Nucleic acid conjugate as well as preparation method and application thereof

Abstract

The invention relates to a compound for forming a conjugate with oligonucleotide. The compound comprises a structure as shown in a formula (I). The invention also provides a corresponding conjugate, and an application of the conjugate in preparation of a medicine for treating and / or preventing liver-derived diseases. According to the delivery molecule, a saturated ring base and a novel connexon are used as a core skeleton to connect a phosphodiester bond, and compared with the prior art, the delivery molecule has excellent animal in-vivo biological activity and drug long-term effect, and meanwhile, the delivery molecule has the advantages that raw materials are cheap, synthesis is simple, the process is easy to develop, and oligonucleotide synthesis efficiency is good. Moreover, by improving a linker between galactosamine and a phosphodiester bond, the metabolic stability of the whole delivery molecule is improved, and the in-vivo delivery efficiency is further improved. # imgabs0 #

Description

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 the target tissue in the body and cannot enter the cell autonomously. siRNA needs to rely on special delivery vectors to achieve the enrichment of siRNA in the target organ and allow it to enter the cell in order to exert the therapeutic effect of siRNA.

[0003] Lipid nanoparticles (LNP) can encapsulate siRNA and effectively achieve liver-targeted delivery. The drug Onpattro using LNP technology is already on the market. However, there are still many shortcomings in the clinical application of LNP technology, such as: the short shelf life of LNP preparations; immune responses are generated during application, and immunosuppressants such as dexamethasone are often required in combination, which complicates the clinical application of LNP.

[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 hepatocytes, 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 delivered to hepatocytes in a targeted manner, allowing siRNA to exert a gene silencing effect on the target gene. Therefore, the development of targeted 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]

[0010] in,

[0011] Linker is -L1-L2-L3-L4-;

[0012] Among them, L1 and L3 are independently selected from one or more connection combinations of the groups represented by the following formulas (A1)-(A14):

[0013]

[0014]

[0015] L2 is selected from the groups represented by (A7), (A10), (A11), (A12) or (A13) above;

[0016] L4 does not exist, or is selected from one or more connection combinations of the groups represented by (A1), (A2), (A7), (A8), (A10), (A11) or (A12) above;

[0017] 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;

[0018] m represents an integer from 0 to 6;

[0019] 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;

[0020] 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;

[0021] Z represents N or CR9, where R9 is selected from H, C1-C20 alkyl or C3-C10 cycloalkyl;

[0022] represents C3-C18 cycloalkyl or C3-C18 heterocyclic group;

[0023] R1 is selected from H, fluorine, hydroxyl, cyano, C1-C20 alkyl, C1-C20 alkoxy, C2-C20 alkenyl or C2-C20 alkynyl;

[0024] n is an integer from 0 to 10;

[0025] represents the site of covalent bond connection of the group;

[0026] R6, R7, and R8 are independently H or K(C=O)-, where K is independently selected from one of methyl, trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, dichloromethyl, chloromethyl, ethyl, n-propyl, isopropyl, phenyl, halogenated phenyl, and alkyl phenyl.

[0027] In this application, the covalent bond connection sites in the groups represented by formula (A1)-(A14) can be interchanged. For example, when L1 is the group represented by formula (A6), the O in formula (A6) can be linked to N-acetylgalactosamine, and C is linked to L2, or the O in formula (A6) can be linked to L2, and C is linked to N-acetylgalactosamine.

[0028] 2. For the compound according to clause 1, L1 and L3 are independently selected from one or more connection combinations of the groups represented by (A1), (A2), (A3), (A5), (A7), (A8), (A9), (A11), (A14); in this clause, L2, L4, R', j1, j2, m, Q, X, R1, n, R6, R7, R8, etc. are all the same as the definitions in clause 1.

[0029] 3. For the compound according to clause 2, L1 and L3 are independently selected from one or more of the groups represented by (A1), (A2), (A3), (A5), (A7), (A14); in this clause, other groups except L1 and L3 are all the same as the definitions in clause 1.

[0030] 4. For the compound according to clause 3, L1 and L3 are independently (A5) or (A14); in this clause, other groups except L1 and L3 are all the same as the definitions in clause 1.

[0031] 5. For the compound according to clause 4, L1 and L3 are independently (A5) or (A14) and 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 all the same as the definitions in clause 1.

[0032] 6. For the compound according to clause 5, L1 and L3 are independently (A5) or (A14) and 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 all the same as the definitions in clause 1.

[0033] 7. The compound according to clause 6, wherein L1 and L3 are independently of each other (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 those defined in clause 1.

[0034] 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 those defined in any one of clauses 1 to 8.

[0035] 9. The compound according to clause 8, wherein R' is hydrogen; in this clause, other groups except R' are the same as those defined in any one of clauses 1 to 8.

[0036] 10. The compound according to clause 1, wherein L4 does not exist or is selected from the groups shown in (A7), (A10) or (A11) above; in this clause, other groups except L4 are the same as those defined in any one of clauses 1 to 9.

[0037] 11. The compound according to clause 1, wherein m represents 0, 1, 2, 3, 4 or 5; preferably, m is 0, 1 or 2; in this clause, other groups except m are the same as those defined in any one of clauses 1 to 10.

[0038] 12. The compound according to clause 1, wherein 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.

[0039] 13. The compound according to clause 1, wherein 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; in this clause, other groups except R4 and R5 are the same as those defined in any one of clauses 1 to 12.

[0040] 14. The compound according to clause 1, wherein p is 1, 2 or 3; in this clause, other groups except p are the same as those defined in any one of clauses 1 to 13.

[0041] 15. The compound according to clause 1, represents C3-C18 cycloalkyl or C3-C18 heterocyclic group; preferably, represents a four- to eight-membered all-carbon or nitrogen-containing saturated ring; in this clause, except All other groups other than those specified in item 0 are the same as those defined in any one of items 1 to 14.

[0042] 16. For the compound according to item 1, R1 is selected from H, fluorine, hydroxyl, cyano, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl or C2-C6 alkynyl; in this item, all other groups other than R1 are the same as those defined in any one of items 1 to 15.

[0043] 17. For the compound according to item 1, n is 0, 1, 2, 3, 4 or 5; preferably, n is 0, 1, 2 or 3; in this item, all other groups other than n are the same as those defined in any one of items 1 to 16.

[0044] 18. For the compound according to item 1, R6, R7, and R8 are acetyl groups; in this item, all other groups other than R6, R7, and R8 are the same as those defined in any one of items 1 to 17.

[0045] 19. For the compound according to item 1, Z is N or CR9, where R9 is selected from H, C1-C6 alkyl or C3-C7 cycloalkyl, preferably, R9 is selected from H; in this item, all other groups other than Z are the same as those defined in any one of items 1 to 18.

[0046] 20. For the compound according to item 1, one of the two " " sites in the structure shown in formula (I) is connected to a phosphoramidite functional group, and the other is connected to a phosphoramidite functional group or a hydroxyl protecting group. In this item, all other groups in the structure shown in formula (I) are the same as those defined in any one of items 1 to 19.

[0047] 21. For the compound according to item 20, the phosphoramidite functional group has the structure shown in formula (G-1),

[0048]

[0049] 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 cyanoethyl; in this item, all other groups other than the phosphoramidite functional group are the same as those defined in any one of items 1 to 20.

[0050] 22. The compound according to clause 20. Generally speaking, a protecting group renders a chemical functional group insensitive to specific reaction conditions and can be attached to and removed from the functional group in the molecule without substantially damaging the rest of the molecule. Representative hydroxyl 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 basic conditions but can be removed under acidic conditions. In some embodiments, non-exclusive examples of hydroxyl 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'-dimethoxytrityl), and 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 hydrocarbyl acyl, trityl, 4-methoxytrityl, 4,4'-bismethoxytrityl (DMTr), and 4,4',4''-trimethoxyphenylmethyl, preferably 4,4'-bismethoxytrityl; in this clause, other groups except the phosphoramidite functional group are the same as those defined in any one of clauses 1 to 18.

[0051] 23. The compound according to any one of clauses 1 to 22 above. Alkyl means straight-chain and branched-chain having a specified number of carbon atoms, and the number is usually 1 to 20 carbon atoms, such as 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-chain alkyls having 1 to 6 carbon atoms. When referring to an alkyl residue having a specific number of carbons, it is intended to cover all branched-chain and straight-chain forms having that number of carbons; thus, for example, "butyl" means including n-butyl, sec-butyl, isobutyl, and tert-butyl; "propyl" includes n-propyl and isopropyl. An alkylene is a subset of alkyl and refers to a residue that is the same as alkyl but has two attachment points.

[0052] 24. The compound according to any one of clauses 1 to 22 above, wherein the alkenyl group refers to an unsaturated branched or straight-chain alkyl group having at least one carbon-carbon double bond, and the carbon-carbon double bond is obtained by removing one hydrogen atom from each of the adjacent carbon atoms of the parent alkyl group. This group can be in the cis or trans configuration of the double bond. Typical alkenyl groups include, but are not limited to: vinyl; 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, for example 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, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl, and so on. In certain embodiments, the alkenyl group has 2 to 20 carbon atoms, while in other embodiments, it has 2 to 10, 2 to 8, or 2 to 6 carbon atoms. An alkenylene is a subset of alkenyl and refers to a residue that is the same as alkenyl but has two attachment points.

[0053] 25. The compound according to any one of clauses 1 to 22 above, wherein the alkynyl group refers to an unsaturated branched or straight-chain alkyl group having at least one carbon-carbon triple bond, and the carbon-carbon triple bond is obtained by removing two hydrogen atoms from each of the adjacent carbon atoms of the parent alkyl group. Typical alkynyl groups include, but are not limited to: ethynyl; propynyl, such as prop-1-yn-1-yl, prop-2-yn-1-yl; butynyl, for example but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc. In certain embodiments, the alkynyl group has 2 to 20 carbon atoms, while in other embodiments, it has 2 to 10, 2 to 8, or 2 to 6 carbons.

[0054] 26. The compound according to any one of clauses 1 to 22 above, wherein the alkoxy group refers to an alkyl group of a specified number of carbon atoms connected by an oxygen bridge. For example, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, pentyloxy, 2-pentyloxy, isopentyloxy, neopentyloxy, hexyloxy, 2-hexyloxy, 3-hexyloxy, 3-methylpentyloxy, etc. The alkoxy group usually has 1 to 10, 1 to 8, 1 to 6, or 1 to 4 carbon atoms connected by an oxygen bridge.

[0055] 27. The compound according to any one of clauses 1 to 22 above, wherein the aryl group refers to a group formed by removing a hydrogen atom from a cyclic carbon atom of an aromatic monocyclic or polycyclic hydrocarbon ring system. The aromatic monocyclic or polycyclic hydrocarbon ring system contains only carbon, hydrogen, and 6 to 18 carbon atoms, and at least one ring in the ring system is completely unsaturated, that is, it contains a cyclic, delocalized (4n + 2)π according to Hückel's theory. -An electronic system. The aryl group includes, but is not limited to, groups such as phenyl, fluorenyl, and naphthyl. The arylene group is a subset of the aryl group and refers to a residue that is the same as the aryl group but has two attachment points.

[0056] 28. For the compound according to any one of the above-mentioned Clauses 1 to 22, the cycloalkyl group refers to a non-aromatic carbocyclic ring, usually having 3 to 7 ring carbon atoms. The ring can be saturated or have one or more carbon-carbon double bonds. Examples of the cycloalkyl group include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, and cyclohexenyl, as well as bridged and cage-like ring groups such as norbornane.

[0057] 29. For the compound according to any one of the above-mentioned Clauses 1 to 22, halogenation refers to fluorination, chlorination, bromination, and iodination, and the term "halogen" includes fluorine, chlorine, bromine, and iodine.

[0058] 30. For the compound according to any one of the above-mentioned Clauses 1 to 22, the heterocyclic group refers to a stable 3- to 18-membered non-aromatic ring group that contains 2 to 12 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, oxygen, and sulfur. Unless otherwise specified in the specification, the heterocyclic group is a monocyclic, bicyclic, tricyclic, or tetracyclic system and may include fused ring or bridged ring systems. The heteroatoms in the heterocyclic group can be optionally oxidized. One or more nitrogen atoms (if present) are optionally quaternized. The heterocyclic group is partially saturated or fully saturated. The heterocyclic group can be attached to the rest of the molecule through any ring atom. Examples of such heterocyclic groups include, but are not limited to: dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolinyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidinone, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxothiomorpholinyl, and 1,1-dioxothiomorpholinyl.

[0059] 31. For the compound according to Clause 1 above, the compound has any one of the following structures:

[0060]

[0061]

[0062] The second aspect of the present invention provides a nucleic acid conjugate, and the characteristics of the conjugate are stated in the following clauses:

[0063] 32. The conjugate contains one or more compounds having the structure shown in formula (II) linked to any position on the oligonucleotide sequence,

[0064]

[0065] wherein Linker, m, Q, Z, X, R1, and n are the same as defined in any one of Clauses 1 to 32;

[0066] One of the two sites shown by "" in the structure shown in formula (II) is linked to a structure shown by (A15), and the other is H or has a structure shown by (A15): ""

[0067]

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

[0069] 33. For the conjugate according to Clause 32, the conjugate contains two, three or four continuously linked compounds having the structure shown in formula (II) linked to any position on the oligonucleotide sequence.

[0070] 34. For the conjugate according to Clause 32, one or more compounds having the structure shown in formula (II) are respectively linked to any one or more positions of the oligonucleotide sequence.

[0071] 35. For the conjugate according to Clause 32, the compound having the structure shown in formula (II) is linked to the 3'-end and / or 5'-end of the oligonucleotide sequence.

[0072] 36. For the conjugate according to Clause 32, the nucleic acid conjugate has any one of the following structures:

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080] wherein Y is O or S, is an oligonucleotide.

[0081] In some embodiments, the oligonucleotide can interact with a target sequence, thereby affecting the normal function of the target sequence molecule, such as causing mRNA cleavage or translational repression or exon skipping to trigger alternative splicing of mRNA, etc. In some embodiments, the oligonucleotide can be substantially complementary to the bases of the target sequence. In some embodiments, the oligonucleotide can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% complementary to 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 a DNA-RNA chimera, or a double-stranded DNA, RNA or DNA-RNA hybrid.

[0082] In some embodiments of the present disclosure, the oligonucleotide is selected from the group consisting of small interfering RNA, microRNA, anti-microRNA, microRNA antagonist, microRNA mimic, decoy oligonucleotide, immunostimulant, G-quadruplex, alternative spliceosome, single-stranded RNA, antisense nucleic acid, aptamer, stem-loop RNA, mRNA fragment, activating RNA; optionally, the oligonucleotide is a single-stranded oligonucleotide or a double-stranded oligonucleotide; optionally, the oligonucleotide is a single-stranded oligonucleotide, and 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 counted from one end of the single-stranded oligonucleotide; optionally, the P atom in formula (A15) is connected to the end of the single-stranded oligonucleotide; 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, the double-stranded oligonucleotide contains 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 counted 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.

[0083] In some embodiments, each nucleotide in the siRNA of 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 contains nucleotide sequence 1, the antisense strand contains nucleotide sequence 2, the lengths of the nucleotide sequence 1 and the nucleotide sequence 2 are each 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 nucleotides, and at least partially reverse complementarily form a complementary double-stranded region, and at least a part of the nucleotide sequence 2 is complementary to a first nucleotide sequence, and the first nucleotide sequence is a nucleotide sequence in the target mRNA.

[0084] In some embodiments, the nucleotide sequence 1 is of the same length as the first nucleotide sequence and has no more than 3 nucleotide differences; the nucleotide sequence 2 is of the same length as the nucleotide sequence B and has no more than 3 nucleotide differences; the nucleotide sequence B is a nucleotide sequence that is completely reverse complementary to the first nucleotide sequence. Without wishing to be bound by theory, these specific nucleotide differences do not significantly reduce the target gene inhibitory ability of the siRNA conjugate, and these siRNA conjugates containing specific nucleotide differences are also within the scope of protection of the present disclosure.

[0085] In some embodiments, the nucleotide sequence 1 and the nucleotide sequence 2 are substantially reverse complementary, substantially completely reverse complementary or completely reverse complementary.

[0086] In some embodiments, the length ratio of the sense strand to the antisense strand 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.

[0087] 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 modified nucleotide groups; in some embodiments, the siRNA of the present disclosure contains modified nucleotide groups.

[0088] Currently, there are various ways available in the art for modifying siRNA, including backbone modification (also known as internucleotide linkage modification, such as phosphate group modification), ribose group modification, base modification, etc. (for example, see Watts, J.K., G.F. Deleavey and M.J. Damha, Chemically modified siRNA: tools and applications. Drug Discov Today, 2008. 13(19-20): p. 842-55, which is incorporated herein by reference in its entirety).

[0089] In the context of the present disclosure, the term "modified nucleotide" refers to a nucleotide in which the ribose group of the nucleotide is modified, such as a nucleotide or nucleotide analogue formed by substituting the 2'-hydroxyl group with another group, or a nucleotide in which the base on the nucleotide is a modified base.

[0090] 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 with a modifying group. In other words, at least a part of the phosphate groups and / or ribose groups in the phospho-sugar backbone of at least one single strand among the sense strand and the antisense strand is a phosphate group with a modifying group and / or a ribose group with a modifying 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.

[0091] In some embodiments, the siRNA of the present disclosure is an siRNA with the following modifications: both the sense strand and the antisense strand contain fluorinated modified nucleotides and non-fluorinated modified nucleotides. The fluorinated modified nucleotides are located in the aforementioned nucleotide sequence 1 and nucleotide sequence 2. The number of fluorinated modified nucleotides in nucleotide sequence 1 is no more than 9. And, in the direction from the 5'-end to the 3'-end, the nucleotides at positions 3, 5, 7, 9, 10, 11, 13, 16, and 18 of nucleotide sequence 1 are fluorinated modified nucleotides; the number of fluorinated modified nucleotides in nucleotide sequence 2 is no more than 8. And, in the direction from the 5'-end to the 3'-end, the nucleotides at positions 2, 3, 5, 8, 10, 14, 16, and 18 of nucleotide sequence 2 are fluorinated modified nucleotides. In some embodiments, the remaining nucleotides in the sense strand are methoxy-modified nucleotides; the remaining nucleotides in the antisense strand are methoxy-modified nucleotides.

[0092] In some specific embodiments of the siRNA of the present disclosure, the nucleotide contains a phosphate group modification. In the context of the present disclosure, the phosphate group modification is phosphorothioate modification in one embodiment, that is, one sulfur atom replaces the non-bridging oxygen atom in the phosphodiester bond, thereby replacing the phosphodiester bond with a phosphorothioate bond. In some embodiments, this modification can stabilize the structure of the siRNA and maintain high specificity and high affinity for base pairing.

[0093] According to some embodiments of the present disclosure, in the siRNA, the phosphorothioate linkage exists in at least one of the groups composed 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 of the above. In some embodiments, the phosphorothioate linkage exists at all the above positions except the 5'-end of the sense strand. In some embodiments, the phosphorothioate linkage exists at all the above positions except the 3'-end of the sense strand. In some embodiments, the phosphorothioate linkage exists in at least one of the following positions:

[0094] The connection between the 1st nucleotide and the 2nd nucleotide at the 5'-end of the sense strand;

[0095] The connection between the 2nd nucleotide and the 3rd nucleotide at the 5'-end of the sense strand;

[0096] The connection between the 1st nucleotide and the 2nd nucleotide at the 3'-end of the sense strand;

[0097] The connection between the 2nd nucleotide and the 3rd nucleotide at the 3'-end of the sense strand;

[0098] The connection between the 1st nucleotide and the 2nd nucleotide at the 5'-end of the antisense strand;

[0099] The connection between the 2nd nucleotide and the 3rd nucleotide at the 5'-end of the antisense strand;

[0100] The connection between the 1st nucleotide and the 2nd nucleotide at the 3'-end of the antisense strand; and

[0101] The connection between the 2nd nucleotide and the 3rd nucleotide at the 3'-end of the antisense strand.

[0102] The third aspect of the present invention provides the use of the above nucleic acid conjugate in the preparation of a medicament for treating and / or preventing liver-derived diseases.

[0103] Due to the application of the above technical solutions, the present invention has the following advantages compared with the prior art:

[0104] The delivery molecule of the present invention patent uses a saturated cyclic group supplemented with a novel linker as the core backbone to connect the phosphodiester bond. Compared with the prior art, it has excellent in vivo biological activity and long-acting drug properties in animals. At the same time, it has the advantages of cheap raw materials, simple synthesis, easy process development, and good oligonucleotide synthesis efficiency. Moreover, the present invention improves the linker between galactosamine and the phosphodiester bond, improves the metabolic stability of the entire delivery molecule, and thus improves the in vivo delivery efficiency. Description of the Drawings

[0105] Figure 1 The activity data of the conjugate in mice in Example 6, where * represents a P value less than 0.05. Detailed Embodiments

[0106] It should be noted that unless otherwise defined, the technical terms or scientific terms used in this application should have the ordinary meaning understood by those skilled in the art. The experimental methods in the following examples are all conventional methods unless otherwise specified. The raw materials, reagent materials, etc. used in the following examples are all commercially available products unless otherwise specified.

[0107] The technical solution provided by the present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and will not limit the protection scope of the present invention.

[0108] Preparation of Compound SA102 (i.e., I-3) in Example 1

[0109] In this embodiment, the synthetic route of compound SA102 (i.e., I-3) is as follows:

[0110]

[0111] 1.1 Preparation of Intermediate 1-1

[0112]

[0113] Place 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) in a clean and dry reaction flask, add 50 mL of acetonitrile, and slowly add tert-butyldimethylchlorosilane (1.3 equiv, 21.9 mmol, 3.31 g) at room temperature. Then stir at room temperature for another 12 hours. After the reaction, add 100 mL of ethyl acetate to the reaction solution, and wash it with 100 mL of saturated sodium bicarbonate solution and 100 mL of saturated brine. Dry the organic phase, filter and concentrate. The obtained crude product is separated and purified by silica gel column chromatography (gradient elution: petroleum ether / ethyl acetate = 20 / 1 - 5 / 1) to obtain colorless oily compound 1-1 (4.9 g, 16.8 mmol, 99% yield). The molecular formula of compound 1-1 is C 17 H 29 ONSi, molecular weight: 291.2, LC-MS found 292.4 (M+H).

[0114] 1.2 Preparation of Intermediate 1-2

[0115]

[0116] Place compound 1-1 (16.8 mmol, 4.9 g) in a clean and dry reaction flask, add 100 mL of methanol, and add palladium on carbon (wet basis, 10% Pd / C) (10% wt, 490.0 mg) under hydrogen at room temperature. Then stir at room temperature for another 12 hours. After the reaction, filter off the palladium on carbon, and concentrate the filtrate to obtain crude product white solid compound 1-2 (3.31 g, 16.5 mmol, 98% yield), which is directly used in the next step without purification. The molecular formula of compound 1-2 is C 10 H 23ONSi, Molecular weight: 201.1, LC-MS found 202.3 (M+H).

[0117] 1.3 Preparation of Intermediate 1-3

[0118]

[0119] Place N-benzyloxycarbonyl-L-serine (commercially available, purchased from Shanghai Titan Scientific Co., Ltd.) (15.0 mmol, 3.58 g) in a clean and dry reaction flask, add 100 mL of dichloromethane, and add benzotriazol-1-yl-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) at room temperature. Then stir at room temperature for 1 hour. After the reaction, add 150 mL of dichloromethane to the reaction solution, and wash with 150 mL of saturated sodium bicarbonate solution and 150 mL of saturated brine. Dry the organic phase, filter and concentrate. The obtained crude product is separated and purified by silica gel column chromatography (gradient elution: petroleum ether / ethyl acetate = 10 / 1 - 1 / 3) to obtain white solid Compound 1-3 (4.5 g, 10.65 mmol, 63% two-step yield). The molecular formula of Compound 1-3 is C 21 H 34 O5N2Si, Molecular weight: 422.2, LC-MS found 423.3 (M+H). 1 H NMR (400 MHz, CDCl3): δ 7.35–7.29 (m, 5H), 5.96 (dd, J = 14.3, 8.3 Hz, 1H), 5.10 (s, 2H), 4.61–4.40 (m, 2H), 3.85–3.68 (m, 2H), 3.65–3.49 (m, 2H), 3.41 (d, J = 12.7 Hz, 1H), 3.31 (s, 1H), 1.95 (qdd, J = 15.0, 11.8, 5.3 Hz, 2H), 1.77 (s, 1H), 0.86 (s, 9H), 0.06 (d, J = 3.1 Hz, 6H).

[0120] 1.4 Preparation of Intermediate 1-4

[0121]

[0122] Compound 1-3 (10.65 mmol, 4.5 g) was placed in a clean and dry reaction flask, 100 mL of pyridine was added, and 4,4'-dimethoxytriphenylmethyl chloride (1.2 equiv, 12.78 mmol, 4.32 g) was added at room temperature. Subsequently, the mixture was stirred at room temperature for another 12 hours. After the reaction, 150 mL of ethyl acetate was added to the reaction solution, and it 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 obtained crude product was separated and purified by silica gel column chromatography (gradient elution: petroleum ether / ethyl acetate = 20 / 1 - 1 / 1), and a pale yellow oily compound 1-4 (7.56 g, 10.43 mmol, 98% yield) was obtained. The molecular formula of compound 1-4 is C 42 H 52 O7N2Si, molecular weight: 724.3, LC-MS found 747.4 (M+Na). 1 1H NMR (400 MHz, CDCl3): δ 7.35–7.31 (m, 1H), 7.28 (d, J = 4.7 Hz, 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, 5.4 Hz, 4H), 5.72 (dd, J = 22.7, 8.3 Hz, 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, 3.7 Hz, 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 = 13.1 Hz, 9H), -0.02 (dd, J = 14.9, 4.2 Hz, 6H).

[0123] 1.5 Preparation of Intermediate 1-5

[0124]

[0125] Compound 1-4 (10.43 mmol, 7.56 g) was placed in a clean and dry reaction flask, 100 mL of methanol was added, and palladium on carbon (wet basis, 10% Pd / C) (10% wt, 750.0 mg) was added under hydrogen at room temperature. Subsequently, the mixture was stirred at room temperature for another 12 hours. After the reaction, the palladium on carbon was removed by filtration, and the filtrate was concentrated to obtain a crude product, a white solid compound 1-5 (6.0 g, 10.22 mmol, 98% yield), which was directly used in the next step of the reaction without purification. The molecular formula of compound 1-5 is C 34 H 46O5N2Si, Molecular weight: 590.3, LC-MS found 591.6 (M+H).

[0126] 1.6 Preparation of Intermediate 1-6

[0127]

[0128] Place compound 1-5 (10.22 mmol, 6.0 g) in a clean and dry reaction flask, add 100 mL of dichloromethane, add 4-dimethylaminopyridine (30 mol%, 3.07 mmol, 374.6 mg) and N,N-diisopropylethylamine (3.0 equiv, 30.66 mmol, 3.96 g) at room temperature, and then add adipic anhydride (1.5 equiv, 15.33 mmol, 1.96 g) to the reaction system. Stir at room temperature for another 4 hours. After the reaction, directly concentrate the reaction solution, and purify the obtained crude product by silica gel column chromatography (gradient elution: dichloromethane / methanol = 50 / 1 - 8 / 1) to obtain white solid compound 1-6 (5.28 g, 7.36 mmol, 72% yield). The molecular formula of compound 1-6 is C 40 H 54 O8N2Si, Molecular weight: 718.3, LC-MS found 717.3 (M-H).

[0129] 1.7 Preparation of Intermediate 1-8

[0130]

[0131] Place compound 1-6 (7.36 mmol, 5.28 g) in a clean and dry reaction flask, add 100 mL of dichloromethane, add 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 WuXi AppTec Co., Ltd., Tianjin) (1.1 equiv, 8.1 mmol, 4.31 g) at room temperature. Stir at room temperature for another 1 hour. After the reaction, add 150 mL of dichloromethane to the reaction solution, wash it with 150 mL of saturated sodium bicarbonate solution and 150 mL of saturated brine, dry the organic phase, filter and concentrate, and purify the obtained crude product by silica gel column chromatography (gradient elution: dichloromethane / methanol = 50 / 1 - 10 / 1) to obtain light yellow solid compound 1-8 (6.75 g, 6.04 mmol, 82% yield). The molecular formula of compound 1-8 is C 58 H 82 O 16 N4Si, Molecular weight: 1118.5, LC-MS found 1117.3 (M-H).

[0132] 1.8 Preparation of Intermediate 1-9

[0133]

[0134] Place compound 1-8 (6.04 mmol, 6.75 g) in a clean and dry reaction flask, add 50 mL of tetrahydrofuran, add tetrabutylammonium fluoride (1.0 M in THF) (2.0 equiv, 12.08 mmol, 12.08 mL) at room temperature, and then continue to stir at room temperature for 2 hours. After the reaction, add 100 mL of ethyl acetate to the reaction solution, wash with 100 mL of saturated sodium bicarbonate solution and 100 mL of saturated brine, dry the organic phase, filter and concentrate. The obtained crude product is separated and purified by silica gel column chromatography (gradient elution: dichloromethane / methanol = 50 / 1 - 8 / 1) to obtain a pale yellow solid compound 1-9 (5.22 g, 5.19 mmol, 86% yield). The molecular formula of compound 1-9 is C 52 H 68 O 16 N4, molecular weight: 1004.4, LC-MS found 1003.3 (M-H).

[0135] 1.9 Preparation of Compound SA102 (i.e., I-3)

[0136]

[0137] Place compound 1-9 (5.19 mmol, 5.22 g) in a clean and dry reaction flask, add 50 mL of anhydrous dichloromethane. Under argon protection at room temperature, add compound 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (2.0 equiv, 10.38 mmol, 3.13 g) and 4,5-dicyanoimidazole (1.5 equiv, 7.79 mmol, 920.2 mg), and continue to stir at room temperature for one hour. After the reaction, add 50 mL of dichloromethane to the reaction solution, wash with 100 mL of saturated sodium bicarbonate solution, dry the organic phase, filter and concentrate. The obtained crude product is prepared by a C18 reverse-phase column (specification: 30 μm; Commercially available, purchased from Shanghai Boyun Biotechnology Co., Ltd.) (MeCN:H2O = 75%:25%) to obtain a white solid SA102 (4.44 g, 3.68 mmol, 71% yield). The molecular formula of compound SA102 is C 61 H 85 O 17 N6P, molecular weight: 1204.5, LC-MS found 1227.3 (M+Na). 11H NMR (400 MHz, DMSO-d6): δ 8.10 (q, J = 8.3 Hz, 1H), 7.80 (d, J = 9.2 Hz, 1H), 7.68 (dd, J = 8.8, 4.9 Hz, 1H), 7.35–7.28 (m, 4H), 7.20 (ddd, J = 11.2, 6.7, 2.2 Hz, 5H), 6.89–6.87 (m, 4H), 5.21 (d, J = 3.3 Hz, 1H), 4.96 (dd, J = 11.2, 3.4 Hz, 1H), 4.85–4.74 (m, 1H), 4.48 (d, J = 8.5 Hz, 2H), 4.04–3.99 (m, 3H), 3.87 (dd, J = 20.0, 8.9 Hz, 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, 4.6 Hz, 2H), 3.06–2.97 (m, 3H), 2.74 (ddd, J = 16.6, 11.8, 5.9 Hz, 1H), 2.61 (td, J = 5.8, 1.8 Hz, 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 = 6.7 Hz, 1H), 1.00 (d, J = 6.7 Hz, 1H). 31 31P NMR (162 MHz, DMSO-d6): δ 147.07 (s), 146.74 (t, J = 32.0 Hz).

[0138] Preparation of Compound SA87 (i.e., I-1) in Example 2

[0139] In this example, the synthetic route of compound SA87 (i.e., I-1) is as follows:

[0140]

[0141] 2.1 Preparation of Intermediate 2-1

[0142]

[0143] Compound 1-5 (10.0 mmol, 5.9 g) was placed in a clean and dry reaction flask, 100 mL of dichloromethane was added, and benzotriazol-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)valeric acid (commercially available, purchased from Shanghai Titan Scientific Co., Ltd.) (1.1 equiv, 11.0 mmol, 2.76 g) were added at room temperature. Stirring was continued at room temperature for 1 hour. After the reaction, 150 mL of dichloromethane was added to the reaction solution, and it 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 obtained crude product was separated and purified by silica gel column chromatography (gradient elution: dichloromethane / methanol = 50 / 1 - 10 / 1), and a pale yellow solid compound 2-1 (7.58 g, 9.2 mmol, 92% yield) was obtained. The molecular formula of compound 2-1 is C 47 H 61 O8N3Si, molecular weight: 823.4, LC-MS found 824.4 (M+H).

[0144] 2.2 Preparation of Intermediate 2-2

[0145]

[0146] Compound 2-1 (9.2 mmol, 7.58 g) was placed in a clean and 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. Subsequently, 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 a crude product, a pale yellow solid compound 2-2 (6.21 g, 9.01 mmol, 98% yield), which was directly used in the next step without purification. The molecular formula of compound 2-2 is C 39 H 55 O6N3Si, molecular weight: 689.3, LC-MS found 690.4 (M+H).

[0147] 2.3 Preparation of Intermediate 2-4

[0148]

[0149] Place compound 2-2 (9.01 mmol, 6.21 g) in a clean and dry reaction flask, add 100 mL of dichloromethane, and add benzotriazol-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-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoic acid (commercially available, purchased from WuXi AppTec Co., Ltd., Tianjin) (1.1 equiv, 9.91 mmol, 4.43 g) at room temperature, and continue to stir at room temperature for 1 hour. After the reaction, add 150 mL of dichloromethane to the reaction solution, wash it with 150 mL of saturated sodium bicarbonate solution and 150 mL of saturated brine, dry the organic phase, filter and concentrate. The obtained crude product is separated and purified by silica gel column chromatography (gradient elution: dichloromethane / methanol = 50 / 1 - 10 / 1) to obtain a pale yellow solid compound 2-4 (8.16 g, 7.3 mmol, 81% yield). The molecular formula of compound 2-4 is C 58 H 82 O 16 N4Si, molecular weight: 1118.5, LC-MS found 1117.3 (M-H).

[0150] 2.4 Preparation of Intermediate 2-5

[0151]

[0152] Place compound 2-4 (7.3 mmol, 8.16 g) in a clean and dry reaction flask, add 50 mL of tetrahydrofuran, add tetrabutylammonium fluoride (1.0 M in THF) (2.0 equiv, 14.6 mmol, 14.6 mL) at room temperature, and then continue to stir at room temperature for 2 hours. After the reaction, add 100 mL of ethyl acetate to the reaction solution, wash it with 100 mL of saturated sodium bicarbonate solution and 100 mL of saturated brine, dry the organic phase, filter and concentrate. The obtained crude product is separated and purified by silica gel column chromatography (gradient elution: dichloromethane / methanol = 50 / 1 - 8 / 1) to obtain a pale yellow solid compound 2-5 (6.38 g, 6.35 mmol, 87% yield). The molecular formula of compound 2-5 is C 52 H 68 O 16 N4, molecular weight: 1004.4, LC-MS found 1003.4 (M-H).

[0153] 2.5 Preparation of Compound SA87 (i.e., I-1)

[0154]

[0155] Place compound 2-5 (6.35 mmol, 6.38 g) in a clean and dry reaction flask, and add 50 mL of anhydrous dichloromethane. Under argon protection at room temperature, add compound 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), and continue stirring at room temperature for one hour. After the reaction, add 50 mL of dichloromethane to the reaction solution, wash it with 100 mL of saturated sodium bicarbonate solution, dry the organic phase, filter and concentrate. The obtained crude product is prepared by a C18 reversed-phase column (specification: 30 μm; commercially available, purchased from Shanghai Boyun Biotechnology Co., Ltd.) (MeCN:H2O = 75%:25%) to obtain white solid SA87 (5.58 g, 4.64 mmol, 73% yield). The molecular formula of compound SA87 is C 61 H 85 O 17 N6P, molecular weight: 1204.5, LC-MS found 1227.3 (M+Na). 11H NMR (400 MHz, DMSO-d6): δ 8.11 (q, J = 7.9 Hz, 1H), 7.81 (d, J = 9.2 Hz, 1H), 7.67 (q, J = 5.5 Hz, 1H), 7.36–7.28 (m, 4H), 7.23–7.17 (m, 5H), 6.88 (dd, J = 8.8, 1.4 Hz, 4H), 5.21 (d, J = 3.4 Hz, 1H), 4.97 (dd, J = 11.2, 3.4 Hz, 1H), 4.80 (dq, J = 21.9, 7.5 Hz, 1H), 4.48 (d, J = 8.5 Hz, 2H), 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, 4.2 Hz, 1H), 3.07–2.97 (m, 3H), 2.78–2.70 (m, 2H), 2.61 (td, J = 5.8, 1.8 Hz, 1H), 2.10 (s, 5H), 2.02 (d, J = 6.9 Hz, 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, 7.2 Hz, 2H), 1.23 (d, J = 2.8 Hz, 1H), 1.20–1.17 (m, 1H), 1.15–1.09 (m, 9H), 1.06 (d, J = 6.7 Hz, 1H), 1.00 (d, J = 6.7 Hz, 1H). 31 31P NMR (162 MHz, DMSO-d6): δ 147.06 (s), 146.76 (t, J = 24.0 Hz).

[0156] Preparation of Compound SA101 (i.e., I-2) in Example 3

[0157]

[0158] According to the synthesis method of Example 2, white solid SA101 (4.43 g, 3.68 mmol, 72% yield) was prepared. The molecular formula of Compound SA101 is C 61 H 85 O 17 N6P, molecular weight: 1204.5, LC-MS found 1227.4 (M + Na). 11H NMR (400 MHz, DMSO-d6): δ 8.14–8.07 (m, 1H), 7.80 (d, J = 9.2 Hz, 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 = 3.3 Hz, 1H), 4.97 (dd, J = 11.2, 3.4 Hz, 1H), 4.90–4.74 (m, 1H), 4.58–4.52 (m, 1H), 4.48 (d, J = 8.5 Hz, 1H), 4.04–3.98 (m, 3H), 3.91–3.83 (m, 1H), 3.74 (s, 6H), 3.70–3.60 (m, 3H), 3.58–3.52 (m, 2H), 3.49–3.44 (m, 2H), 3.39 (dt, J = 10.2, 6.2 Hz, 2H), 3.20 (dt, J = 16.0, 6.2 Hz, 1H), 3.07–2.95 (m, 3H), 2.78–2.73 (m, 2H), 2.67 (t, J = 5.9 Hz, 1H), 2.10–2.06 (m, 5H), 2.02 (d, J = 6.6 Hz, 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 = 6.7 Hz, 1H). 31 13C NMR (162 MHz, DMSO-d6): δ 146.71 (d, J = 12.2 Hz), 146.53 (d, J = 13.2 Hz).

[0159] Preparation of Compound SA123 (i.e., I-4) in Example 4

[0160] In this example, the synthetic route of compound SA123 (i.e., I-4) is as follows:

[0161]

[0162] 4.1 Preparation of Intermediate 4-1

[0163]

[0164] Compound 1-5 (10.0 mmol, 5.9 g) was placed in a clean and dry reaction flask, 100 mL of dichloromethane was added, and benzotriazol-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-carbobenzyloxy-4-aminobutyric acid (commercially available, purchased from Shanghai Titan Scientific Co., Ltd.) (1.1 equiv, 11.0 mmol, 2.61 g) were added at room temperature. Stirring was continued at room temperature for 1 hour. After the reaction, 150 mL of dichloromethane was added to the reaction solution, and it 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 obtained crude product was separated and purified by silica gel column chromatography (gradient elution: dichloromethane / methanol = 50 / 1 - 10 / 1), and a pale yellow solid compound 4-1 (7.04 g, 8.7 mmol, 87% yield) was obtained. The molecular formula of compound 4-1 is C 46 H 59 O8N3Si, molecular weight: 809.4, LC-MS found 810.4 (M+H).

[0165] 4.2 Preparation of Intermediate 4-2

[0166]

[0167] Compound 4-1 (8.7 mmol, 7.04 g) was placed in a clean and dry reaction flask, 100 mL of methanol was added, and palladium on carbon (wet basis, 10% Pd / C) (10% wt, 704.0 mg) was added under hydrogen at room temperature. Subsequently, stirring was continued at room temperature for 12 hours. After the reaction, palladium on carbon was removed by filtration, and the filtrate was concentrated to obtain a crude product, a pale yellow solid compound 4-2 (5.64 g, 8.35 mmol, 96% yield), which was directly used in the next step without purification. The molecular formula of compound 4-2 is C 38 H 53 O6N3Si, molecular weight: 675.3, LC-MS found 674.3 (M-H).

[0168] 4.3 Preparation of Intermediate 4-3

[0169]

[0170] Place compound 4-2 (8.35 mmol, 5.64 g) in a clean and dry reaction flask, add 100 mL of dichloromethane, add N,N-diisopropylethylamine (3.0 equiv, 25.05 mmol, 3.24 g) and triphosgene (0.5 equiv, 4.18 mmol, 1.24 g) at room temperature, continue stirring at room temperature for 0.5 hour, then add compound 1-7 (commercially available, purchased from WuXi AppTec Co., Ltd., Tianjin) (1.1 equiv, 9.19 mmol, 4.89 g) to the reaction system, and continue stirring at room temperature for 1 hour. After the reaction, add 150 mL of dichloromethane to the reaction solution, wash with 150 mL of saturated sodium bicarbonate solution and 150 mL of saturated brine, dry the organic phase, filter and concentrate, and purify the obtained crude product by silica gel column chromatography (gradient elution: dichloromethane / methanol = 50 / 1 - 10 / 1) to obtain pale yellow solid compound 4-3 (3.93 g, 3.51 mmol, 42% yield). The molecular formula of compound 4-3 is C 57 H 81 O 16 N5Si, molecular weight: 1119.5, LC-MS found 1118.3 (M-H).

[0171] 4.4 Preparation of Intermediate 4-4

[0172]

[0173] Place compound 4-3 (3.51 mmol, 3.93 g) in a clean and dry reaction flask, add 50 mL of tetrahydrofuran, add tetrabutylammonium fluoride (1.0 M in THF) (2.0 equiv, 7.02 mmol, 7.02 mL) at room temperature, then continue stirring at room temperature for 2 hours. After the reaction, add 100 mL of ethyl acetate to the reaction solution, wash with 100 mL of saturated sodium bicarbonate solution and 100 mL of saturated brine, dry the organic phase, filter and concentrate, and purify the obtained crude product by silica gel column chromatography (gradient elution: dichloromethane / methanol = 50 / 1 - 8 / 1) to obtain pale yellow solid compound 4-4 (3.11 g, 3.09 mmol, 88% yield). The molecular formula of compound 4-4 is C 51 H 67 O 16 N5, molecular weight: 1005.4, LC-MS found 1004.3 (M-H).

[0174] 4.5 Preparation of Compound SA123 (i.e., I-4)

[0175]

[0176] Compound 4-4 (3.09 mmol, 3.11 g) was placed in a clean and dry reaction flask, and 50 mL of anhydrous dichloromethane was added. Under argon protection at room temperature, compound 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 the mixture was stirred at room temperature for an additional hour. After the reaction, 50 mL of dichloromethane was added to the reaction solution, and the mixture was washed with 100 mL of saturated sodium bicarbonate solution. The organic phase was dried, filtered, and concentrated. The resulting crude product was purified by a C18 reverse-phase column (specification: 30 μm; commercially available, purchased from Shanghai Boyun Biotechnology Co., Ltd.) (MeCN:H2O = 75%:25%) to obtain white solid SA123 (2.57 g, 2.13 mmol, 69% yield). The molecular formula of compound SA123 is C 60 H 84 O 17 N7P, molecular weight: 1205.5, LC-MS found 1228.4 (M+Na). 11H NMR (400 MHz, DMSO-d6): δ 8.18 (q, J = 8.2 Hz, 1H), 7.82 (d, J = 9.2 Hz, 1H), 7.35–7.28 (m, 4H), 7.23–7.17 (m, 5H), 6.88 (dd, J = 8.8, 1.4 Hz, 4H), 5.80–5.74 (m, 1H), 5.21 (d, J = 3.4 Hz, 1H), 4.96 (dd, J = 11.2, 3.4 Hz, 1H), 4.85–4.74 (m, 1H), 4.49 (t, J = 9.6 Hz, 2H), 4.04–3.99 (m, 3H), 3.87 (dt, J = 11.0, 9.0 Hz, 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, 4.2 Hz, 1H), 3.03 (dt, J = 16.6, 8.2 Hz, 1H), 2.95 (dd, J = 12.5, 6.2 Hz, 4H), 2.74 (ddd, J = 16.1, 11.7, 5.9 Hz, 2H), 2.61 (td, J = 5.8, 1.9 Hz, 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, 3.1 Hz, 2H), 1.48–1.41 (m, 2H), 1.34 (dt, J = 13.8, 6.7 Hz, 2H), 1.15–1.08 (m, 10H), 1.05 (d, J = 6.7 Hz, 2H), 0.99 (d, J = 6.7 Hz, 2H). 31 31P NMR (162 MHz, DMSO-d6): δ 147.04 (s), 146.70 (d, J = 32.0 Hz).

[0177] Preparation of the siRNA Conjugate of Example 5

[0178] By the solid-phase phosphoramidite method, using the specially modified compound prepared in the above steps and commercially purchased conventional modified monomers (phosphoramidite monomers of synthetic modified nucleotides dT, Am, Cm, Gm, Um, Af, Cf, Gf, Uf, all purchased from Shanghai Zhaowei Technology Development Co., Ltd.), the nucleoside monomers were connected one by one in the 3'-5' direction according to the nucleotide arrangement order. Among them, the specially modified off-target prevention compound was placed in the seed region of the antisense strand (any position from the 4th to the 8th position counted from the 5' end), and the delivery monomer compound was freely set at the 3' end or the 5' end according to the ordinary monomer. Each connection of a nucleoside monomer included four steps of deprotection, coupling, capping, and oxidation or sulfurization reactions. The synthesis conditions used for the sense strand and the antisense strand.

[0179] Instrument model: MerMade 12 Oligonucleotide syntheizer solid-phase synthesizer, Beijing Haijing 6 mL synthesis column, Cytiva SourceTM 15Q 4.6 / 100PE purification column.

[0180] The reagents used for synthesizing siRNA conjugates were purchased from Suzhou Keloma Biotechnology Co., Ltd.

[0181] The synthesis is briefly described as follows:

[0182] The single-strand synthesis reaction proceeds in the 3’-5’ direction and is completed on a solid-phase synthesizer. It includes four main reaction steps:

[0183] a. DMTr deprotection reaction: The protecting group DMTr on the nucleotide is removed with dichloroacetic acid to obtain the 5’-hydroxyl terminus;

[0184] b. Coupling reaction: The protected nucleotide phosphoramidite monomer is mixed with the activator ethothiotetrazole. The phosphoramidite group is activated, and the 5’-hydroxyl is still protected by DMTr. It undergoes a condensation reaction with the 5’-hydroxyl group attached to the solid support to form a phosphite triester;

[0185] c. Oxidation reaction: Under the action of the oxidant iodine, the phosphite triester obtained from the above condensation reaction is converted into a more stable phosphate ester. (That is, the trivalent phosphorus is oxidized to pentavalent phosphorus);

[0186] d. Sulfurization reaction: Under the action of the thio reagent PADS (phenylacetyl disulfide), the phosphite triester obtained from the above condensation reaction is converted into a phosphorothioate (oxidation or sulfurization is selected according to the sequence design).

[0187] e. Capping reaction: There may be a very small number (less than 2%) of unreacted 5’-hydroxyl groups in the condensation reaction. They react with acetic anhydride and 1-methylimidazole to form an acetate capping that cannot participate in subsequent reactions, preventing further reactions. These short fragments can be separated during purification.

[0188] The above four-step cycle is repeated until the required sequence is synthesized.

[0189] After the last nucleoside monomer is connected, the nucleic acid sequence connected to the solid support is successively cleaved, deprotected, purified, desalted, and then lyophilized to obtain the sense strand and the antisense strand, where:

[0190] The cutting and deprotection conditions are as follows: First, prepare the ammonolysis solution (a mixed solution of ammonia water: ethanol = 3:1 with a volume of 2 mL), add the solid-phase carrier to the above reaction flask, and shake well. Ammonolysis is carried out at 50 °C in a constant-temperature water bath for 16 hours. After 16 hours of ammonolysis, cool the water bath to room temperature (25 °C ± 2 °C), filter with a sintered glass funnel, collect the filtrate in a round-bottom flask, rinse the residue with 50% aqueous ethanol solution, collect the filtrate, concentrate it with a rotary evaporator, then transfer it to a glass bottle, and take a small sample of the crude product to send to the analysis department for LC-MS detection of the crude product. The detection method is as follows: Use Waters AcquityUPLC-LTQ LCMS (column: ACQUITYUPLC BEH C18) to detect the purity of the above sense strand and antisense strand and analyze the molecular weight. The measured value is consistent with the theoretical value, as shown in Table 1.

[0191] The purification and desalting conditions are as follows: Purify using an ion exchange chromatography column and desalt using a Cytiva HiPrepTM 26 / 10 Desalting gel column, and then lyophilize the single strand. After lyophilizing the single strand, it is necessary to take a sample for LC-MS measurement.

[0192] Finally, the obtained sense strand and antisense strand need to be annealed into a double strand.

[0193] The annealing operation is as follows: Dissolve the purified sense strand and antisense strand in water for injection respectively to prepare a solution of 0.1 mg / mL - 40 mg / mL, calibrate and mix them in an equimolar ratio with Thermo Scientific Nanodrop Eight, heat at 90 °C for 5 minutes, and then slowly cool naturally to form a double-stranded structure through hydrogen bonds. Take a sample to send for SEC purity detection of the product, as shown in Table 2, and lyophilize the double-stranded sample.

[0194] Table 1 Delivery molecule conjugated siRNA numbers and sequence information

[0195]

[0196] Table 2 Double-stranded SEC-HPLC purity

[0197] Conjugate number Double-stranded SEC-HPLC purity SD003317 96％ SD004229 94％ SD004362 93％ SD004364 93％

[0198] The capital letters A, C, G, and U in Table 1 above 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'-thiophosphate; Cm represents 2'-O-methylcytidine-3'-phosphate; Cms represents 2'-O-methylcytidine-3'-thiophosphate; Gm represents 2'-O-methylguanosine-3'-phosphate; Gms represents 2'-O-methylguanosine-3'-thiophosphate; Um represents 2'-O-methyluridine-3'-phosphate; Ums represents 2'-O-methyluridine-3'-thiophosphate; Af represents 2'-fluoroadenosine-3'-phosphate; Afs represents 2'-fluoroadenosine-3'-thiophosphate; Cf represents 2'-fluorocytidine-3'-phosphate; Cfs represents 2'-fluorocytidine-3'-thiophosphate; Gf represents 2'-fluoroguanosine-3'-phosphate; Gfs represents 2'-fluoroguanosine-3'-thiophosphate; Uf represents 2'-fluorouridine-3'-phosphate; Ufs represents 2'-fluorouridine-3'-thiophosphate;

[0199]

[0200] Example 6 Testing the Activity of siRNA Conjugates in Mice

[0201] SPF-grade female C57BL / 6J mice aged 6 - 8 weeks were selected, and the body weight of the mice was 20 ± 2 g. Before administration, the above mice were weighed and their status was observed. Animals with uniform body weight and no abnormal status were randomly grouped, with 4 mice in each group. Among them, the mice in the experimental group were given the conjugate, and the mice in the vehicle group were given phosphate-buffered saline (PBS). Subcutaneous administration was performed at a dose of 1 mg / kg conjugate per mouse. 22 days after administration, mouse serum was collected, and the C5 protein level in the serum was detected by the Elisa method.

[0202] The results were expressed as the remaining expression level of the siRNA administration group compared to the vehicle group (the vehicle group was 100%). The siRNA sequences of the conjugates used for injection are shown in Table 1. As Figure 1 and Table 3 show, compared with the positive conjugate SD003317, the conjugates SD004229 and SD004362 showed comparable complement C5 protein silencing activity; the conjugate SD004364 showed significantly better complement C5 protein silencing activity than the positive conjugate SD003317.

[0203] Table 3 Relative Remaining Expression Levels of Complement C5 Protein 22 Days after Administration of the Tested Conjugates

[0204] Conjugate number Relative remaining percentage % Solvent 100.00±24.12 SD003317 13.14±3.17 SD004229 13.91±4.18 SD004362 12.13±4.22 SD004364 6.91±1.05

[0205] The above has described the present invention in detail. The purpose is to enable those skilled in this field of technology to understand the content of the present invention and implement it. However, this cannot limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit and essence of the present invention should be covered within the protection scope of the present invention.

Claims

1. A compound, characterized in that: The compound includes a structure represented by formula (I): Wherein, Linker is -L1-L2-L3-L4-; Wherein, L1 and L3 are each independently selected from one or more connection combinations of the groups represented by the following formulas (A1)-(A14): L2 is selected from the group represented by (A7), (A10), (A11), (A12) or (A13) above; L4 does not exist, or is selected from one or more connection combinations of the groups represented by (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 each independently selected from H, C1-C20 alkyl, C1-C20 alkoxy, C2-C20 alkenyl or C2-C20 alkynyl; X represents wherein, R4 and R5 are each 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 from 1 to 6; Z represents N or CR9, where R9 is selected from H, C1-C20 alkyl or C3-C10 cycloalkyl; represents a C3-C18 cycloalkyl or a 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; Indicates the site where the group is covalently bonded; R6, R7, R8 are independently H or K(C=O)-, wherein, K is independently selected from one of methyl, trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, dichloromethyl, chloromethyl, ethyl, n-propyl, isopropyl, phenyl, halogenated phenyl and alkyl phenyl.

2. The compound according to claim 1, wherein: L1 and L3 are each independently selected from one or more connection combinations of the groups represented by (A1), (A2), (A3), (A5), (A7), (A8), (A9), (A11), (A14) above; preferably, L1 and L3 are each independently selected from one or more of the groups represented by (A1), (A2), (A3), (A5), (A7), (A14) above; 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, j2 is an integer from 1 to 10; preferably, L1 and L3 are each independently (A5) or (A14) and j1 is 2, 3, 4, 5 or 6, j2 is 1, 2, 3, 4 or 5; preferably, L1 and L3 are each independently (A5) or (A14) and j1 is 3, 4, 5 or 6, j2 is 2, 3 or 4; And / or, R’ is hydrogen, C1-C5 alkyl or C3-C6 cycloalkyl; preferably, R’ is hydrogen; And / or, L4 does not exist, or is selected from the group represented by (A7), (A10) or (A11) above.

3. The compound according to claim 1, wherein: m represents 0, 1, 2, 3, 4 or 5; preferably, m is 0, 1 or 2; 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 connected to form a ring; And / or, p is 1, 2 or 3; and / or represents C3-C18 cycloalkyl or C3-C18 heterocyclic group; preferably, represents a four- to eight-membered fully carbon or nitrogen-containing saturated ring; 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; preferably, n is 0, 1, 2 or 3; And / or, Z represents N or CR9, where R9 is selected from H, C1-C6 alkyl or C3-C7 cycloalkyl.

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

5. The compound according to claim 4, characterized in that: The phosphoramidite functional group has the structure shown in 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 cyano, propyl cyano and butyl cyano, preferably, B2 is cyanoethyl; And / or, the hydroxyl protecting group is selected from any one of trityl, 4-methoxytrityl, 4,4'-dimethoxytrityl (DMTr) and 4,4',4'-trimethoxybenzyl, preferably 4,4'-dimethoxytrityl.

6. The compound according to claim 1, characterized in that: The compound has any one of the following structures:

7. A nucleic acid conjugate, characterized in that: The conjugate contains one or more compounds of the structure shown in formula (II) connected at any position on the oligonucleotide sequence, Among them, Linker, m, Q, Z, X, R1, and n are the same as any one of claims 1 to 3; One of the two sites shown is linked to a structure shown by (A15), and the other is H or has a structure shown by (A15): wherein, E1 is OH, SH or BH2; Y is O or S.

8. The nucleic acid conjugate according to claim 7, wherein: The conjugate contains two, three or four consecutively connected compounds of the structure shown in formula (II) connected at any position on the oligonucleotide sequence.

9. The nucleic acid conjugate according to claim 7, wherein: The nucleic acid conjugate has any one of the following structures: wherein Y is O or S, is an oligonucleotide.

10. Use of the nucleic acid conjugate according to any one of claims 7 to 9 in the preparation of a medicament for treating and / or preventing liver diseases.

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