Oligonucleotides comprising lipophilic monomers and their use in non-hepatic delivery

Abstract

An oligonucleotide comprising a lipophilic monomer and its use in non-hepatic delivery. Specifically, the invention relates to an oligonucleotide which comprises an antisense strand complementary to a target gene, a sense strand complementary to the antisense strand, and one or more compounds as shown in a formula (I), and also relates to a pharmaceutical composition containing the oligonucleotide, and medical application of the oligonucleotide and / or the pharmaceutical composition.

Description

Oligonucleotides containing lipophilic monomers and their use in non-hepatic delivery Technical Field

[0001] The present disclosure belongs to the field of biomedicine, and specifically relates to oligonucleotides containing lipophilic monomers. The present disclosure also relates to methods for preparing oligonucleotides and applications. Background Art

[0002] RNA interference (RNAi) is an effective method for silencing gene expression and holds broad application prospects. While RNA drugs offer advantages such as a rich pipeline of candidate targets, a simple and efficient development process, and strong specificity, the number of RNA drugs ultimately authorized by the FDA for clinical treatment remains very limited. These drugs face significant challenges in their clinical application, primarily due to poor stability, short half-life, high immunogenicity, weak targeting, and limited cytoplasmic penetration. Therefore, in addition to improving RNA stability and immunogenicity through chemical modification, it is also necessary to develop efficient and safe in vivo delivery systems to enhance membrane permeability.

[0003] The main delivery technologies for RNA interference drugs include liposome nanodelivery (LNP), N-acetylgalactosamine (GalNAC) modification-mediated liver-targeted delivery, and antibody-mediated targeted delivery. Currently, there are many studies on the use of GalNAC-coupled drug technology. At the end of 2020, two GalNAC-coupled siRNA drugs, lumasirna and inclisiRNA, were approved for clinical use. GalNAC-coupled RNA drugs can achieve efficient and specific delivery to the liver, but there is no effective delivery method for extrahepatic cells that lack ASGPR expression. Therefore, it is necessary to continue to develop efficient in vivo delivery methods so that RNA drugs can play a role in extrahepatic tissues.

[0004] The compound represented by formula (I) of the present disclosure, which contains a lipophilic portion, or its tautomer, has increased hydrophobicity and enhanced membrane permeability, which not only improves the membrane permeability of oligonucleotides, but also enables oligonucleotides containing the compound to be effectively delivered into the cytoplasm and exert RNA effects in non-hepatic tissues.

[0005] Summary of the Invention

[0006] The present disclosure provides an oligonucleotide comprising at least one compound represented by formula (I) or a pharmaceutically acceptable salt thereof, wherein the compound represented by formula (I) has the structure

[0007] in,

[0008] X1 is selected from O, S, N, and C atoms;

[0009] X2 is selected from O and S atoms;

[0010] R1 is selected from C10 -C 30 Straight chain alkyl (such as C 10 、C 11 、C 12 、C 13 、C 14 、C 15 、C 16 、C 17 、C 18 、C 19 、C 20 、C 21 、C 22 、C 23 、C 24 、C 25 、C 26 、C 27 、C 28 、C 29 、C 30 A straight-chain alkyl group) or a C alkyl group interrupted by one or more O or S atoms 10 -C 30 Straight chain alkyl (such as C 10 、C 11 、C 12 、 C 13 、C 14 、C 15 、C 16 、C 17 、C 18 、C 19 、C 20 、C 21 、C 22 、C 23 、C 24 、C 25 、C 26 、C 27 、C 28 、C 29 、C 30 linear alkyl); optionally, the C 10 -C 30 The straight chain alkyl group is replaced by one or more R a Replace, or optionally, the C 10 -C 30 The adjacent two carbon atoms of the straight chain alkyl form a C 3-6 Cycloalkyl (C3, C4, C5, C6 cycloalkyl, including but not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl);

[0011] R2 and R3 are the same or different and are each independently selected from a bond, hydrogen, an activated phosphate group, an activated phosphite group, a phosphoramidite group, a solid support, an internucleotide linker attached to an oligonucleotide, -P(=O)(OH)-O-, -P(=O)(SH)-O-, -P(=O)(OH)-O-nucleoside, -P(=O)(SH)-O-nucleoside, -P(=O)(OH)-O-oligonucleotide fragment, -P(=O)(SH)-O-oligonucleotide fragment, a hydroxyl protecting group (e.g., an ester protecting group, an aryl protecting group, an alkyl protecting group, an alkoxymethyl protecting group, a silyl protecting group, including but not limited to DMTr);

[0012] R a Each is independently selected from hydrogen, deuterium, halogen (e.g., fluorine, chlorine, bromine), hydroxyl, cyano, alkyl (e.g., C1, C2, C3, C4, C5, C6 alkyl, including but not limited to methyl, ethyl, isopropyl), haloalkyl, alkoxy (e.g., C1 alkoxy, C2 alkoxy, C3 alkoxy, C4 alkoxy, C5 alkoxy, C6 alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropoxy), cycloalkyl (including but not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl), heterocycloalkyl;

[0013] n is selected from 0 or 1;

[0014] B is a base;

[0015] The compound represented by formula (I) is not selected from:

[0016] In some embodiments, the compound structure represented by formula (I) is selected from:

[0017] Among them, R b Selected from OH or SH.

[0018] In some embodiments, X1 is selected from O or S atoms, preferably O atoms.

[0019] In some embodiments, X2 is selected from O or S atoms, preferably O atoms.

[0020] In some embodiments, R1 is selected from C 14 -C 24 Straight chain alkyl (such as C 14 、C 15 、C 16 、C 17 、C 18 、C 19 、C 20 、C 21 、C 22 、C 23 、C24 A straight-chain alkyl group) or a C alkyl group interrupted by one or more O or S atoms 14 -C 24 Straight chain alkyl (such as C 14 、C 15 、C 16 、C 17 、C 18 、C 19 、C 20 、C 21 、C 22 、C 23 、C 24 straight-chain alkyl).

[0021] In some embodiments, R a are each independently selected from hydrogen, deuterium, halogen (such as fluorine, chlorine, bromine), C 1-6 Alkyl (e.g., C1, C2, C3, C4, C5, C6 alkyl, including but not limited to methyl, ethyl, isopropyl), C 1-6 Alkoxy (e.g., C1 alkoxy, C2 alkoxy, C3 alkoxy, C4 alkoxy, C5 alkoxy, C6 alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropoxy).

[0022] In some embodiments, R a Each is independently selected from hydrogen, deuterium, fluorine, methyl, and methoxy.

[0023] In some embodiments, R1 is selected from: The a end is connected to X2.

[0024] In some embodiments, B is selected from adenine, guanine, cytosine, uracil, and thymine.

[0025] The present disclosure also provides an oligonucleotide comprising at least one of the following compounds or pharmaceutically acceptable salts thereof, wherein the compound structure is:

[0026] In some embodiments, the compound structure represented by formula (I) is selected from:

[0027] In some embodiments, the oligonucleotide disclosed herein comprises 1, 2, 3 or 4 compounds of formula (I) or pharmaceutically acceptable salts thereof; in some embodiments, the oligonucleotide comprises 1 compound of formula (I) or pharmaceutically acceptable salts thereof.

[0028] In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is located at at least one of the following nucleotide positions:

[0029] Position 1 at the 5' end of the sense strand;

[0030] Position 1 at the 3' end of the sense strand;

[0031] Position 1 at the 5' end of the antisense strand;

[0032] Position 1 at the 3' end of the antisense strand;

[0033] Positions 2-8 in the middle of the sense strand (e.g., position 2, 3, 4, 5, 6, 7, or 8);

[0034] Position 2-8 in the middle of the antisense strand (eg, position 2, position 3, position 4, position 5, position 6, position 7 or position 8).

[0035] The middle position refers to the middle position counted from the first nucleotide at the 5' end or 3' end of the antisense strand and / or the sense strand, for example, "position 6 at the middle position of the sense strand" refers to the sixth nucleotide counted from the first nucleotide at the 5' end or 3' end of the sense strand. Preferably, the middle position refers to the middle position counted from the first nucleotide at the 5' end, for example, "position 6 at the middle position of the sense strand" refers to the position of the sixth nucleotide from the first nucleotide at the 5' end of the sense strand.

[0036] In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is located at at least one of the following nucleotides:

[0037] Position 1 at the 5' end of the sense strand;

[0038] Position 1 at the 3' end of the sense strand;

[0039] The 6th position in the middle of the justice chain.

[0040] In some embodiments, the structure of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is selected from: Among them, the b end is connected to the 5th nucleotide at the 5' end of the sense chain, and the c end is connected to the 7th nucleotide at the 5' end of the sense chain. In formulas (I-1) and (I-2), B represents the base of the 6th nucleotide in the direction from the 5' end to the 3' end of the sense chain.

[0041] In some embodiments, the structure of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is selected from: Among them, the connection end The second nucleotide connected to the 5' end of the sense chain, that is, the first nucleotide located at the 5' end of the sense chain in formula (I-3) and (I-4), where B represents the base of the first nucleotide at the 5' end of the sense chain.

[0042] In some embodiments, the structure of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is selected from: Among them, the connection end The second nucleotide at the 3' end of the sense chain is connected, that is, the first nucleotide at the 3' end of the sense chain in formula (I-5) and (I-6), where B represents the base of the first nucleotide at the 3' end of the sense chain.

[0043] In some embodiments, the oligonucleotide disclosed herein comprises a compound represented by formula (I) or a pharmaceutically acceptable salt thereof; the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is located at the sixth nucleotide at the 5' end of the sense strand, and the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is selected from:

[0044] In some embodiments, the oligonucleotide disclosed herein comprises a compound represented by formula (I) or a pharmaceutically acceptable salt thereof; the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is located at the sixth nucleotide at the 5' end of the sense strand, and the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is selected from:

[0045] In some embodiments, the oligonucleotides disclosed herein are selected from double-stranded RNAi inhibitor molecules; in some embodiments, the oligonucleotides are selected from siRNA, comprising a sense strand and an antisense strand forming a double-stranded region; in some embodiments, the siRNA sense strand is 15-35 nucleotides long, and the antisense strand is 15-30 nucleotides long.

[0046] In some embodiments, the sense strand and the antisense strand are the same or different in length, with the sense strand being 19-23 nucleotides in length and the antisense strand being 19-26 nucleotides in length. Thus, the ratio of the length of the sense strand to the antisense strand of the siRNA 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. In some embodiments, the length ratio of the sense strand to the antisense strand of the siRNA is 19 / 21, 21 / 23, or 23 / 25. In some embodiments, the length ratio of the sense strand to the antisense strand of the siRNA is 21 / 23.

[0047] In some embodiments, the antisense strand is at least partially reverse complementary to the target sequence to mediate RNA interference; in some embodiments, there are no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 mismatch between the antisense strand and the target sequence; in some embodiments, the antisense strand is completely reverse complementary to the target sequence.

[0048] In some embodiments, the sense strand and the antisense strand are at least partially reverse complementary to form a double-stranded region; in some embodiments, there are no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 mismatch between the sense strand and the antisense strand; in some embodiments, the sense strand and the antisense strand are completely reverse complementary.

[0049] In some embodiments, the disclosed siRNAs comprise one or two blunt ends.

[0050] In some specific embodiments, the siRNA comprises an overhang of 1 to 4 unpaired nucleotides, eg, 1, 2, 3, or 4.

[0051] In some embodiments, the disclosed siRNA comprises an overhang located at the 3' end of the antisense strand of the siRNA.

[0052] In some embodiments, in the oligonucleotides described herein, in addition to the compound structure shown in formula (I), at least one additional nucleotide is a modified nucleotide. In some embodiments, the sense strand and antisense strand described herein each contain at least one modified nucleotide.

[0053] In some embodiments, the modified nucleotides are selected from the group consisting of 2'-methoxy modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-amino modified nucleotides, 2'-substituted amino modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy nucleotides, 2'-deoxy-2'-fluoro modified nucleotides, 3'-deoxy-thymine (dT) nucleotides, isonucleotides, LNA, ENA, cET, UNA, GNA.

[0054] In some embodiments, the modified nucleotides are independently selected from the group consisting of: 2'-methoxy modified nucleotides, 2'-fluoro modified nucleotides.

[0055] In some embodiments, the oligonucleotide is selected from a double-stranded RNAi inhibitor molecule, comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand contains three consecutive nucleotides that are 2'-fluoro-modified nucleotides.

[0056] In some embodiments, in the direction from the 5' end to the 3' end, the nucleotides at positions 9, 10, and 11 of the sense strand are each independently a 2'-fluoro-modified nucleotide.

[0057] In some embodiments, in the direction from the 5' end to the 3' end, the nucleotides at positions 9, 10, and 11 of the sense chain are each independently a 2'-fluoro-modified nucleotide, the nucleotide at position 7 of the sense chain is selected from a 2'-fluoro-modified nucleotide, and the nucleotides at other positions of the sense chain are selected from a 2'-methoxy-modified nucleotide.

[0058] In some embodiments, the oligonucleotide is selected from a double-stranded RNAi inhibitor molecule, comprising a sense strand and an antisense strand forming a double-stranded region, wherein, from the 5' end to the 3' end, the nucleotides at positions 2, 6, 8, 9, 14, and 16 of the antisense strand are each independently a 2'-fluoro-modified nucleotide.

[0059] In some embodiments, in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 8, 9, 14, and 16 of the antisense strand are each independently a 2'-fluoro-modified nucleotide, and the nucleotides at other positions of the antisense strand are selected from 2'-methoxy-modified nucleotides.

[0060] In some embodiments, the sense strand comprises or is a sequence represented by the following formula:

[0061] 5'N L N a N a N a N a N a N a Na N b N b N b N a N a N a N a N a N a N a N a N a N a 3’; or,

[0062] 5’N a N a N a N<000010​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​b N b N b N a N a N a N a N a N a N a N a N a N a 3'; or,

[0065] 5'N a N a N a N a N a N a N b N a N b N b N b N a N a N a N a N a N a N a N a N a N L 3'; or,

[0066] 5'N a N a N a N a N a N L N b N a N b N b N b N a N a N a N a N a N a N a N a N a N a 3', where N L Each is independently selected from the compound represented by formula (I) or a pharmaceutically acceptable salt thereof according to the present disclosure; N a 2'-methoxy modified nucleotides, N b It is a 2'-fluoro modified nucleotide.

[0067] In some embodiments, the antisense strand comprises or is a sequence represented by the following formula:

[0068] 5’N L ’N b ’N a ’N a ’N a ’N b ’N a ’N b ’N b ’N a ’N a ’N a ’N a ’N b ’N a ’N b ’N a ’N a ’N a ’N a ’N<00002​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​a 'N b 'N a 'N a 'N a 'N a 'N a 'N a 'N a '3, where N L 'Each independently selected from the compound represented by formula (I) or a pharmaceutically acceptable salt thereof according to the present disclosure; N a ' is a 2'-methoxy modified nucleotide, N b ' is a 2'-fluoro-modified nucleotide.

[0071] In some embodiments, the sense strand comprises or is a sequence represented by the following formula:

[0072] 5'N L NNNNNNNNNNNNNNNNNN3'; or,

[0073] 5'N L N a N a N a N a N a N a N a N b N b N b N a N a N a N a N a N a N a N a N a N a 3'; or,

[0074] 5'N L N a N a N a N a N a N b N a N b N b N b N a N a N a N a N a N a N a N a N a Na 3'; wherein N is a modified or unmodified nucleotide; N a 2'-methoxy modified nucleotides, N b It is a 2'-fluoro-modified nucleotide;

[0075] N L Selected from:

[0076] In some embodiments, the sense strand contains or is a sequence represented by the following formula:

[0077] 5'NNNNNNNNNNNNNNNNNNN L 3'; or,

[0078] 5'N a N a N a N a N a N a N a N a N b N b N b N a N a N a N a N a N a N a N a N a N L 3'; or,

[0079] 5'N a N a N a N a N a N a N b N a N b N b N b N a N a N a N a N a N a N a N a N a N L 3'; wherein N is a modified or unmodified nucleotide; N a 2'-methoxy modified nucleotides, N b It is a 2'-fluoro-modified nucleotide;

[0080] N L Selected from:

[0081] In some embodiments, the sense strand contains or is a sequence represented by the following formula:

[0082] 5'NNNNNN L NNNNNNNNNNNNN3'; or,

[0083] 5'N a N a N a N a N a N L N a N a N b N b N b N a N a N a N a N a N a N a N a N a N a 3'; or,

[0084] 5'N a N a N a N a N a N L N b N a N b N b N b N a N a N a N a N a N a N a N a N a N a 3'; wherein N is a modified or unmodified nucleotide; N a 2'-methoxy modified nucleotides, N b It is a 2'-fluoro-modified nucleotide;

[0085] N L Selected from:

[0086] In some embodiments, the sense strand is a sequence shown in the following formula:

[0087] 5'NNNNNN L NNNNNNNNNNNNNNN3'; or,

[0088] 5'N a N a N a N a N a N L N a N a N b N b N b N a N a N a N a N a N a N a N a N a N a 3'; or,

[0089] 5'N a N a N a N a N a N L N b N a N b N b N b N a N a N a N a N a N a N a N a N a N a 3'; wherein N is a modified or unmodified nucleotide; N a 2'-methoxy modified nucleotides, N b It is a 2'-fluoro-modified nucleotide;

[0090] N L Selected from:

[0091] The b end of the above structure is connected to the 5th nucleotide at the 5' end of the sense chain, and the c end is connected to the 7th nucleotide at the 5' end of the sense chain. B in the above structure represents the base of the 6th nucleotide at the 5' end of the sense chain.

[0092] In some embodiments, at least one phosphate group in the sense strand and / or antisense strand is a phosphate group having a modified group; in some embodiments, at least one phosphate group in the sense strand and / or antisense strand is a phosphorothioate diester group.

[0093] In some embodiments, the phosphorothioate diester group is present in at least one of the following positions:

[0094] between the first and second nucleotides at the 5' end of the sense strand;

[0095] between the second and third nucleotides at the 5' end of the sense strand;

[0096] between the first and second nucleotides at the 3' end of the sense strand;

[0097] between the second and third nucleotides at the 3' end of the sense strand;

[0098] between the first and second nucleotides at the 5' end of the antisense strand;

[0099] between the second and third nucleotides at the 5' end of the antisense strand;

[0100] between the first and second nucleotides at the 3' end of the antisense strand;

[0101] between the second and third nucleotides at the 3' end of the antisense strand.

[0102] In some embodiments, at least one phosphate group in the sense strand and / or the antisense strand is a 5'-vinyl phosphodiester group; in some embodiments, the 5'-vinyl phosphodiester group is present on the first nucleotide from the 5' end of the antisense strand.

[0103] In some embodiments, the oligonucleotides described herein target MUC5B.

[0104] The present disclosure also provides a compound represented by formula (II) or a pharmaceutically acceptable salt thereof:

[0105] in,

[0106] Wherein, the definitions and selection ranges of X1, X2, B and R1 are the same as those in the compound structure shown in formula (I) above;

[0107] n is selected from 1 or 0, R A2 Selected from leaving groups, R A3 Selected from phosphorus-containing active reactive groups, or R A3 Selected from leaving groups, R A2Selected from phosphorus-containing active reactive groups, wherein the compound represented by formula (II) is not selected from:

[0108] In some embodiments, the compound represented by formula (II) or a pharmaceutically acceptable salt thereof is selected from:

[0109] Wherein, the definitions and selection ranges of X1, X2, B, R1, and n are the same as those in the compound structure represented by formula (I) above.

[0110] In some embodiments, X1 is selected from O and S atoms, preferably O atom.

[0111] In some embodiments, X2 is selected from O and S atoms, preferably O atoms.

[0112] In some embodiments, R1 is selected from C 10 -C 30 Straight chain alkyl (such as C 10 、C 11 、C 12 、C 13 、C 14 、C 15 、C 16 、C 17 、C 18 、C 19 、C 20 、C 21 、C 22 、C 23 、C 24 、C 25 、C 26 、C 27 、C 28 、C 29 、C 30 A straight-chain alkyl group) or a C alkyl group interrupted by one or more O or S atoms 10 -C 30 Straight chain alkyl (such as C 10 、C 11 、C 12 、C 13 、C 14 、C 15 、C 16 、C 17 、C 18 、C 19 、C 20 、C 21 、C 22 、C 23 、C 24 、C 25 、C 26 、C27 、C 28 、C 29 、C 30 linear alkyl); optionally, the C 10 -C 30 The straight chain alkyl group may be replaced by one or more R a Replace, or optionally, the C 10 -C 30 The adjacent two carbon atoms of the straight chain alkyl form a C 3-6 Cycloalkyl (C3, C4, C5, C6 cycloalkyl, including but not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl).

[0113] In some embodiments, R1 is selected from C 14 -C 24 Straight chain alkyl (such as C 14 、C 15 、C 16 、C 17 、C 18 、 C 19 、C 20 、C 21 、C 22 、C 23 、C 24 A straight-chain alkyl group) or a C alkyl group interrupted by one or more O or S atoms 14 -C 24 Straight chain alkyl (such as C 14 、C 15 、C 16 、C 17 、C 18 、C 19 、C 20 、C 21 、C 22 、C 23 、C 24 straight-chain alkyl).

[0114] In some specific embodiments, R1 is selected from: The a end is connected to X2.

[0115] In some embodiments, R a are each independently selected from hydrogen, deuterium, halogen (e.g., fluorine, chlorine, bromine), C 1-6 Alkyl (e.g., C1, C2, C3, C4, C5, C6 alkyl, including but not limited to methyl, ethyl, isopropyl), C 1-6Alkoxy (e.g., C1 alkoxy, C2 alkoxy, C3 alkoxy, C4 alkoxy, C5 alkoxy, C6 alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropoxy).

[0116] In some embodiments, R a Each is independently selected from hydrogen, deuterium, fluorine, methyl, and methoxy.

[0117] In some embodiments, B is selected from adenine, guanine, cytosine, uracil, and thymine.

[0118] In some embodiments, the compound represented by formula (II) or a pharmaceutically acceptable salt thereof is selected from:

[0119] The present disclosure also provides a method for preparing the oligonucleotide of the present disclosure, comprising the following steps:

[0120] (1) synthesizing the compound represented by formula (II) or a pharmaceutically acceptable salt thereof as disclosed herein;

[0121] (2) synthesizing the oligonucleotide disclosed herein using the compound represented by formula (II) or a pharmaceutically acceptable salt thereof synthesized in step (1).

[0122] In another aspect, the present disclosure provides a pharmaceutical composition comprising the oligonucleotide described above.

[0123] In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

[0124] In some embodiments, the pharmaceutically acceptable excipient can be, for example, a carrier, a vehicle, a diluent, and / or a delivery polymer.

[0125] In some embodiments, based on the total weight of the pharmaceutical composition, the content of the oligonucleotide may be 0.01-99.99%, further 0.1-99.9%, further 0.5%-99.5%, further 1%-99%, and further 2%-98%.

[0126] In some embodiments, based on the total weight of the pharmaceutical composition, the content of the pharmaceutically acceptable excipient may be 0.01-99.99%, further 0.1-99.9%, further 0.5%-99.5%, further 1%-99%, and further 2%-98%.

[0127] In another aspect, the present disclosure provides a method for inhibiting target gene expression, comprising administering to a subject an effective amount or effective dose of an oligonucleotide of the present disclosure or a pharmaceutical composition comprising the oligonucleotide. In some embodiments, the target gene is selected from one or more of MUC5B, APP, PMP22, or MAPT.

[0128] In some embodiments, the oligonucleotide or pharmaceutical composition may be in a therapeutically effective amount.

[0129] In some embodiments, the unit dose of the oligonucleotide or pharmaceutical composition may be 0.001 mg-1000 mg.

[0130] In some embodiments, the effective amount or effective dose of the oligonucleotide 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.

[0131] In another aspect, the present disclosure provides a use of the aforementioned oligonucleotide or the aforementioned pharmaceutical composition in preparing a drug.

[0132] In some embodiments, the medicament is used to prevent and / or treat diseases related to lung, eye, and central nervous system (CNS) disorders; in some embodiments, the medicament is used to prevent and / or treat diseases related to lung.

[0133] In some embodiments, the medicament is used to prevent and / or treat tumor-related diseases.

[0134] In another aspect, the present disclosure provides a method for delivering an oligonucleotide outside the liver, comprising administering to a subject an effective amount or effective dose of the oligonucleotide or pharmaceutical composition of the present disclosure.

[0135] In some embodiments, the oligonucleotides described herein are delivered to the lungs, eyes, or CNS.

[0136] In some embodiments, the oligonucleotides described herein are delivered to the lungs.

[0137] In some embodiments, the oligonucleotides described herein are delivered to a tumor.

[0138] In some embodiments, the oligonucleotides described herein are delivered to pancreatic cancer, prostate cancer, breast cancer, or lung cancer.

[0139] In the present disclosure, when the above-mentioned oligonucleotides or pharmaceutical compositions are contacted with cells expressing the target gene, the above-mentioned oligonucleotides or pharmaceutical compositions can inhibit 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% when measured by, for example, psiCHECK activity screening and luciferase reporter gene assays, other methods such as PCR or branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence analysis, such as Western Blot or flow cytometry.

[0140] In the present disclosure, when the above-mentioned oligonucleotides or pharmaceutical compositions are contacted with cells expressing the target gene, the percentage of residual target gene mRNA expression caused by the above-mentioned oligonucleotides or pharmaceutical compositions is 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 measured by, for example, psiCHECK activity screening and luciferase reporter gene assay, other methods such as PCR or branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence analysis, such as Western Blot or flow cytometry.

[0141] In the present disclosure, when the above-mentioned oligonucleotides or pharmaceutical compositions are contacted with cells expressing the target gene, the oligonucleotides reduce off-target activity by 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% or at least 75% while maintaining target activity, as determined by, for example, psiCHECK activity screening and luciferase reporter gene assays, other methods such as PCR or branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence analysis, such as Western Blot, or flow cytometry.

[0142] In the present disclosure, when the above-mentioned oligonucleotides or pharmaceutical compositions are contacted with cells expressing the target gene, the oligonucleotides reduce the target activity by 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% or at least 75% while reducing the off-target activity by at least 20%, at least 19%, at most 15%, at most 10%, at most 5% or more than 1%, as measured by, for example, psiCHECK activity screening and luciferase reporter gene assays, other methods such as PCR or branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence analysis, such as Western Blot, or flow cytometry.

[0143] In the present disclosure, the above-mentioned dsRNA or pharmaceutical composition, when contacted with cells expressing the target gene, can increase the target activity by at least 1%, 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% or at least 80%, while reducing off-target activity by 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% or at least 75%, as measured by, for example, psiCHECK activity screening and luciferase reporter gene assays, other methods such as PCR or branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence analysis, e.g., Western Blot, or flow cytometry.

[0144] Without specifying a configuration, the compounds of the present disclosure may exist in specific geometric or stereoisomeric forms. The present disclosure contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the present disclosure. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All of these isomers and their mixtures are included within the scope of the present disclosure.

[0145] The compounds of the present disclosure may be asymmetric, for example, having one or more stereoisomers. Where no configuration is specified, all stereoisomers are included, such as enantiomers and diastereomers. Compounds of the present disclosure containing asymmetric carbon atoms can be isolated in optically pure forms or racemic forms. Optically pure forms can be resolved from racemic mixtures or synthesized using chiral starting materials or chiral reagents.

[0146] Optically active (R)- and (S)-isomers, as well as D and L isomers, can be prepared by chiral synthesis methods, or using chiral reagents or other conventional techniques in the art. If one enantiomer of a compound of the present disclosure is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary groups are cleaved to provide the pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), diastereomeric salts are formed with an appropriate optically active acid or base, followed by diastereomeric resolution by conventional methods known in the art, and then the pure enantiomers are recovered. In addition, separation of enantiomers and diastereoisomers is typically accomplished using chromatography, which employs a chiral stationary phase and is optionally combined with chemical derivatization (e.g., carbamate formation from an amine).

[0147] The present disclosure also includes isotopically labeled compounds of the present disclosure that are identical to those described herein, but where one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into the compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, 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 et al.

[0148] Unless otherwise stated, when a position is specifically designated as deuterium (D), the position is understood to have at least 1000 times the abundance of deuterium greater than the natural abundance of deuterium (which is 0.015%) (i.e., at least 10% deuterium incorporation). In the example, the compound has a natural abundance greater than deuterium that can be at least 1000 times the abundance of deuterium, at least 2000 times the abundance of deuterium, at least 3000 times the abundance of deuterium, at least 4000 times the abundance of deuterium, at least 5000 times the abundance of deuterium, at least 6000 times the abundance of deuterium or more abundant deuterium. The disclosure also includes various deuterated compounds. Each available hydrogen atom connected to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can synthesize deuterated compounds of formula I with reference to relevant literature. Commercially available deuterated starting materials may be used in the preparation of deuterated forms of the compounds of Formula I, or they may be synthesized using conventional techniques using deuterated reagents, including but not limited to deuterated borane, trideuterated borane in tetrahydrofuran, deuterated lithium aluminum hydride, deuterated iodoethane, deuterated iodomethane, and the like.

[0149] In the chemical structures of the compounds disclosed herein, the bond Indicates that the configuration is not specified, that is, if chiral isomers exist in the chemical structure, the bond Can be or include both Although all the above structural formulas are drawn as certain isomers for the sake of simplicity, the present disclosure may include all isomers, such as tautomers, rotational isomers, geometric isomers, diastereomers, racemates and enantiomers. In the chemical structures of the compounds described in the present disclosure, the bonds No configuration is specified, i.e. the bond The configuration can be E-type or Z-type, or include both E and Z configurations. Can

[0150] Explanation of terms

[0151] In order to make the present disclosure more easily understood, some technical and scientific terms are specifically defined below. Unless otherwise clearly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present disclosure belongs.

[0152] As used herein, "RNAi agents" refer to agents containing RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecules that can degrade or inhibit the transcription and translation of target messenger RNA (mRNA) in a sequence-specific manner. RNAi agents in this disclosure can be manipulated by an RNA interference mechanism (i.e., by inducing RNA interference by interacting with the RNA interference pathway machinery of mammalian cells (RNA-induced silencing complex or RISC)), or by any other mechanism or pathway. RNAi agents include, but are not limited to, single-stranded oligonucleotides, single-stranded antisense oligonucleotides, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), and Dicer substrates.

[0153] The RNAi agents described in the present disclosure include oligonucleotides having a strand that is at least partially complementary to a targeted mRNA.

[0154] In this disclosure, the "5' region," also known as the "5' end," or "5' terminus" of the sense or antisense strand may be used interchangeably. For example, nucleotides 2 through 8 in the 5' region of the antisense strand may be replaced with nucleotides 2 through 8 at the 5' terminus of the antisense strand. Similarly, the "3' region," "3' terminus," and "3' terminus" of the sense or antisense strand may be used interchangeably.

[0155] "The sixth position from the 5' end to the 3' end of the sense strand" and "the sixth position from the 5' end of the sense strand" can be used interchangeably. For example, in 5'N A NNNNN C NNNNNNNNNNNNNNN B 3', when the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is located at the first position of the 5' end of the sense chain, that is, N A represents a compound represented by formula (I) or a pharmaceutically acceptable salt thereof; when the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is located at the first position of the 3' end of the sense chain, that is, N B represents a compound represented by formula (I) or a pharmaceutically acceptable salt thereof; when the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is located at the 6th position from the 5' end to the 3' end of the sense chain, that is, N C It represents the compound represented by formula (I) or a pharmaceutically acceptable salt thereof.

[0156] In the present disclosure, “one end is connected to the 5th nucleotide at the 5' end of the sense chain, and the other end is connected to the 7th nucleotide at the 5' end of the sense chain” is intended to indicate the position of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof described in the present disclosure. For example, in 5'NNNNN5N6N7NNNNNNNNNNNN3', the b end in formula (I-1) and (I-2) is connected to N5, the c end is connected to N7, and B in formula (I-1) and (I-2) represents the base of N6; for example, in 5'N1N2NNNNNNNNNNNNNNNN3', the connecting end in formula (I-3) and (I-4) is connected to N6; Connecting N2, in formula (I-3) and (I-4), B represents the base of N1; for example, in 5'NNNNNNNNNNNNNNNNNNNN 2’ N 1’ 3', the connecting end in formula (I-5) and (I-6) Connect N 2’ In formula (I-5) and (I-6), B represents N 1’ bases.

[0157] "Optionally" or "optionally" means that the subsequently described event or circumstance may but need not occur, and the description includes instances where the event or circumstance occurs or does not occur. For example, "C 1-6 The term "alkyl" means that halogen or cyano may but need not be present, and the description includes both the case where the alkyl is substituted by halogen or cyano and the case where the alkyl is not substituted by halogen and cyano.

[0158] The term "lipophilic monomer" or "lipophilic moiety" refers broadly to any 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 log K ow , where K ow is the ratio of the concentration of a chemical substance in the octanol phase to its concentration in the water phase at equilibrium in a two-phase system. In principle, logK ow When it exceeds 0, the chemical is lipophilic. Typically, the logK of the lipophilic moiety is ow More than 1, more than 1.5, more than 2, more than 3, more than 4, more than 5, or more than 10, for example, the log K of 6-aminohexanol ow The logK of cholesteryl N-(hexan-6-ol) carbamate is about 0.7. ow It is 10.7.

[0159] The lipophilicity of a molecule can be altered relative to the functional groups it carries. For example, the addition of a hydroxyl or amine group to the terminus of a lipophilic moiety can increase or decrease the partition coefficient (e.g., log K) of the lipophilic moiety. ow) value. For example, the lipophilic portion can be aliphatic, cyclic such as alicyclic, or polycyclic such as polyalicyclic compounds, such as steroids (e.g., sterols) or straight or branched aliphatic hydrocarbons. The lipophilic portion can generally comprise a hydrocarbon chain, which can be cyclic or acyclic. The hydrocarbon chain can contain various substituents and / or one or more heteroatoms, such as oxygen or sulfur atoms. Such lipophilic aliphatic portions include, but are not limited to, saturated or unsaturated C4-C 30 Hydrocarbons (such as 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 tetraterpenes) and other polyalicyclic hydrocarbons; for example, the lipophilic portion may be optionally substituted C 10-30 For example, the lipophilic portion can be an optionally substituted C 14-24 of a straight chain alkyl group.

[0160] The term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0161] "Pharmaceutically acceptable acid addition salts" refer to salts formed with inorganic or organic acids that retain the biological effectiveness of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobromides, sulfates, nitrates, and phosphates; organic acid salts include, but are not limited to, formates, acetates, 2,2-dichloroacetates, trifluoroacetates, propionates, caproates, octanoates, decanoates, undecylenates, glycolates, gluconates, lactates, sebacates, adipates, glutarates, malonates, oxalates, maleates, succinates, fumarates, tartrates, citrates, palmitates, stearates, oleates, cinnamates, laurates, malates, glutamate, pyroglutamate, aspartate, benzoates, methanesulfonates, benzenesulfonates, p-toluenesulfonates, alginate, ascorbate, salicylates, 4-aminosalicylates, and naphthalene disulfonates. These salts can be prepared by methods known in the art.

[0162] "Pharmaceutically acceptable base addition salts" refer to salts formed with inorganic or organic bases that retain the biological effectiveness of the free acid without other side effects. 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. Preferred inorganic salts are ammonium salts, sodium salts, potassium salts, calcium salts, and magnesium salts, with sodium salts being preferred. Salts derived from organic bases include, but are not limited to, following salts: primary amines, secondary amines and tertiary amines, substituted amines, including natural substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine. These salts can be prepared by methods known in the art.

[0163] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 30 carbon atoms, and in some embodiments is selected from an alkyl group containing 10 to 30 carbon atoms. In some embodiments, it is selected from an alkyl group containing 10 to 20 carbon atoms, for example, an alkyl group containing 14, 15, 16, 17, 18, 19 or 20 carbon atoms. Non-limiting examples include tetradecane, hexadecane, octadecane, eicosane, etc. In some embodiments, the alkyl group is selected from the group consisting of alkyl groups having 1 to 6 carbon atoms, non-limiting examples of which include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and the like. The alkyl group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available point of attachment. The substituent, in some embodiments, is selected from one or more of the following groups, which are independently selected from alkyl, alkoxy, halogen, hydroxy, cyano, cycloalkyl, heterocycloalkyl, and the alkyl group is optionally substituted with halogen. For example, the alkyl group may be a straight chain group containing 1 to 30 carbon atoms, including but not limited to C 10 、C 11 、C 12 、C 13 、C 14 、C15 、C 16 、C 17 、C 18 、C 19 、C 20 、C 21 、C 22 、C 23 、C 24 、C 25 、C 26 、C 27 、C 28 、C 29 、C 30 of a straight chain alkyl group.

[0164] The term "alkoxy" refers to -O-(alkyl), wherein alkyl is as defined above. Non-limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy. Alkoxy may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following 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, wherein the alkyl, alkoxy, cycloalkyl or heterocycloalkyl is optionally substituted by halogen, hydroxy, nitro, cyano or amino.

[0165] Similarly, "cycloalkoxy" and "heterocycloalkoxy" are the same as the above-mentioned "alkoxy".

[0166] The term "cycloalkyl" or "carbocycle" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent, the cycloalkyl ring containing 3 to 20 carbon atoms, in some embodiments selected from 3 to 7 carbon atoms. Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, and the like; polycyclic cycloalkyls include spirocyclic, fused, and bridged cycloalkyls. Cycloalkyls may be substituted or unsubstituted, and when substituted, the substituents may be substituted at any available point of attachment, in some embodiments selected from one or more of the following groups, independently selected from halogen, deuterium, hydroxy, oxo, nitro, cyano, C 1-6 Alkyl, C 1-6 Alkoxy, C 3-6 Cycloalkoxy, 3 to 6 membered heterocycloalkoxy, the C 1-6 Alkyl, C 1-6 Alkoxy, C 3-6 The cycloalkoxy and 3- to 6-membered heterocycloalkoxy groups are optionally substituted by one or more groups selected from halogen, deuterium, hydroxy, oxo, nitro, and cyano.

[0167] The cycloalkyl ring may be fused to an aryl or heteroaryl ring, wherein the ring attached to the parent structure is a cycloalkyl, non-limiting examples of which include indanyl, tetrahydronaphthyl, benzocycloheptanyl, etc. The cycloalkyl may be optionally substituted or unsubstituted, and when substituted, the substituents are in some embodiments selected from one or more of the following groups, which are independently selected from halogen, deuterium, hydroxy, oxo, nitro, cyano, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyloxy, C 2-6 Alkynyloxy, C 3-6 Cycloalkoxy, 3 to 6 membered heterocycloalkoxy, C 3-8 Cycloalkenyloxy, 5 to 6 membered aryl or heteroaryl, the C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyloxy, C 2-6 Alkynyloxy, C 3-6 Cycloalkoxy, 3 to 6 membered heterocycloalkoxy, C 3-8 The cycloalkenyloxy, 5- to 6-membered aryl or heteroaryl is optionally substituted by one or more groups selected from halogen, deuterium, hydroxy, oxo, nitro, cyano.

[0168] The term "heterocycloalkyl" or "heterocycle" or "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which is selected from nitrogen, oxygen or S(O) m (wherein m is an integer from 0 to 2) heteroatoms, but excluding the ring portion of -OO-, -OS- or -SS-, the remaining ring atoms are carbon. In some embodiments, it is selected from 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; in some embodiments, it is selected from 3 to 7 ring atoms. Non-limiting examples of monocyclic heterocycloalkyl groups include pyrrolidinyl, imidazolidinyl, tetrahydrofuranyl, tetrahydrothienyl, dihydroimidazolyl, dihydrofuranyl, dihydropyrazolyl, dihydropyrrolyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc. Polycyclic heterocycloalkyl groups include spirocyclic, fused ring and bridged heterocycloalkyl groups. Non-limiting examples of "heterocycloalkyl" include:

[0169] wait.

[0170] The heterocyclyl ring may be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring attached to the parent structure is a heterocyclyl, non-limiting examples of which include:

[0171] wait.

[0172] The heterocyclyl group may be optionally substituted or unsubstituted. When substituted, the substituents are preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl or carboxylate.

[0173] The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., rings that share adjacent pairs of carbon atoms) group having a conjugated π electron system, preferably 6- to 10-membered, such as phenyl and naphthyl. The aryl ring may be fused to a heteroaryl, heterocyclyl, or cycloalkyl ring, wherein the ring attached to the parent structure is the aryl ring. "Aromatic ring" refers to the ring system in an aryl group. Non-limiting examples of aryl groups include:

[0174] The aryl group may be substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylate, preferably phenyl.

[0175] The term "fused aryl" may be an unsaturated aromatic fused ring structure containing 8-14 ring atoms formed by two or more cyclic structures sharing two adjacent atoms, preferably 8-12 ring atoms. For example, it includes all unsaturated fused aryl rings, such as naphthalene, phenanthrene, etc., and also includes partially saturated fused aryl rings, such as benzo 3-8 membered saturated monocyclic cycloalkyl and benzo 3-8 membered partially saturated monocyclic cycloalkyl. "Fused aromatic ring" refers to the ring system in the fused aryl ring. Specific examples of fused aryl rings include 2,3-dihydro-1H-indenyl, 1H-indenyl, 1,2,3,4-tetrahydronaphthyl, 1,4-dihydronaphthyl, etc.

[0176] The term "heteroaryl" refers to a heteroaromatic system containing 1 to 4 heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen. The heteroaryl group is preferably 5 to 12 members, such as imidazolyl, furyl, thienyl, thiazolyl, pyrazolyl, oxazolyl, pyrrolyl, tetrazolyl, pyridinyl, pyrimidinyl, thiadiazole, pyrazinyl, etc., preferably imidazolyl, pyrazolyl, pyrimidinyl or thiazolyl; more preferably pyrazolyl or thiazolyl. The heteroaryl ring can be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring connected to the parent structure is the heteroaryl ring. "Heteroaromatic ring" refers to the ring system in a heteroaryl group. Non-limiting examples of heteroaryl groups include:

[0177] Heteroaryl groups may be optionally substituted or unsubstituted. When substituted, the substituents are, in some embodiments, selected from one or more of the following groups, which are independently selected from halogen, deuterium, hydroxy, oxo, nitro, cyano, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyloxy, C 2-6 Alkynyloxy, C 3-6 Cycloalkoxy, 3 to 6 membered heterocycloalkoxy, C 3-8 Cycloalkenyloxy, 5 to 6 membered aryl or heteroaryl, the C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyloxy, C 2-6 Alkynyloxy, C 3-6 Cycloalkoxy, 3 to 6 membered heterocycloalkoxy, C 3-8 The cycloalkenyloxy, 5- to 6-membered aryl or heteroaryl is optionally substituted by one or more groups selected from halogen, deuterium, hydroxy, oxo, nitro, and cyano.

[0178] The term "hydroxyalkyl" refers to an alkyl group substituted with one or more hydroxy groups, wherein alkyl is as defined above.

[0179] The term "hydroxy" refers to an -OH group.

[0180] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0181] The term "haloalkyl" refers to an alkyl group substituted with a halogen, wherein alkyl is as defined above.

[0182] The term "haloalkoxy" refers to an alkoxy group substituted with a halogen, wherein alkoxy is as defined above.

[0183] The term "cyano" refers to -CN.

[0184] The term "nitro" refers to -NO2.

[0185] The term "oxo" refers to a =0 group, eg, a carbon atom connected to an oxygen atom via a double bond, where a ketone or aldehyde group is formed.

[0186] The term "amino" refers to -NH2.

[0187] The term "carboxy" refers to -C(O)OH.

[0188] The term "substituted" means that any one or more hydrogen atoms on a designated atom (typically a carbon, oxygen, and nitrogen atom) are replaced by any group as defined herein, provided that the normal valence of the designated atom is not exceeded and the substitution produces a stable compound. Non-limiting examples of substituents include C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, cyano, hydroxyl, oxo, carboxyl, cycloalkyl, cycloalkenyl, heterocyclyl, heteroaryl, aryl, ketone, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, or halogen (e.g., F, Cl, Br, I). When a substituent is ketone or oxo (i.e., =O), then two (2) hydrogen atoms on the atom are replaced.

[0189] "Substituted by one or more..." means that the compound may be substituted by a single or multiple substituents. When substituted by multiple substituents, the substituents may be multiple identical substituents or a combination of one or more different substituents.

[0190] In the chemical structural formulas disclosed herein, the wavy line Indicates the attachment site.

[0191] The term "linked" when referring to a connection between two molecules means that the two molecules are connected by a covalent bond or the two molecules are associated via a non-covalent bond (eg, hydrogen bond or ionic bond), including direct connection and indirect connection.

[0192] The term "directly linked" refers to a first compound or group being linked to a second compound or group without any intervening atoms or groups of atoms.

[0193] The term "indirectly linked" means that a first compound or group is linked to a second compound or group through an intermediate group, compound or molecule (eg, a linking group).

[0194] As used herein, in the context of RNA-mediated gene silencing, the sense strand of siRNA (also referred to as positive strand, SS or SS strand) refers to a strand comprising a sequence identical or substantially identical to the target mRNA sequence; the antisense strand of siRNA (also referred to as AS or AS strand) refers to a strand having a sequence complementary to the target mRNA sequence.

[0195] The term "base" encompasses any known DNA and RNA base, base analogues such as purines or pyrimidines, and also includes the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogues.

[0196] As used herein, "chemical modification" or "modification" includes all changes in a nucleotide by chemical means, such as the addition or removal of a chemical moiety, or the substitution of one chemical moiety for another.

[0197] As used herein, the term "fluoro-modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2'-position of the ribose group of a nucleotide is replaced by fluorine. "Non-fluoro-modified nucleotide" refers to a nucleotide or nucleotide analog in which the hydroxyl group at the 2'-position of the ribose group of a nucleotide is replaced by a non-fluorine group. "Nucleotide analog" refers to a group that can replace a nucleotide in a nucleic acid but has a structure different from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide. Examples include isonucleotides, bridged nucleic acids (BNAs), and acyclic nucleotides. The methoxy-modified nucleotide refers to a nucleotide in which the 2'-hydroxyl group of the ribose group is replaced by a methoxy group. Isonucleotides refer to compounds in which the position of the base on the ribose ring of a nucleotide is altered. In some embodiments, an isonucleotide can be a compound in which the base moves from the 1'-position to the 2'-position or 3'-position of the ribose ring. BNAs refer to constrained or inaccessible nucleotides. BNAs can contain a five-, six-, or seven-membered ring with a "fixed" C3'-endo sugar condensed bridge structure. The bridge is typically incorporated at the 2' or 4' position of the ribose to provide a 2',4'-BNA nucleotide. In some embodiments, BNAs can be LNA, ENA, cET BNA, etc. Acyclic nucleotides are a type of nucleotide formed by opening the sugar ring of the nucleotide. In some embodiments, acyclic nucleotides can be unlocked nucleic acids (UNA) or glycerol nucleic acids (GNA).

[0198] As used herein, the terms "complementary" and "reverse complementary" are used interchangeably and have the meanings known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one chain are paired with bases on the other chain in a complementary manner. In DNA, the purine base adenine (A) is always paired with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (C) is always paired with the pyrimidine base cytosine (G). Each base pair consists of a purine and a pyrimidine. When adenine on one chain is always paired with thymine (or uracil) on the other chain, and guanine is always paired with cytosine, the two chains are considered to be complementary to each other, and the sequence of the chain can be inferred from the sequence of its complementary chain. Accordingly, "mismatch" means in the art that the bases at corresponding positions in a double-stranded nucleic acid are not paired in a complementary manner.

[0199] Unless otherwise specified, in the context of this disclosure, capital letters C, G, U, A, and T represent the base composition of a nucleotide; a lowercase letter d indicates that the nucleotide adjacent to the right of the letter d is a deoxyribonucleotide; a lowercase letter m indicates that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; a lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a fluorine-modified nucleotide; a lowercase letter s indicates that the two nucleotides adjacent to the left and right of the letter s are connected by a thiophosphate group; and a capital letter VP indicates that the two nucleotides adjacent to the left and right of the letter VP are connected by a vinyl phosphate bond. If VP is at the 5' end of the sequence, it means that the nucleotide adjacent to the right of the letter VP is connected by a vinyl phosphate bond.

[0200] A "pharmaceutical composition" comprises an oligonucleotide or double-stranded RNAi inhibitor molecule disclosed herein and a pharmaceutically acceptable excipient and / or adjuvant. The excipient may be one or more of various formulations or compounds conventionally used in the art. For example, the pharmaceutically acceptable excipient may include at least one of a pH buffer, a protective agent, and an osmotic pressure regulator.

[0201] As used herein, the term "inhibit" may be used interchangeably with "reduce," "silence," "downregulate," "repress," or other similar terms, and includes any level of inhibition.

[0202] The terms "blunt end" or "blunt end" are used interchangeably to refer to a given end of an siRNA having no unpaired nucleotides or nucleotide analogs, i.e., no nucleotide overhangs. In most cases, an siRNA having both ends blunt-ended will be double-stranded throughout its entire length.

[0203] The terms "about" and "approximately" refer to values ​​that are within an acceptable error range for a specific value as determined by one of ordinary skill in the art, which depends in part on how it is measured or determined (i.e., the limits of the measurement system). For example, "about" can mean a standard deviation within or exceeding 1. Alternatively, "about" or "substantially comprising" can 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 case in which a number or numerical range is preceded by the term "about" also includes embodiments of the given number. Unless otherwise stated, when a specific value appears in the application and claims, the meaning of "about" or "substantially comprising" should be assumed to be within an acceptable error range for that specific value.

[0204] "Effective amount" or "effective dose" refers to the amount of a drug, compound, or pharmaceutical composition necessary to achieve any one or more beneficial or desired therapeutic results. For prophylactic uses, beneficial or desired results include eliminating or reducing the risk, lessening the severity, or delaying the onset of a condition, including the biochemical, histological, and / or behavioral symptoms of the condition, its complications, and intermediate pathological phenotypes that present during the progression of the condition. For therapeutic applications, beneficial or desired results include clinical results, such as reducing the incidence of various conditions associated with the disclosed target genes, target mRNAs, or target proteins, or ameliorating one or more symptoms of the condition, reducing the dose of another agent required to treat the condition, enhancing the efficacy of another agent, and / or delaying the progression of a condition associated with the disclosed target genes, target mRNAs, or target proteins in a patient.

[0205] In some embodiments, an effective amount or effective dose of siRNA 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.

[0206] As used herein, "subject," "patient," "subject," or "individual" are used interchangeably and include humans or non-human animals, such as mammals, eg, humans or monkeys.

[0207] The siRNA provided herein can be obtained by conventional preparation methods in the art (e.g., solid phase synthesis and liquid phase synthesis methods). Among them, solid phase synthesis already has commercial customization services. Modified nucleotide groups can be introduced into the siRNA described in the present disclosure by using nucleoside monomers with corresponding modifications. Methods for preparing nucleoside monomers with corresponding modifications and methods for introducing modified nucleotide groups into siRNA are also well known to those skilled in the art.

[0208] Some abbreviations used in the present disclosure are defined as follows: DCE: dichloroethane; MeOH: methanol; PE: petroleum ether; EtOAc: ethyl acetate; THF: tetrahydrofuran; DMF: dimethylformamide; TFA: trifluoroacetic acid; LiAlH4: lithium tetrahydrogen aluminum; LiAlD4: ​​lithium deuterated aluminum hydride; TsCl: p-toluenesulfonyl chloride; DMAP: 4-dimethylaminopyridine; MeCN: acetonitrile; NMI: N-methylimidazole; TBDPSCl: tert-butyldiphenylsilyl chloride; HoBt: 1-hydroxybenzotriazole; EDCI: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; DIEA: N,N-diisopropylethylamine; DMTrCl: 4,4'-bis(methoxytrityl)chloride; DMTr: dimethoxytrityl protecting group. BRIEF DESCRIPTION OF THE DRAWINGS

[0209] Figure 1 shows the mRNA expression levels of cells in the drug-treated groups of TJR100072, TJR100073, TJR100344 and TJR100345 at different concentrations. In the figure, *** indicates p<0.001 and ** indicates p<0.01.

[0210] Figure 2 shows the expression of MUC5B mRNA in mice of TJR100072 and TJR100073. Data are presented as mean ± SEM.

[0211] FIG3 shows the expression levels of MUC5B mRNA in mice of TJR100073, TJR100344, and TJR100345. The data are presented as mean ± SEM. *** indicates p < 0.001.

[0212] Figure 4 shows the expression of MAPT mRNA in mice stained with TJR101646 and TJR101647. Data are presented as mean ± SEM. **** indicates p < 0.0001 compared to the blank vector, and *** indicates p < 0.001 compared to the blank vector.

[0213] Figure 5 shows the expression of MAPT mRNA in mice induced by TJR101740. Data are presented as mean ± SEM. **** indicates p < 0.0001 compared to the blank vector, and *** indicates p < 0.001 compared to the blank vector.

[0214] Figure 6 shows the mouse APP mRNA expression levels of TJR102160, TJR102161, and TJR102162. The data are expressed as mean ± SEM. In the figure, **** indicates p < 0.0001 compared with the blank vector, ** indicates p < 0.01 compared with the blank vector, and #### indicates p < 0.0001 compared with TJR102160. DETAILED DESCRIPTION

[0215] The present disclosure is further described below with reference to the following examples, but these examples are not intended to limit the scope of this disclosure. Experimental methods in the examples disclosed herein, where specific conditions are not specified, were generally performed under conventional conditions or as recommended by the raw material or commercial manufacturer. Reagents whose sources are not specified can be obtained from any molecular biology reagent supplier of a quality / purity suitable for molecular biology applications.

[0216] Example

[0217] Unless otherwise specified, the reagents used in the following examples are commercially available products.

[0218] Example 1. Synthesis of lipid-delivered compounds

[0219] Example 1.1: Synthesis of Compound NA0108 (also referred to as "Uhd")

[0220] Compound 1-2:

[0221] Under a nitrogen atmosphere, compound 1-1 (7.00 g, 32.19 mmol) was dissolved in DMF (60 mL), and TDBPSCl (9.70 g, 35.41 mmol) and imidazole (2.40 g, 35.41 mmol) were added, respectively. The mixture was stirred at room temperature for 2 hours. The reaction solution was extracted twice with petroleum ether (60 mL), and the combined organic phases were washed 3-4 times with saturated brine and dried over Na2SO4. The organic phase was concentrated under reduced pressure to obtain compound 1-2 (13.42 g, 96.7% yield), which was used directly in the next reaction without purification.

[0222] Compounds 1-4:

[0223] Compound 1-3 (2.50 g, 11.0 mmol) was added to a mixed solution of compound 1-2 (13.42 g, 27.9 mmol) in DMF (11.8 mL) and diethylene glycol dimethyl ether (7.9 mL). Under a nitrogen atmosphere and in an ice bath, boron trifluoride tetrahydrofuran complex (3.0 g) was slowly added dropwise to the mixed solution. The reaction solution was then heated to 140°C for 16 hours. The reaction solution was cooled to room temperature, quenched by pouring into water (100 mL), and extracted with ethyl acetate (60 mL x 3). The combined organic phases were washed with saturated brine (100 mL), dried over Na2SO4, filtered, and concentrated. The resulting residue was purified by alkaline normal-phase silica gel column chromatography (0.1% Et3N in DCM:MeOH = 10:1, product peak at 5%) to yield compound 1-4 (1.32 g, 25.4% yield).

[0224] LCMS: t R =2.00min, MS(ESI)m / z=467.4[MH] - .

[0225] Compounds 1-5:

[0226] Under a nitrogen atmosphere, compound 1-4 (1.02 g, 2.2 mmol) was dissolved in pyridine (8 mL), and 4A molecular sieves and DMTrCl (1.10 g, 3.2 mmol) were added at 0°C. The mixture was then heated to room temperature and reacted for 16 hours. The reaction solution was quenched by adding water (60 mL) and extracted three times with ethyl acetate (40 mL). The combined organic phases were washed three times with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The resulting residue was purified by alkaline normal silica gel column chromatography (0.1% Et3N in PE:EtOAc = 1:1, product peak at 37%) to obtain compound 1-5 (1.02 g, yield 62.6%).

[0227] LCMS: t R =2.37min, MS(ESI)m / z=769.7[MH] - .

[0228] 1 H NMR(400MHz,DMSO)δppm 8.00(d,J=8.0Hz,1H),7.38(d,J=7.2Hz,2H),7.37-7.18(m,7H),6.84(d,J=8.8Hz,4H),5.86(s,1H),5.17(s,1H),4.46-4.41(m,1H),3. 99-3.93(m,1H),3.80-3.77(m,6H),3.76-3.63(m,2H),3.45(d,J=2.4Hz,2H),1.62-1.54(m,2H),1.35-1.26(m,26H),0.91-0.87(m,3H).

[0229] Compound NA0108:

[0230] At 0°C under a nitrogen atmosphere, 3A molecular sieves were added to a solution of compound 1-5 (630 mg, 0.8 mmol) in dry acetonitrile (6 mL). After stirring for 5 minutes, bis(diisopropylamino)(2-cyanoethoxy)phosphine (540 mg, 1.79 mmol) and a pre-prepared solution of 1H-tetrazole (0.45 M) and N-methylimidazole (0.18 M) in dry acetonitrile (1.8 mL) were added. The mixture was then allowed to react at room temperature for 3 hours. The reaction solution was filtered, and 5% aqueous sodium bicarbonate solution (20 mL) was added. The mixture was extracted twice with dichloromethane (40 mL). The combined organic phases were dried over sodium sulfate, filtered, and concentrated. The resulting residue was purified by C18 reverse-phase column chromatography (H2O / MeCN, elution from 0% to 100%) and lyophilized to provide compound NA0108 (also known as "Uhd") (475 mg, 59.9% yield).

[0231] LCMS: t R =2.67min, MS(ESI)m / z=969.4[MH] - .

[0232] 1 H NMR(400MHz,DMSO-d6)δppm 11.39(s,1H),7.83-7.77(m,1H),7.42-7.24(m,9H),6.94-6.89(m,4H),5.83-5.81 (m,1H),5.32-5.25(m,1H),4.44-4.32(m,1H),4.15-4.06(m,2H),3.76(s,7.3H),3 .65-3.51(m,4.7H),2.79(t,J=6.4Hz,0.7H),2.65-2.58(m,1.3H),1.51-1.49(m,2 H), 1.24 (s, 28H), 1.16-1.11 (m, 10H), 1.01 (d, J = 6.8Hz, 2H), 0.88 (d, J = 6.4Hz, 3H).

[0233] 31 P NMR (100MHz, DMSO-d6) δppm 149.14, 148.63.

[0234] Example 1.2: Synthesis of compound NA0124

[0235] Compound 2-2:

[0236] Under a nitrogen atmosphere, 15.7 mL of 2.5 M LiAlH4 in THF was slowly added dropwise to a dry THF solution (60 mL) of compound 2-1 (10.0 g, 39.3 mmol) in an ice bath. The reaction solution was then warmed to room temperature and stirred for 2 hours. Saturated aqueous ammonium chloride was added to quench the mixture under an ice bath and extracted three times with ethyl acetate (100 mL). The combined organic phase was washed with saturated brine (80 mL), dried over Na2SO4, filtered, and concentrated to give compound 2-2 (8.77 g, 92.8% yield), which was used directly in the next reaction.

[0237] LCMS: t R =1.24min, MS(ESI)m / z=239.3[M+H] + .

[0238] Compound 2-3:

[0239] Under a nitrogen atmosphere, compound 2-2 (8.77 g, 36.5 mmol) was dissolved in DMF (50 mL). TBDPSCl (11.03 g, 40.1 mmol) and imidazole (2.73 g, 40.1 mmol) were added in an ice bath and the mixture was allowed to warm to room temperature for 2 hours. The reaction solution was extracted once with petroleum ether (300 mL). The organic phase was washed three times with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by column chromatography (eluted with pure petroleum ether) to obtain compound 2-3 (14.33 g, 82.0% yield).

[0240] Compound 2-4:

[0241] Under a nitrogen atmosphere, TFA (5.95 g, 52.1 mmol) was slowly added to a 1N solution of ZnEt3 (52.1 mmol) in n-hexane in a dry ice ethanol bath. After stirring for 20 minutes, a 20 mL DCM solution of CH2I2 (22.37 g, 83.5 mmol) was added to the reaction solution over 10 minutes. After stirring for another 20 minutes, a DCM solution of compound 2-3 (10.00 g, 20.8 mmol) was slowly added. The temperature was then raised to room temperature and the reaction was stirred for 4 hours. The reaction solution was quenched by adding a small amount of ice water, and the aqueous phase was extracted three times with dichloromethane (80 mL). The combined organic phases were washed three times with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by column chromatography (eluted with n-hexane) to obtain compound 2-4 (7.21 g, 70.0% yield).

[0242] 1 H NMR(400MHz,DMSO)δppm 7.63-7.60(m,4H),7.47-7.41(m,6H),3.66-3.63(m,2H),1.54-1.50(m,2H),1 .36-1.34(m,20H),1.24(s,2H),1.15(s,8H),1.11(m,4H),0.98-0.54(m,4H).

[0243] Compound 2-6:

[0244] Compound 1-3 (1.80 g, 7.9 mmol) was added to a mixed solution of compound 2-4 (9.81 g, 19.9 mmol) in DMF (8.5 mL) and diglyme (5.7 mL). Under a nitrogen atmosphere and in an ice bath, boron trifluoride tetrahydrofuran complex (2.3 g) was slowly added dropwise to the mixed solution. The reaction solution was then heated to 140°C for 16 hours. The reaction solution was cooled to room temperature, quenched by pouring into water (100 mL), and extracted with EtOAc (60 mL x 3). The combined organic phases were washed with saturated brine (100 mL), dried over Na2SO4, filtered, and concentrated. The resulting residue was purified by alkaline normal phase silica gel column chromatography (0.1% Et3N in DCM:MeOH = 10:1, product peak at 5%) to obtain compound 2-6 (1.08 g, 28.2% yield).

[0245] LCMS: t R =2.02min, MS(ESI)m / z=479.3[MH] - .

[0246] Compound 2-7:

[0247] Under a nitrogen atmosphere, compound 2-6 (1.00 g, 2.1 mmol) was dissolved in pyridine (8 mL), and 4A molecular sieves and DMTrCl (1.06 g, 3.1 mmol) were added at 0°C. The mixture was then heated to room temperature and reacted for 4 hours. The reaction solution was quenched by adding water (60 mL) and extracted three times with ethyl acetate (40 mL). The combined organic phases were washed three times with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by alkaline normal silica gel column chromatography (0.1% Et3N in PE:EtOAc = 1:1, product peak at 37%) to obtain compound 2-7 (0.86 g, yield 52.5%).

[0248] LCMS: t R =2.36min, MS(ESI)m / z=781.5[MH] - .

[0249] 1H NMR(400MHz,DMSO-d6)δppm 11.36(s,1H),7.72(d,J=8.4Hz,1H),7.38-7.32(m,4H),7.24(d,J=8.8Hz,5H),6.90(d,J=8.8Hz, 4H),5.80(d,J=4.0Hz,1H),5.28(d,J=8.4Hz,1H),5.10(d,J=6.4Hz,1H),4.16(q,J=5.6Hz,1H),3. 96-3.89(m,2H),3.74(s,6H),3.60-3.55(m,2H),3.29-3.23(m,2H),1.50-1.32(m,2H),1.32-1.2 3(m,20H),1.13(s,2H),0.86-0.63(m,3H),0.86(s,1.8H),0.62(s,1.2H),-0.33(q,J=4.4Hz,1H).

[0250] Compound NA0124:

[0251] At 0°C under a nitrogen atmosphere, 3A molecular sieves were added to a solution of compound 2-7 (850 mg, 1.0 mmol) in dry acetonitrile (6 mL). After stirring for 5 minutes, bis(diisopropylamino)(2-cyanoethoxy)phosphine (721 mg, 2.4 mmol) and a pre-prepared solution of 1H-tetrazole (0.45 M) and N-methylimidazole (0.18 M) in dry acetonitrile (2.4 mL) were added. The mixture was then warmed to room temperature and reacted for 3 hours. The reaction solution was filtered, and a 5% aqueous sodium bicarbonate solution (20 mL) was added. The mixture was extracted twice with dichloromethane (40 mL). The organic phases were combined, dried over sodium sulfate, filtered, and concentrated. The resulting residue was purified by C18 reverse-phase column chromatography (H2O / MeCN, elution from 0% to 100%) and lyophilized to afford compound NA0124 (841 mg, 79.4% yield).

[0252] LCMS: t R =2.67min, MS(ESI)m / z=981.6[MH] - .

[0253] 1H NMR(400MHz,DMSO-d6)δppm 11.38(s,1H),7.82-7.77(m,1H),7.41(t,J=7.6Hz,2H),7.36-7.24(m,7H),6.94-6.89(m,4H),5.82(t,J=3.2H z,1H),5.31-5.25(m,1H),4.44-4.34(m,1H),4.15-4.06(m,2H),3.83-3.70(m,8H),3.65-3.51(m,5H),3.39-3 .30(m,2H),2.79(t,J=6.4Hz,0.8H),2.64-2.61(m,1.2H),1.54-1.49(m,2H),1.34-1.26(m,20H),1.16-1.11( m,11H),1.00(d,J=6.4Hz,2H),0.87(t,J=7.2Hz,3H),0.66(br,2H),0.57-0.52(m,1H),-0.32(q,J=4.8Hz,1H).

[0254] 31 P NMR (100MHz, DMSO-d6) δppm 149.14, 148.64.

[0255] Example 1.3: Synthesis of Compound NA130

[0256] Compound 3-2:

[0257] Under a nitrogen atmosphere, compound 3-1 (9.00 g, 61.6 mmol) was dissolved in DMF (60 mL). TBDPSCl (18.61 g, 67.7 mmol) and imidazole (4.61 g, 67.7 mmol) were added under an ice bath, and the mixture was allowed to warm to room temperature for 16 hours. The reaction solution was extracted once with petroleum ether (300 mL). The organic phase was washed three times with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The resulting residue was purified by column chromatography (PE:EtOAc = 4:1, product peak at 17%) to obtain compound 3-2 (19.31 g, 81.5% yield).

[0258] LCMS: t R =2.04min, MS(ESI)m / z=385.4[M+H] + .

[0259] Compound 3-3:

[0260] Under a nitrogen atmosphere, LiAlD4 (0.59 g, 14.0 mmol) was added to 10 mL of dry THF in an ice bath. After the solution was stirred for 10 minutes, a THF solution (10 mL) of compound 3-2 (2.61 g, 7.0 mmol) was slowly injected into the LiAlD4 solution. The reaction solution was then warmed to room temperature and stirred for 3 hours. Saturated aqueous ammonium chloride was added to quench the reaction in an ice bath and extracted three times with ethyl acetate (40 mL). The combined organic solution was washed with saturated brine (40 mL), dried over Na2SO4, filtered, and concentrated to give compound 3-3 (2.31 g, 91.8% yield), which was used directly in the next reaction.

[0261] LCMS: t R =2.10min, MS(ESI)m / z=381.3[M+H] + .

[0262] Compound 3-4:

[0263] Under a nitrogen atmosphere, compound 3-3 (13.00 g, 36.2 mmol) was dissolved in dichloromethane (80 mL). Triethylamine (11.8 mL, 79.7 mmol), p-toluenesulfonyl chloride (10.37 g, 54.4), and DMAP (2.21 g, 18.1 mmol) were added sequentially at 0°C. The reaction solution was then warmed to room temperature and stirred overnight. The reaction solution was washed twice with water (60 mL) and saturated brine (100 mL), dried over Na2SO4, filtered, and concentrated. The resulting residue was purified by column chromatography (PE:EtOAc = 20:1, product peak at 5%) to obtain compound 3-4 (14.5 g, 78.00% yield).

[0264] Compound 3-5:

[0265] Under a nitrogen atmosphere, compound 3-4 (14.49 g, 28.2 mmol) was dissolved in tetrahydrofuran (80 mL). Copper chloride (0.19 g, 1.41 mmol), phenyl-1-propyne (0.68 mL, 5.6 mmol), and a 1N solution of decylmagnesium bromide in tetrahydrofuran (84.7 mL, 84.7 mmol) were added sequentially at 0°C. The reaction solution was then warmed to room temperature and stirred for 3 hours. The mixture was quenched by the addition of ice water (60 mL) under an ice bath and extracted three times with ethyl acetate (60 mL). The combined organic solution was washed with saturated brine (40 mL), dried over Na2SO4, filtered, and concentrated. The resulting residue was purified by column chromatography (PE:EtOAc = 20:1, product peak at 0%) to afford compound 3-5 (10.1 g, 74.39% yield).

[0266] 1H NMR(400MHz,DMSO-d6)δppm 7.60-7.58(4H,m),7.33-7.27(m,6H),3.57(t,J=6.4Hz,2H),1.49-1.46(m,2H),1.26-1.16(m,26H),0.97(s,10H),0.82-0.78(m,3H).

[0267] Compound 3-7:

[0268] Compound 1-3 (2.20 g, 9.7 mmol) was added to a mixed solution of compound 3-5 (10.33 g, 21.4 mmol) in DMF (8.5 mL) and diglyme (5.7 mL). Under a nitrogen atmosphere and in an ice bath, boron trifluoride tetrahydrofuran complex (2.30 g) was slowly added dropwise to the mixed solution. The reaction solution was then heated to 140°C for 16 hours. The reaction solution was cooled to room temperature, quenched by pouring into water (60 mL), and extracted with EtOAc (50 mL x 3). The combined organic phases were washed with saturated brine (80 mL), dried over Na2SO4, filtered, and concentrated. The resulting residue was purified by alkaline normal-phase silica gel column chromatography (0.1% Et3N in DCM:MeOH = 10:1, product peak at 6%) to obtain compound 3-7 (0.97 g, 21.19% yield).

[0269] LCMS: t R =2.05&2.20min, MS(ESI)m / z=469.5[MH] - .

[0270] Compound 3-8:

[0271] Under a nitrogen atmosphere, compound 3-7 (0.76 g, 1.6 mmol) was dissolved in pyridine (7 mL), and 4A molecular sieves and DMTrCl (0.82 g, 2.4 mmol) were added at 0 ° C. The mixture was then heated to room temperature and reacted for 4 hours. The reaction solution was quenched by adding water (50 mL) and extracted three times with ethyl acetate (50 mL). The combined organic phases were washed three times with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by alkaline normal silica gel column chromatography (0.1% Et3N in PE:EtOAc = 1:1, product peak at 37%) to obtain compound 3-8 (0.58 g, yield 46.54%).

[0272] LCMS: t R =2.26&2.50min, MS(ESI)m / z=771.7[MH] - .

[0273] 1 H NMR(400MHz,DMSO-d6)δppm 11.36(s,1H)7.72(d,J=8.4Hz,1.3H),7.38(d,J=7.6H,2H),7.33(t,J=7.6Hz,2H),7.28-7.2 4(m,4H),6.90(d,J=8.8Hz,4H),5.80(d,J=4.0Hz,1H),5.29(d,J=8.0Hz,1H),5.11(d,J=6.8H z,1H),4.18(q,J=5.6Hz,1H),3.98-3.95(m,1H),3.90(t,J=4.8Hz,1H),3.79(s,6.7Hz),3.6 1-3.52(m,2.3H),3.30-3.22(m,2H),1.52-1.48(m,2H),1.23(s,27H),0.85(t,J=6.4Hz,3H).

[0274] Compound NA0130:

[0275] At 0°C under a nitrogen atmosphere, 3A molecular sieves were added to a solution of compound 3-8 (580 mg, 0.8 mmol) in dry acetonitrile (5 mL). After stirring for 5 minutes, bis(diisopropylamino)(2-cyanoethoxy)phosphine (500 mg, 1.7 mmol) and a pre-prepared solution of 1H-tetrazole (0.45 M) and N-methylimidazole (0.18 M) in dry acetonitrile (1.7 mL) were added, and the mixture was allowed to react at room temperature for 3 hours. The reaction solution was filtered, and a 5% aqueous sodium bicarbonate solution (20 mL) was added to the reaction solution. The mixture was extracted twice with dichloromethane (40 mL). The organic phases were combined, dried over sodium sulfate, filtered, and concentrated. The resulting residue was purified by C18 reverse-phase column chromatography (H2O / MeCN, elution from 0% to 100%) and lyophilized to obtain compound NA0130 (610 mg, 83.41% yield).

[0276] LCMS: t R =2.46&2.70min,MS(ESI)m / z=971.8[MH] - .

[0277] 1H NMR(400MHz,DMSO-d6)δppm 11.35(s,1H),7.80-7.75(m,1H),7.39(t,J=7.6Hz,2H),7.36-7.23(m,7H),6.93-6.87(m,4H), 5.80(t,J=3.6Hz,1H),5.30-5.24(m,1H),4.43-4.32(m,1H),4.13-4.04(m,2H),3.81-3.63(m, 8H),3.62-3.49(m,5H),3.38-3.34(m,2H),2.77(t,J=6.4Hz,0.8H),2.63-2.61(m,1.3H),1.53 -1.46(m,2H),1.23(s,23H),1.14-1.10(m,10H),0.98(d,J=6.4Hz,2H),0.85(t,J=6.4Hz,3H).

[0278] 31 P NMR (100MHz, DMSO-d6) δppm 149.15, 148.64.

[0279] Example 1.4: Synthesis of compound NA0131

[0280] Compound 4-2:

[0281] Under a nitrogen atmosphere, LiAlD4 (1.64 g, 38.99 mmol) was added to 20 mL of dry THF in an ice bath. After the solution was stirred for 10 minutes, a tetrahydrofuran solution (60 mL) of compound 4-1 (10.0 g, 38.9 mmol) was slowly injected into the LiAlD4 solution. The reaction solution was then warmed to room temperature and stirred for 2 hours. Saturated aqueous ammonium chloride was added to quench the reaction in an ice bath and the mixture was extracted three times with ethyl acetate (100 mL). The combined organic solution was washed with saturated brine (80 mL), dried over Na2SO4, filtered, and concentrated to obtain compound 4-2 (7.32 g, 76.8% yield), which was used directly in the next reaction.

[0282] Compound 4-3:

[0283] Under a nitrogen atmosphere, compound 4-2 (7.00 g, 28.6 mmol) was dissolved in DMF (50 mL). TBDPSCl (8.66 g, 31.5 mmol) and imidazole (2.14 g, 31.5 mmol) were added in an ice bath, and the mixture was allowed to warm to room temperature for 2 hours. The reaction solution was extracted once with petroleum ether (300 mL). The organic phase was washed three times with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by column chromatography (eluted with pure petroleum ether) to obtain compound 4-3 (10.10 g, 73.0% yield).

[0284] LCMS: t R =2.41min, MS(ESI)m / z=483.8[M+H] + .

[0285] Compound 4-5:

[0286] Compound 1-3 (1.35 g, 5.9 mmol) was added to a mixed solution of compound 4-3 (8.65 g, 17.9 mmol) in DMF (5 mL) and diglyme (3.3 mL). Under a nitrogen atmosphere and in an ice bath, boron trifluoride tetrahydrofuran complex (1.60 g) was slowly added dropwise to the mixed solution. The reaction mixture was then heated to 140°C for 16 hours. The reaction solution was cooled to room temperature, quenched by pouring into water (60 mL), and extracted with EtOAc (50 mL x 3). The combined organic phases were washed with saturated brine (80 mL), dried over Na2SO4, filtered, and concentrated. The resulting residue was purified by alkaline normal-phase silica gel column chromatography (0.1% Et3N in DCM:MeOH = 10:1, product peak at 5%) to afford compound 4-5 (0.61 g, 21.72% yield).

[0287] LCMS: t R =2.04min, MS(ESI)m / z=469.6[MH] - .

[0288] Compounds 4-6:

[0289] Under a nitrogen atmosphere, compound 4-5 (0.61 g, 1.3 mmol) was dissolved in pyridine (5 mL), and 4A molecular sieves and DMTrCl (0.75 g, 2.2 mmol) were added at 0°C. The mixture was then heated to room temperature and reacted for 4 hours. The reaction solution was quenched by adding water (50 mL) and extracted three times with ethyl acetate (40 mL). The combined organic phases were washed three times with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by alkaline normal silica gel column chromatography (0.1% Et3N in PE:EtOAc = 1:1, product peak at 37%) to obtain compound 4-6 (0.58 g, yield 58.1%).

[0290] LCMS: t R =2.26&2.37min, MS(ESI)m / z=771.7[MH] - .

[0291] 1 H NMR (400 MHz, DMSO-d6) δ ppm 11.36(s,1H)7.72(d,J=8.4Hz,1.3H),7.38-7.31(m,3.7H),7.25(d,J=8.4Hz ,4H),6.90(d,J=8.8Hz,3.5H),5.79(d,J=3.6Hz,1H),5.28(d,J=8.4Hz,1H), 5.10(d,J=6.8Hz,1H),4.16(q,J=5.6Hz,1.5H),3.96-3.88(m,2.7H),3.74(s ,5.7H),3.28-3.21(m,2H),1.48(s,2H),1.22(s,24H),0.84(t,J=6.0Hz,3H).

[0292] Compound NA0131:

[0293] At 0°C under a nitrogen atmosphere, 3A molecular sieves were added to a solution of compound 4-6 (580 mg, 0.8 mmol) in dry acetonitrile (5 mL). After stirring for 5 minutes, bis(diisopropylamino)(2-cyanoethoxy)phosphine (497 mg, 1.7 mmol) and a pre-prepared solution of 1H-tetrazole (0.45 M) and N-methylimidazole (0.18 M) in dry acetonitrile (1.8 mL) were added, and the mixture was allowed to react at room temperature for 3 hours. The reaction solution was filtered, and a 5% aqueous sodium bicarbonate solution (20 mL) was added. The mixture was extracted twice with dichloromethane (40 mL). The organic phases were combined, dried over sodium sulfate, filtered, and concentrated. The resulting residue was purified by C18 reverse-phase column chromatography (H2O / MeCN, elution from 0% to 100%) and lyophilized to obtain compound NA0131 (630 mg, 86.3% yield).

[0294] LCMS: t R =2.54&2.68min,MS(ESI)m / z=971.8[MH] -

[0295] 1 H NMR(400MHz,DMSO-d6)δppm 11.31(s,1H),7.75-769(m,1H),7.32(t,J=7.6Hz,2H),7.27-7.16(m,7H),6.85-6.80(m,4H), 5.73(t,J=3.6Hz,1H),5.22-5.16(m,1H),4.36-4.21(m,1H),4.06-3.97(m,2H),3.84-3.63(m, 7H),3.60-3.42(m,3H),3.30-3.28(m,2H),2.70(t,J=6.4Hz,0.8H),2.60-2.48(m,1.3H),1.41 -1.36(m,2H),1.15(s,26H),1.08-1.02(m,10H),0.91(d,J=6.4Hz,2H),0.77(t,J=6.4Hz,3H).

[0296] 31 P NMR (100MHz, DMSO-d6) δppm 149.13, 148.60.

[0297] Example 1.5: Synthesis of compound NA0133

[0298] Compound 5-1:

[0299] At 0°C, 1% HCl in methanol (60 mL) was added to a solution of D-ribose (30.0 g, 67 mmol) in methanol (200 mL). The reaction mixture was then warmed to room temperature and stirred for 3 days. The reaction mixture was neutralized with solid sodium bicarbonate, resulting in the formation of a solid. The filtered filtrate was dried and concentrated to yield the crude product. The crude product was redissolved in 500 mL of DMF. Under a nitrogen atmosphere and in an ice bath, NaH (36.0 g, 1500 mmol) was slowly added to the solution. The reaction mixture was stirred for 30 minutes before the addition of 4-benzyl chloride (108.7 g, 675 mmol). The reaction mixture was then warmed to room temperature and stirred overnight. The mixture was quenched by the addition of ice water in an ice bath. The reaction mixture was extracted three times with DCM (150 mL). The combined organic phases were washed with saturated brine, dried over Na2SO4, filtered, and concentrated. The residue was purified by silica gel column chromatography (PE:EA=4 / 1, peak at 10%) to give compound 5-1 (54.5 g, yield 67.55%).

[0300] LCMS: t R =2.09&2.32min,MS(ESI)m / z=561.3[M+Na] + .

[0301] Compound 5-2:

[0302] Under a nitrogen atmosphere, 60 ml of a 10% SnCl₄ solution in DCM was slowly added to a 100 ml solution of compound 5-1 (30.00 g, 55.7 mmol) in DCM. The reaction was stirred at room temperature overnight. The reaction was neutralized and quenched with saturated aqueous sodium bicarbonate solution in an ice bath. The reaction solution was extracted three times with DCM (150 ml). The combined organic phases were washed with saturated brine, dried over Na₂SO₄, filtered, and concentrated. The resulting residue was purified by silica gel column chromatography (PE:EA = 5 / 1, peak at 20%) to afford compound 5-2 (16.00 g, 69.41% yield).

[0303] 1 H NMR(400MHz,DMSO-d6)δppm 7.39(d,J=8.4Hz,2H),7.35(s,4H),7.30(d,J=8.4Hz,2H),4.77(d,J=4.4Hz,1H),4.63(d,J=12.8Hz,1H) ,4.50-4.42(m,3H),4.06-4.05(m,2H),3.67(dd,J=6.4,.3.2Hz,1H),3.45(d,J=4.4Hz,2H),3.31(s,3H).

[0304] LCMS: t R=2.13min, MS(ESI)m / z=435.1[M+Na] + .

[0305] Compound 5-3:

[0306] IBX (40.65 g, 145.2 mmol) was added to a solution of compound 5-2 (20.0 g, 48.4 mmol) in acetonitrile (150 ml). The reaction mixture was heated to 83°C and stirred for 5 hours. The reaction mixture was filtered and extracted three times with ethyl acetate (100 ml). The combined organic phases were washed sequentially with aqueous Na2SO3, saturated aqueous NaHCO3, and saturated brine. The organic phase was dried over Na2SO4, filtered, and concentrated to yield crude compound 5-3 (15.11 g).

[0307] Compound 5-4:

[0308] Under an argon atmosphere, 20 ml of nBuLi (2.5 N in THF) was slowly injected into a THF solution (60 ml) of methyltriphenylphosphonium iodide (19.10 g, 53.2 mmol) at -78°C. After stirring the reaction for 1 hour, a THF solution of compound 5-3 (15.1 g) was added to the reaction mixture, and the reaction was stirred overnight. The reaction mixture was quenched with saturated aqueous ammonium chloride at low temperature. After quenching, it was extracted three times with EA (100 ml). The combined organic phases were washed with saturated brine, dried over Na2SO4, filtered, and concentrated. The resulting residue was purified by silica gel column chromatography (PE:EA = 5 / 1, peak at 20%) to obtain compound 5-4 (10.0 g, 50.49% yield).

[0309] LCMS: t R =1.81min,MS(ESI)m / z=431.3[M+Na] + .

[0310] Compound 5-5:

[0311] Under a nitrogen atmosphere, compound 5-4 (26.2 g, 64.2 mmol) was dissolved in a 0.5 M solution of 9-borabicyclo[3,3,1]-nonane in tetrahydrofuran (384 mL, 192.2 mmol) at room temperature. The reaction mixture was heated to 40°C and stirred for 16 hours. A mixture of sodium perborate (tetrahydrate) (102.4 g, 1152.2 mmol) and water (100 mL) was added to the reaction mixture under an ice bath and stirred for 3-4 hours. The reaction mixture was filtered through celite, the filter cake was washed 2-3 times with ethyl acetate, and the filtrate was extracted three times with ethyl acetate (200 mL). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by silica gel column chromatography (PE:EA = 35%:65%) to afford compound 5-5 (25 g, 91% yield).

[0312] LCMS: t R =1.50min, MS(ESI)m / z=449.4[M+Na] + .

[0313] Compound 5-6:

[0314] Compound 5-5 (2.0 g, 4.6 mmol) was dissolved in anhydrous pyridine (20 mL), and benzoyl chloride (1.09 mL, 9.36 mmol) was added. The mixture was stirred at room temperature overnight. The reaction solution was concentrated and redissolved in dichloromethane (20 mL). The mixture was washed sequentially with saturated aqueous sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by reverse phase column chromatography to afford compound 5-6 (2.3 g, 92% yield).

[0315] LCMS: t R =2.07min,MS(ESI)m / z=553.3[M+Na] + .

[0316] 1 H NMR (400MHz, DMSO) δppm 7.95 (d, J = 1.2Hz, 2H), 7.59 (t, J = 0.8Hz, 1H), 7.46 (t, J = 0.8Hz, 2H), 7.31 (d, J = 0.8Hz, 2H), 7.22-7.15 (m, 6H), 5.07 (d, J = 0.8Hz,1H),4.63-4.57(m,3H),4.52-4.41(m,3H),3.95(d,J=0.8Hz,1H),3.47-3.33(m,5H),2.65-2.55(m,1H).

[0317] Compounds 5-7:

[0318] Uracil (2.7 g, 24.1 mmol) and ammonium sulfate (0.16 g, 0.2 mmol) were added to hexamethyldisilazane (16 mL) and stirred at 140°C until the solution became clear. The reaction was then continued for half an hour and concentrated to yield the tetramethylsilane-protected uracil intermediate. Compound 5-6 (1.6 g, 3.0 mmol) in anhydrous acetonitrile (30 mL) was added to the tetramethylsilane-protected uracil intermediate, followed by tin tetrachloride (1.4 mL, 12.0 mmol) and stirred at room temperature for 16 hours. The reaction mixture was quenched with saturated sodium bicarbonate and extracted three times with dichloromethane (20 mL). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by reverse-phase column chromatography to yield compound 5-7 (1.0 g, 54% yield).

[0319] LCMS: t R =1.79min, MS(ESI)m / z=633.4[M+Na] + .

[0320] 1 H NMR(400MHz,DMSO)δppm 7.89(s,1H),7.87(s,1H),7.65-7.52(m,2H),7.47-7.41(m,2H),7.37-7 .28(m,4H),7.26-7.18(m,4H),6.34(d,J=0.8Hz,1H),5.50(d,J=0.8Hz,1 H),4.74-4.69(m,1H),4.65-4.42(m,6H),4.29(d,J=0.4Hz,1H),4.19(d ,J=0.4Hz,1H),3.73-3.68(m,1H),3.56-3.50(m,1H),2.85-2.79(m,1H).

[0321] Compounds 5-8:

[0322] Compound 5-7 (1.7 g, 2.8 mmol) was dissolved in 7 M ammonia methanol solution (20 mL) and stirred overnight at 60° C. The reaction solution was concentrated and the resulting residue was purified by reverse phase column chromatography to obtain compound 5-8 (1.05 g, yield 74%).

[0323] LCMS: t R =1.35min, MS(ESI)m / z=529.4[M+H] + .

[0324] Compounds 5-9:

[0325] Under ice, sodium hydride (0.89 g) was slowly added to a solution of compound 5-8 (4.50 g, 8.9 mmol) in N,N-dimethylformamide (15 mL). After stirring at room temperature for 30 minutes, iodohexadecane (6.30 g, 17.8 mmol) was added and the reaction was allowed to warm to room temperature and stirred for two hours. The reaction solution was quenched with saturated aqueous ammonium chloride and extracted three times with ethyl acetate (30 mL). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by reverse phase column chromatography to obtain compound 5-9 (3.5 g, 54% yield).

[0326] LCMS: t R =3.83min, MS(ESI)m / z=731.7[M+H] + .

[0327] Compounds 5-10:

[0328] To a solution of compound 5-9 (3.5 g, 4.8 mmol) in methanol (100 mL) was added 10% palladium on carbon (0.5 g), and the mixture was stirred at room temperature overnight under a hydrogen atmosphere. The reaction solution was concentrated, and the resulting residue was purified by reverse-phase column chromatography to afford compound 5-10 (2.2 g, 96% yield).

[0329] LCMS: t R =1.45min, MS(ESI)m / z=481.6[MH] + .

[0330] Compound 5-11:

[0331] To a solution of compound 5-10 (2.2 g, 4.6 mol) in pyridine (20 mL) was added 4,4'-bismethoxytrityl chloride (4.7 g, 13.8 mmol) and stirred at room temperature overnight. The reaction was quenched by the addition of methanol (5 mL) at 0°C. The reaction solution was concentrated, redissolved in water (30 mL), and extracted twice with dichloromethane (50 mL). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by silica gel column chromatography to afford compound 5-11 (2.6 g, 72% yield).

[0332] LCMS: t R =3.22min, MS(ESI)m / z=783.9[MH] + .

[0333] 1H NMR(400MHz,DMSO)δppm 12.11(s,1H),8.38(d,J=8.0Hz,1H),8.24–8.17(m,3H),8.18–8.09(m,3H),8.11–8.03(m,7H),8.07–7.98(m,5H),7.93 –7.86(m,2H),7.75–7.68(m,4H),7.70–7.63(m,2H),7.04(s,1H),6.83(d,J=8.4Hz,1H),6.23(dd,J=8.0,2.0Hz,1H),6. 18(d,J=5.2Hz,1H),5.07–4.99(m,1H),4.75(d,J=6.0Hz,1H),4.56(s,9H),4.55(s,9H),4.48(dd,J=10.0,5.2Hz,1H),4 .29–4.20(m,1H),4.11-4.04(m,4H),3.97(dd,J=10.4,4.0Hz,1H),2.21(s,2H),2.11-2.01(m,25H),1.71–1.63(m,4H).

[0334] Compound NA0133:

[0335] At room temperature and under a nitrogen atmosphere, 3A molecular sieves were added to a solution of compound 5-11 (1.6 g, 2.0 mmol) in dry acetonitrile (15 mL). After stirring at room temperature for 5 minutes, bis(diisopropylamino)(2-cyanoethoxy)phosphine (2.4 g, 8.9 mmol) was added, followed by a pre-prepared solution of 1H-tetrazole (0.45 M, 0.045 mmol) and N-methylimidazole (0.18 M, 0.018 mmol) in dry acetonitrile (7 mL). The reaction was allowed to react at room temperature for 3 hours. After filtration, a 5% aqueous sodium bicarbonate solution (30 mL) was added to the reaction solution, and the mixture was extracted with dichloromethane (30 mL), dried over sodium sulfate, filtered, and concentrated. The residue was purified by C18 reverse-phase column chromatography and lyophilized to obtain compound NA0133 (1.6 g, 80% yield).

[0336] LCMS: t R =3.97min, MS(ESI)m / z=984.1[MH] + .

[0337] 1H NMR(400MHz,DMSO)δppm 11.33(s,1H),7.60(t,J=8.0Hz,1H),7.42-7.35(m,2H),7.34-7.19(m,7H),6.92-6.84(m,4H),5.98(dd,J=16. 0,7.6Hz,1H),5.43(dd,J=11.2,8.0Hz,1H),4.55-4.36(m,1H),4.16-4.03(m,1H),3.74(d,J=2.4Hz,6H),3.70- 3.48(m,5H),3.42(dd,J=9.6,6.8Hz,1H),3.29-3.25(m,4H),2.76(t,J=6.0Hz,1H),2.71-2.64(m,1H),2.60-2. 55(m,1H),1.45-1.35(m,2H),1.31-1.16(m,28H),1.15-1.09(m,8H),1.00(d,J=6.8Hz,2H),0.87-0.82(m,3H).

[0338] Example 2: siRNA synthesis with lipid delivery

[0339] siRNA synthesis follows the conventional phosphoramidite solid-phase synthesis method. For the synthesis of the modified nucleotide at the 5'-6 position of the SS chain, the aforementioned phosphoramidite monomer was used to replace the original nucleoside phosphoramidite monomer in the parent sequence. The synthesis process is briefly described as follows: Nucleoside phosphoramidite monomers were ligated one by one on a Dr. Oligo 48 synthesizer (Biolytic) starting with the Universal CPG vector according to the synthesis protocol. With the exception of the nucleoside phosphoramidite monomer at the 5'-6 position of the SS chain described above, other nucleoside phosphoramidite monomers, such as 2'-F RNA, 2'-O-methyl RNA, Chd, and VPUm monomers, were purchased from Shanghai Zhaowei, Suzhou Jima, or Jiangsu Shenji Biotechnology Co., Ltd. 5-Ethylthio-1H-tetrazole (ETT) was used as the activator (0.6 M in acetonitrile), a 0.22 M PADS solution in a 1:1 volume ratio of acetonitrile and collidine (Suzhou Kelema) was used as the sulfurization reagent, and an iodine pyridine / water solution (Kelema) was used as the oxidant.

[0340] After solid-phase synthesis, the oligoribonucleotides were cleaved from the solid support and soaked in a 3:1 solution of 28% ammonia and ethanol at 50°C for 16 hours. The mixture was then centrifuged, and the supernatant was transferred to another centrifuge tube. After evaporation, the supernatant was purified using C18 reverse-phase chromatography with a mobile phase of 0.1 M TEAA and acetonitrile. DMTr was removed using a 3% trifluoroacetic acid solution. The target oligonucleotides were collected, lyophilized, identified as the desired product by LC-MS, and quantified by UV spectroscopy (260 nm).

[0341] The obtained single-stranded oligonucleotides were annealed according to the complementary pairing in an equal molar ratio, and the resulting double-stranded siRNA was dissolved in 1× PBS and adjusted to the required concentration for the experiment.

[0342] Table 1. siRNA sequences targeting the MUC5B gene

[0343] Table 2. Modified siRNA sequences targeting the MUC5B gene

[0344] Table 3. siRNA sequences targeting the APP gene

[0345] Table 4. Modified siRNA sequences targeting the APP gene

[0346] Table 5. siRNA sequences targeting PMP22 gene

[0347] Table 6. Modified siRNA sequences targeting the PMP22 gene

[0348] Table 7. siRNA sequences targeting MAPT gene

[0349] Table 8. Modified siRNA sequences targeting the MAPT gene

[0350] In the modified siRNA sequence disclosed herein, the structure of Uhd' is:

[0351] The structure of Chd' is:

[0352] The structure of NA0124' is:

[0353] The structure of NA0131' is:

[0354] The structure of NA0133' is:

[0355] The structure of VPUms is:

[0356] Example 3: Evaluation of the Anti-MUC5B Activity of the Oligonucleotides of the Present Disclosure in Vitro Using MLE-12 Cells

[0357] The in vitro anti-MUC5B activity of the four siRNA sequences listed in the table was evaluated in MLE-12 cells using three concentration gradients.

[0358] The specific experimental steps for siRNA anti-MUC5B activity in vitro are as follows:

[0359] In MLE-12 cell lines, four siRNA sequences were tested for their ability to inhibit MUC5B mRNA levels using a transfection reagent transfection assay. The experimental materials and instruments are detailed in Tables 9 and 10.

[0360] Table 9. Experimental consumables and reagents

[0361] Table 10. Experimental instruments

[0362] Experimental steps:

[0363] (1) Cell plating

[0364] 1. Experimental Preparation

[0365] 1.1 Preparation of MLE-12 cells: Purchased from Starfish Biotechnology, adherent cells need to be counted after complete digestion. If the cell viability is greater than or equal to 95%, they can be used;

[0366] 1.2 MLE-12 cell culture medium: purchased from Starfish Biotechnology, stored at 4°C, and equilibrated to room temperature before the experiment;

[0367] 1.3 96-well cell culture plates;

[0368] 1.4 PBS and trypsin were stored at 4°C and taken out to equilibrate to room temperature before the experiment.

[0369] 2. Cell Plating

[0370] One day before transfection, MLE-12 cells were seeded in 96-well plates at a seeding density of 1.5×10 4 cells / well, 100 μL culture medium / well.

[0371] 2.1 Place the culture medium in a 37°C water bath and incubate for 20 minutes.

[0372] 2.2 Wash the cells once with PBS, add trypsin, and place in an incubator for 2 minutes. Add culture medium to terminate the digestion, centrifuge, and count the cells. Pipette 20 μL of the uniform cell suspension and mix with 1 μL of cell counting dye. Let it stand for 1 minute, then pipette 10 μL of the suspension onto a cell counting plate to count the number of viable cells (green).

[0373] 2.3 According to the cell counting results, add an appropriate volume of culture medium and spread 100 μL into a 96-well plate to ensure that the cell count is 1.5x10 4 cells / well, 200 μL PBS was added to the edge of the wells to seal the edges to prevent the volatilization of the culture medium, and the cell plate was cultured in a 37°C, 5% CO2 incubator. Transfection was performed the next day after overnight.

[0374] (II) Cell transfection experiment

[0375] 1. Experimental Preparation

[0376] 1.1 siRNA sample preparation: siRNA samples were quantified to 1 μM and stored at -20°C until use. Brief centrifugation was required before use.

[0377] 1.2 Opti-MEM medium and transfection reagent Lipofectamine RNAi Max stored at 4°C.

[0378] 1.3 8-tube joint.

[0379] 2. Cell transfection experiment

[0380] 2.1 Before transfection, preheat the MLE-12 cell culture medium and replace it with 90 μL / well of the cell plate.

[0381] 2.2 Diluting siRNA: Thaw siRNA from -20°C, mix thoroughly, and dilute to different concentrations according to Table 11 as working solutions for immediate use. Label 8-tube strips (Tube A) and add 40 μL of Opti-MEM to each well. Add 10 μL of siRNA (stock solution) to the well with the highest concentration. Mix thoroughly, then remove 10 μL and perform a serial dilution to obtain different concentrations of siRNA. Incubate at room temperature for 15 minutes and add to a 96-well plate (final concentrations are 10 nM, 1 nM, 0.1 nM, and 0 nM, respectively).

[0382] Table 11. siRNA sample multi-concentration dilution scheme

[0383] 2.3 Preparation of Lipofectamine RNAi Max: Dilute Lipofectamine RNAi Max with Opti-MEM and let it stand for 5 minutes. The specific preparation ratio is 15μL Opti-MEM plus 0.9μL Lipofectamine RNAi Max.

[0384] 2.4 Dispense the prepared Lipofectamine RNAi Max into 8 tube strips, 30 μL / tube, mix thoroughly by pipetting (do not generate bubbles), and then take out 30 μL of siRNA of different concentrations from Tube A and add them to the 8 tube strips containing Lipofectamine RNAi Max. Incubate at room temperature for 20 minutes.

[0385] 2.5 Add the above mixture to the culture plate, 10 μL / well, and set up 3 replicate wells for each concentration.

[0386] 2.6 Incubate at 37°C in a CO2 incubator for 24 hours.

[0387] (3) Sample collection and testing

[0388] 1. Collect cells after 24 hours and use a high-throughput cell RNA extraction kit to extract total cell RNA.

[0389] 2. Reverse transcription and Q-PCR detection.

[0390] In the quantitative real-time PCR detection, SYBR Green detection experiment was used, and the primer information is shown in Table 12.

[0391] Table 12. Primer sequences

[0392] 3. Results Analysis

[0393] After the Q-PCR test is completed, the corresponding Ct value is obtained according to the threshold value automatically set by the system. The expression of a gene can be relatively quantified by comparing the Ct value. Comparative Ct refers to calculating the difference in gene expression by the difference between the Ct value of the internal reference gene, also known as 2 -△△Ct , ΔΔCt = [(Ct experimental group target gene - Ct experimental group internal reference) - (Ct control group target gene - Ct control group internal reference)]. Inhibition rate (%) = (1 - target gene expression residual amount) × 100%. The experimental results are shown in Table 13 and Figure 1.

[0394] The results show that TJR100072, TJR100073, TJR100344 and TJR100345 showed the same level of inhibitory effect on MUC5B mRNA in the cell transfection experiment.

[0395] Table 13. mRNA expression levels of cells in the drug-treated groups at different concentrations

[0396] Example 4: Inhibitory activity of the oligonucleotides disclosed herein on the mouse MUC5B gene

[0397] The specific procedure is as follows: Six-week-old C57BL / 6N female mice (purchased from Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd.) were acclimated for one week before the experiment began. On day 0, the compound was prepared in normal saline before administration, with each test compound at a concentration of 10 mg / kg. Twelve mice were weighed and divided into three groups of four mice each: normal saline, TJR100072-10 mpk, and TJR100073-10 mpk. After isoflurane anesthesia, the drug was administered intratracheally using a mouse pulmonary drug delivery device (purchased from Yuyan Instruments). On day 10, the mice were euthanized, and cardiac blood was collected, followed by whole lung removal, fragmentation, and subsequent RNAi. MUC5B mRNA levels in the whole lung were determined by RT-PCR. The results are shown in Table 14 and Figure 2.

[0398] The results show that compared with the saline group, TJR100072 without lipid delivery did not show obvious inhibitory activity, while the positive molecule TJR100073 showed weaker inhibitory activity.

[0399] Table 14. Expression of MUC5B mRNA in mice

[0400] Example 5: Inhibitory activity of the oligonucleotides disclosed herein on the mouse MUC5B gene

[0401] The specific procedure is as follows: Six-week-old C57BL / 6N female mice (purchased from Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd.) were acclimated for one week before the experiment. On day 0, the compounds were prepared in normal saline prior to dosing, with each test compound at a concentration of 10 mg / kg. Thirty mice were weighed and divided into three groups of 10 mice each, receiving TJR100073-10 mpk, TJR100344-10 mpk, and TJR100345-10 mpk. After isoflurane anesthesia, the drugs were administered intratracheally using a mouse pulmonary drug delivery device (Yuyan Instruments). On day 10, the mice were euthanized, and cardiac blood was collected, followed by whole lung removal, fragmentation, and subsequent RNAi. MUC5B mRNA levels in the whole lungs were determined by RT-PCR. The results are shown in Table 15 and Figure 3.

[0402] The results show that both TJR100344 and TJR100345 exhibited better inhibitory activity than the positive molecule TJR100073.

[0403] Table 15. Expression of MUC5B mRNA in mice

[0404] Example 6: Evaluation of the in vitro anti-APP activity of the oligonucleotides disclosed herein using A172 cells

[0405] The in vitro anti-APP activity of the oligonucleotides of the present disclosure was evaluated in A172 cells (purchased from Yimo Bio) using 7 concentration gradients.

[0406] A172 cells were cultured in Dulbecco's modified eagle medium supplemented with 10% fetal bovine serum at 37°C and 5% CO2. 24 h before transfection, A172 cells were seeded into 96-well plates at a density of 1 × 10 cells per well. 4 Cells were plated in 100 μL of culture medium per well. Referring to the product instruction manual, the oligonucleotides disclosed herein were transfected using Lipofectamine RNAi MAX (ThermoFisher, 13778150), with duplicate wells for each concentration. After 48 hours of treatment, total cell RNA was extracted using a high-throughput cell RNA extraction kit FG0417-L / FG0418-XL (Zhifan Medical, magnetic bead method), RNA reverse transcription experiments (Takara, RR037A) and quantitative real-time PCR (Thermo, 4444557) were performed to determine the mRNA level of human APP, which was corrected according to the GAPDH internal reference gene level.

[0407] The instruments involved in this experiment are shown in Table 16.

[0408] Among them, in the real-time quantitative PCR detection, the probe Q-PCR detection experiment was used, and its primer information is shown in Table 17.

[0409] Table 16. Experimental instruments

[0410] Among them, in the real-time quantitative PCR detection, the probe Q-PCR detection experiment was used, and its primer information is shown in Table 17.

[0411] Table 17. Taqman primer information table

[0412] Result analysis method:

[0413] After the Q-PCR test is completed, the corresponding Ct value is obtained according to the threshold value automatically set by the system. The expression of a gene can be relatively quantified by comparing the Ct value: Comparative Ct refers to calculating the gene expression difference by the difference between the Ct value and the internal reference gene, also known as 2 -△△Ct, △△Ct=[(Ct experimental group target gene - Ct experimental group internal reference) - (Ct control group target gene - Ct control group internal reference)]. Inhibition rate (%)=(1-remaining amount of target gene expression)×100%. The results are expressed as the remaining percentage of human APP mRNA expression relative to the cells of the MOCK group (untreated group). The IC of inhibition rate 50 The results are shown in Table 18.

[0414] It can be seen from the results that the oligonucleotides disclosed in the present invention exhibit a strong inhibitory effect on APP mRNA.

[0415] Table 18. Remaining percentage and IC of APP mRNA expression in cells of drug-treated groups at different concentrations 50

[0416] Example 7: Evaluation of the in vitro anti-PMP22 activity of the oligonucleotides disclosed herein using A172 cells

[0417] The oligonucleotides of the present disclosure were evaluated for in vitro anti-PMP22 activity in A172 cells using 7 concentration gradients.

[0418] A172 cells were cultured in Dulbecco's modified eagle medium supplemented with 10% fetal bovine serum at 37°C and 5% CO2. 24 h before transfection, A172 cells were seeded in 96-well plates at a density of 1 × 10 cells per well. 4 cells, 100 μL culture medium per well.

[0419] According to the product instructions, the oligonucleotides disclosed herein were transfected using Lipofectamine RNAi MAX (ThermoFisher, 13778150) in duplicate wells for each concentration. After 48 hours of treatment, total RNA was extracted using the high-throughput cell RNA extraction kit FG0417-L / FG0418-XL (Zhifan Medical, magnetic bead method). RNA reverse transcription was performed (Takara, RR037A) and quantitative real-time PCR (Thermo, 4444557) to measure human PMP22 mRNA levels. Human PMP22 mRNA levels were corrected for GAPDH internal reference gene levels.

[0420] The instruments involved in this experiment are shown in Table 16 above.

[0421] Among them, in the real-time quantitative PCR detection, the probe Q-PCR detection experiment was used, and its primer information is shown in Table 19.

[0422] Table 19 Taqman primer information table

[0423] Result analysis method:

[0424] After the Q-PCR test is completed, the corresponding Ct value is obtained according to the threshold value automatically set by the system. The expression of a gene can be relatively quantified by comparing the Ct value. Comparative Ct refers to calculating the difference in gene expression by the difference between the Ct value of the internal reference gene, also known as 2 -△△Ct , ΔΔCt = [(Ct experimental group target gene - Ct experimental group internal reference) - (Ct control group target gene - Ct control group internal reference)]. Inhibition rate (%) = (1 - target gene expression residual amount) × 100%. The results are expressed as the percentage of human PMP22 mRNA expression residual in cells relative to the MOCK group (untreated group). The IC of inhibition rate 50 The results are shown in Table 20.

[0425] The results indicate that the oligonucleotides disclosed herein exhibit a strong inhibitory effect on PMP22 mRNA.

[0426] Table 20. Remaining percentages and IC values ​​of PMP22 mRNA expression in the drug-treated groups at different concentrations 50

[0427] Example 8: Evaluation of the in vitro anti-MAPT activity of the oligonucleotides disclosed herein using Neuro2α cell free uptake

[0428] The in vitro anti-MAPT activity of the oligonucleotides of the present invention was evaluated by in vitro free uptake experiments in Neuro2α cells (purchased from Shanghai Jitai Yikesai Biotechnology Co., Ltd.) using 5 concentrations (1000 nM, 300 nM, 30 nM, 3 nM, 0 nM).

[0429] Neuro2α cells were cultured in Eagle's Minimum Essential Medium (EMEM) supplemented with 10% fetal bovine serum at 37°C and 5% CO2. 24 h before free uptake, Neuro2α cells were seeded in 96-well plates at a seeding density of 1.5 × 10 cells per well. 4cells, 100 μL medium per well. On the second day, the oligonucleotides disclosed herein were diluted in 2% fetal bovine serum medium to final concentrations of 1000 nM, 300 nM, 30 nM, 3 nM, and 0 nM, respectively. After incubation of the cells with the oligonucleotides disclosed herein for 72 hours, total cell RNA was extracted using a high-throughput cell RNA extraction kit FG0417-L / FG0418-XL (Zhifan Medical, magnetic bead method), RNA reverse transcription experiments (Takara, RR037A), and quantitative real-time PCR (Thermo, 4444557) were performed to determine the mRNA level of mouse MAPT, and the mRNA level of mouse MAPT was corrected according to the level of the mouse GAPDH internal reference gene.

[0430] The instruments involved in this experiment are shown in Table 16 above.

[0431] Among them, in the real-time quantitative PCR detection, the probe Q-PCR detection experiment was used, and its primer information is shown in Table 21.

[0432] Table 21. Taqman primer information table

[0433] Result analysis method:

[0434] After the Q-PCR test is completed, the corresponding Ct value is obtained according to the threshold value automatically set by the system. The expression of a gene can be relatively quantified by comparing the Ct value. Comparative Ct refers to calculating the difference in gene expression by the difference between the Ct value of the internal reference gene, also known as 2 -△△Ct ΔΔCt = [(Ct experimental group target gene - Ct experimental group internal reference) - (Ct control group target gene - Ct control group internal reference)]. Inhibition rate (%) = (1 - remaining target gene expression) × 100%. Results are expressed as the remaining percentage of mouse MAPT mRNA expression relative to that of cells in the MOCK group (untreated group). The experimental results are shown in Table 22.

[0435] Table 22. Anti-MAPT activity of oligonucleotides of the present disclosure in Neuro2α cells

[0436] It can be seen from the results that the oligonucleotides disclosed herein exhibit a strong inhibitory effect on MAPT mRNA in the cell free uptake experiment.

[0437] Example 9: Inhibitory activity of the oligonucleotides disclosed herein on the mouse MAPT gene

[0438] The specific operation steps are as follows: 8-week-old C57BL / 6J male mice (purchased from Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd.) were acclimated for one week before the experiment. On day 0, the compounds were prepared with normal saline before administration, and the drug concentration of each test substance was 5 mg / kg. The mice were weighed and divided into 3 groups, with 6-10 mice / group, namely the blank vehicle group, TJR101646-5mpk group, and TJR101647-5mpk group. After anesthesia with Zota, unilateral intracerebroventricular (ICV) administration was performed using a stereotactic-guided infusion system (Rayward). Using standard aseptic surgical procedures, a single 2 μl unilateral injection was performed in the right lateral ventricle at the following positions: AP = -0.2 mm (posterior to bregma), ML = -1.0 mm, DV = +2.4 mm. After the midline incision of the scalp skin, burr holes were created at the selected coordinates using stereotactic coordinates and a cranial drill. Afterwards, the dura mater was punctured at the center of the burr hole, and the needle was lowered to the designated depth to reach the lateral ventricle. The glass electrode was filled with a volume slightly larger than 2 μL. The administration volume was 2 μL, and the injection rate was 1 μL / min. After infusion, the needle was left in place for 2 minutes and then slowly withdrawn, pausing for 30 seconds when the needle tip was in the cortical area. On day 14, the mice were euthanized, and the mouse cerebral cortex tissue was obtained, snap-frozen in liquid nitrogen, and then stored at -80°C. RT-PCR was used to detect MAPT mRNA levels in the mouse cerebral cortex. The experimental results are shown in Table 23 and Figure 4.

[0439] The results show that TJR101647 exhibits better inhibitory activity than the positive molecule TJR101646.

[0440] Table 23. TJR101647 inhibitory activity on mouse MAPT mRNA

[0441] Example 10: Inhibitory activity of the oligonucleotides disclosed herein on the mouse MAPT gene

[0442] The specific procedure is as follows: 8-week-old C57BL / 6J male mice (purchased from Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd.) were acclimated for one week before the experiment. On day 0, compounds were prepared in normal saline prior to dosing at a concentration of 5 mg / kg for each test compound. Mice were weighed and divided into two groups of 6-10 mice each: vehicle group and TJR101740-5 mpk. After anesthesia with Zotafenac, intracerebroventricular (ICV) administration was performed using a stereotaxic-guided infusion system (Rayward). Using standard aseptic surgical procedures, a single 2 μl injection was administered unilaterally into the right lateral ventricle at the following locations: AP = -0.2 mm (posterior to the bregma), ML = -1.0 mm, DV = +2.4 mm. After a midline incision of the scalp skin, a burr hole was created at the selected coordinates using stereotaxic coordinates and a cranial drill. The dura mater was then punctured at the center of the burr hole, and the needle was lowered to the designated depth to reach the lateral ventricle. The glass electrode was filled with a volume slightly larger than 2 μL. The administration volume was 2 μL, and the injection rate was 1 μL / min. After infusion, the needle was left in place for 2 minutes to stabilize, then slowly withdrawn, pausing for 30 seconds when the needle tip was in the cortical region. On day 14, mice were euthanized, and brain tissue was obtained, snap-frozen in liquid nitrogen, and stored at -80°C. MAPT mRNA levels in various brain tissues were measured by RT-PCR. The results are shown in Table 24 and Figure 5.

[0443] The results showed that after unilateral intracerebroventricular administration, TJR101740 showed strong inhibitory activity on the MRNA levels of MAPT in various brain tissues of mice.

[0444] Table 24. TJR101740 inhibitory activity on mouse MAPT mRNA

[0445] Example 11: Inhibitory activity of the oligonucleotides disclosed herein on the mouse APP gene

[0446] The specific operation steps are as follows: 8-week-old C57BL / 6J male mice (purchased from Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd.) were acclimated for one week before the experiment. On day 0, the compounds were prepared in normal saline before administration, with each test compound at a concentration of 5 mg / kg. The mice were weighed and divided into 4 groups of 6-8 mice each, namely the blank vehicle group, TJR102160, TJR102161, and TJR102162. After anesthesia with Zota, unilateral intracerebroventricular (ICV) administration was performed using a stereotaxic-guided infusion system (Rayward). Using standard aseptic surgical procedures, a single 2 μl unilateral injection was performed into the right lateral ventricle at the following locations: AP = -0.2 mm (posterior to bregma), ML = -1.0 mm, DV = +2.4 mm. After a midline incision of the scalp skin, burr holes were created at the selected coordinates using stereotaxic coordinates and a cranial drill. Afterward, the dura mater was punctured at the center of the burr hole, and the needle was lowered to the designated depth to reach the lateral ventricle. The glass electrode was filled with a volume slightly larger than 2 μL. The administration volume was 2 μL, and the injection rate was 1 μL / min. After infusion, the needle was left in place for 2 minutes and then slowly withdrawn, pausing for 30 seconds when the needle tip was in the cortical region. On day 7, the mice were euthanized, and the cerebellum tissue was obtained, snap-frozen in liquid nitrogen, and stored at −80°C. APP mRNA levels in the mouse cerebellum were detected by RT-PCR. The results are shown in Table 25 and Figure 6.

[0447] Table 25. Inhibitory activity of oligonucleotides disclosed herein on mouse APP mRNA

[0448] The results indicate that the oligonucleotides disclosed herein exhibit a strong inhibitory effect on APP mRNA in the in vivo mouse experiment.

Claims

1. An oligonucleotide comprising at least one compound represented by formula (I) or a pharmaceutically acceptable salt thereof, wherein the compound represented by formula (I) has the structure in, X1 is selected from O, S, N, and C atoms; X2 is selected from O and S atoms; R1 is selected from C 10 -C 30 A straight-chain alkyl group, or a C 10 -C 30 A straight chain alkyl group; optionally, the C 10 -C 30 The straight chain alkyl group is replaced by one or more R a Replace, or optionally, the C 10 -C 30 The adjacent two carbon atoms of the straight chain alkyl form a C 3-6 Cycloalkyl; R2 and R3 are the same or different and are each independently selected from a bond, hydrogen, an activated phosphate group, an activated phosphite group, a phosphoramidite group, a solid support, an internucleotide linker attached to an oligonucleotide, -P(=O)(OH)-O-, -P(=O)(SH)-O-, -P(=O)(OH)-O-nucleoside, -P(=O)(SH)-O-nucleoside, -P(=O)(OH)-O-oligonucleotide fragment, -P(=O)(SH)-O-oligonucleotide fragment, and a hydroxyl protecting group; R a are each independently selected from hydrogen, deuterium, halogen, hydroxy, cyano, alkyl, haloalkyl, alkoxy, cycloalkyl, heterocycloalkyl; n is selected from 0 or 1; B is a base; Wherein, the compound represented by formula (I) is not selected from:

2. The oligonucleotide according to claim 1, wherein the compound structure represented by formula (I) is selected from: in, R b It is selected from OH or SH, wherein the b and c ends are respectively connected to the adjacent nucleotides on both sides of the compound structure represented by formula (I).

3. The oligonucleotide according to any one of claims 1 to 2, wherein X1 is selected from O or S atoms, preferably O atoms.

4. The oligonucleotide according to any one of claims 1 to 3, wherein X2 is selected from O or S atoms, preferably O atoms.

5. The oligonucleotide according to any one of claims 1 to 4, wherein R1 is selected from C 14 -C 24 A straight-chain alkyl group, or a C 14 -C 24 of a straight chain alkyl group.

6. The oligonucleotide according to any one of claims 1 to 5, wherein R a are each independently selected from hydrogen, deuterium, halogen, C 1-6 Alkyl, C 1-6 Alkoxy; preferably, R a are each independently selected from hydrogen, deuterium, F, methyl and methoxy.

7. The oligonucleotide according to any one of claims 1 to 6, wherein R1 is selected from: The a end is connected to X2.

8. The oligonucleotide according to any one of claims 1 to 7, wherein B is selected from adenine, guanine, cytosine, uracil, and thymine.

9. The oligonucleotide according to any one of claims 1 to 8, wherein The compound structure represented by the formula (1) is selected from: Preferably, the compound structure represented by formula (1) is selected from:

10. The oligonucleotide according to any one of claims 1 to 9, wherein The oligonucleotide comprises 1, 2, 3 or 4 compounds represented by formula (I) or pharmaceutically acceptable salts thereof; preferably, the oligonucleotide comprises 1 compound represented by formula (I) or pharmaceutically acceptable salts thereof.

11. The oligonucleotide according to any one of claims 1 to 10, wherein The oligonucleotide comprises a sense strand and an antisense strand forming a double-stranded region, and the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is located at at least one of the following nucleotide positions: Position 1 at the 5' end of the sense strand; Position 1 at the 3' end of the sense strand; Position 1 at the 5' end of the antisense strand; Position 1 at the 3' end of the antisense strand; Positions 2-8 in the middle of the justice chain; positions 2-8 in the middle of the antisense strand; Preferably, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is located at at least one of the following nucleotide positions: Position 1 at the 5' end of the sense strand; Position 1 at the 3' end of the sense strand; The 6th position in the middle of the justice chain.

12. The oligonucleotide according to any one of claims 2 to 11, wherein The compound represented by formula (I) or a pharmaceutically acceptable salt thereof is located at the sixth position from the 5' end to the 3' end of the sense chain, the b end is connected to the fifth nucleotide from the 5' end of the sense chain, and the c end is connected to the seventh nucleotide from the 5' end of the sense chain, wherein B in formula (I) represents the base of the sixth nucleotide from the 5' end to the 3' end of the sense chain; Alternatively, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is located at the first nucleotide at the 5' end of the sense strand, wherein B represents the base of the first nucleotide at the 5' end of the sense strand; Alternatively, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is located at the first nucleotide at the 3' end of the sense strand, wherein B represents the base of the first nucleotide at the 3' end of the sense strand.

13. The oligonucleotide according to claim 12, wherein The compound structure represented by the formula (I) is selected from:

14. The oligonucleotide according to any one of claims 1 to 13, wherein The oligonucleotide is a double-stranded RNAi inhibitor molecule, comprising a sense strand and an antisense strand forming a double-stranded region; preferably, the length of the sense strand is 15-35 nucleotides, and the length of the antisense strand is 15-30 nucleotides; more preferably, the length ratio of the sense strand to the antisense strand is 19 / 21, 21 / 23 or 23 / 25.

15. The oligonucleotide according to any one of claims 1 to 14, wherein In the oligonucleotide, in addition to the compound structure shown in formula (I), at least one other nucleotide is a modified nucleotide.

16. The oligonucleotide according to any one of 11 to 15, wherein At least one phosphate group in the sense strand and / or the antisense strand is a phosphate group having a modified group, preferably a thiophosphate diester group or a 5'-vinyl phosphodiester group.

17. A compound represented by formula (II) or a pharmaceutically acceptable salt thereof: in, B, X1, X2, R1 and n are defined as in any one of claims 1 to 9; R A2 Selected from leaving groups, R A3 Selected from phosphorus-containing active reactive groups, or R A3 Selected from leaving groups, R A2 Selected from phosphorus-containing active reactive groups; Wherein, the compound represented by formula (II) is not selected from: Preferably, the compound represented by formula (II) is selected from More preferably, the compound represented by formula (II) is selected from:

18. A method for preparing the oligonucleotide according to any one of claims 1 to 17, comprising the following steps: 1) synthesizing the compound represented by formula (II) or a pharmaceutically acceptable salt thereof according to claim 17; Optionally, the method further comprises 2) synthesizing the oligonucleotide according to any one of claims 1 to 16 using the compound of formula (II) or a pharmaceutically acceptable salt thereof synthesized in step 1).

19. A pharmaceutical composition comprising the oligonucleotide according to any one of claims 1 to 16; preferably, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

20. A method for inhibiting target gene expression, comprising administering to a subject an effective amount or an effective dose of the oligonucleotide according to any one of claims 1 to 16 or the pharmaceutical composition according to claim 19.

21. Use of the oligonucleotide according to any one of claims 1 to 16 or the pharmaceutical composition according to claim 19 in the preparation of a medicament for preventing and / or treating diseases related to lung, eye and central nervous system disorders, and tumor-related diseases.

22. A method for delivering an oligonucleotide outside the liver, comprising administering to a subject an effective amount or an effective dose of the oligonucleotide according to any one of claims 1 to 16 or the pharmaceutical composition according to claim 19.

23. The method of claim 22, wherein the oligonucleotide is delivered to the lungs, eyes, or central nervous system; preferably, the oligonucleotide is delivered to the central nervous system.

24. The method of claim 22, wherein the oligonucleotide is delivered to a tumor; preferably, the oligonucleotide is delivered to pancreatic cancer, prostate cancer, breast cancer, or lung cancer; more preferably, the oligonucleotide is delivered to pancreatic cancer and prostate cancer.

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