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

Abstract

The present disclosure relates to oligonucleotides comprising lipophilic monomers and their use in non-liver delivery. Specifically, the present disclosure relates to oligonucleotides comprising an antisense strand complementary to a target gene, a sense strand complementary to the antisense strand, and one or more compounds represented by formula (I). The present disclosure further relates to pharmaceutical compositions comprising the oligonucleotides, and pharmaceutical uses of the oligonucleotides and / or pharmaceutical compositions.

Description

[Technical Field]

[0001] The present disclosure is in the field of biopharmaceuticals, and specifically relates to oligonucleotides containing lipophilic monomers. The present disclosure further relates to methods for preparing and using the oligonucleotides. [Background technology]

[0002] RNA interference (RNAi) is an efficient method for silencing gene expression and has broad prospects for use. While RNA drugs have advantages such as a rich set of target candidates, a simple and efficient development process, and high specificity, only a limited number of RNA drugs have been approved for clinical use by the FDA. The main bottlenecks they face include poor stability, short half-life, high immunogenicity, weak targeting, and limited cytoplasmic penetration. Therefore, in addition to improving RNA stability and immunogenicity through chemical modifications, it is also necessary to provide an efficient and safe in vivo delivery system and improve membrane permeability.

[0003] RNA interference drug delivery technologies mainly include lipid nanoparticle (LNP) delivery, N-acetylgalactosamine (GalNAC) modification-mediated liver-targeted delivery, and antibody-mediated targeting. Currently, there is a relatively large amount of research using GalNAC-conjugated drug technology, and at the end of 2020, two GalNAC-conjugated siRNA drugs, lumasirna and inclisirna, were approved for clinical use. While GalNAC-conjugated RNA drugs can achieve efficient and specific delivery to the liver, there is currently no effective delivery method for extrahepatic cells that lack ASGPR expression. Therefore, there is a need to continue developing efficient in vivo delivery methods to enable RNA drugs to act in extrahepatic tissues.

[0004] The compound of the present disclosure having a lipophilic moiety represented by formula (I) or a tautomer thereof not only has increased hydrophobicity and membrane permeability, thereby improving the membrane permeability of oligonucleotides, but also allows oligonucleotides containing the compound to be effectively delivered into the cytoplasm and exert their RNA function in non-liver tissues. Summary of the Invention

[0005] The present disclosure provides an oligonucleotide comprising at least one compound represented by formula (I) or a pharmaceutically acceptable salt thereof, wherein the structure of the compound represented by formula (I) is: [ka] Among them, X1 is selected from O, S, N, and C atoms; X2 is selected from O and S atoms, R1 is C 10 ~C 30 straight chain alkyl groups (e.g., 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 of C, or C interrupted by one or more O or S atoms 10 ~C 30 straight chain alkyl groups (e.g., C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C18 , C 19 , C 20 , C 21 , C 22 , C 23 , C 24 , C 25 , C 26 , C 27 , C 28 , C 29 , C 30 and optionally, the above C 10 ~C 30 The linear alkyl group of a or optionally substituted with the above C 10 ~C 30 C between two adjacent carbon atoms of the straight-chain alkyl group 3-6 a cycloalkyl group (C3, C4, C5, C6 cycloalkyl group, including but not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl) is formed; R2 and R3 are the same or different and are each independently selected from a bond, hydrogen, an activated phosphate group, an activated phosphate group, a phosphoramidite group, a solid support, an internucleotide linkage group linked 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 hydroxy protecting group (e.g., an ester protecting group, an aryl protecting group, an alkyl protecting group, an alkoxymethyl protecting group, or a silyl protecting group, including, but not limited to, DMTr); R aare each independently selected from hydrogen, deuterium, halogen (e.g., fluorine, chlorine, bromine), hydroxy, cyano, alkyl (e.g., C1, C2, C3, C4, C5, C6 alkyl, including, but not limited to, methyl, ethyl, and isopropyl), haloalkyl, alkoxy (e.g., C1 alkoxy, C2 alkoxy, C3 alkoxy, C4 alkoxy, C5 alkoxy, and C6 alkoxy, including, but not limited to, methoxy, ethoxy, propoxy, and isopropoxy), cycloalkyl (including, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl), and heterocycloalkyl; n is selected from 0 or 1, B is a base, The compound represented by the above formula (I) is [ka] Not selected from.

[0006] In some embodiments, the structure of the compound of Formula (I) is: [ka] Selected from Among them, R b is selected from OH or SH.

[0007] In some embodiments, X1 is selected from an O or S atom, preferably an O atom.

[0008] In some embodiments, X2 is selected from an O or S atom, preferably an O atom.

[0009] In some embodiments, R is C 14 ~C 24 straight chain alkyl groups (e.g., C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , C20 , C 21 , C 22 , C 23 , C 24 a straight chain alkyl group of C, or C interrupted by one or more O or S atoms 14 ~C 24 straight chain alkyl groups (e.g., 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).

[0010] In some embodiments, R a are each independently hydrogen, deuterium, halogen (e.g., fluorine, chlorine, bromine), C 1-6 Alkyl groups (e.g., C1, C2, C3, C4, C5, C6 alkyl groups, including, but not limited to, methyl, ethyl, and isopropyl groups), C 1-6 It is selected from alkoxy groups (e.g., C1 alkoxy groups, C2 alkoxy groups, C3 alkoxy groups, C4 alkoxy groups, C5 alkoxy groups, and C6 alkoxy groups, including, but not limited to, methoxy groups, ethoxy groups, propoxy groups, and isopropoxy groups).

[0011] In some embodiments, R a are each independently selected from hydrogen, deuterium, fluorine, a methyl group, and a methoxy group.

[0012] In some embodiments, R1 is [ka] wherein the a-terminus is linked to X2.

[0013] In some embodiments, B is selected from adenine, guanine, cytosine, uracil, and thymine.

[0014] The present disclosure further provides an oligonucleotide comprising at least one of the following compounds or a pharmaceutically acceptable salt thereof, wherein the structure of the compound is: [ka] [ka] [ka] and In some embodiments, the structure of the compound of formula (I) is: [ka] [ka] [ka] Selected from.

[0015] In some embodiments, the oligonucleotide described herein comprises one, two, three, or four compounds of formula (I) or pharmaceutically acceptable salts thereof, and in some embodiments, the oligonucleotide comprises one compound of formula (I) or pharmaceutically acceptable salts thereof.

[0016] In some embodiments, the position of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is: Position 1 of the 5' end of the sense strand, Position 1 of the 3' end of the sense strand, Position 1 of the 5' end of the antisense strand Position 1 of the 3' end of the antisense strand Positions 2 to 8 (e.g., positions 2, 3, 4, 5, 6, 7, or 8) in the middle of the sense strand, and Positions 2 to 8 (e.g., positions 2, 3, 4, 5, 6, 7, or 8) in the middle of the antisense strand; at least one of the nucleotide positions

[0017] The intermediate position refers to the intermediate position counted from the first nucleotide at the 5'-end or 3'-end of the antisense strand and / or sense strand, for example, "position 6 of the intermediate position of the sense strand" refers to the 6th nucleotide counted from the first nucleotide at the 5'-end or 3'-end of the sense strand. Preferably, the intermediate position refers to the intermediate position counted from the first nucleotide at the 5'-end, for example, "position 6 of the intermediate position of the sense strand" refers to the 6th nucleotide counted from the first nucleotide at the 5'-end of the sense strand.

[0018] In some embodiments, the position of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is: Position 1 of the 5' end of the sense strand, Position 1 of the 3' end of the sense strand, Position 6 in the middle of the sense strand At least one of the nucleotides is

[0019] In some embodiments, the structure of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is: [ka] wherein the b-terminus is linked to the fifth nucleotide at the 5'-end of the sense strand, and the c-terminus is linked to the seventh nucleotide at the 5'-end of the sense strand, and B in formulas (I-1) and (I-2) represents the base of the sixth nucleotide in the direction from the 5'-end to the 3'-end of the sense strand.

[0020] In some embodiments, the structure of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is: [ka] Among them, the connecting terminal JPEG2025541262000013.jpg7170 is linked to the second nucleotide at the 5' end of the sense strand, i.e., formulas (I-3) and (I-4) are at the first nucleotide at the 5' end of the sense strand, and B in formulas (I-3) and (I-4) represents the base of the first nucleotide at the 5' end of the sense strand.

[0021] In some embodiments, the structure of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is: [ka] Among them, the connecting terminal JPEG2025541262000015.jpg7170 is linked to the second nucleotide at the 3' end of the sense strand, i.e., formulas (I-5) and (I-6) are at the first nucleotide at the 3' end of the sense strand, and B in formulas (I-5) and (I-6) represents the base of the first nucleotide at the 3' end of the sense strand.

[0022] In some embodiments, the oligonucleotide described herein comprises a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, wherein the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is located at the 6th nucleotide of the 5'-end of the sense strand, and the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is [ka] Selected from.

[0023] In some embodiments, the oligonucleotide described herein comprises a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, wherein the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is located at the 6th nucleotide of the 5'-end of the sense strand, and the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is [ka] Selected from.

[0024] In some embodiments, the oligonucleotide described herein is selected from double-stranded RNAi inhibitor molecules, and in some embodiments, the oligonucleotide is selected from siRNAs, comprising a sense strand and an antisense strand that form a double-stranded region, and in some embodiments, the siRNA sense strand is 15-35 nucleotides in length, and the antisense strand is 15-30 nucleotides in length.

[0025] In some embodiments, the sense strand and the antisense strand are the same or different in length, and the sense strand is 19 to 23 nucleotides in length, and the antisense strand is 19 to 26 nucleotides in length. Thus, the length ratio of the sense strand to the antisense strand of the siRNA provided in 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.

[0026] In some embodiments, the antisense strand is at least partially reverse complementary to the target sequence to mediate RNA interference, and 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, and in some embodiments, the antisense strand is completely reverse complementary to the target sequence.

[0027] In some embodiments, the sense strand is at least partially reverse complementary to the antisense strand to form a double-stranded region, and 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 and antisense strands, and in some embodiments, the sense strand is completely reverse complementary to the antisense strand.

[0028] In some embodiments, the siRNA of the present disclosure comprises one or two blunt ends.

[0029] In some specific embodiments, the siRNA comprises an overhang of 1 to 4, eg, 1, 2, 3, 4, unpaired nucleotides.

[0030] In some embodiments, the siRNA of the present disclosure comprises an overhang at the 3' end of the siRNA antisense strand.

[0031] In some embodiments, the oligonucleotide described herein has the structure of the compound of formula (I) and at least one other nucleotide is a modified nucleotide. In some embodiments, at least one nucleotide contained in each of the sense strand and the antisense strand described herein is a modified nucleotide.

[0032] In some embodiments, the modified nucleotide is selected from 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, and GNA.

[0033] In some embodiments, the modified nucleotides are independently selected from 2'-methoxy modified nucleotides and 2'-fluoro modified nucleotides.

[0034] In some embodiments, the oligonucleotide is selected from a double-stranded RNAi inhibitor molecule and comprises a sense strand and an antisense strand that form a double-stranded region, wherein the sense strand comprises three consecutive nucleotides that are 2'-fluoro modified nucleotides.

[0035] In some embodiments, in the 5' to 3' direction, the nucleotides at positions 9, 10, and 11 of the sense strand are each independently 2'-fluoro modified nucleotides.

[0036] In some embodiments, from the 5' to the 3' end, the nucleotides at positions 9, 10, and 11 of the sense strand are each independently a 2'-fluoro-modified nucleotide, the nucleotide at position 7 of the sense strand is selected from 2'-fluoro-modified nucleotides, and the nucleotides at other positions of the sense strand are selected from 2'-methoxy-modified nucleotides.

[0037] In some embodiments, the oligonucleotide is selected from a double-stranded RNAi inhibitor molecule, and comprises a sense strand and an antisense strand that form a double-stranded region, and 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 2'-fluoro-modified nucleotides.

[0038] In some embodiments, from the 5' to the 3' end, the nucleotides at positions 2, 6, 8, 9, 14, and 16 of the antisense strand are each independently 2'-fluoro-modified nucleotides, and the nucleotides at other positions of the antisense strand are selected from 2'-methoxy-modified nucleotides.

[0039] In some embodiments, the sense strand is 5'NL 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, 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, 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, 5’N L N a N a N a Na 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 a 3', or 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 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 L are each independently selected from the compounds of formula (I) described herein or pharmaceutically acceptable salts thereof; N ais a nucleotide modified with a 2'-methoxy group, and N b is a 2'-fluoro modified nucleotide.

[0040] In some embodiments, the antisense strand is 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 a 'N a 'N a '3, or 5'N a '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 a 'N a 'N L '3, or 5'N a '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 a 'N a 'N a '3, wherein N L are each independently selected from the compounds of formula (I) described herein or a pharmaceutically acceptable salt thereof; a ' is a nucleotide modified with a 2'-methoxy group, and N b ' is a 2'-fluoro modified nucleotide.

[0041] In some embodiments, the sense strand is 5'N L NNNNNNNNNNNNNNNNNN3', or 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 5'N L N a N a N a N a N a N b N a N b N b N b Na 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; and N a is a nucleotide modified with a 2'-methoxy group, and N b is a 2'-fluoro modified nucleotide, N L teeth, [ka] [ka] Selected from.

[0042] In some embodiments, the sense strand is 5'NNNNNNNNNNNNNNNNNNN L 3', or 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 5'N a N a N a N a N a N a N b N a Nb 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; and N a is a nucleotide modified with a 2'-methoxy group, and N b is a 2'-fluoro modified nucleotide, N L teeth, [ka] Selected from.

[0043] In some embodiments, the sense strand is 5'NNNNNN L NNNNNNNNNNNNN3', or 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 5'N a N a N a N a N a N L N b N a N b Nb 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; and N a is a nucleotide modified with a 2'-methoxy group, and N b is a 2'-fluoro modified nucleotide, N L teeth, [ka] Selected from JPEG2025541262000022.jpg72170.

[0044] In some embodiments, the sense strand is 5'NNNNNN L NNNNNNNNNNNNNNN3', or 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 5'N a N a N a N a N a N L N b 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', wherein N is a modified or unmodified nucleotide, and N a is a nucleotide modified with a 2'-methoxy group, and N b is a 2'-fluoro modified nucleotide, N L teeth, [ka] [ka] Selected from In the above structure, the b-terminus is linked to the 5th nucleotide at the 5'-end of the sense strand, and the c-terminus is linked to the 7th nucleotide at the 5'-end of the sense strand, and B in the above structure represents the base of the 6th nucleotide at the 5'-end of the sense strand.

[0045] In some embodiments, at least one phosphate group in the sense strand and / or the antisense strand is a phosphate group having a modifying group, and in some embodiments, at least one phosphate group in the sense strand and / or the antisense strand is a thiophosphodiester group.

[0046] In some embodiments, the thiophosphodiester group is Between the first and second nucleotides of the 5' end of the sense strand, Between the second and third nucleotides of the 5' end of the sense strand, Between the first and second nucleotides of the 3' end of the sense strand, Between the second and third nucleotides at the 3' end of the sense strand, Between the first and second nucleotides at the 5' end of the antisense strand, Between the second and third nucleotides at the 5' end of the antisense strand, Between the first and second nucleotides at the 3' end of the antisense strand, and Between the second and third nucleotides at the 3' end of the antisense strand, It is in at least one of the following positions:

[0047] In some embodiments, at least one phosphate group in the sense strand and / or the antisense strand is a 5'-vinyl phosphodiester group, and in some embodiments, the 5'-vinyl phosphodiester group is at the first nucleotide at the 5' end of the antisense strand.

[0048] In some embodiments, the oligonucleotides described herein target MUC5B.

[0049] The present disclosure further provides a compound of formula (II) or a pharmaceutically acceptable salt thereof.

[0050] [ka] Among them, In the formula, the definitions and options of X1, X2, B and R1 are the same as those in the structure of the compound represented by formula (I) above; n is selected from 1 or 0, and R A2 is selected from leaving groups, and R A3 is selected from phosphorus-containing active reactive groups, or R A3 is selected from leaving groups, and R A2 is selected from phosphorus-containing active reactive groups, among which the compound represented by formula (II) is [ka] Not selected from.

[0051] In some embodiments, the compound of Formula (II) or a pharmaceutically acceptable salt thereof is [ka] Selected from In the formula, the definitions and ranges of X1, X2, B, R1, and n are the same as those in the structure of the compound represented by formula (I) above.

[0052] In some embodiments, X1 is selected from an O atom and an S atom, preferably an O atom.

[0053] In some embodiments, X2 is selected from an O atom and an S atom, preferably an O atom.

[0054] In some embodiments, R is C 10 ~C 30 straight chain alkyl groups (e.g., 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 of C, or C interrupted by one or more O or S atoms 10 ~C 30 straight chain alkyl groups (e.g., C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C17 , 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 and optionally, the above C 10 ~C 30 The linear alkyl group of a or optionally substituted with the above C 10 ~C 30 C between two adjacent carbon atoms of the straight-chain alkyl group 3-6 Cycloalkyl groups (C3, C4, C5, C6 cycloalkyl groups, including but not limited to cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl groups) are formed.

[0055] In some embodiments, R is C 14 ~C 24 straight chain alkyl groups (e.g., 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 of C, or C interrupted by one or more O or S atoms 14 ~C 24 straight chain alkyl groups (e.g., 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).

[0056] In some specific embodiments, R1 is [ka] wherein the a-terminus is linked to X2.

[0057] In some embodiments, R a are each independently hydrogen, deuterium, halogen (e.g., fluorine, chlorine, bromine), C 1-6 Alkyl groups (e.g., C1, C2, C3, C4, C5, C6 alkyl groups, including, but not limited to, methyl, ethyl, and isopropyl groups), C 1-6 It is selected from alkoxy groups (e.g., C1 alkoxy groups, C2 alkoxy groups, C3 alkoxy groups, C4 alkoxy groups, C5 alkoxy groups, and C6 alkoxy groups, including, but not limited to, methoxy groups, ethoxy groups, propoxy groups, and isopropoxy groups).

[0058] In some embodiments, R a are each independently selected from hydrogen, deuterium, fluorine, a methyl group, and a methoxy group.

[0059] In some embodiments, B is selected from adenine, guanine, cytosine, uracil, and thymine.

[0060] In some embodiments, the compound of Formula (II) or a pharmaceutically acceptable salt thereof is [ka] [ka] Selected from.

[0061] The present disclosure further provides a method for preparing an oligonucleotide described in the present disclosure, comprising: (1) synthesizing a compound of formula (II) or a pharmaceutically acceptable salt thereof as described herein; (2) synthesizing an oligonucleotide according to the present disclosure using the compound of formula (II) or a pharmaceutically acceptable salt thereof synthesized in step (1); Includes.

[0062] In another aspect, the present disclosure provides a pharmaceutical composition comprising an oligonucleotide described in this disclosure.

[0063] In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

[0064] In some embodiments, the pharmaceutically acceptable excipient may be, for example, a carrier, a transporter, a diluent, and / or a delivery polymer.

[0065] In some embodiments, the content of the oligonucleotide may be 0.01% to 99.99%, 0.1% to 99.9%, 0.5% to 99.5%, or even 1% to 99%, or even 2% to 98%, based on the total weight of the pharmaceutical composition.

[0066] In some embodiments, the content of the pharmaceutically acceptable excipients may be 0.01% to 99.99%, 0.1% to 99.9%, 0.5% to 99.5%, 1% to 99%, or even 2% to 98%, based on the total weight of the pharmaceutical composition.

[0067] In another aspect, the present disclosure provides a method for inhibiting expression of a target gene, comprising administering to a subject an effective amount or dose of an oligonucleotide described herein or a pharmaceutical composition comprising the oligonucleotide. In some embodiments, the target gene is one or more selected from MUC5B, APP, PMP22, and MAPT.

[0068] In some embodiments, the oligonucleotide or pharmaceutical composition may be in a therapeutically effective amount.

[0069] In some embodiments, the unit dose of the oligonucleotide or pharmaceutical composition may be 0.001 to 1000 mg.

[0070] In some embodiments, the effective amount or effective dose of the oligonucleotide or pharmaceutical composition is about 0.001 to about 200 mg / kg body weight, about 0.01 to about 100 mg / kg body weight, or about 0.5 to about 50 mg / kg body weight.

[0071] In another aspect, the disclosure provides a use of the oligonucleotide or the pharmaceutical composition in the preparation of a medicament.

[0072] In some embodiments, the medicament is for preventing and / or treating diseases associated with the lungs, eyes, and central nervous system (CNS) disorders, and in some embodiments, the medicament is for preventing and / or treating diseases associated with the lungs.

[0073] In some embodiments, the medicament is for preventing and / or treating a tumor-related disease.

[0074] In another aspect, the present disclosure provides a method for extrahepatic delivery of an oligonucleotide, comprising administering to a subject an effective amount or dose of an oligonucleotide or pharmaceutical composition described in the present disclosure.

[0075] In some embodiments, the oligonucleotides described in this disclosure are delivered to the lung, eye, or CNS.

[0076] In some embodiments, the oligonucleotides described herein are delivered to the lung.

[0077] In some embodiments, the oligonucleotides described in this disclosure are delivered to a tumor.

[0078] In some embodiments, the oligonucleotides described in this disclosure are delivered to pancreatic cancer, prostate cancer, breast cancer, or lung cancer.

[0079] In the present disclosure, when the oligonucleotide or pharmaceutical composition is contacted with a cell expressing a target gene, the oligonucleotide or pharmaceutical composition inhibits expression of the target gene by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, as measured by, for example, psiCHECK activity screening and luciferase reporter gene detection methods, as well as by other methods such as PCR or branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence assays such as Western blot or flow cytometry.

[0080] In the present disclosure, when the oligonucleotide or pharmaceutical composition is contacted with cells expressing a target gene, the percentage of overexpression of target gene mRNA by the oligonucleotide or pharmaceutical composition is 99% or less, 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less, as measured by, for example, psiCHECK activity screening and luciferase reporter gene detection methods, as well as by PCR or branched DNA (bDNA)-based methods, or protein-based methods, such as immunofluorescence analysis methods such as Western blotting and flow cytometry.

[0081] In the present disclosure, when the oligonucleotide or pharmaceutical composition is contacted with cells expressing a target gene, the oligonucleotide retains on-target activity 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 detection methods, as well as by PCR or branched DNA (bDNA)-based methods, or protein-based methods, for example, immunofluorescence assays such as Western Blot or flow cytometry.

[0082] In the present disclosure, when the oligonucleotide or pharmaceutical composition is contacted with cells expressing a target gene, the oligonucleotide reduces on-target activity by up to 20%, up to 19%, up to 15%, up to 10%, up to 5%, or 1% or more, while simultaneously 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 detection methods, as well as by PCR, branched DNA (bDNA)-based methods, or protein-based methods, for example, immunofluorescence assays such as Western Blot and flow cytometry.

[0083] In the present disclosure, when the dsRNA or pharmaceutical composition is contacted with cells expressing a target gene, the oligonucleotide enhances on-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 detection methods, as well as by PCR, branched DNA (bDNA)-based methods, or protein-based methods, for example, immunofluorescence assays such as Western blot and flow cytometry.

[0084] Unless a configuration is explicitly specified, compounds of the present disclosure may exist in particular geometric or stereoisomeric forms. The present disclosure includes cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic and other mixtures thereof, e.g., enantiomerically or diastereomerically enriched mixtures, and all such compounds are intended to be within the scope of the present disclosure. Substituents such as alkyl groups may have additional asymmetric carbon atoms. All such isomers and mixtures thereof are included within the scope of the present disclosure.

[0085] The compounds of the present disclosure may be asymmetric, e.g., have one or more stereoisomers. Unless a configuration is specified, all stereoisomers include, for example, enantiomers and diastereomers. Compounds of the present disclosure containing asymmetric carbon atoms can be isolated in optically pure or racemic form. Optically pure forms can be resolved from racemic mixtures or synthesized using chiral starting materials or chiral reagents.

[0086] Optically active (R)- and (S)-isomers and D- and L-isomers can be prepared by chiral synthetic methods or using chiral reagents or other techniques conventional in the art. A single enantiomer of a compound of the present disclosure can be prepared by asymmetric synthesis or derivatization with chiral auxiliaries, whereby the resulting diastereomeric mixture is isolated and the resulting diastereomeric mixture is cleaved to provide the desired enantiomer in pure form by cleavage of the auxiliaries. Alternatively, if the molecule contains a basic (e.g., amino) or acidic (e.g., carboxy) functional group, diastereomeric salts can be formed with an appropriate optically active acid or base, followed by diastereomeric separation and recovery using conventional methods well known in the art to obtain the enantiomer in pure form. Separation of enantiomers and diastereomers is typically accomplished by chromatography, employing a chiral stationary phase and optionally combined with chemical derivatization (e.g., carbamate formation from an amine).

[0087] The present disclosure further includes some isotopically labeled compounds of the present disclosure that are the same as those described herein, except that one or more atoms have been replaced with an atom having an atomic mass or mass number different from the atomic mass or mass number normally found in nature. Illustrative isotopes that can be attached to compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, for example, 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, etc.

[0088] Unless otherwise specified, when a position is specifically designated as deuterium (D), it should be understood that the position is deuterium (i.e., at least 10% deuterium incorporated) with an abundance at least 1000 times greater than the natural abundance of deuterium (which is 0.015%). For example, a compound having an abundance greater than the natural abundance of deuterium may be at least 1000 times more abundant, at least 2000 times more abundant, at least 3000 times more abundant, at least 4000 times more abundant, at least 5000 times more abundant, at least 6000 times more abundant, or even greater. The present disclosure further includes various deuterated forms of the compound. Each available hydrogen atom attached to a carbon atom may be independently replaced with a deuterium atom. Those skilled in the art can synthesize deuterated forms of the compound of Formula I by referring to the relevant literature. Deuterated forms of the compounds of Formula I, when prepared, may use commercially available deuterated starting materials or may be synthesized by conventional techniques with deuterated reagents, including, but not limited to, deuterated borane, tritiated borane in tetrahydrofuran, lithium aluminum deuterated hydride, deuterated iodoethane, and deuterated iodomethane.

[0089] In the chemical structures of the compounds described in this disclosure, [ka] indicates that the configuration is not specified, i.e., chiral isomers exist in the chemical structure, [ka] The bond [ka] or [ka] For convenience, all of the above structural formulas are depicted in one isomeric form, but the present disclosure also includes all isomers, such as tautomers, rotamers, geometric isomers, diastereomers, racemates, and enantiomers. In the chemical structures of the compounds described in the present disclosure, [ka] The bond is unspecified in configuration, i.e. [ka] The bond configuration may be E or Z, or may include both E and Z configurations simultaneously. [ka] teeth, [ka] may be

[0090] Terminology In order that the present disclosure may be more readily understood, certain technical and scientific terms are specifically defined below. Unless expressly defined otherwise herein, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art.

[0091] As used herein, "RNAi reagent" refers to a reagent comprising an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule capable of sequence-specifically degrading or inhibiting the transcription and translation of a target messenger RNA (mRNA). In the present disclosure, an RNAi reagent may operate via the RNA interference mechanism (i.e., by inducing RNA interference by interacting with the RNA interference pathway component of mammalian cells (RNA-induced silencing complex, or RISC)), or may act via any other mechanism or pathway. RNAi reagents include, but are not limited to, single-stranded oligonucleotides, single-stranded antisense oligonucleotides, small interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), and Dicer substrates.

[0092] The RNAi reagents described in this disclosure include oligonucleotides having strands at least partially complementary to a target mRNA.

[0093] In the present disclosure, the "5' region" of the sense strand or antisense strand, i.e., the "5' end" and "5' terminus" can be used interchangeably. For example, nucleotides 2 to 8 of the 5' region of the antisense strand can be substituted with nucleotides 2 to 8 of the 5' terminus of the antisense strand. Similarly, the "3' region," "3' terminus," and "3' terminus" of the sense strand or antisense strand can be used interchangeably.

[0094] "Position 6 in the direction from the 5' end to the 3' end of the sense strand", i.e., "position 6 at the 5' end of the sense strand" can be used interchangeably, e.g., 5'N A NNNNN C NNNNNNNNNNNNNNN B In the case where the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is located at position 1 of the 5'-end of the sense strand, N A represents a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, and when the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is at position 1 of the 3'-end of the sense strand, NB represents a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, and when the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is at the 6-position in the direction from the 5'-end to the 3'-end of the sense strand, N C represents a compound represented by formula (I) or a pharmaceutically acceptable salt thereof.

[0095] In the present disclosure, the phrase "one end is linked to the 5th nucleotide at the 5' end of the sense strand, and the other end is linked to the 7th nucleotide at the 5' end of the sense strand" is intended to indicate the positions of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof described in the present disclosure, for example, 5'NNNNN5N6N7NNNNNNNNNNNNNN At 3', the b-terminus in formula (I-1) or (I-2) is linked to N5, the c-terminus is linked to N7, and B in formula (I-1) or (I-2) represents the base of N6, for example, 5'N1N2NNNNNNNNNNNNNNNNNNN 3' is the linking terminal in formula (I-3) or (I-4). [ka] is linked to N2, and B in formulas (I-3) and (I-4) represents the base of N1, and for example, 5'NNNNNNNNNNNNNNNNNNNN 2’ N 1’ 3' is the linking terminal in formula (I-5) or (I-6). [ka] N 2’ and B in formula (I-5) and (I-6) is N 1’ The bases are shown.

[0096] "Optionally" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes cases where the event or circumstance occurs and cases where it does not occur. For example, "C optionally substituted with a halogen or cyano group" 1-6The term "alkyl group" refers to the fact that a halogen or cyano group may or may not be present, and this description includes cases where the alkyl group is substituted with a halogen or cyano group and cases where the alkyl group is not substituted with a halogen or cyano group.

[0097] 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 Among them, K ow is the ratio of the concentration of a chemical in the octanol phase to the concentration in the aqueous phase when the two-phase system is in equilibrium. ow is greater than 0, the chemical is lipophilic. Typically, the log K of the lipophilic moiety is ow is greater than 1, greater than 1.5, greater than 2, greater than 3, greater than 4, greater than 5, or greater than 10, e.g., the log K of 6-aminohexanol ow is approximately 0.7, and the log K ow is 10.7.

[0098] The lipophilicity of a molecule can be altered depending on the functional groups it contains. For example, adding a hydroxyl or amine group to the end of the lipophilic moiety can increase the partition coefficient (e.g., logK ow ) can be increased or decreased. For example, the lipophilic moiety may be aliphatic, cyclic, e.g., alicyclic, or polycyclic, e.g., polyalicyclic compounds such as steroids (e.g., sterols), or a straight or branched chain aliphatic hydrocarbon. The lipophilic moiety may generally comprise a hydrocarbon chain, which may be cyclic or acyclic. The hydrocarbon chain may contain various substituents and / or one or more heteroatoms, such as oxygen or sulfur atoms. Such lipophilic aliphatic moieties may be saturated or unsaturated C4-C 30 Hydrocarbons (e.g., C 10 ~C 30hydrocarbons), saturated or unsaturated fatty acids, waxes (e.g., monohydric alcohol esters of fatty acids and fatty diamides), terpenes (e.g., C 10 Terpene, C 15 Sesquiterpene, C 20 Diterpenes, C 30 Triterpenes and C 40 tetraterpenes) and other polyalicyclic hydrocarbons, for example, the lipophilic moiety may be an optionally substituted C 10-30 The lipophilic moiety may be a straight chain alkyl group of, for example, an optionally substituted C 14-24 It may also be a straight chain alkyl group.

[0099] The term "pharmaceutically acceptable salts" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0100] "Pharmaceutically acceptable acid addition salt" refers to a salt formed with an inorganic or organic acid that is capable of retaining the bioavailability of the free base without other adverse effects. Inorganic acid salts include, but are not limited to, hydrochloride, hydrobromide, sulfate, nitrate, phosphate, etc., and organic acid salts include, but are not limited to, formate, acetate, 2,2-dichloroacetate, trifluoroacetate, propionate, caproate, octanoate, decanoate, undecylenate, glycolate, gluconate, lactate, sebacate, adipate, glutarate, malonate, oxalate, maleate, succinate, fumarate, tartrate, citrate, palmitate, stearate, oleate, cinnamate, laurate, malate, glutamate, pyroglutamate, aspartate, benzoate, mesylate, benzenesulfonate, p-toluenesulfonate, alginate, ascorbate, salicylate, 4-aminosalicylate, naphthalenedisulfonate, etc. These salts can be prepared by methods known in the art.

[0101] "Pharmaceutically acceptable base addition salt" refers to a salt formed with an inorganic or organic base that can retain the bioavailability of the free acid without other adverse effects. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts, and the like. Preferred inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts, with sodium salts being preferred. Salts derived from organic bases include, but are not limited to, salts of primary amines, secondary amines, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, glycine betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamide resins, and the like. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. These salts can be prepared by methods known in the art.

[0102] The term "alkyl group" refers to a saturated aliphatic hydrocarbon group that is a straight or branched chain group containing 1 to 30 carbon atoms, and in some embodiments is selected from alkyl groups containing 10 to 30 carbon atoms. In some embodiments, it is selected from alkyl groups containing 10 to 20 carbon atoms, such as alkyl groups of 14, 15, 16, 17, 18, 19, or 20 carbon atoms. Non-limiting examples include tetradecane, hexadecane, octadecane, eicosane, and the like. In some embodiments, alkyl groups are selected from those containing 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, and when substituted, the substituents may be substituted at any available point of attachment, and in some embodiments, the substituents are independently selected from one or more groups selected from alkyl groups, alkoxy groups, halogens, hydroxy groups, cyano groups, cycloalkyl groups, and heterocycloalkyl groups, and the alkyl groups are optionally substituted with halogens. For example, the alkyl group may be a straight chain group containing 1 to 30 carbon atoms, and 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 , C25 , C 26 , C 27 , C 28 , C 29 , C 30 The straight chain alkyl groups include, but are not limited to:

[0103] The term "alkoxy group" refers to an -O-(alkyl group), where alkyl is defined above. Non-limiting examples of alkoxy groups include methoxy, ethoxy, propoxy, and butoxy groups. An alkoxy group may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably halogen, hydroxy, oxo, cyano, amino, C 1-6 Alkyl group, C 1-6 It is one or more groups independently selected from an alkoxy group, a 3- to 7-membered cycloalkyl group, and a 3- to 7-membered heterocycloalkyl group, and the alkyl group, alkoxy group, cycloalkyl group, or heterocycloalkyl group is optionally substituted with a halogen, a hydroxy group, a nitro group, a cyano group, or an amino group.

[0104] Similarly, a "cycloalkoxy group" and a "heterocycloalkoxy group" are defined in the same manner as the above "alkoxy group."

[0105] The term "cycloalkyl group" or "carbocycle" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent, wherein the cycloalkyl group ring contains 3 to 20 carbon atoms, and in some embodiments, 3 to 7 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, and the like, and polycyclic cycloalkyl groups include spirocyclic, fused, and bridged cycloalkyl groups. Cycloalkyl groups can be substituted or unsubstituted, and if substituted, the substituents can be at any available point of attachment, and in some embodiments, can be halogen, deuterium, hydroxy, oxo, nitro, cyano, C 1-6 Alkyl group, C 1-6Alkoxy group, C 3-6 a cycloalkoxy group, a 3- to 6-membered heterocycloalkoxy group, and 1-6 Alkyl group, C 1-6 Alkoxy group, C 3-6 The cycloalkoxy group and the 3- to 6-membered heterocycloalkoxy group are optionally substituted with one or more groups selected from halogen, deuterium, a hydroxy group, an oxo group, a nitro group, and a cyano group.

[0106] The cycloalkyl ring may be fused to an aryl or heteroaryl group, wherein the ring connected to the parent structure is a cycloalkyl group, non-limiting examples of which include indanyl, tetrahydronaphthyl, benzocycloheptanyl, etc. The cycloalkyl group may be optionally substituted or unsubstituted, and when substituted, the substituents may in some embodiments be halogen, deuterium, hydroxy, oxo, nitro, cyano, C 1-6 Alkyl group, C 1-6 Alkoxy group, C 2-6 Alkenyloxy group, C 2-6 Alkynyloxy group, C 3-6 Cycloalkoxy group, 3- to 6-membered heterocycloalkoxy group, C 3-8 a cycloalkenyloxy group, a 5- to 6-membered aryl group, or a heteroaryl group; 1-6 Alkyl group, C 1-6 Alkoxy group, C 2-6 Alkenyloxy group, C 2-6 Alkynyloxy group, C 3-6 Cycloalkoxy group, 3- to 6-membered heterocycloalkoxy group, C 3-8 The cycloalkenyloxy group, 5- to 6-membered aryl group, or heteroaryl group is optionally substituted with one or more groups selected from halogen, deuterium, hydroxy, oxo, nitro, and cyano groups.

[0107] The term "heterocycloalkyl group" or "heterocycle" or "heterocyclyl group" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which may be nitrogen, oxygen, or S(O). m (wherein m is an integer of 0-2), but does not include the -OO-, -OS-, or -SS- ring moieties, with the remaining ring atoms being carbon. In some embodiments, the group is selected from those containing 3 to 12 ring atoms, of which 1 to 4 are heteroatoms, and in some embodiments, those containing 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, and the like. Polycyclic heterocycloalkyl groups include spirocyclic, fused-ring, and bridged-ring heterocycloalkyl groups. Non-limiting examples of "heterocycloalkyl groups" include: [ka] Includes:

[0108] The heterocyclyl ring may be fused to an aryl group, a heteroaryl group, or a cycloalkyl ring, wherein the ring connected to the parent structure is a heterocyclyl group, non-limiting examples of which are: [ka] Includes:

[0109] The heterocyclyl group may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more groups independently selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogens, mercapto groups, hydroxy groups, nitro groups, cyano groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, heterocycloalkylthio groups, oxo groups, carboxy groups, and carboxylic acid ester groups.

[0110] The term "aryl group" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., rings sharing adjacent pairs of carbon atoms) group having a conjugated π-electron system, preferably 6- to 10-membered, such as phenyl and naphthyl groups. The aryl ring may be fused to a heteroaryl group, heterocyclyl group, or cycloalkyl ring, in which the ring connected to the parent structure is an aryl ring. An "aromatic ring" refers to a ring system in an aryl group. Non-limiting examples of aryl groups are: [ka] Includes.

[0111] The aryl group may be substituted or unsubstituted. When the aryl group is substituted, the substituents are preferably one or more groups independently selected from an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an alkylthio group, an alkylamino group, a halogen atom, a mercapto group, a hydroxy group, a nitro group, a cyano group, a cycloalkyl group, a heterocycloalkyl group, an aryl group, a heteroaryl group, a cycloalkoxy group, a heterocycloalkoxy group, a cycloalkylthio group, a heterocycloalkylthio group, a carboxy group, and a carboxylic acid ester group, and are preferably phenyl groups.

[0112] The term "fused-ring aryl group" may refer to an unsaturated fused-ring structure having aromaticity, containing 8 to 14 ring atoms, formed by two or more ring structures linked together by sharing two adjacent atoms, preferably 8 to 12 ring atoms. Examples include fully unsaturated fused-ring aryl groups such as naphthalene and phenanthrene, as well as partially saturated fused-ring aryl groups such as 3- to 8-membered saturated monocyclic benzocycloalkyl groups and 3- to 8-membered partially saturated monocyclic benzocycloalkyl groups. The term "fused aromatic ring" refers to a ring system in a fused-ring aryl group. Specific examples of fused-ring aryl groups include 2,3-dihydro-1H-indenyl, 1H-indenyl, 1,2,3,4-tetrahydronaphthyl, and 1,4-dihydronaphthyl.

[0113] The term "heteroaryl group" refers to a heteroaromatic ring system containing 1 to 4 heteroatoms and 5 to 14 ring atoms, where the heteroatoms are selected from oxygen, sulfur, and nitrogen. The heteroaryl group is preferably 5 to 12-membered, such as imidazolyl, furanyl, thienyl, thiazolyl, pyrazolyl, oxazolyl, pyrrolyl, tetrazolyl, pyridyl, pyrimidine, thiadiazolyl, or pyrazinyl, preferably imidazolyl, pyrazolyl, pyrimidine, or thiazolyl, more preferably pyrazolyl or thiazolyl. The heteroaryl ring may be fused to an aryl, heterocyclyl, or cycloalkyl ring, where the ring connected to the parent structure is the heteroaryl ring. A "heteroaromatic ring" refers to a ring system within the heteroaryl group. Non-limiting examples of heteroaryl groups include: [ka] Includes.

[0114] Heteroaryl groups may be optionally substituted or unsubstituted, and when substituted, the substituents may in some embodiments be halogen, deuterium, hydroxy, oxo, nitro, cyano, C 1-6 Alkyl group, C 1-6 Alkoxy group, C 2-6Alkenyloxy group, C 2-6 Alkynyloxy group, C 3-6 Cycloalkoxy group, 3- to 6-membered heterocycloalkoxy group, C 3-8 a cycloalkenyloxy group, a 5- to 6-membered aryl group, or a heteroaryl group; 1-6 Alkyl group, C 1-6 Alkoxy group, C 2-6 Alkenyloxy group, C 2-6 Alkynyloxy group, C 3-6 Cycloalkoxy group, 3- to 6-membered heterocycloalkoxy group, C 3-8 The cycloalkenyloxy group, 5- to 6-membered aryl group, or heteroaryl group is optionally substituted with one or more groups selected from halogen, deuterium, hydroxy, oxo, nitro, and cyano groups.

[0115] The term "hydroxyalkyl group" refers to an alkyl group substituted with one or more hydroxy groups, wherein the alkyl group is as defined above.

[0116] The term "hydroxy" refers to an -OH group.

[0117] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0118] The term "haloalkyl group" refers to an alkyl group substituted with a halogen, wherein alkyl is as defined above.

[0119] The term "haloalkoxy" refers to an alkoxy group substituted with a halogen, wherein the alkoxy group is as defined above.

[0120] The term "cyano" refers to -CN.

[0121] The term "nitro group" refers to -NO2.

[0122] The term "oxo" refers to the group =0, e.g., a carbon atom and an oxygen atom are linked by a double bond, thereby forming a ketone or aldehyde group.

[0123] The term "amino group" refers to -NH2.

[0124] The term "carboxy" refers to -C(O)OH.

[0125] The term "substituted" refers to the replacement of any one or more hydrogen atoms on a specified atom (usually a carbon, oxygen, or nitrogen atom) with any group defined herein, provided that the replacement does not exceed the normal valence of the specified atom and results in the formation of a stable compound. Non-limiting examples of substituents include a C1-C6 alkyl group, a C2-C6 alkenyl group, a C2-C6 alkynyl group, a cyano group, a hydroxy group, an oxo group, a carboxy group, a cycloalkyl group, a cycloalkenyl group, a heterocyclyl group, a heteroaryl group, an aryl group, a ketone, an alkoxycarbonyl group, an aryloxycarbonyl group, a heteroaryloxycarbonyl group, or a halogen (e.g., F, Cl, Br, I). When the substituent is a ketone or oxo (i.e., =0), two (2) hydrogens on the atom are replaced.

[0126] "Substituted with one or more..." means that the group may be substituted with a single or multiple substituents. When the group is substituted with multiple substituents, the group may be the same or a combination of one or multiple different substituents.

[0127] In the chemical structural formulas of the present disclosure, the wavy line [ka] indicates the linkage site.

[0128] The term "linked," when referring to a connection between two molecules, refers to a covalent bond between the two molecules or a non-covalent bond (e.g., a hydrogen bond or an ionic bond) between the two molecules, and includes a direct link and an indirect link.

[0129] The term "directly linked" means that a first compound or group is connected to a second compound or group without any intervening atoms or groups of atoms.

[0130] The term "indirectly linked" means that a first compound or group and a second compound or group are linked through an intermediate group, compound, or molecule (eg, a linking group).

[0131] As used herein, in the context of RNA-mediated gene silencing, the sense strand (also referred to as the sense strand, SS or SS strand) of an siRNA refers to the strand containing the same or essentially the same sequence as the target mRNA sequence, and the antisense strand (also referred to as the antisense strand, AS or AS strand) of an siRNA refers to the strand having a sequence complementary to the target mRNA sequence.

[0132] The term "base" includes any known DNA and RNA base and base analogs, such as purines and pyrimidines, as well as the naturally occurring compounds adenine, thymine, guanine, cytosine, uracil, inosine, and naturally occurring analogs.

[0133] As used herein, "chemical modification" or "modification" includes any alteration of a nucleotide by chemical means, such as adding or removing a chemical moiety, or substituting one chemical moiety for another.

[0134] As used herein, the term "fluoro-modified nucleotide" refers to a nucleotide formed by substituting the hydroxy group at the 2'-position of the ribosyl group of a nucleotide with a fluoro group; "non-fluoro-modified nucleotide" refers to a nucleotide or nucleotide analog formed by substituting the hydroxy group at the 2'-position of the ribosyl group of a nucleotide with a non-fluoro group; and "nucleotide analog" refers to a group that can substitute for 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 nucleotides (abbreviated as BNA), and acyclic nucleotides. The methoxy-modified nucleotide refers to a nucleotide formed by substituting the 2'-hydroxy group of the ribosyl group with a methoxy group. An isonucleotide refers to a compound formed by changing the position of the base in the ribose ring of a nucleotide. In some embodiments, an isonucleotide may be a compound formed by moving the base from the 1'-position to the 2'-position or the 3'-position of the ribose ring. BNA refers to a restricted or inaccessible nucleotide. BNAs may comprise a 5-, 6-, or 7-membered bridge structure with a "fixed" C3'-endo sugar structure. Typically, the bridge is incorporated at the 2'- or 4'-position of the ribose to provide a 2',4'-BNA nucleotide. In some embodiments, BNAs may be LNAs, ENAs, cET BNAs, etc. Acyclic nucleotides are a type of nucleotide formed by opening the sugar ring of a nucleotide. In some embodiments, acyclic nucleotides may be unlocked nucleic acids (UNAs) or glycerol nucleic acids (GNAs).

[0135] As used herein, the terms "complementary" and "reverse complementary" can be used interchangeably and have the meaning known to those skilled in the art that in a double-stranded nucleic acid molecule, bases in one strand pair with bases in another strand in a complementary manner. In DNA, the purine base adenine (A) always pairs with the pyrimidine base thymine (T) (or uracil (U) in RNA), and the purine base guanine (C) always pairs with the pyrimidine base cytosine (G). Each base pair contains one purine and one pyrimidine. When adenine in one strand always pairs with thymine (or uracil) in the other strand and guanine always pairs with cytosine, the two strands are considered to be complementary to each other, and the sequence of the strand can be deduced from the sequence of the complementary strand. Accordingly, "mismatch," as used in the art, means that bases at corresponding positions in a double-stranded nucleic acid are not present in a complementary pairing.

[0136] Unless otherwise specified, in the context of this disclosure, capital letters C, G, U, A, and T indicate the composition of the nucleotide base, lowercase letter d indicates that one nucleotide adjacent to the right of the letter d is a deoxyribonucleotide, lowercase letter m indicates that one nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide, lowercase letter f indicates that one nucleotide adjacent to the left of the letter f is a fluoro-modified nucleotide, lowercase letter s indicates that the two nucleotides adjacent to both sides of the letter s are linked by a thiophosphate group, and capital letter VP indicates that the two nucleotides adjacent to the left and right of the letter VP are linked by a vinyl phosphate ester bond, and when VP is at the 5' end of the sequence, it indicates that the one nucleotide adjacent to the right of the letter VP is linked by a vinyl phosphate ester bond.

[0137] A "pharmaceutical composition" comprises an oligonucleotide or double-stranded RNAi inhibitor molecule according to the present disclosure and a pharmaceutically acceptable additive and / or adjuvant, which may be one or more of various agents or compounds commonly used in the art. For example, the pharmaceutically acceptable additive may include at least one of a pH buffering agent, a protective agent, and an osmotic pressure adjusting agent.

[0138] As used herein, the term "inhibition" is used interchangeably with "reduction," "silencing," "downregulation," "suppression," or other similar terms, and includes any level of inhibition.

[0139] The terms "flat-ended" and "blunt-ended" can be used interchangeably and refer to the absence of unpaired nucleotides or nucleotide analogs at a given end of an siRNA, i.e., the absence of overhanging nucleotides. In many cases, siRNAs with both blunt ends are double-stranded throughout their entire length.

[0140] The terms "about" and "approximately" mean that a numerical value falls within an acceptable error range of the specified value as determined by one of ordinary skill in the art, the numerical portion being determined by how it is measured or determined (i.e., the limitations of the measurement system). For example, "about" may mean a standard deviation of within 1 or more than 1. Alternatively, "about" or "essentially including" may mean a variation within a range of up to 20%, e.g., 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, the term "about" preceding a number or range of values ​​includes, in each case, the specified number of embodiments. Unless otherwise specified, when a specific value appears in this application and claims, the meaning of "about" or "essentially including" should be assumed to be within an acceptable error range of the specified value.

[0141] An "effective amount" or "effective dose" is the amount of a drug, compound, or pharmaceutical composition required to achieve any one or more beneficial or desired therapeutic results. With respect to prophylactic use, beneficial or desired results include elimination or reduction of the risk, reduction in severity, or delay in the onset of a condition, including biochemical, histological, and / or behavioral manifestations of the condition, its complications, and intermediate pathological phenotypes manifested during the progression of the condition. With respect to therapeutic use, beneficial or desired results include clinical results, such as a reduction in the incidence of or amelioration of one or more symptoms of various target gene, target mRNA, or target protein conditions of the present disclosure, a reduction in the dosage of other drugs required to treat the condition, an improvement in the therapeutic efficacy of another drug, and / or a delay in the progression of a target gene, target mRNA, or target protein condition of the present disclosure in a patient.

[0142] In some embodiments, the effective amount or effective dose of siRNA is about 0.001 to about 200 mg / kg body weight, about 0.01 to about 100 mg / kg body weight, or about 0.5 to about 50 mg / kg body weight.

[0143] As used herein, the terms "subject," "patient," "subject," or "individual" can be used interchangeably and include a human or non-human animal, for example, a mammal such as a human or monkey.

[0144] The siRNAs provided by the present disclosure can be obtained by conventional preparation methods in the art (e.g., solid-phase synthesis and liquid-phase synthesis). Commercial customization services for solid-phase synthesis are already available. Modified nucleotide groups can be introduced into the siRNAs described in the present disclosure using nucleoside monomers with corresponding modifications. Methods for preparing nucleoside monomers with corresponding modifications and methods for introducing modified nucleotide groups into siRNAs are also well known to those skilled in the art.

[0145] Some abbreviations in this disclosure are defined as follows:

[0146] DCE: dichloroethane; MeOH: methanol, PE: petroleum ether; EtOAc: ethyl acetate, THF: tetrahydrofuran; DMF: dimethylformamide, TFA: trifluoroacetic acid; LiAlH4: lithium aluminum hydride, LiAlD4: ​​Lithium aluminum deuteride TsCl: p-toluenesulfonyl chloride, DMAP: 4-dimethylaminopyridine; MeCN: acetonitrile, NMI: N-methylimidazole; TBDPSCl: tert-butyldiphenylchlorosilane, HoBt: 1-hydroxybenzotriazole; EDCI: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; DIEA: N,N-diisopropylethylamine, DMTrCl: 4,4'-dimethoxytrityl chloride; DMTr: dimethoxytrityl protecting group. [Brief explanation of the drawings]

[0147] [Figure 1] The figure shows the cellular mRNA expression levels in the groups administered with different concentrations of TJR100072, TJR100073, TJR100344, and TJR100345. In the figure, *** indicates p<0.001, and ** indicates p<0.01. [Figure 2] The expression levels of mouse MUC5B mRNA in TJR100072 and TJR100073 are shown as mean values ​​± SEM. [Figure 3] The expression levels of mouse MUC5B mRNA in TJR100073, TJR100344, and TJR100345. Data results are shown as mean values ​​± SEM, and in the figure, *** indicates p<0.001. [Figure 4]The expression levels of mouse MAPT mRNA in TJR101646 and TJR101647. Data results are shown as mean ± SEM, where **** indicates p<0.0001 compared to the blank vector, and *** indicates p<0.001 compared to the blank vector. [Figure 5] The expression level of mouse MAPT mRNA in TJR101740. Data results are shown as mean ± SEM, where **** indicates p<0.0001 compared to the blank vector, and *** indicates p<0.001 compared to the blank vector. [Figure 6] Mouse APP mRNA expression levels in TJR102160, TJR102161, and TJR102162. Data results are shown as mean ± SEM. In the figure, **** indicates p<0.0001 compared to blank vector, ** indicates p<0.01 compared to blank vector, and #### indicates p<0.0001 compared to TJR102160. DETAILED DESCRIPTION OF THE INVENTION

[0148] The present disclosure will be further described below in conjunction with examples, but these examples are not intended to limit the scope of the present disclosure. Experimental methods for which specific conditions are not specified in the examples of the present disclosure generally follow conventional conditions or conditions recommended by the manufacturers of raw materials or products. For reagents for which a specific source is not specified, the reagents can be obtained from any molecular biology reagent supplier with quality / purity suitable for molecular biology use.

[0149] Example Unless otherwise specified, all reagents used in the following examples are commercially available products.

[0150] Example 1. Synthesis with lipid delivery compounds Example 1.1: Synthesis of Compound NA0108 (also referred to as "Uhd") [ka] Compound 1-2: Under a nitrogen atmosphere, compound 1-1 (7.00 g, 32.19 mmol) was dissolved in DMF (60 mL), and then TDBPSCl (9.70 g, 35.41 mmol) and imidazole (2.40 g, 35.41 mmol) were added. The mixture was stirred at room temperature for 2 hours. The reaction mixture was extracted twice with petroleum ether (60 mL), and the combined organic phase was washed three to four times with saturated brine and then dried over Na2SO4. The organic phase was concentrated under reduced pressure to give compound 1-2 (13.42 g, 96.7% yield), which was used directly in the next reaction without further purification.

[0151] Compound 1-4: 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 diglyme (7.9 mL). Under a nitrogen gas atmosphere and an ice bath, boron trifluoride tetrahydrofuran complex (3.0 g) was slowly added dropwise to the mixed solution. Then, the reaction solution was heated for 140 o The mixture was heated to 10°C and reacted for 16 hours. The reaction mixture was cooled to room temperature, poured into water (100 mL) to quench the reaction, and extracted with ethyl acetate (60 mL x 3). The combined organic phase was washed with saturated brine (100 mL), dried over Na2SO4, filtered, and concentrated. The resulting residue was purified by basified normal-phase silica gel column chromatography (DCM:MeOH = 10:1 with 0.1% Et3N, product peak at 5%) to give compound 1-4 (1.32 g, 25.4% yield). LCMS:t R =2.00min, MS(ESI)m / z=467.4[MH] - .

[0152] Compound 1-5: Compound 1-4 (1.02 g, 2.2 mmol) was dissolved in pyridine (8 mL) under a nitrogen atmosphere. 4A molecular sieves and DMTrCl (1.10 g, 3.2 mmol) were added at 0 °C, and the mixture was allowed to react for 16 h. The reaction mixture was quenched by adding water (60 mL) and extracted three times with ethyl acetate (40 mL). The combined organic phase was washed three times with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The resulting residue was purified by basified normal-phase silica gel column chromatography (PE:EtOAc = 1:1 with 0.1% EtN, product peak at 37%) to give compound 1-5 (1.02 g, 62.6% yield). LCMS:t R =2.37min, MS(ESI)m / z=769.7[MH] - . 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).

[0153] Compound NA0108: 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 previously 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 warmed to room temperature and reacted for 3 hours. After filtering the reaction mixture, 5% aqueous sodium bicarbonate solution (20 mL) was added to the reaction mixture. After two extractions with dichloromethane (40 mL), the combined organic phases were dried over sodium sulfate, filtered, and concentrated, and the resulting residue was purified by C18 reverse-phase column chromatography (eluted with HO / MeCN, 0% to 100%) and lyophilized to give compound NA0108 (also referred to as "Uhd") (475 mg, 59.9% yield). LCMS:t R =2.67min, MS(ESI)m / z=969.4[MH] - . 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). 31 P NMR (100MHz, DMSO-d6) δppm 149.14, 148.63.

[0154] Example 1.2: Synthesis of Compound NA0124 [ka] Compound 2-2: Under a nitrogen atmosphere and an ice bath, 15.7 mL of a 2.5 M LiAlH solution in THF was slowly added dropwise to a dry THF solution (60 mL) of compound 2-1 (10.0 g, 39.3 mmol). The reaction mixture was then warmed to room temperature and stirred for 2 hours. The mixture was quenched by the addition of saturated aqueous ammonium chloride in 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 NaSO, filtered, and concentrated to give compound 2-2 (8.77 g, 92.8% yield), which was used directly in the next reaction. LCMS:t R =1.24min, MS(ESI)m / z=239.3[M+H] + .

[0155] Compound 2-3: Under a nitrogen atmosphere, compound 2-2 (8.77 g, 36.5 mmol) was dissolved in DMF (50 mL). In an ice bath, TBDPSCl (11.03 g, 40.1 mmol) and imidazole (2.73 g, 40.1 mmol) were added, and the mixture was allowed to warm to room temperature and react for 2 hours. The reaction mixture 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 give compound 2-3 (14.33 g, 82.0% yield).

[0156] Compound 2-4: Under a nitrogen atmosphere and a dry ice ethanol bath, TFA (5.95 g, 52.1 mmol) was slowly added to a 1N n-hexane solution of ZnEt (52.1 mmol). After stirring for 20 min, 20 mL of a DCM solution of CHCl (22.37 g, 83.5 mmol) was added to the reaction mixture within 10 min. After stirring for an additional 20 min, a DCM solution of compound 2-3 (10.00 g, 20.8 mmol) was slowly added. The mixture was then warmed to room temperature and stirred for 4 h. The reaction mixture 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 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 n-hexane) to give compound 2-4 (7.21 g, 70.0% yield). 1 H NMR (400 MHz, 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).

[0157] Compound 2-6: 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 gas atmosphere and an ice bath, boron trifluoride tetrahydrofuran complex (2.3 g) was slowly added dropwise to the mixed solution. Then, the reaction mixture was heated for 140 o The temperature was raised to 0°C and the reaction was continued for 16 hours. The reaction mixture was cooled to room temperature and quenched by pouring into water (100 mL) and then extracted with EtOAc (60 mL x 3). The organic phases were combined, washed with saturated brine (100 mL), dried over NaSO, filtered, and concentrated. The resulting residue was purified by basified normal-phase silica gel column chromatography (DCM:MeOH = 10:1 with 0.1% EtN, product peak at 5%) to give compound 2-6 (1.08 g, 28.2% yield). LCMS:tR =2.02min, MS(ESI)m / z=479.3[MH] - .

[0158] Compound 2-7: Compound 2-6 (1.00 g, 2.1 mmol) was dissolved in pyridine (8 mL) under a nitrogen atmosphere. 4A molecular sieves and DMTrCl (1.06 g, 3.1 mmol) were added at 0 °C, and the mixture was then warmed to room temperature and reacted for 4 h. The reaction mixture was quenched by adding water (60 mL) and extracted three times with ethyl acetate (40 mL). The combined organic phase was washed three times with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by basified normal-phase silica gel column chromatography (PE:EtOAc = 1:1 with 0.1% EtN, product peak at 37%) to give compound 2-7 (0.86 g, 52.5% yield). LCMS:t R =2.36min, MS(ESI)m / z=781.5[MH] - . 1 H 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).

[0159] Compound NA0124: 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 previously 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. After filtering the reaction mixture, 5% aqueous sodium bicarbonate solution (20 mL) was added to the reaction mixture. After two extractions 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 (eluted with HO / MeCN, 0% to 100%) and lyophilized to give compound NA0124 (841 mg, 79.4% yield). LCMS:t R =2.67min, MS(ESI)m / z=981.6[MH] - . 1 H NMR (400MHz, DMSO-d6) δ ppm 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). 31 P NMR (100MHz, DMSO-d6) δppm 149.14, 148.64.

[0160] Example 1.3: Synthesis of Compound NA130 [ka] Compound 3-2: 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 in an ice bath, and the mixture was then warmed to room temperature and reacted for 16 hours. The reaction mixture 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 give compound 3-2 (19.31 g, 81.5% yield). LCMS:t R =2.04min, MS(ESI)m / z=385.4[M+H] + .

[0161] Compound 3-3: Under a nitrogen atmosphere and an ice bath, LiAlD4 (0.59 g, 14.0 mmol) was added to 10 mL of dry THF. After stirring the solution for 10 min, a THF solution (10 mL) of compound 3-2 (2.61 g, 7.0 mmol) was slowly added to the LiAlD4 solution. The reaction mixture was then warmed to room temperature and stirred for 3 h. The mixture was quenched by adding saturated aqueous ammonium chloride solution 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. LCMS:t R =2.10min, MS(ESI)m / z=381.3[M+H] + .

[0162] Compound 3-4: 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 mmol), and DMAP (2.21 g, 18.1 mmol) were added sequentially at 0 °C. The reaction mixture was then warmed to room temperature and stirred overnight. The reaction mixture was washed twice with water (60 mL) and then with 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 give compound 3-4 (14.5 g, 78.00% yield).

[0163] Compound 3-5: Compound 3-4 (14.49 g, 28.2 mmol) was dissolved in tetrahydrofuran (80 mL) under a nitrogen atmosphere. Then, 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 mixture was then warmed to room temperature and stirred for 3 h. The mixture was quenched by adding ice water (60 mL) in 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 give compound 3-5 (10.1 g, 74.39% yield). 1 H 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).

[0164] Compound 3-7: 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 gas atmosphere and an ice bath, boron trifluoride tetrahydrofuran complex (2.30 g) was slowly added dropwise to the mixed solution. Then, the reaction mixture was heated for 140 o The temperature was raised to 0°C and the reaction was continued for 16 hours. The reaction mixture was cooled to room temperature and quenched by pouring into water (60 mL) and then extracted with EtOAc (50 mL x 3). The combined organic phase was washed with saturated brine (80 mL), dried over Na2SO4, filtered, and concentrated. The resulting residue was purified by basified normal-phase silica gel column chromatography (DCM:MeOH = 10:1 with 0.1% Et3N, product peak at 6%) to give compound 3-7 (0.97 g, 21.19% yield). LCMS:t R =2.05&2.20min, MS(ESI)m / z=469.5[MH] - .

[0165] Compound 3-8: Compound 3-7 (0.76 g, 1.6 mmol) was dissolved in pyridine (7 mL) under a nitrogen atmosphere. 4A molecular sieves and DMTrCl (0.82 g, 2.4 mmol) were added at 0 °C, and the mixture was allowed to warm to room temperature and react for 4 h. The reaction mixture was quenched by adding water (50 mL) and then extracted three times with ethyl acetate (50 mL). The combined organic phase was washed three times with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by basified normal-phase silica gel column chromatography (PE:EtOAc = 1:1 with 0.1% EtN, product peak at 37%) to give compound 3-8 (0.58 g, 46.54% yield). LCMS:t R =2.26&2.50min, MS(ESI)m / z=771.7[MH] - . 1H 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).

[0166] Compound NA0130: 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 previously 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 then warmed to room temperature and reacted for 3 hours. After filtering the reaction solution, 5% aqueous sodium bicarbonate solution (20 mL) was added to the reaction solution, and 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 (eluted with HO / MeCN, 0% to 100%) and lyophilized to obtain compound NA0130 (610 mg, yield 83.41%). LCMS:t R =2.46&2.70min, MS(ESI)m / z=971.8[MH] - . 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). 31 P NMR (100MHz, DMSO-d6) δppm 149.15, 148.64.

[0167] Example 1.4: Synthesis of Compound NA0131 [ka] Compound 4-2: Under a nitrogen atmosphere and an ice bath, LiAlD4 (1.64 g, 38.99 mmol) was added to 20 mL of dry THF. After stirring for 10 min, a solution of compound 4-1 (10.0 g, 38.9 mmol) in tetrahydrofuran (60 mL) was slowly added to the LiAlD4 solution. The reaction mixture was then warmed to room temperature and stirred for 2 h. The mixture was quenched by adding saturated aqueous ammonium chloride solution in an ice bath and 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 give compound 4-2 (7.32 g, 76.8% yield), which was used directly in the next reaction.

[0168] Compound 4-3: Compound 4-2 (7.00 g, 28.6 mmol) was dissolved in DMF (50 mL) under a nitrogen atmosphere. Then, TBDPSCl (8.66 g, 31.5 mmol) and imidazole (2.14 g, 31.5 mmol) were added in an ice bath. The mixture was then warmed to room temperature and reacted for 2 hours. The reaction mixture 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 give compound 4-3 (10.10 g, 73.0% yield). LCMS:t R =2.41min, MS(ESI)m / z=483.8[M+H] + .

[0169] Compound 4-5: Compound 1-3 (1.35 g, 5.9 mmol) was added to a 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 an ice bath, boron trifluoride tetrahydrofuran complex (1.60 g) was slowly added dropwise to the mixture. The reaction was then heated to 140 °C and allowed to proceed for 16 h. The reaction was cooled to room temperature and quenched by pouring into water (60 mL) and then extracted with EtOAc (50 mL × 3). The combined organic phase was washed with saturated brine (80 mL), dried over Na2SO4, filtered, and concentrated. The resulting residue was purified by basified normal-phase silica gel column chromatography (DCM:MeOH = 10:1 with 0.1% Et3N, product peak at 5%) to give compound 4-5 (0.61 g, 21.72% yield). LCMS:t R =2.04min, MS(ESI)m / z=469.6[MH] - .

[0170] Compound 4-6: Compound 4-5 (0.61 g, 1.3 mmol) was dissolved in pyridine (5 mL) under a nitrogen atmosphere. 4A molecular sieves and DMTrCl (0.75 g, 2.2 mmol) were added at 0 °C, and the mixture was allowed to warm to room temperature and react for 4 h. The reaction mixture was quenched by adding water (50 mL) and extracted three times with ethyl acetate (40 mL). The combined organic phase was washed three times with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by basified normal-phase silica gel column chromatography (PE:EtOAc = 1:1 with 0.1% EtN, product peak at 37%) to give compound 4-6 (0.58 g, 58.1% yield). LCMS:t R =2.26&2.37min, MS(ESI)m / z=771.7[MH] - . 1 H NMR (400MHz, DMSO-d6)δppm 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).

[0171] Compound NA0131: 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 previously 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 warmed to room temperature and reacted for 3 hours. The reaction mixture was filtered, and then 5% aqueous sodium bicarbonate solution (20 mL) was added to the reaction mixture. After two extractions 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 (eluted with HO / MeCN, 0% to 100%) and lyophilized to give compound NA0131 (630 mg, 86.3% yield). LCMS:t R =2.54&2.68min, MS(ESI)m / z=971.8[MH] - 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). 31 P NMR (100MHz, DMSO-d6) δppm 149.13, 148.60.

[0172] Example 1.5: Synthesis of Compound NA0133 [ka] Compound 5-1: At 0°C, a 1% HCl solution in methanol (60 mL) was added to a 200 mL solution of D-ribose (30.0 g, 67 mmol), followed by stirring for 3 days. The reaction mixture was neutralized and quenched with solid sodium bicarbonate to form a solid. The filtrate was dried and concentrated to give the crude product. The crude product was redissolved in 500 mL of DMF. Under a nitrogen atmosphere and an ice bath, NaH (36.0 g, 1500.0 mmol) was slowly added to the above solution. The reaction mixture was stirred for 30 minutes, followed by the addition of 4-chlorobenzyl (108.7 g, 675.0 mmol). The reaction mixture was then warmed to room temperature and stirred overnight. The mixture was quenched by adding ice water in an ice bath. The reaction mixture was extracted three times with DCM (150 mL). The combined organic phase was washed with saturated brine, dried over Na2SO4, filtered, and concentrated. The resulting 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%). LCMS:t R =2.09&2.32min, MS(ESI)m / z=561.3[M+Na] + .

[0173] Compound 5-2: Under a nitrogen atmosphere, 60 mL of a 10% SnCl solution in DCM was slowly added to a DCM solution (100 mL) of compound 5-1 (30.00 g, 55.7 mmol), and the reaction was stirred overnight at room temperature. The reaction was neutralized and quenched with saturated aqueous sodium bicarbonate in an ice bath. The reaction solution was extracted three times with DCM (150 mL). The combined organic phase was 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 give compound 5-2 (16.00 g, 69.41% yield). 1H 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). LCMS:t R =2.13min, MS(ESI)m / z=435.1[M+Na] + .

[0174] Compound 5-3: 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), and the reaction mixture was 83 o The temperature was raised to C and stirred for 5 hours. The reaction mixture was filtered and extracted three times with ethyl acetate (100 mL). The combined organic phase was washed successively with aqueous NaSO, saturated aqueous NaHCO, and saturated brine. The organic phase was dried over NaSO, filtered, and concentrated to give the crude product, compound 5-3 (15.11 g).

[0175] Compound 5-4: Under argon atmosphere, -78 o At C, 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). After stirring for 1 hour, a THF solution of compound 5-3 (15.1 g) was added to the reaction mixture and allowed to react overnight with stirring. The reaction mixture was quenched at low temperature with saturated aqueous ammonium chloride. After quenching, the mixture was extracted three times with EA (100 mL). The combined organic phase was 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 give compound 5-4 (10.0 g, 50.49% yield). LCMS:t R=1.81min, MS(ESI)m / z=431.3[M+Na] + .

[0176] Compound 5-5: Under a nitrogen gas 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. o The mixture was heated to 1°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 in an ice bath and stirred for 3–4 hours. The reaction mixture was filtered through diatomaceous earth, the filter cake was washed with ethyl acetate two to three times, and the filtrate was extracted three times with ethyl acetate (200 mL). The combined organic phase was 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 give compound 5-5 (25 g, 91% yield). LCMS:t R =1.50min, MS(ESI)m / z=449.4[M+Na] + .

[0177] Compound 5-6: Compound 5-5 (2.0 g, 4.6 mmol) was dissolved in anhydrous pyridine (20 mL), benzoyl chloride (1.09 mL, 9.36 mmol) was added, and the mixture was stirred at room temperature overnight. The reaction mixture was concentrated, redissolved in dichloromethane (20 mL), washed successively with saturated aqueous sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by reverse-phase column chromatography to give compound 5-6 (2.3 g, 92% yield). LCMS:t R =2.07min, MS(ESI)m / z=553.3[M+Na] + . 1H 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).

[0178] Compound 5-7: 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 mixture became clear. The reaction was continued for half an hour and then concentrated to give 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 the addition of tin tetrachloride (1.4 mL, 12.0 mmol) and stirring 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 phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by reverse-phase column chromatography to give compound 5-7 (1.0 g, 54% yield). LCMS:t R =1.79min, MS(ESI)m / z=633.4[M+Na] + . 1H NMR (400 MHz, DMSO) δ ppm 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).

[0179] Compound 5-8: Compound 5-7 (1.7 g, 2.8 mmol) was dissolved in 7 M aqueous ammonia in methanol (20 mL) and stirred overnight at 60° C. The reaction mixture was concentrated, and the resulting residue was purified by reverse-phase column chromatography to give compound 5-8 (1.05 g, 74% yield). LCMS:t R =1.35min, MS(ESI)m / z=529.4[M+H] + .

[0180] Compound 5-9: In an ice bath, sodium hydride (0.89 g) and compound 5-8 (4.50 g, 8.9 mmol) in N,N-dimethylformamide (15 mL) were slowly added. After stirring for 30 minutes, iodohexadecane (6.30 g, 17.8 mmol) was added and the reaction was warmed to room temperature and stirred for 2 hours. The reaction mixture was quenched with saturated aqueous ammonium chloride and extracted three times with ethyl acetate (30 mL). The combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by reverse-phase column chromatography to give compound 5-9 (3.5 g, 54% yield). LCMS:t R =3.83min, MS(ESI)m / z=731.7[M+H] + .

[0181] Compound 5-10: To a solution of compound 5-9 (3.5 g, 4.8 mmol) in methanol (100 mL), 10% palladium on carbon (0.5 g) was added and the mixture was stirred overnight at room temperature under a hydrogen atmosphere. The reaction mixture was concentrated, and the resulting residue was purified by reverse-phase column chromatography to give compound 5-10 (2.2 g, 96% yield). LCMS:t R =1.45min, MS(ESI)m / z=481.6[MH] + .

[0182] Compound 5-11: To a solution of compound 5-10 (2.2 g, 4.6 mol) in pyridine (20 mL) was added 4,4'-dimethoxytrityl chloride (4.7 g, 13.8 mmol) and the mixture was stirred at room temperature overnight. The reaction was quenched by adding methanol (5 mL) at 0 °C. The reaction mixture was concentrated, redissolved in water (30 mL), and extracted twice with dichloromethane (50 mL). The combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by silica gel column chromatography to give compound 5-11 (2.6 g, 72% yield). LCMS:t R =3.22min, MS(ESI)m / z=783.9[MH] + . 1H NMR (400 MHz, 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).

[0183] Compound NA0133: A solution of compound 5-11 (1.6 g, 2.0 mmol) in dry acetonitrile (15 mL) was added with 3A molecular sieves under a nitrogen atmosphere at room temperature. After stirring at room temperature for 5 minutes, bis(diisopropylamino)(2-cyanoethoxy)phosphine (2.4 g, 8.9 mmol) was added, followed by a previously 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 mixture was then allowed to react at room temperature for 3 hours. After filtration, the reaction mixture was added with 5% aqueous sodium bicarbonate (30 mL), 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 give compound NA0133 (1.6 g, 80% yield). LCMS:t R =3.97min, MS(ESI)m / z=984.1[MH] + . 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).

[0184] Example 2: siRNA synthesis with lipid delivery The synthesis of siRNA was performed using standard phosphoramidite solid-phase synthesis. When synthesizing the modified nucleotide at the 5'-6 position of the SS chain, the original nucleoside phosphoramidite monomer in the parent sequence was replaced with the synthesized phosphoramidite monomer. The synthesis process is briefly described below. Starting with the Universal CPG vector, nucleoside phosphoramidite monomers were linked one by one using the synthesis procedure on a Dr. Oligo 48 synthesizer (Biolytic). Except for 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 Jiaowei, Suzhou Jima, or Jiangsu Shenji Biotechnology Co., Ltd. 5-Ethylthio-1H-tetrazole (ETT) was employed as the activator (0.6 M solution in acetonitrile), a solution of 0.22 M PADS in a 1:1 volume ratio of acetonitrile and trimethylpyridine (Suzhou Ke Lema) was used as the vulcanization reagent, and an iodopyridine / water solution (Kelema) was used as the oxidizing agent.

[0185] After solid-phase synthesis was completed, the oligoribonucleotides were dissolved from the solid support and soaked in a 3:1 solution of 28% aqueous ammonia and ethanol at 50°C for 16 hours. After centrifugation, the supernatant was transferred to a separate centrifuge tube, concentrated, and evaporated to dryness. The resulting oligonucleotides were purified by C18 reverse-phase chromatography using a mobile phase of 0.1 M TEAA and acetonitrile, followed by 3% trifluoroacetic acid to remove DMTr. The target oligonucleotides were collected, lyophilized, and identified as the desired products by LC-MS and quantified by UV (260 nm).

[0186] The resulting single-stranded oligonucleotides were complementarily paired and annealed in an equimolar ratio, and the resulting double-stranded siRNA was dissolved in 1x PBS and adjusted to the concentration required for the experiment before use.

[0187] [Table 1] [Table 2] [Table 3] [Table 4] [Table 5] [Table 6] [Table 7] [Table 8]

[0188] In the modified siRNA sequences of the present disclosure, the structure of Uhd' is: [ka] and The structure of Chd' is [ka] and The structure of NA0124' is [ka] and The structure of NA0131' is [ka] and The structure of NA0133' is [ka] and The structure of VPUms is [ka] is.

[0189] Example 3: Evaluation of in vitro anti-MUC5B activity of oligonucleotides according to the present disclosure using MLE-12 cells The four siRNA sequences in the table were evaluated for in vitro anti-MUC5B activity in MLE-12 cells at three concentration gradients.

[0190] The steps of the siRNA in vitro anti-MUC5B activity experiment are specifically as follows:

[0191] The activity of the four siRNA sequences to inhibit MUC5B mRNA levels was detected in the MLE-12 cell line by transfection using a transfection reagent. The experimental materials and equipment are detailed in Tables 9 and 10.

[0192] [Table 9]

[0193] [Table 10]

[0194] Experimental steps: (1) Cell seeding 1. Preparation for the experiment 1.1 Preparation of MLE-12 cells: Purchased from Kaisei Bio. Anchorage-dependent cells must be counted after complete digestion. Cell viability is 95% or higher. 1.2 MLE-12 cell culture medium: Purchased from Kaisei Bio, stored at 4°C, and removed and allowed to equilibrate to room temperature before the experiment. 1.3 96-well cell culture plates, 1.4 PBS and pancreatin were stored at 4°C and removed and allowed to equilibrate to room temperature before the experiment.

[0195] 2. Cell seeding The day before transfection, MLE-12 cells were seeded into 96-well plates at a density of 1.5 × 10 4 Cells / well were seeded in 100 μL of medium / well.

[0196] 2.1 Place the medium in a 37°C water bath and incubate for 20 minutes before use. 2.2 The cells were washed once with PBS, added pancreatin, and then placed in an incubator for 2 minutes to digest. Culture medium was added to terminate the digestion, and the cells were centrifuged and counted. 20 μL of the homogenous cell suspension was aspirated and mixed with 1 μL of cell counting dye. The static staining was then allowed to stand for 1 minute. 10 μL of the suspension was aspirated and injected into a cell counting plate to calculate the viable cell count (green). 2.3 Depending on the cell count result, add an appropriate volume of medium to make the cell volume 1.5 x 10 4 100 μL of cells were placed in a 96-well plate to provide cells / well, and 200 μL of PBS was added to seal the wells around the edges to prevent medium evaporation. The cell plate was then placed in an incubator at 37°C with 5% CO2 for overnight incubation before transfection the next day.

[0197] (2) Cell transfection experiments 1. Preparation for the experiment 1.1 Preparation of siRNA samples: siRNA samples are quantified to 1 μM and stored at −20° C. for use, requiring a brief centrifugation before use.

[0198] 1.2 Opti-MEM medium and transfection reagent Lipofectamine RNAi Max were stored at 4°C.

[0199] 1.3 8 tubes. 2. Cell Transfection Experiments 2.1 Before transfection, pre-warm MLE-12 cell-specific medium and replace it with MLE-12 cell-specific medium in the cell plate, 90 μL / well.

[0200] 2.2 siRNA dilution: The siRNA was removed from -20°C, thawed, mixed uniformly, and diluted to different concentrations according to Table 11 for use as a working solution. An 8-tube strip labeled Tube A was prepared. 40 μL of Opti-MEM was added to each well. 10 μL of siRNA (stock solution) was added to the well with the highest concentration. After mixing uniformly, 10 μL was removed and diluted sequentially in a gradient to obtain siRNAs of different concentrations. These were then incubated at room temperature for 15 minutes and added to a 96-well plate (final concentrations of 10 nM, 1 nM, 0.1 nM, and 0 nM, respectively).

[0201] [Table 11]

[0202] 2.3 Preparation of Lipofectamine RNAi Max: Lipofectamine RNAi Max was diluted with Opti-MEM and allowed to stand for 5 minutes. The specific preparation ratio was 15 μL of Opti-MEM added to 0.9 μL of Lipofectamine RNAi Max.

[0203] 2.4 Dispense 30 μL of the prepared Lipofectamine RNAi Max into 8 tubes, mix thoroughly by pipetting (without creating bubbles), then add 30 μL of siRNA of different concentrations from Tube A to the 8 tubes containing Lipofectamine RNAi Max and incubate at room temperature for 20 minutes.

[0204] 2.5 The above mixture was added to the culture plate at 10 μL / well, and three parallel wells were prepared for each concentration.

[0205] 2.6 The cells were cultured in a CO2 incubator at 37°C for 24 hours.

[0206] (3) Sample collection and detection After 1 and 24 hours, the cells were collected and total cellular RNA was extracted using a high-throughput cellular RNA extraction reagent kit.

[0207] 2. Reverse transcription and Q-PCR detection. Among them, quantitative real-time PCR detection was performed using SYBR Green detection, and the primer information is shown in Table 12.

[0208] [Table 12]

[0209] 3.Result analysis After the Q-PCR experiment, the corresponding Ct value was obtained according to the threshold automatically set by the system. By comparing the Ct values, the expression of a certain gene can be relatively quantified. The Ct comparison is to calculate the difference in gene expression by the difference with the internal reference gene Ct value, and 2 -△△Ct Also known as ΔΔCt, ΔΔCt = [(Ct of target gene in experimental group - Ct of internal standard in experimental group) - (Ct of target gene in control group - Ct of internal standard in control group)]. Inhibition rate (%) = (1 - excess expression of target gene) × 100%. The experimental results are shown in Table 13 and Figure 1.

[0210] As can be seen from the results, TJR100072, TJR100073, TJR100344 and TJR100345 showed the same level of inhibitory effect on MUC5B mRNA in cell transfection experiments.

[0211] [Table 13]

[0212] Example 4: Mouse MUC5B gene inhibitory activity of oligonucleotides according to the present disclosure The specific procedure is as follows. Six-week-old C57BL / 6N female mice (purchased from Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd.) were allowed to adapt for one week before the experiment began. Before administration on day 0, the compound was prepared in saline, with the drug concentration in each subject being 10 mg / kg. Twelve mice were weighed and divided into three groups of four: the saline group, TJR100072-10mpk, and TJR100073-10mpk. After anesthesia with isoflurane, the mice were intratracheally administered using a mouse lung administration device (purchased from Yuxian Instruments). On day 10, the mice were euthanized, and after cardiac blood collection, the whole lungs were collected, cut into small pieces, and then placed in an RNA lab. RT-PCR was used to detect MUC5B mRNA levels in the whole lungs. The experimental results are shown in Table 14 and Figure 2.

[0213] As can be seen from the results, compared to the saline group, TJR100072 without lipid delivery showed no obvious inhibitory activity, while the positive molecule TJR100073 showed relatively weak inhibitory activity.

[0214] [Table 14]

[0215] Example 5: Mouse MUC5B gene inhibitory activity of oligonucleotides according to the present disclosure The specific procedure is as follows. Six-week-old C57BL / 6N female mice (purchased from Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd.) were allowed to adapt for one week before the experiment began. Before administration on day 0, compounds were prepared using saline, with the drug concentration in each subject being 10 mg / kg. Thirty mice were weighed and divided into three groups of 10: TJR100073-10mpk, TJR100344-10mpk, and TJR100345-10mpk. After anesthesia with isoflurane, the mice were intratracheally administered using a mouse lung administration device (Yuanxian Instrument). On day 10, the mice were euthanized, and after cardiac blood collection, the whole lungs were harvested, cut into small pieces, and then placed in an RNA lab. RT-PCR was used to detect MUC5B mRNA levels in the whole lungs. The experimental results are shown in Table 15 and Figure 3.

[0216] As can be seen from the results, both TJR100344 and TJR100345 showed better inhibitory activity than the positive molecule TJR100073.

[0217] [Table 15]

[0218] Example 6: Evaluation of in vitro anti-APP activity of oligonucleotides according to the present disclosure using A172 cells The oligonucleotides according to the present disclosure were evaluated for in vitro anti-APP activity in A172 cells (purchased from Iwakura Bio) at seven gradient concentrations.

[0219] A172 cells were cultured in Dulbecco's modified eagle medium containing 10% fetal bovine serum at 37°C in 5% CO2. 24 h before transfection, A172 cells were seeded into a 96-well plate at a seeding density of 1 × 10 4Cells were transfected at 100 μL of medium per well. Oligonucleotides according to the present disclosure were transfected using Lipofectamine RNAi MAX (ThermoFisher, 13778150) according to the product instructions, with two parallel wells for each concentration. After 48 hours of treatment, total RNA was extracted from the cells using the high-throughput cell RNA extraction reagent kit FG0417-L / FG0418-XL (Bonchi Medical, magnetic bead method). RNA reverse transcription (Takara, RR037A) and quantitative real-time PCR (Thermo, 4444557) were performed to measure human APP mRNA levels. Human APP mRNA levels were normalized based on the GAPDH internal reference gene level.

[0220] The equipment used in this experiment is shown in Table 16.

[0221] Among them, the real-time quantitative PCR detection was performed using probe Q-PCR detection experiments, and the primer information is shown in Table 17.

[0222] [Table 16]

[0223] Among them, the real-time quantitative PCR detection was performed using probe Q-PCR detection experiments, and the primer information is shown in Table 17.

[0224] [Table 17]

[0225] Results analysis method: After the Q-PCR detection experiment is completed, the system automatically sets the threshold value to obtain the corresponding Ct value. By comparing the Ct values, the expression of a certain gene can be relatively quantified. The Ct comparison means calculating the difference in gene expression based on the difference with the internal reference gene Ct value. -△△CtIt is also called △△Ct = [(Ct of target gene in experimental group - Ct of internal standard in experimental group) - (Ct of target gene in control group - Ct of internal standard in control group)]. Inhibition rate (%) = (1 - excess target gene expression) x 100%. The results are shown as the percentage excess human APP mRNA expression compared to the mock group (untreated group) cells. The IC of the inhibition rate 50 The results are shown in Table 18.

[0226] As can be seen from the results, the oligonucleotides according to the present disclosure exhibited a strong inhibitory effect on APP mRNA.

[0227] [Table 18]

[0228] Example 7: Evaluation of in vitro anti-PMP22 activity of oligonucleotides according to the present disclosure using A172 cells Oligonucleotides according to the present disclosure were evaluated for in vitro anti-PMP22 activity in A172 cells at seven gradient concentrations.

[0229] A172 cells were cultured in Dulbecco's modified eagle medium containing 10% fetal bovine serum at 37°C in 5% CO2. 24 h before transfection, A172 cells were seeded into a 96-well plate at a seeding density of 1 × 10 4 Cells / well, 100 μL of medium in each well.

[0230] Oligonucleotides of the present disclosure were transfected using Lipofectamine RNAi MAX (ThermoFisher, 13778150) according to the product instructions, with two parallel wells for each concentration. After 48 hours of treatment, total RNA was extracted using the high-throughput cell RNA extraction reagent kit FG0417-L / FG0418-XL (Bonchi Medical, magnetic bead method), followed by RNA reverse transcription (Takara, RR037A) and quantitative real-time PCR (Thermo, 4444557) to measure human PMP22 mRNA levels. Human PMP22 mRNA levels were normalized based on the GAPDH internal reference gene level.

[0231] The equipment used in this experiment is as shown in Table 16 above.

[0232] Among them, the real-time quantitative PCR detection was performed using probe Q-PCR detection experiments, and the primer information is shown in Table 19.

[0233] [Table 19]

[0234] Results analysis method: After the Q-PCR experiment, the corresponding Ct value was obtained according to the threshold automatically set by the system. By comparing the Ct values, the expression of a certain gene can be relatively quantified. The Ct comparison is to calculate the difference in gene expression by the difference with the internal reference gene Ct value, and 2 -△△Ct It is also called △△Ct = [(Ct of target gene in experimental group - Ct of internal standard in experimental group) - (Ct of target gene in control group - Ct of internal standard in control group)]. Inhibition rate (%) = (1 - excess expression of target gene) x 100%. The results are shown as the percentage excess expression of human PMP22 mRNA compared to the mock group (untreated group). The IC of the inhibition rate 50 The results are shown in Table 20.

[0235] As can be seen from the results, the oligonucleotides according to the present disclosure exhibited a strong inhibitory effect on PMP22 mRNA.

[0236] [Table 20]

[0237] Example 8: Evaluation of in vitro anti-MAPT activity of oligonucleotides according to the present disclosure using free uptake in Neuro2α cells In vitro anti-MAPT activity was evaluated by performing an in vitro free uptake experiment on the oligonucleotides disclosed herein at five concentrations (1000 nM, 300 nM, 30 nM, 3 nM, 0 nM) in Neuro2α cells (purchased from Shanghai Jitai Cosmetics Biotechnology Co., Ltd.).

[0238] Neuro2α cells were cultured in Eagle's Minimum Essential Medium (EMEM) containing 10% fetal bovine serum at 37°C in 5% CO2. 24 h before free uptake, Neuro2α cells were seeded into 96-well plates at a seeding density of 1.5 × 10 4 Cells were plated per well, with 100 μL of medium per well. The next 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 incubating the cells with the oligonucleotides disclosed herein for 72 hours, total cellular RNA was extracted using the high-throughput cellular RNA extraction reagent kit FG0417-L / FG0418-XL (Bonchi Medical, magnetic bead method). RNA reverse transcription (Takara, RR037A) and quantitative real-time PCR (Thermo, 4444557) were performed to measure mouse MAPT mRNA levels. Mouse MAPT mRNA levels were normalized based on the mouse GAPDH internal reference gene level.

[0239] The equipment used in this experiment is as shown in Table 16 above.

[0240] Among them, the real-time quantitative PCR detection was performed using probe Q-PCR detection experiments, and the primer information is shown in Table 21.

[0241] [Table 21]

[0242] Results analysis method: After the Q-PCR experiment, the corresponding Ct value was obtained according to the threshold automatically set by the system. By comparing the Ct values, the expression of a certain gene can be relatively quantified. The Ct comparison is to calculate the difference in gene expression by the difference with the internal reference gene Ct value, and 2 -△△Ct Also known as ΔΔCt, ΔΔCt = [(Ct of target gene in experimental group - Ct of internal standard in experimental group) - (Ct of target gene in control group - Ct of internal standard in control group)]. Inhibition rate (%) = (1 - excess target gene expression) x 100%. The results are shown as the percentage excess expression of mouse MAPT mRNA compared to the mock (untreated) group cells. The experimental results are shown in Table 22.

[0243] [Table 22]

[0244] As can be seen from the results, the oligonucleotides according to the present disclosure exhibited a strong inhibitory effect on MAPT mRNA in the cell free uptake experiment.

[0245] Example 9: Mouse MAPT gene inhibitory activity of oligonucleotides according to the present disclosure The specific procedure is as follows. Eight-week-old C57BL / 6J male mice (purchased from Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd.) were allowed to adapt for one week before the experiment. Before administration on day 0, compounds were prepared in saline, with a drug concentration of 5 mg / kg in each subject. Mice were weighed and divided into three groups of 6–10 mice: the blank vector group, TJR101646-5mpk, and TJR101647-5mpk. After anesthetization with zoletil, the drug was administered intracerebroventricularly (ICV) using a stereotactic injection system (Ruivode). A single 2 μL injection was administered unilaterally into the right lateral ventricle using standard aseptic surgical procedures, at AP = −0.2 mm (posterior to the bregma), ML = −1.0 mm, and DV = +2.4 mm. After a midline incision was made in the scalp, a burr hole was created at the selected coordinates using a stereotaxic and skull drill. The dura mater was then punctured through the center of the burr hole, and the needle was lowered to the desired depth until it reached the lateral ventricle. The fill volume of the glass electrode was slightly larger than 2 μL. The administration volume was 2 μL, and the injection rate was 1 μL / min. After injection, the needle was left in place and allowed to stabilize for 2 minutes, then slowly withdrawn, pausing for 30 seconds when the needle tip was in the cortical region. On day 14, the mice were euthanized, and mouse cerebral cortical tissue was harvested, flash-frozen in liquid nitrogen, and 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.

[0246] As can be seen from the results, TJR101647 showed better inhibitory activity than the positive molecule TJR101646.

[0247] [Table 23]

[0248] Example 10: Mouse MAPT gene inhibitory activity of oligonucleotides according to the present disclosure The specific procedure is as follows. Eight-week-old C57BL / 6J male mice (purchased from Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd.) were allowed to adapt for one week before the experiment. Before administration on day 0, compounds were prepared in saline, with a drug concentration of 5 mg / kg in each subject. Mice were weighed and divided into two groups of 6–10 mice: a blank vector group and TJR101740-5mpk. After anesthetization with zoletil, the compound was administered intracerebroventricularly (ICV) using a stereotactic injection system (Ruivode). Using standard aseptic surgical procedures, a single 2 μL injection was administered intracerebroventricularly into the right lateral ventricle at AP = −0.2 mm (posterior to the bregma), ML = −1.0 mm, and DV = +2.4 mm. After a midline incision of the scalp, a burr hole was created at the selected coordinates using a stereotactic and cranial drill. The dura mater was then punctured through the center of the burr hole, and the needle was lowered to the desired depth until it reached the lateral ventricle. The filling volume of the glass electrode was slightly greater than 2 μL. The administration volume was 2 μL, and the injection rate was 1 μL / min. After injection, the needle was left in place and allowed to stabilize for 2 minutes, then slowly withdrawn, pausing for 30 seconds when the needle tip was in the cortical region. On day 14, the mice were euthanized, and mouse brain tissue was collected, flash-frozen in liquid nitrogen, and stored at -80°C. RT-PCR was used to detect MAPT mRNA levels in each mouse brain tissue. The experimental results are shown in Table 24 and Figure 5.

[0249] As can be seen from the results, TJR101740 showed strong inhibitory activity against the mRNA levels of MAPT in each mouse brain tissue after unilateral intracerebroventricular administration.

[0250] [Table 24]

[0251] Example 11: Mouse APP gene inhibitory activity of oligonucleotides according to the present disclosure The specific procedure is as follows. Eight-week-old C57BL / 6J male mice (purchased from Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd.) were allowed to adapt for one week before the experiment. Before administration on day 0, compounds were prepared in saline, with a drug concentration of 5 mg / kg in each subject. Mice were weighed and divided into four groups of 6–8 mice: blank vector group, TJR102160, TJR102161, and TJR102162. After anesthesia with zoletil, the compounds were administered intracerebroventricularly (ICV) using a stereotactic injection system (Ruivode). Following standard aseptic surgical procedures, a single 2 μL injection was administered into the right lateral ventricle at an AP of −0.2 mm (posterior to the bregma), a ML of −1.0 mm, and a DV of +2.4 mm. After a midline incision was made on the scalp, a burr hole was created at the selected coordinates using a stereotaxic and skull drill. The dura mater was then punctured through the center of the burr hole, and the needle was lowered to the desired depth until it reached the lateral ventricle. The fill volume of the glass electrode was slightly larger than 2 μL. The administration volume was 2 μL, and the injection rate was 1 μL / min. After injection, the needle was left in place and allowed to stabilize for 2 minutes, then slowly withdrawn, pausing for 30 seconds when the needle tip was in the cortical region. On day 7, the mice were euthanized, and cerebellar tissue was harvested, flash-frozen in liquid nitrogen, and stored at -80°C. APP mRNA levels in the mouse cerebellum were detected by RT-PCR. The experimental results are shown in Table 25 and Figure 6.

[0252] [Table 25]

[0253] As can be seen from the results, the oligonucleotides according to the present disclosure exhibited a strong inhibitory effect on APP mRNA in in vivo mouse experiments.

Claims

1. An oligonucleotide comprising at least one compound represented by formula (I) or a pharmaceutically acceptable salt thereof, wherein the structure of the compound represented by formula (I) is 【Chemistry 1】 Among them, X １ is selected from O, S, N and C atoms, X ２ is selected from O and S atoms, R １ is C １０ ~C ３０ or a straight chain alkyl group of C interrupted by one or more O or S atoms １０ ~C ３０ and optionally, the C １０ ~C ３０ The linear alkyl group of ａ or optionally substituted with said C １０ ~C ３０ C between two adjacent carbon atoms of the linear alkyl group ３－６ A cycloalkyl group is formed, R ２ and R ３ are the same or different and are each independently selected from a bond, hydrogen, an activated phosphate group, an activated phosphate group, a phosphoramidite group, a solid support, an internucleotide linkage group 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 hydroxy protecting group; R ａ are each independently selected from hydrogen, deuterium, halogen, a hydroxy group, a cyano group, an alkyl group, a haloalkyl group, an alkoxy group, a cycloalkyl group, and a heterocycloalkyl group; n is selected from 0 or 1; B is a base, Among them, the compound represented by the formula (I) is 【Chemistry 2】 Not selected from Oligonucleotides.

2. The structure of the compound of formula (I) is 【Transformation 3】 Selected from Among them, R ｂ is selected from OH or SH, and the b- and c-terminus of each are linked to adjacent nucleotides on both sides of the structure of the compound represented by formula (I), The oligonucleotide of claim 1.

3. X １ is selected from an O or S atom, preferably an O atom; The oligonucleotide according to any one of claims 1 to 2.

4. X ２ is selected from an O or S atom, preferably an O atom; The oligonucleotide according to any one of claims 1 to 3.

5. R １ is C １４ ~C ２４ or a straight chain alkyl group of C interrupted by one or more O or S atoms １４ ~C ２４ is selected from the linear alkyl groups The oligonucleotide according to any one of claims 1 to 4.

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

7. R １ teeth, 【Chemistry 4】 wherein the a-terminus is X ２ is connected to The oligonucleotide according to any one of claims 1 to 6.

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

9. The structure of the compound represented by formula (1) is 【Transformation 5】 【Transformation 6】 【Transformation 7】 Selected from Preferably, the structure of the compound represented by formula (1) is 【Transformation 8】 【Chemistry 9】 【Chemistry 10】 Selected from The oligonucleotide according to any one of claims 1 to 8.

10. The oligonucleotide comprises one, two, three or four compounds of formula (I) or a pharmaceutically acceptable salt thereof, preferably the oligonucleotide comprises one compound of formula (I) or a pharmaceutically acceptable salt thereof; The oligonucleotide according to any one of claims 1 to 9.

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

12. The compound represented by formula (I) or a pharmaceutically acceptable salt thereof is at the 6th position in the direction from the 5' end to the 3' end of the sense strand, the b-terminus is linked to the 5th nucleotide at the 5' end of the sense strand, and the c-terminus is linked to the 7th nucleotide at the 5' end of the sense strand, wherein in formula (I), B represents the base of the 6th nucleotide in the direction from the 5' end to the 3' end of the sense strand; Alternatively, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is located at the 1st nucleotide of the 5'-end of the sense strand, wherein B represents the base of the 1st nucleotide of the 5'-end of the sense strand; Alternatively, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is located at the 1st nucleotide of the 3'-end of the sense strand, and B represents the base of the 1st nucleotide of the 3'-end of the sense strand. The oligonucleotide according to any one of claims 2 to 11.

13. The structure of the compound represented by formula (I) is 【Chemistry 11】 Selected from The oligonucleotide of claim 12.

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

15. The oligonucleotide has the structure of the compound represented by formula (I) and at least one other nucleotide is a modified nucleotide. An oligonucleotide according to any one of claims 1 to 14.

16. At least one phosphate group in the sense strand and / or the antisense strand is a phosphate group having a modifying group, preferably a thiophosphodiester group or a 5'-vinylphosphodiester group; The oligonucleotide according to any one of claims 11 to 15.

17. A compound of formula (II) or a pharmaceutically acceptable salt thereof, 【Chemistry 12】 Among them, B, X １ , X ２ , R １ and n are defined as in any one of claims 1 to 9, R Ａ２ is selected from leaving groups, R Ａ３ is selected from phosphorus-containing active reactive groups, or R Ａ３ is selected from leaving groups, R Ａ２ is selected from phosphorus-containing active reactive groups; Among them, the compound represented by the formula (II) is 【Chemistry 13】 Not selected from Preferably, the compound of formula (II) 【Chemistry 14】 Selected from More preferably, the compound of formula (II) is 【Chemistry 15】 【Chemistry 16】 Selected from The compound or a pharmaceutically acceptable salt thereof.

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

19. 17. A composition comprising the oligonucleotide of any one of claims 1 to 16, preferably further comprising one or more pharmaceutically acceptable excipients. Pharmaceutical compositions.

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

21. Use of an oligonucleotide according to any one of claims 1 to 16 or a pharmaceutical composition according to claim 19 in the preparation of a medicament for the prevention and / or treatment of diseases associated with disorders of the lungs, eyes and central nervous system, and diseases associated with tumors. use.

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

23. The oligonucleotide is delivered to the lung, eye, or central nervous system, preferably the oligonucleotide is delivered to the central nervous system.

23. The method of claim 22.

24. The oligonucleotide is delivered to a tumor, preferably the oligonucleotide is delivered to pancreatic cancer, prostate cancer, breast cancer, lung cancer, more preferably the oligonucleotide is delivered to pancreatic cancer and prostate cancer; 23. The method of claim 22.

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