Nucleic acid conjugate, preparation method therefor, and use thereof

Abstract

The present invention relates to a compound for forming a conjugate with an oligonucleotide, the compound having a structure as represented by formula (I). Also provided in the present invention are a corresponding conjugate, and the use of the conjugate in preparing a drug for treating and / or preventing hepatogenic diseases. The delivery molecule disclosed in the present invention uses a saturated cyclic group and a novel linker to serve as a core skeleton to link to a phosphodiester bond, and compared with the prior art, exhibits excellent animal in-vivo bioactivity and long-acting drug efficacy, and also achieves the advantages of cheap raw materials, simple synthesis, feasible process development, good oligonucleotide synthesis efficiency, etc. In addition, the present invention improves the linker between galactosamine and the phosphodiester bond, thereby improving the metabolic stability of the whole delivery molecule and further improving the in-vivo delivery efficiency.

Description

Nucleic acid conjugate, preparation method and application thereof Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a nucleic acid conjugate, a preparation method and application thereof. Background Art

[0002] siRNA is a negatively charged macromolecule composed of two oligonucleotide chains. It cannot effectively target target tissues in the body or enter cells on its own. siRNA requires specialized delivery vehicles to enrich siRNA in target organs and allow it to enter cells to exert its therapeutic effects.

[0003] Lipid nanoparticles (LNPs) can encapsulate siRNA and effectively deliver it to the liver. The drug Onpattro, which utilizes LNP technology, is already on the market. However, the clinical application of LNP technology still has many drawbacks, such as the short shelf life of LNP preparations and the potential for immune responses during use, which often requires the coadministration of immunosuppressants such as dexamethasone, complicating the clinical application of LNPs.

[0004] Targeted drug delivery to the interior of cells through cell surface receptor-mediated endocytosis is an effective strategy. The asialoglycoprotein receptor (ASGPR) is a receptor that is highly specifically expressed on the surface of hepatic parenchymal cells and has the characteristics of high abundance and high receptor recycling efficiency. By covalently coupling ligands that can specifically recognize ASGPR, such as monosaccharide and polysaccharide molecules such as galactose, galactosamine, and N-acetylgalactosamine (GalNAc), with siRNA, siRNA can be targeted and delivered to hepatic parenchymal cells, allowing siRNA to exert a gene silencing effect on the target gene. Therefore, the development of targeting ligands suitable for siRNA delivery is of great significance for the clinical application of siRNA. Summary of the Invention

[0005] The purpose of the present invention is to provide a nucleic acid conjugate with a completely new chemical structure and its application.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] The first aspect of the present invention provides a compound characterized by the following clauses:

[0008] 1. The compound comprises a structure as shown in formula (I):

[0009] in,

[0010] Linker is -L1-L2-L3-L4-;

[0011] Wherein, L1 and L3 are independently selected from one or more connected combinations of the groups represented by the following formulae (A1) to (A14):

[0012] L2 is selected from the group represented by (A7), (A10), (A11), (A12) or (A13) above;

[0013] L4 is absent, or is a combination of one or more selected from the groups (A1), (A2), (A7), (A8), (A10), (A11), or (A12);

[0014] wherein R' is hydrogen, a C1-C10 alkyl group, or a C3-C8 cycloalkyl group; j1 is an integer from 1 to 20; j2 is an integer from 1 to 20;

[0015] m represents an integer from 0 to 6;

[0016] Q stands for wherein R2 and R3 are independently selected from H, C1-C20 alkyl, C1-C20 alkoxy, C2-C20 alkenyl or C2-C20 alkynyl;

[0017] X stands for wherein R4 and R5 are independently selected from H, fluorine, hydroxyl, C1-C20 alkyl, C1-C20 alkoxy, C2-C20 alkenyl, C2-C20 alkynyl, or R4 and R5 are directly linked to form a ring, and p is an integer of 1-6;

[0018] Z represents N or CR9, wherein R9 is selected from H, C1-C20 alkyl or C3-C10 cycloalkyl;

[0019] represents a C3-C18 cycloalkyl group or a C3-C18 heterocyclic group;

[0020] R1 is selected from H, fluorine, hydroxy, cyano, C1-C20 alkyl, C1-C20 alkoxy, C2-C20 alkenyl or C2-C20 alkynyl;

[0021] n is an integer from 0 to 10;

[0022] It represents the site of covalent attachment of a group;

[0023] R6, R7, and R8 are independently H or K(C=O)-, wherein K is independently selected from one of methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl, ethyl, n-propyl, isopropyl, phenyl, halogenated phenyl, and alkylphenyl.

[0024] In the present application, the covalent bond connection sites in the groups represented by formulas (A1) to (A14) can be interchanged. For example, when L1 is a group represented by formula (A6), O in formula (A6) can be connected to N-acetylgalactosamine, and C can be connected to L2. Alternatively, O in formula (A6) can be connected to L2, and C can be connected to N-acetylgalactosamine.

[0025] 2. According to the compound described in clause 1, L1 and L3 are independently selected from one or more combinations of the groups represented by (A1), (A2), (A3), (A5), (A7), (A8), (A9), (A11), and (A14); in this clause, L2, L4, R', j1, j2, m, Q, X, R1, n, R6, R7, R8, etc. are the same as those defined in Clause 1.

[0026] 3. According to the compound described in Article 2, L1 and L3 are independently selected from one or more groups represented by (A1), (A2), (A3), (A5), (A7), and (A14); in this article, other groups except L1 and L3 are the same as those defined in Article 1.

[0027] 4. The compound according to clause 3, wherein L1 and L3 are independently (A5) or (A14); in this clause, other groups except L1 and L3 are the same as defined in clause 1.

[0028] 5. The compound according to clause 4, wherein L1 and L3 are independently (A5) or (A14), j1 is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and j2 is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; in this clause, other groups except L1 and L3 are the same as those defined in clause 1.

[0029] 6. The compound according to clause 5, wherein L1 and L3 are independently (A5) or (A14), j1 is 2, 3, 4, 5 or 6, and j2 is 1, 2, 3, 4 or 5; in this clause, other groups except L1 and L3 are the same as those defined in clause 1.

[0030] 7. The compound according to clause 6, wherein L1 and L3 are independently (A5) or (A14), j1 is 3, 4, 5 or 6, and j2 is 2, 3 or 4; in this clause, other groups except L1 and L3 are the same as defined in clause 1.

[0031] 8. The compound according to clause 1, wherein R' is hydrogen, C1-C5 alkyl or C3-C6 cycloalkyl; in this clause, other groups except R' are the same as defined in any one of clauses 1 to 8.

[0032] 9. The compound according to clause 8, wherein R' is hydrogen; in this clause, other groups except R' are the same as defined in any one of clauses 1 to 8.

[0033] 10. The compound according to clause 1, wherein L4 is absent or selected from the group shown in (A7), (A10) or (A11) above; in this clause, the other groups except L4 are the same as defined in any one of clauses 1 to 9.

[0034] 11. The compound according to item 1, wherein m represents 0, 1, 2, 3, 4 or 5; preferably, m is 0, 1 or 2; in this item, other groups except m are the same as defined in any one of items 1 to 10.

[0035] 12. According to the compound described in clause 1, R2 and R3 are independently selected from H, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl or C2-C6 alkynyl; preferably, R2 and R3 are H; in this clause, other groups except R2 and R3 are the same as those defined in any one of clauses 1 to 11.

[0036] 13. In the compound according to clause 1, R4 and R5 are independently selected from H, fluorine, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, C2-C6 alkynyl, or R4 and R5 are directly linked to form a ring; in this clause, other groups except R4 and R5 are the same as those defined in any one of clauses 1 to 12.

[0037] 14. The compound according to item 1, wherein p is 1, 2 or 3; in this item, other groups except p are the same as defined in any one of items 1 to 13.

[0038] 15. The compound according to item 1, represents a C3-C18 cycloalkyl group or a C3-C18 heterocyclic group; preferably, represents a four- to eight-membered all-carbon or nitrogen-containing saturated ring; in this clause, except Other groups except for the above are the same as those defined in any one of Clauses 1 to 14.

[0039] 16. According to the compound described in Article 1, R1 is selected from H, fluorine, hydroxyl, cyano, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl or C2-C6 alkynyl; in this article, other groups except R1 are the same as those defined in any one of Articles 1 to 15.

[0040] 17. The compound according to item 1, wherein n is 0, 1, 2, 3, 4 or 5; preferably, n is 0, 1, 2 or 3; in this item, other groups except n are the same as defined in any one of items 1 to 16.

[0041] 18. The compound according to clause 1, wherein R6, R7, and R8 are acetyl groups; in this clause, other groups except R6, R7, and R8 are the same as defined in any one of clauses 1 to 17.

[0042] 19. According to the compound described in item 1, Z is N or CR9, wherein R9 is selected from H, C1-C6 alkyl or C3-C7 cycloalkyl, preferably, R9 is selected from H; in this item, other groups except Z are the same as those defined in any one of items 1 to 18.

[0043] 20. According to some embodiments of the compound of clause 1, L1 and L3 are independently (A5) or (A14), j1 is 2, 3, 4, 5 or 6, and j2 is 1, 2, 3, 4 or 5;

[0044] L4 is absent or selected from the groups shown in (A7), (A10) or (A11) above;

[0045] m is 0, 1, or 2;

[0046] represents a four- to eight-membered all-carbon or nitrogen-containing saturated ring;

[0047] n is 0, 1, 2 or 3.

[0048] 21. In some more specific embodiments of the compound according to item 1, L1 and L3 are independently (A5) or (A14), j1 is 3, 4, 5 or 6, and j2 is 2, 3 or 4;

[0049] R', R1, R2, R3, and R9 are hydrogen;

[0050] m and n are 0.

[0051] 22. The compound according to item 1, wherein two of the structures represented by formula (I) One of the sites shown is connected to a phosphoramidite functional group, and the other is connected to a phosphoramidite functional group or a hydroxyl protecting group. In this clause, the other groups in the structure shown in formula (I) are the same as those defined in any one of clauses 1 to 21.

[0052] 23. The compound according to item 22, wherein the phosphoramidite functional group has a structure represented by formula (G-1),

[0053] Wherein, B1 is selected from a substituted or unsubstituted C1-C5 hydrocarbon group, preferably, B1 is selected from a methyl group, an ethyl group or an isopropyl group; B2 is selected from one of a C1-C5 alkyl group, an acetonitrile group, a propionitrile group and a butyronitrile group, preferably, B2 is an acetonitrile group; in this clause, except for the phosphoramidite functional group, other groups are the same as those defined in any one of clauses 1 to 22.

[0054] 24. The compound according to clause 22, wherein, in general, the protecting group renders the chemical functional group insensitive to specific reaction conditions and can be added to and removed from the functional group in the molecule without substantially damaging the rest of the molecule. Representative hydroxy protecting groups are disclosed in Beaucage et al., Tetrahedron 1992, 48, 2223-2311, and Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2d ed, John Wiley & Sons, New York, 1991, which are incorporated herein by reference in their entirety. In some embodiments, the protecting group is stable under alkaline conditions but can be removed under acidic conditions. In some embodiments, non-exclusive examples of hydroxy protecting groups that can be used herein include dimethoxytrityl (DMT), monomethoxytrityl, 9-phenylxanthine-9-yl (Pixyl), and 9-(p-methoxyphenyl)xanthine-9-yl (Mox). In some embodiments, non-exclusive examples of hydroxyl protecting groups that can be used herein include Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4'-bismethoxytrityl), TMTr (4,4',4"-trimethoxytrityl) and tert-butyldimethylsilyl (TBS or TBDMS). Non-exclusive examples of hydroxyl protecting groups that can be used herein include any one of alkyl acyl, trityl, 4-methoxytrityl, 4,4'-bismethoxytrityl (DMTr) and 4,4',4'-trimethoxybenzyl, preferably 4,4'-bismethoxytrityl; in this clause, except for the phosphoramidite functional group, other groups are the same as defined in any one of clauses 1 to 21.

[0055] 25. In the compound according to any one of clauses 1 to 24 above, alkyl refers to a straight-chain or branched saturated hydrocarbon radical having a specified number of carbon atoms, typically 1 to 20 carbon atoms, for example 1 to 10 carbon atoms, such as 1 to 8 or 1 to 6 carbon atoms. For example, C1-C6 alkyl includes straight-chain and branched alkyl radicals having 1 to 6 carbon atoms. When an alkyl residue having a specific number of carbon atoms is mentioned, it is intended to encompass all branched and straight-chain forms having that number of carbon atoms; thus, for example, "butyl" is meant to include n-butyl, sec-butyl, isobutyl, and tert-butyl; and "propyl" includes n-propyl and isopropyl. Alkylene is a subset of alkyl and refers to a residue identical to alkyl but having two points of attachment.

[0056] 26. According to any one of clauses 1 to 24 above, the alkenyl group is an unsaturated branched or straight-chain hydrocarbon group having at least one carbon-carbon double bond, wherein the carbon-carbon double bond is obtained by losing one hydrogen atom from each of adjacent carbon atoms of the parent alkyl group. The group may be in the cis or trans configuration about the double bond. Typical alkenyl groups include, but are not limited to, ethenyl; propenyl, such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl), prop-2-en-2-yl; butenyl, such as but-1-en-1-yl, but-1-en-2-yl, 2-methylprop-1-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, but-1,3-dien-1-yl, but-1,3-dien-2-yl, and the like. In certain embodiments, alkenyl groups have 2 to 20 carbon atoms, and in other embodiments, 2 to 10, 2 to 8, or 2 to 6 carbon atoms.Alkenylene is a subset of alkenyl and refers to residues identical to alkenyl but with two points of attachment.

[0057] 27. According to any one of clauses 1 to 24 above, the alkynyl group is an unsaturated branched or straight-chain hydrocarbon group having at least one carbon-carbon triple bond, wherein the carbon-carbon triple bond is obtained by the loss of two hydrogen atoms from adjacent carbon atoms of a parent alkyl group. Typical alkynyl groups include, but are not limited to, ethynyl; propynyl, such as prop-1-yn-1-yl and prop-2-yn-1-yl; butynyl, such as but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, and the like. In certain embodiments, the alkynyl group has 2 to 20 carbon atoms, and in other embodiments, has 2 to 10, 2 to 8, or 2 to 6 carbon atoms.

[0058] 28. In the compound according to any one of clauses 1 to 24 above, the alkoxy group refers to an alkyl group having a specified number of carbon atoms linked via an oxygen bridge, for example, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, pentoxy, 2-pentoxy, isopentoxy, neopentoxy, hexoxy, 2-hexyloxy, 3-hexyloxy, 3-methylpentoxy, etc. The alkoxy group typically has 1 to 10, 1 to 8, 1 to 6, or 1 to 4 carbon atoms linked via the oxygen bridge.

[0059] 29. According to any one of the above clauses 1 to 24, the aryl group is a group derived from an aromatic monocyclic or polycyclic hydrocarbon ring system formed by removing a hydrogen atom from a ring carbon atom, wherein the aromatic monocyclic or polycyclic hydrocarbon ring system contains only hydrogen and 6 to 18 carbon atoms, wherein at least one ring in the ring system is fully unsaturated, i.e., it contains cyclic, delocalized (4n+2)π - Electron system. Aryl groups include, but are not limited to, phenyl, fluorenyl, and naphthyl. Arylene is a subset of aryl, referring to residues identical to aryl but with two points of attachment.

[0060] 30. The compound according to any one of clauses 1 to 24 above, wherein cycloalkyl refers to a non-aromatic carbocyclic ring, typically having 3 to 7 annular carbon atoms. The ring may be saturated or have one or more carbon-carbon double bonds. Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, and cyclohexenyl, as well as bridged and caged ring groups such as norbornane.

[0061] 31. In the compound according to any one of clauses 1 to 24 above, halo refers to fluoro, chloro, bromo and iodo, and the term "halogen" includes fluoro, chloro, bromo and iodine.

[0062] 32. According to any one of clauses 1 to 24 above, the compound wherein the heterocyclic group is a stable 3- to 18-membered non-aromatic ring radical containing 2-12 carbon atoms and 1-6 heteroatoms selected from nitrogen, oxygen, and sulfur. Unless otherwise specified in the specification, the heterocyclic group is a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include fused or bridged ring systems. The heteroatoms in the heterocyclic group may optionally be oxidized. One or more nitrogen atoms, if present, may optionally be quaternized. The heterocyclic group may be partially saturated or fully saturated. The heterocyclic group may be attached to the rest of the molecule via any ring atom. Examples of such heterocyclic groups include, but are not limited to, dioxanyl, thienyl[1,3]dithianyl, decahydroisoquinolinyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxapiperazinyl, 2-oxapiperidinyl, 2-oxapyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuranyl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxothiomorpholinyl, and 1,1-dioxothiomorpholinyl.

[0063] 33. The compound according to item 1 above, which has any one of the following structures:

[0064] A second aspect of the present invention provides a nucleic acid conjugate, the features of which are set out in the following clauses:

[0065] 34. The conjugate contains one or more compounds of the structure shown in formula (II) linked to any position on the oligonucleotide sequence.

[0066] Among them, Linker,m,Q,Z,X, R1 and n are the same as those defined in any of Clauses 1 to 33 and will not be repeated here;

[0067] Two of the structures represented by formula (II) One of the sites shown has the structure shown in (A15), and the other is H or has the structure shown in (A15):

[0068] Wherein, E1 is OH, SH or BH2; Y is O or S.

[0069] 35. The conjugate according to item 34, which contains two, three or four consecutively linked compounds of the structure represented by formula (II) linked to any position on the oligonucleotide sequence.

[0070] 36. The conjugate according to item 34, wherein one or more compounds having the structure represented by formula (II) are linked to any one or more positions of the oligonucleotide sequence.

[0071] 37. The conjugate according to clause 34, wherein the compound represented by formula (II) is linked to the 3' end and / or 5' end of the oligonucleotide sequence.

[0072] end.

[0073] 38. The conjugate according to item 34, wherein the compound of the structure represented by formula (II) is linked to the 3' end and / or 5' end of the sense strand of the oligonucleotide sequence, and the oligonucleotide is siRNA.

[0074] 39. The conjugate according to clause 34, wherein the nucleic acid conjugate has any one of the following structures:

[0075] Where Y is O or S, For oligonucleotides.

[0076] In some embodiments, the oligonucleotide can interact with the target sequence, thereby affecting the normal function of the target sequence molecule, such as causing mRNA fragmentation or translation repression or exon skipping to trigger mRNA alternative splicing. In some embodiments, the oligonucleotide can be substantially complementary to the bases of the target sequence. In some embodiments, the oligonucleotide can be complementary to 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more of the bases of the target sequence, or completely complementary to the target sequence. In some embodiments, the oligonucleotide can contain 1, 2 or 3 bases that are not complementary to the target sequence. In some embodiments, the oligonucleotide includes deoxyribonucleotides or ribonucleotides, as well as nucleotides with modifications. In some embodiments, the oligonucleotide can be single-stranded DNA, RNA, or DNA-RNA chimera, or double-stranded DNA, RNA, or DNA-RNA hybrids.

[0077] In some embodiments of the present disclosure, the oligonucleotide is selected from one of small interfering RNA, microRNA, anti-microRNA, microRNA antagonist, microRNA mimic, decoy oligonucleotide, immunostimulant, G-quadrupole, alternative splicing body, single-stranded RNA, antisense nucleic acid, nucleic acid aptamer, stem-loop RNA, mRNA fragment, and activating RNA; optionally, the oligonucleotide is a single-stranded oligonucleotide or a double-stranded oligonucleotide; optionally, the oligonucleotide is a single-stranded oligonucleotide, the P atom in formula (A15) is connected to the end of the single-stranded oligonucleotide, and the end of the single-stranded oligonucleotide refers to the first 4 nucleotides in the single-stranded oligonucleotide from one end; optionally, the P atom in formula (A15) is connected to the single-stranded oligonucleotide. nucleotide end; optionally, the P atom in formula (A15) is connected to the 3' end of the single-stranded oligonucleotide; optionally, the oligonucleotide is a double-stranded oligonucleotide, and the double-stranded oligonucleotide comprises a sense strand and an antisense strand, and the P atom in formula (A15) is connected to the end of the double-stranded oligonucleotide, and the end of the double-stranded oligonucleotide refers to the first 4 nucleotides from one end of the sense strand or the antisense strand; optionally, the P atom in formula (A15) is connected to the end of the sense strand or the antisense strand; optionally, the P atom in formula (A15) is connected to the 5' end of the antisense strand; optionally, the P atom in formula (A15) is connected to the 2' position, 3' position or 5' position of the nucleotide in the nucleic acid conjugate by forming a phosphodiester bond.

[0078] In some embodiments, each nucleotide in the siRNA in the siRNA conjugate of the present disclosure (hereinafter also referred to as the siRNA of the present disclosure) is independently a modified or unmodified nucleotide, and the siRNA contains a sense strand and an antisense strand, wherein the sense strand comprises a nucleotide sequence 1, the antisense strand comprises a nucleotide sequence 2, the nucleotide sequence 1 and the nucleotide sequence 2 are both 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, and are at least partially reverse-complemented to form a complementary double-stranded region, and at least a portion of the nucleotide sequence 2 is complementary to a first nucleotide sequence, which is a nucleotide sequence in the target mRNA.

[0079] In some embodiments, nucleotide sequence 1 is equal in length to the first nucleotide sequence and differs by no more than 3 nucleotides; nucleotide sequence 2 is equal in length to nucleotide sequence B and differs by no more than 3 nucleotides; and nucleotide sequence B is a nucleotide sequence that is completely reverse complementary to the first nucleotide sequence. Without wishing to be bound by these limitations, these specific nucleotide differences do not significantly reduce the target gene inhibition ability of the siRNA conjugate, and siRNA conjugates containing these specific nucleotide differences are also within the scope of protection of this disclosure.

[0080] In some embodiments, the nucleotide sequence 1 and the nucleotide sequence 2 are substantially reverse complementary, substantially completely reverse complementary, or completely reverse complementary.

[0081] In some embodiments, the ratio of the length of the sense and antisense strands of the siRNA of the present disclosure can be 19 / 20, 19 / 21, 20 / 21, 20 / 22, 21 / 22, 21 / 23, 22 / 23, 22 / 24, 23 / 24, or 23 / 25.

[0082] In some embodiments, the nucleotides in the siRNA of the present disclosure are each independently a modified or unmodified nucleotide. In some embodiments, the siRNA of the present disclosure does not contain a modified nucleotide group; in some embodiments, the siRNA of the present disclosure contains a modified nucleotide group.

[0083] Currently, there are a variety of methods available in the art for modifying siRNA, including backbone modification (also known as internucleotide linkage modification, such as phosphate group modification), ribose group modification, and base modification (for example, see Watts, JK, GF Deleavey and MJ Damha, Chemically modified siRNA: tools and applications. Drug Discov Today, 2008. 13(19-20): p. 842-55, the entire contents of which are incorporated herein by reference).

[0084] In the context of the present disclosure, the term "modified nucleotide" refers to a nucleotide whose ribose group is modified, such as a nucleotide or nucleotide analog formed by replacing the 2' hydroxyl group with other groups, or a nucleotide whose base is a modified base.

[0085] In some embodiments of the present disclosure, at least one nucleotide in the sense strand or the antisense strand is a modified nucleotide, and / or at least one phosphate group is a phosphate group having a modified group. In other words, at least a portion of the phosphate groups and / or ribose groups in the phosphate-sugar backbone of at least one single strand in the sense strand and the antisense strand is a phosphate group having a modified group and / or a ribose group having a modified group (or a modified phosphate group and / or a modified ribose group). In some embodiments of the present disclosure, all nucleotides in the sense strand and / or the antisense strand are modified nucleotides.

[0086] In some embodiments, the siRNA disclosed herein is an siRNA having the following modifications: both the sense strand and the antisense strand contain fluorinated and non-fluorinated nucleotides, wherein the fluorinated nucleotides are located in the aforementioned nucleotide sequence 1 and nucleotide sequence 2, wherein the fluorinated nucleotides in the nucleotide sequence 1 are no more than 9, and, from the 5' end to the 3' end, the nucleotides at positions 3, 5, 7, 9, 10, 11, 13, 16, and 18 of the nucleotide sequence 1 are fluorinated; wherein the fluorinated nucleotides in the nucleotide sequence 2 are no more than 8, and, from the 5' end to the 3' end, the nucleotides at positions 2, 3, 5, 8, 10, 14, 16, and 18 of the nucleotide sequence 2 are fluorinated. In some embodiments, the remaining nucleotides in the sense strand are methoxy-modified nucleotides; and the remaining nucleotides in the antisense strand are methoxy-modified nucleotides.

[0087] In some embodiments of the siRNA disclosed herein, the nucleotides are modified with a phosphate group. In the context of the present disclosure, the phosphate group modification is, in one embodiment, a phosphorothioate modification, i.e., a non-bridging oxygen atom in a phosphodiester bond is substituted with a sulfur atom, thereby replacing the phosphodiester bond with a phosphorothioate diester bond. In some embodiments, this modification can stabilize the structure of the siRNA, maintaining high specificity and high affinity for base pairing.

[0088] According to some embodiments of the present disclosure, in the siRNA, the phosphorothioate linkage is present in at least one of the group consisting of the following positions: between the first and second nucleotides at either end of the sense strand or the antisense strand; between the second and third nucleotides at either end of the sense strand or the antisense strand; or any combination thereof. In some embodiments, the phosphorothioate linkage is present at all of the above positions except the 5' end of the sense strand. In some embodiments, the phosphorothioate linkage is present at all of the above positions except the 3' end of the sense strand. In some embodiments, the phosphorothioate linkage is present at at least one of the following positions:

[0089] the connection between the first nucleotide and the second nucleotide at the 5' end of the sense strand;

[0090] the connection between the second nucleotide and the third nucleotide at the 5' end of the sense strand;

[0091] the connection between the first nucleotide and the second nucleotide at the 3' end of the sense strand;

[0092] the connection between the second nucleotide and the third nucleotide at the 3' end of the sense strand;

[0093] the connection between the first nucleotide and the second nucleotide at the 5' end of the antisense strand;

[0094] the connection between the second nucleotide and the third nucleotide at the 5' end of the antisense strand;

[0095] the connection between the first nucleotide and the second nucleotide at the 3' end of the antisense strand; and

[0096] The connection between the second nucleotide and the third nucleotide at the 3' end of the antisense strand.

[0097] The third aspect of the present invention provides use of the nucleic acid conjugate in the preparation of a medicament for treating and / or preventing hepatic diseases.

[0098] A fourth aspect of the present invention provides a method for treating and / or preventing hepatic diseases, comprising administering the aforementioned nucleic acid conjugate to a subject.

[0099] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0100] The delivery molecule of this patented invention utilizes a saturated ring group supplemented by a novel linker as its core backbone, connected by a phosphodiester bond. Compared to existing technologies, it exhibits superior in vivo biological activity and long-lasting drug efficacy in animals. It also offers advantages such as inexpensive raw materials, simple synthesis, easy process development, and good oligonucleotide synthesis efficiency. Furthermore, by improving the linker between galactosamine and the phosphodiester bond, the invention enhances the metabolic stability of the entire delivery molecule, thereby improving in vivo delivery efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0101] FIG1 shows the activity data of the conjugate of Example 6 in mice, wherein * represents a P value less than 0.05. DETAILED DESCRIPTION

[0102] It should be noted that, unless otherwise defined, the technical or scientific terms used in this application should have the same meaning as those commonly understood by those skilled in the art. The experimental methods in the following examples are all conventional methods unless otherwise specified. The raw materials, reagents, etc. used in the following examples are all commercially available products unless otherwise specified.

[0103] The technical solution provided by the present invention is further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and do not limit the scope of protection of the present invention.

[0104] Example 1 Preparation of Compound SA102 (i.e., I-3)

[0105] In this example, the synthetic route of compound SA102 (i.e., I-3) is as follows:

[0106] 1.1 Preparation of Intermediate 1-1

[0107] Compound (R)-(+)-N-benzyl-3-hydroxypyrrolidine (commercially available, purchased from Shanghai Titan Technology Co., Ltd.) (16.9 mmol, 3.0 g) and imidazole (3.0 equiv, 50.7 mmol, 3.45 g) were placed in a clean, dry reaction flask, 50 mL of acetonitrile was added, and tert-butyldimethylsilyl chloride (1.3 equiv, 21.9 mmol, 3.31 g) was slowly added at room temperature. Stirring was then continued at room temperature for 12 hours. After the reaction, 100 mL of ethyl acetate was added to the reaction solution, and the mixture was washed with 100 mL of saturated sodium bicarbonate solution and 100 mL of saturated brine. The organic phase was dried, filtered, and concentrated. The crude product was separated and purified by silica gel column chromatography (gradient elution: petroleum ether / ethyl acetate = 20 / 1-5 / 1) to obtain compound 1-1 (4.9 g, 16.8 mmol, 99% yield) as a colorless oil. Compound 1-1 Molecular formula: C 17 H 29 ONSi, molecular weight: 291.2, LC-MS found 292.4 (M+H).

[0108] 1.2 Preparation of intermediate 1-2

[0109] Compound 1-1 (16.8 mmol, 4.9 g) was placed in a clean, dry reaction flask, 100 mL of methanol was added, and palladium on carbon (wet basis, 10% Pd / C) (10% wt, 490.0 mg) was added under hydrogen at room temperature. Stirring was continued at room temperature for 12 hours. After the reaction, the palladium on carbon was removed by filtration, and the filtrate was concentrated to obtain the crude product, compound 1-2 (3.31 g, 16.5 mmol, 98% yield), as a white solid. It was used directly in the next reaction without purification. Compound 1-2 Molecular formula: C 10 H 23 ONSi, molecular weight: 201.1, LC-MS found 202.3 (M+H).

[0110] 1.3 Preparation of intermediate 1-3

[0111] The compound N-benzyloxycarbonyl-L-serine (commercially available, purchased from Shanghai Titan Technology Co., Ltd.) (15.0 mmol, 3.58 g) was placed in a clean dry reaction bottle, 100 mL of dichloromethane was added, and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.5 equiv, 22.5 mmol, 8.53 g), compound 1-2 (1.1 equiv, 16.5 mmol, 3.31 g) and N,N-diisopropylethylamine (3.0 equiv, 45.0 mmol, 5.78 g) were added at room temperature, followed by stirring at room temperature for 1 hour. After the reaction, 150 mL of dichloromethane was added to the reaction solution, and the mixture was washed with 150 mL of saturated sodium bicarbonate solution and 150 mL of saturated brine. The organic phase was dried, filtered, and concentrated. The resulting crude product was separated and purified by silica gel column chromatography (gradient elution: petroleum ether / ethyl acetate = 10 / 1-1 / 3) to obtain a white solid compound 1-3 (4.5 g, 10.65 mmol, 63% two-step yield). Compound 1-3 molecular formula: C 21 H 34 O5N2Si, molecular weight: 422.2, LC-MS found 423.3 (M+H). 1 H NMR (400MHz, CDCl3): δ7.35–7.29(m,5H),5.96(dd,J=14.3,8.3Hz,1H),5.10(s,2H),4.61–4.40(m,2H),3.85–3.68(m,2H),3.65–3.4 9(m,2H),3.41(d,J=12.7Hz,1H),3.31(s,1H),1.95(qdd,J=15.0,11.8,5.3Hz,2H),1.77(s,1H),0.86(s,9H),0.06(d,J=3.1Hz,6H).

[0112] 1.4 Preparation of Intermediates 1-4

[0113] Compound 1-3 (10.65 mmol, 4.5 g) was placed in a clean, dry reaction flask, 100 mL of pyridine was added, and 4,4'-bismethoxytrityl chloride (1.2 equiv, 12.78 mmol, 4.32 g) was added at room temperature, followed by stirring at room temperature for 12 hours. After the reaction, 150 mL of ethyl acetate was added to the reaction solution, and the mixture was washed with 150 mL of saturated sodium bicarbonate solution and 150 mL of saturated brine. The organic phase was dried, filtered, and concentrated. The crude product was separated and purified by silica gel column chromatography (gradient elution: petroleum ether / ethyl acetate = 20 / 1-1 / 1) to obtain compound 1-4 (7.56 g, 10.43 mmol, 98% yield) as a light yellow oil. Compound 1-4 molecular formula: C 42 H52 O7N2Si, molecular weight: 724.3, LC-MS found 747.4 (M+Na). 1 HNMR (400MHz, CDCl3): δ7.35–7.31(m,1H),7.28(d,J=4.7Hz,4H),7.27–7.23(m,2H),7.23–7.14(m,5H) ,7.13–7.11(m,2H),6.80–6.78(m,1H),6.76(dd,J=7.7,5.4Hz,4H),5.72(dd,J=22.7,8.3Hz,1H),5.08 –4.99(m,2H),4.69–4.59(m,1H),4.35–4.30(m,1H),3.73(dd,J=4.5,3.7Hz,6H),3.65–3.44(m,2H),3. 36–3.20(m,3H),1.86–1.81(m,1H),1.70(s,1H),0.80(d,J=13.1Hz,9H),-0.02(dd,J=14.9,4.2Hz,6H).

[0114] 1.5 Preparation of Intermediate 1-5

[0115] Compound 1-4 (10.43 mmol, 7.56 g) was placed in a clean, dry reaction flask, and 100 mL of methanol was added. Palladium on carbon (wet basis, 10% Pd / C) (10% wt, 750.0 mg) was then added under hydrogen at room temperature. Stirring was continued at room temperature for 12 hours. After the reaction, the palladium on carbon was removed by filtration, and the filtrate was concentrated to obtain the crude product, compound 1-5 (6.0 g, 10.22 mmol, 98% yield), as a white solid. This was used directly in the next reaction without purification. Compound 1-5 Molecular formula: C 34 H 46 O5N2Si, molecular weight: 590.3, LC-MS found 591.6 (M+H).

[0116] 1.6 Preparation of Intermediate 1-6

[0117] Compound 1-5 (10.22 mmol, 6.0 g) was placed in a clean, dry reaction flask and added with 100 mL of dichloromethane. 4-Dimethylaminopyridine (30 mol%, 3.07 mmol, 374.6 mg) and N,N-diisopropylethylamine (3.0 equiv, 30.66 mmol, 3.96 g) were then added at room temperature. Adipic anhydride (1.5 equiv, 15.33 mmol, 1.96 g) was then added to the reaction system and stirred at room temperature for 4 hours. After the reaction, the reaction solution was directly concentrated, and the resulting crude product was purified by silica gel column chromatography (gradient elution: dichloromethane / methanol = 50 / 1-8 / 1) to obtain compound 1-6 (5.28 g, 7.36 mmol, 72% yield) as a white solid. Compound 1-6 Molecular formula: C 40 H 54 O8N2Si, molecular weight: 718.3, LC-MS found 717.3 (MH).

[0118] 1.7 Preparation of Intermediates 1-8

[0119] Compound 1-6 (7.36 mmol, 5.28 g) was placed in a clean, dry reaction bottle, 100 mL of dichloromethane was added, and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.5 equiv, 11.04 mmol, 4.19 g), N,N-diisopropylethylamine (3.0 equiv, 22.08 mmol, 2.85 g) and compound 1-7 (commercially available, purchased from Tianjin WuXi AppTec New Drug Development Co., Ltd.) (1.1 equiv, 8.1 mmol, 4.31 g) were added at room temperature, and stirring was continued at room temperature for 1 hour. After the reaction, 150 mL of dichloromethane was added to the reaction solution, and the mixture was washed with 150 mL of saturated sodium bicarbonate solution and 150 mL of saturated brine. The organic phase was dried, filtered, and concentrated. The resulting crude product was separated and purified by silica gel column chromatography (gradient elution: dichloromethane / methanol = 50 / 1-10 / 1) to obtain a light yellow solid compound 1-8 (6.75 g, 6.04 mmol, 82% yield). Compound 1-8 molecular formula: C 58 H 82 O 16 N4Si, molecular weight: 1118.5, LC-MS found 1117.3 (MH).

[0120] 1.8 Preparation of Intermediate 1-9

[0121] Compound 1-8 (6.04 mmol, 6.75 g) was placed in a clean, dry reaction flask, and 50 mL of tetrahydrofuran was added. Tetrabutylammonium fluoride (1.0 M in THF) (2.0 equiv, 12.08 mmol, 12.08 mL) was then added at room temperature, and stirring continued at room temperature for 2 hours. After the reaction, 100 mL of ethyl acetate was added to the reaction solution, and the mixture was washed with 100 mL of saturated sodium bicarbonate solution and 100 mL of saturated brine. The organic phase was dried, filtered, and concentrated. The resulting crude product was separated and purified by silica gel column chromatography (gradient elution: dichloromethane / methanol = 50 / 1-8 / 1) to obtain a light yellow solid compound 1-9 (5.22 g, 5.19 mmol, 86% yield). Compound 1-9 molecular formula: C 52 H 68 O 16 N4, molecular weight: 1004.4, LC-MS found 1003.3 (MH).

[0122] 1.9 Preparation of compound SA102 (i.e., I-3)

[0123] Compound 1-9 (5.19 mmol, 5.22 g) was placed in a clean, dry reaction flask and 50 mL of anhydrous dichloromethane was added. Under argon protection at room temperature, 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (2.0 equiv, 10.38 mmol, 3.13 g) and 4,5-dicyanimidazole (1.5 equiv, 7.79 mmol, 920.2 mg) were added and stirred at room temperature for one hour. After the reaction, 50 mL of dichloromethane was added to the reaction mixture, which was then washed with 100 mL of saturated sodium bicarbonate solution. The organic phase was dried, filtered, and concentrated. The resulting crude product was purified by chromatography on a C18 reverse-phase column (specification: 30 μm; Commercially available, purchased from Shanghai Boyun Biotechnology Co., Ltd.) (MeCN:H2O=75%:25%) to prepare white solid SA102 (4.44 g, 3.68 mmol, 71% yield). Compound SA102 molecular formula: C 61 H 85 O 17 N6P, molecular weight: 1204.5, LC-MS found 1227.3 (M+Na). 1HNMR (400MHz, DMSO-d6): δ8.10(q,J=8.3Hz,1H),7.80(d,J=9.2Hz,1H),7.68(d d,J=8.8,4.9Hz,1H),7.35–7.28(m,4H),7.20(ddd,J=11.2,6.7,2.2Hz,5H),6.8 9–6.87(m,4H),5.21(d,J=3.3Hz,1H),4.96(dd,J=11.2,3.4Hz,1H),4.85–4.74( m,1H),4.48(d,J=8.5Hz,2H),4.04–3.99(m,3H),3.87(dd,J=20.0,8.9Hz,1H),3 .74(s,6H),3.72–3.67(m,2H),3.63–3.46(m,4H),3.44–3.38(m,2H),3.18(qd,J =8.8,4.6Hz,2H),3.06–2.97(m,3H),2.74(ddd,J=16.6,11.8,5.9Hz,1H),2.61( td,J=5.8,1.8Hz,1H),2.10(s,5H),1.99(s,6H),1.89(s,3H),1.76(s,3H),1.46 –1.33(m,8H),1.24–1.09(m,12H),1.06(d,J=6.7Hz,1H),1.00(d,J=6.7Hz,1H). 31 P NMR (162MHz, DMSO-d6): δ147.07 (s), 146.74 (t, J = 32.0Hz).

[0124] Example 2 Preparation of Compound SA87 (i.e., I-1)

[0125] In this example, the synthetic route of compound SA87 (i.e., I-1) is as follows:

[0126] 2.1 Preparation of Intermediate 2-1

[0127] Compound 1-5 (10.0 mmol, 5.9 g) was placed in a clean, dry reaction bottle, 100 mL of dichloromethane was added, and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.5 equiv, 15.0 mmol, 5.69 g), N,N-diisopropylethylamine (3.0 equiv, 30.0 mmol, 3.88 g) and 5-(benzyloxycarbonylamino)pentanoic acid (commercially available, purchased from Shanghai Titan Technology Co., Ltd.) (1.1 equiv, 11.0 mmol, 2.76 g) were added at room temperature, and stirring was continued at room temperature for 1 hour. After the reaction, 150 mL of dichloromethane was added to the reaction solution, and the mixture was washed with 150 mL of saturated sodium bicarbonate solution and 150 mL of saturated brine. The organic phase was dried, filtered, and concentrated. The resulting crude product was separated and purified by silica gel column chromatography (gradient elution: dichloromethane / methanol = 50 / 1-10 / 1) to obtain a light yellow solid compound 2-1 (7.58 g, 9.2 mmol, 92% yield). Compound 2-1 molecular formula: C 47 H 61 O8N3Si, molecular weight: 823.4, LC-MS found 824.4 (M+H).

[0128] 2.2 Preparation of intermediate 2-2

[0129] Compound 2-1 (9.2 mmol, 7.58 g) was placed in a clean, dry reaction flask, 100 mL of methanol was added, and palladium on carbon (wet basis, 10% Pd / C) (10% wt, 758.0 mg) was added under hydrogen at room temperature. Stirring was continued at room temperature for 12 hours. After the reaction, the palladium on carbon was removed by filtration, and the filtrate was concentrated to obtain the crude product, compound 2-2 (6.21 g, 9.01 mmol, 98% yield), as a pale yellow solid. This was used directly in the next reaction without purification. Compound 2-2 Molecular formula: C 39 H 55 O6N3Si, molecular weight: 689.3, LC-MS found 690.4 (M+H).

[0130] 2.3 Preparation of intermediate 2-4

[0131] Compound 2-2 (9.01 mmol, 6.21 g) was placed in a clean, dry reaction bottle, 100 mL of dichloromethane was added, and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.5 equiv, 13.52 mmol, 5.13 g), N,N-diisopropylethylamine (3.0 equiv, 27.03 mmol, 3.49 g) and 5-(((2R,3R,4R,5R,6R)-3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoic acid (commercially available, purchased from Tianjin WuXi AppTec New Drug Development Co., Ltd.) (1.1 equiv, 9.91 mmol, 4.43 g) were added at room temperature, and stirring was continued at room temperature for 1 hour. After the reaction, 150 mL of dichloromethane was added to the reaction solution, and the mixture was washed with 150 mL of saturated sodium bicarbonate solution and 150 mL of saturated brine. The organic phase was dried, filtered, and concentrated. The resulting crude product was separated and purified by silica gel column chromatography (gradient elution: dichloromethane / methanol = 50 / 1-10 / 1) to obtain a light yellow solid compound 2-4 (8.16 g, 7.3 mmol, 81% yield). Compound 2-4 molecular formula: C 58 H 82 O 16 N4Si, molecular weight: 1118.5, LC-MS found 1117.3 (MH).

[0132] 2.4 Preparation of Intermediate 2-5

[0133] Compound 2-4 (7.3 mmol, 8.16 g) was placed in a clean, dry reaction flask, and 50 mL of tetrahydrofuran was added. Tetrabutylammonium fluoride (1.0 M in THF) (2.0 equiv, 14.6 mmol, 14.6 mL) was then added at room temperature, and stirring continued for 2 hours at room temperature. After the reaction, 100 mL of ethyl acetate was added to the reaction solution, and the mixture was washed with 100 mL of saturated sodium bicarbonate solution and 100 mL of saturated brine. The organic phase was dried, filtered, and concentrated. The resulting crude product was separated and purified by silica gel column chromatography (gradient elution: dichloromethane / methanol = 50 / 1-8 / 1) to obtain a light yellow solid compound 2-5 (6.38 g, 6.35 mmol, 87% yield). Compound 2-5 molecular formula: C 52 H 68 O 16 N4, molecular weight: 1004.4, LC-MS found 1003.4 (MH).

[0134] 2.5 Preparation of compound SA87 (i.e., I-1)

[0135] Compound 2-5 (6.35 mmol, 6.38 g) was placed in a clean, dry reaction flask and 50 mL of anhydrous dichloromethane was added. Under argon protection at room temperature, 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (2.0 equiv, 12.7 mmol, 3.83 g) and 4,5-dicyanoimidazole (1.5 equiv, 9.53 mmol, 1.13 g) were added and stirred at room temperature for one hour. After the reaction, 50 mL of dichloromethane was added to the reaction mixture, which was then washed with 100 mL of saturated sodium bicarbonate solution. The organic phase was dried, filtered, and concentrated. The resulting crude product was purified by chromatography on a C18 reverse-phase column (specification: 30 μm; Commercially available, purchased from Shanghai Boyun Biotechnology Co., Ltd.) (MeCN:H2O=75%:25%) to prepare white solid SA87 (5.58 g, 4.64 mmol, 73% yield). Compound SA87 molecular formula: C 61 H 85 O 17 N6P, molecular weight: 1204.5, LC-MS found 1227.3 (M+Na). 1 H NMR (400MHz, DMSO-d6): δ8.11(q,J=7.9Hz,1H),7.81(d,J=9.2Hz,1H),7.67(q,J=5.5Hz ,1H),7.36–7.28(m,4H),7.23–7.17(m,5H),6.88(dd,J=8.8,1.4Hz,4H),5.21(d,J=3.4 Hz,1H),4.97(dd,J=11.2,3.4Hz,1H),4.80(dq,J=21.9,7.5Hz,1H),4.48(d,J=8.5Hz,2 H),4.04–3.98(m,3H),3.91–3.83(m,1H),3.74(s,6H),3.72–3.65(m,3H),3.65–3.46(m, 4H),3.42–3.38(m,2H),3.19(qd,J=8.5,4.2Hz,1H),3.07–2.97(m,3H),2.78–2.70(m,2 H),2.61(td,J=5.8,1.8Hz,1H),2.10(s,5H),2.02(d,J=6.9Hz,3H),1.99(s,3H),1.89( s,3H),1.77(s,3H),1.51–1.41(m,6H),1.32(dd,J=13.8,7.2Hz,2H),1.23(d,J=2.8Hz, 1H),1.20–1.17(m,1H),1.15–1.09(m,9H),1.06(d,J=6.7Hz,1H),1.00(d,J=6.7Hz,1H).31 P NMR (162MHz, DMSO-d6): δ147.06 (s), 146.76 (t, J = 24.0Hz).

[0136] Example 3 Preparation of Compound SA101 (i.e., I-2)

[0137] According to the synthesis method of Example 2, a white solid SA101 (4.43 g, 3.68 mmol, 72% yield) was prepared. Compound SA101 molecular formula: C 61 H 85 O 17 N6P, molecular weight: 1204.5, LC-MS found 1227.4 (M+Na). 1 H NMR (400MHz, DMSO-d6): δ8.14–8.07(m,1H),7.80(d,J=9.2Hz,1H),7.66(s,1H),7. 35–7.28(m,4H),7.23–7.17(m,5H),6.89–6.86(m,4H),5.21(d,J=3.3Hz,1H),4.97 (dd,J=11.2,3.4Hz,1H),4.90–4.74(m,1H),4.58–4.52(m,1H),4.48(d,J=8.5Hz,1 H),4.04–3.98(m,3H),3.91–3.83(m,1H),3.74(s,6H),3.70–3.60(m,3H),3.58–3.5 2(m,2H),3.49–3.44(m,2H),3.39(dt,J=10.2,6.2Hz,2H),3.20(dt,J=16.0,6.2Hz ,1H),3.07–2.95(m,3H),2.78–2.73(m,2H),2.67(t,J=5.9Hz,1H),2.10–2.06(m,5 H),2.02(d,J=6.6Hz,2H),1.99(s,3H),1.89(s,3H),1.76(s,3H),1.51–1.41(m,6H ),1.35–1.28(m,2H),1.25–1.19(m,1H),1.18–1.08(m,12H),1.04(d,J=6.7Hz,1H). 31 PNMR (162MHz, DMSO-d6): δ146.71 (d, J = 12.2Hz), 146.53 (d, J = 13.2Hz).

[0138] Example 4 Preparation of Compound SA123 (i.e., I-4)

[0139] In this example, the synthetic route of compound SA123 (i.e., I-4) is as follows:

[0140] 4.1 Preparation of Intermediate 4-1

[0141] Compound 1-5 (10.0 mmol, 5.9 g) was placed in a clean, dry reaction bottle, 100 mL of dichloromethane was added, and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.5 equiv, 15.0 mmol, 5.69 g), N,N-diisopropylethylamine (3.0 equiv, 30.0 mmol, 3.88 g) and N-benzyloxycarbonyl-4-aminobutyric acid (commercially available, purchased from Shanghai Titan Technology Co., Ltd.) (1.1 equiv, 11.0 mmol, 2.61 g) were added at room temperature, and stirring was continued at room temperature for 1 hour. After the reaction, 150 mL of dichloromethane was added to the reaction solution, and the mixture was washed with 150 mL of saturated sodium bicarbonate solution and 150 mL of saturated brine. The organic phase was dried, filtered, and concentrated. The resulting crude product was separated and purified by silica gel column chromatography (gradient elution: dichloromethane / methanol = 50 / 1-10 / 1) to obtain a light yellow solid compound 4-1 (7.04 g, 8.7 mmol, 87% yield). Compound 4-1 Molecular formula: C 46 H 59 O8N3Si, molecular weight: 809.4, LC-MS found 810.4 (M+H).

[0142] 4.2 Preparation of intermediate 4-2

[0143] Compound 4-1 (8.7 mmol, 7.04 g) was placed in a clean, dry reaction flask, and 100 mL of methanol was added. Palladium on carbon (wet basis, 10% Pd / C) (10% wt, 704.0 mg) was then added under hydrogen at room temperature. Stirring was continued at room temperature for 12 hours. After the reaction, the palladium on carbon was removed by filtration, and the filtrate was concentrated to obtain the crude product, compound 4-2 (5.64 g, 8.35 mmol, 96% yield), as a light yellow solid. This was used directly in the next reaction without purification. Compound 4-2 Molecular formula: C 38 H 53 O6N3Si, molecular weight: 675.3, LC-MS found 674.3 (MH).

[0144] 4.3 Preparation of intermediate 4-3

[0145] Compound 4-2 (8.35 mmol, 5.64 g) was placed in a clean, dry reaction bottle, 100 mL of dichloromethane was added, N, N-diisopropylethylamine (3.0 equiv, 25.05 mmol, 3.24 g) and trichloromethyl carbonate (0.5 equiv, 4.18 mmol, 1.24 g) were added at room temperature, and stirring was continued at room temperature for 0.5 hours. Subsequently, compound 1-7 (commercially available, purchased from Tianjin WuXi AppTec New Drug Development Co., Ltd.) (1.1 equiv, 9.19 mmol, 4.89 g) was added to the reaction system, and stirring was continued at room temperature for 1 hour. After the reaction, 150 mL of dichloromethane was added to the reaction solution, and the mixture was washed with 150 mL of saturated sodium bicarbonate solution and 150 mL of saturated brine. The organic phase was dried, filtered, and concentrated. The resulting crude product was separated and purified by silica gel column chromatography (gradient elution: dichloromethane / methanol = 50 / 1-10 / 1) to obtain a light yellow solid compound 4-3 (3.93 g, 3.51 mmol, 42% yield). Compound 4-3 Molecular formula: C 57 H 81 O 16 N5Si, molecular weight: 1119.5, LC-MS found 1118.3 (MH).

[0146] 4.4 Preparation of Intermediate 4-4

[0147] Compound 4-3 (3.51 mmol, 3.93 g) was placed in a clean, dry reaction flask, and 50 mL of tetrahydrofuran was added. Tetrabutylammonium fluoride (1.0 M in THF) (2.0 equiv, 7.02 mmol, 7.02 mL) was then added at room temperature, and stirring continued at room temperature for 2 hours. After the reaction, 100 mL of ethyl acetate was added to the reaction solution, and the mixture was washed with 100 mL of saturated sodium bicarbonate solution and 100 mL of saturated brine. The organic phase was dried, filtered, and concentrated. The crude product was separated and purified by silica gel column chromatography (gradient elution: dichloromethane / methanol = 50 / 1-8 / 1) to obtain a light yellow solid compound 4-4 (3.11 g, 3.09 mmol, 88% yield). Compound 4-4 molecular formula: C 51 H 67 O 16 N5, molecular weight: 1005.4, LC-MS found 1004.3 (MH).

[0148] 4.5 Preparation of compound SA123 (i.e., I-4)

[0149] Compound 4-4 (3.09 mmol, 3.11 g) was placed in a clean, dry reaction flask and 50 mL of anhydrous dichloromethane was added. Under argon protection at room temperature, 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (2.0 equiv, 6.18 mmol, 1.86 g) and 4,5-dicyanoimidazole (1.5 equiv, 4.64 mmol, 547.8 mg) were added and stirred at room temperature for one hour. After the reaction, 50 mL of dichloromethane was added to the reaction solution, which was then washed with 100 mL of saturated sodium bicarbonate solution. The organic phase was dried, filtered, and concentrated. The resulting crude product was purified by chromatography on a C18 reverse-phase column (specification: 30 μm; Commercially available, purchased from Shanghai Boyun Biotechnology Co., Ltd.) (MeCN:H2O=75%:25%) to prepare white solid SA123 (2.57 g, 2.13 mmol, 69% yield). Compound SA123 molecular formula: C 60 H 84 O 17 N7P, molecular weight: 1205.5, LC-MS found 1228.4 (M+Na). 1HNMR (400MHz, DMSO-d6): δ8.18(q,J=8.2Hz,1H),7.82(d,J=9.2Hz,1H),7.35–7.28(m,4H), 7.23–7.17(m,5H),6.88(dd,J=8.8,1.4Hz,4H),5.80–5.74(m,1H),5.21(d,J=3.4Hz,1H),4. 96(dd,J=11.2,3.4Hz,1H),4.85–4.74(m,1H),4.49(t,J=9.6Hz,2H),4.04–3.99(m,3H),3.8 7(dt,J=11.0,9.0Hz,1H),3.73(s,6H),3.72–3.64(m,3H),3.63–3.46(m,4H),3.44–3.38(m, 2H),3.18(qd,J=8.6,4.2Hz,1H),3.03(dt,J=16.6,8.2Hz,1H),2.95(dd,J=12.5,6.2Hz,4H ),2.74(ddd,J=16.1,11.7,5.9Hz,2H),2.61(td,J=5.8,1.9Hz,1H),2.10–2.05(m,5H),1.99 (s,4H),1.89(s,3H),1.77(s,3H),1.55(ddd,J=9.9,6.7,3.1Hz,2H),1.48–1.41(m,2H),1.3 4(dt,J=13.8,6.7Hz,2H),1.15–1.08(m,10H),1.05(d,J=6.7Hz,2H),0.99(d,J=6.7Hz,2H). 31 P NMR (162MHz, DMSO-d6): δ147.04 (s), 146.70 (d, J=32.0Hz).

[0150] Example 5 Preparation of siRNA Conjugates

[0151] By solid-phase phosphoramidite method, using the special modified compounds prepared in the above steps and commercially purchased conventional modified monomers (phosphoramidite monomers for synthesizing modified nucleotides dT, Am, Cm, Gm, Um, Af, Cf, Gf, and Uf, all purchased from Shanghai Zhaowei Technology Development Co., Ltd.), nucleoside monomers are connected one by one from the 3'-5' direction according to the order of nucleotide arrangement. The specially modified anti-off-target compound is placed in the seed region of the antisense chain (any position from the 4th to the 8th starting from the 5' end), and the delivery monomer compound is freely set to be placed at the 3' end or the 5' end according to the common monomer. Each connection of a nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization. The synthesis conditions used for the sense chain and antisense chain.

[0152] Instrument model: MerMade 12Oligonucleotide syntheizer solid phase synthesizer, Beijing Haijing 6mL synthesis column, Si Tuofan SourceTM 15Q 4.6 / 100PE purification column.

[0153] The reagents used to synthesize siRNA conjugates were purchased from Suzhou Kelema Biotechnology Co., Ltd.

[0154] The synthesis is briefly described as follows:

[0155] The single-strand synthesis reaction process is extended from 3' to 5' and is completed on a solid phase synthesizer. It includes four main reaction steps:

[0156] a. DMTr removal reaction: Use dichloroacetic acid to remove the DMTr protecting group on the nucleotide to obtain the 5'-hydroxyl end;

[0157] b. Coupling reaction: The protected nucleotide phosphoramidite monomer is mixed with the activator ethylthiotetrazolium. The phosphoramidite group is activated, and the 5'-hydroxyl group, still protected by DMTr, undergoes a condensation reaction with the 5'-hydroxyl group attached to the solid support to form a phosphite triester.

[0158] c. Oxidation reaction: Under the action of iodine, the phosphite triester obtained in the previous condensation reaction is converted into a more stable phosphate ester (ie, trivalent phosphorus is oxidized to pentavalent phosphorus).

[0159] d. Sulfurization reaction: Under the action of the thio reagent PADS (phenylacetyl disulfide), the phosphite triester obtained in the previous condensation reaction is converted into a thiophosphate (oxidation or thiolation is selected according to the sequence design).

[0160] e. Capping reaction: There may be a very small number of 5'-hydroxyl groups (less than 2%) that do not participate in the condensation reaction. These are reacted with acetic anhydride and 1-methylimidazole to form acetate end-capping groups that cannot participate in subsequent reactions, thereby preventing further reactions. These short fragments can be separated during purification.

[0161] Repeat the above four steps until the desired sequence is synthesized.

[0162] After the last nucleoside monomer is connected, the nucleic acid sequence connected to the solid phase support is cut, deprotected, purified, desalted, and then freeze-dried to obtain the sense chain and antisense chain, wherein:

[0163] Cleavage and deprotection conditions were as follows: First, prepare the aminolysis solution (a mixture of ammonia and ethanol in a ratio of 3:1 to a volume of 2 mL), add the solid support to the reaction flask, and shake thoroughly to mix well. Aminolysis was carried out in a constant temperature water bath at 50°C for 16 hours. After 16 hours of aminolysis, the water bath was cooled to a temperature of 25°C ± 2°C, filtered through a fritted funnel, and the filtrate was collected in a round-bottom flask. The residue was rinsed with 50% aqueous ethanol. The filtrate was collected and concentrated on a rotary evaporator, then transferred to a glass bottle. A small sample of the crude product was sent to the analytical department for analysis by LC-MS. The purity and molecular weight of the sense and antisense strands were determined using a Waters Acquity UPLC-LTQ LCMS (column: ACQUITY UPLC BEH C18). The measured values ​​were consistent with the theoretical values ​​(see Table 1).

[0164] Purification and desalting conditions are as follows: purification using an ion exchange chromatography column and desalting using a Stovepane HiPrep™ 26 / 10 Desalting gel column, followed by single-strand lyophilization. After lyophilization, the single-stranded product needs to be sampled for LC-MS analysis.

[0165] Finally, the obtained sense and antisense strands need to be annealed into double strands.

[0166] Annealing was performed as follows: the purified sense and antisense strands were dissolved in water for injection to prepare solutions ranging from 0.1 mg / mL to 40 mg / mL. The mixture was calibrated with a Thermo Scientific Nanodrop Eight at an equal molar ratio and heated at 90°C for 5 minutes. The mixture was then cooled naturally to allow hydrogen bonding to form a double-stranded structure. Samples were taken for SEC purity testing (see Table 2). The double-stranded samples were lyophilized.

[0167] Table 1 Delivery molecule conjugated siRNA number and sequence information

[0168] Table 2 Double-stranded SEC-HPLC purity

[0169] In Table 1 above, the capital letters A, C, G, and U represent adenosine-3'-phosphate, cytidine-3'-phosphate, guanosine-3'-phosphate, and uridine-3'-phosphate, respectively; dT represents thymidine deoxynucleotide; Am represents 2'-O-methyladenosine-3'-phosphate; Ams represents 2'-O-methyladenosine-3'-phosphorothioate; Cm represents 2'-O-methylcytidine-3'-phosphate; Cms represents 2'-O-methylcytidine-3'-phosphorothioate; Gm represents 2'-O-methylguanosine-3'-phosphate; Gms represents 2'-O-methylguanosine-3'-phosphorothioate ester; Um represents 2'-O-methyluridine-3'-phosphate; Ums represents 2'-O-methyluridine-3'-phosphorothioate; Af represents 2'-fluoroadenosine-3'-phosphate; Afs represents 2'-fluoroadenosine-3'-phosphorothioate; Cf represents 2'-fluorocytidine-3'-phosphate; Cfs represents 2'-fluorocytidine-3'-phosphorothioate; Gf represents 2'-fluoroguanosine-3'-phosphate; Gfs represents 2'-fluoroguanosine-3'-phosphorothioate; Uf represents 2'-fluorouridine-3'-phosphate; Ufs represents 2'-fluorouridine-3'-phosphorothioate.

[0170] Example 6 Testing the Activity of siRNA Conjugates in Mice

[0171] Select 6-8 week old SPF grade female C57BL / 6J mice, the weight of the mice was 20±2g. The mice were weighed and observed before administration. Animals with uniform weight and normal condition were randomly divided into groups of 4 per group. The experimental group mice were given the conjugate, and the vehicle group mice were given phosphate buffered saline (PBS). Each mouse was given a dose of 1 mg / kg of the conjugate for subcutaneous administration. 22 days after administration, the mouse serum was collected and the C5 protein level in the serum was detected by ELISA method.

[0172] The results are expressed as the residual expression level in the siRNA-administered group compared to the vehicle group (vehicle group = 100%). The siRNA sequences of the conjugates used for injection are shown in Table 1. As shown in Figure 1 and Table 3, compared to the positive conjugate SD003317, the conjugates SD004229 and SD004362 exhibited comparable complement C5 protein silencing activity; the conjugate SD004364 exhibited significantly superior complement C5 protein silencing activity to that of the positive conjugate SD003317.

[0173] Table 3 Relative residual expression levels of complement C5 protein 22 days after administration of the tested conjugates

[0174] The above detailed description of the present invention is intended to enable persons familiar with the art to understand the contents of the present invention and implement them. It does not limit the scope of protection of the present invention. Any equivalent changes or modifications made based on the spirit of the present invention should be included in the scope of protection of the present invention.

Claims

1. A compound, characterized in that: The compound includes a structure represented by formula (I): Among them, Linker is -L1-L2-L3-L4-; Among them, L1 and L3 are independently selected from one or more connection combinations of the groups represented by the following formulas (A1)-(A14): L2 is selected from the groups shown in (A7), (A10), (A11), (A12) or (A13) above; L4 does not exist, or is selected from one or more of the groups shown in (A1), (A2), (A7), (A8), (A10), (A11) or (A12) above as a connecting combination; Among them, R' is hydrogen, C1-C10 alkyl or C3-C8 cycloalkyl; j1 is an integer from 1 to 20; j2 is an integer from 1 to 20; m represents an integer from 0 to 6; Q represents Among them, R2 and R3 are independently selected from H, C1-C20 alkyl, C1-C20 alkoxy, C2-C20 alkenyl or C2-C20 alkynyl; X represents Among them, R4 and R5 are independently selected from H, fluorine, hydroxyl, C1-C20 alkyl, C1-C20 alkoxy, C2-C20 alkenyl, C2-C20 alkynyl or R4 and R5 are directly connected to form a ring, and p is an integer from 1 to 6; Z represents N or CR9, where R9 is selected from H, C1-C20 alkyl or C3-C10 cycloalkyl; represents C3-C18 cycloalkyl or C3-C18 heterocyclic group; R1 is selected from H, fluorine, hydroxyl, cyano, C1-C20 alkyl, C1-C20 alkoxy, C2-C20 alkenyl or C2-C20 alkynyl; n is an integer from 0 to 10; represents the site of covalent bond connection of the group; R6, R7, R8 are independently H or K(C=O)-, where K is independently selected from one of methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl, ethyl, n-propyl, isopropyl, phenyl, halogenated phenyl and alkylphenyl.

2. The compound according to claim 1, wherein: L1 and L3 are independently selected from one or more of the groups shown in (A1), (A2), (A3), (A5), (A7), (A8), (A9), (A11), (A14) above as a connecting combination; preferably, L1 and L3 are independently selected from one or more of the groups shown in (A1), (A2), (A3), (A5), (A7), (A14) above; preferably, L1 and L3 are independently (A5) or (A14); preferably, L1 and L3 are independently (A5) or (A14) and j1 is an integer from 1 to 10, j2 is an integer from 1 to 10; and / or, R' is hydrogen, C1-C5 alkyl or C3-C6 cycloalkyl; and / or, L4 does not exist, or is selected from the groups shown in (A7), (A10) or (A11) above; and / or, m represents 0, 1, 2, 3, 4 or 5; and / or, R2 and R3 are independently selected from H, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl or C2-C6 alkynyl; and / or, R4 and R5 are independently selected from H, fluorine, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, C2-C6 alkynyl or R4 and R5 are directly connected to form a ring; and / or, p is 1, 2 or 3; and / or, represents C3-C18 cycloalkyl or C3-C18 heterocyclic group; and / or, R1 is selected from H, fluorine, hydroxyl, cyano, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl or C2-C6 alkynyl; and / or, n is an integer from 0 to 5; and / or, Z represents N or CR9, where R9 is selected from H, C1-C6 alkyl or C3-C7 cycloalkyl.

3. The compound according to claim 1, wherein: L1 and L3 are independently of each other (A5) or (A14) and j1 is 2, 3, 4, 5 or 6, j2 is 1, 2, 3, 4 or 5; L4 does not exist, or is selected from groups as shown in (A7), (A10) or (A11) above; m is 0, 1 or 2; represents a four- to eight-membered all-carbon or nitrogen-containing saturated ring; n is 0, 1, 2 or 3.

4. The compound according to claim 3, wherein: L1 and L3 are independently of each other (A5) or (A14) and j1 is 3, 4, 5 or 6, j2 is 2, 3 or 4; R’, R1, R2, R3, R9 are hydrogen; m and n are 0.

5. The compound according to claim 1, wherein: One of the two sites shown in the structure of formula (I) is linked to a phosphoramidite functional group, and the other is linked to a phosphoramidite functional group or a hydroxyl protecting group. One of the sites shown is linked to a phosphoramidite functional group, and the other is linked to a phosphoramidite functional group or a hydroxyl protecting group.

6. The compound according to claim 5, wherein: The phosphoramidite functional group has a structure represented by formula (G-1), wherein, B1 is selected from substituted or unsubstituted C1-C5 hydrocarbon groups, preferably, B1 is selected from methyl, ethyl or isopropyl; B2 is selected from one of C1-C5 alkyl, ethyl cyanide, propyl cyanide and butyl cyanide, preferably, B2 is ethyl cyanide; and / or, the hydroxyl protecting group is selected from any one of trityl, 4-methoxytrityl, 4,4’-dimethoxytrityl and 4,4’,4’’-trimethoxybenzyl, preferably 4,4’-dimethoxytrityl.

7. The compound according to claim 1, wherein: The compound has any one of the following structures:

8. A nucleic acid conjugate, characterized in that: The conjugate contains one or more compounds of the structure shown in formula (II) attached at any position on the oligonucleotide sequence, wherein, Linker is -L1-L2-L3-L4-; Among them, L1 and L3 are independently selected from one or more connection combinations of the groups represented by the following formulas (A1)-(A14): L2 is selected from groups as shown in (A7), (A10), (A11), (A12) or (A13) above; L4 does not exist, or is selected from one or more connection combinations of groups as shown in (A1), (A2), (A7), (A8), (A10), (A11) or (A12) above; wherein, R’ is hydrogen, C1-C10 alkyl or C3-C8 cycloalkyl; j1 is an integer from 1 to 20; j2 is an integer from 1 to 20; m represents an integer from 0 to 6; Q represents wherein R2 and R3 are independently selected from H, C1-C20 alkyl, C1-C20 alkoxy, C2-C20 alkenyl or C2-C20 alkynyl; X represents wherein, R4 and R5 are independently selected from H, fluorine, hydroxyl, C1-C20 alkyl, C1-C20 alkoxy, C2-C20 alkenyl, C2-C20 alkynyl or R4 and R5 are directly connected to form a ring, p is an integer from 1 to 6; Z represents N or CR9, where R9 is selected from H, C1-C20 alkyl or C3-C10 cycloalkyl; represents C3-C18 cycloalkyl or C3-C18 heterocyclic group; R1 is selected from H, fluorine, hydroxyl, cyano, C1-C20 alkyl, C1-C20 alkoxy, C2-C20 alkenyl or C2-C20 alkynyl; n is an integer from 0 to 10; Two in the structure represented by formula (II) One of the junctions at the indicated site has the structure shown in (A15), and the other is linked to H or has the structure shown in (A15): wherein, E1 is OH, SH or BH2; Y is O or S.

9. The nucleic acid conjugate according to claim 8, wherein: L1 and L3 are each independently selected from one or more linking combinations of the groups shown in (A1), (A2), (A3), (A5), (A7), (A8), (A9), (A11), (A14); preferably, L1 and L3 are each independently selected from one or more of the groups shown in (A1), (A2), (A3), (A5), (A7), (A14); preferably, L1 and L3 are each independently (A5) or (A14); preferably, L1 and L3 are each independently (A5) or (A14) and j1 is an integer from 1 to 10, and j2 is an integer from 1 to 10; and / or, R' is hydrogen, C1-C5 alkyl or C3-C6 cycloalkyl; and / or, L4 is absent, or is selected from the groups shown in (A7), (A10) or (A11) above; and / or, m represents 0, 1, 2, 3, 4 or 5; and / or, R2 and R3 are each independently selected from H, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl or C2-C6 alkynyl; and / or, R4 and R5 are each independently selected from H, fluorine, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, C2-C6 alkynyl or R4 and R5 are directly linked to form a ring; and / or, p is 1, 2 or 3; and / or represents C3-C18 cycloalkyl or C3-C18 heterocyclic group; and / or, R1 is selected from H, fluorine, hydroxyl, cyano, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl or C2-C6 alkynyl; and / or, n is an integer from 0 to 5; and / or, Z represents N or CR9, where R9 is selected from H, C1-C6 alkyl or C3-C7 cycloalkyl.

10. The nucleic acid conjugate according to claim 8, wherein: L1 and L3 are each independently (A5) or (A14) and j1 is 2, 3, 4, 5 or 6, and j2 is 1, 2, 3, 4 or 5; L4 is absent, or is selected from the groups shown in (A7), (A10) or (A11) above; m is 0, 1 or 2; represents a four- to eight-membered all-carbon or nitrogen-containing saturated ring; n is 0, 1, 2 or 3.

11. The nucleic acid conjugate according to claim 9, wherein: L1 and L3 are each independently (A5) or (A14) and j1 is 3, 4, 5 or 6, and j2 is 2, 3 or 4; R', R1, R2, R3, R9 are hydrogen; m and n are 0.

12. The nucleic acid conjugate according to claim 8, wherein: The conjugate contains two, three or four consecutively linked compounds of the structure shown in formula (II) linked to any position on the oligonucleotide sequence.

13. The nucleic acid conjugate according to claim 8, wherein: The nucleic acid conjugate has any one of the following structures: where Y is O or S, is an oligonucleotide.

14. Use of the nucleic acid conjugate according to any one of claims 8 to 13 in the preparation of a medicament for the treatment and / or prevention of liver diseases.

15. A method for treating and / or preventing liver-derived diseases, characterized in that: Administering to a subject the nucleic acid conjugate according to any one of claims 8 to 13.

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