Compound comprising lipophilic monomer for delivery to eyes and central nervous system, and use thereof

Abstract

One aspect of the present invention relates to a compound, wherein the compound is an oligonucleotide, and the compound comprises at least one lipophilic monomer. Another aspect of the present invention relates to a method for gene silencing, wherein the method comprises administering to a cell or a subject in need thereof a therapeutically effective amount of a compound conjugated with a lipophilic monomer.

Description

Compounds containing lipophilic monomers for delivery to the eye and central nervous system and their applications.

[0001] Citation of relevant applications

[0002] This application claims priority and benefits to Chinese Patent Application No. 202410932222.0, filed with the State Intellectual Property Office of the People's Republic of China on July 11, 2024; Chinese Patent Application No. 202411578349.3, filed with the State Intellectual Property Office of the People's Republic of China on November 6, 2024; Chinese Patent Application No. 202510630001.2, filed with the State Intellectual Property Office of the People's Republic of China on May 15, 2025; and Chinese Patent Application No. 202510659691.4, filed with the State Intellectual Property Office of the People's Republic of China on May 21, 2025, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure pertains to the field of biomedicine, specifically relating to compounds containing lipophilic monomers, their preparation methods, and applications. Background Technology

[0004] Modified nucleic acids show great potential as research tools in the laboratory and as therapeutic agents in the clinic for regulating gene expression. Several classes of oligonucleotide or nucleic acid-based therapeutics are in clinical trials, including antisense oligonucleotides (ASO), short interfering RNA (siRNA), double-stranded nucleic acids (dsNA), aptamers, ribozymes, exon skipping and splicing-altering oligonucleotides, immunomodulatory oligonucleotides, mRNA, and CRISPR. Chemical modifications in these molecules play a crucial role in overcoming the challenges of oligonucleotide therapeutics, including improving nuclease stability, RNA binding affinity, and pharmacokinetics.

[0005] GalNAc-conjugated siRNAs have become a major mode of delivery for liver-targeted small nucleic acid drugs. However, the development and clinical application of RNAi oligonucleotides in extrahepatic cells, tissues, and organs (e.g., the central nervous system, eye, or adipose tissue) still face technical hurdles. Therapeutic gene silencing mediated by RNAi oligonucleotides is particularly significant for treating neurological and ocular diseases. This invention provides an oligonucleotide conjugate with high delivery efficiency, capable of effectively delivering oligonucleotides to target organs or tissues, such as the central nervous system and ocular tissues, exhibiting high pharmaceutical activity. Summary of the Invention

[0006] This disclosure relates to hydrophobic-hydrophobic ligand conjugates (e.g., nucleic acid-hydrophobic ligand conjugates and oligonucleotide-hydrophobic ligand conjugates). Specifically, this disclosure relates to nucleic acid-lipid conjugates and oligonucleotide-lipid conjugates or isomers thereof, methods for their preparation, and methods for regulating (e.g., inhibiting or reducing) the expression of target genes in the central nervous system (hereinafter abbreviated as "CNS") using conjugated nucleic acids and oligonucleotides according to the description provided herein (e.g., cells, tissues, or regions of the CNS). This disclosure also provides pharmaceutical compositions comprising the conjugates of this specification and methods for treating various diseases or conditions using said compositions.

[0007] In this invention, novel lipophilic monomers are provided to conjugate lipophilic moieties (e.g., fatty chains) to oligonucleotides (including single-stranded or double-stranded oligonucleotides).

[0008] Specifically, the present invention provides a compound, which is an oligonucleotide, comprising at least one lipophilic monomer selected from the following formula:

[0009] Among them, ring A is selected from 5-6 membered heterocycles;

[0010] D is selected from substituted or unsubstituted chain saturated aliphatic hydrocarbon groups, preferably substituted or unsubstituted straight-chain or branched C. 1-30 Chain-like saturated aliphatic hydrocarbon group, wherein the chain-like saturated aliphatic hydrocarbon group may optionally be converted by hydroxyl, halogen, methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 Hydrocarbon substitution, wherein the methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 The hydrocarbon group may optionally be replaced by a hydroxyl group or a halogen;

[0011] R a Selected from hydrogen, hydroxyl, -C 0-6 Alkylene-OL d ;

[0012] L a Selected from single bonds, reactive phosphorus groups, solid supports, -P(=O)(OH)-O-, or -P(=S)(OH)-O-;

[0013] L b Selected from single bond, C 1-6 Alkyl, substituted C 1-6 Alkyl groups and -C 1-6 An alkyl-benzene ring, wherein the benzene ring forms a fused ring with ring A;

[0014] L c Selected from single bond, C1-6 Alkylene, Alkoxy, -O-, -OC 1-8 Alkylene -OC(=O)-, -OC 1-8 Alkylene-OC(=S)-, -OC 1-8 Alkylene -NH-C(=O)-, -OC 1-8 Alkylene-NH-C(=S)-, -OC 1-8 Alkylene -SC(=O)-, -OC 1-8 Alkylene-SC(=S)-, -OC 1-10 Alkoxy-NH-C(=O)-, -C 1-10 Alkoxy-NH-C(=O)-, -C 1-10 Alkylene -NH-C(=O)-, -OC 1-8 Alkyl-5-6-membered heteroaryl groups, optionally substituted with halogens;

[0015] L d Selected from single bonds, H, reactive phosphorus groups, phosphoramidite groups, solid supports, -P(=O)(OH)-O-, -P(=S)(OH)-O-, hydroxyl protecting groups (e.g., ester protecting groups, aryl protecting groups, alkyl protecting groups, alkoxymethyl protecting groups, silane protecting groups, including but not limited to DMT);

[0016] R b Selected from hydrogen, modified or unmodified base B;

[0017] The lipophilic monomers contained in the compound are not selected from the following:

[0018] B' is a natural base.

[0019] This invention provides compounds that are oligonucleotides, the compounds comprising at least one lipophilic monomer selected from the following formulas:

[0020] Among them, ring A is selected from 5-6 membered heterocycles;

[0021] D is selected from substituted or unsubstituted chain saturated aliphatic hydrocarbon groups, preferably substituted or unsubstituted straight-chain or branched C. 1-30 Chain-like saturated aliphatic hydrocarbon group, wherein the chain-like saturated aliphatic hydrocarbon group may optionally be converted by hydroxyl, halogen, methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 Hydrocarbon substitution, wherein the methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 The hydrocarbon group may optionally be replaced by a hydroxyl group or a halogen;

[0022] R a Selected from hydrogen, hydroxyl, -C 0-6 Alkylene-OL d ;

[0023] L a Selected from single bonds, reactive phosphorus groups, solid supports, -P(=O)(OH)-O-, or -P(=S)(OH)-O-;

[0024] L b Selected from single bond, C 1-6 Alkyl, substituted C 1-6 Alkyl groups and -C 1-6 An alkyl-benzene ring, wherein the benzene ring forms a fused ring with ring A;

[0025] L c Selected from single bond, C 1-6 Alkylene, Alkoxy, -O-, -OC 1-8 Alkylene -OC(=O)-, -OC 1-8 Alkylene-OC(=S)-, -OC 1-8 Alkylene -NH-C(=O)-, -OC 1-8 Alkylene-NH-C(=S)-, -OC 1-8 Alkylene -SC(=O)-, -OC 1-8 Alkylene-SC(=S)-, -OC 1-10 Alkoxy-NH-C(=O)-, -C 1-10 Alkoxy-NH-C(=O)-, -C 1-10 Alkylene -NH-C(=O)-, -OC 1-8 Alkyl-5-6-membered heteroaryl groups, optionally substituted with halogens;

[0026] L d Selected from single bonds, H, reactive phosphorus groups, phosphoramidite groups, solid supports, -P(=O)(OH)-O-, -P(=S)(OH)-O-, hydroxyl protecting groups (e.g., ester protecting groups, aryl protecting groups, alkyl protecting groups, alkoxymethyl protecting groups, silane protecting groups, including but not limited to DMT);

[0027] R b Selected from hydrogen, modified or unmodified nucleobase B;

[0028] The lipophilic monomers contained in the compound are not selected from the following:

[0029] B' is a natural nucleobase.

[0030] In some embodiments, ring A may be selected from a 5- or 6-membered heterocycle containing 1, 2, or 3 heteroatoms selected from N and O. For example, ring A may be selected from tetrahydropyrrole, tetrahydrofuran, tetrahydropyran, morpholino, or triazolyl.

[0031] In some implementations, ring A can be selected from... When ring A is selected from triazole, L is preferred. b Selected from -C 1-6 Alkyl-benzene ring, wherein the benzene ring forms a fused ring with the triazole ring.

[0032] In some embodiments, D is selected from substituted or unsubstituted chain saturated aliphatic hydrocarbon groups, preferably substituted or unsubstituted straight-chain or branched C. 14-24 Chain-like saturated aliphatic hydrocarbon group, wherein the chain-like saturated aliphatic hydrocarbon group may optionally be converted by hydroxyl, halogen, methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 Hydrocarbon substitution, wherein the methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 The hydrocarbon group may optionally be substituted with a hydroxyl group or a halogen. In a preferred embodiment, D may be a self-substituted or unsubstituted C. 12 C 13 C 14 C 15 C 16 C 17 C 18 C 19 C 20 C 21 C 22 C 23 Or C 24 The chain-like saturated aliphatic hydrocarbon group, wherein the chain-like saturated aliphatic hydrocarbon group may optionally be converted by hydroxyl, halogen, methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 Hydrocarbon substitution, wherein the methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 The hydrocarbon group may optionally be replaced by a hydroxyl group or a halogen.

[0033] In a preferred embodiment, D can be selected from C, which is optionally halogenated. 12 C 13 C 14 C 15 C 16 C 17 C 18 C 19 C 20 C 21 C 22C 23 Or C 24 Chain-like saturated aliphatic hydrocarbon groups.

[0034] In one implementation, L a Selected from single bonds, -P(=O)(OH)-O-, or -P(=S)(OH)-O-; or,

[0035] L d Selected from single bonds, H, -P(=O)(OH)-O- or -P(=S)(OH)-O-.

[0036] In some embodiments, the nucleobases are selected from naturally occurring nucleobases, such as cytosine, guanine, thymine, adenine, and uracil; they may also be selected from modified nucleobases. For a definition of modified bases, please refer to the relevant definitions of oligonucleotide chains below.

[0037] In some embodiments, the compound may have one or two Ra.

[0038] In some implementations, -Lc-D can be selected from -OD, for example, D can be selected from one or more of the following:

[0039] In some implementations, -Lc-D can be selected from one or more of the following:

[0040] In some implementations, D may be selected from one or more of the following:

[0041] In some embodiments, the reactive phosphorus group is selected from...

[0042] In some embodiments, the compounds (i.e. oligonucleotides) of the present invention comprise lipophilic monomers having the following structure:

[0043] R1 is defined as described above for D; for example, R1 is the substituted or unsubstituted straight or branched chain C. 1-30 Chain-like saturated aliphatic hydrocarbon group, wherein the chain-like saturated aliphatic hydrocarbon group may optionally be converted by hydroxyl, halogen, methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 Hydrocarbon substitution, wherein the methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 The hydrocarbon group may optionally be replaced by a hydroxyl group or a halogen;

[0044] R a1 Selected from hydrogen, hydroxyl, -C0-6 Alkylene-OL d1 ;

[0045] L d1 Selected from H, reactive phosphorus groups, phosphoramidite groups, solid supports, -P(=O)(OH)-O-, -P(=S)(OH)-O-, hydroxyl protecting groups (e.g., ester protecting groups, aryl protecting groups, alkyl protecting groups, alkoxymethyl protecting groups, silane protecting groups, including but not limited to DMT);

[0046] L2 is independently selected from H, reactive phosphorus groups, hydroxyl protecting groups (e.g., ester protecting groups, aryl protecting groups, alkyl protecting groups, alkoxymethyl protecting groups, silane protecting groups, including but not limited to DMT), -P(=O)(OH)-O-, -P(=S)(OH)-O-, or solid support.

[0047] In some embodiments, the compounds (i.e. oligonucleotides) of the present invention comprise lipophilic monomers having the following structure:

[0048] R1 is defined as described above for D; for example, R1 is the substituted or unsubstituted straight or branched chain C. 1-30 Chain-like saturated aliphatic hydrocarbon group, wherein the chain-like saturated aliphatic hydrocarbon group may optionally be converted by hydroxyl, halogen, methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 Hydrocarbon substitution, wherein the methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 The hydrocarbon group may optionally be replaced by a hydroxyl group or a halogen;

[0049] R a1 Selected from hydrogen, hydroxyl, -C 0-6 Alkylene-OL d1 ;

[0050] L d1 Selected from single bonds, H, reactive phosphorus groups, phosphoramidite groups, solid supports, -P(=O)(OH)-O-, -P(=S)(OH)-O-, hydroxyl protecting groups (e.g., ester protecting groups, aryl protecting groups, alkyl protecting groups, alkoxymethyl protecting groups, silane protecting groups, including but not limited to DMT);

[0051] L2 is independently selected from H, reactive phosphorus groups, hydroxyl protecting groups (e.g., ester protecting groups, aryl protecting groups, alkyl protecting groups, alkoxymethyl protecting groups, silane protecting groups, including but not limited to DMT), -P(=O)(OH)-O-, -P(=S)(OH)-O-, or solid supports.

[0052] In some embodiments, the compounds (i.e. oligonucleotides) of the present invention comprise lipophilic monomers having the following structure:

[0053] R1 is defined as described above for D; for example, R1 is the substituted or unsubstituted straight or branched chain C. 1-30 Chain-like saturated aliphatic hydrocarbon group, wherein the chain-like saturated aliphatic hydrocarbon group may optionally be converted by hydroxyl, halogen, methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 Hydrocarbon substitution, wherein the methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 The hydrocarbon group may optionally be replaced by a hydroxyl group or a halogen;

[0054] R a1 Selected from hydrogen, hydroxyl, -C 0-6 Alkylene-OL d1 ;

[0055] L d1 Selected from single bonds, H, reactive phosphorus groups, phosphoramidite groups, solid supports, -P(=O)(OH)-O-, -P(=S)(OH)-O-, hydroxyl protecting groups (e.g., ester protecting groups, aryl protecting groups, alkyl protecting groups, alkoxymethyl protecting groups, silane protecting groups, including but not limited to DMT);

[0056] L2 is independently selected from single bonds, reactive phosphorus groups, -P(=O)(OH)-O-, -P(=S)(OH)-O-, or solid supports.

[0057] In a preferred embodiment, R1 is a substituted or unsubstituted straight-chain or branched C. 12-24 Chain-like saturated aliphatic hydrocarbon group, wherein the chain-like saturated aliphatic hydrocarbon group may optionally be converted by hydroxyl, halogen, methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 Hydrocarbon substitution, wherein the methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 The hydrocarbon group may optionally be replaced by a hydroxyl group or a halogen.

[0058] In some embodiments, the compounds of the present invention comprise one or more lipophilic monomers having the following structures:

[0059] R1 is defined as described above for D; for example, R1 is the substituted or unsubstituted straight or branched chain C. 1-30 Chain-like saturated aliphatic hydrocarbon group, wherein the chain-like saturated aliphatic hydrocarbon group may optionally be converted by hydroxyl, halogen, methylene group, C 1-4Alkyl groups, or C groups having one or two unsaturated bonds 2-4 Hydrocarbon substitution, wherein the methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 The hydrocarbon group may optionally be replaced by a hydroxyl group or a halogen;

[0060] R a1 Selected from hydrogen, hydroxyl, -C 0-6 Alkylene-OL d1 ;

[0061] L d1 Selected from single bonds, H, -P(=O)(OH)-O- or -P(=S)(OH)-O-;

[0062] L2 is independently selected from single bonds, -P(=O)(OH)-O-, or -P(=S)(OH)-O-.

[0063] In some embodiments, in the case of lipophilic monomers represented by Formula Ia, Formula I-a1, or Formula I-a2, the compounds of the present invention comprise one or more of the lipophilic monomers having the following structure: R1 and L2 are as defined in this paper, where, This indicates a bond that links to the rest of the compound.

[0064] In some embodiments, in the case of lipophilic monomers represented by Formula Ia, Formula I-a1, or Formula I-a2, the compounds of the present invention comprise one or more of the lipophilic monomers having the following structure:

[0065] Where X is selected from O or S, and where R1 and R a1 As defined in this article, where, This indicates a bond linked to the remainder of the compound. Preferably, in the case of lipophilic monomers shown in Formula Ia, Formula I-a1, or Formula I-a2, the compounds of the present invention comprise lipophilic monomers having, for example, the following structure: Where X is selected from O or S, and where R1 and L2 are as defined in this paper, where, This indicates a bond that links to the rest of the compound.

[0066] In some embodiments, the oligonucleotides of the present invention comprise lipophilic monomers having the following structure:

[0067] Where X is selected from O or S, preferably, for example,

[0068] In a preferred embodiment, in the case of the lipophilic monomers represented by formula Ia, formula I-a1, or formula I-a2, R1 is preferably derived from the following structure:

[0069] In some embodiments, in the case of lipophilic monomers represented by Formula Ia, Formula I-a1, or Formula I-a2, the compounds of the present invention comprise one or more of the lipophilic monomers having the following structure: In these structural formulas, Indicates the bonds that link to the rest of the compound.

[0070] In this context, X is independently selected from either O or S.

[0071] L2 is independently selected from single bonds, -P(=O)(OH)-O- or -P(=S)(OH)-O-;

[0072] Ra1 is independently selected from single bonds or hydrogen;

[0073] R1 is independently selected from one or more of the following:

[0074] In some embodiments, in the case of lipophilic monomers represented by Formula Ia, Formula I-a1, or Formula I-a2, the compounds of the present invention comprise one or more of the lipophilic monomers having the following structure:

[0075] In these structural formulas, Indicates the bonds that link to the rest of the compound.

[0076] In this context, X is independently selected from either O or S.

[0077] R a1 Each is independently selected from a single bond or hydrogen;

[0078] R1 is independently selected from one or more of the following:

[0079] In some embodiments, in the case of lipophilic monomers represented by Formula Ia, Formula I-a1, or Formula I-a2, the compounds of the present invention comprise one or more of the lipophilic monomers having the following structure:

[0080] In these structural formulas, Indicates the bonds that link to the rest of the compound.

[0081] In this context, X is independently selected from either O or S.

[0082] R1 is independently selected from one or more of the following:

[0083] In some embodiments, in the case of lipophilic monomers represented by Formula Ia, Formula I-a1, or Formula I-a2, the compounds of the present invention comprise lipophilic monomers having the following structures:

[0084] In this structural formula, Indicates the bonds that link to the rest of the compound.

[0085] In this context, X is independently selected from either O or S.

[0086] R1 is selected from the following:

[0087] In equation Ia, equation I-a1, or equation I-a2, it can be passed through L2 and / or R. a1 It is covalently linked to the rest of the compound.

[0088] In some embodiments, the compounds of the present invention comprise lipophilic monomers having the following structures:

[0089] The definition of -OR5 can be found above regarding -L. c -D is defined as follows: for example, R5 is selected from straight chain or branched chain C. 1-30 Chain-like saturated aliphatic hydrocarbon group, wherein the chain-like saturated aliphatic hydrocarbon group may optionally be converted by hydroxyl, halogen, methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 Hydrocarbon substitution, wherein the methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 The hydrocarbon group may optionally be replaced by a hydroxyl group or a halogen, wherein the straight-chain or branched C 1-30 The chain-like saturated aliphatic hydrocarbon group is substituted with at least one halogen; or R5 is selected from -(CH2-(OCH2CH2)q)-NH-(C(=O)-(C8-C 30 Alkylene), the C8-C 30 The alkylene group is substituted with at least one halogen, and q is selected from 2, 3, 4, 5, and 6; or R5 is selected from -(C 1-8 alkylene)-5-6-membered heteroaryl-(C 12-30 Alkylene), the C 12-30 The alkylene group may optionally be replaced by 1-5 halogens;

[0090] R6 is selected from single bonds, reactive phosphorus groups, -P(=O)(OH)-O-, and -P(=S)(OH)-O-.

[0091] R7 is selected from H, single bond, or hydroxyl protecting group;

[0092] B represents both modified and unmodified nucleobases.

[0093] In some embodiments, the compounds (i.e. oligonucleotides) of the present invention comprise lipophilic monomers having the following structure:

[0094] R5 can be defined as described above regarding -L c -D is defined as follows: for example, R5 is selected from straight chain or branched chain C. 1-30 Chain-like saturated aliphatic hydrocarbon group, wherein the chain-like saturated aliphatic hydrocarbon group may optionally be converted by hydroxyl, halogen, methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 Hydrocarbon substitution, wherein the methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 The hydrocarbon group may optionally be replaced by a hydroxyl group or a halogen, wherein the straight-chain or branched C 1-30 The chain-like saturated aliphatic hydrocarbon group is substituted with at least one halogen; or R5 is selected from -(CH2-(OCH2CH2)q)-NH-(C(=O)-(C8-C 30 Alkylene), the C8-C 30 The alkylene group is substituted with at least one halogen, and q is selected from 2, 3, 4, 5, and 6; or R5 is selected from -(C 1-8 alkylene)-5-6-membered heteroaryl-(C 12-30 Alkylene), the C 12-30 The alkylene group may optionally be replaced by 1-5 halogens;

[0095] R6 is selected from H, reactive phosphorus groups, -P(=O)(OH)-O-, and -P(=S)(OH)-O-.

[0096] R7 is selected from H or a hydroxyl protecting group;

[0097] B represents both modified and unmodified nucleobases.

[0098] In some embodiments, the compounds (i.e. oligonucleotides) of the present invention comprise lipophilic monomers having the following structure:

[0099] The definition of -OR5 can be found above regarding -L. c -D is defined as follows: for example, R5 is selected from straight chain or branched chain C. 1-30 Chain-like saturated aliphatic hydrocarbon group, wherein the chain-like saturated aliphatic hydrocarbon group may optionally be converted by hydroxyl, halogen, methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4Hydrocarbon substitution, wherein the methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 The hydrocarbon group may optionally be replaced by a hydroxyl group or a halogen, wherein the straight-chain or branched C 1-30 The chain-like saturated aliphatic hydrocarbon group is substituted with at least one halogen; or R5 is selected from -(CH2-(OCH2CH2)q)-NH-(C(=O)-(C8-C 30 Alkylene), the C8-C 30 The alkylene group is substituted with at least one halogen, and q is selected from 2, 3, 4, 5, and 6; or R5 is selected from -(C 1-8 alkylene)-5-6-membered heteroaryl-(C 12-30 Alkylene), the C 12-30 The alkylene group may optionally be replaced by 1-5 halogens;

[0100] R6 is selected from H, single bond, reactive phosphorus group, -P(=O)(OH)-O-, -P(=S)(OH)-O-;

[0101] R7 is selected from H, single bond, or hydroxyl protecting group;

[0102] B represents both modified and unmodified nucleobases.

[0103] Optionally, R6 and R7 may not both be H.

[0104] In some embodiments, R5 is defined as described above regarding D. In a preferred embodiment, R5 is selected from -(CH2-(OCH2CH2)q)-NH-(C(=O)-(C8-C 30 Alkylene), the C8-C 30 The alkylene group is substituted with at least one halogen, and q is selected from 2, 3, 4, 5, and 6; or -R5 is selected from -(C 1-8 alkylene)-5-6-membered heteroaryl-(C 12-30 Alkylene), the C 12-30 The alkylene group may optionally be substituted with 1-5 halogens. In a preferred embodiment, R5 is selected from -(C 1-8 alkylene)-5-6-membered heteroaryl-(C 12-30 Alkylene), the C 12-30 The alkylene group may optionally be substituted with 1 to 5 halogens, wherein the 5-6 heteroaryl group is selected from 5-6 heteroaryl groups containing 1, 2 or 3 heteroatoms selected from N or O, for example, the 5-6 heteroaryl group is selected from 5-6 heteroaryl groups containing 1, 2 or 3 N atoms, such as triazolyl.

[0105] In some embodiments, R5 is selected from straight-chain or branched C molecules substituted with at least one halogen. 12-30The chain-like saturated aliphatic hydrocarbon group may optionally be substituted with a methylene group, which may optionally be substituted with a hydroxyl group or a halogen.

[0106] In some embodiments, in the case of lipophilic monomers represented by Formula I-b1 or Formula Ib, the compounds (i.e., oligonucleotides) of the present invention comprise one or more of the lipophilic monomers having the following structures:

[0107] Wherein, X is selected from O or S, R5, R7, and B are each independently as defined herein, wherein the wavy line represents a single bond linked to the remainder of the compound; preferably selected from

[0108] In some embodiments, in the case of lipophilic monomers represented by Formula I-b1 or Formula Ib, the compounds (i.e., oligonucleotides) of the present invention comprise one or more of the lipophilic monomers having the following structures:

[0109] Wherein, X is selected from O or S, R5, R7, and B are each independently as defined herein, wherein the wavy line represents a single bond linked to the remainder of the compound; preferably selected from

[0110] In some implementations, R5 in formula I-b1 or formula Ib can be selected from the following:

[0111] In a specific embodiment, in the case of the lipophilic monomer shown in Formula I-b1 or Formula Ib, it can be covalently linked to the remainder of the compound via R7 and / or R6.

[0112] In some embodiments, the compounds (i.e. oligonucleotides) of the present invention comprise lipophilic monomers having the following structure:

[0113] R8 can be defined as described above -L c The definition of -D, for example, R8 is -(C 1-8 Alkylene-OC(=O)-C 12-30 alkylene), -(C 1-8 Alkylene-OC(=S)-C 12-30 alkylene) or -(C 1-8 Alkylene-NC(=O)-C 12-30 Alkylenes), wherein the alkylene group may optionally be substituted with 1-5 halogens;

[0114] L1 and L2 are each independently selected from H, reactive phosphorus groups, -P(=O)(OH)-O-, -P(=S)(OH)-O-, hydroxyl protecting groups, or solid supports.

[0115] In some embodiments, the compounds (i.e. oligonucleotides) of the present invention comprise lipophilic monomers having the following structure:

[0116] R8 can be defined as described above -L c The definition of -D, for example, R8 is -(C 1-8 Alkylene-OC(=O)-C 12-30 alkylene), -(C 1-8 Alkylene-OC(=S)-C 12-30 alkylene) or -(C 1-8 Alkylene-NC(=O)-C 12-30 Alkylenes), wherein the alkylene group may optionally be substituted with 1-5 halogens;

[0117] L1 and L2 are each independently selected from H, single bonds, reactive phosphorus groups, -P(=O)(OH)-O-, -P(=S)(OH)-O-, hydroxyl protecting groups, or solid supports.

[0118] In some embodiments, in formula Ic, L1 is selected from H or a single bond, and L2 is selected from a single bond, -P(=O)(OH)-O-, or -P(=S)(OH)-O-.

[0119] In some embodiments, in the case of lipophilic monomers represented by Formula Ic, the compounds (i.e., oligonucleotides) of the present invention comprise one or more of the lipophilic monomers having the following structure:

[0120] Wherein, X is selected from O or S, R8 is as defined herein, and the wavy line represents a single bond linked to the rest of the compound.

[0121] In some implementations, R8 in equation Ic can be selected from the following:

[0122] In some embodiments, in the case of lipophilic monomers represented by Formula Ic, the compounds of the present invention comprise one or more of lipophilic monomers having the following structures:

[0123] Wherein, the wavy line represents a single bond linked to the rest of the compound;

[0124] Where X is selected from O or S, and R8 is...

[0125] In a specific embodiment, in the case of the lipophilic monomer shown in Formula Ic, it can be covalently linked to the remainder of the compound via L1 and / or L2.

[0126] In some embodiments, the compounds (i.e. oligonucleotides) of the present invention comprise lipophilic monomers having the following structure:

[0127] R9 can be defined as described above for D, for example, R9 is selected from substituted or unsubstituted straight or branched C. 1-30 Chain-like saturated aliphatic hydrocarbon group, wherein the chain-like saturated aliphatic hydrocarbon group may optionally be converted by hydroxyl, halogen, methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 Hydrocarbon substitution, wherein the methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 The hydrocarbon group may optionally be replaced by a hydroxyl group or a halogen;

[0128] L1 is independently selected from H, reactive phosphorus groups, -P(=O)(OH)-O-, -P(=S)(OH)-O-, hydroxyl protecting groups, or solid supports;

[0129] p is selected from 1, 2, 3, 4, and 5.

[0130] In some embodiments, the compounds (i.e. oligonucleotides) of the present invention comprise lipophilic monomers having the following structure:

[0131] R9 can be defined as described above for D, for example, R9 is selected from substituted or unsubstituted straight or branched C. 1-30 Chain-like saturated aliphatic hydrocarbon group, wherein the chain-like saturated aliphatic hydrocarbon group may optionally be converted by hydroxyl, halogen, methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 Hydrocarbon substitution, wherein the methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 The hydrocarbon group may optionally be replaced by a hydroxyl group or a halogen;

[0132] L1 is independently selected from H, single bond, reactive phosphorus group, -P(=O)(OH)-O-, -P(=S)(OH)-O-, hydroxyl protecting group or solid support;

[0133] p is selected from 1, 2, 3, 4, and 5.

[0134] In some implementations, in formula Id, L1 is independently selected from single bonds, -P(=O)(OH)-O-, or -P(=S)(OH)-O-.

[0135] In some implementations, p in formula Id can be 1 or 2.

[0136] In some embodiments, in the case of the lipophilic monomer shown in Formula Id, the compounds (i.e., oligonucleotides) of the present invention comprise lipophilic monomers having the following structure: Wherein, X is selected from O or S, R9 is as defined herein, and the wavy line represents a single bond linked to the rest of the compound.

[0137] In some implementations, the R9 aliphatic chain in formula Id may be selected from the following:

[0138] In a specific embodiment, in the case of the lipophilic monomer shown in Formula Id, it can be covalently linked to the rest of the compound via L1.

[0139] In some embodiments, the compounds (i.e. oligonucleotides) of the present invention comprise lipophilic monomers having the following structure:

[0140] Among them, R 10 The definition can be as described above -L c The definition of -D, for example, R 10 Selected from straight-chain or branched C atoms substituted with at least one halogen 1-30 Chain-like saturated aliphatic hydrocarbon group, wherein the chain-like saturated aliphatic hydrocarbon group may optionally be converted by hydroxyl, halogen, methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 Hydrocarbon substitution, wherein the methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 The hydrocarbon group may optionally be replaced by a hydroxyl group or a halogen; or R 10 Selected from -(C1-C4 alkylene)-NH-C(=O)-(C 8-30 Alkyl), the C1-C4 alkylene or C 8-30 Alkyl groups may optionally be replaced by 1-5 halogens;

[0141] L1 and L2 are each independently selected from H, reactive phosphorus groups, -P(=O)(OH)-O-, -P(=S)(OH)-O-, hydroxyl protecting groups, or solid supports.

[0142] In some embodiments, the compounds (i.e. oligonucleotides) of the present invention comprise lipophilic monomers having the following structure:

[0143] Among them, R 10 The definition can be as described above -L c The definition of -D, for example, R10 Selected from straight-chain or branched C atoms substituted with at least one halogen 1-30 Chain-like saturated aliphatic hydrocarbon group, wherein the chain-like saturated aliphatic hydrocarbon group may optionally be converted by hydroxyl, halogen, methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 Hydrocarbon substitution, wherein the methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 The hydrocarbon group may optionally be replaced by a hydroxyl group or a halogen; or R 10 Selected from -(C1-C4 alkylene)-NH-C(=O)-(C 8-30 Alkyl), the C1-C4 alkylene or C 8-30 Alkyl groups may optionally be replaced by 1-5 halogens;

[0144] L1 and L2 are each independently selected from H, single bonds, reactive phosphorus groups, -P(=O)(OH)-O-, -P(=S)(OH)-O-, hydroxyl protecting groups, or solid supports.

[0145] In some embodiments, in formula Ie, L1 is selected from H or a single bond, and L2 is selected from a single bond, -P(=O)(OH)-O- or -P(=S)(OH)-O-.

[0146] In some embodiments, the compounds (i.e. oligonucleotides) of the present invention comprise one or more of a lipophilic monomer having the following structure: Where X is selected from O or S, R 10 As defined herein, wavy lines represent single bonds linked to the rest of the compound.

[0147] In some implementations, R 10 Selected from straight-chain or branched C atoms substituted with at least one halogen 12-30 Chain-like saturated aliphatic hydrocarbon group, wherein the chain-like saturated aliphatic hydrocarbon group may optionally be converted by hydroxyl, halogen, methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 Hydrocarbon substitution, wherein the methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 The hydrocarbon group may optionally be replaced by a hydroxyl group or a halogen. In a preferred embodiment, R 10 Selected from -(C1-C4 alkylene)-NH-C(=O)-(C 12-30 Alkyl), the C1-C4 alkylene or C 12-30 The alkyl group may optionally be replaced by 1 to 5 halogens.

[0148] In some implementations, R 10 Selected from

[0149] In a specific embodiment, in the case of the lipophilic monomer shown in Formula Ie, it can be covalently linked to the remainder of the compound via L1 and / or L2.

[0150] On the other hand, the present invention provides a double-stranded RNA having an antisense strand and a sense strand, each strand having 14-30 nucleotides, the nucleotides being linked by phosphate ester groups, thiophosphate ester groups or other linker molecules, wherein the antisense strand is contained in a sequence complementary to the sense strand and the target mRNA, wherein the sense strand or the antisense strand comprises one or more of Formula I.

[0151] In some preferred embodiments, formula I can be selected from formulas Ia, I-a1, I-a2, Ib, I-b1, Ic, Id and Ie.

[0152] In some embodiments, the lipophilic monomer is selected from the group consisting of:

[0153] The wavy lines represent single bonds that are linked to the rest of the compound.

[0154] In some embodiments, this application provides precursors of lipophilic monomers selected from compounds listed below, or pharmaceutically acceptable salts, tautomers, or stereoisomers thereof:

[0155] In this application, the use of the precursor of the lipophilic monomer in the preparation of the compounds of this application (e.g., oligonucleotides) is provided.

[0156] In some embodiments, the lipophilic monomer conjugated to one or more positions on the chain of the compound has the following structure: In the above structure of the lipophilic monomer, the monomer may also contain one or more asymmetric centers, and thus exist in the form of racemic mixtures and racemic mixtures, single enantiomers, individual diastereomers and mixtures of diastereomers. All such isomeric forms of the monomer are explicitly included.

[0157] In some embodiments, the compound is a single-stranded or double-stranded oligonucleotide.

[0158] In some embodiments, the compound is preferably a double-stranded oligonucleotide, such as siRNA.

[0159] The double-stranded oligonucleotide comprises a sense strand and an antisense strand, wherein the antisense strand is complementary to the target gene; and the sense strand is complementary to the antisense strand.

[0160] In some implementations, the antisense strand comprises a sequence B whose inverse complementary sequence to at least 15 consecutive nucleotides in the target sequence differs by no more than 3 nucleotides. Specifically, a start nucleotide is selected in the target sequence along the 5' end to the 3' end, and at least 15 nucleotides extending in the 3' direction, including the start nucleotide, serve as the binding region of the siRNA. The antisense strand comprises the inverse complementary sequence of the nucleotide sequence corresponding to the binding region. It should be noted that the start nucleotide can be a nucleotide at any position in the target sequence, as long as extending in the 3' direction from the start nucleotide yields at least 15 consecutive nucleotides (including the nucleotide at the start position).

[0161] In this invention, the nucleotide sequence of the antisense strand can be completely complementary or substantially complementary to the target sequence. When the nucleotide sequence of the antisense strand is substantially complementary to the target sequence, the nucleotide sequence of the antisense strand contains no more than three mismatched bases with the target sequence. For example, the number of mismatched bases is one, two, or three. When the nucleotide sequence of the antisense strand is completely complementary to the target sequence, the nucleotide sequence of the antisense strand does not contain any mismatched bases with the target sequence.

[0162] Furthermore, the antisense strand consists of at least 15 nucleotides. In some embodiments, the antisense strand consists of 15-28 nucleotides. For example, the length of the antisense strand is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides. Preferably, the antisense strand consists of 18-25 nucleotides, more preferably 18-23 nucleotides, and most preferably 19, 21, or 23 nucleotides.

[0163] In some specific embodiments, the nucleotide that differs from the target sequence is preferably located at the end of the antisense strand. For example, in some specific embodiments, the differing nucleotide is located at the 3' end of the antisense strand. In other specific embodiments, the differing nucleotide is located at the 5' end of the antisense strand.

[0164] In some embodiments, the sense strand comprises a sequence differing from at least 15 consecutive nucleotides in the target sequence by no more than 3 nucleotides. The sense strand includes regions complementary to the antisense strand, and the nucleotide sequence of the sense strand is identical or substantially identical to the sequence of the antisense strand binding region on the target sequence. Therefore, the nucleotide sequence of the sense strand is at least 15 consecutive nucleotides in the target sequence that bind the antisense strand; or, the nucleotide sequence of the sense strand differs from at least 15 consecutive nucleotides in the target sequence that bind the antisense strand by 1, 2, or 3 bases.

[0165] Furthermore, the positive sense strand consists of at least 15 nucleotides. In some embodiments, the positive sense strand consists of 15-28 nucleotides. For example, the length of the positive sense strand is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides. Preferably, the positive sense strand consists of 18-25 nucleotides, more preferably 18-23 nucleotides, and most preferably 19, 21, or 23 nucleotides.

[0166] In some specific embodiments, the nucleotide that differs from the target sequence is preferably located at the end of the sense strand. For example, in some specific embodiments, the differing nucleotide is located at the 3' end of the sense strand. In other specific embodiments, the differing nucleotide is located at the 5' end of the sense strand.

[0167] In this disclosure, the length of the justice chain and the length of the antisense chain may be the same or different.

[0168] In some implementations, the lengths of the justice chain and the antisense chain are the same. Specifically, the ratio of the justice chain to the antisense chain length is 15 / 15, 16 / 16, 17 / 17, 18 / 18, 19 / 19, 20 / 20, 21 / 21, 22 / 22, 23 / 23, 24 / 24, 25 / 25, 26 / 26, 27 / 27, or 28 / 28. Preferably, the ratio of the justice chain to the antisense chain length is 18 / 18, 19 / 19, 20 / 20, 21 / 21, 22 / 22, 23 / 23, 24 / 24, or 25 / 25; more preferably, it is 19 / 19, 20 / 20, 21 / 21, 22 / 22, or 23 / 23; and most preferably, it is 19 / 19, 21 / 21, or 23 / 23.

[0169] In some implementations, the lengths of the justice chain and the antisense chain differ. For example, the justice chain / antisense chain length ratios are 18 / 19, 18 / 20, 18 / 21, 18 / 22, 18 / 23, 18 / 24, 18 / 25, 18 / 26, 19 / 18, 19 / 20, 19 / 21, 19 / 22, 19 / 23, 19 / 24, 19 / 25, 19 / 26, 20 / 19, 20 / 21, 20 / 22, 20 / 23, 20 / 24, 20 / 25, 20 / 26, 21 / 18, 21 / 19 21 / 20, 21 / 22, 21 / 23, 21 / 24, 21 / 25, 21 / 26, 22 / 18, 22 / 19, 22 / 20, 22 / 21, 22 / 23, 22 / 24, 22 / 25, 22 / 26, 23 / 19, 23 / 20, 23 / 21, 23 / 22, 23 / 24, 23 / 25, or 23 / 26, etc.; in some preferred embodiments, the length ratio of the justice chain to the antisense chain is 19 / 21 or 21 / 23.

[0170] In this disclosure, the sense strand and the antisense strand can be fully complementary or substantially complementary. When they are substantially complementary, there are no more than 3 mismatched bases in the double-stranded region formed by the sense strand and the antisense strand.

[0171] In some embodiments, after the sense and antisense strands are at least partially complementary to form a double-stranded region, the sense, antisense, or combination thereof has protruding nucleotides extending out of the double-stranded region. The number of protruding nucleotides can be one or more, for example, one or two. Furthermore, the one or two protruding nucleotides can be located at the 5' end, 3' end, or both ends of any antisense or sense strand, and each protruding nucleotide can be of any type. For example, in the case where the sense strand consists of a 21-unit sequence A+D and the antisense strand consists of a 21-unit sequence B+E, the nucleotides at positions 1-19 of sequence A are completely anticomplementary to those at positions 1-19 of sequence B in the direction from the 5' end to the 3' end, thereby forming protruding nucleotides at the 3' end of sequences D and E, respectively. For example, when the sense strand consists of a 19-position sequence A and the antisense strand consists of a 21-position sequence B+E, the nucleotides 1-19 of sequence A are completely anticomplementary to those 1-19 of sequence B in the direction from the 5' end to the 3' end, thus forming a protruding nucleotide at the 3' end of sequence E on the antisense strand.

[0172] In some embodiments, the sense strand and the antisense strand are at least partially complementary to form the double-stranded region, and the 3' end of the sense strand has 1-2 protruding nucleotides extending out of the double-stranded region, while the 3' end of the antisense strand is blunt. In some embodiments, the sense strand and the antisense strand are at least partially complementary to form the double-stranded region, and the 3' end of the antisense strand has 1-2 protruding nucleotides extending out of the double-stranded region, while the 3' end of the sense strand is blunt. In some embodiments, the sense strand and the antisense strand are at least partially complementary to form the double-stranded region, and both the sense strand and the antisense strand have 1-2 protruding nucleotides extending out of the double-stranded region at their 3' ends. In some embodiments, the sense strand and the antisense strand are at least partially complementary to form the double-stranded region, and both the sense strand and the antisense strand have blunt 3' ends.

[0173] In some embodiments, each nucleotide of the sense strand is independently a modified or unmodified nucleotide. In some embodiments, each nucleotide of the antisense strand is independently a modified or unmodified nucleotide.

[0174] In some embodiments, any two nucleotides linked in the sense strand are connected by a phosphodiester bond or a phosphothiodiester bond. In some embodiments, any two nucleotides linked in the antisense strand are connected by a phosphodiester bond or a phosphothiodiester bond.

[0175] In some embodiments, the ribosome group of the 5' terminal nucleotide of the positive strand is a 5' hydroxyl group.

[0176] In some embodiments, the ribosome of the 5' terminal nucleotide of the sense strand may have a 5' hydroxyl group, a 5' phosphate group, or a 5' phosphate-derived group. In some embodiments, the ribosome of the 5' terminal nucleotide of the antisense strand has a 5' hydroxyl group, a 5' phosphate group, or a 5' phosphate-derived group. The type of group at the 5' position of the ribosome depends on the preparation method used, which is known to those skilled in the art based on the corresponding preparation method.

[0177] For example, the structure of the 5' phosphate group is as follows: The structure of the 5' phosphate derivative group includes, but is not limited to: (EVP), wait.

[0178] Furthermore, the double-stranded oligonucleotide is a siRNA modifier.

[0179] In some embodiments, the siRNA modifier comprises a modification of at least one nucleotide. The nucleotide modification is selected from at least one of ribose group modification and base modification. In some embodiments, "nucleotide modification" refers to a nucleotide or nucleotide derivative formed by replacing the 2' hydroxyl group of the ribose group with another group, or a nucleotide in which the base is a modified base. The nucleotide modification does not result in a significant weakening or loss of the siRNA's ability to suppress gene expression. For example, modified nucleotides disclosed in JKWatts, G.F. Deleavey, and MJDamha, Chemically Modified siRNA: Tools and Applications. Drug Discov Today, 2008, 13(19-20):842-55 can be selected. Nucleotide modification can improve the stability of siRNA and maintain its high repressive efficiency against the SMAD2 gene.

[0180] For example, the modified nucleosides have the following structures:

[0181] Wherein, Base represents a base, such as A, U, G, C, or T. The hydroxyl group at the 2' position of the ribosome is replaced by R. These hydroxyl groups at the 2' position of the ribosome can be replaced by various groups known to those skilled in the art; for example, R can be selected from halogens, alkyl groups, alkoxy groups, substituted alkyl groups, and substituted alkoxy groups. For example, in some specific embodiments, the modified nucleotide includes, but is not limited to, 2'-fluoro(2'-F) modified nucleotides, 2'-alkoxy modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, and 2'-deoxyribonucleotides.

[0182] In some implementations, the 2'-alkoxy modified nucleotide is a 2'-methoxy (2'-OMe, 2'-O-CH3) modified nucleotide, etc.

[0183] In some embodiments, the 2'-substituted alkoxy modified nucleotide is a nucleotide modified with 2'-methoxyethoxy (2'-O-CH2-CH2-O-CH3), a nucleotide modified with 2'-O-CH2-CH=CH2, etc.

[0184] In some embodiments, the 2'-substituted alkyl-modified nucleotide is a 2'-CH2-CH2-CH=CH2 modified nucleotide, etc.

[0185] In some embodiments, the modification of the nucleotide is a modification of the bases. Base modifications can be of various types known to those skilled in the art. For example, base modifications include, but are not limited to, m... 6 A、Ψ、m 1 A、m 5 A, ms 2 i 6 A、i 6 A、m 3 C, m 5 C、ac 4 C, m 7 G, m 2,2 G, m 2 G, m 1 G, Q, m 5 U、mcm 5 U、ncm 5 U、ncm 5 Um, D, mcm 5 s 2 U, Inosine(I), hm 5 C, s 4 U、s 2 U, azobenzene, Cm, Um, Gm, t 6 A, yW, ms 2 t 6 A or its derivatives.

[0186] In some embodiments, a nucleotide derivative refers to a compound that can replace a nucleotide in a nucleic acid but has a structure different from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide. In some embodiments, the nucleotide derivative can be an isonucleotide, a bridged nucleic acid (BNA), or an acyclic nucleotide. A BNA refers to a restricted or inaccessible nucleotide. A BNA can contain a bridging structure with a "fixed" C3'-endoglucan condensation, such as a five-membered, six-membered, or seven-membered ring. This bridge is typically incorporated into the 2',4' position of the ribose to provide a 2',4'-BNA nucleotide, such as LNA, ENA, cET, etc.

[0187] The nucleosides of LNA are shown in formula (1), the nucleosides of ENA are shown in formula (2), and the nucleosides of cET are shown in formula (3):

[0188] In this context, "Base" refers to a base.

[0189] Acyclic nucleotides are a class of nucleotides formed by opening the sugar ring of a nucleotide, such as unopened nucleic acids (UNA) or glycerol nucleic acids (GNA). UNA is shown in formula (4), and GNA is shown in formula (5).

[0190] In formulas (4) and (5) above, Base refers to a base, and R is selected from H, OH or alkoxy (-O-alkyl).

[0191] In some implementations, nucleotide derivative modification refers to the replacement of nucleotides in nucleic acids with nucleotide derivatives. Exemplary examples include isonucleotides, LNA, ENA, cET, UNA, or GNA.

[0192] In some embodiments, the nucleotides in the nucleic acid are replaced with isonucleotides, also referred to as isonucleotide modification in the context of this disclosure. In some embodiments, isonucleotide modification includes incorporating an isonucleotide at one or more sites on the sense and / or antisense strands of the siRNA to be modified, in place of the native nucleoside for coupling at the corresponding position.

[0193] In some embodiments, the isonucleoside modification is D-isonucleoside modification. In other embodiments, the isonucleoside modification is L-isonucleoside modification. In still other embodiments, the isonucleoside modification is a combination of D-isonucleoside and L-isonucleoside modification.

[0194] In some embodiments, the double-stranded ribonucleic acid (siRNA) modifier includes modification of a phosphodiester bond at at least one position. In some embodiments, the modification of the phosphodiester bond refers to the substitution of at least one oxygen atom in the phosphodiester bond by a sulfur atom to form a phosphothiodiester bond. The phosphothiodiester bond can stabilize the double-stranded structure of siRNA and maintain the specificity of base pairing. An exemplary phosphothiodiester bond structure is shown below:

[0195] In some implementations, the siRNA modifier comprises at least one of the following chemical modifications:

[0196] (1) Modification of at least one nucleotide in the positive strand,

[0197] (2) Modification of the phosphodiester bond at at least one position in the positive chain.

[0198] (3) Modification of at least one nucleotide in the antisense strand,

[0199] (4) Modification of phosphodiester bonds at at least one position in the antisense chain.

[0200] Furthermore, the double-stranded RNA modifier is an siRNA modifier containing at least one of the chemical modifications in (1)-(4).

[0201] In some embodiments, the sense strand of the siRNA modifier includes the following modifications along the 5' end to the 3' end: the ribonucleotides at positions 7, 9, 10, and 11 of the sense strand are 2'-fluoromodified ribonucleotides; and the ribonucleotides at other positions of the sense strand are 2'-methoxymodified ribonucleotides.

[0202] In this paper, the 5' nucleotide of the positive strand has a 5' hydroxyl group.

[0203] In this paper, the nucleotide structure of the ribosome with a 5' hydroxyl group at the 5' end of the positive strand is shown in Formula X:

[0204] Wherein, Base represents a base, such as A, U, G, C or T; R is a hydroxyl group or is substituted by various groups known to those skilled in the art, for example, R can be 2'-fluoro(2'-F), 2'-alkoxy, 2'-substituted alkoxy, 2'-alkyl, 2'-substituted alkyl, 2'-amino, 2'-substituted amino, 2'-deoxynucleotide.

[0205] In some embodiments, the positive strand of the siRNA modifier includes phosphothioester bonds at the following positions along the 5' end to the 3' end: between the first and second nucleotides starting at the 5' end, between the second and third nucleotides starting at the 5' end, between the first and second nucleotides starting at the 3' end, and between the second and third nucleotides starting at the 3' end.

[0206] In some implementations, the positive strand of the siRNA modifier includes phosphothioester bonds at the following positions along the 5'-to-3' direction: between the first and second nucleotides starting at the 5' end, and between the second and third nucleotides starting at the 5' end.

[0207] In some embodiments, the antisense strand of the siRNA modifier includes the following modifications along the 5' end to the 3' end: the ribonucleotides at any odd-numbered positions in the antisense strand are 2'-methoxy modified ribonucleotides, and the ribonucleotides at any even-numbered positions in the antisense strand are 2'-fluoro modified ribonucleotides.

[0208] In some embodiments, the antisense strand of the siRNA modifier includes the following modifications along the 5' end to the 3' end: the ribonucleotides at positions 2, 6, 14, and 16 of the antisense strand are 2'-F modified ribonucleotides, and the ribonucleotides at the remaining positions of the antisense strand are 2'-O-CH3 modified ribonucleotides.

[0209] In some embodiments, the antisense strand of the siRNA modifier includes the following modifications along the 5' end to the 3' end: the ribonucleotides at positions 2, 6, 8, 9, 14, and 16 of the antisense strand are 2'-F modified ribonucleotides, and the ribonucleotides at the remaining positions of the antisense strand are 2'-O-CH3 modified ribonucleotides.

[0210] In some embodiments, the antisense strand of the siRNA modifier includes the following modifications along the 5' end to the 3' end: the ribonucleotides at positions 2, 14, and 16 of the antisense strand are 2'-F modified ribonucleotides, the ribonucleotide at position 6 of the antisense strand is a ribonucleotide modified with the nucleotide derivative GNA, and the ribonucleotides at the remaining positions of the antisense strand are 2'-O-CH3 modified ribonucleotides.

[0211] In some embodiments, the antisense strand of the siRNA modifier includes the following modifications along the 5' end to the 3' end: the ribonucleotides at positions 2, 6, 14, and 16 of the antisense strand are 2'-F modified ribonucleotides, the ribonucleotide at position 7 of the antisense strand is a ribonucleotide modified with the nucleotide derivative GNA, and the ribonucleotides at the remaining positions of the antisense strand are 2'-O-CH3 modified ribonucleotides.

[0212] In some embodiments, the antisense strand of the siRNA modifier includes phosphothioester bonds at the following positions along the 5' end to the 3' end: between the first and second nucleotides starting at the 5' end, between the second and third nucleotides starting at the 5' end, between the first and second nucleotides starting at the 3' end, and between the second and third nucleotides starting at the 3' end.

[0213] In embodiments of the present invention, the ribosome of the 5' terminal nucleotide of the antisense strand may have: a 5' hydroxyl group, a 5' phosphate group, or a 5' phosphate derivative group.

[0214] In some embodiments, the oligonucleotide of the present invention comprises 1, 2, 3 or 4 compounds of formula (I) or pharmaceutically acceptable salts thereof; in some embodiments, the oligonucleotide comprises 1 compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0215] In some embodiments, the compound comprises one or more lipophilic monomers having a lipophilic moiety conjugated to the 5' end of the sense chain or the 5' end of the antisense chain.

[0216] In some embodiments, the compound comprises one or more lipophilic monomers having one or more lipophilic moieties conjugated to the 3' end of the sense chain or the 3' end of the antisense chain.

[0217] In some embodiments, the compound comprises one or more lipophilic monomers having one or more lipophilic moieties conjugated to both ends of the positive chain.

[0218] In some embodiments, the compound comprises one or more lipophilic monomers having one or more lipophilic moieties conjugated to the ends of the antisense chain.

[0219] In some embodiments, the compound comprises one or more lipophilic monomers containing one or more lipophilic moieties conjugated to internal positions of the sense or antisense strand. In some embodiments, one or more lipophilic moieties are conjugated to ribose, nucleotides, and / or nucleotides via inter-bonds. In some embodiments, one or more lipophilic moieties are conjugated to ribose at the 2', 3', 4', and / or 5' positions. In some embodiments, one or more lipophilic moieties are conjugated to native nucleotides (such as A, T, G, C, or U) or modified nucleotides as defined herein. In some embodiments, one or more lipophilic moieties are conjugated to phosphate groups or modified phosphate groups as defined herein.

[0220] In some implementations, the compound comprises one or more lipophilic monomers having one or more lipophilic moieties conjugated to the 5' or 3' end of the sense chain, and one or more lipophilic monomers having one or more lipophilic moieties conjugated to the 5' or 3' end of the antisense chain.

[0221] In some embodiments, at least one lipophilic monomer is located at one or more terminal positions of the sense or antisense strand. In one embodiment, at least one lipophilic monomer is located at the 3' or 5' end of the sense strand. In one embodiment, at least one lipophilic monomer is located at the 3' or 5' end of the antisense strand.

[0222] In some embodiments, the lipophilic monomer contains a lipophilic moiety conjugated to one or more internal positions on at least one chain. An internal position on the chain refers to a nucleotide at any position on the chain, excluding the 3' and 5' ends of the chain (e.g., excluding two positions: position 1 counting from the 3' end and position 1 counting from the 5' end).

[0223] In one embodiment, at least one lipophilic monomer is located at one or more internal locations on at least one chain, including all locations except the two terminal locations at each end of the chain (e.g., excluding four locations: locations 1 and 2 counting from the 3' end and locations 1 and 2 counting from the 5' end). In another embodiment, the lipophilic monomer is located at one or more internal locations on at least one chain, including all locations except the three terminal locations at each end of the chain (e.g., excluding six locations: locations 1, 2, and 3 counting from the 3' end and locations 1, 2, and 3 counting from the 5' end).

[0224] In one embodiment, at least one lipophilic monomer is located at one or more positions at at least one end of the double-stranded region, including all positions within the double-stranded region, but excluding overhanging regions or vectors that replace the terminal nucleotide at the 3' end of the positive strand.

[0225] In one embodiment, at least one lipophilic monomer is located on the sense strand within the first five, four, three, two, or one base pairs of the antisense strand 5' end of the double-stranded region.

[0226] In one embodiment, one or more lipophilic monomers are located in one or more of the following internal positions: counting from the 5' end of each chain, positions 4-8 and 13-18 on the sense chain, and positions 6-10 and 15-18 on the antisense chain.

[0227] In one embodiment, one or more lipophilic monomers are located at one or more of the following internal positions: counting from the 5' end of each chain, positions 5, 6, 7, 15 and 17 on the sense chain, and positions 15 and 17 on the antisense chain.

[0228] The present invention also provides a method for preparing the oligonucleotides described herein, comprising the following steps:

[0229] (1) A precursor of the compound represented by formula (I) as disclosed herein, or a pharmaceutically acceptable salt thereof;

[0230] (2) Synthesize the oligonucleotides of the present invention using the precursor synthesized in step (1) or its pharmaceutically acceptable salt.

[0231] On the other hand, the present invention provides a pharmaceutical composition comprising the oligonucleotides described above. In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

[0232] In some embodiments, the pharmaceutically acceptable excipient may be, for example, a carrier, transporter, diluent, and / or delivery polymer.

[0233] On the other hand, this disclosure provides the use of the above-described oligonucleotide or the above-described pharmaceutical composition in the preparation of a medicament.

[0234] In some embodiments, the drug is used to prevent and / or treat diseases associated with eye, central nervous system (CNS) and lung disorders; in other embodiments, the drug is used to prevent and / or treat lung-related diseases.

[0235] In some implementation methods, the drug is used for the prevention and / or treatment of tumor-related diseases.

[0236] The diseases / conditions covered by the publicly available information in this article include, but are not limited to: progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), auricolic granulation disease (AGD), glial tau proteinosis (GGT), age-related tau astrocytosis (ARTAG), familial frontotemporal dementia 17 (FTD-17), tau proteinosis with respiratory failure, dementia with epilepsy, Pick's disease, myotonic dystrophy 1 or 2 (MD1 or MD2), Down syndrome, spastic paraplegia (SP), Niemann-Pick disease type C, Lewy body dementia (DLB), Lewy body dysphagia, Lewy body disease, olivopontocerebellar atrophy, striatum substantia nigra degeneration, Shy-Drager syndrome, spinal muscular atrophy V (SMAV), Huntington's disease (HD), Alzheimer's disease, SCA1, SCA2, SCA3, SCA7, SCA10 (1, 2, 3, 7 or...). Type 10 spinocerebellar ataxia, multiple system atrophy (MSA), spinal and medullary muscular atrophy (SBMA, Kennedy's disease), Friedrich ataxia, fragile X-related tremor / ataxia syndrome (FXTAS), fragile X syndrome (FRAXA), X-linked intellectual disability (XLMR), Parkinson's disease, dystonia, SBMA (spinal medullary muscular atrophy), neuropathic pain disorders, spinal cord injury, dentate nucleus-rubra-pallidus-thalamic nucleus atrophy (DRPLA), latent CNS disorders, ALS (amyotrophic lateral sclerosis), M2DS (MECP2 duplication syndrome), FTD (frontotemporal dementia), prions, adult-onset leukodystrophy, Alexander's disease, Krabbe's disease, chronic traumatic encephalopathy, Pelizaeus-Merzbacher disease (PMD), Lafora's disease, stroke, cerebral amyloid angiopathy (CAA), and metachromatic leukodystrophy (MLD).

[0237] In another aspect, the present invention further provides a method for delivering the compound of the present invention to a specific target in a subject via subcutaneous or intravenous administration. The present invention further provides a method for delivering the compound of the present invention to a specific target in a subject via subcutaneous or intravenous administration.

[0238] Another aspect of the invention relates to a method for reducing the expression of a target gene in a cell, the method comprising contacting the cell with a compound comprising an antisense strand complementary to the target gene; a sense strand complementary to the antisense strand; and one or more lipophilic monomers.

[0239] In the first aspect of the invention relating to compounds, all the above-described embodiments relating to lipophilic monomers, lipophilic moieties, and their conjugation with compounds are suitable for this aspect of the invention in relation to methods for reducing the expression of target genes in cells.

[0240] In one implementation, the cells are extrahepatic cells.

[0241] In one implementation, the cell is not a liver cell.

[0242] Another aspect of the present invention relates to a method for reducing the expression of a target gene in a subject, the method comprising administering a compound to the subject, including contacting the cells with the compound, the compound comprising an antisense strand complementary to the target gene; a sense strand complementary to the antisense strand; and one or more lipophilic monomers.

[0243] In the first aspect of the invention relating to compounds, all the above-described embodiments relating to lipophilic monomers, lipophilic moieties, and their conjugation with compounds are suitable for this aspect of the invention in relation to methods for reducing target gene expression in subjects.

[0244] In some embodiments, the compound is administered extrahepatically.

[0245] In one embodiment, the compound is administered intrathecally or intraventricularly. By administering the compound intrathecally or intraventricularly, this method can reduce the expression of target genes in brain or spinal tissues (e.g., cortex, cerebellum, cervical vertebrae, lumbar vertebrae, and thoracic vertebrae).

[0246] In some embodiments, exemplary target genes are APP, ATXN2, C9orf72, TARDBP, MAPT(Tau), HTT, SNCA, FUS, ATXN3, ATXN1, SCA1, SCA7, SCA8, MeCP2, PRNP, SOD1, DMPK, CTNNB1, MYOC, SCN9A, STAT3, PDL1, and TTR. To reduce the expression of these target genes in a subject, the compound can be applied directly (e.g., intravitreally) to one or more eyes. By applying the compound intravitreally, this method can reduce the expression of target genes in ocular tissues.

[0247] Another aspect of the invention relates to a method of treating a subject suffering from a CNS disorder, the method comprising administering a therapeutically effective amount of an siRNA conjugate to the subject, thereby treating the subject. The siRNA conjugate comprises an antisense strand complementary to a target gene; a sense strand complementary to said antisense strand; and one or more lipophilic monomers. The types of diseases / conditions addressed in the disclosure herein include, but are not limited to: progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), argyrophilic granulation disease (AGD), glial tau proteinosis (GGT), age-related tau astrocytosis (ARTAG), familial frontotemporal dementia 17 (FTD-17), tau proteinosis with respiratory failure, dementia with epilepsy, and Pick's disease. Disease, myotonic dystrophy 1 or 2 (MD1 or MD2), Down syndrome, spastic paraplegia (SP), Niemann-Pick disease type C, Lewy body dementia (DLB), Lewy body dysphagia, Lewy body disease, olivopontocerebellar atrophy, striatum substantia nigra degeneration, Shy-Drager syndrome, spinal muscular atrophy V (SMAV), Huntington's disease (HD), Alzheimer's disease, SCA1, SCA2, SCA3, SCA7, SCA10 (types 1, 2, 3, 7 or 10 spinocerebellar ataxia), multiple system atrophy (MSA), spinal cord and medullary muscular atrophy (SBMA, Kennedy's disease), Friedrich's ataxia, fragile X-related tremor / ataxia syndrome ( Fragile X syndrome (FRAXA), X-linked intellectual disability (XLMR), Parkinson's disease, dystonia, SBMA (spinal medullary muscular atrophy), neuropathic pain disorder, spinal cord injury, dentate nucleus-rubra-pallidus-thalamic atrophy (DRPLA), latent CNS disorders, ALS (amyotrophic lateral sclerosis), M2DS (MECP2 duplication syndrome), FTD (frontotemporal dementia), prions, adult-onset leukodystrophy, Alexander's disease, Krabbe's disease, chronic traumatic encephalopathy, Pelizaeus-Merzbacher disease (PMD), Lafora's disease, stroke, cerebral amyloid angiopathy (CAA), and metachromatic leukodystrophy (MLD).

[0248] definition

[0249] Unless otherwise stated, the following terms as used in this application shall have the following meanings. A particular term should not be considered uncertain or unclear unless specifically defined, but should be understood in accordance with its ordinary meaning in the art. When a trade name appears herein, it is intended to refer to the corresponding product or its active ingredient.

[0250] In this art, “G,” “C,” “A,” “T,” and “U” typically represent the bases of guanine, cytosine, adenine, thymine, and uracil, respectively. However, it is also generally known in the art that each of “G,” “C,” “A,” “T,” and “U” typically also represents a nucleotide containing guanine, cytosine, adenine, thymine, and uracil as a base, respectively. This is a common practice in representing deoxyribonucleic acid (DNA) sequences and / or ribonucleic acid (RNA) sequences. Therefore, in the context of this disclosure, the meanings of “G,” “C,” “A,” “T,” and “U” include all of the above-mentioned possible cases. However, it should be understood that the term “ribonucleotide” or “nucleotide” can also refer to a modified nucleotide (as further detailed below) or one with an alternative substitution. Those skilled in the art will recognize that guanine, cytosine, adenine, and uracil can be substituted with other parts without substantially altering the base-pairing properties of an oligonucleotide (including a nucleotide with such a substitution). For example, without limitation, a nucleotide including inosine as its base can be base-paired with a nucleotide including adenine, cytosine, or uracil. Thus, a nucleotide containing uracil, guanine, or adenine can be replaced in the nucleotide sequence of the dsRNA characterized in this disclosure by a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively, to form a GU swing base pairing with the target mRNA. Sequences containing such replacement portions are suitable for the compositions and methods characterized in this disclosure. The terms “double-stranded oligonucleotide,” “double-stranded RNA (dsRNA) molecule,” and “dsRNA” as used in the context of this disclosure are used interchangeably. The term “dsRNA” refers to a complex of ribonucleic acid molecules having a double-stranded structure comprising two antiparallel and substantially complementary nucleic acid strands, described as having “sense” and “antisense” orientation relative to a target gene, such as the SMAD2 gene. In some embodiments, double-stranded ribonucleic acid (dsRNA) triggers the degradation of target RNA, such as mRNA, through a post-transcriptional gene silencing mechanism (referred to herein as RNA interference or RNAi).

[0251] Typically, the majority of nucleotides in each strand of a dsRNA molecule are ribonucleotides; however, as detailed herein, each or both strands may also include one or more non-ribonucleotides, such as deoxyribonucleotides and / or modified nucleotides. Additionally, as used herein, “double-stranded ribonucleic acid” can include chemically modified ribonucleotides, phosphate backbones, etc. These modifications can include all types of modifications disclosed herein or known in the art.

[0252] As used in the context of this disclosure, the term "isonucleotide" refers to a compound formed by altering the position of a base on the ribose ring, for example, a compound formed by attaching a base to the 2' or 3' position of the ribose ring instead of the 1' position.

[0253] In some embodiments, the double-stranded RNA disclosed herein is siRNA, which interacts with the mRNA sequence transcribed from the target gene (e.g., the mRNA sequence transcribed from the SMAD2 gene) to guide the cleavage of the target RNA. Not wishing to be bound by theory, long double-stranded RNA introduced into the cell is broken down into siRNA by a type III nuclease called Dicer (Sharp et al., Genes Dev. 2001, 15:485). Dicer (a type III ribonuclease-like enzyme) processes dsRNA into short 19–23 base pairs of interfering RNA with a characteristic dibase 3' overhang (Bernstein et al., (2001) Nature 409:363). These siRNAs are then incorporated into the RNA-inducible silencing complex (RISC), where one or more helicases unwind the siRNA duplex, enabling complementary antisense strands to guide target recognition (Nykanen et al., (2001) Cell 107:309). Once bound to a suitable target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir et al., (2001) Genes Dev. 15:188).

[0254] As used in the context of this disclosure, the term "protruding nucleotide" refers to one or more unpaired nucleotides that protrude from the double-stranded structure of a dsRNA when one 3' end of one strand extends beyond the 5' end of the other strand, or vice versa. "Flat-ended" or "blunt-terminated" means that there are no unpaired nucleotides at the said end of the double-stranded ribonucleic acid, i.e., no nucleotide protrusions. A "flat-terminated" double-stranded ribonucleic acid is a dsRNA that is double-stranded throughout its entire length, i.e., without nucleotide protrusions at either end of the molecule.

[0255] The term "antisense strand" refers to a strand of double-stranded RNA that has a region substantially complementary to a target sequence (e.g., derived from human SMAD2 mRNA). Mismatches at the terminal regions are most tolerable when the complementary region is not perfectly complementary to the target sequence, and if mismatches do occur, they are typically within one or more terminal regions, such as within 5, 4, 3, 2, or 1 nucleotides at the 5' and / or 3' ends.

[0256] The term "sense chain" refers to a double-stranded RNA strand containing a region that is substantially complementary to the antisense chain region.

[0257] The term “inhibition” can be used interchangeably with “reduction,” “silence,” “downregulation,” “suppression,” and other similar terms, and includes any level of inhibition.

[0258] The terms "lipophilic monomer" or "lipophilic moiety" broadly refer to any compound or chemical part that has an affinity for lipids. One way to characterize the lipophilicity of a lipophilic moiety is through the octanol-water partition coefficient logK. ow K ow This represents the ratio of the concentration of a chemical substance in the octanol phase to its concentration in the aqueous phase at equilibrium in a two-phase system. In principle, logK... ow When the value exceeds 0, the chemical substance exhibits lipophilicity. Typically, the logK of the lipophilic portion... ow Values ​​exceeding 1, 1.5, 2, 3, 4, 5, or 10, such as the logK of 6-aminohexanol. ow The logK of cholesterol-based N-(hexyl-6-ol)carbamate is approximately 0.7. ow It is 10.7.

[0259] The lipophilicity of a molecule can be altered relative to the functional groups it carries. For example, adding a hydroxyl or amino group to the end of the lipophilic moiety can increase or decrease the partition coefficient (e.g., logK) of the lipophilic moiety. ow The lipophilic moiety can be aliphatic, cyclic (e.g., alicyclic), or polycyclic (e.g., polycyclic alicyclic compounds), such as steroids (e.g., sterols), or straight-chain or branched aliphatic hydrocarbons. The lipophilic moiety may generally contain a hydrocarbon chain, which may be cyclic or acyclic. The hydrocarbon chain may contain various substituents and / or one or more heteroatoms, such as oxygen or sulfur atoms.

[0260] The term "substituted" refers to the substitution of one or more hydrogen atoms on a specific atom by a substituent, provided that the valence state of the specific atom is normal and the resulting compound is stable. When the substituent is oxo (i.e., =O), it means that two hydrogen atoms are substituted; oxo substitution does not occur on aromatic groups.

[0261] The terms “optional” or “optionally” mean that the event or condition subsequently described may or may not occur, including both the occurrence and non-occurrence of said event or condition. For example, the ethyl group “optionally” being halogenated means that the ethyl group can be unsubstituted (-CH2CH3), monosubstituted (e.g., -CH2CH2F), polysubstituted (e.g., -CHFCH2F, -CH2CHF2, etc.), or fully substituted (-CF2CF3). Those skilled in the art will understand that for any group containing one or more substituents, no substitution or substitution pattern that is spatially impossible and / or cannot be synthesized is introduced.

[0262] C in this article m-n This means that the part has an integer number of carbon atoms within a given range. For example, "C 1-6 "" means that the group can have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms or 6 carbon atoms.

[0263] In this invention, "unsaturated bond" refers to carbon-carbon double bonds and carbon-carbon triple bonds. That is, carbon atoms with one or two unsaturated bonds... 2-4 A hydrocarbon group refers to a hydrocarbon group containing an alkenyl or alkynyl group.

[0264] The term "alkenyl" refers to an unsaturated aliphatic hydrocarbon group consisting of a straight or branched chain of carbon and hydrogen atoms, having at least one double bond. Non-limiting examples of alkenyl groups include, but are not limited to, vinyl, 1-propenyl, 2-propenyl, 1-butenyl, isobutenyl, 1,3-butadienyl, etc.

[0265] The term "alkynyl" refers to an unsaturated aliphatic hydrocarbon group consisting of a straight or branched chain of carbon and hydrogen atoms, having at least one triple bond. Non-limiting examples of alkynyl groups include, but are not limited to, ethynyl (-C≡CH), 1-propynyl (-C≡C-CH3), 2-propynyl (-CH2-C≡CH), and 1,3-butyrynyl (-C≡CC≡CH).

[0266] When any variable (e.g., R) appears more than once in the composition or structure of a compound, its definition is independent in each case. Therefore, for example, if a group is substituted by two Rs, each R has an independent option.

[0267] When the number of a linking group is 0, such as -(CH2)0-, it indicates that the linking group is a covalent bond.

[0268] When one of the variables is selected as a covalent bond, it means that the two groups it connects are directly connected. For example, when L in ALZ represents a covalent bond, it means that the structure is actually AZ.

[0269] When a substituent is cross-bonded to two atoms on a ring, it can bond to any atom on that ring. For example, structural units. This indicates that it can be substituted at any position on the cyclohexyl group or cyclohexadiene.

[0270] In this document, ﹏ denotes a site where a group is covalently linked, for example, it may denote a site where the lipophilic monomer is covalently linked to a nucleotide of the double-stranded oligonucleotide. For example, ﹏ denotes a site where the structure is linked to H, or a phosphate group, nucleoside, etc.

[0271] In this invention, the term "reactive phosphorus group" refers to a phosphorus-containing group that can be removed by reaction with other compounds. In this invention, the reactive phosphorus group can be selected from...

[0272] The term "hydroxyl protecting group" refers to the chemical properties of a hydroxyl group by introducing a protecting group onto it. Typically, the introduction of a hydroxyl protecting agent can enhance the stability of a compound, prevent side reactions during the reaction process, and improve the selectivity of the reaction. Common hydroxyl protecting groups include ester protecting groups, aryl protecting groups, alkyl protecting groups, alkoxymethyl protecting groups, and silane protecting groups, such as methyl ethers (ROMe), tert-butyl ethers (ROtBu), benzyl ethers (ROBn), p-methoxybenzyl ethers (ROPMB), and triphenylmethyl ethers (ROTr); trimethylsilyl (TMS), triethylsilyl (TES), dimethyl tert-butylsilyl (TBDMS), triisopropylsilyl (TIPS), and MOM (methoxymethyl ether). Ethers), MEM (methoxyethoxymethyl ether), BOM (benzyloxymethyl ether), SEM (trimethylsilylethoxymethyl ether), THP (tetrahydropyran ether), t-BuCO (Piv, neopentanoyl), PhCO (Bz, benzoyl), MeCO (Ac, acetyl), ClCH2CO (chloroacetyl, and other haloacetyl), benzylidene (also known as benzenemethyl), acetoneide (isopropylidene), alicyclic ketoneide, DMT (4,4'-dimethoxytriphenylmethyl), etc.

[0273] The term "halogen" or "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0274] The term "hydroxyl group" refers to the -OH group.

[0275] The term "alkyl" refers to a compound with the general formula C10. n H 2n+1 The alkyl group is a chain hydrocarbon group. This alkyl group can be straight-chain or branched. For example, the term "C"... 1-6"Alkyl" refers to an alkyl group containing 1 to 6 carbon atoms (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, neopentyl, hexyl, 2-methylpentyl, etc.). Similarly, the alkyl portion (i.e., alkyl) of alkoxy, alkylamino, dialkylamino, alkylsulfonyl, and alkylthio groups has the same definition as above.

[0276] The term "alkylene" refers to a compound with the general formula -C n H 2n -A chain alkylene group. This alkylene group can be straight-chain or branched. For example, the term C1-C... 10 Alkylenes are alkylene groups having 1 to 10 carbon atoms.

[0277] The term "alkoxy" refers to -O-alkyl.

[0278] The term "alkenyl" refers to an unsaturated aliphatic hydrocarbon group consisting of a straight or branched chain of carbon and hydrogen atoms, having at least one double bond. Non-limiting examples of alkenyl groups include, but are not limited to, vinyl, 1-propenyl, 2-propenyl, 1-butenyl, isobutenyl, 1,3-butadienyl, etc.

[0279] The term "heterocyclic group" refers to a non-aromatic ring that is fully saturated or partially unsaturated (but not fully unsaturated) and can exist as a monocyclic, bridged, or spirocyclic ring. Unless otherwise indicated, the heterocycle is typically a 3- to 12-membered ring containing 1 to 3 heteroatoms independently selected from sulfur, oxygen, and / or nitrogen (preferably 1 or 2 heteroatoms). Non-limiting examples of heterocyclic groups include, but are not limited to, ethylene oxide, tetrahydrofuranyl, dihydrofuranyl, pyrrolyl, N-methylpyrrolyl, dihydropyrrolyl, piperidinyl, piperazinyl, pyrazolyl, 4H-pyranyl, morpholinyl, thiomorpholinyl, tetrahydrothiophene, etc.

[0280] The term "heteroaryl" refers to a monocyclic or fused polycyclic system containing at least one ring atom selected from N, O, and S, with the remaining ring atoms being C, and having at least one aromatic ring. Preferred heteroaryl groups have a single 5- to 6-membered ring. Non-limiting examples of heteroaryl groups include, but are not limited to, pyrroleyl, furanyl, thiopheneyl, imidazolyl, oxazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, quinolinyl, isoquinolinyl, tetrazolyl, triazolyl, and triazineyl.

[0281] In this application, the terms "solid support" and "solid-phase carrier" are used interchangeably to refer to the solid-phase carrier used in the synthesis of oligonucleotides by solid-phase synthesis.

[0282] The term "treatment" means administering the compound or preparation described in this application to prevent, improve, or eliminate a disease or one or more symptoms related to said disease, and includes:

[0283] (i) To prevent the occurrence of disease or disease state in mammals, especially when such mammals are susceptible to the disease state but have not yet been diagnosed with the disease state;

[0284] (ii) To suppress the disease or disease state, that is, to curb its development;

[0285] (iii) Alleviate the disease or disease state, even if the disease or disease state subsides.

[0286] The term "therapeutic effective amount" means the amount of the compound of this application used to treat or prevent a particular disease, condition, or disorder; (ii) to reduce, improve, or eliminate one or more symptoms of a particular disease, condition, or disorder; or (iii) to prevent or delay the onset of one or more symptoms of a particular disease, condition, or disorder described herein. The amount of the compound of this application constituting a "therapeutic effective amount" varies depending on the compound, the disease state and its severity, the route of administration, and the age of the mammal to be treated, but may routinely be determined by a person skilled in the art based on their own knowledge and the present disclosure.

[0287] The term "pharmaceutical acceptable" refers to compounds, materials, compositions, and / or dosage forms that, within the bounds of reliable medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0288] As pharmaceutically acceptable salts, for example, metal salts, ammonium salts, salts formed with organic bases, salts formed with inorganic acids, salts formed with organic acids, and salts formed with basic or acidic amino acids may be mentioned.

[0289] The term "pharmaceutical composition" refers to a mixture of one or more compounds of this application or their salts with pharmaceutically acceptable excipients. The purpose of a pharmaceutical composition is to facilitate the administration of the compounds of this application to an organism.

[0290] The term "pharmaceuticalally acceptable excipient" refers to excipients that do not cause significant irritation to the organism and do not impair the biological activity and properties of the active compound. Suitable excipients are well known to those skilled in the art, such as carbohydrates, waxes, water-soluble and / or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, etc.

[0291] The word “comprise” or “include” and its English variants such as comprises or comprising should be understood in an open, non-exclusive sense, meaning “including but not limited to”.

[0292] The compounds and intermediates of this application may also exist in different tautomer forms, and all such forms are included within the scope of this application. The terms "tautomer" or "tautomer form" refer to structural isomers of different energies that can interconvert via low energy barriers. For example, proton tautomers (also known as proton transfer tautomers) include interconversions via proton migration, such as keto-enol and imine-enamine isomerization. A specific example of a proton tautomer is the imidazole moiety, where a proton can migrate between two ring nitrogens. Valence tautomers include interconversions via the recombination of some bonding electrons.

[0293] This application also includes compounds of this application that are identical to those described herein, but with one or more atoms replaced by isotopes whose atomic weights or mass numbers differ from those commonly found in nature. Examples of isotopes that can be incorporated into compounds of this application include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as... 2 H, 3 H, 11 C 13 C 14 C 13 N、 15 N、 15 O、 17 O、 18 O、 31 P, 32 P, 35 S, 18 F, 123 I, 125 I and 36 Cl, etc.

[0294] Certain isotope-labeled compounds of this application (e.g., using...) 3 H and 14 Those labeled with C can be used in the analysis of compound and / or substrate tissue distribution. Tritiumization (i.e. 3 H) and carbon-14 (i.e. 14 C) Isotopes are particularly preferred due to their ease of preparation and detectability. Positron-emitting isotopes, such as... 15 O、 13 N、 11 C and 18 F can be used in positron emission tomography (PET) studies to determine substrate occupancy. The isotopically labeled compounds of this application can typically be prepared by replacing the unlabeled reagent with an isotopically labeled reagent using a procedure similar to those disclosed in the schemes and / or examples below.

[0295] In addition, heavier isotopes (such as deuterium) are used. 2H)) substitution can provide certain therapeutic advantages resulting from higher metabolic stability (e.g., increased in vivo half-life or reduced dose requirement), and may therefore be preferred in certain situations, where deuterium substitution can be partial or complete, with partial deuterium substitution referring to at least one hydrogen being replaced by at least one deuterium.

[0296] The compounds of this application may be asymmetric, for example, having one or more stereoisomers. Unless otherwise stated, all stereoisomers include, such as enantiomers and diastereomers. The compounds containing asymmetric carbon atoms of this application can be isolated in optically active pure form or in racemic form. The optically active pure form can be resolved from a racemic mixture or synthesized using chiral starting materials or chiral reagents.

[0297] The pharmaceutical compositions of this application can be prepared by combining the compounds of this application with suitable pharmaceutically acceptable excipients, for example, in solid, semi-solid, liquid or gaseous formulations, such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, suppositories, injections, inhalers, gels, microspheres and aerosols.

[0298] Typical routes of administration of the compound of this application, its isomers, or pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof, include, but are not limited to, oral, rectal, topical, inhalation, parenteral, sublingual, vaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, and intravenous administration.

[0299] The pharmaceutical composition of this application can be manufactured using methods well known in the art, such as conventional mixing, dissolving, granulation, sugar-coated pill making, grinding, emulsification, freeze drying, etc.

[0300] In some embodiments, the pharmaceutical composition is in oral form. For oral administration, the pharmaceutical composition can be formulated by mixing the active compound with pharmaceutically acceptable excipients well known in the art. These excipients enable the compounds of this application to be formulated into tablets, pills, lozenges, sugar-coated tablets, capsules, liquids, gels, pastes, suspensions, etc., for oral administration to patients.

[0301] Solid oral compositions can be prepared using conventional mixing, filling, or tableting methods. For example, they can be obtained by mixing the active compound with solid excipients, optionally milling the resulting mixture, adding other suitable excipients if necessary, and then processing the mixture into granules to obtain the core of a tablet or sugar-coated formulation. Suitable excipients include, but are not limited to, binders, diluents, disintegrants, lubricants, glidants, sweeteners, or flavoring agents.

[0302] The pharmaceutical composition may also be suitable for parenteral administration, such as in suitable unit dosage forms of sterile solutions, suspensions or lyophilized products.

[0303] For purposes of description and disclosure, all patents, patent applications and other identified publications are expressly incorporated herein by reference. Any reference to these publications herein does not constitute an endorsement that such publication is part of the general knowledge in the art.

[0304] The compounds of this application can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthetic methods, and equivalent substitutions known to those skilled in the art. Preferred embodiments include, but are not limited to, the embodiments of this application.

[0305] The chemical reactions in the specific embodiments of this application are carried out in a suitable solvent, which must be suitable for the chemical changes and the reagents and materials required in this application. In order to obtain the compounds of this application, it is sometimes necessary for those skilled in the art to modify or select the synthesis steps or reaction process based on existing embodiments.

[0306] An important consideration in synthetic route planning in this field is the selection of appropriate protecting groups for reactive functional groups (such as amino groups in this application). For example, see Greene's Protective Groups in Organic Synthesis (4th Ed). Hoboken, New Jersey: John Wiley & Sons, Inc. All references cited in this application are incorporated herein by reference in their entirety.

[0307] Technical effect

[0308] The compounds of this invention can deliver siRNA to the central nervous system and eye tissues, effectively knock down target mRNA, and can be used to treat brain and eye-related diseases, showing broad application prospects. Attached Figure Description

[0309] Figure 1 shows a schematic diagram of the siRNA conjugate according to the present invention used in the experimental examples of the present invention. In the figure, black bases are methoxy, green bases are fluorinated, blue bases are C16 or specially modified, and orange vertical lines represent thiolated bases. Detailed Implementation

[0310] This application uses the following abbreviations:

[0311] AIBN represents azobisisobutyronitrile; n-Bu3SnH represents tributyltinane; iPr2O represents isopropyl ether; PPh3 represents triphenylphosphine; MeCN represents acetonitrile; KF represents potassium fluoride; PE represents petroleum ether; EA represents ethyl acetate; DCM represents dichloromethane; DHP represents 3,4-dihydro-2H-pyran; PPTS represents pyridine 4-methylbenzenesulfonic acid; THF represents tetrahydrofuran; TsOH represents p-methylbenzenesulfonic acid; 2B represents pentafluoroiodoethane; N BS represents N-bromosuccinimide; Et3N*3HF represents triethylamine trihydrofluoride; TFAA represents trifluoroacetic anhydride; TBAHF represents tetra-n-butylammonium difluoride hydrofluoride; DMF represents N,N-dimethylformamide; DAST represents diethylaminosulfur trifluoride; MeOH represents methanol; m-CPBA represents m-chloroperoxybenzoic acid; DMTCl represents 4,4'-bis(methoxy)triphenylmethyl chloride; DBU represents 1,8-diazabicyclo[5.4.0] Undecyl-7-ene; BOMCl represents benzylchloromethyl ether; Pd / C represents palladium on carbon catalyst; Bz represents benzoyl; Fmoc represents 9-fluorenylmethoxycarbonyl; Me represents methyl; TBDPSCl represents tert-butyldiphenylchlorosilane; TBAF represents tetrabutylammonium fluoride; TBDMSO represents TBDMS bonded to oxygen; TBDMSCl represents tert-butyldimethylchlorosilane; ODMT represents oxygen atom bonded to DMT; TEA represents triethylamine; HATU represents... N,N,N',N'-Tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea; DIEA represents N,N-diisopropylethylamine; TsNHBoc represents N-(tert-butyloxycarbonyl)p-toluenesulfonamide; TEBA represents benzyltriethylammonium bromide; TIPSCl2 represents 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane; NIS represents N-iodosuccinimide; NMM represents N-methylmorpholine; DMSO represents dimethyl sulfoxide.

[0312] Compounds artificially or Software naming conventions are used; commercially available compounds use supplier catalog names.

[0313] Example

[0314] The present invention will now be described in further detail through embodiments, but the present invention is not limited to these embodiments.

[0315] All reagents used in this application are commercially available and can be used without further purification.

[0316] Preparation Example 1

[0317] Synthesis of compound P1:

[0318] 1-Iodoperfluoropentane P1-A1 (7.5 g), undecenol P1-A2 (3.0 g) and AIBN (289 mg) were added to a reaction flask, nitrogen was purged three times, the temperature was raised to 80 °C and the reaction was carried out for 16 h. After the reaction was completed, the mixture was cooled to room temperature and concentrated under reduced pressure to obtain compound P1-A3 (10.0 g). 1 H NMR: (400MHz, CDCl3) δppm 4.32 (br d, J = 3.8Hz, 1H), 3.64 (br t, J = 6.4Hz, 2H), 3.06-2.66 (m, 2H), 1.90-1.73 (m, 2H), 1.56 (br d, J = 7.0Hz, 4H), 1.31 (br s,14H).

[0319] Compound P1-A3 (20.0 g), AIBN (579 mg), and 10 mL of toluene were added to a reaction flask and the temperature was raised to 60 °C. Then, n-Bu3SnH (20 g) was added dropwise to the above reaction system. After the addition was complete, the temperature was raised to 80 °C and the reaction was carried out for 16 h. After the reaction was completed, the mixture was cooled to room temperature. The reaction system was diluted with iPr2O (100 mL), washed with saturated brine (50 mL * 3), and the organic phase was separated. KF (20 g) was added to the organic phase and the mixture was stirred for 18 h. The reaction system was filtered, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (PE / EA = 5%-20%, v / v) to obtain compound P1-A4 (9.11 g). 1 H NMR (400MHz, CDCl3) δppm 3.75-3.54(m,2H),2.15-1.91(m,2H),1.62-1.54(m,4H),1.42-1.24(m,14H),1.21(t,J=5.4Hz,1H).

[0320] Compound P1-A4 (9.11 g), PPh3 (5.43 g), and 40 mL of acetonitrile were added to a reaction flask. After purging with nitrogen three times, the temperature was raised to 60 °C. Then, Br2 (3.31 g) was added dropwise to the reaction system, and the reaction was allowed to proceed for 5 h. After the reaction was completed, the mixture was concentrated under pressure and diluted with n-hexane. The solution was then filtered through silica gel (1000 mesh). The organic phase was concentrated under reduced pressure and purified by column chromatography to obtain P1 (12.4 g). 1 H NMR: (400MHz, CDCl3) δppm 3.42 (t, J = 6.9 Hz, 2H), 2.15-1.96 (m, 2H), 1.87 (quin, J = 7.2 Hz, 2H), 1.61 (quin, J = 7.6 Hz, 2H), 1.50-1.21 (m, 14H). 19F NMR: (376MHz, CDCl3) δ-80.68--80.96(m,3F),-114.37--114.45(m,2F),-1 22.66--122.79(m,2F),-123.71--123.82(m,2F),-126.23--126.34(m,2F).

[0321] Synthesis of compound P2:

[0322] 100 g of 11-bromo-1-undecanol P2-A1 was dissolved in DCM (400 mL), followed by the addition of DHP (50.2 g) and PPTS (2.0 g). The reaction was allowed to proceed at room temperature for 1 h, and then quenched by adding 200 mL of saturated sodium bicarbonate solution. The mixture was then diluted with 200 mL of water and extracted with DCM (300 mL x 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound P2-A2, which can be used directly in the next step without further purification. 1 H NMR: (400MHz, CDCl3) δ4.62-4.55(m,1H),3.93-3.85(m,1H),3.79-3.69(m,1H),3.56-3.49(m,1H),3.45-3. 36(m,3H),1.91-1.81(m,3H),1.78-1.69(m,1H),1.65-1.52(m,7H),1.47-1.40(m,2H),1.37-1.29(m,11H).

[0323] Compound P2-A2 (130 g), CuI (14.8 g), and THF (800 mL) were added to a reaction flask. After purging with nitrogen three times, the mixture was cooled to -78 °C. Then, allyl magnesium chloride 2A (2 M, 291 mL) was slowly added dropwise to the reaction system. After the addition was complete, the mixture was kept at this temperature for 2 h, and then heated to room temperature for 1 h. After the reaction was complete, saturated ammonium chloride (400 mL) was added to quench the reaction, and the mixture was diluted with water (500 mL) and extracted with ethyl acetate (300 mL * 3). The organic phases were combined, concentrated under reduced pressure, and purified by column chromatography to obtain compound P2-A3 (107 g). 1H NMR: (400MHz, CDCl3) δ5.90-5.74(m,1H),5.04-4.89(m,2H),4.64-4.51(m,1H),3.92-3.83(m,1H),3.78-3.69(m,1H),3.56-3. 44(m,1H),3.42-3.35(m,1H),2.07-2.01(m,2H),1.90-1.79(m,1H),1.76-1.67(m,1H),1.62-1.53(m,6H),1.36-1.25(m,18H).

[0324] Compound P2-A3 (107 g) was dissolved in methanol (500 mL), and TsOH (62.2 g) was added. The reaction was carried out at room temperature for 16 h. After the reaction was completed, the methanol was removed by concentration under reduced pressure, and the reaction was quenched by adding saturated sodium bicarbonate solution (300 mL). The mixture was then extracted with ethyl acetate (300 mL x 3). The organic phases were combined, washed with saturated brine (300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound P2-A4 (70.0 g), which was used directly in the next step without further purification.

[0325] Compound P2-A4 (35.0 g), AIBN (1.84 g), and pentafluoroiodoethane 2B (55.3 g) were added to a reaction flask and the mixture was heated to 80 °C and reacted for 16 h. After the reaction was complete, the mixture was cooled to room temperature, and 500 mL of water and 500 mL of ethyl acetate were added to the reaction system. The organic phase was separated, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain compound P2-A5 (150 g), which was used directly in the next step without purification. 1 H NMR: (400MHz CDCl3) δ4.30-4.18(m,1H),3.61-3.53(m,2H),2.89-2.62(m,2H),1.80-1.69(m,2H),1.32-1.18(m,20H).

[0326] Compound P2-A5 (150.0 g), AIBN (2.69 g), and 50 mL of toluene were added to a reaction flask and the temperature was raised to 60 °C. Then, n-Bu3SnH (198 g) was added dropwise to the above reaction system. After the addition was complete, the temperature was raised to 80 °C and the reaction was carried out for 16 h. After the reaction was completed, the mixture was cooled to room temperature. The reaction system was diluted with iPr2O (100 mL), washed with saturated brine (50 mL x 3), and the organic phase was separated. KF (20 g) was added to the organic phase and the mixture was stirred for 18 h. The reaction system was filtered, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (PE / EA = 5%-20%) to obtain compound P2-A6 (62 g). 1H NMR: (400MHz, CDCl3)δ=3.65-3.58(m,2H),2.06-1.93(m,2H),1.85-1.77(m,2H),1.56(br d,J=7.3Hz,2H),1.36-1.23(m,20H),0.94-0.75(m,1H).

[0327] Compound P2-A6 (31.0 g), PPh3 (29.4 g), and 300 mL of DCM were added to a reaction flask. After purging with nitrogen three times, the mixture was cooled to 0 °C. Then, NBS (19.9 g) was added to the reaction system, and the reaction was allowed to proceed for 0.5 h. After the reaction was complete, ethanol (500 mL) was added to quench the reaction. The mixture was then concentrated under reduced pressure and recrystallized (at -30 °C in 100 mL of ethanol) to obtain P2 (24.0 g). 1 H NMR: (400MHz, CDCl3) δ3.42 (t, J = 6.9 Hz, 2H), 2.09-1.93 (m, 2H), 1.90-1.82 (m, 2H), 1.63-1.55 (m, 2H), 1.46-1.26 (m, 20H).

[0328] Synthesis of compound P3:

[0329] 1-Hexadecene (1.0 eq.) and Et3N*3HF (0.62 eq.) were dissolved in DCM (1 mmol / 1 mL) and cooled to 0 °C. NBS (1.1 eq.) was added in portions to the reaction system with vigorous stirring. After the addition was complete, the temperature was raised to room temperature. After the reaction was complete, the reaction system was poured into ice water and neutralized with concentrated ammonia. The aqueous phase was extracted three times with DCM, and the organic phases were combined and washed successively with 0.1 M HCl (twice), 5% sodium bicarbonate aqueous solution (three times), and water. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to obtain product P3. 1 H NMR (400MHz, CDCl3) δ4.67–4.46(m,1H),3.80–3.59(m,2H),1.76–1.68(m,2H),1.62–1.43(m,2H),1.26(s,22H),0.88(t,J=6.7Hz,3H). 19 F NMR(376MHz, CDCl3)δ(-189.8)-(-189.4)(m,1F).

[0330] Synthesis of compound P4:

[0331] Weigh 1.0 g of 16-bromo-1-hexadecaneol, 1.5 eq. of pyridine, and 10 mL of anhydrous DCM into a reaction flask. After purging with nitrogen, cool to 0 °C. Add 1.2 eq. of Tf₂O (dissolved in 1 mL of DCM) dropwise to the reaction system. After the addition is complete, slowly heat to 0 °C. After the reaction is complete, add 1 N HCl. Separate the mixture, dry it with anhydrous sodium sulfate, and concentrate it under reduced pressure to obtain the crude product P₄-A₁.

[0332] The crude product P4-A1 was dissolved in DCM, and TBAHF (1.2 eq.) was added. The mixture was reacted at room temperature until the starting material was consumed. The crude product was concentrated under reduced pressure to obtain the crude product. The crude product was extracted three times with petroleum ether. The organic phases were combined, concentrated under reduced pressure, and column chromatography was performed to obtain product P4 (780 mg). 1 H NMR (400MHz, CDCl3) δ4.49(t,J=6.2Hz,1H),4.37(t,J=6.2Hz,1H),3.41(t,J=6.9Hz, 2H),1.85(p,J=7.0Hz,2H),1.75-1.62(m,2H),1.44-1.37(m,4H),1.33-1.26(m,20H). 19 F NMR(376MHz, CDCl3)δ(-218.3)-(-217.6)(m,1F).

[0333] Synthesis of compound P5:

[0334] Weigh 3.0 g of 1-bromohexadecane-6-one (compound P5-A1), 2.0 eq. of difluoromethyl (2-pyridyl) sulfone, and 60 mL of anhydrous DMF into a reaction flask. After purging with nitrogen, cool to -50 °C. Add 1.5 eq. of potassium tert-butoxide (dissolved in 50 mL of DMF) dropwise to the reaction system. After the addition is complete, slowly heat to -20 °C and react overnight. After the reaction is complete, add saturated ammonium chloride (50 mL) and extract with ethyl acetate (100 mL * 3). Combine the organic phases, wash with saturated brine (100 mL * 3), dry with anhydrous sodium sulfate, concentrate under reduced pressure, and purify by column chromatography (pure PE) to obtain product P5 (1.5 g). 1 H NMR(400MHz, CDCl3)δ3.40(t,J=6.8Hz,2H),2.01–1.91(m,4H),1.88-1.85(m,2 H),1.44-1.40(m,4H),1.38-1.34(m,2H),1.26(s,14H),0.88(t,J=6.7Hz,3H). 19 F NMR(376MHz, CDCl3)δ-96.1(s,2F).

[0335] Synthesis of compound P6:

[0336] Concentrated sulfuric acid (36 drops) was added to a reaction system of 16-hydroxyhexadecanoic acid (5.1 g) and methanol (200 mL). The mixture was heated to reflux and reacted overnight. After the reaction was completed, water (140 mL) and diethyl ether (600 mL) were added to the system. The organic phase was obtained by separation. The organic phase was washed successively with water (3 x 100 mL), saturated sodium bicarbonate (100 mL), and saturated brine (100 mL). The mixture was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain product P6-A1 (5.3 g). 1 H NMR(CDCl3):3.66(s,3H),3.63(t,6.66Hz,2H),2.29(t,7.57Hz,2H),1.62-1.59(m,4H),1.26-1.23(m,22H).

[0337] DAST (2.97 g) was dissolved in anhydrous dichloromethane, and nitrogen was purged three times. The reaction system was then cooled to -78°C. P6-A1 (5.3 g, dissolved in anhydrous dichloromethane) was then added dropwise to the above reaction system. The reaction was maintained at this temperature for 10 minutes, then heated to 0°C until the reaction system melted. The temperature was then further increased to room temperature and the reaction was allowed to proceed overnight. After the reaction was complete, water (100 mL) and diethyl ether (80 mL) were added to the system. The organic phase was obtained by separation and washed successively with water (3 x 100 mL), saturated brine (100 mL), and saturated sodium bicarbonate (100 mL). The mixture was then dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (petroleum ether / ethyl acetate = 20 / 1, v / v) to obtain product P6-A2 (3.2 g). 1 H NMR(CDCl3):4.43(dt,47.39,6.25Hz,2H),3.66(s,3H),2.30(t,7.52Hz,2H),1.66-1.65(m,4H),1.39-1.37(m,2H),1.27-1.25(m,22H). 19 F NMR(CDCl3): 1F(-218.1)-(-217.8).

[0338] P6-A2 (2.6 g) was dissolved in methanol (29 mL), dioxane (45 mL) and water (2 mL), and then sodium hydroxide (3.5 g) was added. The mixture was refluxed overnight. After cooling to room temperature, 1 M HCl was added to adjust the pH to 4-5. After the solid precipitated, the mixture was filtered to obtain a filter cake. The filter cake was dried to obtain P6 (1.8 g). 1H NMR(CDCl3):4.46(dt,47.39,6.25Hz,2H),2.31(t,7.52Hz,2H),1.68-1.66(m,4H),1.39-1.37(m,2H),1.27-1.25(m,22H). 19 F NMR(CDCl3): 1F(-218.1)-(-217.8).

[0339] Synthesis of compound P7:

[0340] 6-Amino-1-hexanol (15.0 g, 128.0 mmol, 1.0 eq) was weighed and placed in a reaction flask. Dichloromethane (200 mL), triethylamine (16.8 g, 166.4 mmol, 1.3 eq), and 9-fluorenyl chloroformate (36.4 g, 140.8 mmol, 1.1 eq) were added sequentially. The mixture was stirred overnight at room temperature. The mixture was extracted three times with dichloromethane (200 mL). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (eluent DCM:MeOH = 15:1) to give compound P7-A1 (30.5 g, yield: 70.4%). MS: m / z 340.2 [M+H] + .

[0341] P7-A1 (30.5 g, 89.9 mmol, 1.0 eq) was weighed into a dry reaction flask and dissolved in dichloromethane (300 mL). m-CPBA (48.5 g, 224.8 mol, 2.5 eq) was added under ice bath conditions, and the mixture was stirred at room temperature for 5 h. The reaction was quenched by adding saturated sodium bicarbonate (200 mL) under ice bath conditions. Extraction was performed with dichloromethane (200 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (eluent DCM:MeOH = 15:1) to give compound P7 (20.1 g, yield: 66.3%). MS: m / z 338.2 [M+H] + .

[0342] Example 1: Synthesis of Compound 1

[0343] DMTCl (1.2 eq.) was added in portions to a reaction system of 10 g uracil nucleoside and 50 mL pyridine. The reaction was carried out overnight at room temperature. After the reaction was complete, 10 mL methanol was added to the system. The pyridine was concentrated to dryness under pressure, and then DCM (300 mL) and saturated saline (100 mL) were added. The mixture was then separated and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (DCM / MeOH = 100 / 1-30 / 1) to obtain product 1-A1 (17 g), MS: m / z 547.2 [M+1].+ .

[0344] Weigh 3 g (1.0 eq.) of 1-A1 and 1.2 eq. of DBU, dissolve in 10 mL of anhydrous DMF, and cool to 0 °C. Then, add 1.2 eq. of BOMCl (dissolved in 10 mL of anhydrous DMF) dropwise to the above reaction system. After the addition is complete, heat to room temperature and react for 3 h. TLC monitoring shows that no raw material A1 remains. Add 10 mL of water, and wash the reaction system three times with ethyl acetate (100 mL * 3). Combine the organic phases, wash the organic phase with saturated brine (100 mL * 3) to remove DMF, and then concentrate under reduced pressure and perform column chromatography (DCM / MeOH = 100 / 1-20 / 1) to obtain product 1-A2 (3.5 g). MS: m / z 711.2 [M+45] + .

[0345] 1-A2 (1.0 g, 1.0 eq.), potassium tert-butoxide (1.5 eq.), and compound P1 (1.1 eq.) were weighed sequentially and placed in a reaction flask. 20 mL of anhydrous DMF was added, and the mixture was purged three times with nitrogen. The reaction was carried out at room temperature for 8 h. Then, 10 mL of water was added, and the reaction system was washed three times with ethyl acetate (20 mL * 3). The organic phases were combined and washed with saturated brine (20 mL * 3) to remove DMF. The mixture was then concentrated under reduced pressure and subjected to column chromatography (PE / EA = 10 / 1-2 / 1) to obtain product 1-A3 (600 mg). MS: m / z 1089.4 [M+1] + .

[0346] Weigh 1.2 g of 1-A3 and 30 wt% of Pd / C into a reaction flask, add 20 mL of methanol solvent, purge with hydrogen three times, and heat to 40 °C until all raw materials remain (hydrogen balloons need to be added during the reaction). After the reaction is complete, the reaction system is filtered through diatomaceous earth, concentrated under reduced pressure, and purified by column chromatography (DCM / MeOH = 80 / 1-40 / 1, v / v) to obtain product 1-A4. MS: m / z 667.2 [M+1] + .

[0347] Weigh 300 mg of 1-A4 and 1.1 eq. of silver nitrate into a reaction flask, add 10 mL of DCM, purge with nitrogen three times, and add dropwise DMTCl (1.5 eq., dissolved in 2 mL of DCM). React at room temperature until no 1-A4 remains. After the reaction is complete, concentrate the reaction system under reduced pressure and purify by column chromatography (DCM / MeOH = 80 / 1-40 / 1, v / v) to obtain product 1-A5 (380 mg). MS: m / z 969.3 [M+1] + .

[0348] Tetrazazole (3 eq.) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (6 eq.) were weighed and placed in a reaction flask. 5 mL of DCM was added, and nitrogen gas was purged three times. 1-A5 solution (300 mg, 1.0 eq., dissolved in 2 mL DCM) was added dropwise. The reaction was allowed to proceed at room temperature until no reactants remained. After the reaction was complete, the reaction system was concentrated under reduced pressure and purified by rapid column chromatography (PE / EA = 10 / 1-3 / 1, v / v) to obtain compound 1 (210 mg). MS: m / z 1169.5 [M+1] + .

[0349] Example 2: Synthesis of Compound 2

[0350] Starting from intermediate 1-A2, compound 2 was synthesized under the same conditions as compound 1, except that compound P1 was replaced by compound P2. MS: m / z 1060.5 [M+1] + .

[0351] Example 3: Synthesis of Compound 3

[0352] Starting from intermediate 1-A2, compound 3 was synthesized under the same conditions as compound 1, except that compound P1 was replaced with compound P3. MS: m / z 989.5 [M+1] + .

[0353] Example 4: Synthesis of Compound 4

[0354] Starting from intermediate 1-A2, compound 4 was synthesized under the same conditions as compound 1, except that compound P1 was replaced with compound P4. MS: m / z 989.5 [M+1] + .

[0355] Example 5: Synthesis of Compound 5

[0356] Starting from intermediate 1-A2, compound 5 was synthesized under the same conditions as compound 1, except that compound P1 was replaced with compound P5. MS: m / z 1019.5 [M+1] + .

[0357] Example 6: Synthesis of Compound 6

[0358] Weigh N4-acetylcytidine (5 g, 1.0 eq.) and 4,4'-bismethoxytriphenylmethylchloro (1.2 eq.), add 150 mL of anhydrous pyridine, purge with nitrogen three times, stir overnight at room temperature. After the reaction is complete, quench the reaction with 10 mL of methanol, concentrate to dryness under reduced pressure, and purify by column chromatography (DCM / MeOH / 1% TEA = 100 / 1-10 / 1) to give product 6-A1 5 g, MS: m / z 588.2 [M+1] + . 1 H NMR (400MHz, DMSO-d6) δ10.88(s,1H),8.28(d,J=7.5Hz,1H),7.43–7.36(m,2H),7.33(t,J=7.5Hz,2H),7.29–7.20(m,5H),7.01(d,J=7.5Hz,1 H),6.94–6.86(m,4H),5.63(d,J=4.7Hz,1H),5.10(d,J=7.0Hz,1H),4.16(td,J=7.5,4.7Hz,1H),4.07–3.96(m,2H),3.75(s,6H),2.09(s,3H).

[0359] Weigh 1 g of 6-A1, sodium hydroxide (ground into powder) (3.0 eq.), and anhydrous DMF (20 mL). Purge with nitrogen three times. Add a solution of compound P2 (1.1 eq., dissolved in 2 mL of anhydrous DMF) dropwise to the reaction system. Stir overnight at room temperature (TLC monitoring showed residual starting material). Add 50 mL of water and extract three times with ethyl acetate. Combine the organic phases, wash three times with saturated brine, concentrate under reduced pressure, and purify by column chromatography (DCM / MeOH / 1% TEA = 100 / 1-50 / 1) to obtain 160 mg of product 6-A2. MS: m / z 812.5 [M+1] + . 1H NMR (400MHz, DMSO-d6) δ10.89(s,1H),8.37(d,J=7.5Hz,1H),7.38(d,J=7.2Hz,2H),7.32(t,J=7.5Hz,2H),7.28–7 .23(m,5H),7.02(d,J=7.5Hz,1H),6.90(d,J=8.8Hz,4H),5.73(d,J=1.4Hz,1H),5.52(d,J=5.1Hz,1H),4.27–4.17 (m,1H),4.12–4.06(m,1H),4.04-4.01(m,1H),3.75(s,6H),3.57-3.52(m,1H),3.40–3.35(m,2H),3.31–3.24(m,2 H),2.20-2.11(m,2H),2.10(s,3H),1.52-1.44(m,4H),1.35-1.31(m,2H),1.24-1.21(m,14H),1.03–0.95(m,3H). 19 F NMR(376MHz, CDCl3)δ-84.52(s,3F),(-117.0)-(-116.8)(m,2F).

[0360] Weigh 2.0 eq. of 1H-tetrazole and 4.0 eq. of bis(diisopropylamino)(2-cyanoethoxy)phosphine into a three-necked flask, add 3 mL of freshly distilled anhydrous DCM, purge with nitrogen three times, stir at room temperature for 10 minutes, then add dropwise a solution of 6-A2 (160 mg, 1.0 eq., dissolved in 1 mL of anhydrous DCM). After reacting at room temperature for 3 h, TLC monitoring showed no residue of the starting material and the presence of a product spot. Transfer the product to a single-necked flask and concentrate to dryness under reduced pressure. Purify by column chromatography (PE / TEA = 100 / 1, then PE / EA / TEA = 1 / 1 / 1%) to obtain compound 6 (66 mg). MS: m / z 1102.5 [M+1] + .

[0361] Example 7: Synthesis of Compound 7

[0362] Starting with intermediate 6-A1, compound 7 was synthesized under the same conditions as compound 6, except that compound P2 was replaced with compound P4. MS: m / z 1030.6 [M+1] + .

[0363] Example 8: Synthesis of Compound 8

[0364] Starting with intermediate 6-A1, compound 8 was synthesized under the same conditions as compound 6, except that compound P2 was replaced with compound P5. MS: m / z 1060.6 [M+1] + .

[0365] Example 9: Synthesis of Compound 9

[0366] Weigh N6-benzoyl adenosine (5 g, 1.0 eq.) and 4,4'-bismethoxytriphenylmethyl chloride (1.2 eq.), add 150 mL of anhydrous pyridine, purge with nitrogen three times, stir overnight at room temperature. After the reaction is complete, quench the reaction with 10 mL of methanol, concentrate to dryness under reduced pressure, and purify by column chromatography (DCM / MeOH / 1% TEA = 100 / 1-10 / 1) to give product 9-A1 5.2 g, MS: m / z 674.2 [M+1] + .

[0367] Weigh 1 g of 9-A1, sodium hydroxide (ground into powder) (3.0 eq.), and anhydrous DMF (20 mL). Purge with nitrogen three times. Add a solution of compound P2 (1.1 eq., dissolved in 2 mL of anhydrous DMF) dropwise to the reaction system. Stir overnight at room temperature (TLC monitoring showed residual starting material). Add 50 mL of water and extract three times with ethyl acetate. Combine the organic phases, wash three times with saturated brine, concentrate under reduced pressure, and purify by column chromatography (DCM / MeOH / 1% TEA = 100 / 1-50 / 1) to obtain 145 mg of product 9-A2. MS: m / z 988.5 [M+1] + .

[0368] Weigh 2.0 eq. of 1H-tetrazole and 4.0 eq. of bis(diisopropylamino)(2-cyanoethoxy)phosphine into a three-necked flask, add 3 mL of freshly distilled anhydrous DCM, purge with nitrogen three times, stir at room temperature for 10 minutes, then add dropwise a solution of 9-A2 (130 mg, 1.0 eq., dissolved in 1 mL of anhydrous DCM). After reacting at room temperature for 3 h, TLC monitoring showed no residue of the starting material and the presence of a product spot. Transfer the product to a single-necked flask and concentrate under reduced pressure to dryness. Purify by column chromatography (PE / TEA = 100 / 1 v / v, then PE / EA / TEA = 1 / 1 / 1%) to obtain compound 9 (53 mg). MS: m / z 1188.6 [M+1] + .

[0369] Example 10: Synthesis of Compound 10

[0370] Starting with intermediate 9-A1, compound 10 was synthesized under the same conditions as compound 9, except that compound P2 was replaced with compound P4. MS: m / z 1060.6 [M+1] + .

[0371] Example 11: Synthesis of Compound 11

[0372] Starting with intermediate 9-A1, compound 11 was synthesized under the same conditions as compound 9, except that compound P2 was replaced with compound P5. MS: m / z 1146.6 [M+1] + .

[0373] Example 12: Synthesis of Compound 12

[0374] Weigh 10 g (1.0 eq.) of N2-isobutyrylguanosine monohydrate and 1.2 eq. of 4,4'-bismethoxytriphenylmethyl chloride, add 150 mL of anhydrous pyridine, purge with nitrogen three times, stir overnight at room temperature, and after the reaction is complete, quench the reaction with 10 mL of methanol, concentrate to dryness under reduced pressure, and purify by column chromatography (DCM / MeOH / 1% TEA = 100 / 1-10 / 1) to give product 12-A1 11.2 g, MS: m / z 656.3 [M+1] + .

[0375] Weigh 1 g of 12-A1, sodium hydroxide (ground into powder) (3.0 eq.), and anhydrous DMF (20 mL). Purge the mixture three times with nitrogen. Add a solution of compound P2 (1.1 eq., dissolved in 2 mL of anhydrous DMF) dropwise to the reaction system. Stir overnight at room temperature (TLC monitoring showed residual starting material). Add 50 mL of water and extract three times with ethyl acetate. Combine the organic phases, wash three times with saturated brine, concentrate under reduced pressure, and purify by column chromatography (DCM / MeOH / 1% TEA = 100 / 1-50 / 1) to obtain product 12-A2 280 mg. MS: m / z 970.5 [M+1] + .

[0376] Weigh 2.0 eq. of 1H-tetrazole and 4.0 eq. of bis(diisopropylamino)(2-cyanoethoxy)phosphine into a three-necked flask, add 3 mL of freshly distilled anhydrous DCM, purge with nitrogen three times, stir at room temperature for 10 minutes, then add dropwise a solution of 12-A2 (180 mg, 1.0 eq., dissolved in 1 mL of anhydrous DCM). After reacting at room temperature for 3 h, TLC monitoring showed no residue of the starting material and the presence of a product spot. Transfer the product to a single-necked flask and concentrate under reduced pressure to dryness. Purify by column chromatography (PE / TEA = 100 / 1, then PE / EA / TEA = 1 / 1 / 1%) to obtain compound 12 (73 mg). MS: m / z 1170.6 [M+1] + .

[0377] Example 13: Synthesis of Compound 13

[0378] Starting with intermediate 12-A1, compound 13 was synthesized under the same conditions as compound 12, except that compound P2 was replaced with compound P4. MS: m / z 1098.6 [M+1] + .

[0379] Example 14: Synthesis of Compound 14

[0380] Starting with intermediate 12-A1, compound 14 was synthesized under the same conditions as compound 12, except that compound P2 was replaced with compound P5. MS: m / z 1128.6 [M+1] + .

[0381] Example 15: Synthesis of Compound 15

[0382] Compound (R)-3-pyrrolidone (1.00 g, 11.5 mmol, 1 eq) and anhydrous DMF (10 mL) were added to a reaction flask. After stirring at 0–5 °C for 5 min, imidazole (910.2 mg, 13.8 mmol, 1.2 eq) was added, and stirring continued for 15 min. Then, TBDPSCl dissolved in DMF (2.2 g, 8.1 mmol, 0.7 eq) was added. After the addition was complete, the mixture was moved to room temperature, and the reaction was monitored by TLC until complete. Water (20 mL) was added to the reaction system, and the mixture was extracted with ethyl acetate (300 mL x 3). The organic phases were combined, washed with saturated sodium chloride aqueous solution (50 mL x 3), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: dichloromethane:methanol = 10:1, v / v) to give compound 15-A1 (1.5 g, yield: 40.51%). MS: m / z 326.28 [M+1] + . 1H NMR (400MHz, CDCl3) δ7.67-7.64(m,4H),7.53–7.38(m,6H),4.54-4.50(m,1H),3.56–3 .49(m,1H),3.43-3.38(m,1H),3.24(d,J=3.7Hz,2H),2.06–1.81(m,2H),1.10(s,9H).

[0383] 15-A1 (1.5 g, 4.6 mmol, 1 eq), DMF (15 mL), sodium hydroxide (1.0 g, 25.0 mmol, 4 eq), and hexadecane bromide (2.9 g, 9.5 mmol, 1.5 eq) were added sequentially to the reaction flask. After the reaction was complete as monitored by TLC, water (20 mL) and ethyl acetate (50 mL) were added for extraction three times. The organic phase was washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: dichloromethane:methanol = 10:1, v / v) to obtain 15-A2 (3.2 g, yield: 91.43%). MS: m / z 550.4 [M+1] + ; 1 H NMR(400MHz, CDCl3)δ7.77–7.66(m,4H),7.48-7.39(m,,6H),4.45-4.40(m,1H),2.80-2.76(m,1H),2.67–2.5 4(m,2H),2.52–2.43(m,2H),2.41-2.34(m,1H),2.03-1.95(m,1H),1.87-1.80(m,1H),1.55–1.43(m,2H),1.34 -1.25(m,26H),1.10(s,9H),0.93(t,J=6.6Hz,3H).

[0384] 15-A2 (2.3 g, 4.2 mmol, 1 eq), tetrahydrofuran (30 mL), and TBAF (2.4 g, 7.55 mmol) were added sequentially to a reaction flask and reacted at room temperature under TLC monitoring. After the reaction was complete, water (50 mL) was added, and the mixture was extracted with ethyl acetate (50 mL x 3). The ethyl acetate phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (dichloromethane:methanol = 5:1, v / v) to give 15-A3 (1.1 g, yield: 84.6%). MS: m / z 312.4 [M+1] + ; 1H NMR(400MHz,DMSO-d6)δ5.49(d,J=3.9Hz,1H),4.42(s,1H),3.19(s,6H),3.1 1–3.04(m,2H),1.78–1.57(m,2H),1.31-1.26(s,26H),0.88(t,J=6.6Hz,3H).

[0385] 15-A3 (500.0 mg, 1.6 mmol, 1 eq), anhydrous dichloromethane (10 mL), and triethylamine (487.8 mg, 4.8 mmol, 3 eq) were added sequentially to a reaction flask. Nitrogen gas was purged three times. N,N-diisopropylphosphonamide (2-cyanoethyl) ester (758.4 mg, 3.2 mmol, 2 eq) was added, and the reaction was carried out at room temperature for 2 h. After the reaction was complete as monitored by TLC, the reaction mixture was evaporated to dryness and purified by wet column chromatography with anhydrous dichloromethane (petroleum ether:ethyl acetate = 2:1, v / v) to give compound 15 (800.0 mg, yield: 97.5%). MS: m / z 429.4 [M-(C3H7)2N+(OH)+(H)] + [M-100+17+1] + . 1 H NMR (400MHz, CDCl3) δ4.52-4.45(m,1H),3.93–3.75(m,2H),3.67-3.58(m,2H),3.08-2.96(m,1H),2.70-2.61(m,4H),2.56-2.38 (m,3H),2.24-2.13(m,1H),1.93-1.85(m,1H),1.54-1.48(m,2H),1.35-1.25(m,26H),1.24-1.19(m,12H),0.91(d,J=6.9Hz,3H).

[0386] Example 16: Synthesis of Compound 16

[0387] Starting from intermediate 15-A1, compound 16 was synthesized under the same conditions as compound 15, except that hexadecane bromide was replaced with docosane bromide. MS: m / z 513.6 [M-(C3H7)2N+(OH)+(H)] + [M-100+17+1] + .

[0388] Example 17: Synthesis of Compound 17

[0389] Starting from intermediate 15-A1, compound 17 was synthesized under the same conditions as compound 15, except that hexadecane bromide was replaced with compound P4. MS: m / z 447.4 [M-(C3H7)2N+(OH)+(H)] + [M-100+17+1] + . 1 H NMR (400MHz, CDCl3) δ4.51(t,J=6.2Hz,1H),4.40(t,J=6.2Hz,1H),3.92–3.80(m,2H),3.67-3.59(m,1H),3.02–2.95(m,1H),2.71-2.60(m,4 H),2.56–2.39(m,3H),2.24-2.12(m,1H),1.94–1.84(m,1H),1.79–1. 61(m,2H),1.54-1.49(m,2H),1.34-1.25(m,26H),1.23–1.19(m,12H).

[0390] Example 18: Synthesis of Compound 18

[0391] Weigh 5 g (1.0 eq.) of (3R,5S)-5-{[bis(4-methoxyphenyl)(phenyl)methoxy]methyl}pyrrolidine-3-ol and 5.0 eq. of imidazole and dissolve them in 50 mL of DCM. Then, add 1.2 eq. of TBDMSCl (dissolved in 10 mL of DCM) dropwise to the above reaction system. After the addition is complete, continue the reaction at room temperature for 5 h. When TLC monitoring shows that there is no starting material remaining, add 10 mL of water. Wash the reaction system three times with DCM (100 mL * 3). Combine the organic phases, concentrate under reduced pressure, and then perform column chromatography (EA / PE = 1 / 2-1 / 1, v / v) to obtain product 18-A1 (5.5 g, yield: 86.5%).

[0392] Weigh 2.0 g (1.0 eq.) of 18-A1 and dissolve it in 10 mL of anhydrous acetonitrile. Cool the solution to 0 °C. Slowly add potassium carbonate (2.0 eq.) to the reaction system. Stir at 0 °C for 30 minutes. Then, add hexadecane bromo (2.0 eq., dissolved in 2 mL of anhydrous acetonitrile) dropwise to the reaction system. After the addition is complete, heat to room temperature and react for 10 h. TLC monitoring showed no residue of the starting material. Concentrate under reduced pressure to remove acetonitrile. Add 10 mL of water. Wash the reaction system three times with ethyl acetate (100 mL * 3). Combine the organic phases and wash with saturated brine (100 mL). Concentrate under reduced pressure to obtain crude product 18-A2 (3.0 g).

[0393] Weigh 3.0 g (1.0 equiv.) of crude 18-A2 and dissolve it in 10 mL of tetrahydrofuran. Then, add 5.0 equiv. of TBAF dropwise to the above reaction system and react at room temperature for 18 h. TLC monitoring showed no residue of the starting material. The tetrahydrofuran was removed by concentration under reduced pressure, and column chromatography (EA / PE = 1 / 2-2 / 1, v / v) was used to obtain product 18-A3 (1.58 g, two-step yield: 65.5%).

[0394] Weigh 300 mg (1.0 eq.) of 18-A3 and 1 eq. of tetrazolium and dissolve them in 2 mL of DCM. Replace the nitrogen gas three times. Then add bis(diisopropylamino)(2-cyanoethoxy)phosphine (3 eq.) dropwise to the above reaction system and react at room temperature for 2 h. TLC monitoring showed that there was no residue of the starting material. The reaction system was purified by rapid column chromatography (PE / EA = 1 / 0-10 / 1, v / v) to obtain compound 18 (200 mg, yield: 50.8%).

[0395] Example 19: Synthesis of Compound 19

[0396] Starting from intermediate 15-A1, compound 19 was synthesized under the same conditions as compound 15, except that hexadecane was replaced with tetradecane. MS: m / z 401.4 [M-(C3H7)2N+(OH)+(H)] + [M-100+17+1] + . 1 H NMR (400MHz, CDCl3) δ4.13-4.03(m,1H),3.60-3.54(m,2H),3.29-3.21(m,3H),2.95–2.45(m,6H),2.28-2.18(m,1H),2.11 –1.97(m,1H),1.96–1.72(m,1H),1.67–1.43(m,2H),1.37(s,6H),1.36(s,6H),1.29–1.17(m,22H),0.89(t,J=6.6Hz,3H).

[0397] Example 20: Synthesis of Compound 20

[0398] Starting from intermediate 15-A1, compound 20 was synthesized under the same conditions as compound 15, except that hexadecane bromide was replaced with octadecane bromide. MS: m / z 457.5 [M-(C3H7)2N+(OH)+(H)] + [M-100+17+1] + . 1H NMR (400MHz, CDCl3) δ4.16–3.77(m,2H),3.69–3.51(m,1H),3.46–3.17(m,2H),2.85–2.38(m,7H),2.23(m,J=20.1,13.5,7.0, 6.2Hz,1H),2.14–1.68(m,2H),1.52(q,J=11.5,9.4Hz,2H),1.33(d,J=6.5Hz,12H),1.29–1.16(m,30H),0.90(t,J=6.7Hz,3H).

[0399] Example 21: Synthesis of Compound 21

[0400] Weigh (2R,3S,6R)-6-(aminomethyl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydro-2H-pyran-3-ol (compound 21-A1) (500 mg), hexadecanoic acid (1.2 equiv.), HATU (2.0 equiv.), and DIEA (4.0 eq.) into a reaction flask, add 10 mL of DMF, and react at room temperature until no starting material 21-A1 remains. After the reaction is complete, dilute with water (20 mL), extract with ethyl acetate (50 mL * 3), wash with saturated brine (20 mL * 3), and concentrate the organic phase under reduced pressure and purify by column chromatography (DCM / MeOH = 80 / 1-40 / 1) to obtain product 21-A2 (580 mg). MS: m / z 702.5 [M+1] + .

[0401] Tetrazazole (3 eq.) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (6 eq.) were weighed and placed in a reaction flask. 5 mL of DCM was added, and nitrogen gas was purged three times. A solution of 21-A2 (300 mg, 1.0 eq. dissolved in 2 mL of DCM) was added dropwise. The reaction was allowed to proceed at room temperature until no 21-A2 remained. After the reaction was complete, the reaction system was concentrated under reduced pressure and purified by rapid column chromatography (PE / EA = 10 / 1-3 / 1) to obtain compound 21 (220 mg). MS: m / z 902.6 [M+1] + .

[0402] Example 22: Preparation of compound 22

[0403] 20.0 g (93.8 mmol, 1.0 eq) of methyl 3-nitro-4-fluorophenylacetate was weighed and placed in a reaction flask. DMF (200 mL) and potassium carbonate (13.0 g, 93.8 mmol, 1.0 eq) were added sequentially, and the mixture was purged with nitrogen three times. Hexadecylamine solution (22.7 g, 93.8 mmol, 1.0 eq dissolved in 100 mL DMF) was added dropwise to the reaction mixture, and the mixture was allowed to react overnight at room temperature. After the reaction was complete, 100 mL of water was added, and the mixture was extracted with ethyl acetate (200 mL x 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (eluent PE:EA = 5:1) to give compound 22-A1 (27.7 g, yield 67.9%). 1 H NMR (400MHz, CDCl3) δ8.09(s,1H),8.06(s,1H),7.40(d,J=8.8Hz,1H),6.86(d,J=8.8Hz,1H),3.73(s ,3H),3.57(s,2H),3.37–3.25(m,2H),1.82–1.69(m,2H),1.45–1.23(m,26H),0.91(t,J=6.0Hz,3H).

[0404] Compound 22-A1 (7.2 g, 16.6 mmol, 1.0 eq), methanol (100 mL), and 60% palladium on carbon (4.3 g) were added sequentially to the reaction flask. The mixture was purged with hydrogen three times, and the reaction was allowed to proceed overnight at room temperature. After the reaction was complete, the reaction system was filtered, and the filtrate was evaporated to dryness to obtain compound 22-A2 (7.0 g).

[0405] Compound 22-A2 (7.0 g, 17.3 mmol, 1.0 eq), acetic acid (30 mL), and water (30 mL) were added sequentially to a reaction flask. Sodium nitrite (1.6 g, 22.5 mmol, 1.3 eq) was added at 0 °C, and the mixture was stirred at 0 °C for 2 h. Then, concentrated sulfuric acid (7 mL) was added dropwise, and the reaction was continued at 0 °C for 2 h. After the reaction was complete, the pH was adjusted to 7 with sodium bicarbonate in an ice-water bath. The mixture was extracted three times with ethyl acetate (100 mL), and the organic phases were combined. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (eluent PE:EA = 5:1) to obtain compound 22-A3 (2.8 g, yield 38.9%). 1H NMR (400MHz, DMSO-d6) δ7.91 (s, 1H), 7.83 (d, J = 8.6Hz 1H),7.45(d,J=8.5Hz,1H),4.72–4.64(m,2H),3.87(s,2H),3.63(s,3H),1.95–1.85(m,2H),1.24–1.18(m,26H),0.85(t,J=6.8Hz,3H).

[0406] Compound 22-A3 (2.8 g, 6.7 mmol, 1.0 eq) and anhydrous tetrahydrofuran (50 mL) were added sequentially to the reaction flask. Lithium aluminum hydride (383.8 mg, 10.1 mmol, 1.5 eq) was added at 0 °C, and the reaction was carried out at 0 °C for 1 h. The reaction was quenched by adding saturated ammonium chloride (30 mL) at 0 °C, and extracted with ethyl acetate (50 mL * 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure to obtain the crude product, and purified by column chromatography (eluent PE:EA = 1:1, v / v) to give compound 22-A4 (2.0 g, yield 75.8%). 1 H NMR(400MHz, DMSO-d6)δ7.82(s,1H),7.76(d,J=8.5Hz,1H),7.42(d,J=8.6Hz,1H),4.70–4.62(m,3H) ,3.72–3.63(m,2H),2.93–2.85(m,2H),1.94–1.82(m,2H),1.30–1.16(m,27H),0.84(t,J=6.8Hz,3H).

[0407] Bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.8 g, 6.2 mmol, 6.0 eq), tetrazolium (217 mg, 3.1 mmol, 3.0 eq), and anhydrous dichloromethane (4 mL) were added sequentially to the reaction flask, and nitrogen was purged three times. Compound 22-A4 (400.0 mg, 1.0 mmol, 1.0 eq) was dissolved in anhydrous dichloromethane (1 mL) and added dropwise to the above solution. The mixture was stirred at room temperature for 1 h. After the reaction was complete, the sample was directly loaded onto a wet plate and purified by column chromatography (eluent PE:EA = 1:1 + (1% TEA)) to give compound 22 (320 mg, yield 52.8%). 1HNMR(400MHz,DMSO-d6)δ7.84(s,1H),7.73(d,J=8.5Hz,1H),7.42(d,J=8.3Hz,1 H),4.64(t,J=6.9Hz,2H),3.95–3.75(m,2H),3.75–3.59(m,2H),3.57–3.42(m,2H ),3.01(t,J=6.6Hz,2H),2.71(t,J=5.9Hz,2H),1.92–1.80(m,2H),1.28–1.11(m ,26H),1.08(s,3H),1.07(s,3H),1.01(s,3H),1.0(s,3H),0.81(t,J=6.8Hz,3H). 31 P NMR(162MHz,DMSO-d6)δ146.61(s).

[0408] Example 23: Synthesis of Compound 23

[0409] Compound (S)-benzyloxymethyl ethylene oxide (38.0 g, 231.0 mmol, 10 eq), TsNHBoc (75.4 g, 278.0 mmol, 12 eq), K₂CO₃ (6.40 g, 46.3 mmol, 2.0 eq), and TEBA (5.27 g, 23.1 mmol, 1.0 eq) were added to a three-necked flask at room temperature. The reaction was carried out at 95 °C for 2 hours. After cooling to room temperature, the mixture was diluted with water (500 mL), extracted with dichloromethane (200 mL * 3), and the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (eluent PE:EA = 3:1, v / v) to give compound 23-A1 (40.0 g, yield: 39.7%). MS: m / z 436.2 [M+1] + . 1 H NMR(400MHz, CDCl3)δ7.71(d,J=8.1Hz,2H),7.37–7.32(m,1H),7.32–7.23(m,6H),4.97(t,J=6.4Hz,1H) ,4.79-4.74(m,1H),4.52–4.41(m,2H),3.62–3.48(m,2H),3.34–3.14(m,2H),2.41(s,3H),1.45(s,9H).

[0410] Compound 23-A1 (79.0 g, 181 mmol), (R)-benzyloxymethyl ethylene oxide (24.7 g, 150.8 mmol), potassium carbonate (4.21 g, 30.5 mmol), and TEBA (3.47 g, 15.2 mmol) were reacted at 95 °C for 3 hours. After cooling to room temperature, the reaction mixture was extracted with water (500 mL) and dichloromethane (200 mL x 3). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (eluent PE:EA = 3:1, v / v) to give compound 23-A2 (40.0 g, yield 43.8%). MS: m / z 601.2 [M+1] + . 1 H NMR(400MHz, CDCl3)δ7.74–7.65(m,2H),7.36–7.24(m,12H),5.14-5.08(m,1H),4.59-4.44(m,4H),4.15–4.03 (m,1H),3.70–3.60(m,2H),3.59–3.43(m,3H),3.35–3.23(m,2H),3.19-3.13(m,1H),2.40(s,3H),1.45(s,9H).

[0411] Compound 23-A2 (77.0 g, 128 mmol) and triethylamine (28.6 mL, 205 mmol) were added sequentially to dichloromethane (800 mL) under ice-water bath conditions, followed by the slow addition of methanesulfonyl chloride (24.2 g, 212 mmol). The reaction was carried out at 25 °C for 2 hours, and LC-MS showed that the reaction was complete. The reaction solution was washed with water (800 mL), and the organic phase was concentrated under reduced pressure to give compound 23-A3 (90.0 g). MS: m / z 678.2 [M+1] + .

[0412] Compound 23-A3 (90.0 g, 133 mmol) and potassium carbonate (91.8 g, 664 mmol) were added to methanol (900 mL) at room temperature. The reaction was carried out at 66 °C for 2 hours. TLC (PE / EA = 2 / l, v / v) showed the formation of new spots. The organic phase was concentrated under reduced pressure, and the reaction solution was diluted with water (200 mL). Extraction was performed with dichloromethane (200 mL x 3), and the organic phase was concentrated under reduced pressure. The crude product was purified by column chromatography (PE / EA = 5 / 1-2 / 1, v / v) to give compound 23-A4 (64.0 g, yield 99.9%). MS: m / z 482.2 [M+1] + . 1H NMR (400MHz, DMSO-d6) δ7.62(d,J=8.3Hz,2H),7.47(d,J=8.0Hz,2H),7.33–7.28(m,10H),4.49( s,4H),3.98-3.94(m,2H),3.58-3.52(m,4H),2.94-2.90(m,2H),2.79-2.74(m,2H),2.41(s,3H).

[0413] Compound 23-A4 (69.0 g, 143 mmol) and magnesium filings (54.7 g, 228 mol) were added to methanol (400 mL) and reacted at 66 °C for 1 hour. TLC (DCM / MeOH = 10 / l) showed that the starting material reacted completely and new spots were formed. The reaction solution was diluted with water (3000 mL) and saturated ammonium chloride aqueous solution (3000 mL), extracted with dichloromethane (1000 mL * 3), and the organic phase was washed with saturated sodium bicarbonate (300 mL * 3). The organic phase was concentrated under reduced pressure to give 23-A5 (32.0 g), MS: m / z 328.2 [M+1]. + . 1 H NMR (400MHz, DMSO-d6) δ7.34–7.28(m,10H),4.49(s,4H),3.80-3.75(m,2H),3.61–3.49(m,4H),2.79-2.74(m,2H),2.58-2.53(m,2H).

[0414] Compound 23-A5 (3.00 g, 9.16 mmol), compound P7 (18.3 mmol), and acetic acid (1.01 mL, 18.3 mmol) were added to methanol (30 mL). The reaction was carried out at room temperature for 18 hours, followed by the addition of sodium cyanoborohydride (2.30 g, 36.7 mmol), and the reaction was carried out at 50 °C for 4 hours. TLC (DCM / MeOH = 10 / l) showed the formation of new spots. The reaction solution was diluted with water (50 mL), extracted with dichloromethane (30 mL x 3), and the organic phase was concentrated under reduced pressure. The crude product was purified by column chromatography (dichloromethane / methanol = 99 / 1-5 / 1) to give compound 23-A6 (3.2 g). MS: m / z 649.4 [M+1] +. 1H NMR (400MHz, DMSO-d6) δ7.88(d,J=7.5Hz,2H),7.68(d,J=7.4Hz,2H),7.40(t,J=7.5Hz,2H),7.34–7.26(m,12H),4.50(s,2H),4.31-4.28(m,2H),4.2 3-4.18(m,2H),3.89-3.82(m,2H),3.59-3.54(m,2H),3.53-3.47(m,2H),3 .00-2.94(m,2H),2.23-2.12(m,4H),1.42-1.30(m,4H),1.27-1.22(m,4H).

[0415] Compound 23-A6 (3.00 g) was added to concentrated hydrochloric acid (10 mL, 12 M) and reacted at 50 °C for 18 hours. TLC (DCM / MeOH = 10 / 1) showed the formation of new spots. The reaction solution was concentrated under reduced pressure to give 23-A7 (2.00 g). MS: m / z 469.2 [M+1] + . 1 H NMR (400MHz, DMSO-d6) δ7.88(d,J=7.5Hz,2H),7.68(d,J=7.6Hz,2H),7.41(t, J=7.4Hz,2H),7.33(t,J=7.4Hz,2H),4.30(d,J=6.9Hz,2H),4.24-4.10(m,2H), 4.06-4.00(m,1H),3.66-3.61(m,2H),3.54–3.39(m,4H),2.97(q,J=6.6Hz,2H ),2.38-2.34(m,2H),2.22-2.16(m,5H),1.42-1.37(m,4H),1.28-1.15(m,4H).

[0416] 23-A7 (2.00 g) was dissolved in pyridine (40 mL), and then DMTCl (2.96 g) was added. The reaction was carried out at 25 °C for 18 hours. TLC (PE / EA = 1 / 1) showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, and saturated ammonium chloride aqueous solution (200 mL) was added. Extraction was performed using DCM (100 mL x 3). The organic phase was concentrated under reduced pressure, and the crude product was purified by column chromatography (dichloromethane / methanol = 99 / 1-10 / 1) to give compound 23-A8 (1.40 g). MS: m / z 771.4 [M+1] + .

[0417] 23-A8 (1.4 g) was dissolved in DCM (20 mL), and then DBU (2.0 eq.) was added. The reaction was carried out at 25 °C for 2 hours. TLC (PE / EA = 1 / 1) showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, and the crude product was purified by column chromatography (dichloromethane / methanol = 60 / 1-20 / 1) to give compound 23-A9 (600 mg). MS: m / z 549.3 [M+1] + .

[0418] Weigh 500 mg of 23-A9, 1.2 eq. of hexadecanoic acid, 2.0 eq. of HATU, and 4.0 eq. of DIEA into a reaction flask, add 10 mL of DMF, and react at room temperature until no 23-A9 remains. After the reaction is complete, dilute with 20 mL of water, extract with ethyl acetate (50 mL x 3), wash with saturated brine (20 mL x 3), and concentrate the organic phase under reduced pressure and purify by column chromatography (DCM / MeOH = 80 / 1-40 / 1) to obtain product 23-A10 (600 mg). MS: m / z 787.5 [M+1] + .

[0419] Weigh out tetrazolium (3 eq.) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (6 eq.) and place them in a reaction flask. Add 5 mL of DCM, purge with nitrogen three times, and add dropwise 23-A10 solution (300 mg, 1.0 eq., dissolved in 2 mL of DCM). React at room temperature until no raw material remains. After the reaction is complete, concentrate the reaction system under reduced pressure and purify it by rapid column chromatography (PE / EA+TEA 1% = 10 / 1-3 / 1) to obtain compound 23 (210 mg).

[0420] Example 24: Synthesis of Compound 24

[0421] Weigh 23-A9 (500 mg), docosuccinic acid (1.2 eq.), HATU (2.0 eq.), and DIEA (4.0 eq.) into a reaction flask, add 10 mL of DMF, and react at room temperature until no starting material remains. After the reaction is complete, dilute with water (20 mL), extract with ethyl acetate (50 mL * 3), wash with saturated brine (20 mL * 3), and concentrate the organic phase under reduced pressure and purify by column chromatography (DCM / MeOH = 80 / 1-40 / 1) to obtain product 24-A1 (580 mg).

[0422] Tetrazazole (3 eq.) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (6 eq.) were weighed and placed in a reaction flask. 5 mL of DCM was added, and nitrogen was purged three times. 24-A1 solution (350 mg, 1.0 eq., dissolved in 2 mL of DCM) was added dropwise. The reaction was carried out at room temperature until no raw material remained. After the reaction was completed, the reaction system was concentrated under reduced pressure and purified by rapid column chromatography (PE / EA = 10 / 1-3 / 1) to obtain compound 24 (170 mg).

[0423] Example 25: Synthesis of Compound 25

[0424] Weigh out N4-acetylcytidine (20 g), TIPSCl2 (1.2 eq), and 100 mL of anhydrous pyridine. React overnight at room temperature. Monitor the reaction completion by TLC (dichloromethane:methanol = 30:1, v / v). Quench with 50 mL of methanol, concentrate, and separate and purify by column chromatography (petroleum ether:ethyl acetate = 3:1-1:1, v / v) and (dichloromethane:methanol = 30:1, v / v) to obtain product 25-A1 (10 g). MS: m / z 528.2 [M+1] + .

[0425] Weigh 10 g of 25-A1, add 70 mL of DMSO, 39 mL of acetic anhydride, and 7.9 mL of acetic acid sequentially, react overnight at 30 °C, and monitor the reaction completion by TLC. Dilute with 200 mL of water, extract with dichloromethane (3 x 300 mL), combine the organic phases, and successively pass through 200 mL of saturated sodium bicarbonate solution, 300 mL of saturated sodium chloride solution, and anhydrous sodium sulfate. Filter, concentrate under reduced pressure, and purify by column chromatography (petroleum ether:ethyl acetate = 3:1 to 1:1, v / v) to obtain compound 25-A2 (6 g), MS: m / z 588.2 [M+1] + .

[0426] Weigh 3g of 25-A2, add 30mL of dichloromethane, purge with nitrogen three times, cool to 0℃, then add 3g of 4A molecular sieve, 1.2eq of NIS, 1.2eq of trifluoromethanesulfonic acid, and 1.2eq of N-fluorenemethyloxycarbonyl-diethylene glycol. React at room temperature for 0.5 hours, then heat to 30℃ and react for 2 hours. Monitor the reaction by TLC (petroleum ether:ethyl acetate = 2:1, v / v). Once the reaction is complete, dilute with 200mL of dichloromethane. Wash the organic phase successively with 200mL of saturated sodium carbonate solution and 200mL of saturated sodium sulfite solution. Dry the organic phase with anhydrous sodium sulfate, filter, concentrate, and purify by column chromatography (petroleum ether:ethyl acetate = 6:1 to 1:1, v / v) to obtain 25-A3 (2g). MS: m / z 867.4 [M+1] +.

[0427] 25-A3 (2.0 g) was dissolved in dichloromethane (30 mL) and water (21 mL). DBU (1.8 eq) was added in an ice-water bath, and the reaction was maintained at this temperature for 2 h. Then, the temperature was raised to room temperature and the reaction was continued for another 2 h. After the reaction was completed, the mixture was separated, concentrated under reduced pressure, and purified by column chromatography (dichloromethane:methanol = 10:1-5:1, v / v) to obtain 25-A4 (980 mg). MS: m / z 645.3 [M-1] - .

[0428] 25-A4 (980 mg), compound P5 (2.0 eq.), HATU (2.0 eq.), NMM (2.5 eq.), and DMF (5 mL) were added sequentially to a 50 mL round-bottom flask and reacted at room temperature for 2 h. After the reaction of 25-A4 was complete, water (20 mL) was added to the system, and the mixture was extracted with ethyl acetate (15 mL x 3). The organic phases were combined, washed with saturated sodium chloride aqueous solution (50 mL x 3), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (dichloromethane:methanol = 50:1, v / v) to obtain 25-A5 (950 mg). MS: m / z 901.5 [M+1] + .

[0429] 25-A5 (950 mg) was dissolved in dichloromethane (50 mL), and tetrabutylammonium fluoride trihydrate (2.5 eq) was added. The mixture was reacted at room temperature for 2 hours. The reaction was monitored by TLC (dichloromethane:methanol = 20:1, v / v) until completion. The solution was concentrated under reduced pressure and purified by wet column chromatography (dichloromethane:methanol = 40:1, v / v) to obtain 25-A6 (560 mg). MS: m / z 659.4 [M+1] + .

[0430] 25-A6 (560 mg) was dissolved in dichloromethane (10 mL), N-methylmorpholine (6.0 eq) was added, and DMT-Cl (3.0 eq) was added to the reaction system. The reaction was monitored by TLC (dichloromethane:methanol = 20:1, v / v) until completion. The solution was concentrated under reduced pressure and purified by column chromatography (dichloromethane:methanol = 40:1-20:1, v / v) to obtain 25-A7 (710 mg). MS: m / z 961.5 [M+1] + .

[0431] Weigh 1H-tetrazole (2.0 eq.) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (4.0 eq.) into a three-necked flask, add 3 mL of freshly distilled anhydrous DCM, purge with nitrogen three times, stir at room temperature for 10 minutes, then add dropwise a solution of 25-A7 (217 mg, 1.0 eq., dissolved in 1 mL of anhydrous DCM). After reacting at room temperature for 3 h, TLC monitoring showed no residue of the starting material and the presence of a product spot. Transfer the product to a single-necked flask and concentrate under reduced pressure to dryness. Purify by column chromatography (PE / TEA = 100 / 1, then PE / EA / TEA = 1 / 1 / 1%) to obtain compound 25 (104 mg, 45.26%).

[0432] Example 26: Synthesis of Compound 26

[0433] Except for replacing N4-acetylcytidine with uracil nucleoside, the other conditions were the same as those for the synthesis of compound 25, yielding compound 26.

[0434] Example 27: Synthesis of Compound 27

[0435] Except for replacing N4-acetylcytidine with N6-benzoyladenosine, the other conditions were the same as those for the synthesis of compound 25, yielding compound 27.

[0436] Example 28: Synthesis of Compound 28

[0437] Except for replacing N4-acetylcytidine with N2-isobutyrylguanosine monohydrate, the other conditions were the same as those for the synthesis of compound 25, yielding compound 28.

[0438] Example 29: Synthesis of Compound 29

[0439] Sodium azide (1.5 eq.) and 1-bromohexadecane (2 g) were weighed and placed in a reaction flask. DMSO (10 mL) was added and the mixture was reacted overnight at room temperature. After the reaction was completed, water (50 mL) was added and the mixture was extracted with ethyl acetate (100 mL * 3). The organic phases were combined and washed with saturated brine (50 mL * 3), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (pure PE) to obtain compound P9 (1.9 g).

[0440] Weigh 1 g of 6-A1, NaH (3.0 eq.), and anhydrous DMF (20 mL). Purge with nitrogen three times. Add dropwise 1.1 eq. of 8-bromo-1-octyne solution (dissolved in 2 mL of anhydrous DMF) to the reaction system. Stir overnight at room temperature (TLC monitoring showed residual starting material). Add 50 mL of water and extract three times with ethyl acetate. Combine the organic phases, wash three times with saturated brine, concentrate under reduced pressure, and purify by column chromatography (DCM / MeOH / 1% TEA = 100 / 1-50 / 1) to obtain product 29-A1 380 mg, MS: m / z 696.3 [M+1]. +

[0441] Weigh 1 g of 29-A1, anhydrous copper sulfate (0.6 eq.), and sodium ascorbate (1.2 equiv.). Replace with nitrogen three times, add THF (10 mL), tert-butanol (5 mL), and water (2 mL), then add P9 (1.1 equiv.) dropwise to the reaction system. React at room temperature with stirring overnight. Add 50 mL of water, extract three times with ethyl acetate, combine the organic phases, wash three times with saturated brine, concentrate under reduced pressure, and purify by column chromatography (DCM / MeOH / 1% TEA = 100 / 1-50 / 1) to obtain 1.1 g of product 29-A2. MS: m / z 963.6 [M+1] + .

[0442] Weigh 1H-tetrazole (2.0 eq.) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (4.0 eq.) into a three-necked flask, add 3 mL of anhydrous DCM, purge with nitrogen three times, stir at room temperature for 10 minutes, then add dropwise a solution of 29-A2 (300 mg, 1.0 equiv., dissolved in 1 mL of anhydrous DCM). After reacting at room temperature for 3 h, TLC monitoring showed no residue of the starting material and the presence of a product spot. Transfer the product to a single-necked flask and concentrate under reduced pressure to dryness. Purify by column chromatography (PE / TEA = 100 / 1, then PE / EA / TEA = 1 / 1 / 1%) to obtain compound 29 (280 mg).

[0443] Example 30: Preparation of compound 30

[0444] Except for replacing hexadecylamine with tetradecylamine, the other conditions were the same as those for the synthesis of compound 22, yielding compound 30.

[0445] Compound 30-A1 was obtained. 1H NMR (400MHz, DMSO-d6) δ8.07-8.04(m,1H),7.97(s,1H),7.42-7.39(m,1H),6.97(d,J=8.9Hz,1 H),3.61(s,2H),3.60(s,3H),1.61(q,J=7.1Hz,2H),1.26-1.18(s,24H),0.83(t,J=6.6Hz,3H).

[0446] Compound 30-A3 was obtained. 1 H NMR (400MHz, CDCl3) δ7.94(s,1H),7.48(d,J=8.6Hz,1H),7.43(d,1H),4.60(t,J=7.2Hz,2H ),3.79(s,2H),3.71(s,3H),2.03–1.95(m,2H),1.36–1.20(m,22H),0.88(t,J=6.7Hz,3H).

[0447] Compound 30-A4 was obtained. 1 H NMR (400MHz, DMSO-d6) δ7.82(s,1H),7.76(d,J=8.5Hz,1H),7.42(d,J=8.5Hz,1H),4.67-4.64(m,3H),3 .72–3.63(m,2H),2.89(t,J=7.2Hz,2H),1.92-1.84(m,2H),1.30–1.17(m,22H),0.85(t,J=6.7Hz,3H).

[0448] Compound 30 was obtained. 1 H NMR (400MHz, DMSO-d6) δ7.86 (s, 1H), 7.76 (d, J = 8.5Hz, 1H), 7.44 (d, J = 8.5Hz, 1H),4.66(t,J=6.8Hz,2H),3.98–3.78(m,2H),3.78–3.60(m,2H),3.60–3.42(m ,2H),3.04(t,J=6.8Hz,2H),2.73(t,J=6.8Hz,2H),1.94–1.83(m,2H),1.26–1 .15(m,22H),1.10(d,J=6.8Hz,6H),1.03(J=6.8Hz,6H),0.84(t,J=6.7Hz,3H), 31 P NMR(162MHz,DMSO-d6)δ146.63(s).

[0449] Example 31: Preparation of compound 31

[0450] Except for replacing hexadecylamine with octadecylamine, the other conditions were the same as those for the synthesis of compound 22, yielding compound 31.

[0451] Compound 31-A1 was obtained. 1 H NMR (400MHz, DMSO-d6) δ8.12–8.05(m,1H),7.97(s,1H),7.43(d,J=8.9Hz,1H),7.00(d,J=9.0,3 .6Hz,1H),3.63(s,3H),3.61(s,1H),1.66–1.55(m,2H),1.33–1.17(m,30H),0.88–0.80(m,3H).

[0452] Compound 31-A3 was obtained. 1 H NMR(400MHz, CDCl3)δ7.94(s,1H),7.48(d,J=8.6Hz,1H),7.43(d,1H),4.65–4.57(m,2H),3.79(s, 2H),3.74(s,3H),2.05–1.94(m,2H),1.36–1.30(m,4H),1.28–1.22(m,26H),0.90(t,J=4.8Hz,3H).

[0453] Compound 31-A4 was obtained. 1 H NMR (400MHz, DMSO-d6) δ7.81(s,1H),7.73(d,J=8.5Hz,1H),7.41(d,J=8.5Hz,1H),4.64(t,J=4.8Hz,2H),4.64(t,J=4 .8Hz,1H),3.68(q,J=4.8Hz,2H),2.89(t,J=4.8Hz,2H),1.95–1.83(m,2H),1.27–1.16(m,28H),0.84(t,J=4.8Hz,3H).

[0454] Compound 31 was obtained. 1H NMR (400MHz, DMSO-d6) δ7.83(s,1H),7.73(d,1H),7.41(d,J=7.4Hz,1H),4.63(s,2H),3.93–3.76(m,2H),3.66(dd,J=12.2,5.9Hz,2H),3.5 3–3.41(m,2H),3.01(s,2H),2.74–2.66(m,2H),1.85(s,2H),1.25–1. 12(m,30H),1.11–1.04(m,7H),1.03–0.97(m,6H),0.84–0.77(m,3H). 31 P NMR(162MHz,DMSO)δ146.62(s).

[0455] Example 32: Preparation of compound 32

[0456] Except for replacing hexadecylamine with dodecylamine, the other conditions were the same as those for the synthesis of compound 22, yielding compound 32.

[0457] Compound 32-A1 was obtained. 1 H NMR (400MHz, DMSO-d6) δ7.96 (s, 2H), 7.41 (d, J = 8.9Hz, 1H), 6.97 (J = 8.9Hz, 1H), 3.61 (s, 5H), 3.31 (q, J = 7.0Hz, 2H), 1.65-1.58 (m, 2H), 1.25-1.15 (m, 38H), 0.84 (t, J = 6.5Hz, 3H).

[0458] Compound 32-A3 was obtained. 1 H NMR (400MHz, CDCl3) δ7.97(s,1H),7.52(d,J=8.6Hz,1H),7.46(d,J=8.6Hz,1H),4.65–4.57(m,2H),3.7 9(s,2H),3.74(s,3H),2.05–1.94(m,2H),1.36–1.30(m,4H),1.28–1.22(m,36H),0.90(t,J=4.8Hz,3H).

[0459] Compound 32-A4 was obtained. 1H NMR (400MHz, DMSO-d6) δ7.90(s,1H),7.47(d,J=8.5Hz,1H),7.38(d,J=8.5Hz,1H),4.62(t,J=4.8Hz,2H),3.95(t,J= 4.8Hz,1H),3.05(q,J=4.8Hz,2H),2.02-1.95(m,2H),1.36–1.30(m,4H),1.27–1.16(m,36H),0.84(t,J=4.8Hz,3H).

[0460] Compound 32 was obtained. 1 H NMR (400MHz, CDCl3) δ7.88(s,1H),7.45(d,J=8.5Hz,1H),7.38(d,J=8.5,1H),4.6 1(t,J=7.2Hz,2H),4.01–3.82(m,2H),3.82–3.67(m,2H),3.61-3.52(m,2H),3.09( t,J=6.8Hz,2H),2.57(d,J=6.5,2H),2.01-1.97(m,2H),1.37–1.27(m,4H),1.24- 1.20(m,34H),1.16(d,J=6.8Hz,6H),1.11(d,J=6.8Hz,6H),0.88(t,J=6.7Hz,3H). 31 P NMR (162MHz, CDCl3) δ147.58 (s).

[0461] Example 33: Preparation of compound 33

[0462] Compound (S)-3-pyrrolidone (2.00 g, 23.0 mmol, 1 eq) and anhydrous DMF (25 mL) were added to a reaction flask. After stirring at 0 °C for 5 min, imidazole (1.82 g, 27.6 mmol, 1.2 eq) was added, and stirring continued for 15 min. Then, TBDPSCl dissolved in DMF (4.4 g, 16.2 mmol, 0.7 eq) was added. After the addition was complete, the mixture was allowed to rise to room temperature, and the reaction was monitored by TLC until complete. Water (50 mL) was added to the reaction system, and the mixture was extracted with ethyl acetate (400 mL * 3). The organic phases were combined, washed with saturated sodium chloride aqueous solution (100 mL * 3), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: dichloromethane:methanol = 10:1, v / v) to give compound 33-A1 (2.8 g, yield: 37.8%). MS: m / z 326.28 [M+1] + .

[0463] 33-A1 (2.0 g, 6.1 mmol, 1 eq), DMF (25 mL), sodium hydroxide (1.3 g, 32.5 mmol, 4.0 eq), and hexadecane bromide (3.86 g, 12.6 mmol, 1.5 eq) were added sequentially to the reaction flask. After the reaction was completed as monitored by TLC, water (40 mL) and ethyl acetate (100 mL) were added for extraction three times. The organic phase was washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: dichloromethane:methanol = 10:1, v / v) to obtain 33-A2 (6.26 g, yield: 89.4%).

[0464] 33-A2 (4.6 g, 8.4 mmol, 1 eq), tetrahydrofuran (70 mL), and TBAF (4.8 g, 15.1 mmol) were added sequentially to a reaction flask. The reaction was carried out at room temperature and monitored by TLC. After the reaction was complete, water (100 mL) was added, and the mixture was extracted with ethyl acetate (100 mL * 3). The ethyl acetate phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (dichloromethane:methanol = 5:1, v / v) to give 33-A3 (2.28 g, yield: 87.5%). MS: m / z 312.4 [M+1] + .

[0465] 33-A3 (600.0 mg, 1.9 mmol, 1 eq), anhydrous dichloromethane (15 mL), and triethylamine (585.4 mg, 5.8 mmol, 3 eq) were added sequentially to a reaction flask. Nitrogen gas was purged three times. N,N-diisopropylphosphonamide (2-cyanoethyl) ester (910.0 mg, 3.84 mmol, 2 eq) was added, and the reaction was carried out at room temperature for 2 h. After the reaction was complete as monitored by TLC, the reaction mixture was evaporated to dryness and purified by wet column chromatography with anhydrous dichloromethane (petroleum ether:ethyl acetate = 2:1, v / v) to give compound 33 (948.2 mg, yield: 96.3%). MS: m / z 429.4 [M-(C3H7)2N+(OH)+(H)] + [M-100+17+1] + . 1 H NMR (400MHz, CDCl3) δ4.48-4.41(m,1H),3.86–3.75(m,2H),3.64-3.54(m,2H),3.00-2.92(m,1H),2.67-2.57(m,4H),2.53-2.35 (m,3H),2.21-2.09(m,1H),1.90-1.82(m,1H),1.51-1.46(m,2H),1.31-1.20(m,26H),1.19-1.16(m,12H),0.87(t,J=6.9Hz,3H).

[0466] Example 34: Preparation of compound 34

[0467] Starting from intermediate 33-A1, compound 34 was synthesized under the same conditions as compound 33, except that hexadecane bromide was replaced with compound P4. MS: m / z 447.4 [M-(C3H7)2N+(OH)+(H)] + [M-100+17+1] + . 1 H NMR (400MHz, CDCl3) δ4.48 (t, J=6.2Hz, 1H), 4.46-4.41 (m, 1H), 4.36 (t, J= 6.2Hz,1H),3.87–3.75(m,2H),3.63–3.50(m,2H),3.04–2.91(m,1H),2.68– 2.56(m,4H),2.50–2.34(m,3H),2.20–2.09(m,1H),1.90–1.81(m,1H),1.7 6–1.61(m,2H),1.50–1.45(m,2H),1.32–1.21(m,24H),1.19–1.15(m,12H).

[0468] Preparation Example 2

[0469] 1. Preparation of siRNA

[0470] 1.1 Synthesis of the Justice Chain (SS Chain)

[0471] The oligonucleotide was synthesized using a solid-phase phosphoramide method, employing a universal CPG solid support as the starting cycle. Nucleoside monomers were sequentially linked from the 3'-5' direction according to the nucleotide arrangement of the positive strand. Each linkage of a nucleoside monomer involved four steps: deprotection, coupling, oxidation or thiolation, and capping. The synthesized oligonucleotides were produced in a scale of 1 μmol. The synthetic conditions are as follows:

[0472] Nucleoside monomers were provided in 1.0 g / 10 mL acetonitrile solution. The reaction conditions were identical for each step: 25°C. Deprotection was performed three times using a 3% trichloroacetic acid-dichloromethane solution. The coupling reaction was activated with a 0.25 mol / L 5-ethylthiotetrazole acetonitrile solution for 8 min. Oxidation was performed with a 0.05 mol / L tetrahydrofuran / pyridine / water (70 / 20 / 10, v / v / v) solution for 0.5 min. Thiolation was performed with a 0.05 mol / L N,N-dimethyl-N'-(3-thio-3H-1,2,4-dithiazo-5-yl)formamidin acetonitrile / pyridine (2 / 3, v / v) solution for 8 min. Capping was performed twice per cycle using a 20% acetic anhydride-acetonitrile solution and a 15% N-methylimidazolium / acetonitrile solution.

[0473] 1.2 Synthesis of the antisense chain (AS chain)

[0474] The solid-phase phosphoramide synthesis method utilizes a universal CPG solid-phase support as the starting cycle, linking nucleoside monomers sequentially from the 3'-5' direction according to the nucleotide arrangement sequence of the antisense strand. Each linking of a nucleoside monomer involves four steps: deprotection, coupling, oxidation or thiolation, and capping. The synthesis conditions for 1 μmol of oligonucleotides in the antisense strand are the same as those for the sense strand.

[0475] 1.3 Purification and Annealing of Oligonucleotides

[0476] 1.3.1 Ammonolysis

[0477] The synthesized solid-phase support (sense or antisense chain) was added to a 1.5 mL centrifuge tube, along with 1.0 mL of 50% methylamine / ammonia solution (v / v). The mixture was reacted in a metal bath shaker at 25°C for 10 hours. The supernatant was collected, and the solid-phase support was washed twice with enzyme-free water.

[0478] Each batch of 200 μL was combined with the supernatant, centrifuged and concentrated, and the crude product was then purified.

[0479] 1.3.2 Purification

[0480] Purification was performed using a reverse chromatography system with a Shim-pack GIS C18 column and a gradient elution system of n-hexylamine acetate ions and acetonitrile. The prepared liquid was collected and lyophilized to obtain the single-chain product.

[0481] 1.3.3 Annealing

[0482] The sense chain (SS chain) and the antisense chain (AS chain) were mixed in a molar ratio (SS chain / AS chain = 1 / 1) and reacted in a metal bath shaker at 37°C for 4 hours. After naturally cooling to room temperature, the system was freeze-dried to obtain the product.

[0483] Experimental Example 1: In vivo activity test

[0484] 1. Structures of the siRNAs, their modifications, and conjugates used in the experimental examples.

[0485] In this experimental case, siRNA targeting the rat SOD1 sequence was selected. The sequences of the siRNA or siRNA modifiers used in this experimental case are shown in Tables 1 and 2 below.

[0486] Table 1. Sequences of the sense and antisense strands of siRNA targeting the SOD1 sequence.

[0487] Table 2. Sequences of siRNA conjugates used in the test examples

[0488] The meanings of each abbreviation are as follows:

[0489] A, U, G, and C represent natural adenine ribonucleotide, uracil ribonucleotide, guanine ribonucleotide, and cytosine ribonucleotide, respectively.

[0490] m indicates that the nucleotide to its left is a nucleotide modified by 2'-OCH3. For example, Am, Um, Gm and Cm represent A, U, G and C modified by 2'-OCH3.

[0491] f indicates that the nucleotide adjacent to it on the left is a nucleotide modified by 2'-F. For example, Af, Uf, Gf and Cf represent A, U, G and C modified by 2'-F, respectively.

[0492] “s” indicates that the two adjacent nucleotides and / or delivery vectors are linked by a phosphate dithioester bond;

[0493] VP indicates that the nucleotide adjacent to its right is a vinyl phosphate modified nucleotide;

[0494] Wherein, "(uppercase letter + number)" represents the structure shown in Table 3 below.

[0495] Table 3

[0496] ALN-SOD1 and control molecule 1 were prepared according to the contents disclosed in WO2020 / 257194A1. Based on Preparation Example 2 above, the siRNA conjugates shown in Table 2 were prepared using the following lipophilic monomer precursors. The lipophilic monomer precursors are shown below:

[0497] 2. In vivo efficacy study (brain delivery) experimental protocol

[0498] Experimental steps

[0499] 1. Model: SD rats, 8-10 weeks old, male, purchased from Jicui Yaokang.

[0500] 2. Grouping: After 2-3 days of acclimatization, rats were randomly grouped according to their body weight. The day of grouping was defined as D0.

[0501] 3. Administration: Administration begins on day 0; administration for each group is shown in the table above; route of administration: intrathecal injection; solvent: pH 7.4 phosphate buffer; frequency of administration: single dose.

[0502] 4. Observation: General observation: every 2 days during the trial period; Body weight: twice a week during the trial period;

[0503] 5. Experimental endpoint: 14 days after drug administration, the remaining rats in each group were euthanized, and the following tissues were isolated: cortex, cerebellum, and spinal cord (thoracic vertebral segment). All of the above tissues were divided into two parts, flash-frozen in liquid nitrogen, and then stored at -80℃ for subsequent SOD1 mRNA extraction and qPCR extraction.

[0504] 6. Results Analysis:

[0505] a) Use Quant Studio 7 software with default settings to automatically calculate the Ct value;

[0506] b) Calculate the relative expression level of the gene using the following formula:

[0507] ΔCt=Ct(SOD1 gene)–Ct(GAPDH)

[0508] ΔCt = ΔCt(test sample group) – ΔCt(Mock group), where the Mock group represents the group without siRNA conjugate compared to the test sample group;

[0509] Inhibition rate (%) = (Relative expression level of mRNA in the Mock group – Relative expression level of mRNA in the test sample group) / Relative expression level of mRNA in the Mock group × 100%

[0510] 7. Experimental Results

[0511] The SOD1 mRNA inhibition rate indicated that D-ER-FY003109, D-ER-FY003112, D-ER-FY003116, D-ER-FY003119, D-ER-FY003109S and D-ER-FY003119S exhibited excellent inhibitory ability against SOD1 in the thoracic spinal cord, cerebellum, and cortical tissues.

[0512] 3. In vivo efficacy studies (brain delivery)

[0513] Experimental protocol

[0514] Experimental steps

[0515] 1. Model: SD rats, 8-10 weeks old, male, purchased from Jicui Yaokang.

[0516] 2. Grouping: After 2-3 days of acclimatization, rats were randomly grouped according to their body weight. The day of grouping was defined as D0.

[0517] 3. Administration: Administration begins on day 0; administration for each group is shown in the table above; route of administration: intrathecal injection; solvent: pH 7.4 phosphate buffer; frequency of administration: single dose.

[0518] 4. Observation: General observation: every 2 days during the trial period; Body weight: twice a week during the trial period;

[0519] 5. Experimental endpoint: 14 days after drug administration, the remaining rats in each group were euthanized, and the following tissues were isolated: cortex, cerebellum, and spinal cord (thoracic vertebral segment). All of the above tissues were divided into two parts, flash-frozen in liquid nitrogen, and then stored at -80℃ for subsequent SOD1 mRNA extraction and qPCR extraction.

[0520] 6. Results Analysis:

[0521] a) Use Quant Studio 7 software with default settings to automatically calculate the Ct value;

[0522] b) Calculate the relative expression level of the gene using the following formula:

[0523] ΔCt=Ct(SOD1 gene)–Ct(GAPDH)

[0524] ΔCt = ΔCt(test sample group) – ΔCt(Mock group), where the Mock group represents the group without siRNA conjugate compared to the test sample group;

[0525] Inhibition rate (%) = (Relative expression level of mRNA in the Mock group – Relative expression level of mRNA in the test sample group) / Relative expression level of mRNA in the Mock group × 100%

[0526] 7. Experimental Results

[0527] The SOD1 mRNA inhibition rate indicates that D-ER-FY003123, D-ER-FY003124, and D-ER-FY003125 have excellent inhibitory ability against SOD1 in the thoracic spinal cord, cerebellum, and cortical tissues.

[0528] Experimental Example 2: In vivo activity test (brain delivery)

[0529] 1. Structures of the siRNAs, their modifications, and conjugates used in the experimental examples.

[0530] In this experimental case, siRNA targeting the rat CTNNB1 sequence was selected. The sequences of the siRNA or siRNA modifiers used in this experimental case are shown in Tables 4 and 5 below.

[0531] Table 4. Sequences of the sense and antisense strands of siRNA targeting the CTNNB1 sequence.

[0532] Table 5. Sequences of siRNA conjugates used in the test examples

[0533] The meanings of the abbreviations are the same as above;

[0534] For details on the representations of F05 and F13, please refer to Table 3.

[0535] 2. In vivo efficacy studies (brain delivery)

[0536] Experimental protocol

[0537] Experimental steps

[0538] 1. Model: SD rats, 8-10 weeks old, male, purchased from Jicui Yaokang.

[0539] 2. Grouping: After 2-3 days of acclimatization, rats were randomly grouped according to their body weight. The day of grouping was defined as D0.

[0540] 3. Administration: Administration begins on day 0; administration for each group is shown in the table above; route of administration: intrathecal injection; solvent: pH 7.4 phosphate buffer; frequency of administration: single dose.

[0541] 4. Observation: General observation: every 2 days during the trial period; Body weight: twice a week during the trial period;

[0542] 5. Experimental endpoint: 14 days after drug administration, the remaining rats in each group were euthanized, and the following tissues were isolated: cortex, cerebellum, and spinal cord (thoracic and lumbar vertebrae). All of the above tissues were divided into two longitudinal portions, flash-frozen in liquid nitrogen, and then stored at -80℃ for subsequent CTNNB1 mRNA extraction and qPCR extraction.

[0543] 6. Results Analysis:

[0544] a) Use Quant Studio 7 software with default settings to automatically calculate the Ct value;

[0545] b) Calculate the relative expression level of the gene using the following formula:

[0546] ΔCt=Ct(CTNNB1 gene)–Ct(GAPDH)

[0547] ΔCt = ΔCt(test sample group) – ΔCt(Mock group), where the Mock group represents the group without siRNA conjugate compared to the test sample group;

[0548] Inhibition rate (%) = (Relative expression level of mRNA in the Mock group – Relative expression level of mRNA in the test sample group) / Relative expression level of mRNA in the Mock group × 100%

[0549] 7. Experimental Results

[0550] The CTNNB1 mRNA inhibition results indicate that the compound of this invention, compared with Yangshen AD-77884, exhibits the desired inhibitory ability against CTNNB1 in the thoracic spinal cord, lumbar spinal cord, cerebellum, and cortical tissues. The structure of Yangshen AD-77884 is shown in Figure 1.

[0551] Experimental Example 3: In vivo activity test (ocular delivery)

[0552] 1. Structures of the siRNAs, their modifications, and conjugates used in the experimental examples.

[0553] In this experimental case, siRNA targeting the mouse mTTR sequence was selected. The sequences of the siRNA or siRNA modifiers used in this experimental case are shown in Tables 6 and 7 below.

[0554] Table 6. Sequences of the sense and antisense strands of siRNA targeting the mTTR sequence

[0555] Table 7. Sequences of siRNA conjugates used in the test examples

[0556] The meanings of the abbreviations are the same as above;

[0557] For details on the representations of F05, F13, F20, F21 and F22, please refer to Table 3.

[0558] 2. In vivo efficacy study (ocular delivery)

[0559] Experimental protocol

[0560] Experimental steps

[0561] 1. Model: C57BL / 6 mice, 8-10 weeks old, male, purchased from Jicui Pharmaceutical.

[0562] 2. Grouping: After acclimatization for 2-3 days, mice were randomly grouped according to their body weight. The day of grouping was defined as D0.

[0563] 3. Administration: Administration begins on day 0; administration for each group is shown in the table above; route of administration: intravitreal injection; solvent: pH 7.4 phosphate buffer; administration frequency: single dose.

[0564] 4. Observation: General observation: every 2 days during the trial period; Body weight: twice a week during the trial period;

[0565] 5. Experimental endpoint: 14 days after drug administration, the remaining mice in each group were euthanized, and the whole eyeballs were removed (whole eye examination). After being flash-frozen in liquid nitrogen, they were transferred to -80℃ for subsequent mTTR mRNA detection.

[0566] 6. Results Analysis:

[0567] a) Use Quant Studio 7 software with default settings to automatically calculate the Ct value;

[0568] b) Calculate the relative expression level of the gene using the following formula:

[0569] ΔCt=Ct(mTTR gene)–Ct(GAPDH)

[0570] ΔCt = ΔCt(test sample group) – ΔCt(Mock group), where the Mock group represents the group without siRNA conjugate compared to the test sample group;

[0571] Inhibition rate (%) = (Relative expression level of mRNA in the Mock group – Relative expression level of mRNA in the test sample group) / Relative expression level of mRNA in the Mock group × 100%

[0572] 7. Experimental Results

[0573] The mTTR mRNA inhibition rate results show that the compounds D-ER-FY003150S, D-ER-FY003152, D-ER-FY003154, and D-ER-FY003153 of the present invention have good inhibitory ability on ocular mTTR mRNA.

[0574] Experimental Example 4: In vivo activity test (ocular delivery)

[0575] 1. Structures of the siRNAs, their modifications, and conjugates used in the experimental examples.

[0576] In this experimental case, siRNA targeting the mouse CTNNB1 sequence was selected. The sequences of the siRNA or siRNA modifiers used in this experimental case are shown in Tables 8 and 9 below.

[0577] Table 8. Sequences of the sense and antisense strands of siRNA targeting the CTNNB1 sequence.

[0578] Table 9. Sequences of siRNA conjugates used in the test examples

[0579] The meanings of the abbreviations are the same as above;

[0580] For details on the representations of F20, F13, and F21, please refer to Table 3.

[0581] 2. In vivo efficacy study (ocular delivery)

[0582] Experimental protocol

[0583] Experimental steps

[0584] 1. Model: C57BL / 6 mice, 8-10 weeks old, male, purchased from Jicui Pharmaceutical.

[0585] 2. Grouping: After acclimatization for 2-3 days, mice were randomly grouped according to their body weight. The day of grouping was defined as D0.

[0586] 3. Administration: Administration begins on day 0; administration for each group is shown in the table above; route of administration: intravitreal injection; solvent: pH 7.4 phosphate buffer; administration frequency: single dose.

[0587] 4. Observation: General observation: every 2 days during the trial period; Body weight: twice a week during the trial period;

[0588] 5. Experimental endpoint: 14 days after drug administration, the remaining mice in each group were euthanized, and the whole eyeballs were removed (whole eye examination). After being flash-frozen in liquid nitrogen, they were transferred to -80℃ for subsequent CTNNB1 mRNA detection.

[0589] 6. Results Analysis:

[0590] a) Use Quant Studio 7 software with default settings to automatically calculate the Ct value;

[0591] b) Calculate the relative expression level of the gene using the following formula:

[0592] ΔCt=Ct(CTNNB1 gene)–Ct(GAPDH)

[0593] ΔCt = ΔCt(test sample group) – ΔCt(Mock group), where the Mock group represents the group without siRNA conjugate compared to the test sample group;

[0594] Inhibition rate (%) = (Relative expression level of mRNA in the Mock group – Relative expression level of mRNA in the test sample group) / Relative expression level of mRNA in the Mock group × 100%

[0595] 7. Experimental Results

[0596] The CTNNB1 mRNA inhibition rate results indicate that the compounds of the present invention have the desired inhibitory effect on CTNNB1 mRNA in the eye.

Claims

1. A compound, said compound being an oligonucleotide, said compound comprising at least one lipophilic monomer selected from the following formula: in, Ring A is selected from 5-6 membered heterocycles; D is selected from substituted or unsubstituted chain saturated aliphatic hydrocarbon groups, preferably substituted or unsubstituted straight-chain or branched C. 1-30 Chain-like saturated aliphatic hydrocarbon group, wherein the chain-like saturated aliphatic hydrocarbon group may optionally be converted by hydroxyl, halogen, methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 Hydrocarbon substitution, wherein the methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 The hydrocarbon group may optionally be replaced by a hydroxyl group or a halogen; R a Selected from hydrogen, hydroxyl, -C 0-6 Alkylene-OL d ; L a Selected from single bonds, reactive phosphorus groups, solid supports, -P(=O)(OH)-O-, or -P(=S)(OH)-O-; L b Selected from single bond, C 1-6 Alkyl, substituted C 1-6 Alkyl groups and -C 1-6 An alkyl-benzene ring, wherein the benzene ring forms a fused ring with ring A; L c Selected from single bond, C 1-6 Alkylene, Alkoxy, -O-, -OC 1-8 Alkylene -OC(=O)-, -OC 1-8 Alkylene-OC(=S)-, -OC 1-8 Alkylene -NH-C(=O)-, -OC 1-8 Alkylene-NH-C(=S)-, -OC 1-8 Alkylene -SC(=O)-, -OC 1-8 Alkylene-SC(=S)-, -OC 1-10 Alkoxy-NH-C(=O)-, -C 1-10 Alkoxy-NH-C(=O)-, -C 1-10 Alkylene -NH-C(=O)-, -OC 1-8 Alkyl-5-6-membered heteroaryl groups, optionally substituted with halogens; L d Selected from single bonds, H, reactive phosphorus groups, phosphoramidite groups, solid supports, -P(=O)(OH)-O-, -P(=S)(OH)-O-, and hydroxyl protecting groups; R b Selected from hydrogen, modified or unmodified base B; The lipophilic monomers contained in the compound are not selected from the following: B' is a natural nucleobase.

2. The compound of claim 1, wherein, Ring A is selected from 5-6 membered heterocycles containing 1, 2, or 3 heteroatoms selected from N and O. Preferably, ring A is selected from tetrahydropyrrole, tetrahydrofuran, tetrahydropyran, morpholino, or triazolyl; More preferably, ring A is selected from Optionally, when ring A is selected from triazole, L b Selected from -C 1-6 Alkyl-benzene ring, wherein the benzene ring forms a fused ring with the triazole ring; Alternatively, D is selected from substituted or unsubstituted chain saturated aliphatic hydrocarbon groups, preferably substituted or unsubstituted straight-chain or branched C. 14-24 Chain-like saturated aliphatic hydrocarbon groups, wherein the chain-like saturated aliphatic hydrocarbon groups are optionally replaced by hydroxyl, halogen, methylene group, or C. 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 Hydrocarbon substitution, wherein the methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 The hydrocarbon group may optionally be replaced by a hydroxyl group or a halogen; Alternatively, D can be selected from C, which may be substituted with halogens. 12 C 13 C 14 C 15 C 16 C 17 C 18 C 19 C 20 C 21 C 22 C 23 C 24 The chain-like saturated aliphatic hydrocarbon group, wherein the chain-like saturated aliphatic hydrocarbon group may optionally be converted by hydroxyl, halogen, methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 Hydrocarbon substitution, wherein the methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 The hydrocarbon group may optionally be replaced by a hydroxyl group or a halogen.

3. The compound according to any one of claims 1 or 2, wherein, The compound contains a lipophilic monomer having the following structure: Wherein, R1 is, as defined in claim 1 regarding D, preferably, R1 is a substituted or unsubstituted straight-chain or branched C. 1-30 Chain-like saturated aliphatic hydrocarbon groups, wherein the chain-like saturated aliphatic hydrocarbon groups are optionally replaced by hydroxyl, halogen, methylene group, or C. 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 Hydrocarbon substitution, wherein the methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 The hydrocarbon group may be optionally replaced by a hydroxyl group or a halogen; R a1 Selected from hydrogen, hydroxyl, -C 0-6 Alkylene-OL d1 ; L d1 Selected from single bonds, H, reactive phosphorus groups, phosphoramidite groups, solid supports, -P(=O)(OH)-O-, -P(=S)(OH)-O-, and hydroxyl protecting groups; L2 is independently selected from H, reactive phosphorus groups, hydroxyl protecting groups, -P(=O)(OH)-O-, -P(=S)(OH)-O-, or solid supports; Preferably, the compound comprises a lipophilic monomer having the following structure: Wherein, R1 is the substituted or unsubstituted straight or branched chain C. 1-30 Chain-like saturated aliphatic hydrocarbon group, wherein the chain-like saturated aliphatic hydrocarbon group may optionally be converted by hydroxyl, halogen, methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 Hydrocarbon substitution, wherein the methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 The hydrocarbon group may optionally be replaced by a hydroxyl group or a halogen; R a1 Selected from hydrogen, hydroxyl, -C 0-6 Alkylene-OL d1 ; L d1 Selected from single bonds, H, reactive phosphorus groups, phosphoramidite groups, solid supports, -P(=O)(OH)-O-, -P(=S)(OH)-O-, and hydroxyl protecting groups; L2 is independently selected from single bonds, reactive phosphorus groups, -P(=O)(OH)-O-, -P(=S)(OH)-O-, or solid supports; More preferably, the compound comprises a lipophilic monomer having the following structure: Wherein, R1 is the substituted or unsubstituted straight or branched chain C. 1-30 Chain-like saturated aliphatic hydrocarbon group, wherein the chain-like saturated aliphatic hydrocarbon group may optionally be converted by hydroxyl, halogen, methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 Hydrocarbon substitution, wherein the methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 The hydrocarbon group may optionally be replaced by a hydroxyl group or a halogen; R a1 Selected from hydrogen, hydroxyl, -C 0-6 Alkylene-OL d1 ; L d1 Selected from single bonds, H, -P(=O)(OH)-O- or -P(=S)(OH)-O-; L2 is independently selected from single bonds, -P(=O)(OH)-O- or -P(=S)(OH)-O-; More preferably, in the case of lipophilic monomers represented by Formula Ia, Formula I-a1, or Formula I-a2, the compound comprises one or more lipophilic monomers having the following structure: Wherein, R1 is a straight-chain or branched C that can be optionally substituted with a halogen. 1-30 The chain-like saturated aliphatic hydrocarbon groups, L2, are each independently selected from single bonds, -P(=O)(OH)-O-, or -P(=S)(OH)-O-, wherein, This indicates a bond that links to the rest of the compound.

4. The compound according to claim 1 or 2, wherein, The compound contains a lipophilic monomer having the following structure: Among them, -R5 is selected from straight chain or branched chain C. 1-30 Chain-like saturated aliphatic hydrocarbon group, wherein the chain-like saturated aliphatic hydrocarbon group may optionally be converted by hydroxyl, halogen, methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 Hydrocarbon substitution, wherein the methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 The hydrocarbon group may optionally be replaced by a hydroxyl group or a halogen, wherein the straight-chain or branched C 1-30 The chain-like saturated aliphatic hydrocarbon group is substituted with at least one halogen; or -R5 is selected from -(CH2-(OCH2CH2)q)-NH-(C(=O)-(C8-C 30 Alkylene), the C8-C 30 The alkylene group is substituted with at least one halogen, and q is selected from 2, 3, 4, 5, and 6; or R5 is selected from -(C 1-8 alkylene)-5-6-membered heteroaryl-(C 12-30 Alkylene), the C 12-30 The alkylene group may optionally be replaced by 1-5 halogens; R6 is selected from single bonds, reactive phosphorus groups, -P(=O)(OH)-O-, and -P(=S)(OH)-O-. R7 is selected from H, single bond, or hydroxyl protecting group; B represents both modified and unmodified nucleobases; Alternatively, the compound may contain a lipophilic monomer having the following structure: The definition of -OR5 can be found above regarding -L. c -D is defined as follows: for example, R5 is selected from straight chain or branched chain C. 1-30 Chain-like saturated aliphatic hydrocarbon group, wherein the chain-like saturated aliphatic hydrocarbon group may optionally be converted by hydroxyl, halogen, methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 Hydrocarbon substitution, wherein the methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 The hydrocarbon group may optionally be replaced by a hydroxyl group or a halogen, wherein the straight-chain or branched C 1-30 The chain-like saturated aliphatic hydrocarbon group is substituted with at least one halogen; or R5 is selected from -(CH2-(OCH2CH2)q)-NH-(C(=O)-(C8-C 30 Alkylene), the C8-C 30 The alkylene group is substituted with at least one halogen, and q is selected from 2, 3, 4, 5, and 6; or R5 is selected from -(C 1-8 alkylene)-5-6-membered heteroaryl-(C 12-30 Alkylene), the C 12-30 The alkylene group may optionally be replaced by 1-5 halogens; R6 is selected from H, single bond, reactive phosphorus group, -P(=O)(OH)-O-, -P(=S)(OH)-O-; R7 is selected from H, single bond, or hydroxyl protecting group; B represents both modified and unmodified nucleobases. Optionally, R6 and R7 may not both be H; Preferably, in the case of lipophilic monomers represented by Formula I-b1 or Formula Ib, the compound comprises one or more lipophilic monomers having the following structure: Where X is selected from O or S, and R5 is selected from one or more of them. R7 is selected from hydrogen or a single bond, B is a modified or unmodified nucleobase, where the wavy line represents a single bond linked to the rest of the compound.

5. The compound according to claim 1 or 2, wherein, The compound contains a lipophilic monomer having the following structure: Where R8 is -(C 1-8 Alkylene-OC(=O)-C 12-30 alkylene), -(C 1-8 Alkylene-OC(=S)-C 12-30 alkylene) or -(C 1-8 Alkylene-NC(=O)-C 12-30 Alkylenes), wherein the alkylene group may optionally be substituted with 1-5 halogens; L1 and L2 are each independently selected from H, single bonds, reactive phosphorus groups, -P(=O)(OH)-O-, -P(=S)(OH)-O-, hydroxyl protecting groups, or solid supports; Preferably, in the case of lipophilic monomers represented by Formula Ic, the compound comprises one or more lipophilic monomers having the following structure: Where X is selected from O or S, and R8 is -(C 1-8 Alkylene -OC(=O)-C 12-30 alkylene), -(C 1-8 Alkylene-OC(=S)-C 12-30 alkylene) or -(C 1-8 Alkylene-NC(=O)-C 12-30 Alkylenes, wherein the alkylene group may optionally be substituted with 1 to 5 halogens, wherein the wavy line represents a single bond linked to the rest of the compound.

6. The compound according to claim 1 or 2, wherein, The compound contains a lipophilic monomer having the following structure: R9 is selected from substituted or unsubstituted straight or branched C. 1-30 Chain-like saturated aliphatic hydrocarbon group, wherein the chain-like saturated aliphatic hydrocarbon group may optionally be converted by hydroxyl, halogen, methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 Hydrocarbon substitution, wherein the methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 The hydrocarbon group may optionally be replaced by a hydroxyl group or a halogen; L1 is independently selected from H, single bond, reactive phosphorus group, -P(=O)(OH)-O-, -P(=S)(OH)-O-, hydroxyl protecting group or solid support; p is selected from 1, 2, 3, 4, and 5; Alternatively, in the case of the lipophilic monomer shown in Formula Id, the compound comprises a lipophilic monomer having the following structure: Wherein, X is selected from O or S, and R9 is selected from one or more of the following: The wavy lines represent single bonds that are linked to the rest of the compound.

7. The compound according to claim 1 or 2, wherein, The compound contains a lipophilic monomer having the following structure: Among them, R 10 Selected from straight-chain or branched C atoms substituted with at least one halogen 1-30 Chain-like saturated aliphatic hydrocarbon group, wherein the chain-like saturated aliphatic hydrocarbon group may optionally be converted by hydroxyl, halogen, methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 Hydrocarbon substitution, wherein the methylene group, C 1-4 Alkyl groups, or C groups having one or two unsaturated bonds 2-4 The hydrocarbon group may optionally be replaced by a hydroxyl group or a halogen; or R 10 Selected from -(C1-C4 alkylene)-NH-C(=O)-(C 8-30 Alkyl), the C1-C4 alkylene or C 8-30 Alkyl groups may optionally be replaced by 1-5 halogens; L1 and L2 are each independently selected from H, single bonds, reactive phosphorus groups, -P(=O)(OH)-O-, -P(=S)(OH)-O-, hydroxyl protecting groups, or solid supports; Preferably, the compound comprises one or more lipophilic monomers having the following structures: Where X is selected from O or S, R 10 for The wavy lines represent single bonds that are linked to the rest of the compound.

8. The compound according to any one of claims 1-7, wherein, The lipophilic monomer is selected from the group consisting of: The wavy lines represent single bonds that are linked to the rest of the compound.

9. The compound according to any one of claims 1-8, wherein, The compound is a single-stranded or double-stranded oligonucleotide.

10. The compound according to any one of claims 1-9, wherein, The compound is a double-stranded oligonucleotide, preferably siRNA; preferably, the siRNA comprises a sense strand and an antisense strand, wherein the antisense strand is complementary to the target gene; and the sense strand is complementary to the antisense strand.

11. A pharmaceutical composition comprising the compound of any one of claims 1-10.

12. The use of the compound of any one of claims 1-10 or the pharmaceutical composition of claim 11 in the preparation of a medicament, optionally, said medicament for the prevention and / or treatment of diseases related to the eye and central nervous system (CNS).

13. A compound, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, said compound being selected from the following:

14. Use of the compound of claim 13, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, in the preparation of the compound of any one of claims 1-10.

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