Conjugate and its preparation method and use

Abstract

A compound for forming a conjugate with an active agent, such as an oligonucleotide, having a structure as shown in formula (321). The present disclosure also provides a corresponding conjugate. The conjugate disclosed herein can specifically target hepatocytes, thereby effectively solving the problems associated with the in vivo delivery of oligonucleotide drugs, has low toxicity, and maintains high stability of the delivered oligonucleotide while also having excellent delivery efficiency;

Description

[0001] This application is a divisional application of the invention patent application with application date of November 29, 2018, application number 201880049520.8, and invention name “Conjugates, preparation methods and uses thereof”. Technical Field

[0002] The present disclosure relates to the field of medicine, and in particular to a compound for delivering an active drug by forming a conjugate with the active drug, a preparation method thereof, and a use thereof. The present disclosure also relates to a conjugate formed from the compound. Background Art

[0003] Delivery system is one of the key technologies in the development of small nucleic acid drugs. One small nucleic acid delivery system is targeted conjugation delivery technology for hepatocytes. Summary of the Invention

[0004] According to one aspect of the present invention, the present disclosure provides a compound having a structure shown in formula (321):

[0005]

[0006] in:

[0007] n1 is an integer from 1 to 3, and n3 is an integer from 0 to 4;

[0008] Each m1, m2 and m3 is independently an integer from 2 to 10; R 10 、R 11 、R 12 、R 13 、R 14 and R 15 Each independently selected from H, C1-C 10 Alkyl, C1-C 10 Halogenated alkyl and C1-C 10 alkoxy;

[0009] R4 is a group capable of binding to an active drug or active agent through a covalent bond;

[0010] Each L1 is a straight chain alkylene group having a length of 1 to 70 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more selected from the group consisting of: C(O), NH, O, S, CH=N, S(O)2, C2-C 10 Alkenylene, C2-C 10 Alkynylidene, C6-C 10 Arylene, C3-C 18 Heterocyclylene and C5-C 10Heteroarylene; and wherein L1 optionally has any one or more substituents selected from the group consisting of: C1-C 10 Alkyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, C1-C 10 Haloalkyl, -OC1-C 10 Alkyl, -OC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-OH, -OC1-C 10 Halogenated alkyl, -SC1-C 10 Alkyl, -SC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-SH, -SC1-C 10 Haloalkyl, halogen substituent, -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2, -N(C1-C 10 Alkyl)(C1-C 10 Alkyl), -NH(C1-C 10 Alkyl), -N(C1-C 10 Alkyl)(C1-C 10 Alkylphenyl), -NH(C1-C 10 alkylphenyl), cyano, nitro, -CO2H, -C(O)O(C1-C 10 Alkyl), -CON(C1-C 10 Alkyl)(C1-C 10 alkyl), -CONH(C1-C 10 alkyl), -CONH2, -NHC(O)(C1-C 10 Alkyl), -NHC(O)(phenyl), -N(C1-C 10 alkyl)C(O)(C1-C 10 Alkyl), -N(C1-C 10 alkyl)C(O)(phenyl), -C(O)C1-C 10 Alkyl, -C(O)C1-C 10 Alkylphenyl, -C(O)C1-C 10 Haloalkyl, -OC(O)C1-C 10 Alkyl, -SO2(C1-C 10 Alkyl), -SO2(phenyl), -SO2(C1-C 10 Halogenated alkyl), -SO2NH2, -SO2NH(C1-C 10 Alkyl), -SO2NH(phenyl), -NHSO2(C1-C 10Alkyl), -NHSO2(phenyl) and -NHSO2(C1-C 10 haloalkyl);

[0011] each S1 is independently M1, wherein any reactive hydroxyl groups, if any, are protected by a hydroxyl protecting group;

[0012] Each M1 is independently selected from a ligand capable of binding to a cell surface receptor.

[0013] In some embodiments, each L1 is independently selected from the group consisting of groups A1-A26 and any combination thereof:

[0014]

[0015] as well as

[0016] Wherein, each j1 is independently an integer from 1 to 20;

[0017] Each j2 is independently an integer from 1 to 20;

[0018] Each R' is independently C1-C 10 alkyl;

[0019] Each Ra is independently selected from the group consisting of A27-A45 and any combination thereof:

[0020]

[0021]

[0022] Each Rb is independently C1-C 10 alkyl;

[0023] It indicates the site at which a group is attached to the rest of the molecule;

[0024] In one aspect of the present invention, the present disclosure provides a conjugate having a structure shown in formula (1):

[0025]

[0026] in:

[0027] n1 is an integer from 1 to 3, and n3 is an integer from 0 to 4;

[0028] m1, m2 and m3 are each independently an integer from 2 to 10;

[0029] R 10 、R 11 、R12 、R 13 、R 14 and R 15 Each independently selected from H, C1-C 10 Alkyl, C1-C 10 Halogenated alkyl or C1-C 10 alkoxy;

[0030] R3 is the active drug;

[0031] R2 is a straight chain alkylene group having a length of 1 to 20 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more selected from the group consisting of: C(O), NH, O, S, CH=N, S(O)2, C2-C 10 Alkenylene, C2-C 10 Alkynylidene, C6-C 10 Arylene, C3-C 18 Heterocyclylene and C5-C 10 Heteroarylene; and wherein R2 optionally has any one or more substituents selected from the group consisting of: C1-C 10 Alkyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, C1-C 10 Haloalkyl, -OC1-C 10 Alkyl, -OC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-OH, -OC1-C 10 Halogenated alkyl, -SC1-C 10 Alkyl, -SC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-SH, -SC1-C 10 Haloalkyl, halogen substituent, -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2, -N(C1-C 10 Alkyl)(C1-C 10 Alkyl), -NH(C1-C 10 Alkyl), -N(C1-C 10 Alkyl)(C1-C 10 Alkylphenyl), -NH(C1-C 10 alkylphenyl), cyano, nitro, -CO2H, -C(O)O(C1-C 10 Alkyl), -CON(C1-C 10 Alkyl)(C1-C 10 alkyl), -CONH(C1-C 10alkyl), -CONH2, -NHC(O)(C1-C 10 Alkyl), -NHC(O)(phenyl), -N(C1-C 10 alkyl)C(O)(C1-C 10 Alkyl), -N(C1-C 10 alkyl)C(O)(phenyl), -C(O)C1-C 10 Alkyl, -C(O)C1-C 10 Alkylphenyl, -C(O)C1-C 10 Haloalkyl, -OC(O)C1-C 10 Alkyl, -SO2(C1-C 10 Alkyl), -SO2(phenyl), -SO2(C1-C 10 Halogenated alkyl), -SO2NH2, -SO2NH(C1-C 10 Alkyl), -SO2NH(phenyl), -NHSO2(C1-C 10 Alkyl), -NHSO2(phenyl) and -NHSO2(C1-C 10 haloalkyl);

[0032] Each L1 is independently a linear alkylene group having a length of 1 to 70 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more selected from the group consisting of: C(O), NH, O, S, CH=N, S(O)2, C2-C 10 Alkenylene, C2-C 10 Alkynylidene, C6-C 10 Arylene, C3-C 18 Heterocyclylene and C5-C 10 Heteroarylene; and wherein L1 optionally has any one or more substituents selected from the group consisting of: C1-C 10 Alkyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, C1-C 10 Haloalkyl, -OC1-C 10 Alkyl, -OC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-OH, -OC1-C 10 Halogenated alkyl, -SC1-C 10 Alkyl, -SC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-SH, -SC1-C 10 Haloalkyl, halogen substituent, -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2, -N(C1-C10 Alkyl)(C1-C 10 Alkyl), -NH(C1-C 10 Alkyl), -N(C1-C 10 Alkyl)(C1-C 10 Alkylphenyl), -NH(C1-C 10 alkylphenyl), cyano, nitro, -CO2H, -C(O)O(C1-C 10 Alkyl), -CON(C1-C 10 Alkyl)(C1-C 10 alkyl), -CONH(C1-C 10 alkyl), -CONH2, -NHC(O)(C1-C 10 Alkyl), -NHC(O)(phenyl), -N(C1-C 10 alkyl)C(O)(C1-C 10 Alkyl), -N(C1-C 10 alkyl)C(O)(phenyl), -C(O)C1-C 10 Alkyl, -C(O)C1-C 10 Alkylphenyl, -C(O)C1-C 10 Haloalkyl, -OC(O)C1-C 10 Alkyl, -SO2(C1-C 10 Alkyl), -SO2(phenyl), -SO2(C1-C 10 Halogenated alkyl), -SO2NH2, -SO2NH(C1-C 10 Alkyl), -SO2NH(phenyl), -NHSO2(C1-C 10 Alkyl), -NHSO2(phenyl) and -NHSO2(C1-C 10 haloalkyl);

[0033] Each M1 is selected from one of the ligands capable of binding to a cell surface receptor.

[0034] In some embodiments, each L1 is independently selected from the group consisting of groups A1-A26 and any combination thereof:

[0035]

[0036] as well as

[0037] Wherein, each j1 is independently an integer from 1 to 20;

[0038] Each j2 is independently an integer from 1 to 20;

[0039] Each R' is independently C1-C10 alkyl;

[0040] Each Ra is independently selected from the group consisting of formulae A27-A45 and any combination thereof:

[0041]

[0042]

[0043] Each Rb is independently C1-C 10 alkyl;

[0044] Identifies the point at which a group is attached to the rest of a molecule.

[0045] In one aspect of the present invention, provided herein is a use of the conjugate disclosed herein for preparing a medicament for treating and / or preventing a pathological state or disease caused by the expression of a specific gene in hepatocytes.

[0046] In one aspect of the present invention, provided herein is a method for treating a pathological condition or disease caused by the expression of a specific gene in hepatocytes in a subject in need thereof, the method comprising administering to the subject an effective dose of a conjugate disclosed herein.

[0047] In one aspect of the present invention, provided herein is a method for inhibiting the expression of a specific gene in hepatocytes, the method comprising contacting with a conjugate disclosed herein.

[0048] In one aspect of the present invention, provided herein is a kit comprising a conjugate disclosed herein.

[0049] Without limitation, some and other technical solutions of the present disclosure are shown in the following paragraphs 1 to 112:

[0050] Paragraph 1: A compound having a structure represented by formula (321):

[0051]

[0052] in,

[0053] n1 is an integer from 1 to 3, and n3 is an integer from 0 to 4;

[0054] Each of m1, m2 and m3 is independently an integer from 2 to 10;

[0055] Each R 10 、R 11 、R 12 、R 13 、R 14 and R 15Each independently selected from H, C1-C 10 Alkyl, C1-C 10 Halogenated alkyl and C1-C 10 alkoxy;

[0056] R4 is a group capable of binding to an active drug or active agent through a covalent bond;

[0057] Each L1 is a straight chain alkylene group having a length of 1 to 70 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more selected from the group consisting of: C(O), NH, O, S, CH=N, S(O)2, C2-C 10 Alkenylene, C2-C 10 Alkynylidene, C6-C 10 Arylene, C3-C 18 Heterocyclylene and C5-C 10 Heteroarylene; and wherein L1 optionally has any one or more substituents selected from the group consisting of: C1-C 10 Alkyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, C1-C 10 Haloalkyl, -OC1-C 10 Alkyl, -OC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-OH, -OC1-C 10 Halogenated alkyl, -SC1-C 10 Alkyl, -SC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-SH, -SC1-C 10 Haloalkyl, halogen substituent, -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2, -N(C1-C 10 Alkyl)(C1-C 10 Alkyl), -NH(C1-C 10 Alkyl), -N(C1-C 10 Alkyl)(C1-C 10 Alkylphenyl), -NH(C1-C 10 alkylphenyl), cyano, nitro, -CO2H, -C(O)O(C1-C 10 Alkyl), -CON(C1-C 10 Alkyl)(C1-C 10 alkyl), -CONH(C1-C 10 alkyl), -CONH2, -NHC(O)(C1-C 10Alkyl), -NHC(O)(phenyl), -N(C1-C 10 alkyl)C(O)(C1-C 10 Alkyl), -N(C1-C 10 alkyl)C(O)(phenyl), -C(O)C1-C 10 Alkyl, -C(O)C1-C 10 Alkylphenyl, -C(O)C1-C 10 Haloalkyl, -OC(O)C1-C 10 Alkyl, -SO2(C1-C 10 Alkyl), -SO2(phenyl), -SO2(C1-C 10 Halogenated alkyl), -SO2NH2, -SO2NH(C1-C 10 Alkyl), -SO2NH(phenyl), -NHSO2(C1-C 10 Alkyl), -NHSO2(phenyl) and -NHSO2(C1-C 10 haloalkyl);

[0058] each S1 is independently M1, wherein any reactive hydroxyl groups, if any, are protected by a hydroxyl protecting group;

[0059] Each M1 is independently selected from a ligand capable of binding to a cell surface receptor.

[0060] Paragraph 2: The compound according to Paragraph 1, wherein each L1 is independently selected from the group consisting of groups A1-A26 and any combination thereof:

[0061]

[0062]

[0063] Wherein, each j1 is independently an integer from 1 to 20;

[0064] Each j2 is independently an integer from 1 to 20;

[0065] Each R' is independently C1-C 10 alkyl;

[0066] Each Ra is independently selected from the group consisting of groups A27-A45 and any combination thereof:

[0067]

[0068]

[0069] Each Rb is independently C1-C 10 alkyl;

[0070] Identifies the point at which a group is attached to the rest of a molecule.

[0071] Paragraph 3: The compound according to Paragraph 2, wherein L1 is selected from the group consisting of groups A1, A4, A5, A6, A8, A10, A11, A13 and connected combinations thereof.

[0072] Paragraph 4: The compound according to Paragraph 3, wherein L1 is a linked combination of at least two of the groups A1, A4, A8, A10 and A11.

[0073] Paragraph 5: The compound according to Paragraph 4, wherein L1 is a linked combination of at least two of the groups A1, A8, and A10.

[0074] Paragraph 6: A compound according to any one of paragraphs 1-5, wherein the length of L1 is 3-25 atoms, wherein the length of L1 refers to the number of chain atoms in the longest atomic chain formed by the atom connected to the N atom in the nitrogen-containing skeleton in L1 to the atom connected to S1.

[0075] Paragraph 7: A compound according to Paragraph 6, wherein L1 is 4-15 atoms in length.

[0076] Paragraph 8: The compound according to paragraph 1, wherein j1 is an integer of 2-10, j2 is an integer of 2-10, R' is a C1-C4 alkyl group, Ra is selected from the group consisting of A27, A28, A29, A30 and A31, and Rb is a C1-C5 alkyl group.

[0077] Paragraph 9: The compound according to paragraph 8, wherein j1 is an integer of 3-5, j2 is an integer of 3-5, R' is methyl, ethyl or isopropyl, Ra is A27 or A28, and Rb is methyl, ethyl, isopropyl or butyl.

[0078] Paragraph 10: The compound according to Paragraph 1, wherein n1 is an integer of 1-2, n3 is an integer of 0-1, and n1+n3=2-3.

[0079] Paragraph 11: The compound according to Paragraph 1, wherein each of m1, m2 and m3 is independently an integer of 2-5.

[0080] Paragraph 12: A compound according to Paragraph 11, wherein m1=m2=m3.

[0081] Paragraph 13: The compound according to paragraph 1, wherein the protected hydroxyl group has a YCOO- structure, wherein each Y is independently selected from the group consisting of: C1-C 10 Alkyl and C6-C10 The aryl group optionally has one or more substituents selected from the group consisting of a halogen substituent and a C1-C6 alkyl group.

[0082] Paragraph 14: A compound according to paragraph 13, wherein each Y is independently selected from the group consisting of methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl, ethyl, n-propyl, isopropyl, phenyl, halophenyl and C1-C6 alkylphenyl.

[0083] Paragraph 15: A compound according to Paragraph 1, wherein each M1 is independently a sugar.

[0084] Paragraph 16: A compound according to Paragraph 1, wherein each M1 is independently a monosaccharide, a disaccharide, a trisaccharide or a polysaccharide.

[0085] Paragraph 17: A compound according to Paragraph 1, wherein at least one M1 is modified.

[0086] Paragraph 18: The compound according to paragraph 1, wherein each M1 is independently selected from the group consisting of D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, α-D-mannofuranose, β-D-mannofuranose, α-D-mannopyranose, β-D-mannopyranose, α-D-pyranose, Glucose, β-D-glucopyranose, α-D-glucofuranose, β-D-glucofuranose, α-D-fructofuranose, α-D-fructopyranose, α-D-galactopyranose, β-D-galactopyranose, α-D-galactofuranose, β-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine , N-isobutyrylgalactosamine, 2-amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose, N-glycolyl-α-neuraminic acid, 5-thio-β-D-pyranose Glucose, 2,3,4-tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside methyl ester, 4-thio-β-D-galactopyranose, 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucopyranoside ethyl ester, 2,5-anhydro-D-allosenitrile, ribose, D-ribose, D-4-thioribose, L-ribose, and L-4-thioribose.

[0087] Paragraph 19: A compound according to Paragraph 18, wherein at least one M1 is N-acetylgalactosamine (GalNAc).

[0088] Paragraph 20: A compound according to Paragraph 18, wherein each M1 is N-acetylgalactosamine (GalNAc).

[0089] Paragraph 21: A compound according to Paragraph 1, wherein each S1 is independently one of groups A46-A54:

[0090]

[0091] Paragraph 22: A compound according to Paragraph 21, wherein S1 is A49 or A50.

[0092] Paragraph 23: A compound according to paragraph 21 or 22, wherein Y is methyl.

[0093] Paragraph 24: The compound according to paragraph 1, wherein R 10 、R 11 、R 12 、R 13 、R 14 and R 15 Each is independently H, methyl or ethyl.

[0094] Paragraph 25: A compound according to Paragraph 1, wherein R4 is a group capable of linking to an oligonucleotide via a phosphodiester bond.

[0095] Paragraph 26: A compound according to Paragraph 1, wherein R4 comprises a first functional group that can react with a group on an oligonucleotide or nucleotide to form a phosphate bond.

[0096] Paragraph 27: The compound according to paragraph 26, wherein R4 further includes a second functional group, wherein the second functional group is capable of forming a covalent bond with a hydroxyl group or an amino group, or is a solid phase support connected to the rest of the molecule through a covalent bond formed with a hydroxyl group or an amino group.

[0097] Paragraph 28: The compound according to Paragraph 26, wherein the first functional group is a phosphoramidite, a hydroxyl group, or a protected hydroxyl group.

[0098] Paragraph 29: The compound according to Paragraph 26, wherein the second functional group is phosphoramidite, carboxyl or carboxylate.

[0099] Paragraph 30: The compound according to Paragraph 27, wherein the solid support is linked to the rest of the molecule via a phosphate bond, a carboxylate bond and / or an amide bond.

[0100] Paragraph 31: The compound according to paragraph 30, wherein the solid phase support is a resin.

[0101] Paragraph 32: The compound according to Paragraph 28, wherein the carboxylate is a carboxylate with a metal cation, an ammonium salt, a tertiary amine or a quaternary ammonium ion.

[0102] Paragraph 33: The compound according to paragraph 32, wherein the carboxylate is triethylamine carboxylate or N,N-diisopropylethylamine carboxylate.

[0103] Paragraph 34: The compound according to paragraph 26, wherein R4 comprises hydroxyl, -OR k Or a group represented by formula (C3):

[0104]

[0105] Among them, R k is a hydroxyl protecting group, Identifies the point at which a group is attached to the rest of a molecule.

[0106] Paragraph 35: A compound according to Paragraph 27, wherein R4 comprises a group represented by formula (C1), (C2), (C3), (C1') or (C3'):

[0107]

[0108] In the formula, q1 is an integer of 1-4, X is O or NH, M + is a cation, R k is a hydroxyl protecting group, SPS represents a solid phase support, Identifies the point at which a group is attached to the rest of a molecule.

[0109] Paragraph 36: A compound according to paragraph 26 or 27, wherein R4 has a structure represented by formula (B9), (B10), (B11), (B12), (B9'), (B10'), (B11') or (B12'):

[0110]

[0111]

[0112] Wherein, q1 is an integer of 1-4, q2 is an integer of 1-10, X is O or NH, M + is a cation, SPS represents a solid phase support, R k is a hydroxyl protecting group,

[0113] Identifies the point at which a group is attached to the rest of a molecule.

[0114] Paragraph 37: The compound according to paragraph 36, wherein q2 is an integer of 1-5; q1 is 1 or 2.

[0115] Paragraph 38: The compound according to Paragraph 26, wherein the compound has a structure shown in Formula (403)-(442):

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128] Wherein, X is O or NH, M + is a cation, R k is a hydroxyl protecting group, and SPS represents a solid phase support. Paragraph 39: A compound according to any one of paragraphs 34-38, wherein M + is an alkali metal cation, an ammonium cation, a cation formed by a tertiary amine, or a quaternary ammonium cation, R k is trityl, 4-methoxytrityl, 4,4'-bismethoxytrityl or 4,4',4"-trimethoxytrityl, and SPS represents resin.

[0129] Paragraph 40: The compound according to Paragraph 1, wherein the receptor is a hepatocyte surface receptor.

[0130] Paragraph 41: The compound according to Paragraph 1, wherein the receptor is a receptor on the surface of a mammalian cell.

[0131] Paragraph 42: The compound according to Paragraph 1, wherein the receptor is an asialoglycoprotein receptor on human hepatocytes.

[0132] Paragraph 43: A conjugate having a structure represented by formula (1):

[0133]

[0134] in,

[0135] n1 is an integer from 1 to 3, and n3 is an integer from 0 to 4;

[0136] Each of m1, m2 and m3 is independently an integer from 2 to 10;

[0137] Each R 10 、R 11 、R 12 、R 13 、R 14 and R 15 Each independently selected from H, C1-C 10 Alkyl, C1-C 10 Halogenated alkyl and C1-C 10 alkoxy;

[0138] R3 is the active drug;

[0139] R2 is a straight chain alkylene group having a length of 1 to 20 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more selected from the group consisting of: C(O), NH, O, S, CH=N, S(O)2, C2-C 10 Alkenylene, C2-C 10 Alkynylidene, C6-C 10 Arylene, C3-C 18 Heterocyclylene and C5-C 10 Heteroarylene; and wherein R2 optionally has any one or more substituents selected from the group consisting of: C1-C 10 Alkyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, C1-C 10 Haloalkyl, -OC1-C 10 Alkyl, -OC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-OH, -OC1-C 10 Halogenated alkyl, -SC1-C 10 Alkyl, -SC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-SH, -SC1-C 10Haloalkyl, halogen substituent, -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2, -N(C1-C 10 Alkyl)(C1-C 10 Alkyl), -NH(C1-C 10 Alkyl), -N(C1-C 10 Alkyl)(C1-C 10 Alkylphenyl), -NH(C1-C 10 alkylphenyl), cyano, nitro, -CO2H, -C(O)O(C1-C 10 Alkyl), -CON(C1-C 10 Alkyl)(C1-C 10 alkyl), -CONH(C1-C 10 alkyl), -CONH2, -NHC(O)(C1-C 10 Alkyl), -NHC(O)(phenyl), -N(C1-C 10 alkyl)C(O)(C1-C 10 Alkyl), -N(C1-C 10 alkyl)C(O)(phenyl), -C(O)C1-C 10 Alkyl, -C(O)C1-C 10 Alkylphenyl, -C(O)C1-C 10 Haloalkyl, -OC(O)C1-C 10 Alkyl, -SO2(C1-C 10 Alkyl), -SO2(phenyl), -SO2(C1-C 10 Halogenated alkyl), -SO2NH2, -SO2NH(C1-C 10 Alkyl), -SO2NH(phenyl), -NHSO2(C1-C 10 Alkyl), -NHSO2(phenyl) and -NHSO2(C1-C 10 haloalkyl);

[0140] Each L1 is independently a linear alkylene group having a length of 1 to 70 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more selected from the group consisting of: C(O), NH, O, S, CH=N, S(O)2, C2-C 10 Alkenylene, C2-C 10 Alkynylidene, C6-C 10 Arylene, C3-C 18 Heterocyclylene and C5-C 10 Heteroarylene; and wherein L1 optionally has any one or more substituents selected from the group consisting of: C1-C 10 Alkyl, C6-C10 Aryl, C5-C 10 Heteroaryl, C1-C 10 Haloalkyl, -OC1-C 10 Alkyl, -OC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-OH, -OC1-C 10 Halogenated alkyl, -SC1-C 10 Alkyl, -SC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-SH, -SC1-C 10 Haloalkyl, halogen substituent, -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2, -N(C1-C 10 Alkyl)(C1-C 10 Alkyl), -NH(C1-C 10 Alkyl), -N(C1-C 10 Alkyl)(C1-C 10 Alkylphenyl), -NH(C1-C 10 alkylphenyl), cyano, nitro, -CO2H, -C(O)O(C1-C 10 Alkyl), -CON(C1-C 10 Alkyl)(C1-C 10 alkyl), -CONH(C1-C 10 alkyl), -CONH2, -NHC(O)(C1-C 10 Alkyl), -NHC(O)(phenyl), -N(C1-C 10 alkyl)C(O)(C1-C 10 Alkyl), -N(C1-C 10 alkyl)C(O)(phenyl), -C(O)C1-C 10 Alkyl, -C(O)C1-C 10 Alkylphenyl, -C(O)C1-C 10 Haloalkyl, -OC(O)C1-C 10 Alkyl, -SO2(C1-C 10 Alkyl), -SO2(phenyl), -SO2(C1-C 10 Halogenated alkyl), -SO2NH2, -SO2NH(C1-C 10 Alkyl), -SO2NH(phenyl), -NHSO2(C1-C 10 Alkyl), -NHSO2(phenyl) and -NHSO2(C1-C 10 haloalkyl);

[0141] Each M1 is selected from one of the ligands capable of binding to a cell surface receptor.

[0142] Paragraph 44: The conjugate according to paragraph 43, wherein each L1 is independently selected from the group consisting of groups A1-A26 and any combination thereof:

[0143]

[0144]

[0145] Wherein, each j1 is independently an integer from 1 to 20;

[0146] Each j2 is independently an integer from 1 to 20;

[0147] Each R' is independently C1-C 10 alkyl;

[0148] Each Ra is independently selected from the group consisting of A27-A45 and any combination thereof:

[0149]

[0150]

[0151] Each Rb is independently C1-C 10 alkyl;

[0152] Identifies the point at which a group is attached to the rest of a molecule.

[0153] Paragraph 45: The conjugate according to paragraph 43, wherein L1 is selected from the group consisting of groups A1, A4, A5, A6, A8, A10, A11, A13 and linked combinations thereof.

[0154] Paragraph 46: The conjugate according to paragraph 45, wherein L1 is a linked combination of at least two of groups A1, A4, A8, A10, and A11.

[0155] Paragraph 47: The conjugate according to paragraph 46, wherein L1 is a linked combination of at least two of groups A1, A8, and A10.

[0156] Paragraph 48: The conjugate of any of paragraphs 43-47, wherein L1 is 3-25 atoms in length.

[0157] Paragraph 49: The conjugate of Paragraph 48, wherein L1 is 4-15 atoms in length.

[0158] Paragraph 50: The conjugate according to paragraph 43, wherein j1 is an integer of 2-10, j2 is an integer of 2-10, R' is a C1-C4 alkyl group, Ra is selected from A27, A28, A29, A30 and A31, and Rb is a C1-C5 alkyl group.

[0159] Paragraph 51: The conjugate according to paragraph 50, wherein j1 is an integer of 3-5, j2 is an integer of 3-5, R' is methyl, ethyl or isopropyl, Ra is A27 or A28, and Rb is methyl, ethyl, isopropyl or butyl.

[0160] Paragraph 52: The conjugate according to Paragraph 43, wherein n1 is an integer of 1-2, n3 is an integer of 0-1, and n1+n3=2-3.

[0161] Paragraph 53: The conjugate according to paragraph 43, wherein m1, m2 and m3 are each independently an integer from 2 to 5.

[0162] Paragraph 54: The conjugate of Paragraph 53, wherein m1 = m2 = m3.

[0163] Paragraph 55: The conjugate of Paragraph 43, wherein each M1 is independently a sugar.

[0164] Paragraph 56: The conjugate of Paragraph 43, wherein each M1 is independently a monosaccharide, a disaccharide, a trisaccharide or a polysaccharide.

[0165] Paragraph 57: The conjugate according to Paragraph 43, wherein at least one M1 is modified.

[0166] Paragraph 58: The conjugate according to paragraph 43, wherein each M1 is independently selected from the group consisting of D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, α-D-mannofuranose, β-D-mannofuranose, α-D-mannopyranose, β-D-mannopyranose, α-D-pyranose, Glucopyranose, β-D-glucopyranose, α-D-glucofuranose, β-D-glucofuranose, α-D-fructofuranose, α-D-fructopyranose, α-D-galactopyranose, β-D-galactopyranose, α-D-galactofuranose, β-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactose amine, N-isobutyrylgalactosamine, 2-amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose, N-glycolyl-α-neuraminic acid, 5-thio-β-D-pyranose Glucose, 2,3,4-tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside methyl ester, 4-thio-β-D-galactopyranose, 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucopyranoside ethyl ester, 2,5-anhydro-D-allosenitrile, ribose, D-ribose, D-4-thioribose, L-ribose, and L-4-thioribose.

[0167] Paragraph 59: The conjugate of Paragraph 58, wherein at least one M1 is N-acetylgalactosamine (GalNAc).

[0168] Paragraph 60: The conjugate of Paragraph 58, wherein each M1 is N-acetylgalactosamine (GalNAc).

[0169] Paragraph 61: The conjugate according to paragraph 43, wherein R 10 、R 11 、R 12 、R 13 、R 14 and R 15 Each is independently H, methyl or ethyl.

[0170] Paragraph 62: The conjugate of Paragraph 43, wherein R3 comprises a functional oligonucleotide.

[0171] Paragraph 63: The conjugate according to Paragraph 62, wherein R3 is a group having a structure represented by Formula A59:

[0172]

[0173] Among them, E1 is OH, SH or BH2, and Nu is a functional oligonucleotide.

[0174] Paragraph 64: The conjugate of Paragraph 63, wherein R2 is attached to the P atom on R3.

[0175] Paragraph 65: The conjugate according to Paragraph 63, wherein R2 forms a phosphate bond with the P atom in R3.

[0176] Paragraph 66: The conjugate of paragraph 43, wherein R2 is B5, B6, B5' or B6':

[0177]

[0178] in, represents the site at which the group is attached to the rest of the molecule, and q2 is an integer from 1 to 10.

[0179] Paragraph 67: The conjugate of paragraph 66, wherein the conjugate has a structure represented by formula (3), (4), (5), (6), (7), (8), (9), (10), (11), (12), (13), (14), (15), (16), (17), (18), (19), (20), (21), or (22):

[0180]

[0181]

[0182]

[0183]

[0184]

[0185]

[0186] Paragraph 68: The conjugate according to paragraph 63, wherein the functional oligonucleotide is selected from the group consisting of small interfering RNA, microRNA, anti-microRNA, microRNA antagonist, microRNA mimic, decoy oligonucleotide, immunostimulatory substance, G-quadrupole, alternative spliceosome, single-stranded RNA, antisense nucleic acid, nucleic acid aptamer, stem-loop RNA, mRNA fragment, activating RNA or DNA.

[0187] Paragraph 69: The conjugate according to paragraph 68, wherein the functional oligonucleotide is a single-stranded oligonucleotide or a double-stranded oligonucleotide.

[0188] Paragraph 70: The conjugate according to paragraph 69, wherein the functional oligonucleotide is a single-stranded oligonucleotide, the P atom in formula A59 is connected to the terminal region of the single-stranded oligonucleotide, and the terminal region of the single-stranded oligonucleotide refers to the 4 nucleotides closest to one end of the single-stranded oligonucleotide.

[0189] Paragraph 71: The conjugate of Paragraph 70, wherein the P atom in Formula A59 is attached to the terminus of the single-stranded oligonucleotide.

[0190] Paragraph 72: The conjugate of Paragraph 71, wherein the P atom in Formula A59 is linked to the 3' end of the single-stranded oligonucleotide.

[0191] Paragraph 73: The conjugate according to paragraph 69, wherein the functional oligonucleotide is a double-stranded oligonucleotide, the double-stranded oligonucleotide comprising a sense strand and an antisense strand, the P atom in formula A59 is connected to the terminal region of the double-stranded oligonucleotide, and the terminal region of the double-stranded oligonucleotide refers to the 4 nucleotides closest to one end of the sense strand or the antisense strand.

[0192] Paragraph 74: The conjugate of Paragraph 73, wherein the P atom in Formula A59 is linked to the end of the sense strand or the antisense strand.

[0193] Paragraph 75: The conjugate of Paragraph 74, wherein the P atom in Formula A59 is linked to the 3' end of the sense strand.

[0194] Paragraph 76: The conjugate according to any one of paragraphs 69-75, wherein the P atom in formula A59 is linked to the 2' position, 3' position or 5' position of the nucleotide in the functional oligonucleotide via a phosphodiester bond.

[0195] Paragraph 77: The conjugate of any of paragraphs 69 and 73-75, wherein the double-stranded oligonucleotide is siRNA.

[0196] Paragraph 78: The conjugate according to paragraph 77, wherein each nucleotide in the siRNA is independently a modified or unmodified nucleotide; 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 19 nucleotides in length and are at least partially reverse-complementary to form a double-stranded complementary region; the nucleotide sequence 2 is at least partially complementary to the first nucleotide sequence, which is a segment of nucleotides in the target mRNA; the target mRNA refers to the mRNA of a gene abnormally expressed in hepatocytes.

[0197] Paragraph 79: The conjugate of paragraph 78, wherein the target mRNA is an mRNA corresponding to ApoB, ApoC, ANGPTL3, PCSK9, SCD1, TIMP-1, Col1A1, FVII, STAT3, p53, HBV or HCV.

[0198] Paragraph 80: The conjugate according to paragraph 79, wherein the target mRNA is selected from hepatitis B virus mRNA, mRNA transcribed from angiopoietin-like protein 3 gene, or mRNA transcribed from apolipoprotein C3 gene.

[0199] Paragraph 81: The conjugate according to paragraph 78, wherein the nucleotide sequence 1 is equal to the length of the first nucleotide sequence and differs by no more than 3 nucleotides; the nucleotide sequence 2 is equal to the length of the nucleotide sequence B and differs by no more than 3 nucleotides; and the nucleotide sequence B is a nucleotide sequence that is completely reverse complementary to the first nucleotide sequence.

[0200] Paragraph 82: The conjugate according to paragraph 81, wherein the nucleotide sequence 1 differs from the first nucleotide sequence by no more than 1 nucleotide, and / or the nucleotide sequence 2 differs from the nucleotide sequence B by no more than 1 nucleotide.

[0201] Paragraph 83: The conjugate according to paragraph 81 or 82, wherein the nucleotide difference between the nucleotide sequence 2 and the nucleotide sequence B includes a difference in the Z' position of the first nucleotide on the nucleotide sequence 2 in the direction from the 5' end to the 3' end.

[0202] Paragraph 84: The conjugate according to paragraph 83, wherein, in the direction from the 5' end to the 3' end, the last nucleotide Z on the nucleotide sequence 1 is a nucleotide complementary to Z'.

[0203] Paragraph 85: The conjugate according to Paragraph 78, wherein the nucleotide sequence 1 and the nucleotide sequence 2 are substantially reverse complementary, substantially completely reverse complementary, or completely reverse complementary.

[0204] Paragraph 86: The conjugate according to paragraph 78, wherein the sense strand further contains a nucleotide sequence 3, and the antisense strand further contains a nucleotide sequence 4; the nucleotide sequence 3 and the nucleotide sequence 4 are equal in length and are both 1-4 nucleotides; the nucleotide sequence 3 is connected to the 5' end of the nucleotide sequence 1, and the nucleotide sequence 4 is connected to the 3' end of the nucleotide sequence 2; the nucleotide sequence 4 is complementary to a second segment of nucleotide sequence; the second segment of nucleotide sequence refers to a nucleotide sequence in the target mRNA that is adjacent to the first segment of nucleotide sequence and has the same length as the nucleotide sequence 4; and the nucleotide sequence 3 and the nucleotide sequence 4 are substantially completely reverse complementary or completely reverse complementary.

[0205] Paragraph 87: The conjugate according to paragraph 78 or 86, wherein the siRNA further contains a nucleotide sequence 5 having a length of 1 to 3 nucleotides and is linked to the 3' end of the antisense strand, thereby constituting a 3' overhang of the antisense strand.

[0206] Paragraph 88: The conjugate according to paragraph 87, wherein the nucleotide sequence 5 is 2 nucleotides in length, and in the 5' to 3' direction, the nucleotide sequence 5 is 2 consecutive deoxythymidine nucleotides, 2 consecutive uracil nucleotides, or complementary to a third nucleotide sequence; the third nucleotide sequence refers to a nucleotide sequence in the target mRNA that is adjacent to the first nucleotide sequence or the second nucleotide sequence and has a length equal to that of the nucleotide sequence 5.

[0207] Paragraph 89: The conjugate of any of paragraphs 78-82, 84-86 and 88, wherein 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.

[0208] Paragraph 90: The conjugate according to paragraph 89, wherein each nucleotide in the sense strand and the antisense strand is independently a fluorinated modified nucleotide or a non-fluorinated modified nucleotide, wherein the fluorinated modified nucleotide refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by fluorine; and the non-fluorinated modified nucleotide refers to a nucleotide or a nucleotide analog in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by a non-fluorinated group, and a nucleotide analog is a group that can replace a nucleotide on a nucleic acid and has a structure different from any one of adenine nucleotide, guanine nucleotide, cytosine nucleotide, uracil nucleotide or thymine nucleotide.

[0209] Paragraph 91: The conjugate according to paragraph 90, wherein both the sense strand and the antisense strand contain fluorinated-modified nucleotides and non-fluorinated-modified nucleotides, the fluorinated-modified nucleotides are present in nucleotide sequence 1 and nucleotide sequence 2, the number of fluorinated-modified nucleotides in nucleotide sequence 1 is no more than 5, and, from the 5' end to the 3' end, the 7th, 8th and 9th nucleotides of the nucleotide sequence 1 are fluorinated-modified nucleotides; the number of fluorinated-modified nucleotides in nucleotide sequence 2 is no more than 7, and, from the 5' end to the 3' end, the 2nd, 6th, 14th and 16th nucleotides of the nucleotide sequence 2 are fluorinated-modified nucleotides.

[0210] Paragraph 92: The conjugate according to paragraph 91, wherein, in the direction from the 5' end to the 3' end, in the sense strand, the nucleotides at positions 7, 8 and 9 or positions 5, 7, 8 and 9 of the nucleotide sequence 1 are fluorinated modified nucleotides, and the nucleotides at the remaining positions in the sense strand are non-fluorinated modified nucleotides; and in the direction from the 5' end to the 3' end, in the antisense strand, the nucleotides at positions 2, 6, 14, 16 or positions 2, 6, 8, 9, 14, 16 of the nucleotide sequence 2 are fluorinated modified nucleotides, and the nucleotides at the remaining positions in the antisense strand are non-fluorinated modified nucleotides.

[0211] Paragraph 93: The conjugate according to paragraph 90 or 92, wherein the nucleotide formed by replacing the hydroxyl group at the 2'-ribose group of the nucleotide with a non-fluorinated group is selected from the group consisting of: 2'-alkoxy-modified nucleotides, 2'-substituted alkoxy-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-amino-modified nucleotides, 2'-substituted amino-modified nucleotides and 2'-deoxynucleotides (DNA); and the nucleotide analogues are selected from isonucleotides, LNA, ENA, cET, UNA and GNA.

[0212] Paragraph 94: The conjugate according to paragraph 93, wherein each non-fluorinated modified nucleotide is a methoxy-modified nucleotide, wherein the methoxy-modified nucleotide refers to a nucleotide in which the 2'-hydroxyl group of the ribose group in the nucleotide is replaced by a methoxy group.

[0213] Paragraph 95: The conjugate according to paragraph 89, wherein the phosphate group having a modified group is a phosphorothioate group formed by replacing at least one oxygen atom in the phosphodiester bond in the phosphate group with a sulfur atom.

[0214] Paragraph 96: The conjugate according to paragraph 95, wherein the phosphate group having a modified group is a thiophosphate group having a structure represented by formula (201):

[0215]

[0216] Paragraph 97: The conjugate of paragraph 95, wherein the phosphorothioate linkage in the siRNA is present in at least one of the following:

[0217] between the first and second nucleotides at the 5' end of the sense strand;

[0218] between the second and third nucleotides at the 5' end of the sense strand;

[0219] between the first and second nucleotides at the 3' end of the sense strand;

[0220] between the second and third nucleotides at the 3' end of the sense strand;

[0221] between the first and second nucleotides at the 5' end of the antisense strand;

[0222] between the second and third nucleotides at the 5' end of the antisense strand;

[0223] between the first and second nucleotides at the 3' end of the antisense strand; and

[0224] between the second and third nucleotides at the 3' end of the antisense strand.

[0225] Paragraph 98: The conjugate of paragraph 89, 90 or 95, wherein the 5' terminal nucleotide of the antisense strand is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide.

[0226] Paragraph 99: The conjugate according to paragraph 98, wherein the 5'-phosphate nucleotide or 5'-phosphate analog modified nucleotide is a nucleotide represented by one of formula (202) to formula (206):

[0227]

[0228] Here, R represents a group selected from the group consisting of H, OH, F and methoxy, and Base represents a base selected from A, U, C, G and T.

[0229] Paragraph 100: The conjugate according to paragraph 99, wherein the 5'-phosphate nucleotide or 5'-phosphate analog modified nucleotide is a nucleotide represented by formula (202), formula (203) or formula (205).

[0230] Paragraph 101: Use of the conjugate according to any one of paragraphs 43-100 for the preparation of a medicament for treating and / or preventing a pathological condition or disease caused by the expression of a specific gene in hepatocytes.

[0231] Paragraph 102: The use according to paragraph 101, wherein the specific gene is selected from the group consisting of: hepatitis B virus gene, angiopoietin-like protein 3 gene and apolipoprotein C3 gene.

[0232] Paragraph 103: The use according to paragraph 101, wherein the disease is selected from the group consisting of chronic liver disease, hepatitis, liver fibrosis, liver proliferative disease and dyslipidemia.

[0233] Paragraph 104: The use according to paragraph 103, wherein the dyslipidemia is hypercholesterolemia, hypertriglyceridemia or atherosclerosis.

[0234] Paragraph 105: A method of treating a pathological condition or disease caused by expression of a specific gene in hepatocytes in a subject in need thereof, the method comprising administering to the subject an effective amount of a conjugate of any of paragraphs 43-100.

[0235] Paragraph 106: The method according to Paragraph 105, wherein the specific gene is selected from the group consisting of: hepatitis B virus gene, angiopoietin-like protein 3 gene and apolipoprotein C3 gene.

[0236] Paragraph 107: The method of Paragraph 105, wherein the disease is selected from the group consisting of chronic liver disease, hepatitis, liver fibrosis, liver proliferative disease and dyslipidemia.

[0237] Paragraph 108: The method according to Paragraph 107, wherein the dyslipidemia is hypercholesterolemia, hypertriglyceridemia or atherosclerosis.

[0238] Paragraph 109: A method of inhibiting the expression of a specific gene in a hepatocyte, the method comprising contacting the hepatocyte with the conjugate of any of paragraphs 43-100.

[0239] Paragraph 110: The method according to paragraph 109, wherein the specific gene is ApoB, ApoC, ANGPTL3, PCSK9, SCD1, TIMP-1, Col1A1, FVII, STAT3, p53, HBV or HCV.

[0240] Paragraph 111: The method according to Paragraph 110, wherein the specific gene is selected from the group consisting of: hepatitis B virus gene, angiopoietin-like protein 3 gene and apolipoprotein C3 gene.

[0241] Paragraph 112: A kit comprising the conjugate of any of paragraphs 43-100.

[0242] Other features and advantages of the present disclosure will be described in detail below.

[0243] Incorporated by reference

[0244] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0245] The novel features of the invention are set forth with particularity in the appended claims. The features and advantages of the invention will be better understood from the following detailed description of illustrative embodiments utilizing the principles of the invention, and the accompanying drawings, in which:

[0246] Figure 1A and 1B Shown are semi-quantitative results of the stability test of siRNA conjugates in Tritosomes in vitro.

[0247] Figure 2A and 2B Shown are semi-quantitative results of an in vitro stability assay testing siRNA conjugates in human plasma.

[0248] Figure 3A and 3B Shown are semi-quantitative results of an in vitro stability assay testing siRNA conjugates in monkey plasma.

[0249] Figure 4-11 To show the following PK / TK plasma concentration or tissue concentration over time metabolism curve: At a dose of 10 mg / kg, conjugate 24 in rat plasma ( Figure 4 ); At a dose of 10 mg / kg, conjugate 24 was expressed in rat liver and kidney ( Figure 5 ); At a dose of 50 mg / kg, conjugate 24 in rat plasma ( Figure 6 ); At a dose of 50 mg / kg, conjugate 24 was expressed in rat liver and kidney ( Figure 7 ); At a dose of 10 mg / kg, conjugate 25 in rat plasma ( Figure 8 ); At a dose of 10 mg / kg, conjugate 25 was expressed in rat liver and kidney ( Figure 9 ); At a dose of 50 mg / kg, conjugate 25 in rat plasma ( Figure 10 ); At a dose of 50 mg / kg, conjugate 25 was expressed in rat liver and kidney ( Figure 11 ).

[0250] Figure 12A , 12B, 12C and 12D show the IC values ​​of conjugate 24 for inhibiting GSCM expression, respectively. 50 The values ​​were determined by comparing with GSSM, PSCM and PSSM respectively.

[0251] Figure 13-15 The inhibitory effect of the conjugates of the present disclosure on HBV mRNA in vivo was shown.

[0252] Figure 16 Shown is the time-dependent inhibitory effect of the conjugate of the present disclosure on HBsAg expression in the serum of HBV transgenic mice.

[0253] Figure 17 Shown is the time-dependent inhibitory effect of the conjugate of the present disclosure on HBV DNA expression in the serum of HBV transgenic mice.

[0254] Figure 18 Shown is the time-dependent inhibitory effect of conjugate 25 of the present disclosure on HBsAg expression in the serum of HBV transgenic mice.

[0255] Figure 19 Shown are the time-dependent inhibitory effects of the conjugates of the present disclosure on HBsAg expression in the M-Tg model.

[0256] Figure 20 Shown are the time-dependent inhibitory effects of the conjugates of the present disclosure on HBsAg expression in the M-Tg model.

[0257] Figure 21 Shown are the time-dependent inhibitory effects of the conjugates of the present disclosure on HBsAg expression in the 1.28 copy HBV-Tg model.

[0258] Figure 22-24 The inhibitory effects of the conjugates of the present disclosure on target mRNA relative to off-target mRNA are shown.

[0259] Figures 25-27 The inhibitory effect of the conjugates of the present disclosure on HBV mRNA in vivo was shown.

[0260] Figure 28 Shown is the time-dependent inhibitory effect of the conjugate of the present disclosure on HBsAg expression in the serum of HBV transgenic mice.

[0261] Figure 29 Shown is the time-dependent inhibitory effect of the conjugate of the present disclosure on HBsAg expression in the serum of HBV transgenic mice.

[0262] Figure 30 The in vivo inhibitory effect of the conjugates of the present disclosure on HBV mRNA at day 85 is shown.

[0263] Figure 31 Shown is the time-dependent inhibitory effect of conjugate 43 on HBsAg expression in the serum of HBV transgenic mice.

[0264] Figure 32 Shown is the time-dependent inhibitory effect of conjugate 43 on HBV DNA expression in the serum of HBV transgenic mice.

[0265] Figure 33 and 34 The stability of conjugate 167 in human and murine lysosomal lysates is shown.

[0266] Figure 35 Shown is the time-dependent inhibitory effect of conjugate 168 on HBsAg expression in the 1.28 copy HBV-Tg model.

[0267] Figure 36 Shown is the time-dependent inhibitory effect of conjugate 168 on HBeAg expression in the 1.28 copy HBV-Tg model.

[0268] Figure 37 Shown is the time-dependent inhibitory effect of conjugate 168 on HBV DNA expression in the 1.28 copy HBV-Tg model.

[0269] Figure 38A and 38B The inhibition rates of ANGPTL mRNA expression at day 14 and day 28 are shown.

[0270] Figure 39A and 39B The inhibition rate of the conjugates of the present disclosure on blood lipids is shown, expressed as total cholesterol (CHO) and triglyceride (TG) in serum.

[0271] Figure 40A and 40B Shown are the inhibition rates of conjugate 115 on the changes in blood lipids over time, expressed as serum total cholesterol (CHO) and triglycerides (TG).

[0272] Figure 41A and 41BShown are the inhibition rates of conjugates 115 and 111 on the time-dependent changes in blood lipids, expressed as serum total cholesterol (CHO) and triglycerides (TG).

[0273] Figure 42A 、 42B , 42C and 42D show the inhibition rate of conjugate 111 on the changes in blood lipids over time at different doses, expressed as serum total cholesterol (CHO) and triglycerides (TG).

[0274] Figure 43A and 43B Shown are the inhibition rates of conjugates 25 and 169 on the time-dependent changes in blood lipids, expressed as serum total cholesterol (CHO) and triglycerides (TG); Figure 43C Shown are the inhibition rates of ANGPTL mRNA expression.

[0275] Figure 44A The inhibition rate of APOC3 expression in the liver on day 14 is shown. Figure 44B and 44C The inhibition rates of different doses of conjugate 144 on blood lipids are shown, expressed as serum total cholesterol (CHO) and triglyceride (TG).

[0276] Figure 45A and Figure 45B The inhibition rates of different doses of conjugate 170 on blood lipids are shown, expressed as serum total cholesterol (CHO) and triglyceride (TG).

[0277] Figure 46A 、 46B , 46C and 46D show the inhibition rate of the conjugates of the present disclosure on blood lipids, expressed as serum total cholesterol (CHO) and triglycerides (TG). DETAILED DESCRIPTION

[0278] The following describes the specific embodiments of the present disclosure in detail. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present disclosure, and are not intended to limit the present disclosure in any aspect.

[0279] definition

[0280] In the context of the present disclosure, unless otherwise specified, capital letters C, G, U and A represent the base composition of a nucleotide; a lowercase letter m represents that the nucleotide adjacent to the left of the letter m is a 2'-methoxy-modified nucleotide; a lowercase letter f represents that the nucleotide adjacent to the left of the letter f is a 2'-fluorine-modified nucleotide; a lowercase letter s represents that the two nucleotides adjacent to the left and right of the letter s are connected by a phosphorothioate group; and P1 represents that the nucleotide adjacent to the right of P1 is a 5'-phosphate nucleotide or a 5'-phosphate analogue-modified nucleotide, especially a vinyl phosphate-modified nucleotide (represented by VP in the following examples), a 5'-phosphate nucleotide (represented by P in the following examples) or a 5'-phosphorothioate-modified nucleotide (represented by Ps in the following examples).

[0281] In the context of the present disclosure, the expressions "complementary" or "reverse complementary" are used interchangeably and have the meanings well known in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one chain are each paired with bases on the other chain in a complementary manner. In DNA, the purine base adenine (A) is always paired with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (G) is always paired with the pyrimidine base cytosine (C). Each base pair consists of a purine and a pyrimidine. When adenine on a chain is always paired with thymine (or uracil) on the other chain, and guanine is always paired with cytosine, the two chains are considered complementary and the base sequence of the chain can be inferred from the sequence of its complementary chain. Correspondingly, a "mismatch" refers to the fact that the bases at corresponding positions in a double-stranded nucleic acid do not exist in the form of complementary pairs.

[0282] In the context of the present disclosure, unless otherwise specified, "substantially reverse complementary" means that there are no more than 3 base mismatches in two nucleotide sequences; "substantially completely reverse complementary" means that there is no more than 1 base mismatch in two nucleotide sequences; and "completely reverse complementary" means that there are no base mismatches in two nucleotide sequences.

[0283] In the context of the present disclosure, a "nucleotide difference" between one nucleotide sequence and another nucleotide sequence refers to a change in the base of the nucleotide at the same position between the nucleotide sequences. For example, if the nucleotide base in the second sequence is A, and the base at the same position in the first sequence is U, C, G, or T, the two sequences are considered to have a nucleotide difference at that position. In some embodiments, when a nucleotide at a position is replaced by an abasic nucleotide or a nucleotide analog, a nucleotide difference is also considered to exist at that position.

[0284] In the present disclosure, particularly when describing the method for preparing conjugated molecules or siRNA conjugates of the present disclosure, unless otherwise indicated, nucleoside monomers (nucleoside monomer) refer to, according to the RNA sequence for preparation, described " unmodified or modified RNA phosphoramidite (unmodified or modified RNA phosphoramidite) ", are respectively used for so-called solid phase phosphoramidite synthesis, and solid phase phosphoramidite synthesis is the well-known method for synthesizing RNA in this area. In other places in the text, RNA phosphoramidite is also referred to as nucleoside phosphoramidite (nucleoside phosphoramidite). The nucleoside monomers used in the present disclosure all can be commercially obtained.

[0285] As used herein, a hyphen ("-") that is not between two letters or symbols is used to indicate a point of attachment for a substituent. For example: -C1-C 10 Alkyl-NH2 through C1-C 10 Alkyl group connected.

[0286] As used herein, "optional" or "optionally" means that the event or circumstance described thereafter may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, "optionally substituted alkyl" includes "alkyl" and "substituted alkyl" as defined below. It will be understood by those skilled in the art that for any group containing one or more substituents, these groups are not intended to introduce any substitution or substitution pattern that is sterically unrealistic, synthetically unfeasible, and / or inherently unstable.

[0287] As used herein, "alkyl" refers to straight and branched chains 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 and branched chain alkyl groups of 1 to 6 carbon atoms. When referring to an alkyl residue having a specific number of carbons, it is intended to encompass all branched and straight chain forms having that number of carbons; thus, for example, "butyl" is meant to include n-butyl, sec-butyl, isobutyl, and tert-butyl; "propyl" includes n-propyl and isopropyl. Alkylene is a subset of alkyl and refers to residues that are the same as alkyl, but have two points of attachment.

[0288] As used herein, "alkenyl" refers to an unsaturated branched or straight chain alkyl group having at least one carbon-carbon double bond, wherein the carbon-carbon double bond is obtained by removing a molecule of hydrogen from the adjacent carbon atoms of the parent alkyl group. The group can be in cis or trans configuration with respect to the double bond. Typical alkenyl groups include, but are not limited to, vinyl; propenyl, such as prop-1-ene-1-yl, prop-1-ene-2-yl, prop-2-ene-1-yl (allyl), prop-2-ene-2-yl; butenyl, such as but-1-ene-1-yl, but-1-ene-2-yl, 2-methylprop-1-ene-1-yl, but-2-ene-1-yl, but-2-ene-2-yl, but-1,3-diene-1-yl, but-1,3-diene-2-yl, etc. In certain embodiments, the alkenyl group has 2 to 20 carbon atoms, and in other embodiments, has 2 to 10, 2 to 8, or 2 to 6 carbon atoms. Alkenylene is a subset of alkenyl and refers to residues that are identical to alkenyl but have two points of attachment.

[0289] As used herein, "alkynyl" refers to an unsaturated branched or straight chain alkyl group having at least one carbon-carbon triple bond, obtained by removing two molecules of hydrogen 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, 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, alkynyl groups have 2 to 20 carbon atoms, while in other embodiments, have 2 to 10, 2 to 8, or 2 to 6 carbon atoms. Alkynylene is a subset of alkynyl and refers to residues that are identical to alkynyl, but with two points of attachment.

[0290] As used herein, "alkoxy" refers to an alkyl group of the specified number of carbon atoms attached through 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 attached through the oxygen bridge.

[0291] As used herein, "aryl" refers to a radical derived from an aromatic monocyclic or polycyclic hydrocarbon ring system by removing hydrogen atoms from ring carbon atoms. The aromatic monocyclic or polycyclic hydrocarbon ring system contains only hydrogen and carbon of 6 to 18 carbon atoms, wherein at least one ring in the ring system is fully unsaturated, i.e., it contains a cyclic, delocalized (4n+2)π-electron system according to Hückel's theory. Aryl includes, but is not limited to, radicals such as phenyl, fluorenyl, and naphthyl. Arylene is a subset of aryl and refers to residues identical to aryl but having two points of attachment.

[0292] As used herein, "cycloalkyl" refers to a non-aromatic carbocyclic ring typically having 3 to 7 ring carbon atoms. The ring can 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.

[0293] As used herein, "halogen substituent" or "halogen" refers to fluoro, chloro, bromo and iodo, and the term "halogen" includes fluoro, chloro, bromo and iodine.

[0294] As used herein, "haloalkyl" refers to an alkyl group as defined above in which a specified number of carbon atoms is substituted with one or more, up to the maximum permitted number of halogen atoms. Examples of haloalkyl groups include, but are not limited to, trifluoromethyl, difluoromethyl, 2-fluoroethyl, and pentafluoroethyl.

[0295] "Heterocyclyl" refers to a stable 3 to 18-membered non-aromatic ring radical containing 2 to 12 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, oxygen, and sulfur. Unless otherwise specified in the specification, a heterocyclyl is a monocyclic, bicyclic, tricyclic, or tetracyclic ring system that may include fused or bridged ring systems. The heteroatoms in the heterocyclyl may optionally be oxidized. One or more nitrogen atoms, if present, may optionally be quaternized. The heterocyclyl may be partially saturated or fully saturated. The heterocyclyl may be connected to the remainder of the molecule via any of the ring atoms. 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.

[0296] "Heteroaryl" refers to a group derived from a 3 to 18-membered aromatic ring radical containing 2 to 17 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, oxygen, and sulfur. As used herein, heteroaryl can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system in which at least one ring in the ring system is fully unsaturated, i.e., it contains a cyclic delocalized (4n+2)π-electron system according to Hückel theory. Heteroaryl includes fused or bridged ring systems. The heteroatoms in the heteroaryl are optionally oxidized. One or more nitrogen atoms, if present, are optionally quaternized. The heteroaryl is connected to the rest of the molecule via any annular atom.Examples of heteroaryl groups include, but are not limited to, azacycloheptatrienyl, acridinyl, benzimidazolyl, benzindolyl, 1,3-benzodioxazolyl, benzofuranyl, benzoxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, benzo[b][1,4]oxazinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyrone, benzofuranyl, benzofuranone, benzothiophene, benzothieno[d] ...benzo[b][1,4]oxazinyl, benzo[b][1,4]oxazinyl, benzo[b][1,4]oxazinyl, benzo[b][1,4]oxazinyl, benzo[b][1,4]oxazinyl, benzo[b][1,4]oxazinyl, benzo[b][1,4]oxazinyl, benzo[b][1,4]oxazinyl, benzo[b][1,4]oxazinyl, benzo[b][1,4]oxazinyl, benzo[b][1,4]oxazinyl, benzo[b][1,4]oxazinyl, benzo[b][1,4]oxazinyl, benzo[b][1,4]oxazinyl, 3,2-d]pyrimidinyl, benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl, cyclopenta[d]pyrimidinyl, 6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidinyl, 5,6-dihydrobenzo[h]quinazolinyl, 5,6-dihydrobenzo[h]cinnolinyl, 6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, furano[3,2-c]pyridinyl, 5,6,7,8,9,10-hexahydrocyclooctano[d]pyrimidinyl, 5,6,7,8,9,10-hexahydrocyclooctano[d]pyrimidinyl, ,6,7,8,9,10-hexahydrocyclooctanol[d]pyridinyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, dihydroindolinyl, isodihydroindolinyl, isoquinolinyl, indolizinyl, isoxazolyl, 5,8-methanol-5,6,7,8-tetrahydroquinazolinyl, naphthyridinyl, 1,6-naphthyridinonyl, oxadiazolyl, 2-oxazepinyl, oxazolyl, oxirane, 5,6,6a,7,8,9,10,10a-octahydrobenzo[H]quinazolinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyrazolo[3,4-d]pyrimidinyl, pyridinyl, pyrido[3,2

[0015] In some embodiments, the present invention includes but is not limited to: 1,2-d]pyrimidinyl, 1,4-d]pyrimidinyl, 1,6-d]pyrimidinyl, 1,6-d]pyrimidinyl, 1,6-d]pyrimidinyl, 1,6-d]pyrimidinyl, 1,6-d]pyrimidinyl, 1,6-d]pyrimidinyl, 1,6-d]pyrimidinyl, 1,6-d]pyrimidinyl, 1,6-d]pyrimidinyl, 1,6-d]pyrimidinyl, 1,6-d]pyrimidinyl, 1,6-d]pyrimidinyl, 1,6-d]pyrimidinyl, 1,6-d]pyrimidinyl, 1,6-d]pyrimidinyl, 1,6-d]pyrimidinyl, 1,6-d]pyrimidinyl, 1,6-d]pyrimidinyl, 1,6-d]pyrimidinyl, 1,6-d]pyrimidinyl, 1,6-d]pyrimidinyl, 1,6-d]pyrimidinyl, 1,6-d]pyrimidinyl, 1,6-d]pyrimidinyl, 1,6-d]pyrimidinyl, 1,6-d]pyrimidinyl, 1,6-d]pyrimidinyl, 1,6-d]pyrimidinyl, 1,6-d]pyrimidinyl, 1,6-d]pyrimidinyl,

[0297] Various hydroxy protecting groups can be used in the present disclosure. In general, protecting groups make chemical functional groups insensitive to specific reaction conditions and can be added and removed on 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, Second Edition, Chapter 2, John Wiley & Sons, New York, 1991, each of which is incorporated herein by reference in its 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-phenylxanthen-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthen-9-yl (Mox). In some embodiments, non-exclusive examples of hydroxy protecting groups that may be used herein include Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4'-dimethoxytrityl), and TMTr (4,4',4"-trimethoxytrityl).

[0298] The term "subject," as used herein, refers to any animal, such as a mammal or marsupial. Subjects of the present invention include, but are not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, and any kind of poultry.

[0299] As used herein, "treat," "treat," "alleviate," or "amortize" are used interchangeably herein. These terms refer to an approach for obtaining beneficial or desired results, including but not limited to a therapeutic benefit. "Therapeutic benefit" means eradication or amelioration of the underlying disorder being treated. Furthermore, a therapeutic benefit is achieved by eradication or amelioration of one or more physiological symptoms associated with the underlying disorder, such that an improvement is observed in the patient, although the patient may still be afflicted with the underlying disorder.

[0300] As used herein, "prevent" and "prevention" are used interchangeably. These terms refer to an approach for obtaining a beneficial or desired result, including but not limited to a prophylactic benefit. To obtain a "prophylactic benefit," a conjugate or composition can be administered to a patient at risk for a particular disease, or to a subject reporting one or more physiological symptoms of a disease, even though a diagnosis of the disease may not have yet been made.

[0301] Conjugated molecules

[0302] In one aspect, disclosed herein is a conjugate molecule for delivering an active agent or active drug. In some embodiments, the conjugate molecule disclosed herein is used for tissue-specific targeting. In some embodiments, the conjugate molecule disclosed herein is bound to a cell surface receptor. For this purpose, any cell surface receptor or biomarker or a portion thereof is considered suitable. In some embodiments, the conjugate molecule disclosed herein specifically binds to a unique receptor of a specific tissue, thereby achieving tissue-specific targeting. In some embodiments, the conjugate molecule disclosed herein specifically targets hepatocyte surface receptors, thereby specifically targeting liver tissue. In some embodiments, the conjugate molecule disclosed herein specifically targets cell surface receptors unique to hepatocytes. In some embodiments, the conjugate molecule disclosed herein specifically targets the asialoglycoprotein receptor (ASGPR) on the liver surface.

[0303] As used herein, "active agent" and "active drug" are used interchangeably and refer to molecules that can be delivered by the conjugated molecules disclosed herein. In some embodiments, the active agent is an agent that is desired to be delivered to hepatocytes. Such agents are well known to those skilled in the art and include, but are not limited to, functional nucleotides, such as functional oligonucleotides, particularly those disclosed herein.

[0304] In some embodiments, the present disclosure provides a conjugated molecule having a structure shown in formula (321):

[0305]

[0306] in:

[0307] n1 is an integer from 1 to 3, and n3 is an integer from 0 to 4;

[0308] m1, m2 and m3 are each independently an integer of 2-10; R 10 、R 11 、R 12 、R 13 、R 14 and R 15 Each independently selected from H, C1-C 10 Alkyl, C1-C 10 Halogenated alkyl and C1-C 10 alkoxy;

[0309] R4 is a group capable of binding to an active drug or active agent through a covalent bond;

[0310] Each L1 is a straight chain alkylene group having a length of 1 to 70 carbon atoms, wherein one or more carbon atoms are optionally replaced by one or more selected from the group consisting of: C(O), NH, O, S, CH=N, S(O)2, C2-C 10 Alkenylene, C2-C 10 Alkynylidene, C6-C 10 Arylene, C3-C 18 Heterocyclylene and C5-C 10 Heteroarylene; and wherein L1 optionally has any one or more substituents selected from the group consisting of: C1-C 10 Alkyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, C1-C 10 Haloalkyl, -OC1-C 10 Alkyl, -OC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-OH, -OC1-C 10 Halogenated alkyl, -SC1-C 10 Alkyl, -SC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-SH, -SC1-C 10 Haloalkyl, halogen substituent, -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2, -N(C1-C 10 Alkyl)(C1-C 10 Alkyl), -NH(C1-C 10 Alkyl), -N(C1-C 10 Alkyl)(C1-C 10 Alkylphenyl), -NH(C1-C 10 alkylphenyl), cyano, nitro, -CO2H, -C(O)O(C1-C 10 Alkyl), -CON(C1-C 10 Alkyl)(C1-C 10 alkyl), -CONH(C1-C 10 alkyl), -CONH2, -NHC(O)(C1-C 10 Alkyl), -NHC(O)(phenyl), -N(C1-C 10 alkyl)C(O)(C1-C 10 Alkyl), -N(C1-C 10 alkyl)C(O)(phenyl), -C(O)C1-C 10 Alkyl, -C(O)C1-C 10 Alkylphenyl, -C(O)C1-C 10Haloalkyl, -OC(O)C1-C 10 Alkyl, -SO2(C1-C 10 Alkyl), -SO2(phenyl), -SO2(C1-C 10 Halogenated alkyl), -SO2NH2, -SO2NH(C1-C 10 Alkyl), -SO2NH(phenyl), -NHSO2(C1-C 10 Alkyl), -NHSO2(phenyl) and -NHSO2(C1-C 10 haloalkyl);

[0311] each S1 is independently M1, wherein any reactive hydroxyl groups, if any, are protected by a hydroxyl protecting group;

[0312] Each M1 is independently selected from a ligand capable of binding to a cell surface receptor.

[0313] In some embodiments, n1 can be an integer from 1 to 3, and n3 can be an integer from 0 to 4, thereby ensuring that at least two S1 groups are present in the conjugate molecule. In some embodiments, n1 + n3 ≥ 2, so that the number of M1 ligands in the conjugate formed by the conjugate molecule can be at least 3, thereby making it easier for the M1 ligand to bind to the asialoglycoprotein receptor on the surface of hepatocytes, which can promote the conjugate to enter the cell through endocytosis. Experiments have shown that when the number of M1 ligands is greater than 3, the increased ease of M1 ligand binding to the asialoglycoprotein receptor on the surface of hepatocytes is not significantly increased. Therefore, considering multiple factors such as ease of synthesis, structural / processing costs, and delivery efficiency, in some embodiments, n1 is an integer from 1 to 2, n3 is an integer from 0 to 1, and n1 + n3 = 2-3.

[0314] In some embodiments, when m1, m2 and m3 are each independently selected from an integer of 2-10, it is believed that the spatial positions between multiple M1 ligands in the conjugate formed by the conjugate molecule may be suitable for the binding of the M1 ligand to the asialoglycoprotein receptor on the surface of the hepatocyte. In order to make the conjugate molecules provided by the present disclosure simpler, easier to synthesize and / or reduce costs, according to some embodiments of the present disclosure, m1, m2 and m3 are each independently an integer of 2-5. In one embodiment, m1=m2=m3.

[0315] Those skilled in the art will understand that when R 10 、R 11 、R 12 、R 13 、R 14 and R 15 Each independently selected from H, C1-C 10 Alkyl, C1-C 10Halogenated alkyl and C1-C 10 When R is alkoxy, the properties of the conjugated molecules provided by the present disclosure are not changed, and the purpose of the present disclosure can be achieved. 10 、R 11 、R 12 、R 13 、R 14 and R 15 Each is independently selected from H, methyl or ethyl. In some embodiments, R 10 、R 11 、R 12 、R 13 、R 14 and R 15 Both are H.

[0316] R4 is a group that can bind to an active agent delivered by the conjugated molecules of the present invention. In some embodiments, R4 is a group that can bind to an oligonucleotide to be delivered by the conjugated molecules of the present invention. In some embodiments, R4 is a group that can bind to an oligonucleotide by a covalent bond. In some embodiments, R4 is a group that can bind to an oligonucleotide by a phosphodiester bond. In some embodiments, R4 is selected to achieve connection with the N atom on the nitrogen-containing backbone and provide a suitable reaction site for synthesizing oligonucleotide conjugates. In the context of the present disclosure, "nitrogen-containing backbone" refers to a chain structure to which R is connected. 10 、R 11 、R 12 、R 13 、R 14 and R 15 The carbon atom of the nucleotide sequence of the oligonucleotide is connected to the nitrogen atom of the oligonucleotide. In some embodiments, R4 can be a group that can be connected to the nitrogen atom on the nitrogen-containing backbone in an appropriate manner. In some embodiments, R4 includes a site for connecting to the nitrogen atom on the nitrogen-containing backbone and any functional group that can be reacted to be conjugated to the oligonucleotide through a phosphodiester bond.

[0317] In some embodiments, R4 contains a first functional group that can react with a group on an oligonucleotide or nucleotide to form a phosphate bond, and a second functional group that can form a covalent bond with a hydroxyl or amino group, or contains a solid support connected by the covalent bond. In some embodiments, the first functional group is a phosphoramidite, a hydroxyl or a protected hydroxyl group. In some embodiments, the second functional group is a phosphoramidite, a carboxyl group or a carboxylate. In some embodiments, the second functional group is a solid support that is connected to the rest of the molecule via a covalent bond formed with a hydroxyl or amino group. In some embodiments, the solid support is connected via a phosphate bond, a carboxylate bond or an amide bond. In some embodiments, the solid support is a resin.

[0318] In some embodiments, the first functional group contains hydroxyl, -OR k or a group represented by formula (C3); and / or the second functional group contains a group represented by formula (C1), (C2), (C3), (C1') or (C3'):

[0319]

[0320] In the formula, q1 is an integer of 1-4, X is O or NH, M + is a cation, R k is a hydroxyl protecting group, SPS represents a solid phase support, Indicates the site of covalent attachment of a group.

[0321] In some embodiments, the first functional group contains a phosphoramidite functional group, such as a group shown in formula (C3), which can be coupled to a hydroxyl group at any position on the nucleotide, such as the 2' hydroxyl group or the 3' hydroxyl group, and oxidized to form a phosphodiester bond, thereby conjugating the conjugated molecule to the oligonucleotide. Therefore, even if the second functional group is not present, the conjugated molecule disclosed herein can be conjugated to the nucleotide. In this case, the conjugated molecule is suitable for reacting with the hydroxyl group on the terminal nucleotide in the nucleotide sequence and forming a phosphodiester bond in the subsequent oxidation process, thereby conjugating the conjugated molecule of the present disclosure to the oligonucleotide.

[0322] In some embodiments, the first functional group contains a protected hydroxyl group. In some embodiments, the second functional group contains a group reactive to a solid support to provide a conjugate molecule containing a solid support. In some embodiments, the second functional group contains a carboxyl group, a carboxylate or a phosphoramidite, as shown in formula (C1), (C2) or (C3). The carboxyl group or carboxylate can undergo an esterification reaction or an amidation reaction with a solid support, such as a hydroxyl group or an amino group on a resin, to form a conjugate molecule containing a solid support connected via a carboxylate ester bond or a solid support connected via an amide bond. The phosphoramidite can undergo a coupling reaction with a universal solid support, such as a hydroxyl group on a resin, and be subsequently oxidized to form a solid support connected via a phosphodiester bond. Thus, according to one aspect of the present invention, a method for preparing a conjugate of the present disclosure using such a conjugate molecule is provided. In some embodiments, the method comprises first connecting the conjugate molecule to a solid support by condensation or coupling reaction, and then adding a nucleoside monomer according to a solid phase phosphoramidite synthesis method to obtain a conjugate of the present disclosure comprising the conjugate molecule of the present disclosure conjugated to an oligonucleotide. In some embodiments, during the phosphoramidite solid phase synthesis process, the first functional group is deprotected and then coupled to the phosphoramidite group on the nucleoside under coupling reaction conditions.

[0323] In some embodiments, R 4Contains a first functional group and a second functional group, wherein the first functional group contains a hydroxyl group or a protected hydroxyl group; the second functional group contains a carboxylate bond, an amide bond, or a phosphodiester bond, or a solid phase support connected via a carboxylate bond, an amide bond, or a phosphodiester bond. In some embodiments, the second functional group is a group as shown in formula (C1') or (C3'). In some embodiments, when the second functional group contains a solid phase support, a conjugate molecule containing the solid phase support is used to prepare a conjugate of the present disclosure. Therefore, in one aspect of the present invention, a method for preparing a conjugate of the present disclosure using the conjugate molecule is provided. In some embodiments, the method comprises reacting a conjugate molecule containing a solid phase support with a nucleoside monomer according to a phosphoramidite solid phase synthesis method, thereby conjugating the conjugate molecule of the present disclosure to an oligonucleotide. In some embodiments, the conjugate molecule containing a solid phase support can be obtained internally by reacting a conjugate molecule with a solid phase support, and the conjugate molecule has a carboxyl group, a carboxylate, or a phosphoramidite. In some embodiments, the conjugate molecule can be provided by a supplier.

[0324] In some embodiments, the carboxylate salt can be represented as —COO-M + , where M + is a cation, for example selected from metal cations, ammonium cations NH4 + Or organic ammonium cation. In some embodiments, the metal ion can be an alkali metal ion, such as K + Or Na + In order to improve solubility and facilitate the reaction, in some embodiments, the organic ammonium cation is an ammonium cation formed by a tertiary amine or a quaternary ammonium cation, such as an ammonium ion formed by triethylamine or an ammonium cation formed by N,N-diisopropylethylamine. In some embodiments, the carboxylate is triethylamine carboxylate or N,N-diisopropylethylamine carboxylate.

[0325] In some embodiments of the present disclosure, R4 is a group represented by formula (B9), (B10), (B9'), (B10'), (B11), (B12), (B11'), or (B12'):

[0326]

[0327]

[0328] Wherein, q1 is an integer of 1-4, q2 is an integer of 1-10, X is O or NH, M + is a cation, R k is a hydroxyl protecting group, SPS represents a solid phase support, In some embodiments, R4 comprises a group represented by formula (B9) or (B10). In some embodiments, R4 comprises a group represented by formula (B11) or (B12).

[0329] In some embodiments, R k is one or more of Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4'-dimethoxytrityl), and TMTr (4,4',4'-trimethoxytrityl). k It's DMTr.

[0330] L1 is a linear alkylene group having a length of 1 to 70 carbon atoms, wherein one or more carbon atoms are optionally replaced by one or more selected from the group consisting of: C(O), NH, O, S, CH=N, S(O)2, C2-C 10 Alkenylene, C2-C 10 Alkynylidene, C6-C 10 Arylene, C3-C 18 Heterocyclylene and C5-C 10 Heteroarylene; and wherein L1 optionally has any one or more substituents selected from the group consisting of: C1-C 10 Alkyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, C1-C 10 Haloalkyl, -OC1-C 10 Alkyl, -OC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-OH, -OC1-C 10 Halogenated alkyl, -SC1-C 10 Alkyl, -SC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-SH, -SC1-C 10 Haloalkyl, halogen substituent, -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2, -N(C1-C 10 Alkyl)(C1-C 10 Alkyl), -NH(C1-C 10 Alkyl), -N(C1-C 10 Alkyl)(C1-C 10 Alkylphenyl), -NH(C1-C 10 alkylphenyl), cyano, nitro, -CO2H, -C(O)O(C1-C10 Alkyl), -CON(C1-C 10 Alkyl)(C1-C 10 alkyl), -CONH(C1-C 10 alkyl), -CONH2, -NHC(O)(C1-C 10 Alkyl), -NHC(O)(phenyl), -N(C1-C 10 alkyl)C(O)(C1-C 10 Alkyl), -N(C1-C 10 alkyl)C(O)(phenyl), -C(O)C1-C 10 Alkyl, -C(O)C1-C 10 Alkylphenyl, -C(O)C1-C 10 Haloalkyl, -OC(O)C1-C 10 Alkyl, -SO2(C1-C 10 Alkyl), -SO2(phenyl), -SO2(C1-C 10 Halogenated alkyl), -SO2NH2, -SO2NH(C1-C 10 Alkyl), -SO2NH(phenyl), -NHSO2(C1-C 10 Alkyl), -NHSO2(phenyl) and -NHSO2(C1-C 10 haloalkyl). The skilled artisan will appreciate that although L is defined as a linear alkylene group for convenience, it may not be a linear group or may be named differently, such as an amine or alkenyl group resulting from the above-mentioned replacement and / or substitution. For the purposes of this disclosure, the length of L is the number of atoms in the chain connecting the two points of attachment. For this purpose, a ring resulting from the replacement of a carbon atom of the linear alkylene group (e.g., a heterocyclylene or heteroarylene group) is counted as one atom.

[0331] In some embodiments, the role of L1 is to connect the M1 ligand (or the corresponding S1 group) to the N atom on the nitrogen-containing skeleton, thereby providing liver targeting function for the conjugate of the present disclosure. In some embodiments, L1 is selected from any one of the formula A1-A26 groups or any combination thereof. In some embodiments, L1 is any one or any combination of A1, A4, A5, A6, A8, A10, A11 and A13. In some embodiments, L1 is a combination of at least two of A1, A4, A8, A10 and A11. In some embodiments, L1 is a combination of at least two of A1, A8, A10.

[0332] In some embodiments, L1 may be 3-25, 3-20, 4-15, or 5-12 atoms in length. In some embodiments, L1 is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, or 60 atoms in length. According to some embodiments of the present disclosure, j1 is an integer of 2-10, and in some embodiments, j1 is an integer of 3-5. In some embodiments, j2 is an integer of 2-10, and in some embodiments, j2 is an integer of 3-5. R' is a C1-C4 alkyl group, and in some embodiments, R' is one of methyl, ethyl, and isopropyl. Ra is one of A27, A28, A29, A30, and A31, and in some embodiments, Ra is A27 or A28. Rb is a C1-C5 alkyl group. In some embodiments, Rb is one of a methyl group, an ethyl group, an isopropyl group, and a butyl group. In some embodiments, j1, j2, R', Ra, and Rb in Formulas A1-A26 are selected to achieve, in the oligonucleotide conjugate formed by the conjugated molecule, the attachment of the M1 ligand to the N atom on the nitrogen-containing backbone, and to make the spatial position between the M1 ligands more suitable for binding the M1 ligand to the asialoglycoprotein receptor on the surface of hepatocytes.

[0333] Each M1 is independently selected from a ligand capable of binding to a cell surface receptor. In some embodiments, at least one M1 is a ligand capable of binding to a hepatocyte surface receptor. In some embodiments, at least one M1 is a ligand capable of binding to a mammalian cell surface receptor. In some embodiments, at least one M1 is a ligand capable of binding to a human hepatocyte surface receptor. In some embodiments, at least one M1 is a ligand capable of binding to the liver surface asialoglycoprotein receptor (ASGPR).

[0334] In some embodiments, M1 may be any ligand that has affinity for the asialoglycoprotein receptor (ASGPR) on the surface of mammalian hepatocytes, the types of which are known to those skilled in the art. In some embodiments, at least one M1 is a sugar. In some embodiments, each M1 is a sugar. In some embodiments, at least one M1 is a monosaccharide, disaccharide, trisaccharide, or polysaccharide. In some embodiments, each M1 is a monosaccharide, disaccharide, trisaccharide, or polysaccharide. In some embodiments, at least one M1 is a modified sugar. In some embodiments, each M1 is a modified sugar. In some embodiments, each M1 is independently selected from a polysaccharide, a modified polysaccharide, a monosaccharide, or a monosaccharide derivative. In some embodiments, each or at least one M1 may be independently selected from the group consisting of glucose and its derivatives, mannose and its derivatives, galactose and its derivatives, xylose and its derivatives, ribose and its derivatives, fucose and its derivatives, lactose and its derivatives, maltose and its derivatives, arabinose and its derivatives, fructose and its derivatives, and sialic acid.

[0335] In some embodiments, each or at least one M1 may be independently selected from D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, α-D-mannofuranose, β-D-mannofuranose, α-D-mannopyranose, β-D-mannopyranose, α-D-glucose, β-D-glucose, Glucose, α-D-glucofuranose, β-D-glucofuranose, α-D-fructofuranose, α-D-fructopyranose, α-D-galactopyranose, β-D-galactopyranose, α-D-galactofuranose, β-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-butyrylgalactosamine, N-isobutyrylgalactosamine Galactosamine, 2-amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose, N-glycolyl-α-neuraminic acid, 5-thio-β-D-glucopyranose, 2,3,4-tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside methyl ester, 4-thio-β-D-galactopyranose, 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucopyranoside heptoside ethyl ester, 2,5-anhydro-D-allose nitrile, ribose, D-ribose, D-4-thioribose, L-ribose, L-4-thioribose. In some embodiments, at least one M1 is N-acetylgalactosamine (GalNAc). In some embodiments, each M1 is N-acetylgalactosamine (GalNAc). Ligand selection can refer to, for example, the disclosure of CN105378082A, the entire disclosure of which is incorporated herein by reference.

[0336] CN105378082A discloses a compound comprising a modified oligonucleotide and a conjugated group, wherein the conjugated group comprises at least one phosphorus linking group or a neutral linking group and one or more ligands. Each ligand is selected from polysaccharides, modified polysaccharides, mannose, galactose, mannose derivatives, galactose derivatives, D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, α-D-mannofuranose, β-D-mannofuranose, α-D-mannopyranose, β-D-mannopyranose Sugar, α-D-glucopyranose, β-D-glucopyranose, α-D-glucofuranose, β-D-glucofuranose, α-D-fructofuranose, α-D-fructopyranose, α-D-galactopyranose, β-D-galactopyranose, α-D-galactofuranose, β-D-galactofuranose, glucosamine, sialic acid, α-D-galactosamine, N-acetylgalactosamine, 2-amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose, N-glycolyl-α-neuraminic acid, 5-thio-β-D-glucopyranose, 2,3,4-tris( ...,3,4-tris(2-amino-3-O-[(R)-1-carboxyethyl] The compounds are methyl-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside, 4-thio-β-D-galactopyranose, ethyl-3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucopyranoside, 2,5-anhydro-D-allosenitrile, ribose, D-ribose, D-4-thioribose, L-ribose, or L-4-thioribose. The compounds are said to reduce the amount or activity of nucleic acid transcripts in cells.

[0337] WO2016077321A1 discloses numerous siRNAs specifically targeting the HBV gene and methods for their delivery. The document also discloses siRNA conjugates, specifically several types of siRNA conjugates, by modifying the siRNA nucleotides to enhance their serum stability.

[0338] WO2016168286A1 discloses numerous siRNAs specifically targeting the ANGPTL3 gene and methods for their delivery, and enhances the serum stability of siRNAs by modifying their nucleotides. The document also discloses siRNA conjugates.

[0339] N-acetylgalactosamine (GalNAc) is a ligand that binds to the asialoglycoprotein receptor on the surface of the liver. The asialoglycoprotein receptor (ASGPR) is an endocytic receptor specifically expressed by hepatocytes. In recent years, N-acetylgalactosamine (GalNAc) has been used as a targeting molecule to deliver small RNA to the liver. For example, Alnylam Pharmaceuticals, Inc. first reported that siRNA based on GalNAc conjugation technology exerted interference activity in mice (Nair et al., J.Am.Chem.Soc., 2014, 136, 16958-16961). The article reported that siRNA conjugated to three clusters of GalNAc showed good delivery activity in both in vivo and in vitro experiments. In vivo experiments in mice administered subcutaneously, a single dose of ED 50 The dose was determined to be 1 mg / kg, with a single injection dose of less than 1 ml. In long-term dosing experiments, once-weekly subcutaneous injections achieved stable interfering activity for up to 9 months.

[0340] In some embodiments, S1 is independently M1. In some embodiments, S1 is independently a group in which at least one active hydroxyl group in M1 is protected by a hydroxyl protecting group. In some embodiments, S1 is independently a group in which all active hydroxyl groups (if any) in M1 are protected by hydroxyl protecting groups. In some embodiments, any hydroxyl protecting group well known to those skilled in the art can be used to protect the active hydroxyl groups on M1. In some embodiments, the protected hydroxyl group is represented by the formula YCOO-, wherein each Y is independently selected from C1-C 10 Alkyl and C6-C 10 In some embodiments, each Y is independently selected from the group consisting of methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl, ethyl, n-propyl, isopropyl, phenyl, halophenyl, and C1-C6 alkylphenyl.

[0341] In some embodiments, each S1 is independently selected from the group consisting of groups of formulae A46-A54:

[0342]

[0343]

[0344] In some embodiments, S1 is of formula A49 or A50.

[0345] In some embodiments, each Y is independently one of methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl, ethyl, n-propyl, isopropyl, phenyl, halophenyl, and alkylphenyl. For the purpose of simplifying the conjugate molecules of the present disclosure, in some embodiments, Y is methyl.

[0346] In some embodiments, the conjugate molecules of the present disclosure have a structure represented by Formula (403), (404), (405), (406), (407), (408), (409), (410), (411), (412), (413), (414), (415), (416), (417), (418), (419), (420), (421), or (422):

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353] In the above formulas (403)-(422), X is O or NH, R k is a hydroxyl protecting group, M + M is a metal cation, an ammonium cation, a cation formed by a tertiary amine, or a quaternary ammonium cation. + for

[0354] In some embodiments, the conjugate molecules of the present disclosure may have a structure as shown in Formula (423), (424), (425), (426), (427), (428), (429), (430), (431), (432), (433), (434), (435), (436), (437), (438), (439), (440), (441), or (442):

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361] In the above formulas (423)-(442), wherein X is O or NH, R k is a hydroxyl protecting group, and SPS represents a solid phase support.

[0362] In some embodiments, the conjugate molecules of the present disclosure have a structure represented by formula (503), (504), (505), (506), (507), (508), (509), (510), (511), (512), (513), (514), (515), (516), (517), (518), (519), (520), (521), or (522):

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369] In the above formulas (503)-(522), DMTr represents 4,4'-dimethoxytrityl, and the structure represents the salt formed by the corresponding carboxylic acid and triethylamine.

[0370] In some embodiments, the conjugate molecules of the present disclosure may have a structure as shown in Formula (523), (524), (525), (526), ​​(527), (528), (529), (530), (531), (532), (533), (534), (535), (536), (537), (538), (539), (540), (541), or (542):

[0371]

[0372]

[0373]

[0374]

[0375]

[0376]

[0377] In the above formulae (523) to (542), SPS represents a solid support, and DMTr represents a 4,4'-bismethoxytrityl group.

[0378] Preparation of the Conjugated Molecules of the Present Disclosure

[0379] Those skilled in the art can use any reasonable synthetic route to prepare the conjugate molecules of the present disclosure.

[0380] In some embodiments of the present disclosure, the method for preparing the conjugate molecule represented by formula (321) comprises contacting the compound represented by formula (313) with a cyclic acid anhydride in an organic solvent under esterification reaction conditions and in the presence of a base and an esterification catalyst, performing ion exchange, and isolating the compound represented by formula (321):

[0381]

[0382] in:

[0383] R6 is a group that provides R4 in formula (321). In some embodiments, for example, R6 has a structure shown in formula (A61):

[0384]

[0385] n1, n3, m1, m2, m3, R 10 、R 11 、R 12 、R 13 、R 14 、R 15 , L1, S1, their respective definitions and selectable ranges are as described above, R i To be able to connect with the N atom on the nitrogen-containing skeleton and R k O is connected to any group with a free hydroxyl group, R k is a hydroxy protecting group. In this case, the compound obtained is a compound of formula (321), wherein R4 comprises a hydroxy protecting group as the first functional group and a group represented by formula (C1) or (C2) as the second functional group. In some embodiments, R6 is B7 or B8:

[0386]

[0387] Among them, q2 and R k The respective definitions are as described above.

[0388] The esterification reaction conditions include a reaction temperature of 0-100° C. and a reaction time of 8-48 hours. In one embodiment, the esterification reaction conditions include a reaction temperature of 10-40° C. and a reaction time of 20-30 hours.

[0389] In some embodiments, the organic solvent includes one or more of an epoxy solvent, an ether solvent, a haloalkane solvent, dimethyl sulfoxide, N,N-dimethylformamide and N,N-diisopropylethylamine. In some embodiments, the epoxy solvent is dioxane and / or tetrahydrofuran. In some embodiments, the ether solvent is diethyl ether and / or methyl tert-butyl ether, and the haloalkane solvent is one or more of dichloromethane, chloroform and 1,2-dichloroethane. In some embodiments, the organic solvent is dichloromethane. Relative to the compound represented by formula (313), the amount of the organic solvent used is 3-50 L / mol, and in some embodiments, 5-20 L / mol.

[0390] In some embodiments, the cyclic anhydride is one of succinic anhydride, glutaric anhydride, adipic anhydride, or pimelic anhydride, and in some embodiments, the cyclic anhydride is succinic anhydride. The molar ratio of the cyclic anhydride to the compound of formula (313) is 1:1-10:1, and in some embodiments, 2:1-5:1.

[0391] The esterification catalyst can be any catalyst that catalyzes the esterification reaction, for example, the catalyst can be 4-dimethylaminopyridine. The molar ratio of the catalyst to the compound represented by formula (313) is 1:1-10:1, and in some embodiments, 2:1-5:1.

[0392] In some embodiments, the base can be any inorganic base, organic base or a combination thereof. Considering solubility and product stability, the base is a tertiary amine. In some embodiments, the tertiary amine is triethylamine or N,N-diisopropylethylamine. The molar ratio of the tertiary amine to the compound represented by formula (313) is 1:1-20:1, and in some embodiments is 3:1-10:1.

[0393] The ion exchange is used to convert the compound of formula (321) into the desired carboxylic acid or carboxylate form. The ion exchange method is well known to those skilled in the art. Suitable ion exchange solutions and exchange conditions can be used to obtain a compound having M +In some embodiments, the ion exchange reaction is carried out using a triethylamine phosphate solution. In some embodiments, the concentration of the triethylamine phosphate solution is 0.2-0.8 M, and in some embodiments, the concentration of the triethylamine phosphate solution is 0.4-0.6 M. In some embodiments, the amount of the triethylamine phosphate solution used is 3-6 L / mol relative to the compound of formula (313), and in other embodiments, 4-5 L / mol.

[0394] Any suitable separation method can be used to separate the compound of formula (321) from the reaction mixture. In some embodiments, the solvent can be removed by evaporation and then the compound of formula (321) can be separated by chromatography. For example, the following chromatographic conditions can be used for separation: (1) normal phase purification: 200-300 mesh silica gel filler, using dichloromethane containing 1 wt‰ triethylamine: methanol = 100:18-100:20 gradient elution; or (2) reverse phase purification: C18, C8 reverse phase filler, using methanol: acetonitrile = 0.1:1-1:0.1 gradient elution. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (321), which can be directly used in subsequent reactions.

[0395] In some embodiments, the method for preparing the compound of formula (321) further comprises contacting the ion exchange product with a solid support containing an amino group or a hydroxyl group in an organic solvent in the presence of a condensation agent, a condensation catalyst, and a tertiary amine under condensation reaction conditions. In this case, the compound of formula (321) is obtained, wherein R4 contains a hydroxyl protecting group as the first functional group and a group represented by formula (C1') as the second functional group.

[0396] The solid phase carrier is one of the carriers used in the solid phase synthesis of siRNA, some of which are well known to those skilled in the art. For example, the solid phase carrier can be selected from a solid phase carrier containing an active hydroxyl or amino functional group. In some embodiments, the solid phase carrier is an amino resin or a hydroxyl resin. In some embodiments, the amino or hydroxyl resin has the following parameters: a particle size of 100-400 mesh, and a surface amino or hydroxyl loading of 0.2-0.5 mmol / g. The ratio of the compound represented by formula (321) to the solid phase carrier is 10-400 μmol compound per gram of solid phase carrier (μmol / g). In some embodiments, the ratio of the compound represented by formula (321) to the solid phase carrier is 50-200 μmol / g.

[0397] The organic solvent can be any suitable solvent or mixed solvent known to those skilled in the art. In some embodiments, the organic solvent is acetonitrile, an epoxy solvent, an ether solvent, a haloalkane solvent, dimethyl sulfoxide, N,N-dimethylformamide and N,N-diisopropylethylamine, one or more. In some embodiments, the epoxy solvent is dioxane and / or tetrahydrofuran, the ether solvent is ether and / or methyl tert-butyl ether, and the haloalkane solvent is one or more of dichloromethane, chloroform and 1,2-dichloroethane. In some embodiments, the organic solvent is acetonitrile. Relative to the compound of formula (313), the amount of the organic solvent is 20-200L / mol, and in one embodiment is 50-100L / mol.

[0398] In some embodiments, the condensing agent can be benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, 3-diethoxyphosphoryl-1,2,3-benzotriazole-4(3H)-one and / or O-benzotriazole-tetramethyluronium hexafluorophosphate. In some embodiments, the condensing agent is O-benzotriazole-tetramethyluronium hexafluorophosphate. The molar ratio of the condensing agent to the compound represented by formula (313) is 1:1-20:1, and in other embodiments, it is 1:1-5:1.

[0399] In some embodiments, the tertiary amine is triethylamine and / or N,N-diisopropylethylamine, and in some embodiments, it is N,N-diisopropylethylamine; the molar ratio of the tertiary amine to the compound represented by formula (313) is 1:1-20:1, and in some embodiments, it is 1:1-5:1.

[0400] In some embodiments, the method for preparing the compound of formula (321) further comprises contacting the obtained condensation reaction product with a capping agent and an acylation catalyst in an organic solvent under capping reaction conditions, and isolating to obtain the compound represented by formula (321). The function of the capping reaction is to remove any unreacted active functional groups to avoid the production of unnecessary by-products in subsequent reactions. The conditions of the capping reaction include a reaction temperature of 0-50°C, in some embodiments 15-35°C, and a reaction time of 1-10 hours, in some embodiments 3-6 hours. The capping agent can be a capping agent used in siRNA solid phase synthesis, which is well known to those skilled in the art. In some embodiments, the capping agent consists of capping agent A (capA) and capping agent B (capB). Capping agent A is N-methylimidazole, and in some embodiments is provided in the form of a mixed solution of N-methylimidazole in pyridine / acetonitrile, wherein the volume ratio of pyridine to acetonitrile is 1:10-1:1. In some embodiments, it is 1:3-1:1. In some embodiments, the volume ratio of the total volume of pyridine and acetonitrile to N-methylimidazole is 1:1-10:1, and in some embodiments, 3:1-7:1. In some embodiments, the capping reagent B is acetic anhydride, and in some embodiments, the capping reagent B is provided in the form of an acetonitrile solution of acetic anhydride, wherein the volume ratio of acetic anhydride to acetonitrile is 1:1-1:10, and in other embodiments, 1:2-1:6.

[0401] In some embodiments, the volume ratio of the N-methylimidazole solution in pyridine / acetonitrile to the mass ratio of the compound of formula (313) is 5 ml / g to 50 ml / g, and in some embodiments, 15 ml / g to 30 ml / g. The volume ratio of the acetic anhydride solution in acetonitrile to the mass ratio of the compound of formula (313) is 0.5 ml / g to 10 ml / g, and in some embodiments, 1 ml / g to 5 ml / g.

[0402] In some embodiments, the capping agent is equimolar amounts of acetic anhydride and N-methylimidazole. In some embodiments, the organic solvent is one or more of acetonitrile, epoxy solvents, ether solvents, alkyl halide solvents, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropylethylamine. In some embodiments, the organic solvent is acetonitrile. The amount of the organic solvent used is 10-50 L / mol relative to the compound of formula (313), and in some embodiments, 5-30 L / mol.

[0403] In some embodiments, the acylation catalyst can be selected from any catalyst that can be used for esterification or amide formation, such as a basic heterocyclic compound. In some embodiments, the acylation catalyst is 4-dimethylaminopyridine. The mass ratio of the catalyst to the compound represented by formula (313) is 0.001:1-1:1, and in some embodiments, 0.01:1-0.1:1.

[0404] In some embodiments, the compound of formula (321) can be isolated from the reaction mixture using any suitable method. In some embodiments, the compound of formula (321) can be obtained by washing with an organic solvent and filtering to remove unreacted reactants, excess capping reagent, and other impurities, wherein the organic solvent is selected from acetonitrile, dichloromethane, or methanol. In some embodiments, the organic solvent is acetonitrile.

[0405] In some embodiments, the method for preparing the conjugate molecule of formula (321) comprises contacting the compound of formula (313) with a phosphorodiamidite in an organic solvent under coupling reaction conditions and in the presence of a coupling reagent, and isolating the compound of formula (321). In this case, the compound obtained is the compound of formula (321), wherein R4 contains a hydroxyl protecting group as the first functional group and a group of formula (C3) as the second functional group.

[0406] In some embodiments, the coupling reaction conditions include a temperature of 0-50°C, for example, 15-35°C, and the molar ratio of the compound of formula (313) to the phosphorodiamidite can be 1:1-1:50, for example, 1:5-1:15. The molar ratio of the compound of formula (313) to the coupling reagent can be 1:1-1:100, for example, 1:50-1:80. The reaction time can be 200-3000 seconds, for example, 500-1500 seconds. The phosphorodiamidite can be, for example, bis(diisopropylamino)(2-cyanoethoxy)phosphine, which can be commercially obtained or prepared according to methods known in the art. The coupling reagent is selected from one or more of 1H-tetrazole, 5-ethylthio-1H-tetrazole, and 5-benzylthio-1H-tetrazole, for example, 5-ethylthio-1H-tetrazole. The coupling reaction can be carried out in an organic solvent, and the organic solvent is selected from one or more of anhydrous acetonitrile, anhydrous DMF, and anhydrous dichloromethane, preferably anhydrous acetonitrile. Relative to the compound of formula (313), the amount of the organic solvent can be 3-50 L / mol, for example, 5-20 L / mol. By carrying out the coupling reaction, the hydroxyl group in the compound of formula (313) reacts with the phosphorodiamidite to form a phosphoramidite group. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (321), which can be directly used in subsequent reactions.

[0407] In some embodiments, the method for preparing the compound of formula (321) further comprises: contacting the isolated product with a solid phase support containing a hydroxyl group in an organic solvent and in the presence of a coupling reagent under coupling reaction conditions. Subsequently, the compound of formula (321) is isolated through a capping reaction and an oxidation reaction. In this case, the compound obtained is a compound of formula (321), wherein R4 contains a hydroxyl protecting group as the first functional group and a group represented by formula (C3') as the second functional group.

[0408] In some embodiments, the solid phase carrier is a solid phase carrier that can be used for solid phase synthesis of nucleic acids, for example, it can be a commercially available general solid phase carrier (for example HLUnyLinker™ 300 Oligonucleotide Synthesis Support, Kinovate Life Sciences, structure shown in Formula B80):

[0409]

[0410]

[0411] Deprotection reactions are well known in the art. In some embodiments, the deprotection conditions include a temperature of 0-50°C, for example, 15-35°C; a reaction time of 30-300 seconds, for example, 50-150 seconds. The deprotection reagent can be selected from one or more of trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, and monochloroacetic acid. In some embodiments, the deprotection reagent is dichloroacetic acid. The molar ratio of the deprotection reagent to the -DMTr (4,4'-dimethoxytrityl) protecting group on the solid support can be 2:1-100:1, for example, 3:1-50:1. By performing the deprotection, reactive free hydroxyl groups are obtained on the surface of the solid support, thereby enabling subsequent coupling reactions.

[0412] The coupling reaction conditions and coupling reagents can be selected as described above.By performing the coupling reaction, the free hydroxyl group formed in the deprotection reaction reacts with the phosphoramidite group to form a phosphite linkage.

[0413] In some embodiments, the capping reaction conditions include a temperature of 0-50° C., such as 15-35° C., and a reaction time of 5-500 seconds, such as 10-100 seconds. The capping agent and amount can be selected as described above.

[0414] The oxidation reaction conditions may include a temperature of 0-50° C., for example, 15-35° C., a reaction time of 1-100 seconds, for example, 5-50 seconds, and an oxidizing agent, for example, iodine (in some embodiments, provided in the form of iodine water). In some embodiments, the molar ratio of the oxidizing agent to the nucleic acid sequence attached to the solid support is 1:1-100:1, preferably 5:1-50:1. In some embodiments, the oxidation reaction is carried out in a mixed solvent of tetrahydrofuran: water: pyridine = 3:1:1-1:1:3.

[0415] In some embodiments, R6 is formula B7 or B8. In this case, the compound represented by formula (313) can be obtained by the following preparation method: in an organic solvent, under amide formation reaction conditions, and in the presence of an amide formation reaction condensation agent and a tertiary amine, the compound represented by formula (314) is contacted with the compound represented by formula (A-1) or the compound represented by formula (A-2), and the compound represented by formula (313) is separated:

[0416]

[0417] Among them, n1, n3, m1, m2, m3, R 10 、R 11 、R 12 、R 13 、R 14 、R 15 , L1, S1, q2 and R k The respective definitions and optional ranges are as described above.

[0418] The amide formation reaction conditions may include: a reaction temperature of 0-100° C. and a reaction time of 1-48 hours. In some embodiments, the amide formation reaction conditions include a reaction temperature of 10-40° C. and a reaction time of 2-16 hours.

[0419] In some embodiments, the organic solvent is one or more of an alcohol solvent, an epoxy solvent, an ether solvent, a haloalkane solvent, dimethyl sulfoxide, N,N-dimethylformamide and N,N-diisopropylethylamine. In some embodiments, the alcohol solvent is one or more of methanol, ethanol and propanol, and in other embodiments it is ethanol. In some embodiments, the epoxy solvent is dioxane and / or tetrahydrofuran. In some embodiments, the ether solvent is diethyl ether and / or methyl tert-butyl ether. In some embodiments, the haloalkane solvent is one or more of dichloromethane, chloroform and 1,2-dichloroethane. In some embodiments, the organic solvent is dichloromethane. Relative to the compound of formula (314), the amount of organic solvent used is 3-50 L / mol, and in one embodiment it is 3-20 L / mol.

[0420] In some embodiments, the amide-forming reaction condensation agent is benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, 3-diethoxyphosphoryloxy-1,2,3-benzotriazine-4(3H)-one, 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) or O-benzotriazole-tetramethyluronium hexafluorophosphate, and in a further embodiment, 3-diethoxyphosphoryloxy-1,2,3-benzotriazine-4(3H)-one. The molar ratio of the amide-forming reaction condensation agent to the compound represented by formula (314) can be 1:110:1, and in some embodiments, 2.5:1-5:1.

[0421] In some embodiments, the tertiary amine is triethylamine or N,N-diisopropylethylamine, and in further embodiments, N,N-diisopropylethylamine. The molar ratio of the tertiary amine to the compound of formula (314) is 3:1-20:1, and in one embodiment, 5:1-10:1.

[0422] In some embodiments, the compounds of formula (A-1) and formula (A-2) can be prepared by any suitable means. For example, when R k When it is a DMTr group, the compound of formula (A-1) can be prepared by reacting calcium glycerate with DMTrCl. Similarly, 3-amino-1,2-propanediol can be first contacted with a cyclic anhydride and then reacted with DMTrCl to prepare a compound of formula (A-2), wherein the cyclic anhydride can be a cyclic anhydride having 4-13 carbon atoms, and in one embodiment, 4-8 carbon atoms. It is readily understood by those skilled in the art that the selection of the cyclic anhydride corresponds to different values ​​of q2 in the compound (A-2), for example, when the cyclic anhydride is succinic anhydride, q2=1, and when the cyclic anhydride is glutaric anhydride, q2=2, and so on.

[0423] In some variations, the compound of formula (313) can also be prepared by reacting the compound of formula (314) with the cyclic anhydride, 3-amino-1,2-propanediol, and DMTrCl in sequence. It will be readily understood by those skilled in the art that these variations will not affect the structure and function of the compound of formula (313), and these variations are readily achievable by those skilled in the art based on the above-described methods.

[0424] Similar to the above, any suitable separation method can be used to separate the compound of formula (313) from the reaction mixture. In some embodiments, the solvent can be removed by evaporation and then separated by chromatography. For example, the following two sets of chromatographic conditions can be used for separation: (1) normal phase purification: 200-300 mesh silica gel packing, using petroleum ether: ethyl acetate: dichloromethane: N, N-dimethylformamide = 1:1:1:0.5-1:1:1:0.6 gradient elution; and (2) reverse phase purification: C18, C8 reverse phase packing, using methanol: acetonitrile = 0.1:1-1:0.1 gradient elution. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (313), which can be directly used in subsequent reactions.

[0425] In some embodiments, the compound represented by formula (314) can be obtained by the following preparation method: the method comprises contacting the compound represented by formula (315) with a haloacetic acid in an organic solvent under deprotection reaction conditions, and isolating the compound represented by formula (314):

[0426]

[0427] Wherein, R7 is selected from the group represented by formula (330), (331), (332) or (333). In one embodiment, the structure of R7 is as shown in formula (330):

[0428]

[0429] Among them, n1, n3, m1, m2, m3, R 10 、R 11 、R 12 、R 13 、R 14 、R 15 The definitions and selectable ranges of L1, S1 are as described above.

[0430] The halogenated acetic acid may be selected from one or more of dichloroacetic acid, trichloroacetic acid, monochloroacetic acid and trifluoroacetic acid, and in some embodiments is dichloroacetic acid.

[0431] The deprotection reaction conditions may include a reaction temperature of 0-100° C. and a reaction time of 0.1-24 hours. In one embodiment, the reaction temperature is 10-40° C. and the reaction time is 0.5-16 hours.

[0432] In some embodiments, the organic solvent is one or more of an epoxy solvent, an ether solvent, a haloalkane solvent, dimethyl sulfoxide, N,N-dimethylformamide and N,N-diisopropylethylamine. In one embodiment, the epoxy solvent is dioxane and / or tetrahydrofuran, the ether solvent is ether and / or methyl tert-butyl ether in some embodiments, the haloalkane solvent is one or more of dichloromethane, chloroform and 1,2-dichloroethane in some embodiments, and the organic solvent is dichloromethane. Relative to the compound of formula (315), the amount of organic solvent used is 3-50 L / mol, and in further embodiments, 5-20 L / mol.

[0433] The molar ratio of the haloacetic acid to the compound of formula (315) may be 5:1-100:1, and in some embodiments, 10:1-50:1.

[0434] Similar to the above, any suitable separation method can be used to separate the compound of formula (314) from the reaction mixture. In some embodiments, the solvent can be removed by evaporation and then separated by chromatography. For example, the following two sets of chromatographic conditions can be used for separation: (1) normal phase purification: 200-300 mesh silica gel packing, using dichloromethane: methanol = 100:30-100:40 gradient elution; and (2) reverse phase purification: C18 and C8 reverse phase packing, using methanol: acetonitrile = 0.1:1-1:0.1 gradient elution. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (314), which can be directly used in subsequent reactions.

[0435] The compound represented by formula (315) can be obtained by the following preparation method: the method comprises contacting the compound represented by formula (317) with the compound represented by formula (316) in an organic solvent in the presence of an amide-forming reaction condensation agent and a tertiary amine under condensation reaction conditions, and then separating:

[0436] S1-L1-COOH

[0437] Formula (316)

[0438]

[0439] Among them, n1, n3, m1, m2, m3, R7, R 10 、R 11 、R 12 、R 13 、R 14 、R 15 The definitions and selectable ranges of L1, S1 are as described above.

[0440] The compound of formula (316) can be prepared using, for example, the compounds disclosed in J.Am.Chem.Soc.2014,136,16958-16961, or the compound of formula (316) can be prepared by a person skilled in the art by various methods. For example, certain compounds of formula (316) can be prepared by referring to the method disclosed in Example 1 of US 8,106,022 B2. The entire contents of the above documents are incorporated herein by reference.

[0441] In some embodiments, the condensation reaction conditions include a reaction temperature of 0-100° C. and a reaction time of 0.1-24 hours. In some embodiments, the reaction temperature is 10-40° C. and the reaction time is 0.5-16 hours.

[0442] The molar ratio of the compound represented by formula (316) to the compound represented by formula (317) is 2:1 to 10:1, and in some embodiments, 2.5:1-5:1.

[0443] In some embodiments, the organic solvent is one or more of acetonitrile, epoxy solvent, ether solvent, halogenated alkane solvent, dimethyl sulfoxide, N,N-dimethylformamide and N,N-diisopropylethylamine. In some embodiments, the epoxy solvent is dioxane and / or tetrahydrofuran, the ether solvent is ether and / or methyl tert-butyl ether in some embodiments, the halogenated alkane solvent is one or more of dichloromethane, chloroform and 1,2-dichloroethane in one embodiment, and in some embodiments, the organic solvent is acetonitrile. Relative to the compound of formula (317), the amount of the organic solvent can be 3-50 L / mol, and in one embodiment, 5-20 L / mol.

[0444] In some embodiments, the amide-forming reaction condensation agent is benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, 3-diethoxyphosphoryloxy-1,2,3-benzotriazine-4(3H)-one (DEPBT), O-benzotriazole-tetramethyluronium hexafluorophosphate, or 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride. In further embodiments, it may be 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride. The molar ratio of the amide-forming reaction condensation agent to the compound represented by formula (317) is 2:1-10:1, and in some embodiments, 2.5:1-5:1.

[0445] The tertiary amine may be N-methylmorpholine, triethylamine or N,N-diisopropylethylamine, and in some embodiments, it is N-methylmorpholine; the molar ratio of the tertiary amine to the compound represented by formula (317) may be 3:1-20:1, and in some embodiments, it is 5:1-10:1.

[0446] Similar to the above, any suitable separation method can be used to separate the compound of formula (315) from the reaction mixture. In some embodiments, the solvent can be removed by evaporation and then separated by chromatography. For example, the following two sets of chromatographic conditions can be used for separation: (1) normal phase purification: 200-300 mesh silica gel filler, using dichloromethane: methanol = 100:5-100:7 gradient elution; and (2) reverse phase purification: C18 and C8 reverse phase filler, using methanol: acetonitrile = 0.1:1-1:0.1 gradient elution. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (315), which can be directly used in subsequent reactions.

[0447] In some embodiments, the compound of formula (317) is reacted once with a sufficient amount of a compound of formula (316) to produce the desired compound of formula (315), wherein each S1-L1 group of the compound of formula (315) is identical. In some embodiments, the compound of formula (317) can be reacted in batches with different compounds of formula (316), i.e., compounds of formula (316) having different L1 and / or S1, so that the generated compound of formula (315) contains two or more S1 and / or L1, as needed. For example, 1 eq of the compound of formula (317) can be first contacted with 2 eq of the first compound of formula (316), and the first S1-L1 group can be connected to the two terminal primary amine groups in the compound of formula (317). Subsequently, it can be contacted with (n3+n1-1) eq of the second compound of formula (316) (the definitions and value ranges of n3 and n1 are as described above), thereby connecting the second S1-L1 group to the (n3+n1-1) secondary amine groups in the compound of formula (317).

[0448] In some embodiments, the compound represented by formula (317) can be obtained by the following preparation method: the method comprises contacting the compound represented by formula (318) with a methylamine aqueous solution in the presence of an organic solvent under deprotection reaction conditions, and isolating the compound represented by formula (317):

[0449]

[0450] Among them, n1, n3, m1, m2, m3, R7, R 10 、R 11 、R 12 、R 13 、R 14 and R 15 The respective definitions and optional ranges are as described above.

[0451] The deprotection reaction conditions may include a reaction temperature of 0-150° C. and a reaction time of 5-72 hours. In some embodiments, the deprotection reaction conditions may include a reaction temperature of 20-80° C. and a reaction time of 10-30 hours.

[0452] The organic solvent can be selected from alcohol, and in some embodiments, it is one of methanol, ethanol and isopropanol, and in some embodiments, it is methanol; relative to the compound of formula (318), the amount of the organic solvent used can be 1-20 L / mol, and in one embodiment, 1.5-10 L / mol.

[0453] The concentration of the methylamine aqueous solution may be 30-40% by mass, and the molar ratio of methylamine to the compound represented by formula (318) may be 10:1-500:1, and in some embodiments, 50:1-200:1.

[0454] Similar to the above, any suitable separation method can be used to separate the compound of formula (317) from the reaction mixture. In some embodiments, the solvent can be removed by evaporation and then separated by chromatography. For example, the following two sets of chromatographic conditions can be used for separation: (1) normal phase purification: 200-300 mesh silica gel filler, using dichloromethane: methanol: ammonia water (25wt%) = 1:1:0.05-1:1:0.25 gradient elution; and (2) reverse phase purification: C18 and C8 reverse phase filler, using methanol: acetonitrile = 0.1:1-1:0.1 gradient elution. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (317), which can be directly used in subsequent reactions.

[0455] In some embodiments, the compound represented by formula (318) can be obtained by the following preparation method: the method comprises contacting the compound represented by formula (319) with triphenylmethane (TrCl), diphenylethylphenylmethane, phenyldiethylphenylmethane or triethylphenylmethane, in some embodiments, with triphenylmethane (TrCl) under substitution reaction conditions in the presence of an organic solvent, and then separating:

[0456]

[0457] Among them, n1, n3, m1, m2, m3, R 10 、R 11 、R 12 、R 13 、R 14 and R 15 The respective definitions and optional ranges are as described above.

[0458] The substitution reaction conditions may include a reaction temperature of 0-100° C. and a reaction time of 5-72 hours. In one embodiment, the reaction temperature is 10-40° C. and the reaction time is 10-30 hours.

[0459] Triphenylmethane (TrCl), diphenylethylphenylmethane, phenyldiethylphenylmethane or triethylphenylmethane are commercially available. The molar ratio of triphenylmethane (TrCl), diphenylethylphenylmethane, phenyldiethylphenylmethane or triethylphenylmethane to the compound represented by formula (319) can be 1:1-10:1, and in some embodiments, 1:1-3:1.

[0460] The organic solvent may be one or more of an epoxy solvent, an ether solvent, a haloalkane solvent, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropylethylamine. In some embodiments, the epoxy solvent is dioxane and / or tetrahydrofuran, in one embodiment, the ether solvent is diethyl ether and / or methyl tert-butyl ether, and in one embodiment, the haloalkane solvent is one or more of dichloromethane, chloroform, and 1,2-dichloroethane; in one embodiment, the organic solvent is dichloromethane. The amount of the organic solvent used relative to the compound of formula (319) is 3-50 L / mol, and in one embodiment, 5-20 L / mol.

[0461] Similar to the above, any suitable separation method can be used to separate the compound of formula (318) from the reaction mixture. In some embodiments, the solvent can be removed by evaporation and then separated by chromatography. For example, the following two sets of chromatographic conditions can be used for separation: (1) normal phase purification: 200-300 mesh silica gel packing, using methanol: dichloromethane = 0.01:1-0.5:1 gradient elution; or using methanol: dichloromethane: ethyl acetate: petroleum ether = 0.1:1:1:1-1:1:1:1 gradient elution; and (2) reverse phase purification: C18 and C8 reverse phase packing, using methanol: acetonitrile = 0.1:1-1:0.1 gradient elution. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (318), which can be directly used in subsequent reactions.

[0462] In some embodiments, the compound represented by formula (319) can be obtained by the following preparation method: the method comprises contacting the compound represented by formula (320) with ethyl trifluoroacetate in an organic solvent under substitution reaction conditions, and isolating the compound represented by formula (319):

[0463]

[0464] Among them, n1, n3, m1, m2, m3, R 10 、R11 、R 12 、R 13 、R 14 and R 15 The respective definitions and optional ranges are as described above.

[0465] In some embodiments, the organic solvent is one or more of acetonitrile, epoxy solvent, ether solvent, alkyl halide solvent, dimethyl sulfoxide, N,N-dimethylformamide and N,N-diisopropylethylamine. In some embodiments, the epoxy solvent is dioxane and / or tetrahydrofuran in some embodiments, the ether solvent is ether and / or methyl tert-butyl ether in one embodiment, the alkyl halide solvent is one or more of dichloromethane, chloroform and 1,2-dichloroethane in one embodiment, and the organic solvent is acetonitrile. The amount of the organic solvent used relative to the compound of formula (320) is 1-50 L / mol, and in some embodiments is 1-20 L / mol.

[0466] The substitution reaction conditions may include a reaction temperature of 0-100° C. and a reaction time of 5-72 hours. In some embodiments, the reaction temperature is 10-40° C. and the reaction time is 10-30 hours.

[0467] The compound of formula (320) is commercially available or can be obtained by a person skilled in the art using known methods. For example, when m1=m2=m3=3, n1=1, n3=2, and R 10 、R 11 、R 12 、R 13 、R 14 、R 15 When both are H, the compound of formula (320) is commercially available from Alfa Aesar Inc.

[0468] The molar ratio of ethyl trifluoroacetate to the compound of formula (320) may be 2:1-10:1, and in one embodiment, 3:1-5:1.

[0469] Similar to the above, any suitable separation method can be used to separate the compound of formula (319) from the reaction mixture. In some embodiments, the solvent can be removed by evaporation and then separated by chromatography. For example, the following two sets of chromatographic conditions can be used for separation: (1) normal phase purification: 200-300 mesh silica gel packing, using methanol: dichloromethane = 0.01:1-0.5:1 gradient elution; or using methanol: dichloromethane: ethyl acetate: petroleum ether = 0.1:1:1:1-1:1:1:1 gradient elution; and (2) reverse phase purification: C18 and C8 reverse phase packing, using methanol: acetonitrile = 0.1:1-1:0.1 gradient elution. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (319), which can be directly used in subsequent reactions.

[0470] Conjugate

[0471] In another aspect, the present disclosure provides a conjugate having a structure as shown in formula (1):

[0472]

[0473] in:

[0474] n1 is an integer from 1 to 3, and n3 is an integer from 0 to 4;

[0475] Each of m1, m2 and m3 is independently an integer from 2 to 10;

[0476] Each R 10 、R 11 、R 12 、R 13 、R 14 and R 15 Each independently selected from H, C1-C 10 Alkyl, C1-C 10 Halogenated alkyl or C1-C 10 Alkoxy, in some embodiments, R 10 、R 11 、R 12 、R 13 、R 14 and R 15 Each independently selected from H, methyl or ethyl;

[0477] R3 is an active drug. In some embodiments, R3 comprises a functional oligonucleotide;

[0478] R2 is a straight chain alkylene group having a length of 1 to 20 carbon atoms, wherein one or more carbon atoms are optionally replaced by one or more of the following groups: C(O), NH, O, S, CH=N, S(O)2, C2-C 10 Alkenylene, C2-C 10 Alkynylidene, C6-C 10 Arylene, C3-C 18 Heterocyclylene and C5-C 10 Heteroarylene; and wherein R2 is optionally substituted by any one or more groups selected from the following: C1-C 10 Alkyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, C1-C 10 Haloalkyl, -OC1-C 10 Alkyl, -OC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-OH, -OC1-C 10 Halogenated alkyl, -SC1-C 10 Alkyl, -SC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-SH, -SC1-C 10 Haloalkyl, halogen substituent, -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2, -N(C1-C 10 Alkyl)(C1-C 10 Alkyl), -NH(C1-C 10 Alkyl), -N(C1-C 10 Alkyl)(C1-C 10 Alkylphenyl), -NH(C1-C 10 alkylphenyl), cyano, nitro, -CO2H, -C(O)O(C1-C 10 Alkyl), -CON(C1-C 10 Alkyl)(C1-C 10 alkyl), -CONH(C1-C 10 alkyl), -CONH2, -NHC(O)(C1-C 10 Alkyl), -NHC(O)(phenyl), -N(C1-C 10 alkyl)C(O)(C1-C 10 Alkyl), -N(C1-C 10 alkyl)C(O)(phenyl), -C(O)C1-C 10 Alkyl, -C(O)C1-C 10 Alkylphenyl, -C(O)C1-C 10Haloalkyl, -OC(O)C1-C 10 Alkyl, -SO2(C1-C 10 Alkyl), -SO2(phenyl), -SO2(C1-C 10 Halogenated alkyl), -SO2NH2, -SO2NH(C1-C 10 Alkyl), -SO2NH(phenyl), -NHSO2(C1-C 10 Alkyl), -NHSO2(phenyl) and -NHSO2(C1-C 10 haloalkyl);

[0479] Each L1 is a straight chain alkylene group having a length of 1 to 70 carbon atoms, wherein one or more carbon atoms are optionally replaced by one or more selected from the group consisting of: C(O), NH, O, S, CH=N, S(O)2, C2-C 10 Alkenylene, C2-C 10 Alkynylidene, C6-C 10 Arylene, C3-C 18 Heterocyclylene and C5-C 10 Heteroarylene; and wherein L1 optionally has any one or more substituents selected from the group consisting of: C1-C 10 Alkyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, C1-C 10 Haloalkyl, -OC1-C 10 Alkyl, -OC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-OH, -OC1-C 10 Halogenated alkyl, -SC1-C 10 Alkyl, -SC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-SH, -SC1-C 10 Haloalkyl, halogen substituent, -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2, -N(C1-C 10 Alkyl)(C1-C 10 Alkyl), -NH(C1-C 10 Alkyl), -N(C1-C 10 Alkyl)(C1-C 10 Alkylphenyl), -NH(C1-C 10 alkylphenyl), cyano, nitro, -CO2H, -C(O)O(C1-C 10 Alkyl), -CON(C1-C 10 Alkyl)(C1-C10 alkyl), -CONH(C1-C 10 alkyl), -CONH2, -NHC(O)(C1-C 10 Alkyl), -NHC(O)(phenyl), -N(C1-C 10 alkyl)C(O)(C1-C 10 Alkyl), -N(C1-C 10 alkyl)C(O)(phenyl), -C(O)C1-C 10 Alkyl, -C(O)C1-C 10 Alkylphenyl, -C(O)C1-C 10 Haloalkyl, -OC(O)C1-C 10 Alkyl, -SO2(C1-C 10 Alkyl), -SO2(phenyl), -SO2(C1-C 10 Halogenated alkyl), -SO2NH2, -SO2NH(C1-C 10 Alkyl), -SO2NH(phenyl), -NHSO2(C1-C 10 Alkyl), -NHSO2(phenyl) and -NHSO2(C1-C 10 In some embodiments, L1 can be selected from the group consisting of formula A1-A26 or any combination thereof, wherein the structures and definitions of A1-A26 are as described above.

[0480] n1,n3,m1,m2,m3,R 10 ,R 11 ,R 12 ,R 13 ,R 14 ,R 15 and M1 are as defined above.

[0481] In some embodiments, R2 is a linking group formed by reacting the R4 group in the compound of formula (321) to an active drug. In some embodiments, R2 is a linking group formed by reacting the R4 group in the compound of formula (321) to a functional oligonucleotide. In some embodiments, the R2 group contains both a linking site connected to the N atom on the nitrogen-containing backbone and a linking site connected to the P atom in R3. In some embodiments, the site in R2 that is connected to the N atom on the nitrogen-containing backbone forms an amide bond with the N atom, and the site that is connected to the P atom in R3 forms a phosphate bond with the P atom. In some embodiments, R2 can be B5, B6, B5' or B6':

[0482]

[0483] in, It represents the site where the group is covalently bonded. The selection and value range of q2 are as described above.

[0484] In some embodiments, R3 is a group represented by the structure of A59:

[0485]

[0486] Wherein, E1 is OH, SH or BH2. In some embodiments, E1 is OH or SH; Nu is a functional oligonucleotide.

[0487] In the context of the present disclosure, unless otherwise specified, a "conjugate" group or molecule refers to a group or molecule that can form a covalent bond with a corresponding ligand, and the conjugate group or molecule and its ligand have specific functions. Accordingly, a "conjugate" refers to a compound formed by covalently linking the chemical group and its ligand. Furthermore, an "oligonucleotide conjugate" refers to a compound formed by covalently linking one or more conjugate groups with specific functions to an oligonucleotide. In some embodiments, the conjugate disclosed herein is an oligonucleotide conjugate. In the context of the present disclosure, a "conjugate molecule" can be a specific compound that is conjugated to an oligonucleotide through a reaction to ultimately form the oligonucleotide conjugate of the present disclosure. In some embodiments, the oligonucleotide is an siRNA, in which case the conjugate of the present disclosure is an siRNA conjugate.

[0488] In some embodiments, the conjugate has a structure represented by formula (3), (4), (5), (6), (7), (8), (9), (10), (11), (12), (13), (14), (15), (16), (17), (18), (19), (20), (21), or (22):

[0489]

[0490]

[0491]

[0492]

[0493]

[0494]

[0495] In some embodiments, the oligonucleotide in the oligonucleotide conjugate of the present disclosure is a functional oligonucleotide. Functional oligonucleotide refers to such oligonucleotide: the oligonucleotide can be by producing stable and specific hybridization between the target sequence, utilizing the principles such as RNA activation (RNA activation, RNAa), RNA interference (RNA interference, RNAi), antisense nucleic acid technology, exon skipping (exon skipping) technology, to increase or decrease the expression of the target gene, or cause mRNA variable splicing. In some aspects, the functional oligonucleotide can also be a nucleic acid structure that is stably and specifically combined with the target protein. In addition, it is readily understood by those skilled in the art that polynucleotides (such as mRNA itself or its fragments) are also applicable to be conjugated with the conjugate molecule provided by the present disclosure to form a conjugate to achieve targeted delivery, such as liver-targeted delivery, so as to regulate the expression of the protein transcribed by mRNA. Therefore, in this context, the concept of "functional oligonucleotide" may also encompass mRNA or its fragments.

[0496] In some embodiments, the functional oligonucleotide is capable of interacting 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 functional oligonucleotide is complementary to the bases of the target sequence. In some embodiments, the functional oligonucleotide can be complementary to more than 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the bases in the target sequence, or completely complementary to the target sequence. In some embodiments, the functional oligonucleotide can contain 1, 2 or 3 bases that are not complementary to the target sequence. In some embodiments, the functional oligonucleotide includes deoxyribonucleotides or ribonucleotides, as well as nucleotides with modifications. In some embodiments, the functional oligonucleotide is a single-stranded DNA, RNA, or DNA-RNA chimera, or a double-stranded DNA, RNA, or DNA-RNA hybrid.

[0497] Thus, in some embodiments, the functional oligonucleotide suitable for the oligonucleotide conjugate of the present disclosure can be one of small interfering RNA (siRNA), microRNA (microRNA), anti-microRNA (antimiR), microRNA antagonist (antagomir), microRNA mimics (microRNA mimics), decoy oligonucleotide (decoy), immune stimulatory (immune stimulatory), G-quadruplex (G-quadruplex), alternative splicing body (splice altering), single-stranded RNA (ssRNA), antisense nucleic acid (antisense), nucleic acid aptamer (Nucleic Acid Aptamer), small activating RNA (saRNA), stem-loop RNA (stem-loop RNA) or DNA. WO2015 / 006740A2 discloses a conjugate of different ligands conjugated to oligonucleotides, wherein the ligand is connected to the oligonucleotide via a linker. The oligonucleotide is selected from one of small interfering RNA (siRNA), microRNA (microRNA), anti-microRNA (antimiR), microRNA antagonist (antagomir), microRNA mimics (microRNA mimics), decoy oligonucleotide (decoy), immunostimulatory, G-quadruplex, splicing alteration, single-stranded RNA (ssRNA), antisense nucleic acid (antisense), aptamer, stem-loop RNA (stem-loop RNA) or DNA. These conjugates show good stability in the in vivo delivery of oligonucleotides. In a further embodiment, the functional oligonucleotide suitable for the oligonucleotide conjugates of the present disclosure is the oligonucleotide disclosed in WO2009082607A2, WO2009073809A2 or WO2015006740A2, the entire contents of which are incorporated herein by reference.

[0498] The oligonucleotide conjugates disclosed herein can modulate the abnormal expression of specific genes in specific cells, such as hepatocytes, by improving the liver-targeted delivery efficiency of active agents, such as functional oligonucleotides, thereby enhancing the interaction between the functional oligonucleotide and the targeting sequence in the cell. In some embodiments, the specific gene can be an endogenous gene expressed in the liver or a gene of a pathogen that propagates in the liver. Genes abnormally expressed in hepatocytes can be, for example, ApoB, ApoC, ANGPTL3, PCSK9, SCD1, TIMP-1, Col1A1, FVII, STAT3, p53, HBV, HCV, and the like. In some embodiments, the gene abnormally expressed in hepatocytes is an HBV gene, an ANGPTL3 gene, or an APOC3 gene. In the context of the present disclosure, the HBV gene refers to a gene having a sequence as shown in Genbank Accession No. NC_003977.1; the ANGPTL3 gene refers to a gene having an mRNA sequence as shown in Genbank Accession No. NM_014495.3; and the APOC3 gene refers to a gene having an mRNA sequence as shown in Genbank Accession No. NM_000040.1.

[0499] In some embodiments, the "target sequence" is a target mRNA. In the context of the present disclosure, "target mRNA" refers to mRNA corresponding to a gene abnormally expressed in, for example, hepatocytes. It can be mRNA corresponding to an overexpressed gene or mRNA corresponding to an underexpressed gene. In some embodiments, since most diseases arise from overexpression of mRNA, the target mRNA is preferably mRNA corresponding to an overexpressed gene. In some embodiments of the present disclosure, the target mRNA corresponding to the above-mentioned abnormally expressed genes can be mRNA corresponding to genes such as ApoB, ApoC, ANGPTL3, PCSK9, SCD1, TIMP-1, Col1A1, FVII, STAT3, p53, HBV, and HCV. In some embodiments, the target mRNA can be mRNA transcribed from the corresponding HBV gene, ANGPTL3 gene, or APOC3 gene.

[0500] The P atom in formula A59 can be connected to any possible position in the oligonucleotide sequence, for example, can be connected on any Nucleotide of oligonucleotide.In some embodiments, the functional oligonucleotide in oligonucleotide conjugates of the present disclosure is a single-stranded oligonucleotide (for example, single-stranded RNA or aptamer).Now, the P atom in formula A59 can be connected to the terminal region of described single-stranded oligonucleotide, and the terminal region of described single-stranded oligonucleotide refers to first 4 Nucleotide counting from one end in described single-stranded oligonucleotide.In some embodiments, the P atom in formula A59 is connected to any end of described single-stranded oligonucleotide.

[0501] In some embodiments, the functional oligonucleotide in the oligonucleotide conjugate of the present disclosure is a double-stranded oligonucleotide (e.g., siRNA, microRNA or DNA), and the double-stranded oligonucleotide includes a sense strand and an antisense strand. In some embodiments, the P atom in the formula A59 is connected to the terminal region of the sense strand or antisense strand in the double-stranded oligonucleotide, and the terminal region refers to the first 4 nucleotides from one end in the sense strand or the antisense strand. In some embodiments, the P atom in the formula A59 is connected to any end of the sense strand or the antisense strand; In some embodiments, the P atom in the formula A59 is connected to the 3' end of the sense strand. In the case where the P atom in the formula A59 is connected to the above-mentioned position of the sense strand of the double-stranded oligonucleotide, after the oligonucleotide conjugate provided by the present disclosure enters the cell, when unwinding, a separate double-stranded oligonucleotide antisense strand can be released to block the process of protein translation by the target mRNA and inhibit the expression of specific genes.

[0502] The P atom in formula A59 can be connected to any possible position on the nucleotide in the oligonucleotide sequence, for example, at the 5' position of the nucleotide, the 2' position of the nucleotide, the 3' position of the nucleotide or the base of the nucleotide. In some embodiments, the P atom in formula A59 can be connected to the 2' position, 3' position or 5' position of the nucleotide in the oligonucleotide sequence by forming a phosphodiester bond. In some specific embodiments, the P atom in formula A59 is connected to the oxygen atom formed after dehydrogenation of the 3' hydroxyl group of the nucleotide at the 3' terminal end of the sense strand in the double-stranded oligonucleotide sequence, or the P atom in formula A59 is connected to the nucleotide by replacing the hydrogen in the 2'-hydroxyl group of the nucleotide in the sense strand in the double-stranded oligonucleotide sequence, or the P atom in formula A59 is connected to the nucleotide by replacing the hydrogen atom in the 5' hydroxyl group of the nucleotide at the 5' terminal end of the sense strand in the double-stranded oligonucleotide sequence.

[0503] Without wishing to be limited, the following embodiments and examples describe in detail the case where the functional oligonucleotide in the oligonucleotide conjugate of the present invention is a small interfering RNA (siRNA). In this case, the oligonucleotide conjugate of the present invention is an siRNA conjugate. In the context of this article, for the convenience of description, the siRNA conjugates in these embodiments are also referred to as the siRNA conjugate of the present invention. This does not mean that the oligonucleotide in the oligonucleotide conjugate of the present invention can only be siRNA. On the contrary, the oligonucleotide or even the active drug may be other alternatives disclosed herein or well known to those skilled in the art. Based on the detailed description of the siRNA conjugate, it can be imagined that other active drugs or functional oligonucleotides will have similar effects when conjugated to the conjugate molecules provided by the present invention.

[0504] As is well known to those skilled in the art, siRNAs contain nucleotide groups as structural units, each of which comprises a phosphate group, a ribose group, and a base. Typically, active, i.e., functional, siRNAs are approximately 12-40 nucleotides in length, and in some embodiments, approximately 15-30 nucleotides in length. Each nucleotide in the siRNA can independently be a modified or unmodified nucleotide. To increase stability, at least one nucleotide in the siRNA is a modified nucleotide.

[0505] The inventors of the present disclosure have discovered that the siRNA described in the following embodiments has high activity and / or stability, and thus can be used as the siRNA for the purpose of the invention in the present disclosure.

[0506] 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 comprises a sense strand and an antisense strand, wherein the sense strand comprises nucleotide sequence 1, and the antisense strand comprises 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-complementary 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.

[0507] In some embodiments, the siRNA disclosed herein is capable of inhibiting HBV gene expression, ANGPTL3 gene expression, or APOC3 gene expression by at least 50% at a concentration of 3 mg / kg. In some embodiments, the siRNA disclosed herein is capable of inhibiting HBV gene expression, ANGPTL3 gene expression, or APOC3 gene expression by at least 55%, 60%, 65%, 70%, 75%, or 80% at a concentration of 3 mg / kg.

[0508] 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; 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 inhibitory ability of the siRNA conjugate, and siRNA conjugates containing these specific nucleotide differences are also within the scope of protection of this disclosure.

[0509] In some embodiments, the nucleotide sequence 1 and the nucleotide sequence 2 are substantially reverse complementary, substantially completely reverse complementary, or completely reverse complementary.

[0510] In some embodiments, the nucleotide sequence 1 differs from the first nucleotide sequence by no more than one nucleotide, and / or the nucleotide sequence 2 differs from the nucleotide sequence B by no more than one nucleotide. In some embodiments, the nucleotide difference between the nucleotide sequence 2 and the nucleotide sequence B comprises a difference in the position of the first nucleotide Z' in the nucleotide sequence 2, as viewed from the 5' end to the 3' end. In some embodiments, the last nucleotide Z in the nucleotide sequence 1, as viewed from the 5' end to the 3' end, is complementary to Z'.

[0511] In some embodiments, the sense strand further comprises a nucleotide sequence 3, and the antisense strand further comprises a nucleotide sequence 4, wherein the nucleotide sequence 3 and the nucleotide sequence 4 are of equal length and are both 1-4 nucleotides, the nucleotide sequence 3 is connected to the 5' end of the nucleotide sequence 1, and the nucleotide sequence 4 is connected to the 3' end of the nucleotide sequence 2, and the nucleotide sequence 4 is complementary to the second segment of nucleotide sequence, which refers to a nucleotide sequence in the target mRNA that is adjacent to the first segment of nucleotide sequence and has the same length as the nucleotide sequence 4. In some embodiments, the nucleotide sequence 3 and the nucleotide sequence 4 are substantially completely reverse complementary or completely reverse complementary. Therefore, the length of the sense strand and the antisense strand can be 19-23 nucleotides.

[0512] In some embodiments, the siRNA of the present disclosure further comprises a nucleotide sequence 5 having a length of 1 to 3 nucleotides and attached to the 3' end of the antisense strand, thereby constituting a 3' overhang of the antisense strand; in some embodiments, the nucleotide sequence 5 has a length of 1 or 2 nucleotides. Thus, in some embodiments, the ratio of the lengths 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.

[0513] In one embodiment, the nucleotide sequence 5 is 2 nucleotides in length, and from the 5' end to the 3' end, the nucleotide sequence 5 is 2 consecutive deoxythymidine nucleotides, or 2 consecutive uracil nucleotides, or is complementary to a third nucleotide sequence, wherein the third sequence refers to a nucleotide sequence in the target mRNA that is adjacent to the first nucleotide sequence or adjacent to the second nucleotide sequence and has a length equal to that of the nucleotide sequence 5. In some embodiments, the ratio of the length of the sense strand to the antisense strand of the siRNA of the present disclosure is 19 / 21 or 21 / 23. In this case, the siRNA of the present disclosure has significant hepatocyte mRNA silencing activity.

[0514] 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.

[0515] 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).

[0516] In the context of the present disclosure, the term "modified nucleotide" includes nucleotides whose ribose groups are modified, such as nucleotides in which the 2'-hydroxyl group is replaced by other groups, nucleotide analogs, or nucleotides having modified bases.

[0517] 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 modified group. In other words, at least a portion of the phosphate group and / or ribose group in the phosphate-ribose backbone of at least one single strand in the sense strand and the antisense strand is a phosphate group with a modified group and / or a ribose group with 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.

[0518] In some embodiments, each nucleotide in the sense strand and the antisense strand is independently a fluorinated modified nucleotide or a non-fluorinated modified nucleotide.

[0519] The "fluorinated modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2'-position of the ribose group of the nucleotide is substituted with fluorine, and has a structure represented by the following formula (207).

[0520] "Non-fluorinated modified nucleotides" refer to nucleotides or nucleotide analogs in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by a non-fluorinated group. In some embodiments, each non-fluorinated modified nucleotide is independently a nucleotide or nucleotide analog in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by a non-fluorinated group.

[0521] Nucleotides in which the hydroxyl group at the 2' position of the ribose group is replaced by a non-fluorinated group are well known to those skilled in the art. These nucleotides are, for example, 2'-alkoxy-modified nucleotides, 2'-substituted alkoxy-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-amino-modified nucleotides, 2'-substituted amino-modified nucleotides or 2'-deoxynucleotides.

[0522] In some embodiments, the 2'-alkoxy modified nucleotide is a methoxy modified nucleotide, as shown in formula (208). In some embodiments, the 2'-substituted alkoxy modified nucleotide is a 2'-O-methoxyethyl modified nucleotide, as shown in formula (209). In some embodiments, the 2'-amino modified nucleotide is as shown in formula (210).

[0523] In some embodiments, the 2'-deoxynucleotide (DNA) is represented by formula (211).

[0524]

[0525] Nucleotide analogs are groups that can replace nucleotides in nucleic acids but are different from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, or thymine. In some embodiments, the nucleotide analogs can be, for example, isonucleotides, bridged nucleic acid (BNA) nucleotides, or acyclic nucleotides.

[0526] BNA nucleotides are constrained or inaccessible nucleotides. BNAs can contain five-, six-, or seven-membered rings with a "fixed" C3'-endo sugar condensed bridge structure. The bridge is usually incorporated into the 2'- and 4'-positions of the ribose ring to provide 2',4'-BNA nucleotides, such as LNA, ENA, and cET BNA, where LNA is shown in formula (212), ENA is shown in formula (213), and cET BNA is shown in formula (214).

[0527]

[0528] Acyclic nucleotides are nucleotides formed by opening the sugar ring of a nucleotide, such as unlocked nucleic acid (UNA) nucleotides and glycerol nucleic acid (GNA) nucleotides, where UNA is shown in formula (215) and GNA is shown in formula (216).

[0529]

[0530] Wherein, R is H, OH or alkoxy (O-alkyl).

[0531] Isonucleotides are nucleotides formed by changing the position of the base on the ribose ring, for example, compounds formed by moving the base from the 1'-position to the 2'-position or 3'-position of the ribose ring, as shown in formula (217) or (218).

[0532]

[0533] Wherein, Base represents a nucleic acid base, such as A, U, G, C or T; R is H, OH, F or a non-fluorinated group as described above.

[0534] In some embodiments, the nucleotide analog is an isonucleotide, LNA, ENA, cET, UNA or GNA. In some embodiments, each non-fluorinated modified nucleotide is a methoxy-modified nucleotide, wherein the methoxy-modified nucleotide is a nucleotide in which the 2'-hydroxyl group of the ribose group is replaced by a methoxy group.

[0535] In the above and below, "fluorinated nucleotides", "2'-fluorinated nucleotides", "nucleotides in which the 2'-hydroxyl group of the ribose group is replaced by fluorine" and "nucleotides having a 2'-fluorinated ribose group" have the same meaning, all referring to the 2'-hydroxyl group of the nucleotide being replaced by fluorine to form a structure as shown in formula (207); "methoxy-modified nucleotides", "2'-methoxy-modified nucleotides", "nucleotides in which the 2'-hydroxyl group of the ribose group is replaced by a methoxy group" and "nucleotides having a 2'-methoxyribose group" have the same meaning, all referring to the 2'-hydroxyl group of the ribose group of the nucleotide being replaced by a methoxy group to form a structure as shown in formula (208).

[0536] In some embodiments, the siRNA of the present invention is an siRNA with the following modifications: in the direction from the 5' end to the 3' end, the nucleotides at positions 7, 8, and 9 of the nucleotide sequence 1 in the sense strand of the siRNA are fluorine-modified nucleotides, and the nucleotides at the remaining positions in the sense strand are methoxy-modified nucleotides and / or in the antisense strand, the nucleotides at positions 2, 6, 14, and 16 of the nucleotide sequence 2 are fluorine-modified nucleotides, and the nucleotides at the remaining positions in the antisense strand are methoxy-modified nucleotides; in some embodiments, the siRNA of the present invention is an siRNA with the following modifications: in the direction from the 5' end to the 3' end, the nucleotides at positions 5, 7, 8, and 9 of the nucleotide sequence 1 in the sense strand of the siRNA are fluorine-modified nucleotides, and the nucleotides at the remaining positions in the sense strand are methoxy-modified nucleotides; and / or in the antisense strand, the nucleotides at positions 2, 6, 8, 9, 14, and 16 of the nucleotide sequence 2 are fluorine-modified nucleotides, and the nucleotides at the remaining positions in the antisense strand are 2'-methoxy-modified nucleotides. In some embodiments, the siRNA disclosed herein is an siRNA having the following modifications: in the direction from the 5' end to the 3' end, the nucleotides at positions 7, 8, and 9 of the nucleotide sequence 1 in the sense strand of the siRNA are fluorine-modified nucleotides, and the nucleotides at the remaining positions in the sense strand are methoxy-modified nucleotides, and / or in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 14, and 16 of the nucleotide sequence 2 in the antisense strand of the siRNA are fluorine-modified nucleotides, and the nucleotides at the remaining positions in the antisense strand are methoxy-modified nucleotides.

[0537] In some embodiments of the siRNA disclosed herein, the nucleotides are modified with a phosphate group. In the context of the present disclosure, the modification on the phosphate group is, in one embodiment, a phosphorothioate modification as shown in formula (201), 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 stabilizes the structure of the siRNA, maintaining high specificity and high affinity for base pairing.

[0538]

[0539] According to some embodiments of the present disclosure, in the siRNA, the phosphorothioate linkage is present in at least one 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:

[0540] between the first and second nucleotides at the 5' end of the sense strand;

[0541] between the second and third nucleotides at the 5' end of the sense strand;

[0542] between the first and second nucleotides at the 3' end of the sense strand;

[0543] between the second and third nucleotides at the 3' end of the sense strand;

[0544] between the first and second nucleotides at the 5' end of the antisense strand;

[0545] between the second and third nucleotides at the 5' end of the antisense strand;

[0546] between the first and second nucleotides at the 3' end of the antisense strand; and

[0547] between the second and third nucleotides at the 3' end of the antisense strand.

[0548] According to some embodiments of the present disclosure, the 5'-terminal nucleotide of the antisense strand sequence of the siRNA molecule is a 5'-phosphate nucleotide or a 5'-phosphate analog-modified nucleotide.

[0549] In some embodiments, the 5'-phosphate nucleotide may have the structure shown in formula (202):

[0550]

[0551] The types of commonly used 5'-phosphate analogue-modified nucleotides are well known to those skilled in the art, for example, the four nucleotides shown in formulas (203) to (206) disclosed in Anastasia Khvorova and Jonathan K. Watts, The chemical evolution of oligonucleotide therapies of clinical utility. Nature Biotechnology, 2017, 35(3): 238-48:

[0552]

[0553]

[0554] wherein R represents a group selected from the group consisting of H, OH, F and methoxy;

[0555] Base represents a base selected from A, U, C, G or T.

[0556] In some embodiments, the 5'-phosphate nucleotide or 5'-phosphate analog modified nucleotide is a vinylphosphonate (VP) modified nucleotide as shown in formula (203), a 5'-phosphate nucleotide as shown in formula (202), or a 5'-thiophosphate modified nucleotide as shown in formula (205).

[0557] The inventors of the present disclosure unexpectedly discovered that the siRNA conjugates of the present disclosure, while having significantly improved serum stability, also exhibited no significantly reduced target mRNA silencing activity and excellent in vivo gene expression inhibition effect. This shows that the siRNA conjugates of the present disclosure have high in vivo delivery efficiency. According to some embodiments of the present disclosure, the oligonucleotide conjugates of the present disclosure are siRNA conjugates comprising siRNAs such as those shown in Tables 1A to 1F:

[0558] Table 1 siRNA sequences

[0559] Table 1A

[0560]

[0561]

[0562] Table 1B

[0563]

[0564] Table 1C

[0565]

[0566]

[0567] Table 1D

[0568]

[0569] Table 1E

[0570]

[0571]

[0572] Table 1F

[0573]

[0574]

[0575] *S: sense strand; AS: antisense strand

[0576] In the above table, capital letters C, G, U, and A represent the base composition of the nucleotide; lowercase letter m indicates that the nucleotide adjacent to the left of the letter m is a 2'-methoxy-modified nucleotide; lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a 2'-fluoro-modified nucleotide; lowercase letter s indicates that the connection between the two nucleotides adjacent to the left and right of the letter s is a phosphorothioate connection; P1 indicates that the nucleotide adjacent to the right of P1 is a 5'-phosphate nucleotide or a 5'-phosphate analogue-modified nucleotide, and in some embodiments, it is a vinyl phosphate-modified nucleotide (represented by VP in the following examples), a 5'-phosphate nucleotide (represented by P in the following examples), or a phosphorothioate-modified nucleotide (represented by Ps in the following examples).

[0577] It is well known to those skilled in the art that modified nucleotide groups can be introduced into the siRNA described herein by using nucleoside monomers having corresponding modifications, and methods for preparing nucleoside monomers having corresponding modifications and methods for introducing modified nucleotide groups into siRNA are also well known to those skilled in the art. All modified nucleoside monomers can be purchased commercially or prepared using known methods.

[0578] Preparation of oligonucleotide conjugates

[0579] The oligonucleotide conjugates of the present disclosure can be prepared using any reasonable synthetic route. For example, the oligonucleotide conjugates of the present disclosure can be prepared using the following method, which includes sequentially connecting nucleoside monomers in the 3' to 5' direction according to the nucleotide types and sequence of the oligonucleotide under phosphoramidite solid phase synthesis conditions, wherein the connection of each nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization; in some embodiments, the method further includes contacting the compound represented by formula (321) with the nucleoside monomer or the nucleotide sequence connected to the solid phase support under coupling reaction conditions and in the presence of a coupling reagent, thereby connecting the compound represented by formula (321) to the nucleotide sequence through a coupling reaction.

[0580] In some embodiments, the method further comprises the steps of removing the protecting group, cleaving from the solid phase support, and separating and purifying.

[0581] In some embodiments, the oligonucleotide is a double-stranded oligonucleotide, and the preparation method comprises the following steps: contacting the compound represented by formula (321) with the nucleoside monomer at the 3' end of the sense chain or antisense chain under coupling reaction conditions and in the presence of a coupling reagent, so that the compound represented by formula (321) is connected to the first nucleotide in the sequence, and the nucleoside monomers are sequentially connected in the direction from 3' to 5' to synthesize the sense chain or antisense chain of the double-stranded oligonucleotide; wherein, the compound (321) contains a hydroxyl group protected as the first functional group in R4, and a hydroxyl group as the second functional group as shown in formula (C1') or The compound of formula (321) with the group shown in (C3') is deprotected before being connected to the first nucleoside monomer; the connection of each nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization; the sense chain or antisense chain of the nucleic acid connected with the conjugate molecule is obtained; the nucleoside monomers are sequentially connected in the direction of 3' to 5' to synthesize the other chain of the double-stranded oligonucleotide, and the connection of each nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization; the protecting group is removed and cut with the solid phase carrier, the sense chain and the antisense chain are separated and purified, and annealed.

[0582] In some embodiments, the oligonucleotide is a double-stranded oligonucleotide, and the preparation method comprises the following steps: sequentially connecting nucleoside monomers in the 3' to 5' direction to synthesize the sense chain and antisense chain of the double-stranded oligonucleotide, wherein the connection of each nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization to obtain a sense chain connected to a solid phase support and an antisense chain connected to a solid phase support; under coupling reaction conditions and in the presence of a coupling reagent, contacting the compound represented by formula (321) with the sense chain connected to the solid phase support or the antisense chain connected to the solid phase support, thereby connecting the compound of formula (321) to the sense chain or the antisense chain, wherein the compound of formula (321) is a compound of formula (321) containing phosphoramidite as the first functional group in R4; removing the protecting group and cutting with the solid phase support, obtaining the sense chain or antisense chain of the oligonucleotide by separation and purification, and annealing, wherein the sense chain or antisense chain of the oligonucleotide is connected to a conjugate molecule.

[0583] In some embodiments, the P atom in formula A59 is linked to the 3' end of the sense strand in the siRNA, and the method for preparing the siRNA conjugate of the present disclosure comprises:

[0584] (1) Removal of the protecting group R in the compound of formula (321) connected to the solid phase support (hereinafter also referred to as the L-conjugated molecule connected to the solid phase support) k ; Under coupling reaction conditions and in the presence of a coupling reagent, contacting the L-conjugated molecule connected to the solid phase support with a nucleoside monomer to obtain a nucleoside monomer connected to the solid phase support through the L-conjugated molecule;

[0585] (2) starting with the nucleoside monomer connected to the solid phase support via the L conjugated molecule, synthesizing the positive strand of the siRNA by the phosphoramidite solid phase synthesis method in the 3'-5' direction;

[0586] (3) synthesizing the antisense strand of siRNA by phosphoramidite solid phase synthesis;

[0587] (4) Separating the sense strand and antisense strand of siRNA and annealing them to obtain the siRNA conjugate disclosed herein.

[0588] Wherein, in step (1), the protecting group R in the L conjugated molecule connected to the solid phase carrier is removed kThe method comprises contacting a compound of formula (321) with a deprotecting agent under deprotection conditions. The deprotection conditions include a temperature of 0-50° C., in some embodiments 15-35° C., a reaction time of 30-300 seconds, in some embodiments 50-150 seconds, and a deprotecting agent selected from one or more of trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, and monochloroacetic acid, in some embodiments dichloroacetic acid. The molar ratio of the deprotecting agent to the compound of formula (321) may be 10:1 to 1000:1, in some embodiments 50:1 to 500:1.

[0589] The coupling reaction conditions and coupling reagents can use any conditions and reagents suitable for the above coupling reaction. In some embodiments, the same conditions and reagents as those used in the coupling reaction in the solid phase synthesis method are used.

[0590] In some embodiments, the conditions of the coupling reaction include a reaction temperature of 0-50°C, in some embodiments 15-35°C. The molar ratio of the compound of formula (321) to the nucleoside monomer can be 1:1-1:50, in some embodiments 1:2-1:5; the molar ratio of the compound of formula (321) to the coupling reagent can be 1:1 to 1:50, in some embodiments 1:3-1:10, and the reaction time can be 200-3000 seconds, in some embodiments 500-1500 seconds. The coupling reagent can be selected from one or more of 1H-tetrazole, 5-ethylthio 1H-tetrazole, 5-benzylthio 1H-tetrazole, and in some embodiments 5-ethylthio 1H-tetrazole. The coupling reaction can be carried out in an organic solvent, and the organic solvent can be selected from one or more of anhydrous acetonitrile, anhydrous DMF, and anhydrous dichloromethane, and in some embodiments anhydrous acetonitrile. The amount of the organic solvent used may be 3-50 L / mol relative to the compound of formula (321), and in some embodiments, 5-20 L / mol.

[0591] In step (2), the sense strand S of the siRNA conjugate is synthesized in the 3'-5' direction starting from the nucleoside monomers attached to the solid phase support by the phosphoramidite nucleic acid solid phase synthesis method. At this time, the L conjugated molecule is attached to the 3' end of the obtained sense strand.

[0592] Other conditions for the solid phase synthesis described in steps (2) and (3), including nucleoside monomer deprotection conditions, deprotection reagent type and amount, coupling reaction conditions, coupling reagent type and amount, capping reaction conditions, capping reagent type and amount, oxidation reaction conditions, oxidation reagent type and amount, sulfurization reaction conditions, sulfurization reagent type and amount, various reagents, amounts and conditions conventionally used in the art are adopted herein.

[0593] For example, in some embodiments, the solid phase synthesis in steps (2) and (3) may use the following conditions:

[0594] Nucleoside monomer deprotection conditions include a temperature of 0-50°C, in some embodiments 15-35°C, a reaction time of 30-300 seconds, in some embodiments 50-150 seconds, and a deprotection reagent selected from one or more of trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, and monochloroacetic acid, in some embodiments dichloroacetic acid. The molar ratio of the deprotection reagent to the 4,4'-dimethoxytrityl protecting group on the solid support can be 2:1-100:1, in some embodiments 3:1-50:1.

[0595] The coupling reaction conditions include a temperature of 0-50°C, in some embodiments 15-35°C, a molar ratio of the nucleic acid sequence attached to the solid support to the nucleoside monomer of 1:1-1:50, in some embodiments 1:5-1:15; a molar ratio of the nucleic acid sequence attached to the solid support to the coupling reagent of 1:1-1:100, in some embodiments 1:50-1:80, and the reaction time and the choice of coupling reagent are the same as described above.

[0596] The capping reaction conditions include a temperature of 0-50°C, in some embodiments 15-35°C, a reaction time of 5-500 seconds, in some embodiments 10-100 seconds, and the selection of the capping reagent is the same as described above. The molar ratio of the total amount of the capping reagent to the nucleic acid sequence attached to the solid support can be 1:100-100:1, in some embodiments 1:10-10:1. When equimolar amounts of acetic anhydride and N-methylimidazole are used as the capping reagent, the molar ratio of acetic anhydride, N-methylimidazole, and the nucleic acid sequence attached to the solid support can be 1:1:10-10:10:1, in some embodiments 1:1:2-2:2:1.

[0597] The oxidation reaction conditions include a temperature of 0-50°C, in some embodiments 15-35°C, a reaction time of 1-100 seconds, in some embodiments 5-50 seconds, and the oxidizing agent is iodine in some embodiments (in further embodiments, provided in the form of iodine water). The molar ratio of the oxidizing agent to the nucleic acid sequence connected to the solid phase support in the coupling step can be 1:1-100:1, in some embodiments 5:1-50:1. In some embodiments, the oxidation reaction is carried out in a mixed solvent of tetrahydrofuran: water: pyridine = 3:1:1-1:1:3. The sulfurization reaction conditions include a temperature of 0-50°C, in some embodiments 15-35°C, a reaction time of 50-2000 seconds, in some embodiments 100-1000 seconds, and the sulfurizing agent is hydrogenated xanthan in some embodiments. The molar ratio of the sulfurizing agent to the nucleic acid sequence connected to the solid phase support in the coupling step can be 10:1–1000:1, in some embodiments 10:1-500:1. In some embodiments, the sulfurization reaction is carried out in a mixed solvent of acetonitrile:pyridine=1:3-3:1.

[0598] According to the method provided herein, after all nucleoside monomers are linked and before annealing, the method further includes separating the sense and antisense strands of the siRNA. Separation methods are well known to those skilled in the art and generally include cleaving the synthesized nucleotide sequence from the solid support, removing protecting groups on the bases, phosphate groups, and ligands, and purifying and desalting.

[0599] The synthesized nucleotide sequence is cleaved from the solid support, and the protecting groups on the bases, phosphate groups, and ligands are removed according to conventional cleavage and deprotection methods used in siRNA synthesis. For example, the obtained nucleotide sequence attached to the solid support is contacted with concentrated ammonia water; during the deprotection process, the protecting groups YCOO- of the A46-A54 groups are converted to hydroxyl groups, and the S1 groups are converted to the corresponding M1 groups, thereby generating the conjugate represented by formula (1). The concentrated ammonia water can refer to ammonia water with a concentration of 25-30% by weight, and the amount of concentrated ammonia water used can be 0.2 ml / μmol-0.8 ml / μmol relative to the target siRNA sequence.

[0600] When at least one 2'-TBDMS protection is present on the synthesized nucleotide sequence, the method further comprises contacting the nucleotide sequence, after removal of the solid support, with triethylamine trihydrofluoride to remove the 2'-TBDMS protection. In this case, the resulting target siRNA sequence has a free 2'-hydroxyl group of the corresponding nucleoside. The amount of pure triethylamine trihydrofluoride used can be 0.4 ml / μmol to 1.0 ml / μmol relative to the target siRNA sequence. This yields the siRNA conjugate of the present disclosure.

[0601] Purification and desalting methods are well known to those skilled in the art. For example, nucleic acids can be purified using a preparative ion chromatography column with a gradient elution of NaBr or NaCl. After the product is collected and combined, it can be desalted using a reversed-phase chromatography column.

[0602] During the synthesis process, the purity and molecular weight of the nucleic acid sequence can be tested at any time to better control the synthesis quality. Testing methods are well known to those skilled in the art. For example, nucleic acid purity can be tested by ion exchange chromatography, and molecular weight can be determined by liquid chromatography-mass spectrometry (LC-MS).

[0603] Annealing methods are also well known to those skilled in the art. For example, the synthesized sense strand (S strand) and antisense strand (AS strand) can be simply mixed in an equimolar ratio in water for injection, heated to 70-95°C, and then cooled to room temperature to form a double-stranded structure through hydrogen bonding. This can produce the siRNA conjugate of the present disclosure.

[0604] After obtaining the conjugate of the present invention, in some embodiments, the synthesized siRNA conjugate can be characterized by molecular weight detection, etc., using methods such as liquid chromatography-mass spectrometry, to confirm that the synthesized siRNA conjugate is the target designed siRNA conjugate and that the sequence of the synthesized siRNA is consistent with the sequence of the siRNA to be synthesized, for example, consistent with one of the sequences listed in Table 1 above.

[0605] Applications of the conjugates disclosed herein

[0606] As shown in the present disclosure, the conjugates can deliver an active agent to cells for the treatment or prevention of a disease or condition that may require such delivery. Without wishing to be bound by any theory, we believe that the spatial arrangement of the conjugated molecules is particularly effective in targeting cell surface receptors, thereby allowing the loaded active agent to contact the cell. In some embodiments, the conjugate is an oligonucleotide conjugate targeted to hepatocytes.

[0607] In some embodiments, the oligonucleotide conjugates disclosed herein have excellent liver-targeting specificity, enabling efficient delivery of the conjugated functional oligonucleotide to the liver, thereby effectively regulating the expression of specific genes in hepatocytes. Consequently, the oligonucleotide conjugates disclosed herein have broad application prospects.

[0608] According to some embodiments of the present disclosure, the present disclosure provides the use of the oligonucleotide conjugates of the present disclosure in the preparation of a medicament for treating and / or preventing pathological conditions or diseases caused by the expression of specific genes in liver cells. The specific gene can be an endogenous gene expressed in the liver or a pathogen gene that propagates in the liver. In some embodiments, the specific gene is, for example, ApoB, ApoC, ANGPTL3, PCSK9, SCD1, TIMP-1, Col1A1, FVII, STAT3, p53, HBV, or HCV. In some embodiments, the specific gene is a hepatitis B virus gene, an angiopoietin-like protein 3 gene, or an apolipoprotein C3 gene. Accordingly, the disease is selected from chronic liver disease, hepatitis, liver fibrosis, liver proliferative disease, and dyslipidemia. In some embodiments, the dyslipidemia is hypercholesterolemia, hypertriglyceridemia, or atherosclerosis.

[0609] In some embodiments, provided herein are methods for treating pathological conditions or diseases caused by the expression of specific genes in hepatocytes, comprising administering an oligonucleotide conjugate of the present disclosure to a patient in need thereof. In some embodiments, the specific genes are, for example, ApoB, ApoC, ANGPTL3, PCSK9, SCD1, TIMP-1, Col1A1, FVII, STAT3, p53, HBV, or HCV. In some embodiments, the specific genes are selected from hepatitis B virus genes, angiopoietin-like protein 3 genes, and apolipoprotein C3 genes. Accordingly, the disease is selected from chronic liver disease, hepatitis, liver fibrosis, liver proliferative diseases, and dyslipidemia. In some embodiments, the dyslipidemia is hypercholesterolemia, hypertriglyceridemia, or atherosclerosis. In some embodiments, the conjugates provided herein can also be used to treat other liver diseases, including diseases characterized by unwanted cell proliferation, blood diseases, metabolic diseases, and diseases characterized by inflammation. Proliferative diseases of the liver can be benign or malignant diseases, such as cancer, hepatocellular carcinoma (HCC), liver metastasis or hepatoblastoma. Hepatic hematological or inflammatory diseases can be diseases involving coagulation factors, complement-mediated inflammation or fibrosis. Metabolic diseases of the liver include dyslipidemia and irregularities in glucose regulation. In some embodiments, the method includes administering one or more oligonucleotides with a high degree of homology to a gene sequence involved in liver disease.

[0610] According to some embodiments of the present disclosure, the present disclosure provides a method for inhibiting the expression of a specific gene in a hepatocyte, the method comprising contacting the hepatocyte with the siRNA conjugate of the present disclosure.

[0611] By administering the oligonucleotide conjugates of the present disclosure to patients in need, the purpose of preventing and / or treating pathological conditions or diseases caused by the expression of specific genes in liver cells can be achieved through the mechanism of regulating gene expression. Therefore, the oligonucleotide conjugates of the present disclosure can be used to prevent and / or treat the pathological conditions or diseases disclosed herein, or to prepare medicaments for preventing and / or treating the pathological conditions or diseases disclosed herein.

[0612] As used herein, the term "administration / administration" refers to the delivery of a conjugate, such as an oligonucleotide conjugate, into a subject by a method or route that at least partially localizes the conjugate, such as an oligonucleotide conjugate, to a desired site to produce a desired effect. Routes of administration suitable for the methods of the present disclosure include, but are not limited to, local administration and systemic administration. In general, local administration results in more oligonucleotide conjugate being delivered to a specific site than in the subject's systemic circulation; whereas systemic administration results in the delivery of the oligonucleotide conjugate to the patient's systemic circulation. Given that the present disclosure is intended to provide a means of preventing and / or treating pathological conditions or diseases caused by the expression of specific genes in hepatocytes, in some embodiments, an administration method that is capable of delivering the drug to the liver is employed.

[0613] The drug can be administered to a patient by any suitable route known in the art, including but not limited to oral or parenteral routes, such as intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, airway administration (aerosol), pulmonary administration, nasal administration, rectal administration, and topical administration (including buccal administration and sublingual administration). The frequency of administration can be once or more per day, per week, per two weeks, per month, or per year.

[0614] The dosage of the oligonucleotide conjugates disclosed herein can be conventional dosages in the art, which can be determined based on various parameters, particularly the patient's age, weight, and sex. Toxicity and efficacy can be determined by standard pharmaceutical procedures in cell culture or experimental animals, such as determining the LD50 (lethal dose that kills 50% of the population) and the ED50 (the dose that elicits 50% of the maximum response in quantitative responses and the dose that elicits a positive response in 50% of the subjects in qualitative responses). A range of human dosages can be derived based on data obtained from cell culture assays and animal studies.

[0615] When administering the conjugates of the present disclosure, for example, for male or female, 6-12 week old, 18-25 g C57BL / 6J or C3H / HeNCrlVr mice, based on the amount of the oligonucleotide in the oligonucleotide conjugate: for the delivery of the oligonucleotide conjugate formed by the functional oligonucleotide and the conjugate molecule, the amount of the oligonucleotide delivered by the conjugate can be 0.001-100 mg / kg body weight, in some embodiments 0.01-50 mg / kg body weight, in some embodiments 0.05-20 mg / kg body weight, in other embodiments 0.1-15 mg / kg body weight, in other embodiments 0.1-10 mg / kg body weight. When administering the oligonucleotide conjugates of the present disclosure, reference may be made to the above dosages.

[0616] In addition, by introducing the oligonucleotide conjugates of the present invention into liver cells in which a specific gene is abnormally expressed, the purpose of inhibiting the expression of the specific gene in the liver cells can also be achieved through the mechanism of gene expression regulation. In some embodiments, the liver cells are hepatitis cells, and in some embodiments, they are HepG2.2.15 cells. In some embodiments, the liver cells can be selected from liver cancer cell lines such as Hep3B, HepG2, Huh7, or isolated primary liver cells, and in some embodiments, they are Huh7 liver cancer cells.

[0617] In the case where the method provided by the present disclosure is used to suppress specific gene expression in hepatocytes, the dosage of the functional oligonucleotide in the oligonucleotide conjugate provided is that those skilled in the art easily determine according to the effect obtained by expectation. For example, in some embodiments, the oligonucleotide conjugate is a siRNA conjugate, and the siRNA dosage in the siRNA conjugate provided is such amount: it is enough to reduce the expression of the target gene, and causes the extracellular concentration of 1pM to 1 μM or 0.01nM to 100nM or 0.05nM to 50nM or 0.05nM to about 5nM. The amount required for reaching this local concentration will vary with various factors, and the factors include delivery method, delivery site, the number of the cell layer between delivery site and target cell or tissue, delivery route (local or whole body) etc. The concentration at the delivery site can be significantly higher than the concentration at the surface of target cell or tissue.

[0618] Beneficial effects

[0619] In some embodiments, the conjugates provided by the present disclosure have higher oligonucleotide delivery efficiency, lower toxicity, better stability and / or higher activity in vivo. In some embodiments, the conjugates of the present disclosure show at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% inhibition of target gene expression. In some embodiments, the conjugates of the present disclosure show at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% inhibition of HBV gene expression. In some embodiments, the conjugates of the present disclosure show at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% inhibition of HBV gene expression in the liver. In some embodiments, the conjugates of the present disclosure show at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% inhibition of HBV gene expression in the liver in animal models. In some embodiments, the conjugates of the present disclosure exhibit at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% inhibition of HBV expression in human subjects. In some embodiments, the conjugates of the present disclosure exhibit at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% inhibition of HBV surface antigen expression. In some embodiments, the siRNA, siRNA composition, or siRNA conjugate provided herein exhibits at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% inhibition of ANGPTL3 gene expression. In some embodiments, the conjugates of the present disclosure exhibit at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% inhibition of ANGPTL3 gene expression in the liver. In some embodiments, the conjugates of the present disclosure exhibit at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% inhibition of ANGPTL3 gene expression in an animal model. In some embodiments, the conjugates of the present disclosure exhibit at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% inhibition of ANGPTL3 gene expression in a human subject. In some embodiments, the conjugates of the present disclosure exhibit at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% inhibition of APOC3 gene expression. In some embodiments, the conjugates of the present disclosure exhibit at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% inhibition of APOC3 gene expression in the liver.In some embodiments, the conjugates of the present disclosure exhibit at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% inhibition of APOC3 gene expression in the liver in an animal model. In some embodiments, the conjugates of the present disclosure exhibit at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% inhibition of APOC3 gene expression in human subjects. In some embodiments, the conjugates of the present disclosure do not exhibit significant off-target effects. An off-target effect can be, for example, inhibition of gene expression of a non-target gene. An off-target effect is considered insignificant if the binding / inhibition of off-target gene expression is less than 50%, 40%, 30%, 20%, or 10% of the effect on the on-target gene.

[0620] According to some embodiments of the present disclosure, the conjugates of the present disclosure can effectively deliver siRNA to the liver and exhibit excellent properties of inhibiting HBV gene expression. For example, while having low off-target effects, 87.4%-92.2% of HBV gene expression in the liver of hepatitis B model mice is inhibited at a dose of 1 mg / kg. In some embodiments, the siRNA conjugates of the present disclosure effectively reduce HBV surface antigen expression in hepatitis B model mice, and at a dose of 3 mg / kg, a 96.9% HBV surface antigen expression inhibition rate and a 95.4% HBV DNA inhibition rate can be achieved. In some embodiments, the conjugates provided by the present invention exhibit excellent inhibitory effects on HBV expression at low doses for up to 140 days.

[0621] According to one embodiment of the present disclosure, the conjugate provided by the present disclosure can effectively deliver siRNA to the liver and exhibit excellent properties of inhibiting HBV gene expression, for example, while having low off-target effects, it inhibits at least 69.3% or 78.1-89.1% of HBV gene expression in the liver of hepatitis B model mice at a dose of 1 mg / kg. In some embodiments, the conjugate of the present disclosure effectively reduces HBV surface antigen expression in hepatitis B model mice, reaching a 98.4% HBV surface antigen expression inhibition rate and a 95.8% HBV DNA inhibition rate at a dose of 3 mg / kg. In some embodiments, compared to the reference conjugate, the specific conjugate provided by the present invention exhibits excellent inhibitory effects on HBV expression at low doses for up to 84 days.

[0622] According to one embodiment of the present disclosure, the conjugate provided by the present disclosure can effectively deliver siRNA to the liver and exhibit excellent properties of inhibiting HBV gene expression. For example, while having low off-target effects, it can inhibit at least 48.5% or 76.8-80.0% of HBV gene expression in the liver of hepatitis B model mice at a dose of 1 mg / kg. In some embodiments, the conjugate of the present disclosure also effectively reduces HBV surface antigen expression in hepatitis B model mice, even reaching an 84.6% HBV surface antigen expression inhibition rate and an 85.6% HBV DNA inhibition rate at a dose of 3 mg / kg. In some embodiments, compared to the reference conjugate, the conjugate provided by the present invention exhibits a higher inhibitory effect on HBV expression at a low dose for up to 21 days.

[0623] According to one embodiment of the present disclosure, the conjugates provided herein can effectively deliver siRNA to the liver and exhibit excellent properties for inhibiting HBV gene expression. For example, at a dose of 1 mg / kg, they can inhibit gene expression of the HBV X gene region in the liver of hepatitis B model mice by at least 60.1%, or in one embodiment, 80.7-84.5%, while having minimal off-target effects. In some embodiments, the conjugates of the present disclosure also effectively reduce HBV surface antigen expression in hepatitis B model mice, even achieving a 94.8% inhibition rate of HBV surface antigen expression and a 95.8% inhibition rate of HBV DNA at a dose of 3 mg / kg. In some embodiments, compared to a reference conjugate, the conjugates provided herein exhibit superior inhibitory effects on HBV expression at low doses for up to 56 days.

[0624] According to one embodiment of the present disclosure, the conjugates provided herein can effectively deliver siRNA to the liver and exhibit excellent properties for inhibiting ANGPTL3 gene expression. For example, at a dose of 1 mg / kg, ANGPTL3 gene expression in the liver of hyperlipidemia model mice was inhibited by at least 57.3%; at a dose of 3 mg / kg, the gene inhibition rate reached a maximum of 90.4%. In some embodiments, compared to a reference conjugate, the conjugates provided herein exhibit superior ANGPTL3 expression inhibition and lipid-lowering effects at low doses and low administration frequencies for up to 49 days.

[0625] According to one embodiment of the present disclosure, the conjugates provided herein can effectively deliver siRNA to the liver and exhibit excellent properties for inhibiting ApoC3 gene expression. For example, at a dose of 3 mg / kg, the conjugates inhibited at least 75% of APOC3 gene expression in the liver of high-fat model mice. In some embodiments, compared to a reference conjugate, the conjugates provided herein exhibited excellent lipid-suppressing effects at low doses and low administration frequencies for up to 65 days.

[0626] In certain embodiments, the conjugates of the present disclosure exhibit low toxicity in animal models, indicating a good safety profile. For example, in some embodiments, no significant toxicity was observed in C57BL / 6J mice even when the conjugates of the present disclosure were administered at 100 times the effective concentration (based on an effective concentration of 3 mg / kg).

[0627] The above examples illustrate that the conjugates provided herein are effective for targeting cell surface receptors and delivering the loaded active agent to cells expressing the receptor. It is envisioned that the conjugate molecules can be adapted to additional cell surface receptors and additional active agents to target the active agent to cells expressing these receptors.

[0628] Reagent test kit

[0629] In another aspect, provided herein is a kit comprising a conjugate as described above.

[0630] In some embodiments, the kits provided herein contain a conjugate in a container. In some embodiments, the kits provided herein contain a container containing a pharmaceutically acceptable excipient. In some embodiments, the kits provided herein further contain a pharmaceutically acceptable excipient, such as a stabilizer or a preservative. In some embodiments, the kits provided herein contain at least one additional therapeutic agent. In some embodiments, the kit contains an additional therapeutic agent in at least one container that is different from the therapeutic agent containing the conjugate of the present disclosure. In some embodiments, the kit contains instructions for mixing the conjugate with a pharmaceutically acceptable excipient or other ingredients (if present).

[0631] In the kits of the present disclosure, the conjugate and / or pharmaceutically acceptable excipients can be provided in any form, such as liquid form, dry form, or lyophilized form. In some embodiments, the conjugate and / or pharmaceutically acceptable excipients are substantially pure and / or sterile. In some embodiments, sterile water is provided in the kits of the present disclosure.

[0632] Example

[0633] The present disclosure is described in detail below through examples. Unless otherwise stated, the reagents and culture media used in the following examples are commercially available products, and the nucleic acid electrophoresis, real-time PCR, and other procedures used are performed according to the methods described in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)).

[0634] HEK239A cells were provided by the Laboratory of Nucleic Acid Technology, Institute of Molecular Medicine, Peking University and cultured in DMEM complete medium (Hyclone) containing 20% ​​fetal bovine serum (FBS, Hyclone) and 0.2v% blueberry double antibody (penicillin-streptomycin, Gibco, Invitrogen) at 37°C in an incubator containing 5% CO2 / 95% air.

[0635] Huh7 cells were purchased from the Stem Cell Bank of the Chinese Academy of Sciences and cultured in DMEM complete medium (Hyclone) containing 10% fetal bovine serum (FBS, Hyclone) and 1% non-essential amino acids (NEAA, Corning) at 37°C in an incubator containing 5% CO2 / 95% air.

[0636] Unless otherwise specified, when cells were transfected with the siRNA conjugates synthesized in the following Preparation Examples 12 to 15, Lipofectamine™ 2000 (Invitrogen) was used as the transfection reagent. Specific operations were performed according to the instructions provided by the manufacturer.

[0637] Unless otherwise stated, the reagent ratios provided below are calculated by volume (v / v).

[0638] Unless otherwise stated, the animal models used were as follows:

[0639] C57BL / 6J mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.

[0640] SD rats: provided by Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.

[0641] HBV transgenic mice C57B / 6N-Tg(1.28HBV) / Vst(genotype A) were purchased from Beijing Weitongda Biotechnology Co., Ltd. Before the experiment, COI>10 was selected. 4 mice (hereinafter referred to as 1.28copy mice);

[0642] HBV transgenic mice C57BL / 6J-Tg(Alb1HBV)44Bri / J were purchased from the Department of Laboratory Animal Science, Peking University Health Science Center. Mice with an S / CoV ratio > 10 were selected before the experiment.

[0643] HBV transgenic mice: designated M-TgHBV, purchased from the Animal Department of Shanghai Public Health Center. The transgenic mice were prepared as described by Ren J. et al., J. Medical Virology. 2006, 78: 551-560.

[0644] AAV-HBV transgenic mice were prepared according to the literature method (Dong Xiaoyan et al., Chin J Biotech 2010, May 25; 26(5): 679-686). rAAV8-1.3HBV, type D (ayw) virus (purchased from Beijing Wujiahe Molecular Medicine Research Institute Co., Ltd., 1×10 12 Viral genome (vg) / mL, batch number 2016123011) rAAV8-1.3HBV was diluted to 5×10 11 vg / mL, and each mouse was injected with 200 μL of diluted rAAV8-1.3HBV (i.e., each mouse was injected with 1×10 11 On day 28 after virus injection, all mice were bled (approximately 100 μL) via orbital bleeding for serum collection to detect HBsAg and HBV DNA.

[0645] Low-concentration AAV-HBV transgenic mice: The modeling method was basically the same as above, except that the virus was diluted to 1×10 with sterile PBS before the experiment. 11 vg / mL, and each mouse was injected with 100 μL of virus, that is, each mouse was injected with 1×10 10 vg;

[0646] BALB / c mice, 6–8 weeks old, were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.

[0647] ob / ob mice: 6-8 weeks old, purchased from Changzhou Cavens Laboratory Animal Co., Ltd.

[0648] Human APOC3 transgenic mice: B6;CBA-Tg(APOC3)3707Bres / J, purchased from Jackson Lab;

[0649] Metabolic syndrome monkeys: All were male and provided by the Nonhuman Primate Research Center, Institute of Molecular Medicine, Peking University.

[0650] Preparation Example 1 Preparation of L-9 Conjugated Molecule (Conjugated Molecule 1)

[0651] In this preparation example, conjugated molecule 1 (hereinafter also referred to as L-9 conjugated molecule) was synthesized according to the following method.

[0652] (1-1) Synthesis of GAL-5 (L-9 conjugated terminal molecule)

[0653]

[0654] (1-1a) Synthesis of GAL-2

[0655] Dissolve 100.0 g of GAL-1 (N-acetyl-D-galactosamine hydrochloride, CAS No. 1772-03-8, purchased from Ningbo Hongxiang Biochemical Co., Ltd., 463.8 mmol) in 1000 ml of anhydrous pyridine. Add 540 ml of acetic anhydride (purchased from Enox, 5565.6 mmol) under ice-water bath. Stir and react at room temperature for 1.5 hours. Pour the reaction solution into 10 L of ice water and filter under reduced pressure. Wash the filter cake with 2 L of ice water and add a mixed solvent of acetonitrile / toluene (volume ratio of acetonitrile:toluene = 1:1) until completely dissolved. Evaporate the solvent to obtain 130.0 g of GAL-21 as a white solid.

[0656] (1-1b) Synthesis of GAL-3

[0657] GAL-2 (35.1 g, 90.0 mmol) obtained in step (1-1a) was dissolved in 213 ml of anhydrous 1,2-dichloroethane. 24.0 g of TMSOTf (CAS No.: 27607-77-8, purchased from MacLean, 108.0 mmol) was added in an ice-water bath under nitrogen protection, and the reaction was carried out at room temperature overnight.

[0658] 400 ml of dichloromethane was added to the reaction solution to dilute it, and the mixture was filtered through diatomaceous earth. 1 L of saturated aqueous sodium bicarbonate solution was added and stirred evenly. The organic phase was separated and the aqueous phase was extracted twice with 300 ml of dichloroethane each time. The organic phases were combined and washed with 300 ml of saturated aqueous sodium bicarbonate solution and 300 ml of saturated brine, respectively. The washed organic phase was separated and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure to obtain 26.9 g of a light yellow viscous syrupy product GAL-3.

[0659] Synthesis of (1-1c)GAL-4

[0660] GAL-3 (26.9 g, 81.7 mmol) obtained in step (1-1b) was dissolved in 136 ml of anhydrous 1,2-dichloroethane and dried 30g of molecular sieve powder was added, and then 9.0g of 5-hexen-1-ol (CAS No.: 821-41-0, purchased from Adamas-beta, 89.9mmol) was added, and stirred at room temperature for 30 minutes. 9.08g of TMSOTf (40.9mmol) was added under ice bath and nitrogen protection, and the reaction was stirred at room temperature overnight. Filter and remove Molecular sieve powder, add 300 ml of dichloroethane to the filtrate to dilute, filter with diatomaceous earth, add 500 ml of saturated sodium bicarbonate aqueous solution and stir for 10 minutes to wash, separate the organic phase, and extract the remaining aqueous phase once with 300 ml of dichloroethane. Combine all the organic phases and wash them with 300 ml of saturated sodium bicarbonate aqueous solution and 300 ml of saturated brine, respectively. Separate the washed organic phase, dry over anhydrous sodium sulfate, and evaporate the solvent under reduced pressure to obtain 1.3 g of yellow syrupy product GAL-44, which is directly used for the next oxidation reaction without purification.

[0661] Synthesis of (1-1d)GAL-5

[0662] GAL-4 (14.9 g, 34.7 mmol) obtained by the method described in step (1-1c) was dissolved in a mixed solvent of 77 ml of dichloromethane and 77 ml of acetonitrile, and 103 ml of deionized water and 29.7 g of sodium periodate (CAS No.: 7790-28-5, purchased from Aladdin, 138.8 mmol) were added respectively. The mixture was stirred under an ice-water bath for 10 minutes, and ruthenium trichloride (CAS No.: 14898-67-0, purchased from Anaiji, 238 mg, 1.145 mmol) was added. The reaction mixture was reacted at room temperature overnight. The reaction solution was diluted and stirred with 300 ml of water, and saturated sodium bicarbonate was added to adjust the pH to about 7.5. The organic phase was separated and discarded, and the remaining aqueous phase was extracted three times with dichloromethane, each time with 200 ml, and the extracted organic phase was discarded. The aqueous phase obtained by extraction was adjusted to pH about 3 with solid citric acid and extracted three times with 200 ml of dichloromethane each time. The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain 6.85 g of white foamy solid product GAL-5.

[0663] Next, the L-9 conjugate molecule was synthesized using the GAL-5 compound obtained according to the above method through the following process route:

[0664]

[0665] (1-2) Synthesis of M-11-T3:

[0666]

[0667] J-0 (1.883 g, 10 mmol, purchased from Alfa Aesar) was dissolved in 25 ml of acetonitrile, triethylamine (4.048 g, 40 mmol) was added, and the mixture was cooled to 0°C in an ice-water bath. Ethyl trifluoroacetate (5.683 g, 40 mmol) was added and the mixture was reacted at room temperature for 22 h. The solvent was evaporated under reduced pressure, and the residue was evaporated and dried for 18 h using a vacuum oil pump to obtain 5.342 g of crude solid M-11-T3, which was used directly in the subsequent reaction without further purification. MS m / z: C15H22F9N4O3, [M+H]+ , theoretical value: 477.35, measured value: 477.65.

[0668] (1-3) Synthesis of M-11-T3-Tr:

[0669]

[0670] The crude product M-11-T3 (5.342 g, 10 mmol) was dissolved in 50 ml of dichloromethane. TrCl (3.345 g, 12 mmol) and triethylamine (1.518 g, 15 mmol) were added to the resulting reaction solution and stirred at room temperature for 20 h. The reaction solution was washed twice with 20 ml of saturated sodium bicarbonate each time and once with 20 ml of saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and the organic solvent was evaporated under reduced pressure. The residue was foamed using a vacuum oil pump and dried overnight to obtain 7.763 g of crude solid M-11-T3-Tr. MS m / z: C 34 H 36 F9N4O3, [M+Na] + , theoretical value: 741.25, found value: 741.53. The crude solid M-11-T3-Tr was used in the next step of synthesizing M-18-Tr without purification.

[0671] (1-4) Synthesis of M-18-Tr:

[0672]

[0673] The crude product of M-11-T3-Tr (7.763 g, 10 mmol) obtained in step (1-3) was dissolved in 100 ml of methanol, and then 100 ml of methylamine aqueous solution (40 mass %) was added, and the reaction was stirred at 50 ° C for 23 h. The insoluble particles were filtered out, and the solvent was evaporated under reduced pressure. 200 ml of a DCM: methanol mixed solvent with a volume ratio of 1:1 was added to the residue, and the mixture was washed with 50 ml of saturated sodium bicarbonate. The aqueous phase was extracted with dichloromethane three times, 50 ml each time, and the mixture was combined. All the organic phases were dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. The residue was foamed with a vacuum oil pump and dried overnight. It was purified by a 200-300 mesh normal phase silica gel column, packed with petroleum ether, and the acidity of the silica gel was neutralized with 1 wt% triethylamine. The product was eluted with a gradient of dichloromethane: methanol: ammonia water (25 wt%) = 1:1:0.05 to 1:1:0.25. The eluate was collected and the solvent was evaporated under reduced pressure. The residue was foamed with a vacuum oil pump and dried to obtain 2.887 g of pure M-18-Tr. 1H NMR (400 MHz, DMSO) δ 7.47–7.39 (m, 6H), 7.32–7.24 (m, 6H), 7.19–7.12 (m, 3H), 2.60–2.47 (m, 4H), 2.46–2.19 (m, 13H), 1.70–1.55 (m, 4H), 1.40 (p, J = 6.8 Hz, 2H). MS m / z: C28H39N4, [M+H]+, calcd: 431.65, found: 432.61.

[0674] Synthesis of (1-5)L-5-Tr:

[0675]

[0676] M-18-Tr (2.02 g, 4.69 mmol) obtained in step (1-4) and GAL-5 (6.93 g, 15.48 mmol) obtained in step (1-1) were mixed and dissolved in 47 ml of acetonitrile. N-methylmorpholine (3.13 g, 30.96 mmol) and 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (DMTMM, 4.28 g, 15.48 mmol) were added, and the mixture was stirred at room temperature for 2 h. The reaction solution was diluted with 200 ml of dichloromethane, and the organic phase was washed with 100 ml of saturated sodium bicarbonate solution and 100 ml of saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure to obtain a crude product. The product was purified by a 200-300 mesh normal phase silica gel column packed with petroleum ether. The silica gel was acidified with 1 wt% triethylamine and eluted with a gradient of dichloromethane:methanol = 100:5 to 100:7. The product eluate was collected and evaporated to dryness under reduced pressure to obtain 7.49 g of pure L-5-Tr. 1 H NMR (400MHz, DMSO) δ7.83–7.10(m,4H),7.67–7.60(m,1H),7.44–7.34(m,6H),7.33–7.24(m,6 H),7.20–7.15(m,3H),5.22(s,3H),4.97(d,J=11.3Hz,3H),4.49(d,J=8.4Hz,3H),4.06–3.07 (m,9H),3.95–3.83(m,3H),3.77–3.64(m,3H),3.45–3.35(m,3H),3.12–2.87(m,8H),2.30–2. 15(m,3H),2.11–1.98(m,22H),1.95–1.84(m,11H),1.81–1.61(m,14H),1.54–1.36(m,14H).MS m / z:C 85 H 119 N7O 30, [M+H]+, theoretical value: 1718.81, found value: 1718.03.

[0677] (1-6) Synthesis of L-8:

[0678]

[0679] L-5-Tr (5.94 g, 3.456 mmol) obtained in step (1-5) was dissolved in 69 ml of dichloromethane, and dichloroacetic acid (13.367 g, 103.67 mmol) was added. The reaction mixture was allowed to react at room temperature for 2 h. The resulting reaction solution was diluted with 100 ml of dichloromethane and then washed with saturated sodium bicarbonate solution to adjust the pH to 7-8. The separated aqueous phase was extracted with dichloromethane six times, 30 ml each time. All organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure to obtain a crude product. Purification was performed using 200-300 mesh normal phase silica gel. The silica gel acidity was neutralized with 10 wt% triethylamine, and the column was equilibrated with 1 wt‰ triethylamine. The column was eluted with a gradient of dichloromethane:methanol = 100:30-100:40. The product eluate was collected and the solvent was evaporated under reduced pressure to obtain 4.26 g of pure L-8. 1 H NMR (400MHz, DMSO) δ7.84(d,J=9.0Hz,3H),7.27–7.23(m,1H),7.13–7.18(m,1H),5.22(d,J=3.1Hz,3 H),4.97(dd,J=11.3,3.1Hz,3H),4.48(d,J=8.4Hz,3H),4.09–3.98(m,9H),3.88(dd,J=19.3,9.3Hz, 3H),3.75–3.66(m,3H),3.44–3.38(m,3H),3.17–3.30(m,4H),3.10–2.97(m,4H),2.35–2.20(m,6H), 2.15–2.08(m,9H),2.07–1.98(m,13H),1.94–1.87(m,9H),1.81–1.74(m,9H),1.65–1.42(m,18H).MS m / z:C 85 H 119 N7O 30 , [M+H] + , theoretical value: 1477.59, measured value: 1477.23.

[0680] Synthesis of (1-7a)A-1

[0681]

[0682] DMTrCl (4,4'-bismethoxytrityl chloride, 38.12 g, 112.5 mmol) was dissolved in 450 ml of anhydrous pyridine, and DL-calcium glycerate hydrate (12.88 g, 45.0 mmol) was added. The mixture was reacted at 45°C for 22 h. The reaction solution was filtered and the filter cake was washed with 200 ml of water. The residue was washed with DCM, and the filtrate was concentrated to dryness under reduced pressure. The residue was redissolved in 500 ml of dichloromethane and washed twice with 0.5 M triethylamine phosphate (pH = 7-8), 200 ml each time. The separated aqueous phase was extracted twice with dichloromethane, 200 ml each time. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. The residue was purified on a 200-300 mesh normal phase silica gel column and eluted with petroleum ether: ethyl acetate: dichloromethane: methanol = 1:1:1:0.35-1:1:1:0.55 gradient. The product eluate was collected, the solvent was evaporated under reduced pressure, the residue was redissolved in 500 ml of dichloromethane, and 200 ml The mixture was washed once with 0.5 M triethylamine phosphate, and the aqueous phase was extracted twice with 200 ml of dichloromethane each time. All the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. The residue was dried under reduced pressure using a vacuum oil pump overnight to obtain 20.7 g of white solid product A-1. 1 H NMR (400 MHz, DMSO-d6) δ 7.46 (ddd, J = 6.5, 2.3, 1.1 Hz, 1H), 7.40–7.28 (m, 7H), 6.89–6.81 (m, 4H), 4.84 (d, J = 5.0 Hz, 1H), 4.36–4.24 (m, 1H), 4.29 (s, 6H), 3.92 (dd, J = 12.4, 7.0 Hz, 1H), 3.67 (dd, J = 12.3, 7.0 Hz, 1H), 2.52 (q, J = 6.3 Hz, 6H), 1.03 (t, J = 6.3 Hz, 9H). MS m / z: C24H23O6, [MH]-, calcd: 407.15, found: 406.92.

[0683] (1-7b) Synthesis of L-7:

[0684]

[0685] L-8 (2.262 g, 1.532 mmol) obtained in step (1-6) and A-1 (2.342 g, 4.596 mmol) obtained in step (1-7a) were mixed and dissolved in 16 ml of dichloromethane. 3-diethoxyphosphoryloxy-1,2,3-benzotriazine-4(3H)-one (DEPBT, 1.375 g, 4.596 mmol) was added, followed by diisopropylethylamine (1.188 g, 9. The reaction was stirred at 25°C for 2 h, and the organic phase was washed with 10 ml of saturated sodium bicarbonate. The aqueous phase was extracted three times with 10 ml of dichloromethane. All organic phases were combined and washed with 10 ml of saturated brine. The separated aqueous phase was extracted twice with 10 ml of dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. The residue was dried overnight using a vacuum oil pump to obtain 4.900 g of crude product. The crude product was purified by column purification using 120 g of 200-300 mesh normal phase silica gel. The silica gel acidity was neutralized with 20 ml of triethylamine. The column was equilibrated with petroleum ether containing 1 wt% triethylamine and eluted with a gradient of petroleum ether: ethyl acetate: dichloromethane: N,N-dimethylformamide = 1:1:1:0.5 to 1:1:1:0.6. The product eluate was collected and the solvent was evaporated under reduced pressure to obtain 2.336 g of pure product L-7. 1 H NMR(400MHz,DMSO)δ7.90–7.78(m,4H),7.75–7.64(m,1H),7.38–7.18(m,9H),6 .91–6.83(m,4H),5.25–5.10(m,4H),4.97(dd,J=11.2,3.2Hz,3H),4.48–4.30(m ,4H),4.02(s,9H),3.93–3.84(m,3H),3.76–3.66(m,9H),3.45–3.35(m,3H),3. 24–2.98(m,10H),2.30–2.20(m,2H),2.11–1.88(m,31H),1.80–1.40(m,28H).MS m / z: C90H128N7O35, [M-DMTr]+, theoretical value: 1564.65, found value: 1564.88.

[0686] (1-8) Synthesis of L-9:

[0687]

[0688] L-7 (2.300 g, 1.26 mmol), succinic anhydride (0.378 g, 3.78 mmol), and 4-dimethylaminopyridine (DMAP, 0.462 g, 3.78 mmol) obtained in step (1-7b) were dissolved in 13 ml of dichloromethane. DIPEA (0.814 g, 6.30 mmol) was then added and stirred at 25°C for 24 h. The resulting reaction solution was washed with 5 ml of 0.5 M triethylamine phosphate. The separated aqueous phase was extracted three times with 5 ml of dichloromethane each time. All organic phases were combined and evaporated to dryness under reduced pressure to yield 2.774 g of crude product. The crude product was purified by column chromatography using 60 g of 200-300 mesh normal phase silica gel. The acidity of the silica gel was neutralized with 1 wt% triethylamine. The column was equilibrated with dichloromethane and eluted with a gradient of dichloromethane containing 1 wt% triethylamine:methanol = 100:18 to 100:20. The product eluate was collected and the solvent was evaporated under reduced pressure to obtain 1.874 g of pure L-9 conjugated molecule (conjugated molecule 1). 1 H NMR (400MHz, DMSO) δ8.58(d,J=4.2Hz,1H),7.94–7.82(m,3H),7.41–7.29(m,5H),7.22(d,J=8.1Hz,5H),6.89(d,J=8.3Hz,4H ),5.49–5.37(m,1H),5.21(d,J=3.0Hz,3H),4.97(d,J=11.1Hz,3H),4.49(d,J=8.2Hz,3H),4.02(s,9H),3.88(dd,J=19.4,9. 4Hz,3H),3.77–3.65(m,9H),3.50–3.39(m,6H),3.11–2.90(m,5H),2.61–2.54(m,4H),2.47–2.41(m,2H),2.26–2.17(m,2H),2.15–1.95(m,22H),1.92–1.84(m,9H),1.80–1.70(m,10H),1.65–1.35(m,17H),1.31–1.19(m,4H),0.96(t,J=7.1Hz,9H).MS m / z:C94H132N7O38,[M-DMTr]+,theor.: 1664.72, found: 1665.03. The structure of the resulting L-9 conjugated molecule is shown in formula (503).

[0689] Preparation Example 2 Preparation of P-9 Conjugated Molecule (Conjugated Molecule 2)

[0690] In this preparation example, conjugated molecule 2 (hereinafter also referred to as P-9 conjugated molecule) was synthesized according to the following method:

[0691]

[0692] (2-1) Synthesis of GAL5-C4-1

[0693] To 40 ml of N,N-dimethylformamide were added GAL-5 (13.43 g, 30.0 mmol), 4-amino acid tert-butyl ester hydrochloride (5.87 g, 30.0 mmol), O-benzotriazole-tetramethyluronium hexafluorophosphate (13.65 g, 36.0 mmol), and diisopropylethylamine (11.63 g, 90.0 mmol), uniformly dissolved, and stirred at room temperature for 5 hours. To the resulting reaction solution was added 300 ml of saturated aqueous sodium bicarbonate solution. The separated aqueous phase was extracted with ethyl acetate three times, 200 ml each time. The organic phases obtained by washing were combined and washed once with 200 ml of saturated brine. The washed organic phase was separated and dried over anhydrous sodium sulfate. The solvent was evaporated to dryness under reduced pressure to obtain 30.3 g of crude oily product GAL5-C4-1, which was directly used in the next reaction.

[0694] (2-2) Synthesis of GAL5-C4-2

[0695] The crude GAL5-C4-1 product (30.3 g, 30 mmol) obtained in step (2-1) was dissolved in 180 ml of formic acid and stirred at room temperature for 16 hours. The solvent was evaporated to dryness, and the crude product was purified by column chromatography (200-300 mesh normal phase silica gel, gradient elution of dichloromethane:methanol = 100:18 to 100:20). The eluate was collected and concentrated to remove the solvent to obtain 14.84 g of the target product GAL5-C4-2.

[0696] (2-3) Synthesis of P-6:

[0697] M-18-Tr (2.02 g, 4.69 mmol) obtained according to the method described in step (1-4) was mixed with GAL5-C4-2 (8.24 g, 15.48 mmol) obtained in step (2-2) and dissolved in 47 ml of acetonitrile. N-methylmorpholine (3.13 g, 30.96 mmol) was added, and finally 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM, 4.28 g, 15.48 mmol) was added, and the reaction was stirred at room temperature for 2 h. The resulting reaction solution was diluted with 200 ml of dichloromethane, and the organic phase was washed with 100 ml of saturated sodium bicarbonate solution and 100 ml of saturated brine, respectively. All organic phases were combined and dried over anhydrous sodium sulfate. After filtration, the solvent was evaporated under reduced pressure to obtain a crude product, which was purified on a 200-300 mesh normal phase silica gel column packed with petroleum ether. The silica gel was acidified with 1 wt% triethylamine and eluted with a gradient of dichloromethane:methanol = 100:5-100:7. The product eluate was collected and evaporated to dryness under reduced pressure to obtain 8.27 g of pure P-6.

[0698] (2-4) Synthesis of P-7:

[0699] P-6 (6.82 g, 3.456 mmol) obtained in (2-3) above was dissolved in 69 ml of dichloromethane, and dichloroacetic acid (13.367 g, 103.67 mmol) was added. The reaction mixture was allowed to react at room temperature for 2 h. 100 ml of dichloromethane was added to dilute the resulting reaction solution, which was then washed with saturated sodium bicarbonate solution and adjusted to pH 7-8. The separated aqueous phase was extracted with dichloromethane six times, 30 ml each time. All organic phases were combined, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure to obtain a crude product. The product was purified using 200-300 mesh normal phase silica gel. The silica gel was acidified with 10 wt% triethylamine, and the column was equilibrated with 1 wt‰ triethylamine. A gradient elution of dichloromethane:methanol = 100:30 to 100:40 was performed. The product eluate was collected and the solvent was evaporated to dryness under reduced pressure to obtain 4.82 g of P-7. MS m / z: C78H127N10O33, [M+H]+, calcd: 1732.91, found: 1735.73.

[0700] (2-5) Synthesis of P-8:

[0701]

[0702] P-7 (2.653 g, 1.532 mmol) and A-1 (2.342 g, 4.596 mmol) were mixed and dissolved in 16 ml of dichloromethane. 3-Diethoxyphosphoryl-1,2,3-benzotriazol-4(3H)-one (DEPBT) (1.375 g, 4.596 mmol) was added, followed by diisopropylethylamine (1.188 g, 9.191 mmol). The mixture was stirred at 25°C for 2 h. The organic phase was washed with 10 ml of saturated sodium bicarbonate, and the aqueous phase was extracted three times with 10 ml of dichloromethane each time. All organic phases were combined and washed with 10 ml of saturated brine. The separated aqueous phase was extracted twice with 10 ml of dichloromethane each time. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. The residue was dried overnight using a vacuum oil pump to obtain the crude product. The crude product was purified by column chromatography using 120 g of 200-300 mesh normal phase silica gel. The acidity of the silica gel was neutralized with 20 ml of triethylamine. The column was equilibrated with petroleum ether containing 1 wt% triethylamine. The column was eluted with a gradient of petroleum ether:ethyl acetate:dichloromethane:N,N-dimethylformamide = 1:1:1:0.5 to 1:1:1:0.6. The eluate was collected and the solvent was evaporated under reduced pressure to obtain 2.793 g of pure P-8.

[0703] (2-6) Synthesis of P-9:

[0704] P-8 (490 mg, 0.231 mmol), succinic anhydride (69 mg, 0.693 mmol), and 4-dimethylaminopyridine (DMAP, 68 mg, 0.554 mmol) were dissolved in 2.3 ml of dichloromethane. Diisopropylethylamine (DIPEA, 149 mg, 1.155 mmol) was then added and stirred at 25°C for 21 h. The resulting reaction solution was diluted with 50 ml of dichloromethane and washed with 100 ml of 0.5 M triethylamine phosphate. The separated aqueous phase was extracted with dichloromethane three times (10 ml each time). All organic phases were combined and evaporated to dryness under reduced pressure to obtain the crude product. The crude product was purified by column chromatography using 80 g of 200-300 mesh normal phase silica gel. The acidity of the silica gel was neutralized with 1 wt% triethylamine. The column was equilibrated with dichloromethane and eluted with a gradient of dichloromethane containing 1 wt% triethylamine in a ratio of 100:18 to 100:20. The eluate was collected and the solvent evaporated under reduced pressure to yield 200 mg of the pure P-9 conjugate molecule (conjugate molecule 2). MS m / z: C₁₀H₁₀N₁₀O₄₁₀, [M-DMTr]⁺, theoretical: 1921.05, measured: 1920.97. The structure of the resulting P-9 conjugate molecule is shown in Formula (504).

[0705] Preparation Example 3 Preparation of R-4 Conjugated Molecule (Conjugated Molecule 3)

[0706] In this preparation example, conjugated molecule 3 (hereinafter also referred to as R-4 conjugated molecule) was synthesized according to the following method.

[0707]

[0708] (3-1) Synthesis of GAL-C7-1

[0709] GAL-3 (26.4 g, 80.2 mmol) obtained by the method described in step (1-1b) was dissolved in 134 ml of anhydrous 1,2-dichloroethane and added 60g of molecular sieve powder was added to 7-octen-1-ol (11.3g, 88.2mmol), and the mixture was stirred at room temperature for 10 minutes. Trimethylsilyl trifluoromethanesulfonate (TMSOTf, 8.9g, 40.1mmol) was added under ice bath and nitrogen protection, and the mixture was stirred at room temperature for 24 hours. Filter and remove Molecular sieve powder, add 500 ml of saturated sodium bicarbonate aqueous solution to the filtrate for washing, separate the organic phase, and extract the remaining aqueous phase once with 100 ml of dichloromethane. All organic phases are combined and washed once with 250 ml of saturated brine. The washed organic phase is separated and dried over anhydrous sodium sulfate. The solvent is evaporated to dryness under reduced pressure to obtain 33.3 g of yellow syrupy product GAL-C7-1, which is directly used for the next oxidation reaction without purification.

[0710] (3-2) Synthesis of GAL-C7-2

[0711] GAL-C7-1 (33.3 g, 72.8 mmol) obtained in step (3-1) was dissolved in a mixed solvent of 160 ml of dichloromethane and 160 ml of acetonitrile. 216 ml of water and solid sodium periodate (62.3 g, 291.2 mmol) were added respectively. The mixture was stirred in an ice-water bath for 10 minutes. The catalyst ruthenium trichloride (498 mg, 2.4 mmol) was added and the temperature was naturally raised to room temperature and stirred for 23 hours. The resulting reaction solution was diluted with 200 ml of water and stirred. The pH was adjusted to 7.5 with saturated sodium bicarbonate. The organic phase was separated, and the remaining aqueous phase was extracted three times with dichloromethane. The organic phase was discarded, and the aqueous phase was adjusted to a pH of approximately 3 with solid citric acid. The extracted phase was extracted three times with 200 ml of dichloromethane each time. The combined organic phases were dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The product was then purified by column chromatography (200-300 mesh normal phase silica gel, gradient elution of dichloromethane:methanol = 100:18 to 100:20) to obtain 22.4 g of the product GAL-C7-2 as a white foamy solid. MS m / z: C21H32NO11, [M+H]+, Required: 476.50, Found: 475.94.

[0712] (3-3) Synthesis of R-1:

[0713] M-18-Tr (2.02 g, 4.69 mmol) obtained according to the method described in step (1-4) was mixed with GAL-C7-2 (7.36 g, 15.48 mmol) and dissolved in 47 ml of acetonitrile. N-methylmorpholine (3.13 g, 30.96 mmol) was added, and finally 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM, 4.28 g, 15.48 mmol) was added, and the reaction was stirred at room temperature for 2 h. The resulting reaction solution was diluted with 200 ml of dichloromethane, and the organic phase was washed with 100 ml of saturated sodium bicarbonate solution and 100 ml of saturated brine, respectively. All organic phases were combined and dried over anhydrous sodium sulfate. After filtration, the solvent was evaporated under reduced pressure to obtain a crude product, which was purified on a 200-300 mesh normal phase silica gel column packed with petroleum ether. The silica gel was acidified with 1 wt% triethylamine and eluted with a gradient of dichloromethane:methanol = 100:5-100:7. The product eluate was collected and evaporated to dryness under reduced pressure to obtain 7.82 g of pure R-1.

[0714] (3-4) Synthesis of R-2:

[0715] R-1 (6.23 g, 3.456 mmol) was dissolved in 69 ml of dichloromethane, followed by the addition of dichloroacetic acid (13.367 g, 103.67 mmol) and the reaction was allowed to react at room temperature for 2 h. The resulting reaction solution was diluted with 100 ml of dichloromethane, washed with saturated sodium bicarbonate solution, and the pH was adjusted to 7-8. The separated aqueous phase was extracted with dichloromethane six times, 30 ml each time. All organic phases were combined and dried over anhydrous sodium sulfate. After filtration, the solvent was evaporated under reduced pressure to obtain the crude product. The crude product was purified using 200-300 mesh normal phase silica gel. The silica gel was acidified with 10 wt% triethylamine, the column was equilibrated with 1 wt‰ triethylamine, and a gradient elution of dichloromethane:methanol = 100:30 to 100:40 was performed. The solvent was evaporated under reduced pressure to obtain 4.49 g of pure R-2.

[0716] (3-5) Synthesis of R-3:

[0717] R-2 (2.391 g, 1.532 mmol) and A-1 (2.342 g, 4.596 mmol) were mixed and dissolved in 16 ml of dichloromethane. 3-Diethoxyphosphoryloxy-1,2,3-benzotriazine-4(3H)-one (DEPBT, 1.375 g, 4.596 mmol) was added, followed by diisopropylethylamine (1.188 g, 9.191 mmol). The mixture was stirred at 25°C for 2 h. The organic phase was washed with 10 ml of saturated sodium bicarbonate, and the separated aqueous phase was extracted three times with 10 ml of dichloromethane each time. All organic phases were combined and washed with 10 ml of saturated brine. The separated aqueous phase was extracted two times with 10 ml of dichloromethane each time. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. The residue was dried overnight under reduced pressure using an oil vacuum pump to obtain the crude product. The crude product was purified by column chromatography using 120 g of 200-300 mesh normal phase silica gel. The acidity of the silica gel was neutralized with 20 ml of triethylamine. The column was equilibrated with petroleum ether containing 1 wt% triethylamine. The elution was performed using a gradient ratio of petroleum ether:ethyl acetate:dichloromethane:N,N-dimethylformamide = 1:1:1:0.5 to 1:1:1:0.6. The solvent was evaporated under reduced pressure to obtain 2.642 g of pure R-3.

[0718] (3-6) Synthesis of R-4:

[0719] R-3 (795 mg, 0.4074 mmol), succinic anhydride (82 mg, 0.8148 mmol), and 4-dimethylaminopyridine (DMAP, 100 mg, 0.8148 mmol) were dissolved in 4 ml of dichloromethane. Diisopropylethylamine (DIPEA, 100 mg, 0.8148 mmol) was added and stirred at 25°C for 18 h. The resulting reaction solution was washed with 5 ml of 0.5 M triethylamine phosphate. The aqueous phase was extracted with dichloromethane three times (5 ml each time). All organic phases were combined and the solvent was evaporated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography using 30 g of 200-300 mesh normal phase silica gel. The acidity of the silica gel was neutralized with 1 wt% triethylamine. The column was equilibrated with dichloromethane and eluted with a gradient of dichloromethane containing 1 wt% triethylamine:methanol (100:18 to 100:20). The eluate was collected and the solvent evaporated under reduced pressure to obtain 505 mg of pure R-4 conjugated molecule (conjugated molecule 3). The structure of the resulting R-4 conjugated molecule is shown in Formula (507).

[0720] Preparation Example 4 Preparation of LA-4 Conjugated Molecule (Conjugated Molecule 4)

[0721] According to the following process route, it is expected that conjugated molecule 4 (hereinafter also referred to as LA-4 conjugated molecule) can be synthesized, and the structure of the obtained LA-4 conjugated molecule is shown in formula (512).

[0722]

[0723] Conjugated molecules Conjugated molecules

[0724] Preparation Example 5 Preparation of LB-4 Conjugated Molecule (Conjugated Molecule 5)

[0725] In this preparation example, conjugate molecule 5 (hereinafter also referred to as LB-4 conjugate molecule) was synthesized according to the following method:

[0726]

[0727] (5-1) Synthesis of LB-1:

[0728] L-8 (5.0 g, 3.386 mmol), adipic anhydride (870 mg, 6.772 mmol), and 4-dimethylaminopyridine (DMAP, 827 mg, 6.772 mmol), obtained according to the method described in step (1-6), were dissolved in 130 ml of dichloromethane. Diisopropylethylamine (DIPEA, 2.2 g, 16.931 mmol) was then added, and the mixture was stirred at 25°C for 4 h. The resulting reaction solution was diluted with 70 ml of dichloromethane and washed with 0.5 M triethylamine phosphate. The separated aqueous phase was extracted with dichloromethane four times, 10 ml each time. All organic phases were combined, and the solvent was evaporated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography using 120 g of 200-300 mesh normal-phase silica gel. The silica was acidified with 1 wt% triethylamine, and the column was equilibrated with dichloromethane. Elution was performed using a gradient of petroleum ether:ethyl acetate:dichloromethane:methanol (1:1:1:0.2 to 1:1:1:1). The solvent was evaporated under reduced pressure to yield 4.267 g of pure LB-1.

[0729] (5-2) Synthesis of LB-2:

[0730] LB-1 (4.697 g, 2.753 mmol, obtained from two batches of product) obtained according to the method described in step (5-1), 3-amino-1,2-propanediol (313 mg, 3.442 mmol), 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (DMTMM, 953 mg, 3.442 mmol), and N-methylmorpholine (700 mg, 6.884 mmol) were added sequentially to a mixture of 30 mL of acetonitrile and 3 mL of methanol. The mixture was stirred at room temperature overnight. The solvent was evaporated to dryness, and the residue was purified by column chromatography (200-300 mesh normal phase silica gel, gradient elution of dichloromethane:methanol = 1:0.07 to 1:0.5). The eluate was collected and concentrated to remove the solvent to obtain 3.27 g of the target product LB-2.

[0731] (5-3) Synthesis of LB-3:

[0732] Dissolve LB-2 (2.27 g, 1.353 mmol) in 14 ml of anhydrous pyridine. Add 4,4'-bismethoxytrityl chloride (688 mg, 2.03 mmol) and stir at room temperature overnight. Quench the reaction with 150 ml of methanol and evaporate the solvent to dryness. The residue is purified by column chromatography (200-300 mesh normal phase silica gel, gradient elution of dichloromethane:methanol = 1:0.05-1:0.2). The eluate is collected and concentrated to remove the solvent to obtain 1.647 g of the desired product, LB-3.

[0733] (5-4) Synthesis of LB-4:

[0734] LB-3 (822 mg, 0.415 mmol), succinic anhydride (83 g, 0.83 mmol), and 4-dimethylaminopyridine (DMAP, 102 mg, 0.83 mmol) were dissolved in 4 ml of dichloromethane. DIPEA (270 mg, 2.075 mmol) was then added and stirred at 25°C overnight. The resulting reaction solution was washed three times with 0.5 M triethylamine phosphate. The separated aqueous phase was extracted three times with 2 ml of dichloromethane each time. All organic phases were combined and the solvent was evaporated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography using 200-300 mesh normal phase silica gel. The silica was acidified with 5 wt% triethylamine. The column was equilibrated with petroleum ether and eluted with a gradient of dichloromethane:methanol (100:5 to 100:20) containing 1 wt% triethylamine. The solvent was evaporated under reduced pressure to obtain 787 mg of pure LB-4 conjugated molecule (conjugated molecule 5). The structure of the resulting LB-4 conjugated molecule is shown in formula (513).

[0735] Preparation Example 6V-7 Synthesis of Conjugated Molecule (Conjugated Molecule 6)

[0736] According to the following process route, it is expected that conjugated molecule 6 (hereinafter also referred to as V-7 conjugated molecule) can be synthesized, and the structure of the obtained V-7 conjugated molecule is shown in formula (514).

[0737]

[0738] Preparation Example 7W-7 Preparation of Conjugated Molecule (Conjugated Molecule 7)

[0739] In this preparation example, conjugated molecule 7 (hereinafter also referred to as W-7 conjugated molecule) was synthesized according to the following method.

[0740]

[0741] (7-1) Synthesis of W-1:

[0742] Dissolve W-0 (2.024 g, 10 mmol) in 25 ml of acetonitrile, add triethylamine (4.048 g, 40 mmol), cool to approximately 0°C in an ice-water bath, add ethyl trifluoroacetate (5.683 g, 40 mmol), and react at room temperature for 22 h. Evaporate the solvent under reduced pressure, and dry the residue using a vacuum oil pump for 18 h to obtain 5.835 g of crude solid W-1.

[0743] (7-2) Synthesis of W-2:

[0744] The crude product W-1 (5.835 g, 10 mmol) was dissolved in 50 ml of dichloromethane. TrCl (3.345 g, 12 mmol) and triethylamine (1.518 g, 15 mmol) were added to the reaction solution and stirred at room temperature for 20 h. The resulting reaction solution was washed twice with 20 ml of saturated sodium bicarbonate and once with 20 ml of saturated brine. All organic phases were combined and dried over anhydrous sodium sulfate. After filtration, the organic solvent was evaporated under reduced pressure and the residue was dried overnight using a vacuum oil pump to obtain 8.012 g of crude solid W-2. The crude solid W-2 was not treated and was subjected to the next deprotection reaction.

[0745] (7-3) Synthesis of W-3:

[0746] The crude product W-2 (8.012 g, 10 mmol) was dissolved in 100 ml of methanol, and 100 ml of a 40 wt% aqueous methylamine solution was added. The reaction was stirred at 50°C for 23 h. Insoluble particulate matter was removed by filtration, and the solvent was evaporated under reduced pressure. The residue was added to 200 ml of a 1:1 (volume ratio) DCM-methanol mixture. The resulting organic phase was washed with 50 ml of saturated sodium bicarbonate. The separated aqueous phase was extracted three times with 50 ml of dichloromethane. All organic phases were combined and dried over anhydrous sodium sulfate. After filtration, the solvent was evaporated under reduced pressure. The residue was dried overnight using a vacuum oil pump. The product was purified using a 200-300 mesh normal phase silica gel column packed with petroleum ether. The acidity of the silica gel was neutralized with 1 wt % triethylamine and the product was eluted with a gradient of dichloromethane:methanol:ammonia water (25 wt %) = 1:1:0.05 to 1:1:0.25. The product eluate was collected and the solvent was evaporated under reduced pressure. The residue was dried using a vacuum oil pump to obtain 3.062 g of pure W-3.

[0747] (7-4) Synthesis of W-4:

[0748] W-3 (675 mg, 1.517 mmol) and GAL-C7-2 (2.60 g, 5.46 mmol) were mixed and dissolved in 47 ml of acetonitrile. Diisopropylethylamine (1.57 g, 12.14 mmol) was then added, followed by 3-diethoxyphosphoryloxy-1,2,3-benzotriazine-4(3H)-one (DEPBT, 1.816 g, 6.04 mmol). The mixture was stirred at room temperature for 2.5 h. The resulting reaction solution was diluted with 100 ml of dichloromethane. The organic phase was washed with 80 ml of saturated sodium bicarbonate solution and 80 ml of saturated brine, respectively. All organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure to obtain the crude product. The crude product was purified on a 200-300 mesh normal phase silica gel column packed with petroleum ether. The silica gel was acidified with 1 wt% triethylamine and eluted with a gradient of dichloromethane:methanol = 100:5-100:7. The eluate was collected and evaporated to dryness under reduced pressure to obtain 1.610 g of pure W-4.

[0749] (7-5) Synthesis of W-5:

[0750] W-4 (1.61 g, 0.886 mmol) was dissolved in 125 ml of dichloromethane, and dichloroacetic acid (3.5 ml, 42.43 mmol) was added. The reaction mixture was allowed to react at room temperature for 1 h. 150 ml of pyridine was added to neutralize the resulting reaction solution, and the solvent was evaporated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography using 200-300 mesh normal phase silica gel, 10 wt% triethylamine to neutralize the silica acidity, and 1 wt‰ triethylamine to balance the column. Elution was performed using a gradient of dichloromethane:methanol = 100:30 to 100:40. The product eluate was collected and the solvent was evaporated under reduced pressure to obtain 1.26 g of pure W-5.

[0751] (7-6) Synthesis of W-6:

[0752] W-5 (1.25 g, 0.793 mmol) and A-1 (1.21 g, 2.38 mmol), obtained according to the method described in step (1-7a), were mixed and dissolved in 12 ml of dichloromethane. 3-Diethoxyphosphoryloxy-1,2,3-benzotriazine-4(3H)-one (DEPBT, 0.712 g, 2.38 mmol) was added, followed by diisopropylethylamine (0.615 g, 4.76 mmol). The reaction was stirred at 25°C for 3 h. The organic phase was washed with 80 ml of saturated sodium bicarbonate, and the separated aqueous phase was extracted three times with 10 ml of dichloromethane each time. All organic phases were combined and washed with 10 ml of saturated brine. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. The residue was dried overnight using a vacuum pump to obtain the crude product. The crude product was purified by column chromatography using 185 g of 200-300 mesh normal phase silica gel and 20 ml of triethylamine to neutralize the acidity of the silica. The column was equilibrated with petroleum ether containing 1 wt% triethylamine and eluted with a gradient of petroleum ether:ethyl acetate:dichloromethane:N,N-dimethylformamide = 1:1:1:0.1 to 1:1:0.7. The eluate was collected and the solvent was evaporated under reduced pressure to obtain 1.57 g of pure W-6.

[0753] (7-7) Synthesis of W-7:

[0754] W-6 (1.238 g, 0.63 mmol), succinic anhydride (0.189 g, 1.89 mmol), and 4-dimethylaminopyridine (DMAP, 0.231 g, 1.89 mmol) were dissolved in 7 ml of dichloromethane. DIEA (0.407 g, 3.15 mmol) was added and stirred at 25°C for 24 h. The resulting reaction solution was washed with 5 ml of 0.5 M triethylamine phosphate, and the separated aqueous phase was extracted three times with 5 ml of dichloromethane. All organic phases were combined and evaporated to dryness under reduced pressure to obtain the crude product. The crude product was purified by column chromatography using 30 g of 200-300 mesh normal phase silica gel. The silica was acidified with 1 wt% triethylamine and the column was equilibrated with dichloromethane. The column was eluted with a gradient of dichloromethane containing 1 wt% triethylamine and methanol (100:18 to 100:20). The product eluate was collected and the solvent evaporated under reduced pressure to yield 1.033 g of pure W-7 conjugated molecule (conjugated molecule 7). MS m / z: C101H146N7O38, [M-DMTr]+, theoretical: 1763.92, found: 1763.21. The structure of the resulting W-7 conjugated molecule is shown in Formula (515).

[0755] Preparation Example 8X-7 Preparation of Conjugated Molecule (Conjugated Molecule 8)

[0756] According to the following process route, it is expected that conjugated molecule 8 (hereinafter also referred to as X-7 conjugated molecule) can be synthesized, and the structure of the obtained X-7 conjugated molecule is shown in formula (521).

[0757]

[0758] Preparation Example 9 Preparation of K-3 Conjugated Molecule (Comparative Conjugated Molecule 1)

[0759] In this preparation example, a K-3 conjugated molecule (hereinafter also referred to as comparative conjugated molecule 1) was synthesized according to the following method.

[0760]

[0761] (9-1) Synthesis of K-1:

[0762] To 60 mL of dichloromethane were added A-1 (3.0 g, 6.0 mmol), obtained according to the method described in step (1-7a), PyBOP (6.2 g, 12.0 mmol), HOBt (1.6 g, 2.0 mmol), and diisopropylethylamine (DIPEA, 3.9 g, 30.0 mmol). The mixture was stirred at room temperature for 10 minutes. The resulting solution was then added to K-0 (5.6 g, 30.0 mmol) and allowed to react at room temperature for 1 hour and 50 minutes. The reaction solution was poured into 30 mL of saturated sodium bicarbonate solution, and the separated aqueous phase was extracted three times with 30 mL of dichloromethane each time. All organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column purification using a 200-300 mesh normal phase silica gel column with a gradient elution ratio of dichloromethane:methanol:ammonia water (25 wt%) = 10:2:0.1 to 4:4:1. The product eluate was collected and concentrated to remove the solvent. The residue was dried using a vacuum oil pump to obtain 2.2 g of white solid product K-1. 1 H NMR (400 MHz, DMSO-d6) δ 8.02 (s, 1H), 7.43 (d, J = 7.8 Hz, 2H), 7.34–7.17 (m, 7H), 6.87 (d, J = 8.6 Hz, 4H), 4.05 (d, J = 5.2 Hz, 1H), 3.74 (s, 6H), 3.20–3.01 (m, 5H), 2.60–2.38 (m, 12H), 1.60–1.39 (m, 8H), 1.24 (s, 1H). MS m / z: C33H47N4O5, [M+H]+, calcd: 579.35, found: 579.26.

[0763] (9-2) Synthesis of K-2:

[0764] To 3 mL of dichloromethane were added GAL-5 (483 mg, 1.08 mmol), 3-diethoxyphosphoryloxy-1,2,3-benzotriazin-4(3H)-one (359 mg, 1.2 mmol), and diisopropylethylamine (DIPEA, 310 mg, 2.4 mmol), obtained according to the method described in (1-1). The mixture was stirred at room temperature for 30 minutes, followed by the addition of K-1 (174 mg, 0.3 mmol). The reaction was allowed to react at room temperature for 16 hours. The reaction solution was poured into 10 mL of saturated sodium bicarbonate solution. The separated aqueous phase was extracted three times with 10 mL of dichloromethane. All organic phases were combined and washed with 10 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The product was purified on a 200-300 mesh normal phase silica gel column using a dichloromethane:methanol ratio of 20:1. The eluate was collected, concentrated to remove the solvent, and dried under vacuum to obtain 205 mg of K-2, a yellow solid.

[0765] (9-3) Synthesis of K-3:

[0766] K-2 (205 mg, 0.11 mmol), succinic anhydride (22 mg, 0.22 mmol), 4-dimethylaminopyridine (DMAP, 27 mg, 0.22 mmol), and diisopropylethylamine (DIPEA, 71 mg, 0.55 mmol) were added to 1.1 ml of dichloromethane and stirred at room temperature overnight. The reaction solution was washed three times with 0.5 ml of 0.5 M triethylamine phosphate solution, and the aqueous phase obtained after each wash was back-extracted once with 0.5 ml of dichloromethane. All organic phases were combined and dried over anhydrous sodium sulfate, concentrated to remove the solvent, and dried under vacuum with an oil pump to obtain 218 mg of the light yellow solid product K-3 conjugated molecule (comparative conjugated molecule 1).

[0767] Preparation Example 10 This preparation example is used to illustrate the synthesis of (GalNAc)3 conjugated molecules (comparative conjugated molecules 2)

[0768] In this preparation example, compound 47 was synthesized according to the preparation method described in Examples 1-4 of WO2014025805A1. The structure of compound 47 is shown in the following formula, which is referred to herein as (GalNAc)3 conjugated molecule (comparative conjugated molecule 2):

[0769]

[0770] Preparation Example 11 This preparation example is used to illustrate the preparation of FIN-2 conjugate molecule (comparative conjugate molecule 3)

[0771] In this preparation example, referring to the preparation method described in Rajeev et al., ChemBioChem 2015, 16, 903–908, the FIN-2 conjugate molecule (comparative conjugate molecule 3) was synthesized according to the following process route:

[0772] (11-1) Synthesis of PRO-10 Compound

[0773]

[0774] (11-1-1) Synthesis of PRO-7

[0775] 2.93 g PRO-6 (L-hydroxyproline, CAS No.: 51-35-4, purchased from Anage, 22.4 mmol) was dissolved in 22.5 ml 1,4-dioxane, and 34 ml 10% (w / w) Na2CO3 aqueous solution was added. 6.954 g Fmoc-Cl (9-fluorenylmethyl chloroformate, CAS No.: 28920-43-6, purchased from Anage, 26.8 mmol) was dissolved in 56.5 ml 1,4-dioxane and added to the reaction mixture under ice bath, and the temperature was naturally raised to room temperature for reaction overnight. The reaction solution was poured into 150 ml of ice water and extracted three times with 100 ml of methyl tert-butyl ether each time. The resulting organic phase was discarded, and the remaining aqueous phase was adjusted to pH ≤ 5 with concentrated HCl and extracted twice with 100 ml of ethyl acetate. The combined organic phases were dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain 7.83 g of PRO-7 as a white foamy solid product. 1 H NMR(400MHz, DMSO-d6)δ7.91(t,J=7.2Hz,2H),7.67(d,J=7.5Hz,2H),7.48–7.39(m,2H),7.38–7.27(m,2H),5.17 (s,1H),4.27(s,2H),4.23–4.11(m,2H),3.55–3.41(m,3H),2.31–2.10(m,1H),2.08–1.88(m,1H).HRMS(ESI)m / z calcd forC 20 H 19 NO5[MH]-352.1190, measured value 352.1033.

[0776] (11-1-2) Synthesis of PRO-8

[0777] 7.83 g of PRO-7 (22.2 mmol) was dissolved in 80 ml of THF and heated in an oil bath to 65°C. 36.6 ml of a 2 mol / L solution of BH₃-Me₂S in THF (CAS No. 13292-87-0, purchased from J&K Pharmaceuticals, 73.2 mmol) was added under reflux, and the reaction was continued under reflux for 3 hours. The reaction solution was decanted, and the remaining solid was dissolved with methanol. Methanol was then added with stirring until the reaction solution ceased gas evolution and stirring was continued for 30 minutes. The solvent was evaporated under reduced pressure, and the residue was purified three times with petroleum ether to obtain 7.1 g of PRO-8 as a white solid. 1HNMR(400MHz, DMSO-d6)δ7.91(t,J=6.7Hz,2H),7.67(d,J=7.2Hz,2H),7.49–7.39(m,2H),7.38–7.26(m,2H),5.18(dd,J= 6.1,3.8Hz,1H),4.28(s,2H),4.23–4.13(m,2H),3.55–3.38(m,2H),2.32–2.11(m,1H),2.08–1.89(m,1H).HRMS(ESI)m / z calcd forC 20 H 21 NO4[M+Na] + 362.1368, measured value 362.1012.

[0778] (11-1-3) Synthesis of PRO-9

[0779] 7.1 g PRO-8 (21 mmol) was dissolved in 100 ml pyridine, and 14.2 g DMTr-Cl (4,4'-bis(methoxytrityl)chloride, 42 mmol) was added. The reaction was stirred at room temperature for 5 hours. The solvent was evaporated under reduced pressure, the crude product was dissolved in ethyl acetate and filtered to remove salt impurities. After the solvent was evaporated under reduced pressure, the residue was purified by silica gel column. For purification, the crude product dissolved in DCM was loaded onto a silica gel column pretreated with pyridine to alkalize. DMTr-Cl was first eluted with DCM containing 1% (v / v) pyridine, and then the product was eluted with ethyl acetate. The product eluate was collected and the solvent was evaporated under reduced pressure to obtain 8.2 g of white solid product PRO-9; HRMS (ESI) m / z calcd for C 41 H 39 NO6[M+Na] + 664.2675, found 664.2348; C18 RP-HPLC (batch number JJS160324-1), purity 94.20%.

[0780] (11-1-4) Synthesis of PRO-10

[0781] 8.2 g PRO-9 (12.8 mmol) was dissolved in 64 ml DMF, and 40 ml piperidine (384 mmol) was added. The reaction mixture was stirred at room temperature for 30 minutes. The reaction mixture was poured into 300 ml of ice water and extracted three times with 150 ml of ethyl acetate. The resulting organic phases were combined and washed with 200 ml of saturated brine. The washed organic phases were dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the residue was purified on a silica gel column. For purification, the crude product dissolved in DCM was loaded onto a silica gel column pretreated with pyridine to alkalize. The Fmoc residue was first eluted with DCM containing 1% (v / v) pyridine, followed by elution of the product with ethyl acetate. The product eluate was collected and the solvent was evaporated under reduced pressure to yield 4.65 g of PRO-10 as a white solid. 1 H NMR(400MHz,DMSO-d6)δ7.40(d,J=7.2Hz,2H),7.35–7.18(m,7H),6.93–6.84(m ,4H),4.56(d,J=3.9Hz,1H),4.12(s,1H),3.74(s,6H),3.46–3.37(m,1H),2.88( ddd,J=18.5,10.0,5.5Hz,2H),2.75(dd,J=8.7,5.8Hz,1H),2.62(dd,J=11.0,2 .7Hz,1H),1.74–1.65(m,1H),1.40(ddd,J=12.9,8.5,5.9Hz,1H); HRMS(ESI)m / z calcd for C 26 H 29 NO4[M+Na] + 442.1994, found 442.1999; C18 RP-HPLC (batch number JJS160329-1), purity 97.07%.

[0782] (11-2) Synthesis of FIN-1

[0783]

[0784] GAL-5 (4.5 g, 10 mmol) obtained according to the method described in (1-1) was dissolved in 40 ml of DMF. 3.9 g of DIPEA (N,N-diisopropylethylamine, CAS No. 7087-68-5, purchased from Aladdin, 30 mmol) and 3.8 g of HBTU (benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, CAS No. 94790-37-2, purchased from Aladdin, 11 mmol) were added sequentially, and the mixture was stirred at room temperature for 10 minutes to obtain a reaction solution. PRO-10 (4.2 g, 10 mmol) obtained in step (12-4) was dissolved in 40 ml of DMF and then added to the reaction solution. Anhydrous sodium sulfate was added to the reaction solution, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was poured into 120 ml of ice water and extracted three times with 60 ml of ethyl acetate each time. The resulting organic phases were combined and washed with 20 ml of water and 20 ml of saturated brine, respectively. The organic phase obtained from the washes was separated and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the residue was purified on a silica gel column. For purification, the sample was loaded onto a silica gel column pretreated with pyridine to alkalize it. Elution was performed with a solution of 1% (v / v) triethylamine and 1% (v / v) methanol in dichloromethane (DCM). The eluate was collected and the solvent was evaporated under reduced pressure to obtain 6.5 g of the product FIN-1 as a light yellow foamy solid. 1 HNMR(400MHz,DMSO-d6)δ7.83(d,J=9.2Hz,1H),7.32(t,J=6.6Hz,4H),7.20(td,J=8.9,3.5Hz,5H),6.93–6.84(m,4H),5.21( d,J=3.2Hz,1H),5.04–4.90(m,2H),4.49(s,1H),4.40(d,J=4.4Hz,0.8H),4.31(d,J=5.0Hz,0.2H),4.15(s,1H),4.03(s,3H) , 3.93 (s, 1H), 3.74 (s, 7H), 3.59 (dt, J = 12.0, 6.0 Hz, 1H), 3.50–3.40 (m, 1H), 3.39–3.25 (m, 3H), 3.13 (dd, J = 8.9, 5.2 Hz, 1H), 3.00 (dq, J = 9.3, 5.3, 4.3 Hz, 1H), 2.22 (s, 2H), 2.07 (s, 3H), 1.99 (s, 3H), 1.90 (s, 4H), 1.74 (s, 3H), 1.50 (s, 3H), 1.36 (s, 1H). Purity: 95.45% by C18 RP-HPLC (Batch No. LJ160422).

[0785] (11-3) Synthesis of FIN-2

[0786]

[0787] FIN-1 (3.0 g, 3.53 mmol) obtained in step (11-2) was dissolved in 10 ml of DMF. Under nitrogen protection, 2.13 g of PA (bis(diisopropylamino)(2-cyanoethoxy)phosphine, Adamas, product number 11356B, 7.06 mmol) and 346 mg of tetrazole (CAS number: 288-94-8, purchased from Aladdin, 4.94 mmol) were added. The reaction was stirred at room temperature. 10 ml of DMF was added and the stirring reaction was continued for 1 hour. After the solvent was evaporated under reduced pressure, the residue was purified by silica gel column chromatography. For purification, the crude product dissolved in DCM was loaded onto a silica gel column pretreated with pyridine to make it alkaline, eluted with ethyl acetate, and the eluate was collected and the solvent was evaporated under reduced pressure to obtain 4.5 g of a colorless syrupy crude product. The crude product was dissolved in 50% (v / v) acetonitrile aqueous solution until completely dissolved and then added with (C-18, 330 g, ) The sample was purified by a medium-pressure purification column. The column was pre-alkalinized with an acetonitrile solution containing 1% (v / v) pyridine. The product was collected by gradient elution. The solvent was evaporated under reduced pressure to obtain 2.2 g of a white powder product FIN-2 conjugated molecule (comparative conjugated molecule 3). 31 P NMR (162 MHz, CDCl3) δ 148.04, 147.94, 147.62, 147.19, phosphorus spectrum purity 92%; C18 RP-HPLC purity 90.54%.

[0788] Preparation Example 12 This preparation example is used to illustrate the preparation of Z-4 conjugate molecule (conjugate molecule 153)

[0789] In this preparation example, the conjugated molecule 153 (hereinafter also referred to as Z-4 conjugated molecule) was synthesized according to the following method.

[0790]

[0791] Synthesis of (12-1)Z-1:

[0792] W-3 (1.50 g, 3.37 mmol) obtained according to the method described in step (7-3) and GAL5-C4-2 (7.18 g, 13.48 mmol) obtained according to the method described in step (2-2) were mixed and dissolved in 34 ml of dichloromethane, and diisopropylethylamine (3.48 g, 26.96 mmol) was added, followed by 3-diethoxyphosphoryloxy-1,2,3-benzotriazine-4(3H)-one (DEPBT, 4.04 g, 13.48 mmol), and the reaction was stirred at room temperature for 4.5 hours. The resulting reaction solution was diluted with 100 ml of dichloromethane, and the organic phase was washed with 80 ml of saturated sodium bicarbonate solution and 80 ml of saturated brine, respectively. All organic phases were combined and dried over anhydrous sodium sulfate. After filtration, the solvent was evaporated under reduced pressure to obtain a crude product. The crude product was purified on a 200-300 mesh normal phase silica gel column packed with petroleum ether. The silica gel was acidified with 1 wt% triethylamine, and the column was eluted with a gradient of dichloromethane:methanol = 30:1 to 15:1. The eluate was collected and evaporated to dryness under reduced pressure to obtain 3.97 g of pure Z-1. MS m / z: C98H143N10O33, [M+H]+, calcd: 1987.98, found: 1987.90.

[0793] (12-2) Synthesis of Z-2:

[0794] Z-1 (3.97 g, 2.00 mmol) was dissolved in 250 ml of dichloromethane, followed by the addition of dichloroacetic acid (10.941 g, 84.85 mmol). The reaction was allowed to react at room temperature for 1 hour. Pyridine was added to neutralize the resulting reaction solution until neutral, and the solvent was evaporated under reduced pressure to obtain the crude product. The column was packed with 200 g of 200-300 mesh normal phase silica gel, acidified with 10% pyridine, and equilibrated with 1‰ pyridine. Elution was performed using a gradient of dichloromethane:methanol (10:1 to 2:1). The eluate was collected and the solvent was evaporated under reduced pressure to obtain 3.49 g of pure Z-2. MS m / z: C79H129N10O33, [M+H]+, theoretical: 1746.94, found: 1746.90.

[0795] Synthesis of (12-3)Z-3:

[0796] Z-2 (3.49 g, 2.0 mmol) and A-1 (3.06 g, 6.0 mmol) obtained by the method described in step (1-7a) were mixed and dissolved in 30 ml of dichloromethane. 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT, 1.80 g, 6.0 mmol) was added, followed by diisopropylethylamine (1.55 g, 12.0 mmol). The reaction mixture was stirred at 25°C for 3 hours. 100 ml of dichloromethane was added to dilute the resulting reaction solution. The organic phase was washed twice with saturated sodium bicarbonate (30 ml each time), and the aqueous phase was extracted with 10 ml of dichloromethane. All organic phases were combined and washed with 50 ml of saturated brine. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. The residue was dried overnight using a vacuum oil pump to obtain the crude product. The crude product was purified by column chromatography using 200 g of 200-300 mesh normal-phase silica gel. 20 ml of triethylamine was used to neutralize the acidity of the silica. The column was equilibrated with petroleum ether containing 1 wt% triethylamine. Elution was performed using a gradient of dichloromethane:methanol (25:1 to 15:1). The eluate was collected and the solvent evaporated under reduced pressure to yield 2.2 g of pure product Z-3. MS m / z: C₁₃H₁₅N₁₃O₃₈, [M+H]⁺, calcd: 2136.02, found: 2136.20.

[0797] Synthesis of (12-4)Z-4:

[0798] Z-3 (2.10 g, 0.983 mmol) was dissolved in 14.8 mL of dichloromethane containing DIEA (635 mg, 4.915 mmol). 4-Dimethylaminopyridine (DMAP, 240 mg, 1.966 mmol) was added and stirred to clarify. Succinic anhydride (197 mg, 1.966 mmol) was then added and stirred at 25°C for 18 hours. The resulting reaction solution was diluted with 50 mL of dichloromethane, and the organic phase was washed with 80 mL of 0.5 M triethylamine phosphate. The aqueous phase was extracted twice with 50 mL of dichloromethane each time. All organic phases were combined and evaporated to dryness under reduced pressure to yield the crude product. The crude product was purified by column chromatography using 188 g of 200-300 mesh normal phase silica gel. The silica was acidified with 1 wt% triethylamine and the column was equilibrated with dichloromethane. The column was eluted with a gradient of dichloromethane containing 1 wt% triethylamine and methanol (10:1 to 3:1). The eluate was collected and the solvent evaporated under reduced pressure to yield 1.95 g of pure Z-4 ​​conjugated molecule (conjugated molecule 12). MS m / z: C₁₀H₁₅N₁₀O₄₁₁, [M+H]⁺, theoretical: 1935.07, found: 1935.29. The structure of the resulting Z-4 conjugated molecule is shown in Formula (422).

[0799] Preparation Example 13 Preparation of L10-siHBa1 Conjugate (Conjugate 9)

[0800] In this preparation example, starting from the L-9 conjugate molecule (conjugate molecule 1), the L10-siHBa1 conjugate (hereinafter also referred to as conjugate 9) was prepared according to the following method.

[0801] (13-1) Synthesis of compound L-10:

[0802]

[0803] In this step, the L-10 compound was prepared by linking the L-9 conjugate molecule to a solid support.

[0804] The L-9 conjugate molecule obtained in step (1-8) (0.233 g, 0.1126 mmol), O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU, 0.064 g, 0.1689 mmol), and diisopropylethylamine (DIPEA, 0.029 g, 0.2252 mmol) were mixed and dissolved in 19 ml of acetonitrile. The mixture was stirred at room temperature for 5 minutes. Aminomethyl resin (0.901 g, 100-200 mesh, amino loading 400 μmol / g, purchased from Nankai Hecheng Company) was added to the reaction solution. The reaction was carried out on a shaker at 220 rpm at 25°C for 15 hours and then filtered. The residue was washed twice with 30 ml of DCM, three times with 30 ml of acetonitrile, and once with 30 ml of diethyl ether. The mixture was then dried under vacuum for 2 hours. The capping reaction was then carried out according to the feed ratios shown in Table 2.

[0805] Table 2 Capping reaction feed ratio

[0806] raw material Dosage Specification batch number Manufacturer Cap1 20ml —— —— —— Cap2 2.3ml —— —— —— DMAP 0.01g analytically pure I1422139 Aladdin Acetonitrile 2.3ml Spectrally pure O15161001 Shanghai Xingke

[0807] Cap1 and Cap2 are capping reagent solutions. Cap1 is a solution of 20% (v / v) N-methylimidazole in a pyridine / acetonitrile mixture, with a volume ratio of pyridine to acetonitrile of 3:5; Cap2 is a solution of 20% (v / v) acetic anhydride in acetonitrile.

[0808] Cap1, Cap2, 4-dimethylaminopyridine (DMAP), and acetonitrile were added to the above reaction mixture and reacted on a shaker at 25°C, 200 rpm, for 5 hours. The reaction solution was filtered, and the residue was rinsed with acetonitrile three times, 30 ml each time. The solvent was evaporated to dryness, and the mixture was dried under reduced pressure using a vacuum oil pump overnight to obtain 1.100 g of compound L-10 (i.e., L-9 conjugated molecule attached to a solid support) with a loading of 90.8 μmol / g. The structure of compound L-10 is shown in Formula (523).

[0809] (13-2) Synthesis of the positive chain of L10-siHBa1 conjugate

[0810] In this step, the siRNA of the siRNA conjugate is the sequence numbered siHBa1:

[0811] siHBa1

[0812] Sense strand: 5'-CCUUGAGGCAUACUUCAAA-3' (SEQ ID NO: 1),

[0813] Antisense strand: 5′-UUUGAAGUAUGCCUCAAGGUU-3′ (SEQ ID NO: 2);

[0814] Starting with compound L-10 prepared in the above steps, nucleoside monomers were linked one by one in the 3'-5' direction according to the above sequence. Each linking of nucleoside monomers involved four steps: deprotection, coupling, capping, and oxidation. The synthesis conditions are given as follows:

[0815] The nucleoside monomer was provided as a 0.1 M acetonitrile solution. The deprotection reaction conditions for each step were the same, namely, a temperature of 25° C., a reaction time of 70 seconds, a deprotection reagent of 3% v / v dichloroacetic acid in dichloromethane, and a molar ratio of 5:1 between dichloroacetic acid and the 4,4'-dimethoxytrityl protecting group on the solid support.

[0816] The coupling reaction conditions were the same for each step, including a temperature of 25°C, a molar ratio of the nucleic acid sequence attached to the solid support to the nucleoside monomer of 1:10, a molar ratio of the nucleic acid sequence attached to the solid support to the coupling reagent of 1:65, a reaction time of 600 seconds, and a 0.5 M acetonitrile solution of 5-ethylthio-1H-tetrazole as the coupling reagent.

[0817] The capping conditions were identical for each step, including a temperature of 25°C and a reaction time of 15 seconds. The capping reagent solution was a mixture of Cap1 and Cap2 at a 1:1 molar ratio, and the molar ratio of capping reagent to the nucleic acid sequence attached to the solid support was 1:1:1 acetic anhydride:N-methylimidazole:nucleic acid sequence attached to the solid support.

[0818] The oxidation reaction conditions for each step were identical, including a temperature of 25°C, a reaction time of 15 seconds, and the oxidizing agent being 0.05 M iodine solution. The molar ratio of iodine to the nucleic acid sequence attached to the solid support during the coupling step was 30:1. The reactions were performed in a mixture of tetrahydrofuran:water:pyridine (3:1:1).

[0819] Cleavage and deprotection conditions were as follows: The synthesized nucleotide sequence linked to the carrier was added to 25 wt% ammonia water at a concentration of 0.5 ml / μmol, reacted at 55°C for 16 h, the liquid was removed, and the residue was concentrated to dryness in vacuo. After ammonia treatment, the product was dissolved in 0.4 ml / μmol N-methylpyrrolidone for the amount of single-stranded nucleic acid, followed by the addition of 0.3 ml / μmol triethylamine and 0.6 ml / μmol triethylamine trihydrofluoride to remove the 2'-TBDMS protection on the ribose. Purification and desalting: Nucleic acid purification was achieved using a preparative ion chromatography column (Source 15Q) by gradient elution with NaCl. Specifically, the following steps were used: eluent A: 20 mM sodium phosphate (pH 8.1), solvent: water / acetonitrile = 9:1 (volume ratio); eluent B: 1.5 M sodium chloride, 20 mM sodium phosphate (pH 8.1), solvent: water / acetonitrile = 9:1 (volume ratio); elution gradient: eluent A:eluent B = 100:0-50:50 gradient elution. The product eluates were collected and combined, and desalted using a reversed-phase chromatography column. Specific conditions included desalting using a Sephadex column with Sephadex-G25 as the filler, and eluting with deionized water.

[0820] Detection: The purity detected by ion exchange chromatography (IEX-HPLC) was 92.4%. The molecular weight was analyzed by liquid chromatography-mass spectrometry (LC-MS), with a theoretical value of 7253.96 and an actual value of 7253.12.

[0821] Thus, in this step, the L-9 conjugated molecule is linked to the 3' end of the obtained sense strand, thereby obtaining the siRNA sense strand S in which the L-9 conjugated molecule is conjugated to the 3' end of the siRNA.

[0822] (13-3) Synthesis of antisense strand

[0823] In this step, a universal solid phase carrier (UnyLinker TM loaded The antisense strand of the L10-siHB1 conjugate was synthesized by HL Solid Supports (Kinovate Life Sciences Inc.). The antisense strand of the siRNA, AS, was obtained using the same conditions as the sense strand synthesis, including deprotection, coupling, capping, oxidation, deprotection, cleavage, and separation conditions used in solid-phase synthesis.

[0824] Testing: The purity was 93.2% as determined by ion exchange chromatography (IEX-HPLC); the molecular weight was analyzed by liquid chromatography-mass spectrometry (LC-MS). The theoretical value was 6675.04, and the measured value was 6674.50.

[0825] (13-4) Synthesis of L10-siHBa1 Conjugate

[0826] The S chain and AS chain were dissolved in an aqueous solution for injection to obtain a 40 mg / ml solution. They were mixed in an equimolar ratio, heated at 50°C for 15 minutes, and cooled to room temperature to form a double-chain structure through hydrogen bonding.

[0827] After the above synthesis was completed, the conjugate was diluted to a concentration of 0.2 mg / mL using ultrapure water (Milli-Q ultrapure water instrument, resistivity 18.2 MΩ*cm (25°C)). Molecular weight was determined using a liquid chromatography-mass spectrometry (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters, model: LCT Premier). The results showed that the theoretical molecular weight S was 7253.96 and the measured molecular weight S was 7253.24 and the measured molecular weight S was 6674.61. These values ​​were consistent with the theoretical values, confirming that the synthesized conjugate was the target designed double-stranded nucleic acid sequence with the L-9 conjugate molecule. The structure of the L10-siHBa1 conjugate (conjugate 9) is shown in formula (3).

[0828] Preparation Example 14 Preparation of Conjugates 16-18, 24-26, 42-43, 62, 78, 105, 109, 111, 115, 144, and 154-171

[0829] The title conjugate was prepared using the same method as in Preparation Example 13, except that: 1) the conjugated siRNA had the sequences corresponding to conjugates 16-18, 24-26, 42-43, 62, 78, 105, 109, 111, 115, 144, 157-163, and 167-171 shown in Tables 3A-3G; 2) when two nucleotides in the target sequence were linked by a phosphorothioate, the oxidation step in the latter nucleotide connection was replaced by the following sulfurization step; the conditions for each sulfurization step were identical, including a temperature of 25°C, a reaction time of 300 seconds, and the sulfurization reagent was hydroxanthin. The molar ratio of the sulfurization reagent to the nucleic acid sequence attached to the solid support in the coupling step was 120:1. The reaction was carried out in a 1:1 acetonitrile:pyridine mixed solvent; and 3) when all nucleotides in the target sequence have modified hydroxyl groups at the 2'-position, the cleavage and deprotection conditions did not include a step to remove the 2'-TBDMS protection from the ribose sugar. Thus, conjugates 16-18, 24-26, 42-43, 62, 78, 105, 109, 111, 115, 144, 157-163, and 167-171 of the present disclosure were prepared.

[0830] In addition, conjugates 154-156 and 164-166 were prepared using the same method as in Preparation Example 13, except that the following conjugate molecules were used instead of the L-9 conjugate molecule: the P-9 conjugate molecule (conjugate molecule 2) obtained in Preparation Example 2 was used to prepare conjugate 164, the W-7 conjugate molecule (conjugate molecule 7) obtained in Preparation Example 7 was used to prepare conjugates 155, 156 and 165, and the Z-4 ​​conjugate molecule (conjugate molecule 12) obtained in Preparation Example 12 was used to prepare conjugates 154 and 166, and the conjugated siRNAs were the sequences corresponding to conjugates 154-156 and 164-166 shown in Tables 3A-3G, respectively. The above conjugates were numbered according to Tables 3A-3G, and their structures were shown in Formula (3), Formula (4), Formula (15) and Formula (22), respectively. The molecular weight of the conjugates was determined by liquid chromatography-mass spectrometry. The results were as follows: the theoretical value S of conjugate 142 was 7649.55, AS: 6991.46; the measured value S was 7649.1, AS: 6991; the theoretical value S of conjugate 170 was 7649.55, AS: 6995.47, the measured value S was 7648.8, AS: 6994.8; the theoretical value S of conjugate 171 was 7649.55, AS: 7011.53, the measured value S was 7648.8, AS: 7010.9; The theoretical value of conjugate 115 is S: 7584.5, AS: 7007.46, and the measured value is S: 7584, AS: 7006.2; the theoretical value of conjugate 109 is S: 7584.5, AS: 6931.47, and the measured value is S: 7584, AS: 6930.9; the theoretical value of conjugate 106 is S: 7572.47, AS: 6907.41, and the measured value is S: 7571.8, AS: 6906.9; the theoretical value of conjugate 113 is S: 7584.5, AS: 7011.4 7, found S: 7584, AS: 7011.3; conjugate 17 theoretical value S: 7504.34, AS: 6961.52, found S: 7503.4, AS: 6960.9; conjugate 25 theoretical value S: 7504.34, AS: 7037.51, found S: 7503.6, AS: 7036.9; conjugate 18 theoretical value S: 8218.83, AS: 7703.05, found S: 8218, AS: 7702.5; conjugate 1 The theoretical value of 6 is S: 7516.37, AS: 6985.58, the measured value is S: 7516.5, AS: 6984.9; the theoretical value of conjugate 159 is S: 7504.34, AS: 7057.58, the measured value is S: 7503.6, AS: 7057; the theoretical value of conjugate 160 is S: 7504.34, AS: 7041.52, the measured value is S: 7503.6, AS: 7040.8; the theoretical value of conjugate 161 is S: 7516.37, AS: 7065.58, found S: 7516.6, AS: 7064.5; conjugate 162 theoretical value S: 7504.34, AS: 7139.68, found S: 7515.6, AS: 7138.9; conjugate 163 theoretical value S: 7516.37, AS: 7081.64, found S: 7515.6, AS: 7080.9; conjugate 62 theoretical value S: 7485.3, AS: 7161.7, found S: 7484.4, AS : 7160.9; the theoretical value of conjugate 78 is S: 7423.22, AS: 7207.78, the measured value is S: 7422.6, AS: 7207.2; the theoretical value of conjugate 42 is S: 7407.22, AS: 7208.77, the measured value is S: 7406.4, AS: 7208.1; the theoretical value of conjugate 43 is S: 7407.22, AS: 7170.72, the measured value is S: 7406.5, AS: 7170.1, the measured value is consistent with the theoretical value.

[0831] Table 3 siRNA conjugates

[0832] Table 3A

[0833]

[0834]

[0835]

[0836] Table 3B

[0837]

[0838]

[0839] Table 3C

[0840]

[0841]

[0842] Table 3D

[0843]

[0844]

[0845] Table 3E

[0846]

[0847]

[0848]

[0849] Table 3F

[0850]

[0851]

[0852]

[0853] Table 3G

[0854]

[0855]

[0856] *S: sense strand; AS: antisense strand

[0857] Note: Capital letters C, G, U, and A represent the base composition of the nucleotide; dT represents deoxythymidine nucleotide; lowercase letter m represents that the nucleotide to the left of the letter m is a 2'-methoxy-modified nucleotide; lowercase letter f represents that the nucleotide to the left of the letter f is a 2'-fluorine-modified nucleotide; lowercase letter s represents that the connection between the two nucleotides adjacent to the letter s is a phosphorothioate connection; VP represents that the nucleotide to the right of the letter VP is a vinyl phosphate-modified nucleotide; P represents that the nucleotide to the right of the letter P is a phosphate nucleotide; Ps represents that the nucleotide to the right of the letter Ps is a phosphorothioate-modified nucleotide, and Tmoe represents a 2'-methoxyethoxy-modified thymidine nucleotide.

[0858] Vinyl phosphate and 2'-methoxy modified uridine monomers (VP-Um) were synthesized as follows:

[0859]

[0860] (14-1) Synthesis of VP-U-2

[0861] The VP-U-2 molecule was synthesized according to the following method:

[0862]

[0863] 2'-Methoxy-modified uridine (2'-OMe-U, 51.30 g, 91.6 mmol), tert-butyldiphenylsilyl chloride (TBDPSCl, 50.35 g, 183.2 mmol), and imidazole (12.47 g, 183.2 mmol) were dissolved in 450 ml of N,N-dimethylformamide (DMF) and stirred at room temperature for 20 hours. The DMF was evaporated, and the residue was dissolved in 600 ml of dichloromethane and washed with 300 ml of saturated sodium bicarbonate. The separated aqueous phase was extracted three times with 300 ml of dichloromethane (DCM). All organic phases were combined and washed with 5% oxalic acid until the aqueous phase had a pH <5. The solvent was evaporated to dryness to obtain crude VP-U-1, which was directly used in the subsequent synthesis of VP-U-2.

[0864] Dissolve the crude VP-U-1 in 100 ml of dichloromethane and stir in an ice bath for 10 minutes. Add 450 ml of a 2% p-toluenesulfonic acid solution (pre-cooled at 4°C) (a 3:7 volume ratio methanol-dichloromethane mixture) and allow to react for 10 minutes. Add 200 ml of saturated sodium bicarbonate to quench the reaction. Wash the resulting organic phase with saturated sodium bicarbonate solution until the pH reaches 8. Combine the aqueous phases and extract twice with 200 ml of dichloromethane. Combine all organic phases and wash once with 200 ml of saturated brine. Evaporate the solvent to dryness. Purify the residue using a 200-300 mesh normal-phase silica gel column packed with petroleum ether and elute with a gradient of petroleum ether:ethyl acetate:dichloromethane:methanol = 1:1:1:0.05 to 1:1:1:0.25. Collect the product eluate, evaporate the solvent under reduced pressure, and dry the residue using a vacuum pump to obtain 40.00 g of pure VP-U-2. 1H NMR(400MHz,DMSO-d6)δ7.96(d,J=7.8Hz,1H),7.64(dtd,J=5.1,4.0,2.2Hz,4H),7 .41–7.30(m,6H),6.79(d,J=4.7Hz,1H),5.73(d,J=7.6Hz,1H),4.94(t,J=7.0Hz,1H ),4.12(td,J=4.6,3.9Hz,1H),4.05(dd,J=4.8,4.0Hz,1H),3.96(t,J=4.7Hz,1H),3.68(ddd,J=11.8,7.0,4.6Hz,1H),3.57–3.46(m,1H),3.39(s,3H),1.05(s,8H).MS m / z:C26H33N2O6Si,[M+H]+,theoretical value: 497.21, found: 497.45.

[0865] (14-2) Synthesis of VP-U-4:

[0866]

[0867] VP-U-2 (19.84 g, 40.0 mmol), dicyclohexylcarbodiimide (DCC, 16.48 g, 80.0 mmol), pyridine (4.20 g, 53.2 mmol), and trifluoroacetic acid (6.61 g, 53.2 mmol) were dissolved in 200 ml of dimethyl sulfoxide (DMSO) and stirred at room temperature for 20 hours to obtain a reaction solution. Separately, tetraethyl methylene diphosphate (21.44 g, 74.4 mmol) was dissolved in 120 ml of THF and cooled in an ice bath. t-BuOK (11.36 g, 101.2 mmol) was added in an ice bath and allowed to react for 10 minutes, then at room temperature for 0.5 hours. The mixture was then added to the reaction solution over approximately 1 hour. The reaction was continued in an ice bath for 1 hour, then at room temperature for 18 hours. The reaction was quenched by adding water, and the separated aqueous phase was extracted three times with 200 ml of dichloromethane. Combine all organic phases, wash once with 200 ml of saturated brine, and evaporate the solvent to dryness. Purify on a 200-300 mesh normal phase silica gel column, packed with petroleum ether, and elute with a gradient of petroleum ether: ethyl acetate = 1:1-1:4. The product eluate is collected, the solvent is evaporated under reduced pressure, and the residue is dried by foaming with a vacuum oil pump to obtain 14.00 g of pure VP-U-4. 1HNMR (400 MHz, DMSO-d6) δ7.96 (d, J = 7.8 Hz, 1H), 7.64 (dtd, J = 5.1, 4.0, 2.2 Hz, 4H), 7.41–7.30 (m, 6H), 6.82–6.71 (m, 2H), 5.90 (ddd, J = 25.9, 15.0, 1.0 Hz, 1H), 5.73 (d, J =7.6Hz,1H),4.36–4.21(m,3H),4.18(t,J=4.9Hz,1H),4.05(ddq,J=9.7,8.5,6.9Hz,2H),3.87(t,J=4.8Hz,1H),3.39(s,3H),1.32(td,J=6.9,0.7Hz,6H),1.05(s,8H).MS m / z:C31H42N2O8PSi,[M+H]+,theor.:629.24,found:629.51.

[0868] (14-3) Synthesis of VP-U-5:

[0869]

[0870] VP-U-4 (14.00 g, 22.29 mmol) was dissolved in 100 ml of tetrahydrofuran, and triethylamine trihydrofluoride (17.96 g, 111.45 mmol) was added. The mixture was stirred at room temperature for 20 hours until the reaction was complete. The solvent was directly evaporated to dryness, and the mixture was dissolved in dichloromethane and then evaporated to dryness twice, using 50 ml of dichloromethane each time, to obtain a crude product. The crude product was purified on a 200-300 mesh normal phase silica gel column, packed with petroleum ether, and eluted with a gradient of petroleum ether: ethyl acetate: dichloromethane: methanol = 1:1:1:0.05 to 1:1:1:0.25. The product eluate was collected, the solvent was evaporated under reduced pressure, and the residue was dried by foaming with a vacuum oil pump to obtain 6.70 g of pure VP-U-5. 1HNMR(400MHz,DMSO-d6)δ7.96(d,J=7.8Hz,1H),6.77(dd,J=15.0,6.2Hz,1 H),5.99–5.82(m,2H),5.73(d,J=7.6Hz,1H),5.27(d,J=5.1Hz,1H),5.10(dd ,J=5.3,4.7Hz,1H),4.29(ddq,J=9.8,8.6,7.0Hz,2H),4.17(ddd,J=6.2,5.2 ,1.0Hz,1H),4.12–3.98(m,3H),3.39(s,2H),1.32(td,J=6.9,0.6Hz,6H).MS m / z: C15H24N2O8P, [M+H]+, theoretical value: 391.13, found value: 391.38.

[0871] (14-4) Synthesis of VP-U-6:

[0872]

[0873] VP-U-5 (391 mg, 1.0 mmol), pyridinium trifluoroacetate (0.232 g, 1.2 mmol), N-methylimidazole (0.099 g, 1.2 mmol), and bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.452 g, 1.5 mmol) were added to 10 ml of anhydrous dichloromethane under argon protection. The mixture was stirred at room temperature for 5 hours. The solvent was evaporated to dryness, and the residue was purified by column chromatography (200-300 mesh normal phase silica gel, gradient elution of dichloromethane:acetonitrile (containing 0.5 wt% triethylamine) = 3:1 to 1:3). The product eluate was collected and concentrated to remove the solvent to obtain 508 mg of the target product VP-U-6. 31P NMR (161 MHz, DMSO-d6) δ 150.34, 150.29, 17.07, 15.50. MS m / z: C24H41N4O9P2, [M+H]+, calcd: 591.23, found: 591.55. This indicates that VP-U-6 is the target product VP-Um and participates in RNA chain synthesis as a nucleoside monomer.

[0874] A 5'-phosphate modification was attached to the 5' end of the antisense strand using the following method:

[0875] The raw material is a phosphorylated structural monomer with the following CPR-I structure, provided by Suzhou Jima, Cat#13-2601-XX:

[0876]

[0877] After all nucleoside monomers in the antisense strand are connected, a CPR-I monomer is attached to the 5' end of the antisense strand via a four-step reaction process of deprotection, coupling, capping, and oxidation, following the phosphoramidite nucleic acid solid-phase synthesis method. Cleavage and deprotection are then performed under the following conditions to obtain the antisense strand:

[0878] The synthesized nucleotide sequence linked to the vector was added to 25 wt% ammonia at a concentration of 0.5 ml / μmol relative to the nucleotide sequence. The reaction was incubated at 55°C for 16 hours, the liquid was removed, and the residue was concentrated to dryness in vacuo. After ammonia treatment, the product was dissolved in 0.4 ml / μmol N-methylpyrrolidone relative to the amount of single-stranded nucleic acid, followed by the addition of 0.3 ml / μmol triethylamine and 0.6 ml / μmol triethylamine trihydrofluoride to remove the 2'-TBDMS protection on the ribose. Purification and Desalting: The nucleic acid was purified using a preparative ion chromatography column (Source 15Q) by gradient elution with NaCl. Specifically, the following steps were used: eluent A: 20 mM sodium phosphate (pH 8.1), solvent: water / acetonitrile = 9:1 (volume ratio); eluent B: 1.5 M sodium chloride, 20 mM sodium phosphate (pH 8.1), solvent: water / acetonitrile = 9:1 (volume ratio); elution gradient: eluent A:eluent B = 100:0-50:50 gradient elution. The product eluates were collected and combined, and desalted using a reversed-phase chromatography column. Specific conditions included desalting using a Sephadex column with Sephadex-G25 as the filler, and eluting with deionized water.

[0879] In the case where the target product has a 5'-phosphorothioate modification, the same steps as above are used, except that during the ligation, the sulfidation reaction is performed under sulfidation reaction conditions instead of the oxidation reaction conditions described above.

[0880] The synthesized sense and antisense strands were tested for purity using ion exchange chromatography (IEX-HPLC) and their molecular weights were analyzed using liquid chromatography-mass spectrometry (LC-MS) to confirm that the synthesized nucleic acid sequences corresponded to the siRNAs of the Examples and Comparative Examples in Table 5.

[0881] Preparation Example 15 Preparation of the remaining conjugates in Tables 3A-3G

[0882] Alternatively, the same method as in Preparation Example 13 is used to prepare the remaining siRNA conjugates in Tables 3A-3G, except that: 1) for conjugates 31-37, 52-58, 68-74, 84-90, 121-127 and 146-152 and comparative conjugates 4-5, 7-8 and 10-13, the conjugate molecules 2-8 or 12 obtained in the above Preparation Examples 2-10 or 12 and comparative conjugate molecules 1-2 are used instead of the L-9 conjugate molecules (for example, when the P-9 conjugate molecule (conjugate molecule 2) is used instead of the L-9 conjugate molecule, the conjugates 31, 52, 68, 84, 121 and 146, which are numbered with P10, are expected to be obtained, and when the R-4 conjugate molecule (conjugate molecule 3) is used instead of the L-9 conjugate molecule, the conjugates 32, 53, 69, 85, 122 and 147, which are numbered conjugates labeled with R5, and so on); 2) the conjugated siRNA has the sequence shown in Tables 3A-3G corresponding to conjugates 10-15, 19-23, 27-41, 44-61, 63-77, 79-104, 106-108, 110, 112-114, 116-141 and 143-152, and comparative conjugates 4-5, 7-8 and 10-17; 3) when the target sequence has a phosphorothioate linkage between two nucleotides, the sulfurization reaction step described in Preparation Example 13 is used instead of the oxidation reaction step in the linkage of the latter nucleotide of the two nucleotides; and 4) when the 2'-position of all nucleotides in the target sequence is a modified hydroxyl group, the cleavage and deprotection conditions do not include a step for removing the 2'-TBDMS protection on the ribose. Thus, it is expected that conjugates 10-15, 19-23, 27-41, 44-61, 63-77, 79-104, 106-108, 110, 112-114, 116-141 and 143-152 and comparative conjugates 4-5, 7-8 and 10-17 can be prepared, which are numbered according to Tables 3A-3G. The structures of these conjugates of the present disclosure are respectively represented by formula (3), (4), (7), (12), (13), (14), (15), (21) or (22), and the structures of the comparative conjugates are respectively represented by formula (901) and formula (902):

[0883]

[0884] where Nu is the siRNA in comparative conjugates 4-5, 7-8, and 10-13.

[0885] Preparation Example 16 Comparative Synthesis of Conjugates 6 and 9

[0886] The FIN-2 obtained in the above step (11-3) was used to synthesize the comparative conjugates 6 and 9. The RNA solid phase synthesis conditions and deprotection conditions were the same as those of the nucleic acid solid phase synthesis described in the above step (11-2), except that a universal solid phase carrier (UnyLinker) was required. TM loaded HL Solid Supports), first conjugating FIN_FIN_FIN to the 3' end of the RNA sense strand through three reaction cycles of FIN-2, followed by solid-phase synthesis.

[0887] The FIN-FIN-FIN conjugate group is attached using the method described in Rajeev et al., ChemBioChem 2015, 16, 903-908. Specifically, the hydroxyl protecting group on the universal solid support is first removed. The conjugate is then coupled to a FIN-2 conjugate molecule under coupling reaction conditions and in the presence of a coupling reagent. After capping and oxidation, a FIN conjugate molecule attached to the solid support is obtained. The hydroxyl protecting group DMTr on the FIN conjugate molecule attached to the solid support is then removed. The conjugate is then coupled to another FIN-2 conjugate molecule, followed by capping and oxidation. The deprotection-coupling-capping-oxidation steps are repeated once more, and a third FIN-2 conjugate molecule is attached to obtain a conjugate group (FIN-FIN-FIN) attached to the solid support. Subsequently, nucleotide monomers are attached, starting with the conjugate group attached to the solid support, ultimately yielding an RNA sense strand with a conjugate group (FIN-FIN-FIN) attached to its 3' terminus.

[0888] In the above reaction, the conditions, solvents and reagents for the deprotection, coupling, capping and oxidation reactions are the same as those in the nucleic acid solid phase synthesis method described in step (11-2).

[0889] Subsequently, cleavage and deprotection, purification and desalting, detection, and annealing were performed using the same methods as in Preparation Example 12, ultimately yielding comparative conjugates 6 and 9 (hereinafter sometimes referred to as FIN conjugates). Molecular weights were determined using a liquid chromatography-mass spectrometry (LC-MS, purchased from Waters Corp., model: LCT Premier). The measured values ​​were consistent with the theoretical values, confirming that the synthesized FIN-siHBa1M1SP conjugate was the target compound, with the structural formula shown below in Formula (903):

[0890]

[0891] Wherein, Nu is the siRNA in comparative conjugate 6 or 9.

[0892] After the above conjugate is prepared, it is lyophilized using standard means to be stored as a solid powder. When needed, it can be re-dissolved into a solution of the desired concentration using, for example, water for injection or physiological saline.

[0893] Experimental Example 1 This experiment illustrates the toxicity of the conjugates disclosed herein.

[0894] In C57BL / 6J mice, a single subcutaneous dose of 100 mg / kg or 200 mg / kg (calculated as siRNA in a 0.9% sodium chloride aqueous solution, administered at a concentration of 10 mg / mL or 20 mg / mL, respectively, in a 0.9% sodium chloride solution, administered at 10 mL / kg) of conjugate 24 was administered. During the continuous observation period, no animal deaths occurred, and no clinical symptoms related to adverse drug reactions were observed. Clinical pathology and gross autopsy findings were also unremarkable 24 hours after administration. These results demonstrate that the conjugates disclosed herein exhibit low animal-level toxicity.

[0895] Experimental Example 2 This experiment illustrates the stability of the conjugate disclosed herein

[0896] Experimental Example 2-1 Stability in in vitro lysosomal lysis solution

[0897] Conjugates 24, 25, 42, 43, 62, 78, and comparative sequence 1 (provided as siRNAs at a concentration of 20 μM in 0.9% sodium chloride aqueous solution, 12 μl per group) were mixed with 27.2 μL of sodium citrate aqueous solution (pH 5.0), 4.08 μL of deionized water, and 2.72 μL of mouse Tritosomes (purchased from Xenotech Inc., Catalog No. R0610LT, No. 1610069, final concentration 0.2 mU / μL) and incubated at 37°C. 5 μl of sample was removed at each time point of 0 h, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, and 8 h and denatured in 15 μL of 9 M urea aqueous solution. 4 μl of loading buffer (Solar Bio, Catalog No. 20160830) was then added and immediately frozen at -80°C to terminate the reaction. 0 hours indicates the time immediately after the test sample is mixed with the lysosomal lysis buffer and removed. Reference samples not treated with the lysosomal lysis buffer were prepared by mixing 1.5 μl of each equimolar amount of the above conjugate (20 μM) with 7.5 μl of sodium citrate (pH 5.0) and 1 μl of deionized water. Denature each solution by adding 15 μl of 9 M urea solution. Then, add 4 μl of loading buffer (Solarbau, Cat. No. 20160830) and mix thoroughly. The reaction was terminated by immediately freezing in a -80°C freezer. Each conjugate reference sample is labeled "Con" in the electropherogram.

[0898] A 16 wt% non-denaturing polyacrylamide gel was prepared. All samples from each frozen sample group were mixed with 4 μl of loading buffer (commercially available from Solebro Inc., containing 20 mM EDTA, 36 wt% glycerol, and 0.06 wt% bromophenol blue in water). Then, 20 μl of the mixture was loaded onto the gel and electrophoresed at 20 mA for 10 minutes, followed by 40 mA for 30 minutes. Following electrophoresis, the gel was stained with Gelred dye (BioTium, Cat. 13G1203) for 10 minutes and then phased. The results are shown in Figure 2. Figure 1A shown.

[0899] The comparison sequence 1 is as follows:

[0900] Sense strand: 5′-CCUUGAGGCAUACUUCAAA-3′ (SEQ ID NO: 250);

[0901] Antisense strand: 5'-UUUGAAGUAUGCCUCAAGGUU-3' (SEQ ID NO: 251).

[0902] The stability of conjugates 105, 109, 111 and 115 was measured within 6 h using the same method. The results are shown in Figure 1B middle.

[0903] Figures 1A-1B The semi-quantitative test results of the stability of siRNA conjugates in Tritosomes in vitro are shown. The results show that the conjugates disclosed in the present invention can be maintained in Tritosomes for a long time without degradation, showing good stability.

[0904] Experimental Example 2-2 In vitro stability in human plasma

[0905] Conjugates 24, 25, 42, 43, 62, 78, and comparative sequence 1 (provided as siRNAs at a 20 μM concentration in a 0.9 wt% sodium chloride aqueous solution, 12 μl per group) were mixed with 108 μL of 90% human plasma (diluted with PBS) and incubated at 37°C. A 10 μL sample was removed at each time point of 0, 2, 4, 6, 8, 24, 48, and 72 hours, immediately snap-frozen in liquid nitrogen, and stored at -80°C. Simultaneously, an equimolar amount of each conjugate (2 μM, 2 μl) was mixed with 8 μl of 1× PBS to obtain a 10 μL sample not treated with human plasma (denoted as Con). A 20 wt% non-denaturing polyacrylamide gel was prepared. All samples from each frozen sample group were mixed with 4 μL of loading buffer (20 mM EDTA, 36 wt% glycerol, 0.06 wt% bromophenol blue in water). The samples were then loaded onto the gel and electrophoresed at a constant current of 80 mA for 60 minutes. After electrophoresis, the gel was stained with 1× Sybr Gold dye (Invitrogen, Cat. 11494) for 15 minutes and then phased. The results are shown in Figure 2. Figure 2A The comparison sequence 1 is as follows:

[0906] Sense strand: 5′-CCUUGAGGCAUACUUCAAA-3′ (SEQ ID NO: 250);

[0907] Antisense strand: 5'-UUUGAAGUAUGCCUCAAGGUU-3' (SEQ ID NO: 251).

[0908] The stability of conjugates 105, 109, 111 and 115 was measured within 72 h using the same method. The results are shown in Figure 2B middle.

[0909] Depend on Figure 2A and 2B The semi-quantitative test results of the stability of the tested siRNA conjugates in human plasma in vitro are shown. The results show that the conjugates of the present disclosure are not degraded in human plasma for up to 72 hours, showing excellent stability in human plasma.

[0910] Experimental Example 2-3 In vitro stability in monkey plasma

[0911] Conjugates 24, 25, 42, 43, 62, 78 and comparative sequence 2 (respectively provided in the form of a 0.9wt% sodium chloride aqueous solution with a siRNA concentration of 20 μM, 12 μl per group, the comparative sequence 2 being a siRNA whose sense and antisense chains have the sequences shown in SEQ ID NO: 80 and SEQ ID NO: 81, respectively, but not conjugated to any conjugated molecule) were mixed with 108 μL of 90% cynomolgus monkey plasma (Monkey plasma, purchased from Hongquan Biology, HQ70082, diluted with PBS) and incubated at 37°C. 10 μL of sample was taken out at each time point of 0, 2, 4, 6, 8, 24, 48, and 72 hours, immediately frozen in liquid nitrogen and stored in a -80°C refrigerator. After sampling at each time point, each sample was diluted 5 times with 1×PBS (pH 7.4) and 10 μL was taken for standby use. At the same time, an equimolar amount of each of the above conjugates (2 μM, 2 μl) was mixed with 8 μl of 1×PBS to obtain a 10 μl sample not treated with monkey plasma (denoted as Con). A 20 wt% non-denaturing polyacrylamide gel was prepared, and all of the samples in each diluted sample group were mixed with 4 μl of loading buffer (20 mM EDTA, 36 wt% glycerol, 0.06 wt% bromophenol blue in water). The gel was then loaded and electrophoresed at a constant current of 80 mA for 60 minutes. After electrophoresis, the gel was stained with 1× Sybr Gold dye (Invitrogen, Cat. 11494) for 15 minutes and then phased. The results are shown in Figure 2. Figure 3A shown.

[0912] The stability of conjugates 105, 109, 111 and 115 and comparative sequence 2 was measured within 72 hours using the same method. The results are shown in Figure 3B middle.

[0913] Figure 3A and 3B The semi-quantitative test results of the stability of the tested siRNA in monkey plasma in vitro are shown. It can be seen that the siRNA conjugates disclosed in the present invention are not degraded in cynomolgus monkey plasma for up to 72 hours, showing excellent stability in monkey plasma.

[0914] Experimental Example 3 This experiment illustrates the pharmacokinetics of conjugates 24 and 25 in rats

[0915] In this experimental example, conjugates 24 and 25 were subcutaneously injected into experimental groups of rats (10 rats per group, half male and half female), with single doses of 10 mg / kg and 50 mg / kg, respectively. Plasma drug concentrations and liver and kidney tissue drug concentrations were then measured at various time points.

[0916] First, SD rats were randomized by sex based on body weight using the PRISTIMA version 7.2.0 data system and then administered the conjugate according to the designed dose. All animals received a single subcutaneous dose of 10 and 50 mg / kg, calculated based on body weight, in the form of 1 mg / ml and 5 mg / ml 0.9% sodium chloride aqueous solution in a volume of 10 ml / kg. Whole blood was collected from the jugular vein before and 5 minutes (±30 seconds), 30 minutes (±1 minute), 1 hour (±2 minutes), ...

Claims

1. A conjugate having a structure represented by formula (1): in, n1 is an integer from 1 to 3, and n3 is an integer from 0 to 4; Each of m1, m2 and m3 is independently an integer from 2 to 5; Each R 10 、R 11 、R 12 、R 13 、R 14 and R 15 Each independently selected from H, methyl or ethyl; R3 is a group having a structure represented by formula A59: wherein E1 is OH or SH, Nu is a functional oligonucleotide, the functional oligonucleotide is siRNA, the siRNA comprises a sense strand and an antisense strand, and the P atom in the formula A59 is connected to the 3' end of the sense strand; R2 is B5, B6, B5' or B6': in, represents the site where the group is attached to the rest of the molecule, and q2 is an integer from 1 to 10; Each L1 is independently a linked combination of at least two of the groups A1, A8, and A10, and L1 is 4 to 15 atoms in length; Wherein, each j1 is independently an integer from 1 to 20; Each M1 is N-acetylgalactosamine (GalNAc).

2. The conjugate according to claim 1, wherein n1 is an integer of 1-2, n3 is an integer of 0-1, and n1+n3=2-3.

3. The conjugate according to claim 1, wherein m1=m2=m3.

4. The conjugate according to claim 1, wherein R2 forms a phosphate bond with the P atom in R3.

5. The conjugate of claim 1, wherein the conjugate has a structure represented by formula (3), (4), (5), (6), (7), (8), (9), (10), (11), (12), (13), (14), (15), (16), (17), (18), (19), (20), or (21): in, The Nu represents siRNA.

6. The conjugate according to claim 1 or 5, wherein Each nucleotide in the siRNA is independently a modified or unmodified nucleotide, the positive 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 19 nucleotides in length and are at least partially reverse-complementary to form a double-stranded complementary region; the nucleotide sequence 2 is at least partially complementary to the first nucleotide sequence, which is a segment of nucleotides in the target mRNA; the target mRNA refers to the mRNA of a gene overexpressed in hepatocytes.

7. The conjugate according to claim 6, wherein The target mRNA is selected from hepatitis B virus mRNA, mRNA transcribed from angiopoietin-like protein 3 gene, or mRNA transcribed from apolipoprotein C3 gene.

8. The conjugate according to claim 6, wherein The nucleotide sequence 1 is equal in length to the first nucleotide sequence and differs by no more than 3 nucleotides; the nucleotide sequence 2 is equal in length to the nucleotide sequence B and differs by no more than 3 nucleotides; the nucleotide sequence B is a nucleotide sequence that is completely reverse complementary to the first nucleotide sequence.

9. The conjugate according to claim 8, wherein The nucleotide sequence 1 differs from the first nucleotide sequence by no more than 1 nucleotide, and / or the nucleotide sequence 2 differs from the nucleotide sequence B by no more than 1 nucleotide.

10. The conjugate according to claim 8 or 9, wherein The nucleotide difference between the nucleotide sequence 2 and the nucleotide sequence B includes the difference in the Z' position of the first nucleotide in the nucleotide sequence 2 in the direction from the 5' end to the 3' end.

11. The conjugate according to claim 10, wherein In the direction from the 5' end to the 3' end, the last nucleotide Z in the nucleotide sequence 1 is a nucleotide complementary to Z'.

12. The conjugate according to claim 8, wherein The nucleotide sequence 1 and the nucleotide sequence 2 are substantially reverse complementary, substantially completely reverse complementary, or completely reverse complementary.

13. The conjugate according to claim 8, wherein The positive sense chain further contains a nucleotide sequence 3, and the antisense chain further contains a nucleotide sequence 4; the nucleotide sequence 3 and the nucleotide sequence 4 are equal in length and are both 1-4 nucleotides; the nucleotide sequence 3 is connected to the 5' end of the nucleotide sequence 1, and the nucleotide sequence 4 is connected to the 3' end of the nucleotide sequence 2; the nucleotide sequence 4 is complementary to the second segment of nucleotide sequence; the second segment of nucleotide sequence refers to a nucleotide sequence in the target mRNA that is adjacent to the first segment of nucleotide sequence and has the same length as the nucleotide sequence 4; and the nucleotide sequence 3 and the nucleotide sequence 4 are substantially completely reverse complementary or completely reverse complementary.

14. The conjugate according to claim 8 or 13, wherein The siRNA further contains a nucleotide sequence 5, which has a length of 1 to 3 nucleotides and is connected to the 3' end of the antisense strand, thereby forming a 3' overhang of the antisense strand.

15. The conjugate according to claim 14, wherein The nucleotide sequence 5 is 2 nucleotides in length, and in the 5' to 3' direction, the nucleotide sequence 5 is 2 consecutive deoxythymidine nucleotides, 2 consecutive uracil nucleotides, or complementary to the third nucleotide sequence; the third nucleotide sequence refers to a nucleotide sequence in the target mRNA that is adjacent to the first nucleotide sequence or the second nucleotide sequence and has a length equal to that of the nucleotide sequence 5.

16. The conjugate according to claim 8, wherein 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, wherein the phosphate group having a modified group is a thiophosphate group having a structure represented by formula (201):

17. The conjugate according to claim 16, wherein Each nucleotide in the sense strand and the antisense strand is independently a fluorinated modified nucleotide or a non-fluorinated modified nucleotide, wherein the fluorinated modified nucleotide refers to a nucleotide in which the hydroxyl group at the 2'-ribose group of the nucleotide is replaced by fluorine; the non-fluorinated modified nucleotide refers to a nucleotide or a nucleotide analog in which the hydroxyl group at the 2'-ribose group of the nucleotide is replaced by a non-fluorinated group, wherein the nucleotide analog is a group that can replace a nucleotide on a nucleic acid and has a structure different from any one of adenine nucleotide, guanine nucleotide, cytosine nucleotide, uracil nucleotide or thymine nucleotide; wherein the nucleotide in which the hydroxyl group at the 2'-ribose group of the nucleotide is replaced by a non-fluorinated group is selected from the group consisting of: a 2'-methoxy-modified nucleotide, a 2'-O-methoxyethyl-modified nucleotide and a 2'-deoxynucleotide (DNA); and the nucleotide analog is selected from the compounds represented by formula (217), formula (218), LNA, ENA, cET, UNA and GNA: Wherein, Base represents a base selected from A, U, G, C or T; R is H, OH, F, methoxy or 2'-O-methoxyethyl.

18. The conjugate according to claim 16, wherein In the siRNA, the phosphorothioate linkage is present in at least one of the following: between the first and second nucleotides at the 5' end of the sense strand; between the second and third nucleotides at the 5' end of the sense strand; between the first and second nucleotides at the 3' end of the sense strand; between the second and third nucleotides at the 3' end of the sense strand; between the first and second nucleotides at the 5' end of the antisense strand; between the second and third nucleotides at the 5' end of the antisense strand; between the first and second nucleotides at the 3' end of the antisense strand; and between the second and third nucleotides at the 3' end of the antisense strand.

19. The conjugate according to claim 16, wherein The 5'-terminal nucleotide of the antisense strand is a 5'-phosphate nucleotide or a 5'-phosphate analog-modified nucleotide.

20. The conjugate according to claim 19, wherein The 5'-phosphate nucleotide or 5'-phosphate analogue modified nucleotide is a nucleotide represented by one of formulas (202) to (206): Here, R represents a group selected from the group consisting of H, OH, F and methoxy, and Base represents a base selected from A, U, C, G and T.

21. Use of the conjugate according to any one of claims 1 to 20 in the preparation of a medicament for treating and / or preventing a pathological condition or disease caused by the expression of a specific gene in hepatocytes, wherein the specific gene is an endogenous gene expressed in the liver or a gene of a pathogen that propagates in the liver.

22. The use according to claim 21, wherein The specific gene is selected from the group consisting of: hepatitis B virus gene, angiopoietin-like protein 3 gene and apolipoprotein C3 gene.

23. The use according to claim 22, wherein The disease is selected from the group consisting of chronic liver disease, hepatitis, liver fibrosis, liver proliferative disease and dyslipidemia.

24. A kit comprising the conjugate of any one of claims 1-20.

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