl optionally substituted with halogen, hydroxyl, nitro, cyano, or amino.

[0224] The term "heterocyclyl" or "heterocycloalkyl" refers to saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituents, containing 3 to 20 ring atoms, one or more of which are heteroatoms selected from nitrogen, oxygen, or S(O) m (O)m, wherein m is an integer from 0 to 2, ring members other than -O-O-, -O-S-, or -S-S-, and the remainder of the ring atoms are carbon. Preferably, 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably, 3 to 7 ring atoms. Non-limiting examples of "heterocyclyl" include:

[0225] and the like.

[0226] The heterocyclyl ring can be fused to an aryl or heteroaryl ring, where the ring that is attached to the parent structure is heterocyclyl, non-limiting examples of which include:

[0227] and the like.

[0228] Heterocycloalkyl groups can be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more groups independently selected from halogen, hydroxyl, oxo, cyano, amino, C1-C6 alkyl, C1-C6 alkoxy, 3- to 6-membered cycloalkyl, or 3- to 6-membered heterocycloalkyl, said alkyl, alkoxy, cycloalkyl, or heterocycloalkyl optionally substituted with halogen, hydroxyl, nitro, cyano, or amino. 1-6 alkyl, C 1-6Alkoxy, 3- to 6-membered cycloalkyl, or 3- to 6-membered heterocycloalkyl, wherein the alkyl, alkoxy, cycloalkyl, or heterocycloalkyl may optionally be substituted with halogen, hydroxyl, nitro, cyano, or amino.

[0229] The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., a ring sharing adjacent carbon atom pairs) group having a conjugated π-electron system, preferably 6- to 12-membered, such as phenyl and naphthyl. The aryl ring may be fused to a heteroaryl, heterocyclic, or cycloalkyl ring, wherein the ring attached to the parent structure is an aryl ring, and non-limiting examples include:

[0230] The aryl group can be substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from halogen, hydroxyl, oxo, nitro, cyano, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 alkenyloxy group, C 2-6 Alkyne group, 3- to 6-membered cycloalkoxy group, 3- to 6-membered heterocycloalkoxy group, C 3-8 Cycloalkenyloxy, 5- to 6-membered aryl or heteroaryl, wherein the C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 alkenyloxy group, C 2-6 The alkynyl group, 3 to 6 cycloalkoxy group, 3 to 6 heterocycloalkoxy group, 3 to 8 cycloalkenyl group, 5 to 6 aryl group, or heteroaryl group may be selected from one or more halogens, hydroxyl groups, cyano groups, amino groups, C6 groups, etc. 1-6 Alkyl or C 1-6 Alkyl-substituted.

[0231] The term "heteroaryl" refers to a heteroaryl system comprising 1 to 4 heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur, and nitrogen. Heteroaryl groups are preferably 5 to 12-membered, more preferably 5- or 6-membered. For example, non-limiting examples include: imidazolyl, furanyl, thiophene, thiazolyl, pyrazolyl, oxazolyl, pyrroleyl, tetrazolyl, pyridyl, pyrimidinyl, thiadiazole, and pyrazine. wait.

[0232] The heteroaryl ring may be fused to an aryl, heterocyclic, or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring, and non-limiting examples include:

[0233] The heteroaryl group can be optionally substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from halogens, hydroxyl groups, cyano groups, amino groups, C4 groups, etc. 1-6 Alkyl or C 1-6 Alkyl group.

[0234] The term "alkoxy" refers to an -O-(alkyl) group, wherein alkyl is as defined above. Non-limiting examples of alkoxy groups include methoxy, ethoxy, propyloxy, butyloxy. The alkoxy group can be optionally substituted or unsubstituted, and when substituted, the substituent(s) are preferably one or more groups independently selected from halogen, hydroxy, oxo, cyano, amino, C 1-6 alkyl, C 1-6 alkoxy, 3- to 7-membered cycloalkyl, or 3- to 7-membered heterocycloalkyl, said alkyl, alkoxy, cycloalkyl, or heterocycloalkyl being optionally substituted with halogen, hydroxy, nitro, cyano, or amino.

[0235] The term "hydroxy" refers to an -OH group.

[0236] The term "oxo" refers to an =O substituent.

[0237] The term "cyano" refers to -CN.

[0238] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0239] The term "amino" refers to -NH2.

[0240] The term "protecting group" is used in its conventional chemical sense of a group that reversibly inactivates a functional group under the conditions of a desired reaction. Following the desired reaction, the protecting group can be removed to deprotect the protected functional group. All protecting groups should be removable without degrading a significant proportion of the synthetically produced molecule.

[0241] The present disclosure "hydroxy protecting group" is a group known in the art that can be used to protect a hydroxy group, see the hydroxy protecting groups in the text (Protective Groups in Organic Synthesis, 5 Th Ed. T. W. Greene & P. G. M. Wuts). By way of example, but not limitation, the hydroxy protecting group can be, by way of example, (C 1-10 alkyl or aryl) silyl, for example: triethylsilyl, triisopropylsilyl, t-butyldimethylsilyl, t- butyldiphenylsilyl, and the like; can be (C 1-10 alkyl or substituted alkyl, for example: methyl, t-butyl, allyl, benzyl, methoxymethyl, ethoxyethyl, 2-tetrahydropyranyl (THP), and the like; can be (C 1-10 alkyl or aryl) acyl, for example: formyl, acetyl, benzoyl, and the like; can be (C 1-6 alkyl or C 6-10 aryl) sulfonyl; can also be (C 1-6 alkoxy or C 6-10The aryloxy) carbonyl group can be acetyl (Ac), 2-methoxyethoxymethyl ether (MEM), methoxymethyl ether (MOM), p-methoxybenzyl ether (PMB), or methyl thiomethyl ether (MTM).

[0242] Some abbreviations in this disclosure are defined as follows: THF: Tetrahydrofuran; TFA: Trifluoroacetic acid; DMF: Dimethylformamide; EDCI: 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; DMTrCl: 4,4'-Dimethoxytriphenylmethyl chloride; DMTr: Dimethoxytriphenylmethyl protecting group; MMTr: Methoxyphenyldiphenylmethyl; Piv: Pivaloyl; TBSCl: Tert-butyldimethylchlorosilane; py: Pyridine; DMSO: Dimethyl sulfoxide; NMI: N-methylimidazolium; TBAF: Tetrabutylammonium fluoride; NMM: N-methylmorpholine; DCI: 4,5-Dicyanoimidazolium; DCM: Dichloromethane; CsF: Cesium fluoride; Ag2OTf: Silver trifluoromethanesulfonate. Detailed Implementation

[0243] The present disclosure is further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the disclosure. Experimental methods in the embodiments of this disclosure that do not specify specific conditions are generally performed under conventional conditions or as recommended by the raw material or product manufacturer. Reagents whose specific source is not specified can be obtained from any supplier of molecular biology reagents at the quality / purity required for molecular biology applications.

[0244] Example 1. Synthesis of Compound 1

[0245] Compound 1 was synthesized from starting compound 1-1 using the following route:

[0246] Step 1: Synthesis of Compounds 1-2

[0247] Under ice bath conditions, 1H-imidazole (39 mL, 580.88 mmol) and tert-butyldimethylchlorosilane (83 mL, 484.06 mmol) were added to a solution of compound 1-1 (50.00 g, 193.62 mmol) in dimethylformamide (300 mL), and the reaction mixture was stirred for 18 hours. The reaction mixture was diluted with water (1 L), extracted with ethyl acetate (3 × 500 mL), the organic phases were combined, washed with saturated brine (3 × 200 mL), dried over anhydrous sodium sulfate, and the organic layer was dried and concentrated to give title compound 1-2 (109.0 g).

[0248] LCMS: m / z(ESI) 487.2 [M+H] + .

[0249] Step 2: Synthesis of compound 1-3

[0250] To a solution of compound 1-2 (100.00 g, 205.44 mmol) in tetrahydrofuran (500 mL) was added a mixture of deionized water (250 mL) and trifluoroacetic acid (250 mL) under ice bath. The reaction was stirred at 0 °C for 2 h. The reaction was neutralized with ammonia water, extracted with ethyl acetate (1000 mL) twice, the organic phase was combined, washed with saturated brine (3 x 200 mL), dried over anhydrous sodium sulfate, concentrated, and the residue was purified by column chromatography to give the title compound 1-3 (25.00 g, yield 33%).

[0251] LCMS: m / z (ESI) 373.0 [M+H] + .

[0252] Step 3: Synthesis of compound 1-4

[0253] To a solution of compound 1-3 (18.50 g, 49.67 mmol) in dimethyl sulfoxide (220 mL) was added EDCI (28.50 g, 148.99 mmol), pyridine (4 mL, 49.66 mmol) and trifluoroacetic acid (1.8 mL, 24.83 mmol) under ice bath. The reaction was stirred at room temperature for 1.5 h. The reaction was added to water (500 mL), extracted with ethyl acetate (300 mL) twice, the organic phase was combined, washed with saturated brine (3 x 200 mL), dried over anhydrous sodium sulfate, concentrated to give the title compound 1-4 (22.00 g).

[0254] LCMS: m / z (ESI) 371.2 [M+H] + .

[0255] Step 4: Synthesis of compound 1-5

[0256] To a solution of compound 1-4 (14.30 g, 38.60 mmol) in dimethyl sulfoxide (130 mL) and tetrahydrofuran (70 mL) was added sodium hydride (3.09 g, 60%, 77.20 mmol) under ice bath. The reaction was stirred at room temperature for 1 h, then a solution of trimethylsulfoxonium iodide (25.4 g, 115.60 mmol) in tetrahydrofuran (70 mL) was added dropwise at 0 °C. The reaction was stirred at ice bath for 1.5 h. The reaction was added to ammonium chloride (20 mL), extracted with ethyl acetate (300 mL) twice, the organic phase was combined, washed with saturated brine (3 x 50 mL), dried over anhydrous sodium sulfate, concentrated, and the residue was purified by column chromatography to give the title compound 1-5 (9.00 g, yield 61%).

[0257] LCMS: m / z (ESI) 385.2 [M+H] + .

[0258] 1 H NMR (400 MHz, CDC13) δ 8.70 (s, 1H), 7.61 (d, J = 8.2 Hz, 1H), 7.43 (d, J = 8.2 Hz, 1H), 5.82 (d, J = 3.2 Hz, 1H), 5.76 (d, J = 2.8 Hz, 1H), 5.63 (dd, J = 8.2, 2.1 Hz, 1H), 4.13 - 4.05 (m, 2H), 3.51 (t, J = 3.5 Hz, 1H), 3.38 (d, J = 2.8 Hz, 3H), 3.09 (t, J = 3.1 Hz, 1H), 2.84 - 2.73 (m, 2H), 0.83 - 0.75 (m, 9H), -0.01 (dd, J = 9.9, 7.0 Hz, 6H).

[0259] Fifth Step: Synthesis of compound 1-6

[0260] To a solution of compound 1-5 (1.10 g, 2.86 mmol) and lithium bromide (0.45 g, 5.15 mmol) in tetrahydrofuran (10.0 mL) was added dropwise boron trifluoride etherate (0.92 g, 3.15 mmol, 48% content) at -70 °C. The reaction was stirred at room temperature for 1 h. The reaction was added to saturated aqueous sodium bicarbonate solution (20 mL), extracted with dichloromethane (20 mL) three times, the organic phase was combined, washed with saturated brine (3 x 50 mL), dried over anhydrous sodium sulfate, concentrated and the residue was purified by column chromatography to give the title compound 1-6 (1.50 g).

[0261] LCMS: m / z (ESI) 465.07 / 467.02 [M+H] + .

[0262] 1 H NMR (400 MHz, CDC13) δ 8.70 (s, 1H), 7.61 (d, J = 8.2 Hz, 1H), 7.43 (d, J = 8.2 Hz, 1H), 5.82 (d, J = 3.2 Hz, 1H), 5.76 (d, J = 2.8 Hz, 1H), 5.63 (dd, J = 8.2, 2.1 Hz, 1H), 4.13 - 4.05 (m, 2H), 3.51 (t, J = 3.5 Hz, 1H), 3.38 (d, J = 2.8 Hz, 3H), 3.09 (t, J = 3.1 Hz, 1H), 2.84 - 2.73 (m, 2H), 0.83 - 0.75 (m, 9H), -0.01 (dd, J = 9.9, 7.0 Hz, 6H).

[0263] Sixth Step: Synthesis of compound 1-7

[0264] To a solution of compound 1-6 (1.10 g, 2.36 mmol) and triazole (0.24 g, 3.55 mmol) in anisole (10.0 mL) was added cesium carbonate (1.54 g, 4.73 mmol) at room temperature. The reaction was stirred at 80 °C for 16 h. The reaction was added to water solution (20 mL), extracted with ethyl acetate (20 mL) for three times, combined organic phase, washed with saturated brine (3 x 50 mL), dried over anhydrous sodium sulfate, concentrated and the residue was purified by column chromatography to give the title compound 1-7 (0.86 g, yield 80%).

[0265] LCMS: m / z (ESI) 454.22 [M+H] + .

[0266] 1 H NMR (400 MHz, CDC13) δ 9.72 (s, 1H), 8.17 (s, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.80 (s, 1H), 5.79 (d, J = 3.2 Hz, 1H), 5.77 (s, 1H), 4.39 (t, J = 5.6 Hz, 1H), 4.35-4.29 (m, 2H), 4.20-4.17 (m, 1H), 4.03 (d, J = 5.6 Hz, 1H), 3.87 (t, J = 4.4 Hz, 1H), 3.51 (s, 3H), 2.07 (br, 1H), 0.89 (s, 9H), 0.08 (d, J = 10.8 Hz, 6H).

[0267] Seventh step: synthesis of compound 1-8

[0268] To a solution of compound 1-7 (0.40 g, 0.88 mmol) in pyridine (4.0 mL) was added 4,4'-dimethoxytrityl chloride (1.50 g, 4.41 mmol) at room temperature. The reaction was stirred at 55 °C for 18 h. The reaction was added to water solution (20 mL), extracted with dichloromethane (50 mL) for three times. The combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated and the residue was purified by column chromatography to give the title compound 1-8 (0.21 g, yield 32%).

[0269] LCMS: m / z (ESI) 778.49 [M+Na] + .

[0270] Eighth step: synthesis of compound 1-9

[0271] To a solution of compound 1-8 (0.48 g, 0.64 mmol) in tetrahydrofuran (5.0 mL) was added TBAF (0.34 g, 1.28 mmol) at room temperature. The reaction was stirred at room temperature for 2 hours. The reaction was added to water solution (20 mL) and extracted with dichloromethane (50 mL) for three times. The organic phase was combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated and the residue was purified by column chromatography to give the title compound 1-9 (0.40 g, yield 98%).

[0272] LCMS: m / z (ESI) 664.29 [M+Na] + .

[0273] Ninth step: synthesis of compound 1

[0274] To a solution of compound 1-9 (0.26 g, 0.41 mmol) in dry dichloromethane (4 mL) was added 3A molecular sieves under nitrogen atmosphere at room temperature. After stirring for 5 minutes at room temperature, bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.36 g, 1.20 mmol) was added, followed by the pre-prepared solution of 1H-tetrazole (0.45 M) and N-methylimidazole (0.18 M) in dry acetonitrile (2.7 mL). The reaction was stirred at room temperature for 2 hours. After filtration, 5% sodium bicarbonate aqueous solution (30 mL) was added to the reaction. Dichloromethane (30 mL) was used to extract, dried over sodium sulfate, filtered, concentrated and purified by C18 reverse phase column to give compound 1 (0.23 g, yield 67%).

[0275] LCMS: MS (ESI) m / z = 840.52 [M-H] - .

[0276] 1H NMR (400 MHz, DMSO-d6) δ 11.51 (d, J = 10.4 Hz, 1H), 8.12 (d, J = 18.8 Hz, 1H), 7.94 (d, J = 8.0 Hz, 1H), 7.84 (d, J = 14.2 Hz, 1H), 7.48 - 7.24 (m, 9H), 6.94 (dd, J = 8.4, 4.0 Hz, 4H), 5.79 (dd, J = 22.4, 12.0 Hz, 1H), 5.71 (dd, J = 27.2, 8.0 Hz, 1H), 4.41 - 4.30 (m, 1H), 4.20 - 4.07 (m, 2H), 4.03 - 3.91 (m, 1H), 3.88 - 3.79 (m, 1H), 3.76 (s, 6H), 3.71 - 3.65 (m, 1H), 3.62 - 3.60 (m, 1H), 3.45 - 3.37 (m, 3H), 3.29 (s, 1H), 2.74 (t, J = 6.0 Hz, 1H), 2.50 - 2.48 (m, 2H), 2.45 - 2.39 (m, 1H), 1.08 - 1.00 (m, 9H), 0.74 (d, J = 6.8 Hz, 3H).

[0277] 31 P NMR (162 MHz, DMSO-d6) δ 150.41, 149.84.

[0278] Example 2. Synthesis of compound 2

[0279] First step: synthesis of compound 2-1

[0280] To a solution of compound 1-5 (2.0 g, 5.20 mmol) in dimethylformamide (10 mL) and methanol (20 mL) was added morpholine (0.90 mL, 10.40 mmol) at room temperature. The reaction was stirred at 40 °C for 18 h. The reaction was concentrated and diluted with water (30 mL), extracted with ethyl acetate (3 x 50 mL), the organic phase was combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate and concentrated to give the title compound 2-1 (2.20 g, yield 89%).

[0281] LCMS: m / z (ESI) 472.2 [M+H] + .

[0282] Second step: synthesis of compound 2-2

[0283] To a solution of compound 2-1 (1.00 g, 2.12 mmol) in dichloromethane (30 mL) was added NMM (2.14 g, 21.20 mmol) and DMTrCl (7.18 g, 21.20 mmol) sequentially at room temperature. The reaction was stirred at room temperature for 18 h. The reaction was added to water (20 mL) and extracted with dichloromethane (3 x 20 mL), the organic phase was combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, concentrated and the residue was purified by column chromatography to give the title compound 2-2 (1.00 g, yield 63%).

[0284] LCMS: m / z (ESI) 796.2 [M+Na] + .

[0285] Third Step: Synthesis of compound 2-3

[0286] To a solution of compound 2-2 (2.30 g, 2.97 mmol) in dimethylformamide (40 mL) was added cesium fluoride (1.35 g, 8.92 mmol) at room temperature. The reaction was stirred at room temperature for 4 h. The reaction was diluted with ethyl acetate (60 mL), washed with saturated brine (3 x 50 mL), dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography to give the title compound 2-3 (0.70 g, yield 36%).

[0287] LCMS: m / z (ESI) 682.2 [M+Na] + .

[0288] 1 H NMR (400 MHz, CDC13) δ 8.14 (s, 1H), 7.42 (d, J = 4.0 Hz, 2H), 7.33-7.31 (m, 5H), 7.26-7.28 (m, 2H), 6.80-6.84 (m, 4H), 5.87 (s, 1H), 5.55 (s, 1H), 4.32 (s, 1H), 3.79-3.83 (m, 10H), 3.62 (s, 3H), 3.55 (s, 3H), 2.61-2.55 (m, 1H), 2.0-2.16 (m, 5H).

[0289] Fourth Step: Synthesis of compound 2

[0290] To a solution of compound 2-3 (0.60 g, 0.91 mmol) in dichloromethane (6 mL) was added bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.68 g, 2.27 mmol) followed by DCI (0.16 g, 1.36 mmol) under nitrogen atmosphere at room temperature. The reaction was stirred at room temperature for 1 h. The reaction was quenched with saturated sodium bicarbonate solution (10 mL) and extracted with dichloromethane (2 x 10 mL). The organic layers were combined and concentrated. The crude was purified by reverse phase column chromatography to afford the title compound 2 (0.72 g, 93% yield).

[0291] LCMS: m / z (ESI) 861.1 [M+H] + .

[0292] 1 H NMR (400 MHz, CD3CN) δ 8.90 (br, 1H), 8.11 (dd, J = 12.8, 8.0 Hz, 1H), 7.47 - 7.24 (m, 9H), 6.88 (ddd, J = 9.2, 5.2, 2.0 Hz, 4H), 5.90 (dd, J = 20.0, 4.0 Hz, 1H), 5.59 (dd, J = 32.0, 8.0 Hz, 1H), 4.44 - 4.31 (m, 2H), 4.10 - 4.04 (m, 1H), 3.77 (d, J = 3.6 Hz, 6H), 3.74 - 3.69 (m, 1H), 3.64 - 3.49 (m, 4H), 3.43 - 3.34 (m, 6H), 2.66 - 2.57 (m, 2H), 2.32 (td, J = 6.0, 2.4 Hz, 1H), 2.10 - 2.07 (m, 2H), 2.02 - 1.94 (m, 2H), 1.92 - 1.84 (m, 2H), 1.16 - 1.13 (m, 6H), 1.09 (d, J = 6.8 Hz, 3H), 1.03 (d, J = 6.8 Hz, 3H).

[0293] 31 P NMR (162 MHz, CD3CN) δ 150.51, 149.48.

[0294] Example 3. Synthesis of compound 3

[0295] Compound 3 was synthesized from starting material compound 3-1 following the route below:

[0296] First Step: Synthesis of compound 3-2

[0297] Compound 3-1 (25 g, 37.328 mmol) was dissolved in dichloromethane (200 mL), imidazole (5.08 g, 74.656 mmol) was added at 0 °C, then tert-butyl dimethylsilyl chloride (11.25 g, 74.656 mmol) was added, slowly returned to room temperature and stirred overnight. LC-MS monitoring of the conversion of the raw material was complete. Water was added, dichloromethane was extracted, the organic phase was combined, anhydrous sodium sulfate was dried, filtered, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain the target compound 3-2 (25 g, yield 85.4%).

[0298] LCMS: MS (ESI) m / z = 800.3 [M-H] - .

[0299] Second step: synthesis of compound 3-3

[0300] Compound 3-2 (25 g, 31.888 mmol) was dissolved in dichloromethane (100 mL), dichloroacetic acid (395.3 mL, 95.663 mmol) was added, and stirred at room temperature for 3 hours. LC-MS monitoring of the conversion of the raw material was complete, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain the target compound 3-3 (15 g, 31.144 mmol, 97.67%).

[0301] LCMS: MS (ESI) m / z = 498.2 [M-H] - .

[0302] Third step: synthesis of compound 3-4

[0303] To the solution of compound 3-3 (8.7 g, 17.41 mmol) in dimethyl sulfoxide (80 mL), EDCI (16.69 g, 87.06 mmol) was added, under nitrogen atmosphere, pyridine (4.82 g, 60.94 mmol) and trifluoroacetic acid (0.99 g, 8.706 mmol) were added at about 10 °C, and the reaction was stirred at room temperature for 1 hour. The reaction liquid was added to ice water (300 mL), extracted twice with ethyl acetate (150 mL), the organic phase was combined, washed with water (150 ml), saturated brine (150 mL), dried with anhydrous sodium sulfate, concentrated to obtain the title compound 3-4 (8.66 g, yield 100.4%).

[0304] LCMS: MS (ESI) m / z = 496.2 [M-H] - .

[0305] Fourth step: synthesis of compound 3-5

[0306] To a solution of compound 3-5 (4 g, 7.82 mmol) and triazole (0.81 g, 11.73 mmol) in DMF (40.0 mL) was added cesium carbonate (4.58 g, 14.07 mmol) at room temperature. The reaction was stirred at 80 °C for 2 h. The reaction was added to aqueous solution (200 mL) and extracted with dichloromethane (100 mL) twice, combined organic phase, concentrated and the residue was purified by column chromatography to give the title compound 3-6 (2 g, yield 44.05%).

[0307] LCMS: MS (ESI) m / z = 510.2 [M-H] - .

[0308] Fifth step: Synthesis of compound 3-6

[0309] To a solution of compound 3-5 (4 g, 7.82 mmol) and triazole (0.81 g, 11.73 mmol) in DMF (40.0 mL) was added cesium carbonate (4.58 g, 14.07 mmol) at room temperature. The reaction was stirred at 80 °C for 2 h. The reaction was added to aqueous solution (200 mL) and extracted with dichloromethane (100 mL) twice, combined organic phase, concentrated and the residue was purified by column chromatography to give the title compound 3-6 (2 g, yield 44.05%).

[0310] LCMS: MS (ESI) m / z = 579.2 [M-H] - .

[0311] Sixth step: Synthesis of compound 3-7

[0312] To a solution of compound 3-6 (2.5 g, 4.29 mmol) in pyridine (20.0 mL) and 2,6- dimethylpyridine (20.0 mL) was added silver trifluoromethanesulfonate (11.02 g, 42.90 mmol) and 4,4'-dimethoxybenzhydryl chloride (14.54 g, 42.90 mmol) at room temperature. The reaction was stirred at 60 °C for 3 h. Cooled, the reaction was added to dichloromethane (200 mL), concentrated and the residue was purified by column chromatography to give the title compound 3-7 (1.7 g, yield 44.77%).

[0313] LCMS: MS (ESI) m / z = 881.3 [M-H] - .

[0314] Seventh step: Synthesis of compound 3-8

[0315] To a solution of compound 3-7 (810 mg, 0.92 mmol) in tetrahydrofuran (8 mL) was added TBAF (1 M in THF) (2.1 ml, 2.1 mmol) at room temperature. The reaction was stirred at room temperature for 2 h. The reaction was concentrated, taken in water (20 mL) and extracted with ethyl acetate (20 mL) once. The residue after concentration was purified by column chromatography to get the title compound 3-8 (670 mg, yield 94.37%).

[0316] LCMS: MS (ESI) m / z = 767.4 [M-H] - .

[0317] Eighth step: synthesis of compound 3

[0318] Compound 3-8 (1200 mg, 1.598 mmol) was dissolved in dichloromethane, molecular sieves were added, nitrogen was replaced, 3-({bis[bis(prop-2-yl)amino]phosphoryl}oxy)propanenitrile (2.538 mL, 7.991 mmol) was added under nitrogen atmosphere, followed by the addition of DCI (0.835 mL, 5.594 mmol) and stirred at room temperature for half an hour. LC-MS monitoring was done for the completion of the conversion of starting material, aqueous sodium bicarbonate solution was added, dichloromethane was extracted, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure and the crude was purified by reverse phase column chromatography to get the target compound 3 (1.26 g, 1.325 mmol, 82.89%).

[0319] LCMS: MS (ESI) m / z = 969.4 / 970.4 [M+H] +

[0320] 31 P NMR (400 MHz, CD3CN) δ 150.69 (d, J = 47.6 Hz).

[0321] 1H NMR (400 MHz, CD3CN) δ 9.33 (s, 1H), 8.72 (d, J = 3.8 Hz, 1H), 8.47 (d, J = 11.0 Hz, 1H), 8.04 (d, J = 5.6 Hz, 2H), 7.79 - 7.63 (m, 3H), 7.60 - 7.51 (m, 4H), 7.43 (dd, J = 18.8, 8.3 Hz, 4H), 7.25 (dt, J = 14.4, 7.7 Hz, 3H), 6.89 - 6.81 (m, 4H), 6.04 (dd, J = 6.2, 4.1 Hz, 1H), 5.15 (dt, J = 36.1, 5.7 Hz, 1H), 4.56 - 4.45 (m, 1H), 4.31 (ddd, J = 21.0, 13.7, 9.2 Hz, 1H), 4.03 (ddd, J = 23.9, 13.9, 4.1 Hz, 1H), 3.86 - 3.74 (m, 9H), 3.59 - 3.45 (m, 3H), 3.35 (d, J = 8.8 Hz, 3H), 2.62 (td, J = 6.2, 2.5 Hz, 1H), 2.38 - 2.21 (m, 1H), 1.15 - 1.07 (m, 9H), 0.86 (d, J = 6.8 Hz, 3H).

[0322] Example 4. Synthesis of compound 4

[0323] Compound 4 was synthesized from starting material compound 4-1 according to the following route:

[0324] First step: synthesis of compound 4-2

[0325] Compound 4-1 (50 g, 194.363 mmol) was dissolved in pyridine (500 mL), replaced with nitrogen, and DMTrCl (131.70 g, 388.727 mmol) was added under a nitrogen atmosphere, and the temperature was maintained for 16 hours. LC-MS was used to monitor the complete conversion of the starting material. Methanol was added, and the mixture was concentrated under reduced pressure. The crude product was dissolved in dichloromethane, petroleum ether was added, and a solid precipitated. The supernatant was decanted to obtain the target compound 4-2 (100 g, yield: 91.9%).

[0326] LCMS: m / z (ESI) 560.2 [M+H] + .

[0327] Second step: synthesis of compound 4-3

[0328] Compound 4-2 (100 g, 178.693 mmol) was dissolved in dichloromethane, imidazole (24.3 g, 357.38 mmol) and tert-butyldimethylsilyl chloride (53.97 g, 357.38 mmol) were added at 0 °C and the stirring was resumed slowly to room temperature overnight. LC-MS monitoring of the conversion of the starting material was complete. Water was added and the organic phase was collected, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was dissolved in dichloromethane and petroleum ether was added slowly until the solid precipitated. The supernatant was decanted and the target compound 4-3 (67.2 g, yield: 51.7%) was obtained.

[0329] LCMS: m / z (ESI-NEG) 672.2 [M-H] - .

[0330] Third step: synthesis of compound 4-4

[0331] Compound 4-3 (50 g, 74.197 mmol) was dissolved in dichloromethane and dichloroacetic acid (95.67 g, 741.972 mmol) was added at room temperature and stirred for 3 hours at room temperature. LC-MS monitoring of the conversion of the starting material was complete, water was added and the organic phase was extracted with dichloromethane, the combined organic phases were washed with saturated aqueous sodium bicarbonate solution, the combined organic phases were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel to obtain the target compound 4-4 (16 g, 43.067 mmol, 58.04%).

[0332] LCMS: m / z (ESI-NEG) 370.1 [M-H] - .

[0333] Fourth step: synthesis of compound 4-5

[0334] Compound 4-4 (20 g, 53.834 mmol) was dissolved in dichloromethane and triethylamine (22.448 mL, 161.503 mmol) was added at 0 °C, followed by the addition of isobutyric anhydride (9.374 mL, 56.526 mmol) and the stirring was resumed slowly to room temperature for 3 hours. LC-MS monitoring of the conversion of the starting material was complete. The reaction was quenched with water and extracted with dichloromethane, the combined organic phases were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel to obtain the target compound 4-5 (16 g, 36.232 mmol, 67.30%).

[0335] LCMS: m / z (ESI-NEG) 440.6 [M-H] + .

[0336] Fifth step: synthesis of compound 4-6

[0337] Compound 4-5 (10 g, 22.645 mmol) was dissolved in dimethyl sulfoxide, EDCI (13.02 g, 67.935 mmol), pyridine (1.832 mL, 22.645 mmol), trifluoroacetic acid (0.841 mL, 11.322 mmol) were added at 0 °C, the reaction was allowed to warm up slowly to room temperature and stirred for 2 h. LC-MS monitoring showed the starting material was consumed completely. The reaction was poured into water, extracted with ethyl acetate, the organic layers were combined, washed with saturated brine, the organic layer was collected, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, the crude was dissolved in 100 mL of toluene, concentrated under reduced pressure and used directly in the next step (9.9 g, 99.5% yield).

[0338] LCMS: m / z (ESI) 440.1 [M+H]+.

[0339] Sixth step: synthesis of compound 4-7

[0340] Trimethylsulfoxonium iodide (8.729 mL, 67.706 mmol) was dissolved in DMSO, replaced with nitrogen, sodium hydride (2.7 g, 67.500 mmol) was added at room temperature, the reaction was stirred at room temperature for 1 h until the reaction was clear. Compound 4-6 (9.9 g, 22.521 mmol) was dissolved in dimethyl sulfoxide and added to the reaction. The reaction was stirred at room temperature for 2 min. LC-MS monitoring showed the starting material was consumed completely. The reaction was poured into water, extracted with ethyl acetate, the organic layers were combined, washed with saturated brine, the organic layer was collected, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, the crude was purified by silica gel column chromatography (0-40% ethyl acetate / petroleum ether) to give the target compound 4-7 (5 g, 11.023 mmol, 48.92%).

[0341] LCMS: m / z (ESI-NEG) 452.2 [M-H] + .

[0342] Seventh step: synthesis of compound 4-8

[0343] Compound 4-7 (5 g, 11.023 mmol) was dissolved in DMF, 1H-1,2,4-triazole (1.324 mL, 22.045 mmol) K2CO3 (3.05 g, 22.045 mmol) was added, stirred at 60 °C for 1 hour (need to control the reaction time, the longer the reaction time, the more impurities). LC-MS monitoring of the complete conversion of raw materials. The reaction was poured into water, extracted with ethyl acetate, the organic phase was combined, washed with saturated brine, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain the target compound 4-8 (2.5 g, 4.783 mmol, 43.39%).

[0344] LCMS: m / z (ESI-NEG) 521.2 [M-H] + .

[0345] Eighth step: synthesis of compound 4-9

[0346] Compound 4-8 (2.4 g, 4.592 mmol), DMTrCl (15.56 g, 45.917 mmol), silver nitrate (7.76 g, 45.917 mmol), 4A molecular sieve were placed in a microwave tube, THF (48 mL), pyridine (12 mL), 2,4,6-trimethylpyridine (12 mL) were added, nitrogen was replaced, stirred at 80 °C overnight. LC-MS monitoring of the remaining ~ 10% of raw materials, methanol was added to quench the reaction, filtered, the filter cake was washed with methanol, the filtrate was collected, concentrated under reduced pressure, the residue was dissolved in a small amount of tetrahydrofuran, the solid was not dissolved, filtered, the filtrate was collected, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (dichloromethane / 5% methanol in dichloromethane: 0-40%) to obtain the target compound 4-9 (3.0 g, 3.636 mmol, yield: 79.19%).

[0347] LCMS: m / z (ESI) 752.0 [M+H] + .

[0348] Ninth step: synthesis of compound 4-10

[0349] Compound 4-9 (3.0 g, 3.636 mmol) was dissolved in THF (30 mL), TBAF solution 1M (7.2 mL, 7.272 mmol) was added at room temperature, and stirred at room temperature for 0.5 hours. LC-MS monitoring of the complete conversion of raw materials. Concentrated under reduced pressure, the crude product was purified by silica gel column chromatography (0-60%:5% MeOH in DCM / DCM) to obtain the target compound 4-10 (2.8 g, yield: 72%).

[0350] LCMS: MS (ESI) m / z = 711.3 [M+H] - .

[0351] Tenth step: synthesis of compound 4

[0352] Compound 4-10 (2.5 g, 3.517 mmol) was dissolved in dichloromethane, molecular sieves were added, nitrogen was replaced, 3-({bis[bis(prop-2-yl)amino]phosphoryl}oxy)propanenitrile (5.30 g, 17.586 mmol) was added at room temperature, then DCI (1.45 g, 12.310 mmol) was added, stirred at room temperature for 1 hour. LC-MS monitoring of the complete conversion of the starting material. Saturated aqueous sodium bicarbonate solution was added, dichloromethane was added to extract, the organic phase was combined, anhydrous sodium sulfate was added to dry, filtered, concentrated under reduced pressure, and the crude product was purified by reverse phase column chromatography (0-80%: water / acetonitrile) to obtain the target compound 4 (2.6 g, yield: 81.14%).

[0353] LCMS: MS (ESI) m / z = 911.4 [M+H] +

[0354] 31P NMR (400 MHz, DMSO-d6) δ 150.11 (s), 148.92 (s).

[0355] 1H NMR (400 MHz, DMSO-d6) δ 10.97 (d, J = 2.0 Hz, 1H), 8.51 (dd, J1 = 7.6 Hz, J2 = 38.8 Hz, 1H), 8.06 (d, J = 17.2 Hz, 1H), 7.85 (d, J = 11.2 Hz, 1H), 7.45-7.41 (m, 2H), 7.38-7.26 (m, 7H), 7.24-7.17 (m, 1H), 6.95-6.90 (m, 4H), 5.92-5.88 (m, 1H), 4.41-4.29 (m, 2H), 4.08-4.03 (m, 1H), 3.94-3.81 (m, 2H), 3.76 (s, 3H), 3.75 (s, 3H), 3.71-3.61 (m, 2H), 3.46-3.37 (m, 6H), 2.76-2.69 (m, 2H), 2.41-2.37 (m, 1H), 1.08-1.03 (m, 12H), 0.95 (d, J = 6.4 Hz, 3H), 0.80 (d, J = 6.4 Hz, 3H).

[0356] Example 5. Synthesis of compound 5

[0357] Compound 5 was synthesized from the starting material compound 5-1 according to the following route:

[0358] First Step: Synthesis of compound 5-2

[0359] Compound 5-1 (25 g, 37.328 mmol) was dissolved in dichloromethane (200 mL), imidazole (5.08 g, 74.656 mmol) was added at 0 °C, then tert-butyl dimethylchlorosilane (11.25 g, 74.656 mmol) was added, slowly returned to room temperature and stirred overnight. LC-MS monitoring of the conversion of the raw material was complete. Water was added, dichloromethane was extracted, the organic phase was combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was directly used in the next step reaction.

[0360] LCMS: m / z (ESI) 782.4 [M-H] -

[0361] Second Step: Synthesis of compound 5-3

[0362] Compound 5-2 (25 g, 31.888 mmol) was dissolved in DCM (100 mL), dichloroacetic acid (395.347 mL, 95.663 mmol) in dichloromethane was added, and stirred at room temperature overnight. LC-MS monitoring of the conversion of the raw material was complete, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain the title compound 5-3 (15 g, yield: 97.7%).

[0363] LCMS: m / z (ESI) 480.2 [M-H] - .

[0364] Third Step: Synthesis of compound 5-4

[0365] EDCI (17.08 g, 89.07 mmol) was added to a solution of compound 5-3 (14.3 g, 29.69 mmol) in dimethyl sulfoxide (140 mL), under a nitrogen atmosphere, pyridine (8.22 g, 103.92 mmol) and trifluoroacetic acid (1.69 g, 14.85 mmol) were added at about 10 °C, and the reaction was stirred at room temperature for 1 hour. The reaction was added to ice water (500 mL), extracted twice with ethyl acetate (300 mL), the organic phase was combined, washed with water (200 ml), saturated brine (200 mL), dried over anhydrous sodium sulfate, concentrated to obtain the title compound 5-4 (14.3 g, yield 100.4%).

[0366] LCMS: m / z (ESI) 480.7 [M+H] + .

[0367] Step 4: Synthesis of compound 5-5

[0368] To a solution of trimethylsulfoxonium iodide (13.12 g, 59.63 mmol) in dimethyl sulfoxide (130 mL) was added sodium hydride (2.39 g, 60%, 59.63 mmol) at room temperature. After the reaction was stirred at room temperature for 1 h, a solution of compound 5-4 (14.30 g, 29.82 mmol) in tetrahydrofuran (100 mL) was added dropwise at 10 °C. The reaction was stirred at room temperature for 0.5 h. The reaction was added to ammonium chloride (600 mL), extracted with ethyl acetate (300 mL) twice, the organic phases were combined, concentrated and the residue was purified by column chromatography to give the title compound 5-5 (9.20 g, yield 62.5%).

[0369] LCMS: m / z (ESI) 494.8 [M+H] + .

[0370] Step 5: Synthesis of compound 5-6

[0371] To a solution of compound 5-5 (9.20 g, 18.64 mmol) and lithium bromide (2.91 g, 33.55 mmol) in tetrahydrofuran (90.0 mL) was added boron trifluoride etherate (2.91 g, 20.5 mmol) dropwise at -70 °C. The reaction was stirred at room temperature for 1 h. The reaction was added to saturated aqueous sodium bicarbonate solution (100 mL), extracted with dichloromethane (50 mL) twice, the organic phases were combined, concentrated and the residue was purified by column chromatography to give the title compound 5-6 (7.5 g, yield 70.04%).

[0372] LCMS: m / z (ESI) 574.7 / 576.7 [M+H] + .

[0373] Step 6: Synthesis of compound 5-7

[0374] To a solution of compound 5-6 (7.5 g, 13.05 mmol) and triazole (1.35 g, 19.58 mmol) in anisole (50.0 mL) was added cesium carbonate (8.51 g, 26.11 mmol) at room temperature. The reaction was stirred at 80 °C for 2 h. The reaction was added to aqueous solution (100 mL), extracted with dichloromethane (50 mL) twice, the organic phases were combined, concentrated and the residue was purified by column chromatography to give the title compound 5-7 (5.2 g, yield 70.75%).

[0375] LCMS: m / z (ESI) 563.8 [M+H] + .

[0376] Seventh step: Synthesis of compound 5-8

[0377] To a solution of compound 5-7 (5.2 g, 9.24 mmol) in pyridine (50.0 mL) and 2,6-dimethylpyridine (50.0 ml) was added silver triflate (23.74 g, 92.41 mmol) and 4,4'-dimethoxytrityl chloride (31.31 g, 92.41 mmol) at room temperature. The reaction was stirred at 60 °C for 3 h. After cooling, the reaction was added to dichloromethane (200 mL) and the residue was purified by column chromatography to give the title compound 5-8 (5 g, yield 62.58 %).

[0378] LCMS: m / z (ESI) 866.2 [M+H] + .

[0379] Eighth step: Synthesis of compound 5-9

[0380] To a solution of compound 5-8 (2.90 g, 3.35 mmol) in tetrahydrofuran (29 mL) was added TBAF (6.7 ml, 6.70 mmol) at room temperature. The reaction was stirred at room temperature for 2 h. The reaction was concentrated and added to water solution (70 mL) and extracted with ethyl acetate (50 mL) once. The residue was purified by column chromatography to give the title compound 5-9 (2.20 g, yield 87.41 %).

[0381] LCMS: m / z (ESI) 752.0 [M+H] + .

[0382] 1H NMR (400 MHz, DMSO-d6) δ 12.09 (s, 1H), 11.55 (s, 1H), 8.29 (s, 1H), 8.18 (s, 1H), 7.82 (s, 1H), 7.37 - 7.32 (m, 2H), 7.28 - 7.18 (m, 7H), 6.82 (dd, J = 9.0, 2.6 Hz, 4H), 5.78 (d, J = 6.5 Hz, 1H), 5.23 (d, J = 5.9 Hz, 1H), 4.40 (td, J = 5.6, 3.6 Hz, 1H), 4.36 - 4.30 (m, 1H), 4.19 (d, J = 6.0 Hz, 2H), 3.81 (td, J = 6.0, 3.9 Hz, 1H), 3.73 (d, J = 1.2 Hz, 6H), 3.64 (t, J = 3.8 Hz, 1H), 3.35 (s, 3H), 2.73 (p, J = 6.8 Hz, 1H), 1.11 (dd, J = 6.8, 5.7 Hz, 6H).

[0383] Ninth step: synthesis of compound 5

[0384] To a solution of compound 5-9 (1.57 g, 2.09 mmol) in dry dichloromethane (15 mL) was added 4A molecular sieves at room temperature under nitrogen protection. After stirring for 5 minutes at room temperature, bis(diisopropylamino)(2-cyanoethoxy) phosphine (3.15 g, 10.46 mmol) and 4,5-dicyanoimidazole (0.86 g, 7.32 mmol) were added. The reaction was allowed to proceed for 1 hour at room temperature. Saturated aqueous sodium bicarbonate solution (20 mL) was added to the reaction mixture. Dichloromethane (30 mL) was added and the mixture was concentrated. The residue was purified by C18 reverse phase column and lyophilized to give compound 5 (1.5 g, yield 75.38%).

[0385] LCMS: MS (ESI) m / z = 952.3 [M+H] +

[0386] 31 P NMR (162 MHz, CD3CN) δ 150.69 (d, J = 68.6 Hz).

[0387] 1H NMR (400 MHz, CD3CN) δ 8.04 (s, 1H), 8.02 (s, 1H), 7.86 (s, 1H), 7.78 (s, 1H), 7.76 (s, 1H), 7.71 (s, 1H), 7.57 - 7.49 (m, 4H), 7.39 (ddt, J = 12.2, 10.0, 3.0 Hz, 8H), 7.31 (td, J = 7.4, 5.7 Hz, 4H), 7.26 - 7.22 (m, 2H), 6.85 (ddd, J = 9.0, 6.6, 2.0 Hz, 8H), 5.83 (d, J = 6.7 Hz, 1H), 5.78 (d, J = 6.1 Hz, 1H), 4.79 (dd, J = 6.7, 5.1 Hz, 1H), 4.65 (t, J = 5.7 Hz, 1H), 4.51 - 4.45 (m, 2H), 4.38 (dd, J = 13.9, 8.1 Hz, 1H), 4.30 (dd, J = 13.9, 8.9 Hz, 1H), 4.15 (dd, J = 13.9, 5.1 Hz, 1H), 4.07 (dd, J = 13.8, 4.5 Hz, 1H), 3.94 - 3.84 (m, 4H), 3.82 (dd, J = 8.3, 4.1 Hz, 1H), 3.76 (s, 12H), 3.62 - 3.45 (m, 7H), 3.39 (s, 3H), 3.35 (s, 3H), 2.63 (d, J = 12.1 Hz, 2H), 2.37 (ddt, J = 12.2, 9.4, 6.1 Hz, 4H), 2.22 (p, J = 6.8 Hz, 1H), 1.25 - 1.18 (m, 2H), 1.16 - 1.10 (m, 16H), 1.05 (dt, J = 18.4, 6.9 Hz, 12H), 0.95 (d, J = 6.8 Hz, 2H), 0.85 (d, J = 6.8 Hz, 4H).

[0388] Synthesis of oligonucleotides of the disclosure

[0389] The synthesis of oligonucleotides of the disclosure is no different from the general phosphoramidite solid-phase synthesis method, and the synthesis process is briefly described as follows: starting with a universal CPG carrier on a Dr. Oligo 48 synthesizer (Biolytic), according to the synthesis procedure, sequentially connecting unmodified nucleoside phosphoramidite monomers or nucleoside phosphoramidite monomers with modifications at the corresponding positions in the sequence. The nucleoside phosphoramidite monomers used in the examples of the disclosure are purchased from Shanghai Zhiwei Company and Suzhou Jimar Company (adjust according to the actual supplier). 5-ethylthio-1H-tetrazole (ETT) is used as an activator (0.6M acetonitrile solution), 0.22M PADS dissolved in a 1:1 volume ratio of acetonitrile and 3- pyridine (Shanghai Lingjiang) solution is used as a sulfurizing reagent, and iodonium / water solution (Shanghai Lingjiang) is used as an oxidizing agent.

[0390] The oligonucleotide containing NA0167 was prepared according to the same method as the preparation example of WO2025067317A1, and the oligonucleotide containing NAG0052’ was prepared according to the same method as the preparation example of WO2023274395A. The oligonucleotides containing (NA0218)(U), (NA0218)(A) and (NA0218)(G) were prepared by using the phosphoramidite monomer compounds 1, 3 and 5 prepared in Example 1, Example 3 and Example 5, respectively, at the corresponding positions in the sequence during solid-phase synthesis.

[0391] After the completion of solid-phase synthesis, the oligoribonucleotide was cleaved from the solid support and soaked in a 3:1 solution of 28% ammonia and ethanol at 50°C for 16 hours. Then, centrifugation was performed, and the supernatant was transferred to another centrifuge tube. After concentration and evaporation, C18 reverse phase chromatography was used for purification with 0.1M TEAA and acetonitrile as the mobile phase. The target oligonucleotide was collected, freeze-dried, and identified as the target product by LC-MS, and then quantified by UV (260 nm). The obtained single-stranded oligonucleotide was annealed according to the complementary pairing in an equimolar ratio, and the final double-stranded oligonucleotide was dissolved in 1xPBS and adjusted to the required concentration for experiments. The oligonucleotides prepared in this example have the sequences shown in Table 2 below:

[0392] Table 2. Oligonucleotide sequences

[0393] In Table 2, in the same nucleic acid sequence, G, C, A, U from left to right represent nucleosides with guanine, cytosine, adenine, and uracil, respectively, and the sequence from left to right represents the 5’ end to the 3’ end direction. The lowercase letter m indicates that the nucleoside adjacent to the left of the letter m is a 2’-methoxy-modified nucleoside; the lowercase letter f indicates that the nucleoside adjacent to the left of the letter f is a 2’-fluorinated-modified nucleoside; the lowercase letter s indicates that the two nucleosides adjacent to the letter s are connected by a phosphorothioate diester group. Unless otherwise specified, the two adjacent nucleosides or the nucleoside and the delivery group NA0167 are connected by a phosphodiester group; the capital letter VP indicates that the 5’ position of the sugar ring of the nucleoside adjacent to the right is an E-vinyl phosphate group; unless otherwise specified, the 3’ position of the first nucleotide at the 3’ end of each strand is a hydroxyl group; the 5’ position of the first nucleotide at the 5’ end of each strand is a hydroxyl group; and NA0218 indicates that the nucleoside adjacent to the right is a nucleoside with the structure shown in formula (I-5).

[0394] The 2'-methoxy modified nucleoside, 2'-fluoro modified nucleoside, E-vinyl phosphate group, phosphorodithioate group, phosphodiester group, nucleoside having the structure shown in formula (I-5) are shown in Table 3 below, and when the RNAi agent of the present disclosure exists in the form of a salt, for example, in the form of a sodium salt, the structure of the salt form corresponding to the structure in Table 3 below is also within the protection scope of the present disclosure:

[0395] Table 3

[0396] Example 7. psiCHECK validation at target and off-target levels

[0397] In vitro molecular level simulation of the oligonucleotide of the present disclosure in HEK293A cells was carried out using 9 concentration gradients to screen the activity at the target.

[0398] The psi-CHECK plasmid used in this example was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0399] The construction rules of the target plasmid are as follows: the oligonucleotide corresponding to the target sequence of INHBE or SARM1 gene is inserted into the psiCHECK-2 plasmid, i.e. GSCM target plasmid. The plasmid contains Renilla luciferase gene and firefly luciferase gene. As a dual reporter gene system, the GSCM target sequence of the oligonucleotide is inserted into the 3'UTR region of the Renilla luciferase gene, and the activity of the oligonucleotide of the present disclosure to the GSCM target sequence can be reflected by the detection of the Renilla luciferase expression calibrated by the firefly luciferase, and the detection uses Dual-Luciferase Reporter Assay System (Promega, E2940).

[0400] The construction rules of the off-target plasmid are as follows: the off-target plasmid (GSSM) which is completely complementary to the 1-8 positions of the 5' end of the antisense strand of the oligonucleotide and completely mismatched at other positions is constructed, and the base mismatching corresponds to the rules of A and C intermatching, G and T intermatching; in order to improve the detection sensitivity, the GSSM-5hits off-target plasmid is constructed, i.e. 5 identical GSSM sequences are connected by TTCC to form a group, which is inserted into the psiCHECK plasmid, and the plasmid contains Renilla luciferase gene and firefly luciferase gene. As a dual reporter gene system, the GSSM target sequence is inserted into the 3'UTR region of the Renilla luciferase gene. The activity to the GSSM target sequence can be reflected by the detection of the Renilla luciferase expression calibrated by the firefly luciferase, and the detection uses Dual-Luciferase Reporter Assay System (Promega, E2940).

[0401] HEK293A cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum at 37°C and 5% CO2. 24 hours before transfection, HEK293A cells were seeded into 96-well plates at a density of 1 × 10⁴ cells per well with 100 μL of medium per well.

[0402] Following the instructions, Lipofectamine 2000 (ThermoFisher, 11668019) was used to co-transfect cells with oligonucleotides and corresponding plasmids, with 0.2 μL of Lipofectamine 2000 per well. The plasmid transfection volume was 20 ng per well. Nine concentration points were set for the target sequence plasmid, with the highest concentration being 20 nM. Four-fold serial dilutions were used: 20 nM, 5 nM, 1.25 nM, 0.3125 nM, 0.0781 nM, 0.0195 nM, 0.0049 nM, 0.0012 nM, and 0.0003 nM. Target levels were detected 24 h post-transfection using the Dual-Luciferase Reporter Assay System (Promega, E2940).

[0403] The experimental materials and instruments used in this embodiment are detailed in Tables 4 and 5.

[0404] The experimental results are shown in Tables 6 and 7. Tables 6 and 7 show that oligonucleotides targeting both INHBE and SARM1 exhibited significantly reduced off-target activity compared to oligonucleotides TJR100532 and TJR103388, which contained the structure shown in Formula (I) and did not contain the structure shown in Formula (I), while maintaining essentially the same target activity. Furthermore, oligonucleotides TJR103687 and TJR103688, containing the structure shown in Formula (I) at different sequence positions, showed a significant improvement in off-target activity.

[0405] Table 4. Consumables and Reagents for the psi-CHECK Experiment

[0406] Table 5. Psi-CHECK Test Apparatus

[0407] Table 6. Target and off-target activities of INHBE target psi-CHECK

[0408] Table 7. On-target and off-target activity of SARM1 target psi-CHECK

Claims

1. An oligonucleotide comprising at least one structure as shown in formula (I): wherein, X is selected from O or C(R X )2; Each R X Each is independently selected from hydrogen, halogen, and C. 1-6 Alkyl, the C 1-6 Alkyl groups are optionally surrounded by one or more components selected from R A The substituents replaced by R A Selected from halogens, hydroxyl groups, cyano groups, and oxo groups; R 1A , R 1B each independently is selected from hydrogen, hydroxyl, C 1-6 1-6alkyl, C 1-6 1-6alkoxy, amino or halogen, said C 1-6 1-6alkyl, C 1-6 1-6alkoxy, amino optionally being substituted with one or more substituents selected from R A , R A selected from halogen, hydroxyl, cyano, oxo; or R 1A , R 1B form a 3- to 6-membered heterocyclyl, which is optionally substituted by one or more substituents selected from R 1A , R 1B form a 3- to 6-membered heterocyclyl, which is optionally substituted by one or more substituents selected from R A , R A is selected from halogen, hydroxy, cyano, oxo; R2, R3are each independently selected from hydrogen or C 1-6 alkyl, said C 1-6 alkyl is optionally substituted with one or more substituents selected from R A A selected from halogen, hydroxy, cyano, oxo;​ m is selected from 0, 1, 2 or 3; said ring A is selected from 5- to 12-membered heteroaryl or 3- to 12-membered heterocyclyl; R4is each independently selected from the group consisting of hydrogen, cyano, halogen, hydroxyl, amino, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, -S-C 1-6 alkyl, C 1-6 alkoxy, -N(C 1-6 alkyl)2, oxo, 3- to 6-membered cycloalkyl, 6- to 10-membered aryl, 5- to 12-membered heteroaryl, 3- to 12-membered heterocyclyl, -NH-(C=0)-C 1-6 alkyl, -NH-(C=0)-C 3-6 cycloalkyl, -NH(C=0)-OC 1-6 alkyl, -NH(C=0)-OC 3-6 cycloalkyl, -O(C=0)NHC 1-6 alkyl, -O(C=0)NH-C 3-6 cycloalkyl, -(C=0)NH-C 1-6 alkyl, -(C=0)-NH-C 3-6 cycloalkyl, -(C=0)-C 1- 6alkyl, -(C=0)-C 3-6 cycloalkyl, -SO2-C 1-6 alkyl, -SO2-C 3-6 cycloalkyl, -SO2-NH2, -SO2-NH-C 1-6 alkyl, -SO2-NH-C 3-6 cycloalkyl, -SO2-N(C 1-6 alkyl)2, -SO2-NH(C 3-6 cycloalkyl)2, -S(O)(NH)-C 1-6 alkyl, -S(O)(NH)-C 3-6 cycloalkyl; n is selected from 0, 1, 2, 3, 4 or 5; Base represents a base; * represents the direction towards the 5' end of the sequence and # represents the direction towards the 3' end of the sequence; Preferably, n is selected from 0 or 1; Preferably, the structure as shown in formula (I) is a structure as shown in formula (I-1): More preferably, the structure as shown in formula (I) is a structure as shown in formula (I-2) or as shown in formula (I-3):

2. The oligonucleotide of claim 1, said structure as Formula (I) is a structure as Formula (I-4):

3. The oligonucleotide according to claim 1 or 2, wherein: R 1A selected from hydroxy, C 1-6 alkoxy or halogen, said C 1-6 alkoxy being optionally substituted with one or more substituents selected from R A , said R A being selected from halogen, hydroxy, cyano, oxo; More preferably, R 1A selected from C 1-6 alkyl, alkoxy or halogen, said C 1-6 alkyl is optionally substituted with one or more substituents selected from R A selected from halogen, hydroxy, cyano, oxo; and A selected from halogen, hydroxy, cyano, oxo; and Most preferably, R 1A is selected from methoxy or fluoro.

4. The oligonucleotide according to any one of claims 1-3, wherein ring A is selected from 5- to 6-membered heteroaryl or 3- to 6-membered heterocyclyl; More preferably, ring A is selected from 1,2,4-triazole ring, morpholine ring or azetidine; Most preferably, ring A is selected from 5. The oligonucleotide according to any one of claims 1 to 4, wherein each R4 is independently selected from hydrogen, cyano, halogen, hydroxyl, amino, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 alkenyl, C 1-6 alkoxy, -N(C 1-6 alkyl)2; R4is each independently selected from the group consisting of hydrogen, C 1-6 alkyl, -S-C 1-6 alkyl, C 1-6 alkoxy, -N(C 1-6 alkyl)2; More preferably, each R4is independently hydrogen or -N(C 1-6 alkyl)2.

6. The oligonucleotide of any one of claims 1-10, wherein, The structure as shown in formula (I) is a structure as shown in any one of formula (I-5) to formula (I-12): wherein, Base represents a base; Preferably, the structure as shown in formula (I) is a structure as shown in formula (I-5) or (I-6): More preferably, the structure according to Formula (I) is a structure according to any one of Formulae (I-5A), (I-5B), (I-6A), or (I-6B):

7. The oligonucleotide according to any one of claims 1-6, which is a double stranded RNAi agent comprising a sense strand and an antisense strand which are at least partially complementary to form a double stranded region; Preferably, only one nucleotide in the oligonucleotide contains one structure as shown in formula (I); More preferably, the nucleotide at position 7 or 8 from the 5' end of the antisense strand contains one structure as shown in formula (I).

8. The oligonucleotide according to claim 7, wherein the length of the sense strand is 15-35 nucleotides and the length of the antisense strand is 15-35 nucleotides; Preferably, the length ratio of the sense strand and the antisense strand is 19 / 21, 21 / 21, 21 / 23 or 23 / 25; More preferably, the length ratio of the sense strand and the antisense strand is 19 / 21 or 21 / 21.

9. The oligonucleotide according to claim 7 or 8, further comprising one or more delivery groups attached to the sense strand and / or the antisense strand.

10. The oligonucleotide according to any one of claims 7-9, wherein at least one additional nucleotide is a modified nucleotide; Preferably, all nucleotides in the oligonucleotide are modified nucleotides; More preferably, the modified nucleotide is selected from: 2'-methoxy modified nucleotide, 2'-substituted alkoxy modified nucleotide, 2'-alkyl modified nucleotide, 2'-substituted alkyl modified nucleotide, 2'-amino modified nucleotide, 2'-substituted amino modified nucleotide, 2'-fluoro modified nucleotide, 2'-deoxy nucleotide, 2'-deoxy-2'-fluoro modified nucleotide, 3'-deoxy-thymine nucleotide, iso-nucleotides, LNA, ENA, cET, UNA, GNA.

11. The oligonucleotide according to any one of claims 7-10, wherein at least one phosphodiester group in the oligonucleotide is a phosphodiester group with a modification group; Preferably, at least one phosphodiester group in the oligonucleotide is a phosphorothioate group; More preferably, the oligonucleotide is TJR102840, TJR103687 or TJR103688.

12. A compound of formula (II) or a tautomer thereof: ###00006### (II) wherein R 1A , R 1B , R2, R3, R4, m, n, ring A, Base, X are as defined in claim 1; W is H or a hydroxyl protecting group; Q is a phosphorus-containing active reactive group; Preferably, Q is Preferably, W is selected from MMTr, DMTr or trityl.

13. The compound of formula (II) or a tautomer thereof according to claim 12, which is a compound of formula (II-1) or (II-2) or a tautomer thereof: In formula (II-1), R 1A , R2, R3, R4, m, n, ring A, Base are as defined in claim 1. W, Q are as defined in claim 12; In formula (II-2), R 1A , R4, m, n, ring A, Base are as defined in claim 1. W, Q are as defined in claim 12.

14. The compound of formula (II) or a tautomer thereof according to claim 13, which is a compound of formula (II-3) or a tautomer thereof: wherein, R 1A , R2, R3, R4, n, ring A, Base are as defined in claim 1 ; W, Q are as defined in claim 12; Preferably, it is a compound of formula (II-4) or a tautomer thereof: wherein R 1A , R4, n, ring A, Base are as defined in claim 1 ; W, Q are as defined in claim 12.

15. A compound of formula (II) or a tautomer thereof according to any one of claims 12 to 14, wherein R 1A is selected from hydroxy, C 1-6 alkoxy or halogen, said C 1-6 alkoxy being optionally substituted with one or more substituents selected from R A , said R A being selected from halogen, hydroxy, cyano, oxo; Preferably, R 1A Selected from C 1-6 alkoxy or halogen, the C 1-6 Alkyl groups are optionally surrounded by one or more R groups. A The substituents replaced by R A Selected from halogens, hydroxyl groups, cyano groups, and oxo groups; More preferably, R 1A is selected from methoxy or fluoro.

16. The compound of formula (II) or a tautomer thereof according to any one of claims 12-15, wherein ring A is selected from 5- to 6-membered heteroaryl or from 3- to 6-membered heterocyclyl; More preferably, ring A is selected from 1,2,4-triazole ring, morpholine ring or azetidine; Most preferably, ring A is selected from 17. A compound of formula (II) or a tautomer thereof according to any one of claims 12 to 16, wherein each R4 is independently selected from hydrogen, cyano, halogen, hydroxy, amino, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 alkenyl, C 1-6 alkoxy, -N(C 1-6 alkyl)2; R4is each independently selected from the group consisting of hydrogen, C 1-6 alkyl, -S-C 1-6 alkyl, C 1-6 alkoxy, -N(C 1-6 alkyl)2; More preferably, each R4is independently selected from hydrogen or -N(C 1-6 alkyl)2.

18. The compound of formula (II) according to any one of claims 12-17, wherein the compound of formula (II) is selected from the compounds of formulas (II-5) to (II-12): wherein, Base is as defined in claim 1.

19. A method for preparing the oligonucleotide of any one of claims 1-11, comprising the steps of: (1) synthesizing the compound of formula (II) or a tautomer thereof according to any one of claims 12-18; (2) synthesizing the oligonucleotide of any one of claims 1-11 using the compound of formula (II) or a tautomer thereof obtained in step (1).

20. A pharmaceutical composition comprising the oligonucleotide of any one of claims 1-11. Optionally, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

21. Use of the oligonucleotide of any one of claims 1-11 or the pharmaceutical composition of claim 20 for the manufacture of a medicament.

22. A method for inhibiting the expression of a target gene or mRNA thereof, comprising administering to a subject the oligonucleotide of any one of claims 1-11 or the pharmaceutical composition of claim 20.

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