Modified nucleoside compound and use thereof in oligonucleotides

Abstract

The present invention relates to the field of biopharmaceuticals, and specifically relates to a modified nucleoside compound and the use thereof in oligonucleotides. Provided in the present invention is a nucleoside compound represented by formula (I). The compound can be used for preparing an oligonucleotide. The oligonucleotide modified with the above nucleoside compound has no cytotoxicity or immunogenicity, and has a better biocompatibility, stability and safety. By means of such modification, the safety and efficacy of the oligonucleotide can be significantly improved, which is expected to promote the development of gene therapy technology. Moreover, by means of conjugating same with different ligands, this class of compounds can achieve target gene silencing in CNS tissue, adipose tissue, and muscle tissue, thereby enabling delivery to the CNS tissue, adipose tissue, and muscle tissue.

Description

A modified nucleoside compound and its application in oligonucleotides Technical Field

[0001] This invention relates to the field of biopharmaceuticals, and more specifically to a modified nucleoside compound and its application in oligonucleotides. Background Technology

[0002] Oligonucleotides are polymers of nucleotides that, as nucleic acid inhibitor molecules, can regulate the levels of target mRNAs within cells and have shown early promise in the treatment of genetic diseases, metabolic diseases, inflammatory diseases, cancer, and viral infections. Nucleic acid inhibitor molecules can regulate mRNA expression through a variety of mechanisms, including RNA interference (RNAi).

[0003] RNAi is a conserved pathway found in most eukaryotes, in which a double-stranded RNA molecule (dsRNA) inhibits the expression of a target mRNA with a complementary sequence to the dsRNA. In one typical RNAi pathway, the longer dsRNA is cleaved by a nuclease (Dicer) into a shorter RNA duplex called small interfering RNA (“siRNA”). siRNA has been shown to associate with the nuclease, trans-activating response RNA-binding protein (TRBP), and Argonaute 2 (“Ago2”) to form a complex, sometimes referred to as the RNA-induced silencing complex (“RISC”). Ago2 is a nuclease that uses the antisense strand (also known as the guide strand) of the siRNA to guide the sequence-specific cleavage of the target mRNA.

[0004] Various double-stranded RNAi inhibitor molecular structures have been developed over the years. For example, early work on RNAi inhibitor molecules focused on double-stranded nucleic acid molecules mimicking natural siRNA, where each strand has 19-25 nucleotides and includes at least one 3' overhang with 1 to 6 nucleotides (see, for example, U.S. Patent No. 8,372,968). Subsequently, longer double-stranded RNAi inhibitor molecules were developed, which are cleaved in vivo by endonucleases into active RNAi inhibitor molecules (see, for example, U.S. Patent No. 8,883,996). Subsequent work developed extended double-stranded nucleic acid inhibitor molecules in which at least one end of at least one strand extends beyond the double-stranded target region of the molecule, one of the strands comprising a thermodynamically stable tetracyclic structure (see, for example, U.S. Patent Nos. 8,513,207, 8,927,705, WO 2010 / 033225, and WO 2016 / 100401). These structures include single-strand extensions (on one or both sides of the molecule) and double-strand extensions.

[0005] In recent years, many modified nucleosides have been used for RNAi, such as common modifications like 2'-fluorine, 2'-methoxy, 2'-methoxyethyl, UNA, LNA, BNA, GNA, and 5'-(E)-vinyl phosphate. In some cases, chemical modifications are introduced into nucleic acid inhibitor molecules to obtain potentially desired properties after in vivo administration. These modifications include those designed to, for example, antagonize nucleases or other enzymes that interfere with the structure or activity of oligonucleotides, increase cellular uptake of oligonucleotides, or improve the pharmacokinetic properties of oligonucleotides. Introducing modified nucleosides into the corresponding oligonucleotide sequences improves the pharmacokinetic properties and bioavailability of oligonucleotide drugs in vivo. Further research is needed on the chemical modification of oligonucleotide drugs to obtain oligonucleotide drugs with superior properties in all aspects. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention aims to develop a nucleoside compound with a 1'-position extension, which possesses more stable conformation and metabolic stability. When applied to oligonucleotide molecules (including but not limited to siRNA, antisense nucleic acids, saRNA, miRNA aptamers, and lncRNA), it can modify the pharmacokinetics of oligonucleotides while simultaneously improving the activity and metabolic stability of the oligonucleotide molecules.

[0007] One objective of this invention is to provide a nucleoside compound (nucleoside phosphoramide compound, as shown in Formula 1, for modifying nucleotides, i.e., a nucleoside backbone structure with 1' position extension modification)

[0008] This invention first provides a nucleoside phosphoramidide compound as shown in Formula 1, its stereoisomers, and deuterated compounds:

[0009] in,

[0010] R 1 R 2 R 3 R 4 R 5 R 9 R 10 Each is independently selected from hydrogen, deuterium, halogen, cycloalkyl, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl.

[0011] R 6 Selected from hydrogen, deuterium, halogen, cycloalkyl, substituted or unsubstituted C1-C 30 Alkyl, substituted or unsubstituted C1-C 30 Alkoxy, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C 20 alkynyl group, OR 14 NR 14 R 15 SR 14 CR 14 R 15 ;where R 14 R 15 Each C1-C group is independently selected from hydrogen, acyl, cycloalkyl, substituted or unsubstituted groups. 30 Alkyl, substituted or unsubstituted C1-C 30 Alkoxy, substituted or unsubstituted C2-C 20 alkenyl, substituted or unsubstituted C2-C 10 alkynyl group;

[0012] R 7 R 8 Each of the following is independently selected from hydrogen, deuterium, halogen, cycloalkyl, substituted or unsubstituted C1-C 30 Alkyl, substituted or unsubstituted C1-C 30 Alkoxy, substituted or unsubstituted C2-C 20 alkenyl, substituted or unsubstituted C2-C 10 Alkyne group, -OR 14 -NR 14 R 15 -SR 14 -CR 14 R 15 , where R 14 R 15 Each C1-C group is independently selected from hydrogen, acyl, cycloalkyl, substituted or unsubstituted groups. 30 Alkyl, substituted or unsubstituted C1-C 30 Alkoxy, substituted or unsubstituted C2-C 20 alkenyl, substituted or unsubstituted C2-C 10 alkynyl group;

[0013] Or, R 7 R 8 Each is independently selected from -(CH2) n OP 2 -(CH2) n SP 2 ;where P 2 The components are P(M)2, P(OM)(N(M)2), and P(M)(N(M)2); where M is selected from hydrogen, C1-C 20 Alkyl, wherein the C1-C 20 Alkyl groups can be substituted with one or more halogen, deuterium, or cyano groups; n is any integer from 0 to 10;

[0014] Z represents -O-, -S-, -Se-, -NR 11 or -CR 11 R 12 , where R 11 and R 12 Each of them is independently hydrogen, halogen, acyl, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic or substituted or unsubstituted cycloalkyl;

[0015] W is independently selected from -(CH2). n O- or -(CH2) n S-, where n is any integer from 0 to 10;

[0016] Base can be a natural nucleobase, a modified nucleobase, a universal base, or an aryl base;

[0017] P 1 It is a hydroxyl protecting group; preferably, P 1 For DMTr and MMTr.

[0018] In some embodiments of the present invention, the compound represented by Formula 1 has the structure represented by Formula 2a or Formula 2b:

[0019] in,

[0020] R 1 R 2 R 3 R 4 R 5 R 9 R 10 Each is independently selected from hydrogen, deuterium, halogen, cycloalkyl, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl.

[0021] R 6 R 7 R 8 Each is independently selected from hydrogen, deuterium, halogen, cycloalkyl, substituted or unsubstituted C1-C 30 Alkyl, substituted or unsubstituted C1-C 30 Alkoxy, substituted or unsubstituted C2-C 20 alkenyl, substituted or unsubstituted C2-C 10 alkynyl group, OR 14 NR 14 R 15 SR 14 CR 14 R15 ;where R 14 R 15 Each is independently selected from hydrogen, acyl, cycloalkyl, substituted or unsubstituted C1-C 30 Alkyl, substituted or unsubstituted C1-C 30 Alkoxy, substituted or unsubstituted C2-C 20 alkenyl, substituted or unsubstituted C2-C 10 alkynyl group;

[0022] Z represents -O-, -S-, -Se-, -NR 11 or -CR 11 R 12 , where R 11 and R 12 Each of them is independently hydrogen, halogen, acyl, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic or substituted or unsubstituted cycloalkyl;

[0023] X and W are each independently selected from -(CH2). n O- or -(CH2) n S-, where n is any integer from 0 to 10;

[0024] Base can be a natural nucleobase, a modified nucleobase, a universal base, or an aryl base;

[0025] P 1 It is a hydroxyl protecting group; preferably, P 1 For DMTr and MMTr;

[0026] P 2 The components are P(M)2, P(OM)(N(M)2), and P(M)(N(M)2); where M is selected from hydrogen, C1-C 20 Alkyl, wherein the C1-C 20 Alkyl groups can be replaced by one or more halogen, deuterium, or cyano groups.

[0027] In some embodiments of the present invention, the compound represented by Formula 1 has a structure as shown in Formulas 3a and 3b:

[0028] in,

[0029] R 1 R 2 R 9 R 10 Each is independently selected from hydrogen, deuterium, halogen, cycloalkyl, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl.

[0030] R 6 R 7 R 8 Each is independently selected from hydrogen, deuterium, halogen, cycloalkyl, substituted or unsubstituted C1-C 30 Alkyl, substituted or unsubstituted C1-C 30 Alkoxy, substituted or unsubstituted C2-C 20 alkenyl, substituted or unsubstituted C2-C 10 alkynyl group, OR 14 NR 14 R 15 SR 14 CR 14 R 15 ;where R 14 R 15 Each is independently selected from hydrogen, acyl, cycloalkyl, substituted or unsubstituted C1-C 30 Alkyl, substituted or unsubstituted C1-C 30 Alkoxy, substituted or unsubstituted C2-C 20 alkenyl, substituted or unsubstituted C2-C 10 alkynyl group;

[0031] Z represents -O-, -S-, -Se-, -NR 11 or -CR 11 R 12 , where R 11 and R 12 Each of them is independently hydrogen, halogen, acyl, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic or substituted or unsubstituted cycloalkyl;

[0032] X and W are each independently selected from -(CH2). n O- or -(CH2) n S-, where n is any integer from 0 to 10;

[0033] Base can be a natural nucleobase, a modified nucleobase, a universal base, or an aryl base;

[0034] P 1 It is a hydroxyl protecting group; preferably, P 1 For DMTr and MMTr;

[0035] P 2 The components are P(M)2, P(OM)(N(M)2), and P(M)(N(M)2); where M is selected from hydrogen, C1-C 20 Alkyl, wherein the C1-C 20 Alkyl groups can be replaced by one or more halogen, deuterium, or cyano groups.

[0036] In some embodiments of the present invention, the compound represented by Formula 1 has a structure as shown in Formulas 4a and 4b:

[0037] in,

[0038] R 2 Selected from hydrogen, deuterium, halogen, cycloalkyl, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl.

[0039] R 6 R 7 R 8 Each is independently selected from hydrogen, deuterium, halogen, cycloalkyl, substituted or unsubstituted C1-C 30 Alkyl, substituted or unsubstituted C1-C 30 Alkoxy, substituted or unsubstituted C2-C 20 alkenyl, substituted or unsubstituted C2-C 10 alkynyl group, OR 14 NR 14 R 15 SR 14 CR 14 R 15 ;where R 14 R 15 Each is independently selected from hydrogen, acyl, cycloalkyl, substituted or unsubstituted C1-C 30 Alkyl, substituted or unsubstituted C1-C 30 Alkoxy, substituted or unsubstituted C2-C 20 alkenyl, substituted or unsubstituted C2-C 10 alkynyl group;

[0040] Z represents -O-, -S-, -Se-, -NR 11 or -CR 11 R 12 , where R 11 and R 12 Each of them is independently hydrogen, halogen, acyl, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic or substituted or unsubstituted cycloalkyl;

[0041] X and W are each independently selected from -(CH2). n O- or -(CH2) n S-, where n is any integer from 0 to 10;

[0042] Base can be a natural nucleobase, a modified nucleobase, a universal base, or an aryl base;

[0043] P 1 It is a hydroxyl protecting group; preferably, P 1 For DMTr and MMTr;

[0044] P 2 The components are P(M)2, P(OM)(N(M)2), and P(M)(N(M)2); where M is selected from hydrogen, C1-C 20 Alkyl, wherein the C1-C 20 Alkyl groups can be replaced by one or more halogen, deuterium, or cyano groups.

[0045] In some embodiments of the present invention, the compound represented by Formula 1 has a structure as shown in Formula 5a or Formula 5b:

[0046] in,

[0047] R 7 R 8 Each is independently selected from hydrogen, deuterium, halogen, cycloalkyl, substituted or unsubstituted C1-C 30 Alkyl, substituted or unsubstituted C1-C 30 Alkoxy, substituted or unsubstituted C2-C 20 alkenyl, substituted or unsubstituted C2-C 10 alkynyl group, OR 14 NR 14 R 15 SR 14 CR 14 R 15 ;where R 14 R 15 Each is independently selected from hydrogen, acyl, cycloalkyl, substituted or unsubstituted C1-C 30 Alkyl, substituted or unsubstituted C1-C 30 Alkoxy, substituted or unsubstituted C2-C 20 alkenyl, substituted or unsubstituted C2-C 10 alkynyl group;

[0048] Z represents -O-, -S-, -Se-, -NR 11 or -CR 11 R 12 , where R 11 and R 12 Each of them is independently hydrogen, halogen, acyl, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic or substituted or unsubstituted cycloalkyl;

[0049] X is selected from -(CH2) n O- or -(CH2) nS-, where n is any integer from 0 to 10;

[0050] Base can be a natural nucleobase, a modified nucleobase, a universal base, or an aryl base;

[0051] P 1 It is a hydroxyl protecting group; preferably, P 1 For DMTr and MMTr;

[0052] P 2 The components are P(M)2, P(OM)(N(M)2), and P(M)(N(M)2); where M is selected from hydrogen, C1-C 20 Alkyl, wherein the C1-C 20 Alkyl groups can be replaced by one or more halogen, deuterium, or cyano groups.

[0053] In some embodiments of the present invention, the compound represented by Formula 1 has a structure as shown in Formula 6a or Formula 6b:

[0054] in,

[0055] R 7 R 8 Each is independently selected from hydrogen, deuterium, halogen, cycloalkyl, substituted or unsubstituted C1-C 30 Alkyl, substituted or unsubstituted C1-C 30 Alkoxy, substituted or unsubstituted C2-C 20 alkenyl, substituted or unsubstituted C2-C 10 alkynyl group, OR 14 NR 14 R 15 SR 14 CR 14 R 15 ;where R 14 R 15 Each is independently selected from hydrogen, acyl, cycloalkyl, substituted or unsubstituted C1-C 30 Alkyl, substituted or unsubstituted C1-C 30 Alkoxy, substituted or unsubstituted C2-C 20 alkenyl, substituted or unsubstituted C2-C 10 alkynyl group;

[0056] Z represents -O-, -S-, -Se-, -NR 11 or -CR 11 R 12 , where R 11 and R 12Each of them is independently hydrogen, halogen, acyl, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic or substituted or unsubstituted cycloalkyl;

[0057] Base can be a natural nucleobase, a modified nucleobase, a universal base, or an aryl base;

[0058] P 2 The components are P(M)2, P(OM)(N(M)2), and P(M)(N(M)2); where M is selected from hydrogen, C1-C 20 Alkyl, wherein the C1-C 20 Alkyl groups can be replaced by one or more halogen, deuterium, or cyano groups.

[0059] In some embodiments of the present invention, the compound represented by Formula 1 has a structure as shown in Formula 7a or Formula 7b:

[0060] in,

[0061] R 7 R 8 Each is independently selected from hydrogen, deuterium, halogen, cycloalkyl, substituted or unsubstituted C1-C 30 Alkyl, substituted or unsubstituted C1-C 30 Alkoxy, substituted or unsubstituted C2-C 20 alkenyl, substituted or unsubstituted C2-C 10 alkynyl group, OR 14 NR 14 R 15 SR 14 CR 14 R 15 ;where R 14 R 15 Each is independently selected from hydrogen, acyl, cycloalkyl, substituted or unsubstituted C1-C 30 Alkyl, substituted or unsubstituted C1-C 30 Alkoxy, substituted or unsubstituted C2-C 20 alkenyl, substituted or unsubstituted C2-C 10 alkynyl group;

[0062] Base can be a natural nucleobase, a modified nucleobase, a universal base, or an aryl base.

[0063] In some embodiments of the present invention, the Base is selected from the following structures:

[0064] In some embodiments of the present invention, the nucleoside phosphoramidide compound of Formula 1, wherein...

[0065] R1 Selected from hydrogen;

[0066] R 2 Selected from hydrogen, halogens, and methoxy groups;

[0067] R 3 Selected from hydrogen, deuterium, and methyl;

[0068] R 4 Selected from hydrogen and deuterium;

[0069] R 5 Selected from hydrogen;

[0070] R 6 Selected from hydrogen, deuterium, halogens, and methyl groups;

[0071] R 7 Selected from hydrogen, halogens, -OR 14 , where R 14 Selected from acyl, cyclopropane, substituted or unsubstituted C1-C 22 Alkyl, substituted or unsubstituted C3 alkoxy, C3 alkynyl;

[0072] Or, R 7 Selected from -OP 2 ;where P 2 P(OM)(N(M)2); where M is selected from C2-C3 alkyl groups, wherein the C2-C3 alkyl group may be substituted with a cyano group;

[0073] R 8 Selected from -OR 14 , where R 14 Selected from C1-C 22 alkyl;

[0074] Or, R 8 Selected from -OP 2 ;where P 2 P(OM)(N(M)2) and P(M)(N(M)2); wherein M is selected from C1-C3 alkyl groups, and the C1-C3 alkyl groups may be substituted with cyano groups;

[0075] R 9 Selected from hydrogen, deuterium, and methyl;

[0076] R 10 Selected from hydrogen, deuterium, and methyl;

[0077] Z is -O-, -S-, or -CR 11 R 12 , where R 11 and R 12 Each is independently selected from hydrogen;

[0078] W is selected from oxygen;

[0079] Base is selected from the following structure:

[0080] P 1 For DMTr and MMTr.

[0081] In some embodiments of the present invention, the nucleoside compound represented by Formula 1 of the present invention includes, but is not limited to, the following nucleoside phosphoramide compounds:

[0082] The present invention also provides the use of the above-mentioned nucleoside phosphoramide compound in the preparation of oligonucleotides.

[0083] The present invention also provides the use of the above-mentioned nucleoside phosphoramide compound as an intermediate in the preparation of oligonucleotides.

[0084] Furthermore, the oligonucleotide is selected from siRNA, antisense nucleic acid, saRNA, miRNA, nucleic acid aptamer, and lncRNA.

[0085] The present invention also provides an oligonucleotide comprising at least one structure shown in Formula 8:

[0086] in,

[0087] R 1 R 2 R 3 R 4 R 5 R 9 R 10 Each is independently selected from hydrogen, deuterium, halogen, cycloalkyl, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl.

[0088] R 6 Selected from hydrogen, deuterium, halogen, cycloalkyl, substituted or unsubstituted C1-C 30 Alkyl, substituted or unsubstituted C1-C 30 Alkoxy, substituted or unsubstituted C2-C 20 alkenyl, substituted or unsubstituted C2-C 10 alkynyl group, OR 14 NR 14 R 15 SR 14 CR 14 R 15;where R 14 R 15 Each C1-C group is independently selected from hydrogen, acyl, cycloalkyl, substituted or unsubstituted groups. 30 Alkyl, substituted or unsubstituted C1-C 30 Alkoxy, substituted or unsubstituted C2-C 20 alkenyl, substituted or unsubstituted C2-C 10 alkynyl group;

[0089] R 17 R 18 Each of the following is independently selected from hydrogen, deuterium, halogen, cycloalkyl, substituted or unsubstituted C1-C 30 Alkyl, substituted or unsubstituted C1-C 30 Alkoxy, substituted or unsubstituted C2-C 20 alkenyl, substituted or unsubstituted C2-C 10 Alkyne group, -OR 14 -NR 14 R 15 -SR 14 -CR 14 R 15 , where R 14 R 15 Each C1-C group is independently selected from hydrogen, acyl, cycloalkyl, substituted or unsubstituted groups. 30 Alkyl, substituted or unsubstituted C1-C 30 Alkoxy, substituted or unsubstituted C2-C 20 alkenyl, substituted or unsubstituted C2-C 10 alkynyl group;

[0090] Or, R 17 R 18 Each is independently selected from -(CH2) n OP 4 -、-(CH2) n SP 4 -, where P4 is Where P 3 OH, substituted or unsubstituted C1-C 20 Alkyl, substituted or unsubstituted C1-C 20 Alkyl group; Y is S, O, or BH2;

[0091] Z represents -O-, -S-, -Se-, -NR 11 or -CR 11 R 12 , where R 11 and R 12Each of them is independently hydrogen, halogen, acyl, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic or substituted or unsubstituted cycloalkyl;

[0092] W is selected from -(CH2) n O- or -(CH2) n S-, where n is any integer from 0 to 10;

[0093] Base can be a natural nucleobase, a modified nucleobase, a universal base, or an aryl base.

[0094] The present invention also provides a siRNA comprising at least one structure shown in formula 9a or 9b:

[0095] in,

[0096] R 1 R 2 R 3 R 4 R 5 R 9 R 10 Each is independently selected from hydrogen, deuterium, halogen, cycloalkyl, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl.

[0097] R 6 R 7 R 8 Each is independently selected from hydrogen, deuterium, halogen, cycloalkyl, substituted or unsubstituted C1-C 30 Alkyl, substituted or unsubstituted C1-C 30 Alkoxy, substituted or unsubstituted C2-C 20 alkenyl, substituted or unsubstituted C2-C 10 alkynyl group, OR 14 NR 14 R 15 SR 14 CR 14 R 15 ;where R 14 R 15 Each is independently selected from hydrogen, acyl, cycloalkyl, substituted or unsubstituted C1-C 30 Alkyl, substituted or unsubstituted C1-C 30 Alkoxy, substituted or unsubstituted C2-C 20 alkenyl, substituted or unsubstituted C2-C 10 alkynyl group;

[0098] Z represents -O-, -S-, -Se-, -NR 11 or -CR 11 R 12 , where R 11 and R 12 Each of them is independently hydrogen, halogen, acyl, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic or substituted or unsubstituted cycloalkyl;

[0099] X and W are each independently selected from -(CH2). n O- or -(CH2) n S-, where n is any integer from 0 to 10;

[0100] Base can be a natural nucleobase, a modified nucleobase, a universal base, or an aryl base;

[0101] P 3 OH, substituted or unsubstituted C1-C 20 Alkyl, substituted or unsubstituted C1-C 20 Alkoxy;

[0102] Y represents S, O, and BH2.

[0103] Preferably, the siRNA is a double-stranded conjugate having a sense strand and an antisense strand; the nucleotide length of the sense strand is 14-40; and the nucleotide length of the antisense strand is 14-40.

[0104] Preferably, at least one of the following sites of the siRNA comprises the structure described in Formula 9a or 9b: positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 starting from the 5' end of the sense strand, and positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, and 23 starting from the 5' end of the antisense strand.

[0105] Unless otherwise stated, the terms used in the specification and claims have the following meanings.

[0106] The carbon, hydrogen, oxygen, sulfur, nitrogen, or F, Cl, Br, I mentioned in the groups and compounds described in this application include their isotopes, and the carbon, hydrogen, oxygen, sulfur, or nitrogen mentioned in the groups and compounds described in this application may optionally be further replaced by one or more of their corresponding isotopes, wherein the isotopes of carbon include 12 C 13 C and14 C, the isotopes of hydrogen include protium (H), deuterium (D, also called heavy hydrogen), and tritium (T, also called superheavy hydrogen), and the isotopes of oxygen include 16 O、 17 O and 18 O, isotopes of sulfur include 32 S, 33 S, 34 S and 36 S, nitrogen isotopes include 14 N and 15 N, isotopes of fluorine include 17 F and 19 F, isotopes of chlorine include 35 Cl and 37 Cl, isotopes of bromine include 79 Br and 81 Br.

[0107] In this invention, "oligonucleotide" refers to a polymer of linked nucleosides, each nucleoside being independently modified or unmodified, comprising an oligonucleotide sequence of approximately 10-50 single-stranded or double-stranded nucleotide base pairs. In some embodiments, the oligonucleotide has a nucleobase sequence at least partially complementary to the core sequence of a target gene expressed in cells. In some embodiments, the oligonucleotide, upon delivery to a cell expressing the gene, regulates the expression of the corresponding target gene. Target gene expression can be regulated in vitro or in vivo. "Oligonucleotide" includes, but is not limited to: single-stranded antisense oligonucleotides, short interfering RNA (nucleic acid), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), non-coding RNA (lncRNA), ribozymes, interfering RNA molecules, and dicer enzyme substrates.

[0108] In this invention, "nucleic acid drug" refers to an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule that can reduce or inhibit the translation of messenger RNA (mRNA) in a sequence-specific manner.

[0109] Nucleic acid molecules can exert their effects through RNA interference mechanisms (e.g., by inducing mRNA degradation through interaction with the mRNA interference pathway mechanism (RNA-induced silencing complex RISC) in mammalian cells) or any other mechanism or pathway. While the term "nucleic acid drug" as used in this invention is considered to primarily exert its effects through RNA interference mechanisms, the nucleic acid drug is not limited to or restricted to any particular pathway or mechanism of action. Types of nucleic acid drug molecules include, but are not limited to: single-stranded antisense oligonucleotides, short interfering RNA (nucleic acid), double-stranded RNA (dsRNA), microRNA (miRNA), non-coding RNA (lncRNA), short hairpin RNA (shRNA), and dicer enzyme substrates. The nucleic acid drug of this invention comprises an oligonucleotide chain having at least a partial complementarity to the mRNA serving as the target. In some embodiments, the nucleic acid drug of this invention is double-stranded and comprises an antisense strand and a sense strand at least partially complementary to the antisense strand.

[0110] The terms “silence,” “reduction,” “inhibition,” “downregulation,” or “knockdown” refer to a reduction or decrease in the expression level of a given gene when it is directly administered into cells, tissues, organs, or animals treated with the nucleic acid drug molecules described in this invention, compared to administration into cells, tissues, organs, or animals that have not been so treated.

[0111] The term "sequence" or "nucleotide sequence" refers to the order or sequence of nucleobases or nucleotides, expressed alphabetically using standard nucleotide nomenclature.

[0112] The term "heterocyclic base" is a nucleobase or modified nucleobase as defined in this invention. In some embodiments, the heterocyclic base moiety is a pyrimidine, a substituted pyrimidine, a purine, or a substituted purine, as well as a nitrogen-containing heterocycle. In some embodiments, the heterocyclic base moiety is an unconventional purine or a substituted purine. In some embodiments, the heterocyclic base moiety is an unconventional pyrimidine or a substituted pyrimidine. In some embodiments, the heterocyclic base moiety contains a non-natural five-membered nitrogen-containing heterocycle. In some embodiments, the heterocyclic base moiety may include one or more protecting groups.

[0113] In this invention, "nucleotide base" or "nucleobase" refers to heterocyclic pyrimidine or purine compounds, wherein "conventional nucleoside base" includes the bases of conventional nucleotides such as adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U). Nucleobases can be modified or substituted with novel unconventional nucleobases. Nucleotide bases include naturally occurring nucleotide bases as well as non-naturally occurring nucleotide bases. It should be obvious to those skilled in the art that various nucleotide bases previously considered "non-naturally occurring" have later been discovered in nature. Therefore, "nucleotide base" includes not only known purine and pyrimidine heterocycles but also their heterocyclic analogs and tautomers. Exemplary examples of nucleotide bases include adenine, guanine, thymine, cytosine, uracil, purine, xanthine, diaminopurine, 8-oxo-N6-methyladenine, 7-diazaxanthine, 7-diazaguanine, N4,N4-bridged ethylidene cytosine, N6,N6-bridged ethylidene-2,6-diaminopurine, 5-methylcytosine, 5-(C3-C6)-alkynylcytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolpyridine, isocytosine, isoguanine, inosine, and the “non-naturally occurring” nucleotide bases described in U.S. Patent No. 5,432,272 to Benner et al. The term “nucleotide base” includes each and all of these examples and their analogues and tautomers. Particularly important nucleotide bases include adenine, guanine, thymine, cytosine, and uracil, which are considered to be naturally occurring nucleotide bases relevant to human therapeutic and diagnostic applications.

[0114] The term "complementarity," used to describe the relationship between a first nucleotide sequence (e.g., the sense strand of a nucleic acid drug or target mRNA) and a second nucleotide sequence (e.g., a single-stranded antisense oligonucleotide or a double-stranded antisense strand of a nucleic acid drug), refers to the ability of an oligonucleotide or oligonucleotide containing the first nucleotide sequence to hybridize with an oligonucleotide or oligonucleotide containing the second nucleotide sequence under certain conditions (under mammalian physiological conditions or similar in vitro conditions) to form base pairs and a double-stranded or double-helix structure. A complementary sequence contains either a Watson-Crick base pair or a non-Watson-Crick base pair and contains native or modified nucleotides or nucleotide analogs to a degree sufficient to satisfy the hybridization requirements described above. For example, for the purpose of determining identity or complementarity, monomers a and Af are complementary to U (or T) and are equivalent to A.

[0115] The term "sense strand" refers to the nucleotide sequence on an RNA molecule that carries the information of the amino acids that encode the protein. It is also called the coding strand, sense strand, or positive strand, while the other nucleotide sequence that is complementary to it is called the antisense strand.

[0116] The term "antisense strand" refers to a nucleotide sequence in the mRNA of the target gene that is substantially anticomplementary or substantially anticomplementary to the antisense strand.

[0117] In the structure shown in Formula 9a or Formula 9b, the W at the 5' end of the pentose sugar is connected to the P of the phosphate group at the 3' end of the preceding nucleotide, and one end of the lipid-modified phosphate backbone is connected to the C at the 5' end of the following nucleotide. The position of the nucleotides is determined according to their position in the oligonucleotide, with the position closer to the 5' end of the oligonucleotide being the front and the position closer to the 3' end being the back.

[0118] The terms “optional” or “optionally” mean that the event or environment described below may, but does not have to, occur, and the description includes the possibility that the event or environment may or may not occur. For example, “optionally alkyl-substituted heterocyclic group” means that an alkyl group may, but does not have to, be present, and the description includes cases where the heterocyclic group is substituted with an alkyl group and cases where the heterocyclic group is not substituted with an alkyl group.

[0119] The term "substitution" refers to one or more hydrogen atoms in a group, preferably up to five, and more preferably one to three hydrogen atoms, being independently substituted by the corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, which can be substituted, whether experimentally or theoretically, by means of existing technology and experimental conditions. For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom having an unsaturated bond (such as an alkene).

[0120] The minimum and maximum carbon atom content in hydrocarbon groups are indicated by a prefix, for example, the prefix C. a～ C b Alkyl groups indicate any alkyl group containing "a" to "b" carbon atoms. Therefore, for example, C 1～6 Alkyl groups are straight-chain or branched alkyl groups containing 1 to 6 carbon atoms.

[0121] Alkyl refers to a straight-chain or branched hydrocarbon group in an alkane molecule, such as methyl-CH3, ethyl-CH2CH3, or methylene-CH2-. The alkyl group can also be part of other groups, such as C1-C6 alkoxy groups or C1-C6 alkylamino groups. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, and n-hexyl groups. Alkyl groups can be substituted or unsubstituted. When substituted, the substituent can be substituted at any usable connection point. The substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxy, carboxyl, or carboxylic acid ester groups.

[0122] "Alkenyl" refers to a straight-chain or branched hydrocarbon group having at least two carbon atoms and at least one vinyl unsaturation site (>C=C<). For example, C a-b Alkenyl refers to an alkenyl group having a to b carbon atoms and is intended to include, for example, vinyl, propenyl, isopropenyl, 1,3-butadienyl, etc.

[0123] "Alynyl" refers to a straight-chain monovalent hydrocarbon group or a branched monovalent hydrocarbon group containing at least one triple bond. The term "alkynyl" is also intended to include those hydrocarbon groups having one triple bond and one double bond. For example, C 2-6 The term "alkynyl" is intended to include ethynyl, propynyl, etc.

[0124] "Alkoxy" refers to -O-(alkyl) or -O-(cycloalkyl), where alkyl is defined as described above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentoxy, and cyclohexoxy. Alkoxy groups can be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl, or carboxylic acid ester group.

[0125] "Halogen" refers to fluorine, chlorine, bromine, or iodine.

[0126] "Halogenated alkyl" refers to an alkyl group that is substituted with one or more halogens, wherein the alkyl group is as defined above.

[0127] "Ester group" refers to -C(O)O (alkyl) or -C(O)O (cycloalkyl), where alkyl and cycloalkyl are as defined above.

[0128] "Acyl" refers to a compound containing a -C(O)R group, where R is an alkyl, cycloalkyl, heterocyclic, aryl, or heteroaryl group.

[0129] A heteroatom is an atom in an organic compound that is other than carbon and hydrogen. It generally refers to an atom that replaces carbon in the molecular skeleton (especially in ring systems). Nitrogen, oxygen, sulfur, phosphorus, boron, chlorine, bromine, and iodine are common heteroatoms. If an organic compound contains a ring that includes heteroatoms, it is called a heterocyclic compound.

[0130] "Heteroaryl" refers to an aromatic ring group having a conjugated planar ring system and containing heteroatoms. It can be a 3- to 8-membered (e.g., 3, 4, 5, 6, 7, 8-membered) monocyclic, a 5- to 12-membered (e.g., 5, 6, 7, 8, 9, 10, 11, 12-membered) bicyclic, or a 10- to 15-membered (e.g., 10, 11, 12, 13, 14, 15-membered) tricyclic system, and contains 1 to 6 (e.g., 1, 2, 3, 4, 5, 6) heteroatoms selected from N, O, or S. Non-limiting examples of heteroaryl include triazolyl, pyridyl, furanyl, thiophene, pyranyl, pyrroloyl, pyrimidinyl, pyrazinyl, pyridazinyl, imidazolyl, piperidinylbenzimidazolyl, benzopyridyl, and pyrrolopyridyl. The heteroaryl group may optionally be further substituted by one or more substituents.

[0131] "Heterocyclic group" or "heterocycle" refers to a saturated or unsaturated aromatic heterocycle or a non-aromatic heterocycle. When it is an aromatic heterocycle, its definition is the same as the definition of "heteroaryl" above. When it is a non-aromatic heterocycle, it can be a 3- to 10-membered (e.g., 3, 4, 5, 6, 7, 8, 9, 10-membered) monocyclic, a 4- to 12-membered (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12-membered) bicyclic, or a 10- to 15-membered (e.g., 10, 11, 12, 13, 14, 15-membered) tricyclic system, and contains 1 to 4 (e.g., 1, 2, 3, 4) heteroatoms selected from N, O, or S, preferably a 3- to 8-membered heterocyclic group. Non-limiting examples of "heterocyclic group" or "heterocycle" include oxoheterobutyl, azaheterobutyl, thioheterobutyl, 1,3-dioxopentyl, 1,4-dioxopentyl, 1,3-dioxahexacycloyl, azaheptanyl, oxoheptanyl, thioheptanyl, triazolyl, pyridyl, piperidinyl, furanyl, thiophene, pyranyl, N-alkylpyrroleyl, pyrimidinyl, pyrazinyl, pyridazinyl, piperazinyl, homopiperazinyl, imidazolyl, piperidinyl, morpholinyl, thiomorpholinyl, thiaxylyl, 1,3-dithiaalkyl, dihydrofuranyl, dithiapentylyl Cycloyl, tetrahydrofuranyl, tetrahydrothiophenyl, tetrahydropyranyl, tetrahydrothiophenyl, tetrahydropyrroleyl, tetrahydroimidazoyl, tetrahydrothiazoyl, tetrahydropyranyl, benzimidazolyl, benzopyridyl, pyrrolopyridyl, benzodihydrofuranyl, 2-pyrrolinyl, 3-pyrrolinyl, dihydroindolyl, 2H-pyranyl, 4H-pyranyl, dioxacyclohexyl, 1,3-dioxapentyl, pyrazolinyl, dithiaalkyl, dithiamonyl, dihydrothiophenyl, pyrazolyl, imidazolinyl, imidazolinyl, 1,2,3,4-tetrahydroisoquinolinyl. The "heterocyclic group" or "heterocycle" may optionally be further substituted with one or more substituents.

[0132] In this invention, a "protecting group" refers to an unstable chemical moiety known in the art for preventing reactive groups (e.g., hydroxyl, amino, carboxyl, and thiol groups) from undergoing undesirable reactions during synthesis. Protecting groups are typically used selectively and / or orthogonally to protect other reactive sites in the reaction and are subsequently removed to release unprotected groups, making them available for further reactions. In some embodiments, "substituted" groups or substituents contain protecting groups.

[0133] Representative hydroxyl protecting groups used in this invention are disclosed in Beaucage et al., Tetrahedron 1992, 48, 2223-2311, and Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2d ed., John Wiley & Sons, New York, 1991, all of which are incorporated herein by reference in their entirety. In some embodiments, the protecting group is stable under basic conditions but can be removed under acidic conditions. In some embodiments, non-exclusive examples of hydroxyl protecting groups that may be used in this invention include dimethoxytriphenylmethyl (DMT), monomethoxytriphenylmethyl, 9-phenyloxanthracene-9-yl (Pixyl), and 9-(p-methoxyphenyl)oxanthracene-9-yl (Mox). In some embodiments, non-exclusive examples of hydroxyl protecting groups that may be used in this invention include Tr (triphenylmethyl), MMTr (4-methoxytriphenylmethyl), DMTr (4,4'-dimethoxytriphenylmethyl), and TMTr (4,4',4”-trimethoxytriphenylmethyl).

[0134] The compounds and compositions described in this invention may have certain atoms (e.g., N, O, or S atoms) in a protonated or deprotonated state, depending on the environment in which the compound or composition is situated. Therefore, as used in this invention, the structures described herein take into account that certain functional groups, such as OH, SH, or NH, can be protonated or deprotonated. The disclosure of this invention is intended to cover the compounds and compositions described above, regardless of their protonation state based on environmental pH, as will be readily understood by those skilled in the art.

[0135] The terms "salt" and "available salt" refer to acidic and / or basic salts formed by the above-described compounds or their stereoisomers with inorganic and / or organic acids and bases, including zwitterionic salts (internal salts) and quaternary ammonium salts, such as alkyl ammonium salts. These salts can be obtained directly during the final separation and purification of the compounds. Alternatively, they can be obtained by mixing the above-described compounds, or their stereoisomers, with an appropriate (e.g., equimolar) amount of acid or base. These salts may be obtained by precipitating in solution and collecting by filtration, or by recovery after solvent evaporation, or by freeze-drying after reaction in an aqueous medium.

[0136] This invention provides a nucleoside compound of Formula I, which can be used to prepare oligonucleotides. Oligonucleotides modified with the above-mentioned nucleoside compound are non-cytotoxic, non-immunogenic, and exhibit better biocompatibility, stability, and safety. This modification can significantly improve the safety and efficacy of oligonucleotides, and is expected to promote the development of gene therapy technology. Furthermore, by conjugating different ligands, these compounds can achieve target gene silencing in CNS tissues, adipose tissues, and muscle tissues, enabling delivery to CNS tissues and adipose / muscle tissues.

[0137] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0138] Figure 1 shows the in vivo activity results of siRNA conjugates with hB-modified nucleosides. Detailed Implementation

[0139] Unless otherwise specified, the instruments used in this invention are not conventional instruments, and the reagents used are all conventional reagents.

[0140] The structure of a compound is determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS).

[0141] NMR shift (δ) with 10 -6 The unit (ppm) is given. NMR was determined using a Broker Avance III 400 NMR spectrometer. The solvents used were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD). The internal standard was tetramethylsilane (TMS). MS was determined using an Agilent 6120B (ESI) and Agilent 6120B (APCI). Thin-layer chromatography (TLC) used Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates. The TLC plates used had a diameter of 0.15 mm-0.20 mm, and the purified products used for TLC had a diameter of 0.4 mm-0.5 mm.

[0142] Example 1: Synthesis of compound hB-9

[0143] Synthesis of Compound 9: Compound 9 was synthesized with reference to known literature (Pryde, DC, Middleton, DS, Stephenson, PT, Wainwright, P., Maddaford, A., Zhang, X., Leese, D., Glen, R., Hart, J., Forrest, N., & Guyot, T. (2011). Practical synthetic routes to carbon-substituted nucleosides. Tetrahedron Letters, 52(48), 6415-6419. https: / / doi.org / 10.1016 / j.tetlet.2011.09.074).

[0144] Synthesis of Compound 10: Substrate 9 (2.0 g, 7.11 mmol) dissolved in anhydrous DMSO (30 mL) was added to a dry 100 mL single-necked flask. Then, under nitrogen protection, CH3I (0.91 g, 6.40 mmol) and KOH (798 mg, 14.22 mmol) were added. After the addition was complete, the reaction system was stirred at room temperature for 24 hours. The reaction was considered complete when the starting material was completely eliminated by LCMS. The reaction solution was then directly concentrated to obtain a crude product, which was resolved by SFC to give compound 10 (750 mg, 2.56 mmol, 36% yield).

[0145] Synthesis of Compound 11: Substrate 10 (750 mg, 2.56 mmol) was dissolved in anhydrous Pyridine (10 mL) in a dry 25 mL single-necked flask. Then, B2Cl (1.44 g, 10.24 mmol) was added under nitrogen protection at 0 °C. After the addition was complete, the reaction mixture was stirred at room temperature for 15 hours. LC-MS analysis confirmed the complete disappearance of the starting material, indicating the end of the reaction. Then, 100 mL of ethyl acetate was added to the reaction mixture, followed by washing with water (100 mL x 3) and then with saturated brine (100 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated to obtain crude compound 11 (2.4 g), which was used directly in the next reaction without purification.

[0146] Synthesis of Compound 12: Crude substrate 11 (2.4 g) was dissolved in pyridine (30 mL) in a dry 500 mL single-necked flask, and a few drops of pyridine were added dropwise. Then, 2 M NaOH in MeOH / H2O (24 mL) was added at 0 °C. After the addition was complete, the reaction system was stirred at 0 °C for half an hour. LC-MS analysis showed complete disappearance of the starting material, indicating the end of the reaction. 100 mL of ethyl acetate was added to the reaction solution, and the mixture was washed with saturated NH4Cl solution (100 mL x 3) and then with saturated brine (100 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated to obtain a crude product. This crude product was purified by column chromatography (petroleum ether:ethyl acetate = 3:1) and concentrated to give Compound 12 (0.91 g, 2.28 mmol, 89% yield).

[0147] Synthesis of Compound 13: Substrate 12 (220 mg, 0.55 mmol) dissolved in pyridine (5 mL) was added to a dry 10 mL single-necked flask, followed by the addition of DMTrCl (242 mg, 0.71 mmol). After the addition was complete, the reaction system was stirred overnight at room temperature. The reaction was considered complete when the starting material was detected by LCMS. Then, 20 mL of ethyl acetate was added to the reaction solution, and the mixture was washed with water (50 mL * 3) and then with saturated brine (50 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated to obtain a crude product. This crude product was purified by column chromatography (petroleum ether: ethyl acetate = 1:2), and then separated and concentrated by HPLC to obtain Compound 13 (200 mg, 0.28 mmol, 50.9% yield). 1 H NMR (400MHz, DMSO-d6): δ11.14(s,1H),10.20(s,1H),8.73(s,1H),8.33(s,1H),8 .09–7.99(m,2H),7.68–7.50(m,3H),7.40–7.18(m,9H),6.93–6.84(m,4H),5.17–5 .10(m,1H),4.591–4.49(m,1H),4.41–4.29(m,1H),4.20–4.11(m,1H),4.02-3.86 (m,2H),3.72(s,6H),3.55–3.48(m,1H),3.25(s,3H),3.06–3.99(m,2H).ESI-LCMS m / z 700.1[MH] - .

[0148] Synthesis of compound hB-9: Substrate 13 (200 mg, 0.28 mmol) was dissolved in anhydrous DCM (5 mL) in a dry 10 mL single-necked flask. Then, under nitrogen protection, DCI (26 mg, 0.22 mmol) and CEP[N(iPr)2]2 (101 mg, 0.34 mmol) were added. After the addition was complete, the reaction system was stirred at room temperature for 2 hours. LCMS confirmed the complete disappearance of the starting material, indicating the end of the reaction. Then, 5 mL of dichloromethane was added to the reaction solution, followed by washing with water (10 mL * 2) and then with saturated brine (10 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated to obtain the crude product. The crude product was then concentrated using a reversed-phase C18 (CH3CN / DCM = 1 / 0) to obtain the transparent oily product hB-9 (190 mg, 0.21 mmol, 75.0% yield). 1 H NMR (400MHz, DMSO-d6): δ11.2(m,1H),8.74-8.72(m,1H),8.36-8.35(m,1H),8.05 -8.03(m,2H),7.68-7.62(m,1H),7.57-7.53(m,2H),7.41-7.20(m,9H),6.92-6.88 (m,4H),4.65-4.48(m,1H),4.45-4.30(m,3H),3.98-3.90(m,1H),3.89-3.50(m,1 1H),3.30-3.22(m,3H),3.20-3.00(m,2H),2.83-2.73(m,2H),1.15-1.01(m,12H). 31 P NMR (162MHz, DMSO-d6): δ149.71,148.61.ESI-LCMS m / z 903.4[M+H] + .

[0149] The synthesis of compounds hB-6, hB-7, hB-8, and hB-10 follows the same synthetic route as hB-9.

[0150] Example 2: Synthesis of compound hB-114

[0151] Synthesis of compound 14: Substrate 9 (1.5 g, 5.33 mmol) was dissolved in anhydrous DMSO (30 mL) in a dry 100 mL single-necked flask, followed by the addition of C under nitrogen protection. 16 H 35Br (2.0 g, 6.40 mmol), KOH (600 mg, 10.67 mmol). After addition, the reaction system was stirred at room temperature for 24 hours. LCMS analysis showed complete disappearance of the starting material, indicating the end of the reaction. The reaction solution was then directly concentrated to obtain a crude product, which was resolved to give compound 14 (900 mg, 1.78 mmol, 33% yield). 1 H NMR(400MHz, DMSO-d6)δ8.09(d,J=13.9Hz,2H),7.19(s,2H),4.92–4.79(m,1H),4.77–4.61(m,1H),4.34–4.13(m,2H),4.10–3.99(m,1H),3.9 2–3.82(m,1H),3.75–3.61(m,1H),3.58–3.41(m,2H),3.40–3.32(m,2H),3.30–3.16(m,1H),1.45–1.13(m,28H),0.94–0.75(m,3H).ESI-LCMS m / z 506.3[M+H] - .

[0152] Synthesis of Compound 15: Substrate 14 (900 mg, 1.78 mmol) was dissolved in anhydrous Pyridine (10 mL) in a dry 25 mL single-necked flask. Then, B2Cl (872 mg, 6.23 mmol) was added under nitrogen protection at 0 °C. After the addition was complete, the reaction mixture was stirred at room temperature for 15 hours. LC-MS analysis confirmed the complete disappearance of the starting material, indicating the end of the reaction. Then, 100 mL of ethyl acetate was added to the reaction mixture, and the mixture was washed with water (100 mL x 3) and then with saturated brine (100 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated to obtain crude compound 15 (1.3 g).

[0153] Synthesis of Compound 16: Substrate 15 (1.3 g) dissolved in pyridine (13 mL) was added to a dry 500 mL single-necked flask, followed by a few drops of pyridine. Then, 2 M NaOH in MeOH / H2O (13 mL) was added at 0 °C. After the addition was complete, the reaction mixture was stirred at 0 °C for half an hour. LCMS analysis confirmed the complete disappearance of the starting material, indicating the end of the reaction. 100 mL of ethyl acetate was added to the reaction mixture, and the mixture was washed with saturated NH4Cl solution (100 mL x 3) and then with saturated brine (100 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated to obtain a crude product. This crude product was purified by column chromatography (petroleum ether:ethyl acetate = 3:1) and concentrated to give compound 16 (1.0 g, 1.64 mmol, 92% yield). ESI-LCMS m / z 610.4 [M+H] + .

[0154] Synthesis of Compound 17: Substrate 16 (1.0 g, 1.64 mmol) was dissolved in anhydrous Pyridine (10 mL) in a dry 50 mL single-necked flask, followed by the addition of DMTrCl (833 mg, 2.46 mmol) under nitrogen protection. After the addition was complete, the reaction system was stirred at room temperature for 15 hours. The reaction was considered complete when the starting material was detected by LCMS. The mixture was filtered, and the filtrate was diluted with ethyl acetate (100 mL), washed with water (100 mL x 3), and washed with saturated brine (100 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated to obtain a crude product. This crude product was purified by column chromatography (petroleum ether: ethyl acetate = 5:1) and concentrated to give a yellow solid product 17 (1.3 g, 1.43 mmol, 87% yield). 1 H NMR(400MHz,DMSO-d6)δ11.15(s,1H),8.72(s,1H),8.35(s,1H),8.13–8.02(m,2 H),7.69–7.53(m,9H),6.92–6.86(m,4H),4.86–4.81(m,1H),4.55–4.36(m,2H),4 .24–4.16(m,1H),3.92–3.70(m,8H),3.60–3.45(m,2H),3.28–3.18(m,1H),3.10– 2.96(m,2H),1.47–1.38(m,2H),1.24–1.16(m,26H),0.85–0.82(m,3H).ESI-LCMS m / z 912.5[M+H] - .

[0155] Synthesis of compound hB-114: Substrate 17 (1.3 g, 1.43 mmol) was dissolved in anhydrous DCM (15 mL) in a dry 100 mL single-necked flask. Then, under nitrogen protection, DCI (143 mg, 1.21 mmol) and CEP[N(iPr)2]2 (560 mg, 1.86 mmol) were added. After the addition was complete, the reaction system was stirred at room temperature for 2 hours. LCMS analysis confirmed the complete disappearance of the starting material, indicating the end of the reaction. Then, 50 mL of dichloromethane was added to the reaction solution, followed by washing with water (100 mL x 3) and then with saturated brine (100 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated to obtain a crude product. This crude product was purified using a reverse-phase C18 (CH3CN / H2O (0.05% NH4HCO3) = 1 / 0) reactor and concentrated to obtain product hB-114 (1.4 g, 1.26 mmol, 88% yield). 1H NMR(400MHz,DMSO-d6)δ11.15(s,1H),8.70(s,1H),8.39–8.30(m,1H),8.08–8.01(m,2H),7.68–7.5 1(m,3H),7.40–7.16(m,9H),6.97–6.80(m,4H),4.59–4.40(m,2H),4.30–4.15(m,1H),4.10–3.91(m ,2H),3.75–3.39(m,12H),3.30–3.15(m,1H),3.12–2.92(m,2H),2.76–2.67(m,1H),2.58–2.50(m,1 H),2.48–2.31(m,2H),2.29–2.18(m,26H),1.15–1.06(m,9H),0.95–0.89(m,3H),0.87–0.80(m,3H). 31 P NMR(162MHz,DMSO-d6)δ148.86,148.32.ESI-LCMS:m / z 1111.6[M+H] + .

[0156] The synthesis of compounds hB-111, hB-112, hB-113, and hB-115 follows the same synthetic route as hB-114.

[0157] Example 3: Synthesis of compound hB-119

[0158] Synthesis of compound 18: Substrate 9 (1.5 g, 5.33 mmol) was dissolved in anhydrous DMSO (30 mL) in a dry 100 mL single-necked flask, followed by the addition of C under nitrogen protection. 16 H 35 Br (2.0 g, 6.40 mmol), KOH (600 mg, 10.67 mmol). After addition, the reaction system was stirred at room temperature for 24 hours. LCMS analysis showed complete disappearance of the starting material, indicating the end of the reaction. The reaction solution was then directly concentrated to obtain a crude product, which was resolved to give compound 18 (500 mg, 0.99 mmol, 19% yield). 1H NMR(400MHz, DMSO-d6)δ8.10(d,J=15.0Hz,2H),7.21(s,2H),5.05–4.94(m,1H),4.90–4.81(m,1H),4.38–4.24(m,1H),4.18–4.05(m,1H),4.0 5–3.95(m,1H),3.94–3.84(m,1H),3.80–3.69(m,1H),3.61–3.47(m,2H),3.46–3.33(m,3H),1.53–1.16(m,28H),0.90–0.79(m,3H).ESI-LCMS m / z 506.3[M+H] - .

[0159] Synthesis of Compound 19: Substrate 18 (500 mg, 0.99 mmol) was dissolved in anhydrous Pyridine (10 mL) in a dry 25 mL single-necked flask. Then, B2Cl (485 mg, 3.47 mmol) was added under nitrogen protection at 0 °C. After the addition was complete, the reaction mixture was stirred at room temperature for 15 hours. LC-MS analysis confirmed the complete disappearance of the starting material, indicating the end of the reaction. Then, 100 mL of ethyl acetate was added to the reaction mixture, and the mixture was washed with water (100 mL x 3) and then with saturated brine (100 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated to obtain crude compound 19 (0.9 g).

[0160] Synthesis of Compound 20: Substrate 19 (0.9 g) dissolved in pyridine (10 mL) was added to a dry 50 mL single-necked flask, followed by a few drops of pyridine. Then, 2 M NaOH in MeOH / H2O (10 mL) was added at 0 °C. After the addition was complete, the reaction system was stirred at 0 °C for half an hour. LCMS analysis showed complete disappearance of the starting material, indicating the end of the reaction. 10 mL of ethyl acetate was added to the reaction solution, and the mixture was washed with saturated NH4Cl solution (10 mL x 3) and then with saturated brine (10 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated to obtain a crude product. This crude product was purified by column chromatography (petroleum ether:ethyl acetate = 3:1) and concentrated to obtain Compound 20 (550 mg, 0.90 mmol, 91% yield). ESI-LCMS m / z 610.4 [M+H] + .

[0161] Synthesis of Compound 21: Substrate 20 (550 mg, 0.90 mmol) was dissolved in anhydrous Pyridine (10 mL) in a dry 50 mL single-necked flask, followed by the addition of DMTrCl (366 mg, 1.08 mmol) under nitrogen protection. After the addition was complete, the reaction system was stirred at room temperature for 15 hours. The reaction was considered complete when the starting material was detected by LCMS. The mixture was filtered, and the filtrate was diluted with ethyl acetate (100 mL), washed with water (100 mL x 3), and washed with saturated brine (100 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated to obtain a crude product, which was purified by column chromatography (petroleum ether: ethyl acetate = 5:1). The crude product was concentrated to give a yellow solid, product 21 (600 mg, 0.66 mmol, 73% yield). 1 H NMR(400MHz,DMSO-d6)δ11.15(s,1H),8.83(s,1H),8.35(s,1H),8.09–8.01(m,2H),7.69– 7.51(m,3H),7.36–7.18(m,9H),6.92–6.86(m,4H),5.03–4.96(m,1H),4.54–4.31(m,2H),4 .22–4.13(m,1H),3.92–3.80(m,2H),3.77–3.65(m,6H),3.60–3.45(m,2H),3.28–2.20(m,1 H),3.10–2.96(m,2H),1.40–1.35(m,2H),1.30–1.18(m,26H),0.87–0.81(m,3H).ESI-LCMS m / z 912.5[M+H] - .

[0162] Synthesis of compound hB-119: Substrate 21 (600 mg, 0.66 mmol) was dissolved in anhydrous DCM (10 mL) in a dry 25 mL single-necked flask. Then, under nitrogen protection, DCI (66 mg, 0.56 mmol) and CEP[N(iPr)2]2 (258 mg, 0.86 mmol) were added. After the addition was complete, the reaction system was stirred at room temperature for 2 hours. LCMS analysis confirmed the complete disappearance of the starting material, indicating the end of the reaction. Then, 50 mL of dichloromethane was added to the reaction solution, followed by washing with water (20 mL x 3) and then with saturated brine (20 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated to obtain a crude product. This crude product was purified using a reverse-phase C18 (CH3CN / H2O (0.05% NH4HCO3) = 1 / 0) reactor and concentrated to obtain product hB-119 (600 mg, 0.54 mmol, 82% yield). 1H NMR(400MHz,DMSO-d6)δ11.15(s,1H),8.76–8.69(m,1H),8.42–8.34(m,1H),8.09 –8.01(m,2H),7.80–7.49(m,3H),7.45–7.16(m,9H),6.92–6.83(m,4H),4.70–4.2 5(m,4H),3.96–3.85(m,1H),3.82–3.35(m,2H),3.31–3.01(m,3H),2.87–2.68(m, 2H),1.46–1.35(m,2H),1.27–1.10(m,6H),1.04–2.97(m,2H),1.89–1.81(m,3H), 31 P NMR(162MHz,DMSO-d6)δ149.48,148.32.ESI-LCMS:m / z 1111.6[M+H] + .

[0163] Example 4: Synthesis of compound hB-134

[0164] The synthesis of compounds hB-131, hB-132, hB-133, and hB-135 follows the same synthetic route as compound hB-134.

[0165] Example 5: Synthesis of compound hB-139

[0166] Example 6: Synthesis of compounds hB-71 to hB-75

[0167] Example 7 Synthesis of compounds hB-31 to hB-35

[0168] Example 8: Synthesis of compounds hB-76 to hB-80

[0169] Example 9: Synthesis of compounds hB-151 to hB-165

[0170] The preparation methods in Examples 1, 2, and 4 are the same, except that Z and R in compounds hB-151 to hB-165 are different. 7 The choice varies, and adaptive adjustments are made based on the structure of compounds hB-151 to hB-165.

[0171] Example 10: Synthesis of siRNA sequences modified with the nucleoside compounds of the present invention

[0172] The synthesis of siRNA is no different from the usual phosphoramidite solid-phase synthesis method. When synthesizing nucleotides modified at various positions of the SS and AS chains, the original nucleotides in the parent sequence are replaced by the above-synthesized hB phosphoramidite monomer.

[0173] The synthesis process is briefly described as follows: For the sense and antisense strands of the siRNA sequence of the present invention, as well as the sense and antisense strands of the modified double strands, CPG is used as a solid-phase carrier; N-acetylgalactosamine (GalNAc) modified CPG is used as the starting cycle for the synthesis of the sense strand, and universal CPG is used as the starting cycle for the antisense strand.

[0174] Using a YB-192S synthesizer, a solid-phase synthesis method of phosphoramidite was employed. Starting with a solid support, nucleoside monomers were sequentially linked in the 3'-5' direction to achieve a synthesis scale of 0.2 μmol.

[0175] The phosphorus amide monomer is linked through a continuous cycle of four chemical reactions: deprotection, coupling, oxidation / sulfidation, and capping. The phosphorus amide monomer is prepared as a 0.05 M acetonitrile solution, with 0.3 M 1H-benzotriazole (BTT) in acetonitrile as the activator, a 3% trichloroacetic acid / dichloromethane solution as the deprotecting agent, a 0.05 M iodine / pyridine / tetrahydrofuran / aqueous solution (v / v / v = 2 / 1 / 7) as the oxidizing agent, acetic anhydride / acetonitrile solution as capping agent A (v / v = 2 / 8), pyridine / aziridine-methylimidazolium / tetrahydrofuran solution as capping agent B (v / v / v = 10 / 16 / 74) as the capping agent, and a 0.05 M dibenzothiophene-2-sulfonylthioacetic acid (DDTT) in pyridine / acetonitrile solution (v / v = 4 / 6) as the thiochemical agent.

[0176] After solid-phase synthesis, the support was transferred to a 2 mL centrifuge tube, and 0.8 mL of concentrated ammonia was added. The mixture was then sealed and reacted at 55 °C for 16 h. After cooling to room temperature, the solution was transferred to a 2 mL centrifuge tube and concentrated to dryness. 0.2 mL of anhydrous DMSO was added to dissolve the solution, followed by 0.25 mL of triethylamine trihydrofluoride. The reaction was carried out at 65 °C for 2 h. After the reaction was complete, the mixture was cooled to room temperature, and the crude sequence was obtained by ethanol precipitation.

[0177] The crude product was purified by reversed-phase HPLC, and the collected fraction was lyophilized. Ethanol precipitation was performed by adding 0.3 mL of 1M sodium acetate solution and 0.9 mL of ethanol to replace the sequence with sodium salt. Then, desalting was carried out using a 3KD ultrafiltration tube to remove excess free salt.

[0178] The sense and antisense chains were prepared into an aqueous solution of a certain concentration. The sense and antisense chains were mixed at a molar ratio of 1:1.05, incubated at 95°C for 5 minutes, and then naturally cooled to room temperature. The product was then freeze-dried to obtain the target product.

[0179] The modification schemes used in Table 1 are as follows:

[0180] In this context, uppercase letters A, C, G, U, and I represent adenosine-3'-phosphate, cytidine-3'-phosphate, guanosine-3'-phosphate, uridine-3'-phosphate, and inosine-3'-phosphate, respectively; lowercase letter m indicates that the nucleotide adjacent to the right of m is 2'-methoxy modified; lowercase letter f indicates that the nucleotide adjacent to the right of f is 2'-fluoro modified; the two nucleotides adjacent to each other by an asterisk (*) are linked by a thiophosphate group; hB-1 to hB-180 indicate that the nucleotide at that position is one of the aforementioned modified nucleosides; GalNAc is GalNAc-L96.

[0181] Table 1: siRNA conjugate sequences

[0182] The effects of the present invention are illustrated below through experimental examples:

[0183] Experimental Example 1: In vitro activity assay (in vitro silencing activity of ANGPTL3 target mRNA and hB-modified siRNA)

[0184] I. Experimental Methods

[0185] Activity screening steps:

[0186] Cell culture and transfection

[0187] Cell culture: Hep3B cells (ATCC) were cultured at 37°C and 5% CO2 in MEM complete medium (Gibco, with 10% FBS) until near confluence. Then, the cells were digested with trypsin and seeded into 96-well plates. 2.0 × 103 Hep3B cells and 1.0 mL of MEM complete medium (Gibco, with 10% FBS) were added to each well. After culturing at 37°C and 5% CO2 for 16-24 h, transfection was performed.

[0188] Cell transfection: Add 0.15 μL of lipofectamine RNAiMax (Invitrogen) to each well of Opti-MEM, then add 5.0 μL of siRNA and mix. Add this mixture to a PCR tube and incubate at room temperature for 5 minutes. Finally, add this siRNA mixture to the cells and continue culturing for 24 hours before RNA extraction. Single-dose experiments were performed using 10 nM and 0.1 nM or 0.1 nM and 0.01 nM siRNA duplex concentrations. IC50 50The tests were conducted at siRNA duplex concentrations of 10 nM, 1.0 nM, 0.1 nM, 0.01 nM, 0.001 nM, 0.0001 nM, and 0.00001 nM.

[0189] RNA extraction

[0190] Using the Total RNA Isolation Kit (Omega, CAT: R6834-02): Collect cells, wash with 1% PBS, then add lysis buffer (containing 2% β-mercaptoethanol) to lyse the cells. Follow the instructions for the RNA isolation kit. Finally, add 30 μL of RNase-free water, let stand for 2 minutes, and then centrifuge at 14000g for 2 minutes to collect RNA.

[0191] cDNA synthesis

[0192] cDNA synthesis was performed using the TransGold gDNA Removal and cDNA Synthesis Kit (TransGold Biotechnology Co., Ltd., Beijing, China, Cat#AE311-03). 1 μg of total RNA was added to each sample, and cDNA synthesis was performed using a gradient thermal cycler (LongGene, A600) following the manufacturer's instructions.

[0193] Real-time quantitative PCR

[0194] Add the synthesized cDNA and the mixed stock solution (containing primers, qPCR premix and ultrapure water) to a 384-well plate (Bokcom Biosystems Cat#PC-0040-9U) to make the final real-time quantitative PCR system contain 0.25 μM each of upstream and downstream primers of the target gene (AGT) or internal reference gene (GADPH) and 1×SYBR Green premix (Applied Biosystems Cat#A25742).

[0195] The ΔΔCt measurement method was used in ABI QuantStudio. TM 6. Perform real-time fluorescence PCR in a real-time fluorescence PCR system. Perform 3-4 independent transfection tests for each double-stranded strain, with 3-4 assays per transfection.

[0196] Human / cynomolgus monkey primary liver cells

[0197] Free access: Resuscitate cryopreserved primary liver cells from humans or cynomolgus monkeys, perform live cell counting, and adjust to an appropriate density (6 × 10⁻⁶). 5 10 μL of the 10× compound was added to collagen-coated cell culture plates, and 90 μL (5.4 × 10⁻⁶ cells / mL) was dispensed into each well. 4Cell suspensions ( / well) were transferred to 96-well collagen-coated plates. The plates were incubated at 37°C in a 5% CO2 incubator for 48 hours. Single-dose experiments were performed at 10 nM and 0.1 nM siRNA duplex concentrations, with IC50... 50 The tests were conducted at siRNA duplex concentrations of 10 nM, 3.33 nM, 1.11 nM, 0.37 nM, 0.12 nM, 0.041 nM, 0.014 nM, and 0.0046 nM.

[0198] cDNA synthesis: After 48 hours of free uptake, the culture medium was removed and cells were lysed for RNA extraction. Use according to the kit instructions. Total RNA was extracted using a 96 Kit (QIAGEN-74182). cDNA was then synthesized using the FastKing RT Kit (With gDNase) (Tiangen-KR116-02) according to the manufacturer's instructions.

[0199] Real-time quantitative PCR: Add the synthesized cDNA and mixed stock solution (containing primers, qPCR premix and ultrapure water) to a 384-well plate, so that the final real-time quantitative PCR system contains 0.25 μM each of upstream and downstream primers of the target gene (AGT) or internal reference gene (GADPH), 0.125 μM probe, and 1× Universal Probe Master premix (Roche, catalog number 04914058001).

[0200] The ΔΔCt measurement method was used in ABI QuantStudio. TM 6. Perform real-time fluorescence PCR in a real-time fluorescence PCR system. Perform 3-4 independent transfection tests for each double-stranded strain, with 3-4 assays per transfection.

[0201] Experimental Example 2: Cytotoxicity Test

[0202] I. Experimental Methods

[0203] Cell culture: Hep3B cells (ATCC) were cultured at 37°C and 5% CO2 in MEM complete medium (Gibco, with 10% FBS added) until near confluence. Cells were then trypsinized and seeded into 24-well plates, with 0.5 × 10⁶ cells added to each well. 5 Hep3B cells were cultured in 0.5 mL of MEM complete medium (Gibco, with 10% FBS) at 37°C and 5% CO2 for 16-24 h before transfection.

[0204] Cell transfection: Add 0.75 μL of lipofectamine RNAiMax (Invitrogen) to each well of 24.25 μL opt-MEM, then add 25 μL of the siRNA conjugate from Example 10 and mix. Add the mixture to a PCR tube and incubate at room temperature for 5 minutes. Finally, add this siRNA mixture to the cells and continue culturing for 48 or 96 hours before performing cytotoxicity testing. This experiment was performed at 10 nM and 1 nM siRNA duplex concentrations.

[0205] Cytotoxicity test:

[0206] Cytotoxicity was tested using a CCK-8 assay kit (Beyotime, CAT: #C0040). After 48 / 96 hours of cell transfection, the culture medium was removed, and 500 μL of the corresponding complete culture medium (containing 10% CCK-8) was added to each well. The cells were incubated at 37°C for 30-60 minutes in the dark. The OD values ​​of the samples were detected using a microplate reader (Tecan CAT: #spark 20M) with a wavelength of 450 nm and a reference wavelength of 620 nm.

[0207] II. Experimental Results

[0208] siRNA conjugates with hB-modified nucleosides did not show cytotoxicity.

[0209] Experimental Example 3: siRNA Immunogenicity Test

[0210] I. Experimental Methods

[0211] Human peripheral blood cells (PBMCs) were resuscitated one day before siRNA transfection, at a dose of 1*10-1. 6 PBMCs were seeded into 24-well plates at a density equal to the well density. An appropriate amount of siRNA (final concentration 100 nM) was diluted in 125 μL of opti-MEM, and 4 μL of transfection reagent GenePORTER reagent (Genlantis Cat#T202007) was diluted in 125 μL of opti-MEM. The two were mixed 1:1 and incubated at room temperature for 15 min. Cells were resuspended in 250 μL of the transfection complex and added to 24-well plates. Cells were cultured in a 5% CO2 incubator at 37°C for 4 h. Then, 250 μL of 1640 medium (Hyclone Cat#SH30809.01) containing 10% FBS was added. After 24 h of culture, the supernatant and cells were collected separately. The supernatant was used for ELISA to detect changes in the expression of Type I IFN-related proteins: IL-6 (R&D Cat#41100) and INF-α (thermo Cat#BMS216INST). Cells were used for RNA extraction, and quantitative real-time PCR was used to detect changes in the expression of Type I IFN genes (P56 and OSA1).

[0212] P56 forward primer: 5'-GCCTCCTTGGGTTCGTCTATA-3'; (SEQ ID NO 105)

[0213] P56 reverse primer: 5'-CTCAGGGCCCGCTCATAGTA-3'; (SEQ ID NO 106)

[0214] OSA1 forward primer: 5'-CGAGGGAGCATGAAAACACATTT-3'; (SEQ ID NO 107)

[0215] OSA1 reverse primer: 5'-GCAGAGTTGCTGGTAGTTTATGAC-3'. (SEQ ID NO 108)

[0216] II. Experimental Results

[0217] siRNA conjugates with hB-modified nucleosides did not exhibit immunogenic activation compared to the parent sequence.

[0218] Experiment Example 4: In vivo activity assay of ANGPTL3 target mRNA conjugate with hB-modified nucleoside

[0219] I. Experimental Methods

[0220] Evaluation of siRNA sequence activity in wild-type mice:

[0221] The activity of siRNA with hB-modified nucleosides in vivo was evaluated using wild-type C57BL / 6 mice.

[0222] C57BL / 6 mice aged 6-8 weeks were used, and the mice were placed in the facility for acclimatization for more than 3 days before administration. Each group of 3-5 mice was injected subcutaneously with a single dose of 1 mg / kg of the compound (compounds in Table 2). Blood samples were collected via the orbital sinus before administration and on days 7 and 14 after administration. After the blood samples were left at room temperature for 2 hours, they were centrifuged at 5500 rpm for 10 min at 4°C to separate and collect serum, and the expression level of ANGPTL3 protein in the animal serum was detected.

[0223] Table 2. Dosing regimens for in vivo activity evaluation of siRNA conjugates

[0224] II. Experimental Results

[0225] The experimental results are shown in Figure 1. The ANGPTL3 protein expression levels of conjugate 1, conjugate 8 and unmodified siRNA are similar, indicating that the siRNA conjugates containing hB-modified nucleosides are active in vivo, and the effects of different conjugates vary.

[0226] Experimental Example 5: In vivo activity assay of SOD1 target mRNA conjugate with hB-modified nucleoside I. Experimental Methods

[0227] Evaluation of siRNA sequence activity in wild-type mice:

[0228] Six- to eight-week-old C57BL / 6J mice were randomly divided into groups of 3-5 mice each, based on their body weight. The drug dosage for each group was calculated based on body weight and administered according to Table 3. The first administration day was Day 1.

[0229] Lateral ventricle administration: Experimental mice were anesthetized by inhalation of isoflurane. The mouse head was prepared and disinfected with povidone-iodine solution. The anesthetized mice were then fixed in a stereotaxic apparatus. Specifically, the upper incisors were inserted into the horizontal bar, and the knob was adjusted to press the nose bar firmly. The ear bars were inserted into the mouse's ear canal, and the left and right ear bars were adjusted to ensure that the line connecting the two ears was aligned with the ear bars. After ensuring that the scale positions of the left and right ear bars were the same, the knob was adjusted to lock the ear bars. The criteria for proper mouse fixation were: nose aligned with the midline, head still, tail not falling off when lifted, and skull level. Isoflurane was continuously inhaled throughout the entire procedure.

[0230] Use ophthalmic scissors to make a longitudinal incision of about 1 cm in the mouse's head skin. Use forceps to peel off and remove the periosteal connective tissue along the surface of the skull. Use a sterile cotton ball to remove any oozing blood, clean the surface of the skull, and expose the anterior and posterior fontanelles.

[0231] Adjust the x and y coordinate knobs on the brain localization instrument to position the dental micro-drill above the anterior fontanelle and lightly touch it, using the Bregma point as the reference point (zero point) of the three-dimensional coordinate system. Adjust the X and Y axis knobs to move the operating arm to the coordinate position and slowly drill a hole using the micro-drill. A noticeable feeling of emptiness will be felt when drilling through the skull; handle this carefully to avoid damaging the dura mater. Fix the microinjector and adjust the Z-axis knob to insert the needle into the right ventricle. Process the brain tissue: After cutting open the mouse skull with scissors, separate the mouse brain tissue and collect the hippocampus, frontal cortex, hypothalamus, and other remaining brain tissue (preserving the left and right sides separately). Store in RNAlater for PCR detection. Collect the remaining brain tissue from both sides, cerebrospinal fluid, heart, liver (left lobe), and kidneys, flash-freeze in liquid nitrogen, and then transfer to -80℃ for storage. Administer a slow injection using a microinjection pump at a rate of 1 μL / min. After injection, stop the injection for 30 minutes before removing the tissue.

[0232] Mice were anesthetized with isoflurane on days 8 and 22, their abdominal cavities were opened, the abdominal aorta was severed, and the mice were euthanized. The mouse skull was then cut open with scissors, and the brain tissue was separated. The hippocampus, frontal cortex, hypothalamus, and other remaining brain tissue (left and right sides were preserved separately) were collected and stored in RNAlater for PCR detection. The remaining brain tissue from both sides, cerebrospinal fluid, heart, liver (left lobe), and kidneys were collected, flash-frozen in liquid nitrogen, and then stored at -80°C. The remaining expression levels of SOD1 mRNA and CTNNB1 mRNA in each tissue were detected separately.

[0233] Table 3: Administration routes, dosages, and regimens in animal models

[0234] II. Experimental Results

[0235] siRNA conjugates modified with hB-117 to hB-118 nucleoside compounds can deliver siRNA into the CNS tissues. They exhibit good mRNAKD activity in the hippocampus, frontal cortex, hypothalamus, and other remaining brain tissues, while showing lower mRNAKD in peripheral tissues such as the heart and liver, and no KD in the kidneys. This indicates that the siRNA conjugates have good targeting properties, specifically acting on CNS tissues with minimal toxicity or interference to other peripheral tissues.

[0236] Experimental Example 6: In vivo activity assay of SOD1 target mRNA conjugate with hB-modified nucleoside

[0237] Evaluation of siRNA sequence activity in wild-type mice:

[0238] The activity of siRNA with hB-modified nucleosides in vivo was evaluated using wild-type C57BL / 6 mice.

[0239] C57BL / 6 mice aged 6-8 weeks were used. Before administration, the mice were acclimatized in the facility for more than 3 days and divided into groups of 3 mice each according to their weight. The mice were administered a single injection of the compound (compounds in Table 4) at a dose of 2 mg / kg via subcutaneous or intravenous injection. The mice were euthanized before administration and on day 7 after administration. The inguinal adipose tissue, periepididymal adipose tissue, scapular subcutaneous adipose tissue, perirenal adipose tissue, heart, liver, quadriceps femoris muscle, and kidney were collected from the mice, and the SOD1 mRNA in each tissue was detected.

[0240] Table 4: In vivo administration of the conjugate to WT mice

[0241] II. Experimental Results

[0242] siRNA conjugates with hB-137 to hB-139 modified nucleoside compounds can achieve good mRNAKD in adipose and muscle tissues. This indicates that the hB-137 to hB-139 modified nucleoside compounds, when conjugated to siRNA, can achieve delivery to adipose and muscle tissues.

Claims

1. A nucleoside phosphoramidite compound as represented by Formula 1 and stereoisomers, deuterated compounds thereof: ###00001### Formula 1 wherein, R 1 , R 2 , R 3 , R 4 , R 5 , R 9 , R 10 each independently is selected from the group consisting of hydrogen, deuterium, halogen, cycloalkyl, substituted or non-substituted C1-C6alkyl, substituted or non-substituted C1-C6alkoxy, substituted or non-substituted C2-C6alkenyl, substituted or non-substituted C2-C6alkynyl; R 6 selected from the group consisting of hydrogen, deuterium, halogen, cycloalkyl, substituted or non-substituted C1-C 30 alkyl, substituted or non-substituted C1-C 30 alkoxy, substituted or non-substituted C2-C 20 alkenyl, substituted or non-substituted C2-C 20 alkynyl, OR 14 , NR 14 R 15 , SR 14 , CR 14 R 15 ; wherein R 14 , R 15 are each independently selected from the group consisting of hydrogen, acyl, cycloalkyl, substituted or non-substituted C1-C 30 alkyl, substituted or non-substituted C1-C 30 alkoxy, substituted or non-substituted C2-C 20 alkenyl, substituted or non-substituted C2-C 10 alkynyl; R 7 , R 8 are each independently selected from the group consisting of hydrogen, deuterium, halogen, cycloalkyl, substituted or non-substituted C1-C 30 alkyl, substituted or non-substituted C1-C 30 alkoxy, substituted or non-substituted C2-C 20 alkenyl, substituted or non-substituted C2-C 10 alkynyl, -OR 14 , -NR 14 R 15 , -SR 14 , -CR 14 R 15 , wherein R 14 , R 15 are each independently selected from the group consisting of hydrogen, acyl, cycloalkyl, substituted or non-substituted C1-C 30 alkyl, substituted or non-substituted C1-C 30 alkoxy, substituted or non-substituted C2-C 20 alkenyl, substituted or non-substituted C2-C 10 alkynyl; or R 7 , R 8 are each independently selected from the group consisting of -(CH2) n O-P 2 , -(CH2) n S-P 2 ; wherein P 2 is P(M)2, P(OM)(N(M)2), P(M)(N(M)2); wherein M is selected from the group consisting of hydrogen, C1-C 20 alkyl, wherein said C1-C 20 alkyl can be substituted with one or more halogen, deuterium, cyano; n is any integer between 0 and 10; Z is -0-, -S-, -Se-, -NR 11 or -CR 11 R 12 wherein R 11 and R 12 are each independently hydrogen, halogen, acyl, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle, or substituted or unsubstituted cycloalkyl; W are each independently selected from -(CH2) n O- or -(CH2) n S-, wherein n is any integer from 0 to 10; Base is a natural nucleobase, a modified nucleobase, a universal base, an aryl group; P 1 is a hydroxyl protecting group; preferably, P 1 is DMTr, MMTr.

2. The nucleoside phosphoramidite compound of claim 1, wherein: The compounds of Formula 1 have the structure of Formula 2a or Formula 2b: wherein, R 1 , R 2 , R 3 , R 4 , R 5 , R 9 , R 10 each independently is selected from hydrogen, deuterium, halogen, cycloalkyl, substituted or non-substituted C1-C6alkyl, substituted or non-substituted C1-C6alkoxy, substituted or non-substituted C2-C6alkenyl, substituted or non-substituted C2-C6alkynyl; R 6 , R 7 , R 8 are each independently selected from the group consisting of hydrogen, deuterium, halogen, cycloalkyl, substituted or non-substituted C1-C 30 alkyl, substituted or non-substituted C1-C 30 alkoxy, substituted or non-substituted C2-C 20 alkenyl, substituted or non-substituted C2-C 10 alkynyl, OR 14 , NR 14 R 15 , SR 14 , CR 14 R 15 ; wherein R 14 , R 15 are each independently selected from the group consisting of hydrogen, acyl, cycloalkyl, substituted or non-substituted C1-C 30 alkyl, substituted or non-substituted C1-C 30 alkoxy, substituted or non-substituted C2-C 20 alkenyl, substituted or non-substituted C2-C 10 alkynyl; Z is -0-, -S-, -Se-, -NR 11 or -CR 11 R 12 wherein R 11 and R 12 are each independently hydrogen, halogen, acyl, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle, or substituted or unsubstituted cycloalkyl; X, W are each independently selected from -(CH2) n O- or -(CH2) n S-, wherein n is any integer from 0 to 10; Base is a natural nucleobase, a modified nucleobase, a universal base, an aryl group; P 1 is a hydroxyl protecting group; preferably, P 1 is DMTr, MMTr; P 2 P(M)2, P(OM)(N(M)2), P(M)(N(M)2); wherein M is selected from the group consisting of hydrogen, C1-C 20 alkyl, wherein said C1-C 20 alkyl can be substituted by one or more halogen, deuterium, cyano.

3. The nucleoside phosphoramidide compound according to claim 1, characterized in that: The compounds of Formula 1 have structures as shown in Formula 3a Formula 3b: wherein, R 1 , R 2 , R 9 , R 10 each independently is selected from hydrogen, deuterium, halogen, cycloalkyl, substituted or non-substituted C1-C6alkyl, substituted or non-substituted C1-C6alkoxy, substituted or non-substituted C2-C6alkenyl, substituted or non-substituted C2-C6alkynyl; R 6 R 7 R 8 Each is independently selected from hydrogen, deuterium, halogen, cycloalkyl, substituted or unsubstituted C1-C 30 Alkyl, substituted or unsubstituted C1-C 30 Alkoxy, substituted or unsubstituted C2-C 20 alkenyl, substituted or unsubstituted C2-C 10 alkynyl group, OR 14 NR 14 R 15 SR 14 CR 14 R 15 ;where R 14 R 15 Each is independently selected from hydrogen, acyl, cycloalkyl, substituted or unsubstituted C1-C 30 Alkyl, substituted or unsubstituted C1-C 30 Alkoxy, substituted or unsubstituted C2-C 20 alkenyl, substituted or unsubstituted C2-C 10 alkynyl group; Z is -0-, -S-, -Se-, -NR 11 or -CR 11 R 12 wherein R 11 and R 12 are each independently hydrogen, halogen, acyl, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle, or substituted or unsubstituted cycloalkyl; X, W are each independently selected from -(CH2) n O- or -(CH2) n S-, wherein n is any integer from 0 to 10; Base is a natural nucleobase, a modified nucleobase, a universal base, an aryl group; P 1 is a hydroxyl protecting group; preferably, P 1 is DMTr, MMTr; P 2 P(M)2, P(OM)(N(M)2), P(M)(N(M)2); wherein M is selected from the group consisting of hydrogen, C1-C 20 alkyl, wherein said C1-C 20 The alkyl group can be substituted by one or more halogen, deuterium, cyano.

4. The nucleoside phosphoramidite compound of claim 1, wherein: ###0002### The compounds of Formula 1 have structures as shown in Formula 4a Formula 4b: wherein, R 2 selected from hydrogen, deuterium, halogen, cycloalkyl, substituted or non- substituted Ci-C6alkyl, substituted or non-substituted Ci-C6alkoxy, substituted or non- substituted C2-C6alkenyl, substituted or non-substituted C2-C6alkynyl; R 6 , R 7 , R 8 are each independently selected from the group consisting of hydrogen, deuterium, halogen, cycloalkyl, substituted or non-substituted C1-C 30 alkyl, substituted or non-substituted C1-C 30 alkoxy, substituted or non-substituted C2-C 20 alkenyl, substituted or non-substituted C2-C 10 alkynyl, OR 14 , NR 14 R 15 , SR 14 , CR 14 R 15 ; wherein R 14 , R 15 are each independently selected from the group consisting of hydrogen, acyl, cycloalkyl, substituted or non-substituted C1-C 30 alkyl, substituted or non-substituted C1-C 30 alkoxy, substituted or non-substituted C2-C 20 alkenyl, substituted or non-substituted C2-C 10 alkynyl; Z is -0-, -S-, -Se-, -NR 11 or -CR 11 R 12 wherein R 11 and R 12 each independently is hydrogen, halogen, acyl, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle, or substituted or unsubstituted cycloalkyl; X, W are each independently selected from -(CH2) n O- or -(CH2) n S-, wherein n is any integer from 0 to 10; Base is a natural nucleobase, a modified nucleobase, a universal base, an aryl group; P 1 is a hydroxyl protecting group; preferably, P 1 is DMTr, MMTr; P 2 P(M)2, P(OM)(N(M)2), P(M)(N(M)2); wherein M is selected from the group consisting of hydrogen, C1-C 20 alkyl, wherein said C1-C 20 alkyl can be substituted by one or more halogen, deuterium, cyano.

5. The nucleoside phosphoramidide compound according to claim 1, characterized in that: The compound of Formula 1 has a structure as shown in Formula 5a or Formula 5b: wherein, R 7 R 8 Each is independently selected from hydrogen, deuterium, halogen, cycloalkyl, substituted or unsubstituted C1-C 30 Alkyl, substituted or unsubstituted C1-C 30 Alkoxy, substituted or unsubstituted C2-C 20 alkenyl, substituted or unsubstituted C2-C 10 alkynyl group, OR 14 NR 14 R 15 SR 14 CR 14 R 15 ;where R 14 R 15 Each is independently selected from hydrogen, acyl, cycloalkyl, substituted or unsubstituted C1-C 30 Alkyl, substituted or unsubstituted C1-C 30 Alkoxy, substituted or unsubstituted C2-C 20 alkenyl, substituted or unsubstituted C2-C 10 alkynyl group; Z is -0-, -S-, -Se-, -NR 11 or -CR 11 R 12 wherein R 11 and R 12 each independently is hydrogen, halogen, acyl, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle, or substituted or unsubstituted cycloalkyl; X is selected from -(CH2) n O- or -(CH2) n S-, wherein n is any integer from 0 to 10; Base is a natural nucleobase, a modified nucleobase, a universal base, an aryl group; P 1 is a hydroxyl protecting group; preferably, P 1 is DMTr, MMTr; P 2 P(M)2, P(OM)(N(M)2), P(M)(N(M)2); wherein M is selected from the group consisting of hydrogen, C1-C 20 alkyl, wherein said C1-C 20 alkyl can be substituted by one or more halogen, deuterium, cyano.

6. The nucleoside phosphoramidide compound according to claim 1, characterized in that: The compound of Formula 1 has a structure as shown in Formula 6a or Formula 6b: wherein, R 7 , R 8 each independently is selected from the group consisting of hydrogen, deuterium, halogen, cycloalkyl, substituted or non-substituted C1-C 30 alkyl, substituted or non-substituted C1-C 30 alkoxy, substituted or non-substituted C2-C 20 alkenyl, substituted or non-substituted C2-C 10 alkynyl, OR 14 , NR 14 R 15 , SR 14 , CR 14 R 15 ; wherein R 14 , R 15 each independently is selected from the group consisting of hydrogen, acyl, cycloalkyl, substituted or non-substituted C1-C 30 alkyl, substituted or non-substituted C1-C 30 alkoxy, substituted or non-substituted C2-C 20 alkenyl, substituted or non-substituted C2-C 10 alkynyl; Z is -0-, -S-, -Se-, -NR 11 or -CR 11 R 12 wherein R 11 and R 12 each independently is hydrogen, halogen, acyl, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle, or substituted or unsubstituted cycloalkyl; Base is a natural nucleobase, a modified nucleobase, a universal base, an aryl group; P 2 P(M)2, P(OM)(N(M)2), P(M)(N(M)2); wherein M is selected from the group consisting of hydrogen, C1-C 20 alkyl, wherein said C1-C 20 alkyl can be substituted by one or more halogen, deuterium, cyano.

7. The nucleoside phosphoramidide compound according to claim 1, characterized in that: The compound shown in Formula 1 has a structure as shown in Formula 7a or Formula 7b: wherein, R 7 R 8 Each is independently selected from hydrogen, deuterium, halogen, cycloalkyl, substituted or unsubstituted C1-C 30 Alkyl, substituted or unsubstituted C1-C 30 Alkoxy, substituted or unsubstituted C2-C 20 alkenyl, substituted or unsubstituted C2-C 10 alkynyl group, OR 14 NR 14 R 15 SR 14 CR 14 R 15 ;where R 14 R 15 Each is independently selected from hydrogen, acyl, cycloalkyl, substituted or unsubstituted C1-C 30 Alkyl, substituted or unsubstituted C1-C 30 Alkoxy, substituted or unsubstituted C2-C 20 alkenyl, substituted or unsubstituted C2-C 10 alkynyl group; Base is a natural nucleobase, a modified nucleobase, a universal base, an aryl group.

8. The nucleoside phosphoramidite compound according to claims 1-7, characterized by: said Base is selected from the following structures:

9. The nucleoside phosphoramidite compound of claim 1, wherein: R 1 selected from hydrogen; R 2 selected from hydrogen, halogen, methoxy; R 3 selected from hydrogen, deuterium, methyl; R 4 selected from hydrogen, deuterium; R 5 selected from hydrogen; R 6 selected from hydrogen, deuterium, halogen, methyl; R 7 selected from hydrogen, halogen, -OR 14 wherein R 14 is selected from acyl, cyclopropane, substituted or non-substituted C1-C 22 alkyl, substituted or non-substituted C3alkoxy, C3alkynyl; or R 7 is selected from -O-P 2 ; wherein P 2 is P(OM)(N(M)2); wherein M is selected from C2-C3alkyl, wherein said C2-C3alkyl can be substituted by a cyano group; R 8 selected from -OR 14 wherein R 14 is selected from C1-C 22 alkyl; or R 8 is selected from -O-P 2 ; wherein P 2 is P(OM)(N(M)2), P(M)(N(M)2); wherein M is selected from C1-C3 alkyl, wherein said C1-C3 alkyl can be substituted by cyano; R 9 selected from hydrogen, deuterium, methyl; R 10 selected from hydrogen, deuterium, methyl; Z is -0-, -S-, or -CR 11 R 12 wherein R 11 and R 12 are each independently selected from hydrogen; W is selected from oxygen; Base is selected from the following structures: P 1 DMTr, MMTr.

10. The nucleoside phosphoramidite compound of claim 1, wherein: The compounds of Formula I are one of the following structures:

11. Use of the nucleoside phosphoramidite compound of claims 1-10 for the manufacture of an oligonucleotide.

12. Use of the nucleoside phosphoramidite compound of claims 1-10 as an intermediate for the manufacture of an oligonucleotide.

13. Use according to claims 11-12, characterized in that: the oligonucleotide is selected from siRNA, antisense nucleic acid, saRNA, miRNA, aptamer, lncRNA.

14. An oligonucleotide comprising at least one structure according to Formula 8: wherein, R 1 , R 2 , R 3 , R 4 , R 5 , R 9 , R 10 each independently is selected from hydrogen, deuterium, halogen, cycloalkyl, substituted or non-substituted C1-C6alkyl, substituted or non-substituted C1-C6alkoxy, substituted or non-substituted C2-C6alkenyl, substituted or non-substituted C2-C6alkynyl; R 6 selected from the group consisting of hydrogen, deuterium, halogen, cycloalkyl, substituted or non-substituted C1-C 30 alkyl, substituted or non-substituted C1-C 30 alkoxy, substituted or non-substituted C2-C 20 alkenyl, substituted or non-substituted C2-C 10 alkynyl, OR 14 , NR 14 R 15 , SR 14 , CR 14 R 15 ; wherein R 14 , R 15 are each independently selected from the group consisting of hydrogen, acyl, cycloalkyl, substituted or non-substituted C1-C 30 alkyl, substituted or non-substituted C1-C 30 alkoxy, substituted or non-substituted C2-C 20 alkenyl, substituted or non-substituted C2-C 10 alkynyl; R 17 , R 18 are each independently selected from the group consisting of hydrogen, deuterium, halogen, cycloalkyl, substituted or non-substituted C1-C 30 alkyl, substituted or non-substituted C1-C 30 alkoxy, substituted or non-substituted C2-C 20 alkenyl, substituted or non-substituted C2-C 10 alkynyl, -OR 14 , -NR 14 R 15 , -SR 14 , -CR 14 R 15 , wherein R 14 , R 15 are each independently selected from the group consisting of hydrogen, acyl, cycloalkyl, substituted or non-substituted C1-C 30 alkyl, substituted or non-substituted C1-C 30 alkoxy, substituted or non-substituted C2-C 20 alkenyl, substituted or non-substituted C2-C 10 alkynyl; or R 17 , R 18 are each independently selected from -(CH2) n O-P 4 -, -(CH2) n S-P 4 -, wherein P4 is wherein P 3 is OH, substituted or unsubstituted C1-C 20 alkyl, substituted or unsubstituted C1-C 20 alkoxy; Y is S, O, BH2; Z is -0-, -S-, -Se-, -NR 11 or -CR 11 R 12 wherein R 11 and R 12 are each independently hydrogen, halogen, acyl, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle, or substituted or unsubstituted cycloalkyl; W is selected from -(CH2) n O- or -(CH2) n S-, wherein n is any integer from 0 to 10; Base is a natural nucleobase, a modified nucleobase, a universal base, an aryl group.

15. An siRNA comprising at least one structure according to Formula 9a or 9b: ###00039### 9a 9b wherein, R 1 , R 2 , R 3 , R 4 , R 5 , R 9 , R 10 each independently is selected from hydrogen, deuterium, halogen, cycloalkyl, substituted or non-substituted C1-C6alkyl, substituted or non-substituted C1-C6alkoxy, substituted or non-substituted C2-C6alkenyl, substituted or non-substituted C2-C6alkynyl; R 6 , R 7 , R 8 are each independently selected from the group consisting of hydrogen, deuterium, halogen, cycloalkyl, substituted or non-substituted C1-C 30 alkyl, substituted or non-substituted C1-C 30 alkoxy, substituted or non-substituted C2-C 20 alkenyl, substituted or non-substituted C2-C 10 alkynyl, OR 14 , NR 14 R 15 , SR 14 , CR 14 R 15 ; wherein R 14 , R 15 are each independently selected from the group consisting of hydrogen, acyl, cycloalkyl, substituted or non-substituted C1-C 30 alkyl, substituted or non-substituted C1-C 30 alkoxy, substituted or non-substituted C2-C 20 alkenyl, substituted or non-substituted C2-C 10 alkynyl; Z is -0-, -S-, -Se-, -NR 11 or -CR 11 R 12 wherein R 11 and R 12 are each independently hydrogen, halogen, acyl, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle, or substituted or unsubstituted cycloalkyl; X, W are each independently selected from -(CH2) n O- or -(CH2) n S-, wherein n is any integer from 0 to 10; Base is a natural nucleobase, a modified nucleobase, a universal base, an aryl group; P 3 OH, substituted or non-substituted C1-C 20 alkyl, substituted or non-substituted C1-C 20 alkoxy; Y is S, O, BH2.

16. The siRNA of claim 15, wherein the siRNA is a double-stranded conjugate having a sense strand, an antisense strand; the sense strand has a nucleotide length of 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40; the antisense strand has a nucleotide length of 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40.

17. The siRNA according to claim 15 or 16, characterized in that, the siRNA comprises at least one of the following positions with a structure as described in formula 9a or 9b: 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st from the 5’ end of the sense strand, 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd from the 5’ end of the antisense strand.

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