Liver targeting compound, conjugate thereof, preparation method and application

Abstract

The invention provides a compound capable of forming a conjugate with an active molecule such as oligonucleotide. The compound has a structure as shown in formula (I). The invention further provides application of the corresponding conjugate or the pharmaceutically acceptable salt of the conjugate to regulation and control of gene expression in hepatocytes, preparation of the conjugate or the pharmaceutically acceptable salt of the conjugate and application of the conjugate or the pharmaceutically acceptable salt of the conjugate to drugs for preventing and / or treating related diseases. The conjugate or the pharmaceutically acceptable salt thereof disclosed by the invention can specifically target hepatocytes, so that the in-vivo delivery difficulty of oligonucleotide drugs is effectively solved. The compound not only has excellent delivery efficiency, but also has the advantages of low toxicity and simple preparation method.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical chemistry, and in particular relates to a liver-targeted compound and a conjugate thereof, a preparation method and use thereof. Background Art

[0002] Oligonucleotides and their analogs have been shown to be effective methods for treating many different types of diseases. Oligonucleotides and their analogs include small interfering RNA (siRNA), antisense oligonucleotides (ASO) and microRNA (miRNA), which mainly degrade specific messenger RNA (mRNA) through complementary pairing, thereby downregulating the expression of target genes. However, how to effectively deliver oligonucleotides to specific tissues in patients remains a challenge. The asialoglycoprotein receptor (ASGP-R) is an endocytic receptor highly expressed on the surface of hepatocytes. This receptor is hardly found in extrahepatic cells and is an ideal approach for hepatocyte-targeted therapy. The mammalian asialoglycoprotein receptor consists of two homologous subtypes, H1 and H2, of which the trimer consisting of two H1s and one H2 is the most common of all identified ASGP-R structures. The literature reports that many ligands can deliver oligonucleotides to the liver by binding to the asialoglycoprotein receptor (ASGP-R). Among them, N-acetylgalactosamine (GalNAc) showed the highest affinity, and the corresponding GalNAc-siRNA conjugates have been used by Alnylam pharmaceuticals, Inc. in drugs such as Givosiran, Lumasiran and Inclisiran. Summary of the invention

[0003] The purpose of the present invention is to provide a compound that can specifically target liver cells and help deliver oligonucleotide drugs in vivo.

[0004] The first aspect of the present invention provides a conjugate or a pharmaceutically acceptable salt thereof, wherein the conjugate contains a liver cell-targeting structure represented by the following formula II, an isomer or an isotopic variant thereof:

[0005]

[0006] In the formula, two monovalent groups among R1', R2' and R3' independently contain a ligand having affinity for the asialoglycoprotein receptor on the surface of mammalian liver cells, and the remaining one is a divalent group containing a structure for covalently linking to a nucleic acid molecule; wherein:

[0007] The monovalent groups are each independently selected from the following structures: -L1-X1-L1'-E, -CH(L2-X2-L2'-E)(X3-L3-E), -L4-X4-CH(X4-L4'-E)(L4'-X4-L4"-E), and -L5-X5-CH(X5-L5'-E)(L5'-X5-L5"-E);

[0008] The divalent group is -L6-X6-L7-X7-L8'-R4'-;

[0009] L1, L1', L2, L2', L3, L4, L4', L4", L5, L5' and L5" are each independently C1-C 20 Alkylene;

[0010] X1, X2, X3, X4 and X5 are each independently selected from -CO-, -NH-, -CO-NH-, -NH-CO-, -CH=N-, -S(O)2-, C2-C 10 Alkenylene, C2~C 10 Alkynylidene, C6~C 14 Arylene, C3~C 18 Heterocyclylene and C5~C 10 heteroarylene;

[0011] Each E is independently the ligand having affinity for the asialoglycoprotein receptor on the surface of mammalian liver cells;

[0012] L6 is C1-C 20 Alkylene;

[0013] X6 is selected from -CO-, -NH-, -CO-NH-, -NH-CO-, -CH=N-, -S(O)2-, C2~C 10 Alkenylene, C2~C 10 Alkynylidene, C6~C 14 Arylene, C3~C 18 Heterocyclylene and C5~C 10 heteroarylene;

[0014] L7 is a bond, C1~C 20 Alkylene or carbonyl C1~C 20 Alkylene;

[0015] X7 is a bond, -CO-, -NH-, -CO-NH-, -NH-CO-, -CH=N-, -S(O)2-, C2~C 10 Alkenylene, C2~C 10 Alkynylidene, C6~C 14 Arylene, C3~C 18 Heterocyclylene or C5~C10 heteroarylene;

[0016] L8' is a bond or an optionally substituted C1-C 20 Alkylenecarbonyl;

[0017] R4' is hydroxy, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl or optionally substituted heterocyclic, and at least one of L8' and R4' contains an -O- group for covalently linking to the phosphate group of the nucleic acid molecule via this group.

[0018] A second aspect of the present invention provides a pharmaceutical composition, which contains the conjugate described in any embodiment herein or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0019] The third aspect of the present invention provides a compound described in the following formula I, an isomer or isotopic variant thereof:

[0020]

[0021] In the formula, two of R1, R2 and R3 are groups containing ligands having affinity for asialoglycoprotein receptors on the surface of mammalian liver cells, and the remaining one is a group containing a structure for covalently linking to an oligonucleotide; wherein:

[0022] The groups containing ligands having affinity for asialoglycoprotein receptors on the surface of mammalian liver cells have structures independently selected from the following groups: -L1-X1-L1'-E, -CH(L2-X2-L2'-E)(X3-L3-E), -L4-X4-CH(X4-L4'-E)(L4'-X4-L4"-E) and -L5-X5-CH(X5-L5'-E)(L5'-X5-L5"-E);

[0023] The group comprising a structure for covalently linking to an oligonucleotide is -L6-X6-L7-X7-L8-R4;

[0024] The L1, L1', L2, L2', L3, L4, L4', L4", L5, L5' and L5" are each independently C1-C 20 Alkylene;

[0025] The X1, X2, X3, X4 and X5 are each independently selected from -CO-, -NH-, -CO-NH-, -NH-CO-, -CH=N-, -S(O)2-, C2-C 10 Alkenylene, C2~C 10 Alkynylidene, C6~C 14 Arylene, C3~C 18 Heterocyclylene and C5~C10 Heteroarylene, each E is independently a ligand having affinity for the asialoglycoprotein receptor on the surface of mammalian liver cells;

[0026] The L6 is C1-C 20 Alkylene;

[0027] The X6 is selected from -CO-, -NH-, -CO-NH-, -NH-CO-, -CH=N-, -S(O)2-, C2~C 10 Alkenylene, C2~C 10 Alkynylidene, C6~C 14 Arylene, C3~C 18 Heterocyclylene and C5~C 10 heteroarylene;

[0028] The L7 is a bond, C1~C 20 Alkylene or carbonyl C1~C 20 Alkylene;

[0029] The X7 is a bond, -CO-, -NH-, -CO-NH-, -NH-CO-, -CH=N-, -S(O)2-, C2~C 10 Alkenylene, C2~C 10 Alkynylidene, C6~C 14 Arylene, C3~C 18 Heterocyclylene or C5~C 10 heteroarylene;

[0030] The L8 is a bond or an optionally substituted C1-C 20 Alkylenecarbonyl;

[0031] The R4 is hydroxy, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl or optionally substituted heterocyclyl.

[0032] A fourth aspect of the present invention provides an application selected from the following:

[0033] (1) Use of the structure of formula (II), its isomers or isotopic variants, or the compound represented by formula (I), its isomers or isotopic variants, as described in any embodiment herein, in the preparation of a conjugate targeting hepatocytes;

[0034] (2) Use of the conjugate or a pharmaceutically acceptable salt thereof according to any embodiment of the present invention in the preparation of a drug, especially a drug for inhibiting the expression of a target nucleic acid; and

[0035] (3) Use of the conjugate or a pharmaceutically acceptable salt thereof according to any embodiment of the present invention in the preparation of a medicament for treating or preventing a TTR-mediated disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 It is the inhibition rate of TTR gene mRNA in the experimental group, vehicle control group and positive control group.

[0037] Figure 2 is the inhibition rate of TTR protein in the experimental group, vehicle control group and positive control group. DETAILED DESCRIPTION

[0038] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a preferred technical solution.

[0039] the term

[0040] As used herein, "alkyl" refers to a linear or branched monovalent saturated hydrocarbon group. The alkyl group may have 1 to 20 carbon atoms. In some embodiments, the alkyl group may have 1 to 12 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbon atoms, 1 to 4 carbon atoms, or 1 to 3 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, and n-hexyl.

[0041] As used herein, "alkylene" refers to a divalent alkyl group, such as C1-C 20 Alkylene, C1-C 12 Alkylene, C1-C8 alkylene, C1-C6 alkylene, C1-C4 alkylene, etc. Examples of the alkylene group include methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), butylene (-CH2CH2CH2CH2-), hexylene, heptylene, etc.

[0042] Herein, "alkenyl" refers to an unsaturated hydrocarbon group having one or more carbon-carbon double bonds, and the carbon chain length is usually 2 to 20 carbon atoms, such as 2 to 10, 2 to 8, 2 to 6 or 2 to 4 carbon atoms. The alkenyl group can be a chain alkenyl group or an alkenyl group having an unsaturated ring. The chain alkenyl group can be a straight chain or a branched chain.

[0043] Herein, "alkynyl" refers to an unsaturated hydrocarbon group having one or more carbon-carbon triple bonds, and its carbon chain length is usually 2 to 20 carbon atoms, such as 2 to 10, 2 to 8, 2 to 6 or 2 to 4 carbon atoms. Alkynyl groups can be straight or branched.

[0044] As used herein, "alkenylene" and "alkynylene" refer to divalent alkenyl and divalent alkynyl groups.

[0045] Herein, "amino" is represented by -NR'R", wherein R' and R" are each independently hydrogen or a C1-C6 alkyl group (preferably a C1-C4 alkyl group) which may be optionally substituted, such as a halogenated C1-C6 alkyl group. In some of the schemes, the amino group described herein is -NH2, C1-C6 alkyl-NH- or (C1-C6 alkyl)2N-.

[0046] Herein, "carbocyclic group" includes cycloalkyl and partially saturated carbocyclic groups. The number of ring carbon atoms is usually 3 to 8, such as 3 to 6. Exemplary cycloalkyl is C3 to C8 cycloalkyl, including cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl. Partially saturated carbocyclic groups include cycloalkenyl, such as C3 to C8 cycloalkenyl, for example cyclopentenyl, cycloheptenyl and cyclooctenyl, etc.

[0047] Herein, "aryl" refers to a monocyclic, bicyclic or tricyclic aromatic group containing 6 to 14 carbon atoms (C6-14). Preferred aryl groups are C6-10 aryl groups. Typical aryl groups include phenyl, naphthyl, phenanthrenyl, anthracenyl, indenyl, azulenyl, biphenyl, biphenylene and fluorenyl.

[0048] Herein, "arylene" refers to a divalent aromatic group, that is, two hydrogen atoms are missing from the aromatic ring to form a divalent group.

[0049] Herein, "heterocyclyl" refers to a saturated or partially saturated 3-7-membered monocyclic ring, or a 7-10-membered bicyclic ring system, consisting of carbon atoms and 1-4 heteroatoms selected from O, N and S. Preferably, the number of ring atoms of the heterocyclyl is 4-10. The heterocyclyl can be a 5- to 8-membered heterocycloalkyl, i.e., one or more ring C atoms in the cycloalkyl are replaced by heteroatoms selected from N, O and S. Herein, examples of heterocyclyl include tetrahydrofuranyl, tetrahydropyranyl, pyranyl, piperidinyl, piperazinyl, oxetanyl, azetidinyl 1,4-diazepanyl, pyrrolidinyl, imidazolidinyl, imidazolinyl, dihydroindole, isoindole, quinuclidinyl, morpholinyl, isochromanyl, chromanyl, pyrazolidinyl, pyrazolinyl and tetrahydroisoquinolinyl, etc.

[0050] As used herein, "heterocyclylene" refers to a divalent heterocyclic group in which two hydrogen atoms on the ring are missing to form a divalent group.

[0051] Herein, "heteroaryl" refers to a group containing 5-14, preferably 5-10 ring atoms, and having 6, 10 or 14 π electrons shared in the ring system, wherein the ring atoms are carbon atoms and 1 to 3 heteroatoms selected from O, S and N. Exemplary heteroaryl groups include thienyl, benzisothiazolyl, benzothienyl, naphthiophenyl, thianthrenyl, furanyl, pyranyl, isobenzofuranyl, chromenyl, xanthrenyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, indolyl, indazolyl, purinyl, 4H-quinolizinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, quinazolinyl, cinnolinyl, pteridinyl, carbazolyl, β-carbolinyl, phenanthridinyl, acridinyl, peridinyl, phenanthrolinyl, Phenazinyl, isothiazolyl, phenothiazinyl, isoxazolyl, furazanyl, phenoxazinyl, 7-aminoisocoumarin, benzisoxazolyl such as 1,2-benzisoxazol-3-yl, benzimidazolyl, 2-hydroxyindolyl, thiadiazo, 2-oxobenzimidazolyl, imidazopyridazinyl, imidazopyridyl, imidazotriazine, triazolopyrimidine, triazolopyridyl, triazolopyrazinyl, triazolopyridazinyl, triazolotriazine, pyrazolopyrimidine, pyrazolotriazine, pyrrolopyrimidine, pyrrolopyridinyl, pyrrolopyrazinyl, pyrrolotriazine and triazolopyrazinyl, etc.

[0052] As used herein, "heteroarylene" refers to a divalent heteroaryl group in which two hydrogen atoms on the ring are missing to form a divalent group.

[0053] Herein, the term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0054] "Pharmaceutically acceptable acid addition salts" refer to salts formed with inorganic or organic acids that retain the biological effectiveness of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobromides, sulfates, nitrates, phosphates, and the like; organic acid salts include, but are not limited to, formates, acetates, 2,2-dichloroacetates, trifluoroacetates, propionates, caproates, caprylates, decanoates, undecylenates, glycolates, gluconates, lactates, sebacates, adipates, glutarates, malonates, oxalates, maleates, succinates, fumarates, tartrates, citrates, palmitates, stearates, oleates, cinnamates, laurates, malates, glutamate, pyroglutamate, aspartate, benzoates, methanesulfonates, benzenesulfonates, p-toluenesulfonates, alginate, ascorbate, salicylates, 4-aminosalicylates, naphthalene disulfonates, and the like. These salts may be prepared by methods known in the art.

[0055] "Pharmaceutically acceptable base addition salt" refers to a salt formed with an inorganic base or an organic base that can maintain the biological effectiveness of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, and the like. Preferred inorganic salts are ammonium salts, sodium salts, potassium salts, calcium salts, and magnesium salts. The salt derived from organic base includes but is not limited to the following salt: primary amines, secondary amines and tertiary amines, substituted amines, including natural substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins etc. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine. These salts can be prepared by methods known in the profession.

[0056] Herein, "pharmaceutical composition" refers to a preparation of a compound of the present invention and a medium generally accepted in the art for delivering a biologically active compound to a mammal (e.g., a human). The medium includes a pharmaceutically acceptable carrier. The purpose of the pharmaceutical composition is to promote administration of an organism, facilitate the absorption of the active ingredient, and thus exert biological activity.

[0057] As used herein, "pharmaceutically acceptable" refers to a substance (such as a carrier or diluent) that does not affect the biological activity or properties of the compounds of the present invention and is relatively non-toxic, i.e., the substance can be administered to a subject without causing adverse biological reactions or interacting in an adverse manner with any components contained in the composition.

[0058] As used herein, "pharmaceutically acceptable excipients" include, but are not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier approved by the relevant governmental regulatory authorities as acceptable for human or livestock use.

[0059] As used herein, the terms "prophylactic," "prevention," and "prevention" include reducing the likelihood of a disease or condition occurring or becoming worse in a patient.

[0060] As used herein, the term "treatment" and other similar synonyms include the following meanings:

[0061] (i) preventing a disease or condition from occurring in a mammal, particularly where such mammal is susceptible to the disease or condition but has not yet been diagnosed as having the disease or condition;

[0062] (ii) inhibiting a disease or condition, i.e. arresting its development;

[0063] (iii) alleviate the disease or condition, that is, cause regression of the disease or condition; or

[0064] (iv) alleviating the symptoms caused by the disease or condition.

[0065] As used herein, the terms "administering", "administering", "dosing", etc. refer to methods that can deliver a compound or composition to the desired site for biological action. These methods include, but are not limited to, oral routes, intraduodenal routes, parenteral injections (including intravenous, subcutaneous, intraperitoneal, intramuscular, intraarterial injection or infusion), topical administration, and rectal administration. Those skilled in the art are familiar with administration techniques that can be used for the compounds and methods described herein, such as those discussed in Goodman and Gilman, The Pharmacological Basis of Therapeutics, current ed.; Pergamon; and Remington's, Pharmaceutical Sciences (current edition), Mack Publishing Co., Easton, Pa. In preferred embodiments, the compounds and compositions discussed herein are administered orally.

[0066] It will also be appreciated by those skilled in the art that in the methods described herein, the functional groups of the intermediate compounds may need to be protected by appropriate protecting groups. Such functional groups include hydroxyl, amino, sulfhydryl and carboxylic acid. Suitable hydroxyl protecting groups include trialkylsilyl or diarylalkylsilyl (e.g. tert-butyldimethylsilyl, tert-butyldiphenylsilyl or trimethylsilyl), tetrahydropyranyl, benzyl, etc. Suitable amino, amidino and guanidino protecting groups include tert-butyloxycarbonyl, benzyloxycarbonyl, etc. Suitable sulfhydryl protecting groups include -C(O)-R" (wherein R" is alkyl, aryl or aralkyl), p-methoxybenzyl, trityl, etc. Suitable carboxyl protecting groups include alkyl, aryl or aralkyl esters.

[0067] Protecting groups can be introduced and removed according to standard techniques known to those skilled in the art and as described herein. The use of protecting groups is described in detail in Greene, TW and PGM Wuts, Protective Groups in OrganiSynthesis, (1999), 4th Ed., Wiley. The protecting group can also be a polymer resin.

[0068] Herein, "target cell" refers to a cell that is expressing a target nucleic acid. In some embodiments, the target cell can be in vivo or in vitro. In some embodiments, the target cell is a mammalian cell, such as a rodent cell, such as a mouse cell or a human cell, particularly a hepatocyte.

[0069] As used herein, "reduced expression" of a gene refers to a decrease in the amount of RNA transcript or protein encoded by the gene and / or a decrease in the amount of activity of the gene in a cell or subject, as compared to an appropriate reference cell or subject. For example, the act of treating a cell with a drug combination or a double-stranded oligonucleotide (e.g., an oligonucleotide having an antisense strand complementary to a TTR mRNA sequence) may result in a decrease in the amount of RNA transcript, protein, and / or enzyme activity (e.g., encoded by the TTR gene) as compared to a cell not treated with the drug combination or double-stranded oligonucleotide, respectively. Similarly, as used herein, "reduced expression" refers to an act that results in a decrease in the expression of a gene (e.g., a TTR gene).

[0070] Liver-targeted compounds

[0071] The present invention provides a compound containing a structure that specifically targets liver cells, and the compound has a structure shown in the following formula I:

[0072]

[0073] In the formula, two of R1, R2 and R3 are groups containing ligands having affinity for asialoglycoprotein receptors on the surface of mammalian liver cells, and the remaining one is a group containing a structure for covalently linking to an oligonucleotide;

[0074] The groups containing ligands having affinity for asialoglycoprotein receptors on the surface of mammalian liver cells are independently selected from the following structures: -L1-X1-L1'-E, -CH(L2-X2-L2'-E)(X3-L3-E), -L4-X4-CH(X4-L4'-E)(L4'-X4-L4"-E), -L5-X5-CH(X5-L5'-E)(L5'-X5-L5"-E); wherein L1, L1', L2, L2', L3, L4, L4', L4", L5, L5' and L5" are independently C1 to C 20 Alkylene, X1, X2, X3, X4 and X5 are each independently selected from -CO-, -NH-, -CO-NH-, -NH-CO-, -CH=N-, -S(O)2-, C2~C 10 Alkenylene, C2~C 10 Alkynylidene, C6~C 14 Arylene, C3~C 18 Heterocyclylene and C5~C 10Heteroarylene, each E is independently a ligand having affinity for the asialoglycoprotein receptor (ASGPR) on the surface of mammalian liver cells;

[0075] The group containing a structure for covalently linking to an oligonucleotide is -L6-X6-L7-X7-L8-R4, wherein L6 is C1-C 20 Alkylene, X6 is selected from -CO-, -NH-, -CO-NH-, -NH-CO-, -CH=N-, -S(O)2-, C2~C 10 Alkenylene, C2~C 10 Alkynylidene, C6~C 14 Arylene, C3~C 18 Heterocyclylene and C5~C 10 Heteroarylene, L7 is a bond, C1~C 20 Alkylene or carbonyl C1~C 20 Alkylene, X7 is a bond, -CO-, -NH-, -CO-NH-, -NH-CO-, -CH=N-, -S(O)2-, C2~C 10 Alkenylene, C2~C 10 Alkynylidene, C6~C 14 Arylene, C3~C 18 Heterocyclylene or C5~C 10 Heteroarylene, L8 is a bond or an optionally substituted C1-C 20 Alkylenecarbonyl, R4 is hydroxy, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl or optionally substituted heterocyclyl.

[0076] Herein, E can be selected from a monosaccharide group, a monosaccharide derivative, a polysaccharide group or a polysaccharide derivative. Exemplary E is selected from mannosyl, L-mannopyranosyl, L-galactosyl, D-galactosyl, alpha-D-galactopyranosyl, beta-D-galactopyranosyl, alpha-D-galactofuranosyl, beta-D-galactofuranosyl, D-arabinosyl, D-glucosyl, L-glucosyl, alpha-D-glucopyranosyl, beta-D-glucopyranosyl, alpha-D-glucopyranosyl, beta-D-glucopyranosyl, D-glucofuranosyl, fructosyl, alpha-D-fructofuranosyl, alpha-D-fructopyranosyl, xylosyl, L-xylofuranosyl, D-xylofuranosyl, alpha-D-mannofuranosyl, beta-D-mannofuranosyl, alpha-D-mannopyranosyl, beta-D-mannopyranosyl, glucosamine, sialyl, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactose amine, N-butyrylgalactosamine, N-isobutyrylgalactosamine, 2-amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-beta-D-glucopyranosyl, 2-deoxy-2-methylamino-L-glucopyranosyl, 2-deoxy-2-sulfoamino-D-glucopyranosyl, 4,6-dideoxy-4-carboxamido-2,3-di-O-methyl-D-mannopyranosyl, N-acetylneuraminic acid, 5-thio-beta-D-pyranosyl pyranosyl, 2,3,4-tri-O-acetyl-1-thio-6-O-trityl-alpha-D-glucopyranosyl, 4-thio-beta-D-galactopyranosyl, 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-alpha-D-glucopyranosyl, 2,5-anhydro-D-allosenitrile, D-ribosyl, D-4-thioribosyl, L-ribosyl or L-4-thioribosyl.

[0077] Preferably, E is acetylgalactosamine.

[0078] In some embodiments, the active hydrogen-containing group of E may be protected by a protecting group. Preferably, the hydroxyl group of E is protected by a protecting group. In some embodiments, when E has both a hydroxyl group and an amino group, the hydroxyl group and the amino group are protected by a protecting group. Preferably, an acyl group (such as an acetyl group) is used to protect the amino group and the hydroxyl group of E. In some embodiments, E is an acetylgalactosamine group with a protected hydroxyl group.

[0079] Herein, E is usually covalently linked to the rest of Formula I via the oxygen on its hydroxyl group; preferably, E is covalently linked to the rest of Formula I via the oxygen on its 1-hydroxyl group. Preferably, E has the following structure:

[0080]

[0081] The wavy line indicates the position where E is attached to the rest of Formula I.

[0082] In some embodiments, R1 and R3 are the groups containing a ligand having affinity for the asialoglycoprotein receptor on the surface of mammalian liver cells, and R2 is the group containing a structure for covalently linking to an oligonucleotide.

[0083] In other embodiments, R1 and R2 are the groups containing a ligand having affinity for the asialoglycoprotein receptor on the surface of mammalian liver cells, and R3 is the group containing a structure for covalently linking to an oligonucleotide.

[0084] In some embodiments, the two groups containing a ligand having affinity for an asialoglycoprotein receptor on the surface of a mammalian liver cell are: -L1-X1-L1'-E and -CH(L2-X2-L2'-E)(X3-L3-E), -L1-X1-L1'-E and -L4-X4-CH(X4-L4'-E)(L4'-X4-L4"-E), -L1-X1-L1'-E and -L5-X5-CH(X5-L5'-E)(L5'-X5-L5"-E), -C H(L2-X2-L2'-E)(X3-L3-E) and -L4-X4-CH(X4-L4'-E)(L4'-X4-L4”-E), -CH(L2-X2-L2'-E)(X3-L3-E) and -L5-X5-CH (X5-L5'-E)(L5'-X5-L5"-E), or -L4-X4-CH(X4-L4'-E)(L4'-X4-L4"-E) and -L5-X5-CH(X5-L5'-E)(L5'-X5-L5"-E).

[0085] In some embodiments, R1 is -L1-X1-L1'-E, R3 is -CH(L2-X2-L2'-E)(X3-L3-E), -L4-X4-CH(X4-L4'-E)(L4'-X4-L4"-E), or -L5-X5-CH(X5-L5'-E)(L5'-X5-L5"-E).

[0086] In some embodiments, R1 and R2 are each independently -CH(L2-X2-L2'-E)(X3-L3-E), -L4-X4-CH(X4-L4'-E)(L4'-X4-L4"-E), or -L5-X5-CH(X5-L5'-E)(L5'-X5-L5"-E).

[0087] In some embodiments, L1, L1', L2, L2', L3, L4, L4', L4", L5, L5' and L5" are each independently C1-C 12 The alkylene groups are either independently C1-C8 alkylene groups, or independently C1-C6 alkylene groups, or independently C3-C8 alkylene groups, or independently C3-C6 alkylene groups.

[0088] In some embodiments, the arylene groups defined in X1, X2, X3, X4 and X5 include but are not limited to phenylene, naphthylene and biphenylene; the heterocyclic groups include divalent groups containing O and / or N heterocyclic groups, including but not limited to oxetanediyl, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, tetrahydropyranyl and morpholinyl, etc.; the heteroarylene groups include divalent groups containing nitrogen heteroaryl groups, such as imidazolylene, triazolyl, pyridinyl and pyrazinyl, etc.

[0089] In some embodiments, X1, X2, X3, X4 and X5 are each independently -CO-, -NH-, -CO-NH-, -NH-CO-, -CH=N- or -S(O)2-. In some embodiments, X1, X2, X3, X4 and X5 are each independently -CO-, -NH-, -CO-NH- or -NH-CO-. In some embodiments, X1, X2, X3, X4 and X5 are each independently -CO-, -NH-, -CO-NH- or -NH-CO-.

[0090] In some embodiments, the two groups containing a ligand having affinity for the asialoglycoprotein receptor on the surface of mammalian liver cells are: -L1-X1-L1'-E and -CH(L2-X2-L2'-E)(X3-L3-E); wherein L1, L1', L2, L2' and L3 are each independently a C3-C8 alkylene group, X1, X2 and X3 are each independently -CO-NH- or -NH-CO-, and E is the ligand having affinity for the asialoglycoprotein receptor (ASGPR) on the surface of mammalian liver cells, preferably an acetylgalactosamine group with protected hydroxyl groups.

[0091] In some embodiments, the two groups containing a ligand having affinity for the asialoglycoprotein receptor on the surface of mammalian liver cells are: -L4-X4-CH(X4-L4'-E)(L4'-X4-L4"-E) and -L5-X5-CH(X5-L5'-E)(L5'-X5-L5"-E); wherein L4, L4', L4", L5, L5' and L5" are each independently C3-C8 alkylene, X4 and X5 are each independently -CO-NH- or -NH-CO-, and E is the ligand having affinity for the asialoglycoprotein receptor (ASGPR) on the surface of mammalian liver cells, preferably an acetylgalactosamine group with protected hydroxyl groups.

[0092] In some embodiments, L6 is C1-C 12 The alkylene groups are either independently C1-C8 alkylene groups, or independently C1-C6 alkylene groups, or independently C3-C8 alkylene groups, or independently C3-C6 alkylene groups.

[0093] In some embodiments, the arylene group defined in X6 and X7 includes but is not limited to phenylene, naphthylene and biphenylene; the heterocyclic group includes a divalent group containing O and / or N heterocyclic groups, including but not limited to oxetane, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, tetrahydropyranyl and morpholinyl, etc.; the heteroaryl group includes a divalent group containing nitrogen heteroaryl, such as imidazolyl, triazolyl, pyridinyl and pyrazinyl, etc.

[0094] In some embodiments, X6 is -CO-, -NH-, -CO-NH-, -NH-CO-, -CH=N-, -S(O)2-, or C3~C 18 In some embodiments, X6 is -CO-, -NH-, -CO-NH-, -NH-CO-, or C4-C 10 In some embodiments, X6 is -CO-NH-, -NH-CO- or C4~C 10 Heterocyclylene. The heterocyclylene is preferably a divalent group of a nitrogen-containing heterocyclic group, such as a pyrrolidinylene, a piperazinylene or a piperidinylene; preferably, the heterocyclylene is covalently linked to other parts of the compound through the ring nitrogen atom it contains.

[0095] In some embodiments, L7 is a bond, C1-C 12 Alkylene or carbonyl C1~C 12 The alkylene group is preferably a bond, a C1-C8 alkylene group or a carbonyl C1-C8 alkylene group, and more preferably a bond, a C3-C8 alkylene group or a carbonyl C3-C8 alkylene group.

[0096] In some embodiments, X7 is each independently a bond, -CO-, -NH-, -CO-NH-, -NH-CO-, -CH=N- or -S(O)2-. In some embodiments, X7 is each independently a bond, -CO-, -NH-, -CO-NH- or -NH-CO-. In some embodiments, X7 is each independently a bond, -CO-, -NH-, -CO-NH- or -NH-CO-.

[0097] In some embodiments, L8 is a bond or an optionally substituted C1-C 12 The alkylenecarbonyl group is preferably a bond or an optionally substituted C1-C8 alkylenecarbonyl group, more preferably a bond or an optionally substituted C1-C6 alkylenecarbonyl group, more preferably a bond or an optionally substituted C1-C4 alkylenecarbonyl group. In some embodiments, the alkylene group is substituted with a hydroxyl group, and the hydroxyl group is protected by a protecting group.

[0098] In some embodiments, R4 is hydroxy, optionally substituted C3-C8 cycloalkyl, optionally substituted C6-C 14 Aryl, optionally substituted C5~C 10 Heteroaryl or optionally substituted C4~C 10 Heterocyclic group. The cycloalkyl group is preferably a cycloheptyl or cyclohexyl group; the aryl group is preferably a phenyl group; the heteroaryl group is preferably a nitrogen-containing heteroaryl group, including but not limited to imidazolyl, pyrazolyl, triazolyl, pyridyl, pyrazinyl, indolyl, etc.; the heterocyclic group is preferably a nitrogen- and / or oxygen-containing heterocyclic group, such as azetidinyl, oxetanyl, pyrrolidinyl, piperazinyl, piperidinyl, tetrahydropyranyl, tetrahydrofuranyl, azepanyl and morpholinyl, etc. Preferably, the cycloalkyl, aryl, heterocyclic group and heteroaryl group may be optionally substituted by 1 to 3 substituents selected from carboxyl (-COOH), C1 to C8 alkyl, hydroxyl, hydroxyl-substituted C1 to C8 alkyl and carboxyl and / or amino-substituted C1 to C8 alkyl. The hydroxyl group present in R4, including the hydroxyl group as R4 or the hydroxyl group as a substituent, may be optionally protected by a protecting group. Preferably, the heteroaryl and heterocyclic group are covalently linked to L8 through their ring nitrogen atoms.

[0099] In some embodiments, the group comprising a structure for covalent attachment to an oligonucleotide is:

[0100] -L6-X6-L7-R4, wherein L6 is C1-C6 alkylene, X6 is -CO-NH- or -NH-CO-, L7 is C3-C8 alkylene, and R4 is a 5-10 membered nitrogen-containing heteroaryl group optionally substituted with 1 or 2 hydroxyl groups and substituted with a C1-C8 alkyl group; the hydroxyl group is optionally protected by a protecting group;

[0101] -L6-X6-L7-X7-L8-R4, wherein L6, L7 and L8 are each independently a C1-C6 alkyl group, X6 and X7 are each independently -CO-NH- or -NH-CO-, and R4 is a 5-10 membered nitrogen-containing heteroaryl group optionally substituted with a C1-C8 alkyl group which is optionally substituted with 1 or 2 hydroxyl groups; the hydroxyl group is optionally protected by a protecting group;

[0102] -L6-X6-L7-X7-R4, wherein L6 is C1-C6 alkylene, X6 is -CO-NH- or -NH-CO-, L7 is C3-C8 alkylene, X7 is -CO-, R4 is a 4-10 membered nitrogen-containing heterocyclic group optionally substituted by 1 or 2 substituents selected from hydroxyl and C1-C8 alkyl substituted by hydroxyl, and the hydroxyl group is optionally protected by a protecting group;

[0103] -L6-X6-L7-X7-L8-R4, wherein L6 is C1-C6 alkylene, X6 and X7 are each independently -CO-NH- or -NH-CO-, L7 is C3-C8 alkylene, L8 is a C1-C4 alkylene carbonyl group optionally substituted with 1 to 3 hydroxyl groups, and R4 is a 4-10 membered nitrogen-containing heterocyclic group optionally substituted with 1 or 2 hydroxyl groups, and the hydroxyl groups are optionally protected by a protecting group;

[0104] -L6-X6-R4, wherein L6 is a C1-C6 alkylene group, X6 is -CO-, and R4 is a hydroxyl group; the hydroxyl group is optionally protected by a protecting group;

[0105] -L6-X6-L7-X7-L8-R4, wherein L6 is a C1-C6 alkylene group; X6 is the C3-C 18 The heterocyclic group is preferably a divalent group containing a nitrogen-containing heterocyclic group, more preferably selected from a pyrrolidinyl group, a piperazinyl group or a piperidinyl group, preferably covalently linked to L6 through its nitrogen-containing atom; L7 is a carbonyl group C3-C 12 Alkylene; X7 is -CO-, -CO-NH- or -NH-CO-; L8 is a bond, or is a C1-C4 alkylene carbonyl group optionally substituted by 1 to 3 hydroxyl groups; R4 is a 4-10 membered nitrogen-containing heterocyclic group optionally substituted by 1 or 2 substituents selected from hydroxyl and C1-C8 alkyl substituted by hydroxyl; wherein the hydroxyl group is optionally protected by a protecting group.

[0106] In some embodiments, the compound of Formula I described herein has the structure shown below:

[0107]

[0108] wherein L4, L4', L4", L5, L5', L5", L6, L7, L8, X4, X5, X6 and E are as described in any of the preceding embodiments; preferably:

[0109] L4, L4', L4", L5, L5' and L5" are each independently C1-C 12 Alkylene, preferably C3-C8 alkylene;

[0110] X4 and X5 are each independently -CO-, -NH-, -CO-NH-, -NH-CO-, -CH=N- or -S(O)2-; preferably, X4 and X5 are each independently -CO-, -NH-, -CO-NH- or -NH-CO-; more preferably, X4 and X5 are each independently -CO-NH- or -NH-CO-;

[0111] E is the ligand having affinity for the asialoglycoprotein receptor (ASGPR) on the surface of mammalian liver cells, preferably an acetylgalactosamine group with a protected hydroxyl group, more preferably:

[0112]

[0113] L6 is a C1-C6 alkylene group;

[0114] X6 is C3~C 18 a heterocyclylene group, preferably a divalent group of a nitrogen-containing heterocyclyl group, more preferably selected from a pyrrolidinylene group, a piperazinylene group or a piperidinylene group, preferably covalently linked to L6 via its nitrogen-containing atom;

[0115] L7 is a carbonyl group C3~C 12 Alkylene;

[0116] X7 is -CO-, -CO-NH- or -NH-CO-;

[0117] L8 is a bond, or a C1-C4 alkylene carbonyl group optionally substituted by 1-3 hydroxyl groups; the hydroxyl groups are optionally protected by a protecting group

[0118] R4 is a 4- to 10-membered nitrogen-containing heterocyclic group optionally substituted by 1 or 2 substituents selected from hydroxyl and C1-C8 alkyl substituted by hydroxyl; wherein the hydroxyl group is optionally protected by a protecting group.

[0119] In some embodiments, the -X7-L8-R4 is selected from the following groups:

[0120]

[0121] Wherein, the wavy line indicates the position where the group is connected to the rest of the compound, and R5 is a C1-C6 alkyl group optionally substituted with 1, 2 or 3 hydroxyl groups. In some embodiments, the hydroxyl group on the pyrrolidinyl group is located at the meta position, and the hydroxyl group on the piperidinyl group is located at the para position or the meta position. The hydroxyl group (including the hydroxyl group on the heterocyclic group and the hydroxyl group on the alkyl group) can be protected by a hydroxyl protecting group.

[0122] In some embodiments, the -X7-L8-R4 is selected from the following groups:

[0123]

[0124] Wherein, the wavy line indicates the position where the group is connected to the rest of the compound, and n is an integer from 1 to 6. In some embodiments, the hydroxyl group on the pyrrolidinyl group is located at the meta position, and the hydroxyl group on the piperidinyl group is located at the para position or the meta position. The hydroxyl group (including the hydroxyl group on the heterocyclic group and the hydroxyl group on the alkyl group) can be protected by a hydroxyl protecting group.

[0125] Herein, the protecting group of hydroxyl can be selected from C1-C6 alkyl, 4,4'-dimethoxytrityl, acetyl (AcO-), tert-butyldiphenylsilyl, trimethylsilyl or tert-butyldimethylsilyl. In some embodiments, the protecting group is selected from 4,4'-dimethoxytrityl (DMTr), acetyl (AcO-) and tert-butyldiphenylsilyl (TBDPS). In some embodiments, hydroxyl is protected by DMTr group.

[0126] In some embodiments, the compound of formula I is selected from:

[0127]

[0128]

[0129]

[0130]

[0131]

[0132] Also included herein are stereoisomers of the compounds of formula I, such as tautomers, geometric isomers, mesoforms, racemates, enantiomers, diastereomers, or mixtures thereof.

[0133] Conventional techniques for preparing / isolating isomers include chiral synthesis from suitable optically pure precursors, or resolution of racemates (or racemates of salts or derivatives) using, for example, chiral high performance liquid chromatography, see, for example, Gerald Gübitz and Martin G. Schmid (Eds.), Chiral Separations, Methods and Protocols, Methods in Molecular Biology, Vol. 243, 2004; AMStalcup, Chiral Separations, Annu. Rev. Anal. Chem. 3: 341-63, 2010; Fumiss et al. (eds.), VOGEL'S ENCYCLOPEDIA OF PRACTICAL ORGANIC CHEMISTRY 5. sup. TH ED., Longman Scientific and Technical Ltd., Essex, 1991, 809-816; Heller, Acc. Chem. Res. 1990, 23, 128.

[0134] Also included herein are all suitable isotopic variants of the compounds of formula I, their pharmaceutically acceptable salts or isomers. The isotopic variant is defined as a compound in which at least one atom in the compound of formula I, its pharmaceutically acceptable salts or isomers is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass commonly found in nature. Isotopes that can be incorporated into the compounds of the present invention, their pharmaceutically acceptable salts and isomers include, but are not limited to, isotopes of H, C, N and O, for example 2 H. 3 H. 11 C. 13 C. 14 C. 15 N. 17 O. 18 O. 35 S. 18 F. 36 Cl and 125 I. Isotopic variations of the compounds of the present invention or pharmaceutically acceptable salts thereof can be prepared by conventional techniques using appropriate isotopic variations of suitable reagents.

[0135] Liver targeting constructs

[0136] In some embodiments, provided herein is a structure that can target liver cells, which is shown in the following formula II:

[0137]

[0138] wherein two of R1', R2' and R3' are monovalent groups containing a ligand having affinity for an asialoglycoprotein receptor on the surface of mammalian liver cells, and the remaining one is a divalent group containing a structure for covalently linking to an oligonucleotide;

[0139] The monovalent groups containing the ligand having affinity for the asialoglycoprotein receptor on the surface of mammalian liver cells are each independently selected from the following structures: -L1-X1-L1'-E, -CH(L2-X2-L2'-E)(X3-L3-E), -L4-X4-CH(X4-L4'-E)(L4'-X4-L4"-E), -L5-X5-CH(X5-L5'-E)(L5'-X5-L5"-E); wherein L1, L1', L2, L2', L3, L4, L4', L4", L5, L5' and L5" are each independently C1 to C 20 Alkylene, X1, X2, X3, X4 and X5 are each independently selected from -CO-, -NH-, -CO-NH-, -NH-CO-, -CH=N-, -S(O)2-, C2~C 10 Alkenylene, C2~C 10 Alkynylidene, C6~C 14 Arylene, C3~C 18 Heterocyclylene and C5~C 10 Heteroarylene, each E is independently a ligand having affinity for the asialoglycoprotein receptor (ASGPR) on the surface of mammalian liver cells;

[0140] The divalent group containing a structure for covalently linking with an oligonucleotide is -L6-X6-L7-X7-L8'-R4'-, wherein L6 is C1-C 20 Alkylene, X6 is selected from -CO-, -NH-, -CO-NH-, -NH-CO-, -CH=N-, -S(O)2-, C2~C 10 Alkenylene, C2~C 10 Alkynylidene, C6~C 14 Arylene, C3~C 18 Heterocyclylene and C5~C 10 Heteroarylene, L7 is a bond, C1~C 20 Alkylene or carbonyl C1~C 20 Alkylene, X7 is a bond, -CO-, -NH-, -CO-NH-, -NH-CO-, -CH=N-, -S(O)2-, C2~C 10 Alkenylene, C2~C 10 Alkynylidene, C6~C 14 Arylene, C3~C 18 Heterocyclylene or C5~C10 Heteroarylene, L8' is a bond or an optionally substituted C1-C 20 Alkylenecarbonyl, R4' is hydroxyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl or optionally substituted heterocyclyl, and at least one of L8' and R4' contains an -O- group for covalently linking to the phosphate group of the oligonucleotide via this group.

[0141] Preferably, the above groups are as described in any of the above embodiments, and L8' and R4' correspond to L8 and R4 described above, respectively, and are divalent groups of L8 and R4, and the hydroxyl groups contained therein are changed to -O- groups. For example, in some embodiments, L8' is a C1-C 20 Alkylenecarbonyl, preferably -O-substituted C1-C 12 Alkylene carbonyl, more preferably -O-substituted C1-C8 alkylene carbonyl, more preferably -O-substituted C1-C6 alkylene carbonyl; R4' is a 5-10-membered nitrogen-containing heteroaryl group optionally substituted by 1 or 2 hydroxyl groups of C1-C8 alkyl, or a 4-10-membered nitrogen-containing heterocyclic group optionally substituted by 1 or 2 substituents selected from hydroxyl groups and hydroxyl-substituted C1-C8 alkyl, and the hydrogen of one hydroxyl group is removed to form an -O- group. The liver targeting structure can be covalently linked to the RNA molecule of interest through the -O- group on L8' or R4', preferably by forming a phosphate group.

[0142] In some embodiments, the structure of the liver targeting structure is shown in the structure numbered 1#, 2#, 3#, 4#, 5#, 6#, 7#, 8#, 9# or 10# in Table 1.

[0143] In some embodiments, the present invention also provides compounds containing liver targeting moieties described herein.

[0144] Also included herein are stereoisomers of the structure represented by Formula II, such as tautomers, geometric isomers, meso- and racemates, enantiomers, diastereomers, or mixtures thereof.

[0145] Also included herein are isotopic variants of the structure of Formula II. The isotopic variant is defined as a structure in which at least one atom in the structure of Formula II is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass commonly found in nature. Isotopes that may be incorporated into the structure include, but are not limited to, isotopes of H, C, N, and O, such as 2 H. 3 H. 11 C. 13 C. 14 C. 15 N. 17 O.18 O. 35 S. 18 F. 36 Cl and 125 I. Isotopic variations of the compounds of the present invention or pharmaceutically acceptable salts thereof can be prepared by conventional techniques using appropriate isotopic variations of suitable reagents.

[0146] Conjugate

[0147] The present invention provides a conjugate or a pharmaceutically acceptable salt thereof, comprising a liver targeting structure as described herein, an isomer or isotopic variant thereof and a nucleic acid molecule. The nucleic acid molecule is an RNA molecule. In some embodiments, the nucleic acid molecule is an oligonucleotide. The connection between the liver targeting structure and the biomolecule can be connected by a covalent bond well known in the art according to the type of the biomolecule. For example, the liver targeting structure and the nucleic acid molecule can be covalently connected through a phosphate group.

[0148] RNA molecules suitable for use herein can be various types of oligonucleotides and analogs thereof having therapeutic or prophylactic activity. Herein, the term "oligonucleotide" has a well-known meaning herein, generally referring to a molecule comprising two or more covalently linked nucleosides. The length of an oligonucleotide is generally less than 100 nucleotides. An oligonucleotide can be single-stranded or double-stranded. An oligonucleotide may or may not have a duplex region. Exemplary oligonucleotides include, but are not limited to, small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), dicer substrate interfering RNA (dsiRNA), antisense oligonucleotides, short siRNA or single-stranded siRNA. In some embodiments, a double-stranded oligonucleotide is an RNAi oligonucleotide. Oligonucleotides can be prepared using techniques well known in the art.

[0149] Herein, "RNAi oligonucleotide" refers to (a) a double-stranded oligonucleotide having a sense strand and an antisense strand, wherein the antisense strand or a portion of the antisense strand is used by Argonaute 2 (Ago2) endonuclease to cleave target mRNA, or (b) a single-stranded oligonucleotide having a single antisense strand, wherein the antisense strand or a portion of the antisense strand is used by Ago2 endonuclease to cleave target mRNA. RNAi oligonucleotides mediate targeted cleavage of RNA transcripts through the RNA-induced silencing complex (RISC) pathway.

[0150] Herein, "antisense strand" comprises a region that is completely, fully or substantially complementary to the target sequence, and "sense strand" comprises a region that is completely, fully or substantially complementary to the region of the antisense strand. The term "complementary region" refers to a region on the antisense strand that is completely, fully or substantially complementary to the target mRNA sequence. In the case where the complementary region is not completely complementary to the target sequence, the mismatch can be located in the interior or terminal region of the molecule. Typically, the most tolerant mismatch is located in the terminal region, for example, within 5, 4, 3, 2 or 1 nucleotides at the 5' and / or 3' ends. The antisense strand portion that is most sensitive to mismatch is referred to as a "seed region". The term "complementary" refers to the ability of a polynucleotide to hybridize with another polynucleotide under certain conditions, such as stringent conditions. For example, stringent conditions may include 400mM NaCl, 40mM PIPES pH 6.4, 1mM EDTA at 50°C or 70°C for 12-16 hours. The "complementary" sequence may also include or be completely formed from non-Watson-Crick base pairs and / or base pairs formed from non-natural and modified nucleotides. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble base pairing or Hoogstein base pairing.

[0151] Herein, siRNA refers to small interfering ribonucleic acid, which is a class of double-stranded RNA molecules, also referred to as short interfering RNA or silencing RNA. siRNA generally comprises a sense strand and an antisense strand, wherein the length of each strand is 17-30 nucleotides, typically 19-25 nucleotides, wherein the antisense strand is complementary (e.g., completely complementary) to the target nucleic acid (specifically a mature mRNA sequence), and the sense strand is complementary to the antisense strand, so that the sense strand and the antisense strand form a duplex or duplex region. The siRNA strand may form a flat-ended duplex, or the 3' ends of the sense and antisense strands may form a 3' overhang. In certain embodiments, both the sense strand and the antisense strand have a 3' overhang of 2 nucleotides. Once inside the cell, the antisense strand is incorporated into the RISC complex, which mediates target degradation or target inhibition of the target nucleic acid.

[0152] The siRNA may comprise modified nucleotides. In some embodiments, all nucleotides of the siRNA strand may be modified.

[0153] Herein, the modification in the modified nucleotide can be directed to the modification of base, sugar group and / or phosphate group. In some embodiments, the modified base can be selected from isocytosine, pseudoisocytosine, 5-methylcytosine, 5-thiazole-cytosine, 5-propynyl-cytosine, 5-propynyl-uracil, 5-bromouracil, 5-thiazole-uracil, 2-thio-uracil, 2'-thio-thymine, inosine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine and 2-chloro-6-aminopurine. Modification for phosphate group includes but is not limited to phosphorothioate (phosphorothioate, PS) modification. Modification for sugar group includes but is not limited to locked nucleic acid (locked nucleic acid, LNA) modification, 2' methoxy (2'OMe) modification, 2' ethoxy (2'OET) and 2' fluoro (2'F) modification.

[0154] Herein, the modified nucleotides can be selected from: deoxynucleotides, 3'-terminal deoxythymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, unlocked nucleotides, conformationally restricted nucleotides, constrained ethyl nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides, 2'-hydroxy modified nucleotides, 2'-methoxyethyl modified nucleotides, Modified nucleotides, 2'-O-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, non-natural bases containing nucleotides, unlinked nucleotides, tetrahydropyran modified nucleotides, 1,5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing thiophosphate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, nucleotides containing 5'-phosphate mimetics, ethylene glycol modified nucleotides and 2-O-(N-methylacetamide) modified nucleotides, and combinations thereof.

[0155] In some embodiments, the oligonucleotide comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.The phosphorothioate or methylphosphonate internucleotide linkage may be at the 3' terminus and / or at the 5' terminus of one or both strands.

[0156] In some embodiments, the nucleotide sequence of the antisense strand of the siRNA described herein is as shown in SEQ ID NO: 2, and the sense strand is complementary to the antisense strand. In some embodiments, the nucleotide sequence of the sense strand of the siRNA described herein is as shown in SEQ ID NO: 1.

[0157] In some embodiments, the modifications in the oligonucleotide sequences described herein include 2'-methoxy modification, 2'-fluorine modification, and phosphorothioate linkage between two adjacent nucleotides. In some embodiments, the nucleotide sequence of the antisense strand of the siRNA described herein is:

[0158] UmsUfsAmUfAmGfAmGfCmAfAmGmAmAfCmAfCmUfGmUfUmsUmsUm,

[0159] The nucleotide sequence of the sense strand is:

[0160] AfsAmsCfAmGfUmGfUmUfCfUmUfGfCmUfCmUfAmUfAmAf;

[0161] Among them, the lowercase letter m indicates that the nucleotide adjacent to the left of the letter m is a 2′-methoxy-modified nucleotide; the lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a 2′-fluorine-modified nucleotide; the 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.

[0162] Herein, "antisense oligonucleotide" is an oligonucleotide that can regulate the expression of a target gene by hybridizing with a target nucleic acid, particularly with an adjacent sequence on the target nucleic acid. Antisense oligonucleotides are not substantially double-stranded and are therefore not siRNA or shRNA. Preferably, antisense oligonucleotides do not include RNA nucleosides because this will reduce nuclease resistance. Antisense oligonucleotides may include one or more modified nucleosides or nucleotides, such as 2' sugar-modified nucleosides. Preferably, unmodified nucleosides are DNA nucleosides.

[0163] Herein, the target nucleic acid may be a target nucleic acid related to the occurrence and development of a disease well known in the art. The target nucleic acid may be a gene, RNA, mRNA or cDNA sequence.

[0164] An exemplary target nucleic acid is the TTR gene, which is a nucleic acid encoding a thyroid transporter protein, whose mutation causes hereditary transthyretin-mediated amyloidosis (hATTR amyloidosis). The disease is predominantly male (the male-to-female ratio is approximately 3:1), and diagnosis usually occurs around the age of 70. The main manifestations of hATTR amyloidosis are polyneuropathy and cardiomyopathy. More than 95% of TTR in the circulation comes from the liver. The main physiological role of TTR is to act as a carrier of retinol (also known as vitamin A), forming a binding protein (RBP): vitamin A complex with retinol. TTR also acts as a minor carrier of thyroxine (T4). Mutations in the TTR gene cause instability of the tetrameric protein, with TTR subunits dissociating into dimers and individual mutants and wild-type monomers, followed by misfolding. These misfolded TTR monomers can self-assemble into oligomers and form amyloid fibrils and plaques in the extracellular space of various tissues, including the peripheral nervous system, heart, gastrointestinal tract, kidney, central nervous system (CNS), and eye, leading to cellular damage and organ dysfunction with corresponding clinical manifestations.

[0165] For in vivo or in vitro applications, the oligonucleotides of the invention are generally capable of inhibiting the expression of a TTR target nucleic acid in a cell. The contiguous sequence of nucleobases of the oligonucleotides of the invention are generally complementary to the TTR target nucleic acid, as measured over the length of the entire oligonucleotide, optionally except for one or two mismatches, and optionally excluding nucleotide-based linkers that may link the oligonucleotide to optional functional groups, such as conjugates or other non-complementary terminal nucleotides (e.g., region D' or D").

[0166] Preparation of compounds of formula I

[0167] The compounds of Formula I described herein can be prepared by using the following exemplary reaction schemes and selecting appropriate reaction conditions with reference to the reactions disclosed in the Examples. The definitions of the various groups in the formula are as described above.

[0168]

[0169] The synthesis of the compounds in some embodiments can be carried out using commercially available 5-chloromethyl-2,4-dihydro[1,2,4]triazol-3-one as a starting material, first converting the chloro group into a piperazine or azide group, and then using sodium hydride as a base to alkylate the halogenated chain to obtain a trisubstituted triazolone. The trisubstituted triazolone first exposes the active group, condenses with the ligand E, and then connects with a linker to obtain the compound of formula I.

[0170] Preparation of conjugates

[0171] The conjugates herein can be synthesized by phosphoramidite solid phase synthesis methods well known in the art. According to the nucleotide types and sequence of the oligonucleotides in the conjugates, the nucleoside monomers are sequentially connected in the 3' to 5' direction, and the connection of each nucleoside monomer must go through an identical cycle, including four steps of deprotection, coupling, capping, oxidation or sulfurization.

[0172] In the phosphoramidite solid phase synthesis method, the liver targeting structure of the present invention can be connected to the CPG solid phase carrier, and the nucleoside monomers are sequentially connected from 3' to 5' according to the sequence of the oligonucleotide. Usually, the sense strand or antisense strand of the oligonucleotide is connected to the liver targeting structure.

[0173] In some specific embodiments, the acetonitrile solution of the nucleoside monomer is prepared, and the concentration thereof may be 0.01-0.10 mol / L.

[0174] In the deprotection step, 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 deprotection reagent solvent can be dissolved in an appropriate organic solvent. A suitable organic solvent is dichloromethane. In some embodiments, a dichloromethane solution of the deprotection reagent is used for deprotection, and the concentration of the deprotection reagent in the solution can be 1 to 5% (v / v). Typically, 1 to 5 deprotection treatments can be performed, and the reaction time for each treatment can be 20 to 40 seconds.

[0175] Herein, the coupling agent used in the coupling step can be selected from one or more of 1H-tetrazole, 5-ethylthio 1H-tetrazole and 5-benzylthio 1H-tetrazole. In some embodiments, the coupling agent is 5-ethylthio 1H-tetrazole. The coupling agent can be dissolved in a suitable organic solvent. In some embodiments, the organic solvent is acetonitrile. In some embodiments, an acetonitrile solution of the coupling agent is used for coupling, and the concentration of the coupling agent in the solution can be 0.1 to 0.5 mol / L. The coupling reaction can be carried out 1 to 5 times, and the reaction time of each time can be 1 to 5 minutes. The coupling reaction can be carried out in an organic solvent. Suitable organic solvents include, but are not limited to, one or more of anhydrous acetonitrile, anhydrous DMF and anhydrous dichloromethane. In some embodiments, the organic solvent is anhydrous acetonitrile.

[0176] Herein, the capping step can be performed in an acetic anhydride-acetonitrile solution and an N-methylimidazole / pyridine / acetonitrile solution. In the acetic anhydride-acetonitrile solution, the concentration of acetic anhydride can be 10 to 30% (v / v). In the N-methylimidazole / pyridine / acetonitrile solution, the volume concentration of N-methylimidazole can be 15 to 25%, the volume concentration of pyridine can be 25 to 35%, and the volume concentration of acetonitrile can be 45 to 55%. The number of capping can be 1 to 3 times.

[0177] Herein, the oxidation step can use a pyridine / water solution (90 / 10, v / v) of iodine at a concentration of 0.01 to 0.10 mol / L, and the thiolation step can use a 5-imino-1,2,4-dithiazolidine-3-thione / pyridine solution at a concentration of 0.10 to 0.30 mol / L. The thiolation can be performed 1 to 3 times.

[0178] After the last nucleoside is connected, aminolysis is performed. The conjugate can be separated from the solid phase support by aminolysis. In the aminolysis step, concentrated ammonia (e.g., a concentration of 25-28%) can be used to mix with the reaction product, and then reacted at a temperature of 60-80° C. for 1-8 hours, and filtered to obtain a filtrate containing the conjugate. The filtrate can be concentrated to obtain a crude product.

[0179] When there is at least one 2′-TBDMS protection on the synthesized nucleotide sequence, the 2′-TBDMS protection needs to be removed. The reagent used for removal can be triethylamine trihydrofluoride. In some embodiments, the filtrate or crude product is contacted with N-methylimidazole / triethylamine hydrofluoride / triethylamine solution and reacted at 60 to 70° C. for 1 to 4 hours. In the N-methylimidazole / triethylamine hydrofluoride / triethylamine solution, the volume proportions of N-methylimidazole, triethylamine hydrofluoride and triethylamine can be 55 to 65%, 25 to 35% and 35 to 45%, respectively.

[0180] After deprotection, purification and desalting can be performed. The purification and desalting methods known in the art can be used. For example, a preparative ion chromatography purification column can be used to elute with a gradient of NaBr or NaCl to purify the nucleic acid; after the products are collected and combined, a reverse phase chromatography purification column can be used for desalting. The fractions with a purity greater than 95% are collected and concentrated to dryness.

[0181] The above method can be used to prepare the sense chain and the antisense chain respectively, and then the sense chain and the antisense chain are mixed and annealed to prepare the conjugate of the present invention. Usually, the sense chain and the antisense chain are dissolved in PBS solution respectively, and then the PBS solutions of the two are mixed and annealed. The mixing molar ratio of the sense chain and the antisense chain can be 1 to 1.2:1, preferably 1.1 to 1.2:1. Annealing can be carried out at 83 to 87°C, and the annealing time can be 3 to 5 minutes. After the annealing is completed, it can be naturally cooled to room temperature and can be freeze-dried as needed.

[0182] Pharmaceutical composition

[0183] The present invention provides a pharmaceutical composition comprising (1) a conjugate of the present invention or a pharmaceutically acceptable salt thereof and (2) a pharmaceutically acceptable carrier.

[0184] As used herein, "pharmaceutical" or "pharmaceutically acceptable" means a substance that is not biologically or otherwise undesirable, for example, the substance can be incorporated into a pharmaceutical composition administered to a patient without causing any significant undesirable biological consequences or causing harmful interactions with any other components of the composition containing it. Pharmaceutical carriers preferably have met the necessary standards for toxicological testing and pharmaceutical testing and / or are included in the "Inert Ingredients Guide" compiled by the U.S. Food and Drug Administration.

[0185] The term "pharmaceutically acceptable carrier" used in the present application refers to an inert or inactive substance that can be used to produce a drug or medicament (such as a tablet containing a compound of the present invention as an active ingredient). The term carrier can include a variety of substances, including, but not limited to, any substance used as a binder, disintegrant, coating, compression / encapsulation aid, cream or lotion, lubricant, solution for parenteral administration, substance for chewable tablets, sweeteners or flavoring agents, suspending agents / gelling agents or wet granulation agents. Binders include, for example, carbomer, povidone, xanthan gum, etc.; coatings include, for example, cellulose acetate, ethylcellulose, gellangum, maltodextrin, enteric coatings, etc.; compression / encapsulation aids include, for example, calcium carbonate, dextrose, fructose dc (dc = "directly compressible"), honey dc, lactose (anhydrous or monohydrate; optionally in combination with aspartame, cellulose or microcrystalline cellulose), starch dc, sucrose, etc.; disintegrants include, for example, cross-linked carboxymethyl cellulose sodium, gellan gum, sodium starch glycolate, etc.; creams Or lotions include, for example, maltodextrin, carrageenan, etc.; lubricants include, for example, magnesium stearate, stearic acid, sodium stearyl fumarate, etc.; substances for chewable tablets include, for example, dextrose, fructose DC, lactose (monohydrate, optionally in combination with aspartame or cellulose), etc.; suspending agents / gelling agents include, for example, carrageenan, sodium starch glycolate, xanthan gum, etc.; sweeteners include, for example, aspartame, dextrose, fructose DC, sorbitol, sucrose DC, etc.; and wet granulating agents include, for example, calcium carbonate, maltodextrin, microcrystalline cellulose, etc.

[0186] Uses and methods

[0187] In some embodiments, provided herein is the use of a compound represented by formula (I) herein, an isomer or isotopic variant thereof in the preparation of a conjugate targeting hepatocytes. In some embodiments, provided herein is a conjugate as described in any embodiment herein targeting hepatocytes.

[0188] In some embodiments, provided herein is a liver targeting structure shown in formula (II) as described herein, an isomer or isotopic variant thereof for use in improving the serum stability of a conjugate and / or for use in improving the in vivo delivery efficiency of a conjugate, and a liver targeting structure shown in formula (II), an isomer or isotopic variant thereof for use in preparing a conjugate with improved serum stability and / or improved in vivo delivery efficiency. In some embodiments, provided herein is a liver targeting structure shown in formula (II) for use in improving the serum stability of a conjugate and / or for use in improving the in vivo delivery efficiency of a conjugate, an isomer or isotopic variant thereof.

[0189] In some embodiments, the present invention also provides the use of the conjugate described herein or a pharmaceutically acceptable salt thereof in the preparation of a drug for inhibiting the expression of a target nucleic acid. In some embodiments, the present invention also provides the use of the conjugate described herein or a pharmaceutically acceptable salt thereof in the preparation of a drug.

[0190] In some embodiments, the present invention also provides a method for inhibiting expression of a target nucleic acid, the method comprising contacting a conjugate described herein or a pharmaceutically acceptable salt thereof with a target nucleic acid or a cell or tissue containing the target nucleic acid. In some embodiments, the method comprises administering an effective amount of a conjugate described herein to an individual in need.

[0191] In some embodiments, also provided herein is a method for treating or preventing a disease, the method comprising administering to an individual in need thereof a therapeutically effective amount or a preventively effective amount of a conjugate described herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0192] Herein, the individual can be any individual of interest, such as a mammal. In some embodiments, the individual can be a human, a pet such as a dog and a cat, livestock such as a pig, a sheep and a cow, and the like.

[0193] The disease may be different depending on the nucleic acid molecule in the conjugate or its pharmaceutically acceptable salt. For example, in some embodiments, the nucleic acid molecule in the conjugate or its pharmaceutically acceptable salt is an siRNA that specifically inhibits the expression of the TTRmRNA gene. Therefore, the conjugate or its pharmaceutically acceptable salt can be used to prepare a drug for treating or preventing TTR-mediated diseases. In some embodiments, the TTR-mediated disease includes hATTR amyloidosis, including polyneuropathy and cardiomyopathy associated with hATTR amyloidosis, and misfolded TTR monomers in the extracellular space of tissues (including the peripheral nervous system, heart, gastrointestinal tract, kidneys, central nervous system and eyes) to form amyloid fibrils and plaques, resulting in cell damage and organ dysfunction. In some embodiments, the present invention also provides the use of the conjugate described herein in the preparation of a drug for treating or preventing TTR-mediated diseases.

[0194] As used herein, a "therapeutically effective amount" refers to an amount that is effective to achieve the desired therapeutic outcome (e.g., reduced hATTR amyloidosis, increased lifespan, or increased life expectancy) at the necessary dose and for the necessary period of time. The therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the subject, and the ability of the compound to elicit the desired response in the subject. The dosing regimen may be adjusted to provide the optimal therapeutic response. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the compound are exceeded by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount that is effective to achieve the desired preventive outcome (e.g., reduced hATTR amyloidosis, increased lifespan, increased life expectancy) at the necessary dose and for the necessary period of time. Typically, a prophylactic dose is used in a subject before or in the early stages of the disease, so that the prophylactic effective amount may be less than the therapeutically effective amount.

[0195] "Administration" as used herein refers to a method that can deliver a conjugate or its pharmaceutical composition to a desired site for biological action. Methods of administration known in the art can be used in the present invention. These methods include, but are not limited to, oral routes, intraduodenal routes, parenteral injections (including intrapulmonary, intranasal, intrathecal, intravenous, subcutaneous, intraperitoneal, intramuscular, intraarterial injection or infusion), topical administration, and rectal administration. Those skilled in the art are familiar with the administration techniques that can be used for the compounds and methods described herein, such as those discussed in Goodman and Gilman, The Pharmacological Basis of Therapeutics, current ed.; Pergamon; and Remington's, Pharmaceutical Sciences (current edition), Mack Publishing Co., Easton, Pa.

[0196] The liver-targeting structure of the present invention can significantly improve the serum stability of the conjugate, significantly reduce the expression level of the target nucleic acid in the target cells, and improve the in vivo delivery efficiency of the nucleic acid molecule of interest.

[0197] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0198] Preparation Example 1: Preparation of Compound L1

[0199] 1.1 Synthesis of TZ-1

[0200]

[0201] Boc protected lysine (80.0 g, 231.2 mmol) and potassium carbonate (47.9 g, 346.8 mmol, 1.5 equivalents) were added to anhydrous N, N-dimethylformamide (230 ml), and iodomethane (39.4 g, 277.4 mmol, 1.2 equivalents) was slowly added dropwise under stirring in an ice bath. After the addition was complete, the temperature was raised to room temperature and stirred overnight. After the reaction was completed by TLC detection, water was added to quench the reaction. The solvent N, N-dimethylformamide was removed by high vacuum and reduced pressure distillation, and the residue was dissolved in dichloromethane. The organic phase was washed once with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and the organic phase was concentrated to obtain a crude product. The crude product was recrystallized from petroleum ether / ethyl acetate (v / v, 3:1) to obtain the target product as a white solid with a mass of 79.0 g and a yield of 95%. The product was identified by LC-MS: MS m / z: C 17 H 32 N2O6, [M+1] + , theoretical: 361.23, measured: 361.18. 1 H NMR (500MHz, CDCl3) δ5.10 (brs, 1H), 4.59 (brs, 1H), 4.28–4.24 (m, 2H), 3.72 (s, 3H), 3. 10–3.06 (m, 2H), 2.01–1.82 (m, 2H), 1.55–1.52 (m, 2H), 1.42 (s, 18H), 1.27–1.23 (m, 2H).

[0202] 1.2 Synthesis of TZ-2

[0203]

[0204] TZ-1 (79.0 g, 220 mmol) was dissolved in methanol (220 ml), and hydrazine hydrate (50% wt, 220 mmol, 1.0 equivalent) was slowly added dropwise under stirring in an ice bath. The reaction was heated to room temperature and stirred overnight, and the methanol was removed by distillation under reduced pressure. The mixture was extracted with dichloromethane three times, and the combined organic phase was washed once with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product. The crude product was recrystallized from petroleum ether / ethyl acetate (2:1) to obtain the target product as a white solid with a mass of 69.5 g and a yield of 88%. The product was identified by LC-MS: MS m / z: C 16 H 32 N4O5, [M+1] + , theoretical: 361.24, measured: 361.18. 1H NMR (500MHz, DMSO) δ8.99 (s, 1H), 6.80–6.67 (m, 2H), 4.18 (s, 2H), 3.83 (td, J=8.4, 5.6Hz, 1H), 2.87 (qd, J=6.7, 3.6Hz, 2H), 1.47 (dddd, J=18.3, 13.5, 9.2, 6.0Hz, 2H), 1.37 (s, 18H), 1.28–1.11 (m, 4H).

[0205] 1.3 Synthesis of TZ-3

[0206]

[0207] Mono-Boc protected hexanediamine (9.7 g, 45 mmol) was dissolved in dichloromethane (200 ml), and saturated sodium bicarbonate aqueous solution (100 ml) and triphosgene (4.5 g, 15 mmol) were added in sequence under ice bath stirring. The reaction solution was stirred under ice bath for 1 hour, the organic phase was separated, the aqueous phase was extracted twice with dichloromethane, the combined organic phase was washed once with saturated brine, dried over anhydrous sodium sulfate, and filtered to obtain a dichloromethane solution of the product TZ-3, which could be directly used in the next step without purification.

[0208] 1.4 Synthesis of TZ-4

[0209]

[0210] Dissolve hydrazide TZ-2 (10.9 g, 30 mmol) in dichloromethane (100 ml), slowly add the isocyanate TZ-3 solution (45 mmol, 1.5 equivalents) obtained in the previous step under stirring in an ice bath for 1.5 hours. After the addition is complete, the reaction solution continues to stir for 15 minutes. After TLC detection, hydrazide TZ-2 is completely consumed, and after vacuum distillation, it is purified by silica gel flash column chromatography (dichloromethane / methanol = 100:1-20:1 gradient elution) to obtain the target product as a colorless transparent oil with a mass of 17.2 g and a yield of 95%. The product is identified by LC-MS: MS m / z: C 28 H 54 N6O8, [M+1] + , theoretical: 603.40, measured: 603.37.

[0211] 1.5 Synthesis of TZ-5

[0212]

[0213] TZ-4 (8.65 g, 14.4 mmol) was dissolved in a mixed solvent of ethanol / water (v / v, 1:1, 30 ml), and potassium hydroxide (0.81 g, 14.4 mmol, 1 equivalent) was added. The reaction solution was heated to 90 degrees in an oil bath and refluxed for 24 hours. After the reaction was cooled in an ice bath, concentrated hydrochloric acid was added dropwise to neutralize the reaction solution. After ethanol was removed by vacuum concentration, dichloromethane was added and extracted three times. The combined organic phase was washed once with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was recrystallized from acetonitrile to obtain the target product as a white solid with a mass of 7.6 g and a yield of 91%. The product was identified by LC-MS: MS m / z: C 28 H 52 N6O7, [M+1] + , theoretical: 585.39, measured: 585.41.

[0214] 1.6 Synthesis of TZ-6

[0215]

[0216] TZ-5 (1.5 g, 2.6 mmol) and benzyl 4-bromobutyrate (1.0 g, 3.9 mmol, 1.5 eq) were dissolved in anhydrous N,N-dimethylformamide (26 ml), and potassium carbonate (2.0 g, 14.8 mmol, 5.7 eq) was added. The reaction was heated to 60°C and stirred for 24 hours. After TLC detection of complete consumption of TZ-5, the reaction solution was cooled to room temperature, and the inorganic salts in the reaction solution were filtered to remove the inorganic salts. Dichloromethane was added to the filtrate, and the solution was washed once with water and saturated brine, then dried over anhydrous sodium sulfate, filtered, and concentrated by vacuum distillation. The residue was purified by silica gel flash column chromatography (dichloromethane / methanol = 50:1–20:1 gradient elution) to obtain the target product as a white solid with a mass of 2.05 g and a yield of 75%. The product was identified by LC-MS: MS m / z: C 39 H 64 N6O9, [M+1] + , theoretical: 761.47, measured: 761.06.

[0217] 1H NMR (500MHz, DMSO) δ7.40–7.20 (m, 6H), 6.72 (t, J=5.7Hz, 2H), 4.50 (td, J=8.8, 5.5Hz, 1H), 4.43 (s, 2H), 3.62 (t, J=7.0Hz, 2H), 3.54 (t, J=7.7Hz , 2H), 3.40 (t, J=6.5Hz, 2H), 2.95–2.83 (m, 4H), 1.85–1.65 (m, 2H), 1.65 –1.53(m, 2H), 1.53–1.45(m, 4H), 1.45–1.32(m, 27H), 1.27–1.18(m, 8H); 13 C NMR (126MHz, DMSO) δ155.5, 155.3, 153.3, 146.1, 138.7, 128.1, 127.3, 127.2, 78.2, 77.2, 77.2, 71.8, 69.5, 46.0, 44.2, 40.8, 39.6, 31.2, 29.4, 29.1, 28.6, 28.2, 28.1, 28.1, 26.0, 25.9, 25.7, 25.2, 22.6.

[0218] 1.7 Synthesis of TZ-7

[0219]

[0220] The intermediate TZ-6 (1.2 g, 1.6 mmol) was dissolved in anhydrous dichloromethane (16 ml), and the reaction system was placed in a nitrogen atmosphere by vacuum pump replacement. Trimethylsilyl trifluoromethanesulfonate (1.3 ml, 5.4 mmol, 3.3 equivalents) was slowly added dropwise under ice bath stirring. After the addition was completed, the temperature was raised to room temperature and the reaction was stirred overnight. After TLC detection, the raw material TZ-6 was completely consumed, and the solvent was removed by vacuum concentration to obtain a brown oil, which could be directly used in the next step without purification. The product was identified by LC-MS: MSm / z: C 24 H 40 N6O3, [M+1] + , theoretical: 461.32, measured: 461.25.

[0221] 1.8 Synthesis of TZ-8

[0222]

[0223] Gal-5 (purchased from Suzhou Ruiting Chemical Co., Ltd., 2.00 g, 4.47 mmol), benzotriazole-N, N, N', N'-tetramethyluronium hexafluorophosphate (HBTU, 1.88 g, 4.96 mmol, 1.1 equivalents) and 1-hydroxybenzotriazole (HOBt, 0.67 g, 4.96 mmol, 1.1 equivalents) were dissolved in anhydrous N, N-dimethylformamide (25 ml) in sequence, and the reaction system was placed in a nitrogen atmosphere by vacuum pump replacement, and N, N-diisopropylethylamine (DIPEA, 2.17 ml, 1.25 mmol) was added dropwise under stirring in an ice bath. After the reaction was heated to room temperature and stirred for 0.5 hours, TZ-7 was dissolved in DMF (5 ml) and added dropwise to the reaction solution, and stirred at room temperature overnight. After TLC detection, the reaction was completed, and the solvent N,N-dimethylformamide was removed by high vacuum pump concentration, and dichloromethane was added to dissolve the residue, and then washed once with saturated sodium bicarbonate and saturated brine, respectively, and then dried over anhydrous sodium sulfate, filtered, and concentrated by vacuum distillation. The residue was purified by silica gel flash column chromatography (dichloromethane / methanol = 50:1-20:1 gradient elution) to obtain the target product as a white foamy solid with a mass of 2.04 g and a yield of 73%. The product was identified by LC-MS: MS m / z: C 81 H 121 N9O 33 , [M+1] + , theoretical: 1748.81, measured: 1748.79.

[0224] 1.9 Synthesis of TZ-9

[0225]

[0226] TZ-8 (1.00 g, 0.57 mmol) was dissolved in anhydrous methanol (10 ml), and palladium carbon (100 mg, 10% palladium loading) was added. The reaction system was placed in a hydrogen atmosphere by vacuum pump replacement and stirred at room temperature overnight. The palladium carbon was removed by diatomaceous earth filtration, and the filtrate was concentrated to obtain the target product TZ-9 (i.e., compound L6) as a white solid with a mass of 0.94 g and a yield of about 99%. It can be directly used in the next step without purification. The product was identified by LC-MS: MS m / z: C 74 H 115 N9O 33 , [M+1] + , theoretical: 1658.76, measured: 1658.72.

[0227] 1.10 Synthesis of TZ-10

[0228]

[0229] Dissolve octanoic acid monomethyl ester (25.00 g, 133.29 mmol) in anhydrous tetrahydrofuran (133 ml), replace the reaction system with a vacuum pump to place it in a nitrogen atmosphere, and add borane / tetrahydrofuran solution (1M, 134 ml, 1 equivalent) dropwise while stirring in an ice bath. After the addition is complete, return to room temperature and stir overnight. Concentrate under reduced pressure to remove the solvent, and purify the residue by silica gel rapid column chromatography (petroleum ether / ethyl acetate = 20:1-10:1 gradient elution) to obtain the target product as a colorless transparent oil with a mass of 21.01 g and a yield of 91%.

[0230] 1.11 Synthesis of TZ-11

[0231]

[0232] TZ-10 (21.01 g, 120.60 mmol) and imidazole (16.4 g, 241.20 mmol, 2 equivalents) were dissolved in anhydrous N,N-dimethylformamide (120 ml), and the reaction system was placed in a nitrogen atmosphere by vacuum pump replacement. Tert-butyldiphenylsilyl chloride (39.70 g, 144.72 mmol, 1.2 equivalents) was added dropwise under stirring in an ice bath. After the addition was complete, the temperature was restored to room temperature and the reaction was allowed to react overnight. After TLC detection, the reaction was quenched by adding water, and dichloromethane was added to extract three times. The combined organic phase was washed once with water and saturated brine, then dried over anhydrous sodium sulfate, filtered, and concentrated by vacuum distillation. The residue was purified by silica gel flash column chromatography (petroleum ether / ethyl acetate = 50:1–10:1 gradient elution) to obtain the target product as a colorless oil with a mass of 15 g and a yield of 30%. The product was identified by LC-MS: MS m / z: C 25 H 36 O3Si, [M+1] + , theoretical: 413.24, measured: 413.22.

[0233] 1.12 Synthesis of TZ-12

[0234]

[0235] TZ-11 (7.42 g, 18.01 mmol) was dissolved in methanol (35 ml), and hydrazine hydrate (50-60% aqueous solution, 15 ml, 8.2 equivalents) was added dropwise to the reaction through a dropping funnel, and then the reaction was heated under reflux overnight. After the solvent was removed by vacuum distillation, ethyl acetate (50 ml) was added, and then washed twice with saturated brine, dried over anhydrous sodium sulfate, filtered, and vacuum distilled and concentrated to obtain compound TZ-12 as a colorless oily liquid with a mass of 7.40 g and a yield of about 100%. This product can be directly used in the next step without purification.

[0236] 1.13 Synthesis of TZ-13

[0237]

[0238] Benzyl N-(6-hydroxyhexyl)carbamate (10.00 g, 39.82 mmol) and p-toluenesulfonic acid (0.68 g, 3.98 mmol, 0.1 eq) were dissolved in dichloromethane (120 ml). 3,4-dihydropyrone (5.42 ml, 59.73 mmol, 1.5 eq) was added dropwise under stirring in an ice bath.

[0239] The reaction mixture was slowly added dropwise to the reaction mixture through a funnel. After stirring overnight at room temperature, the reaction mixture was quenched with water, and then extracted three times with dichloromethane. The combined organic phase was washed twice with saturated brine, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure and concentrated. The residue was purified by silica gel flash column chromatography (petroleum ether / ethyl acetate = 100:1-50:1 gradient elution). After drying with a vacuum pump, TZ-13 was obtained as a brown-yellow oil with a mass of 10.81 g and a yield of 81%. The product was identified by LC-MS: MS m / z: C 19 H 29 NO4, [M+1] + , theoretical: 335.21, measured: 335.20.

[0240] 1.14 Synthesis of TZ-14

[0241]

[0242] Compound TZ-13 (11.35 g, 33.86 mmol) and palladium / carbon (1.10 g, 10% palladium loading) were added to anhydrous methanol (55 ml), and the reaction system was placed in a hydrogen atmosphere by vacuum pump replacement. Stirring was continued overnight at room temperature, and then palladium / carbon was removed by diatomaceous earth filtration, and after vacuum distillation and concentration, compound TZ-14 was obtained by vacuum pump drying as a white foamy solid with a mass of 6.60 g and a yield of 97%, which could be directly used in the next step without purification.

[0243] 1.15 Synthetic TZ-15

[0244]

[0245] Compound TZ-14 (4.10 g, 20.38 mmol) and triethylamine (5.67 ml, 40.76 mmol, 2.0 eq) were dissolved in dichloromethane (45 ml), and the reaction system was placed in a nitrogen atmosphere by vacuum pump replacement. Under ice bath stirring, a dichloromethane solution of triphosgene (2.4 g, 8.13 mmol, 0.4 eq, 45 ml) was slowly added to the reaction through a dropping funnel. The reaction was then heated to room temperature and stirred for 15 minutes. 1N dilute hydrochloric acid (50 ml) was added to quench the reaction, and then extracted three times with dichloromethane. The combined organic phase was washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated by vacuum distillation to obtain the product TZ-15, with a mass of 4.61 g and a yield of 99%. This product can be directly used in the next step without purification.

[0246] 1.16 Synthetic TZ-16

[0247]

[0248] Compound TZ-12 (3.51 g, 8.51 mmol) was dissolved in anhydrous tetrahydrofuran (17 ml), and the reaction system was placed in a nitrogen atmosphere by vacuum pump replacement. Under ice bath stirring, a tetrahydrofuran solution of TZ-15 (1.93 g, 8.51 mmol, 1.0 equivalent, 17 ml) was added dropwise to the reaction through a dropping funnel. The reaction was then heated to room temperature and stirred for 1 hour. After reduced pressure distillation, the residue was purified by silica gel flash column chromatography (dichloromethane / methanol = 100:1-20:1 gradient elution), and after vacuum pump drying, TZ-16 was obtained as a bright yellow oil with a mass of 4.90 g and a yield of 90%.

[0249] 1.17 Synthesis of TZ-17

[0250]

[0251] Compound TZ-16 (6.40 g, 10.00 mmol) and KOH (4.48 g, 80.00 mmol, 8 equivalents) were added to methanol (40 ml), and the reaction was heated to 60°C and stirred for 24 hours. After cooling in an ice bath, the reaction system was adjusted to neutral with 6N hydrochloric acid, and then extracted three times with dichloromethane. The combined organic phase was washed once with saturated sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, filtered, and distilled and concentrated under reduced pressure to obtain TZ-17 as a light yellow oil with a mass of 3.80 g and a yield of 99%. This product can be directly used in the next step without purification. The product was identified by LC-MS: MS m / z: C 20 H 37 N3O4, [M+1] + , theoretical: 384.28, measured: 384.26.

[0252] 1.18 Synthetic TZ-18

[0253]

[0254] Compound TZ-17 (3.80 g, 10.00 mmol) and imidazole (1.02 g, 15.00 mmol, 1.5 equivalents) were dissolved in N,N-dimethylformamide (50 ml), and then the reaction system was placed in a nitrogen atmosphere by vacuum pump replacement. Tert-butyldiphenylsilyl chloride (3.01 g, 11.00 mmol, 1.1 equivalents) was added under ice bath stirring. The reaction was then heated to room temperature and stirred overnight. Water was then added to quench the reaction, and dichloromethane was added to extract three times. The combined organic phase was washed once with saturated sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure and concentrated. The residue was purified by silica gel rapid column chromatography (dichloromethane / methanol = 100:1-20:1 gradient elution), and TZ-18 was obtained as a white foam solid after being dried by a vacuum pump, with a mass of 6.10 g and a yield of 98%. The product was identified by LC-MS: MS m / z: C 36 H 55 N3O4Si, [M+1] + , theoretical: 622.40, measured: 622.43.

[0255] 1.19 Synthesis of TZ-19

[0256]

[0257] 5-(Z-amino)-1-pentanol (purchased from Anaiji Company, 9.42 g, 39.74 mmol) and carbon tetrabromide (3.35 g, 43.67 mmol, 1.1 equivalents) were dissolved in anhydrous dichloromethane (80 ml), and then the reaction system was placed in a nitrogen atmosphere by vacuum pump replacement. Triphenylphosphine (15.60 g, 59.49 mmol, 1.5 equivalents) was added under stirring in an ice bath, and then the reaction system was allowed to rise to room temperature and stirred overnight. Dichloromethane was added to dilute the reaction, and then washed once with saturated sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure and concentrated. The residue was purified by silica gel rapid column chromatography (petroleum ether / ethyl acetate = 100:1-20:1 gradient elution) to obtain 10.60 g of TZ-19 with a yield of 89%. The product was identified by LC-MS: MS m / z: C 13 H 19 BrNO2, [M+1] + , theoretical: 300.05, 302.05, measured: 300.04, 302.04.

[0258] 1.20 Synthetic TZ-20

[0259]

[0260] TZ-18 (6.21 g, 10.00 mmol) and TZ-19 (2.99 g, 10.00 mmol, 1.0 equivalent) were dissolved in anhydrous N,N-dimethylformamide (30 ml), and then anhydrous potassium carbonate (7.94 g, 57.50 mmol, 5.7 equivalent) was added, and then the reaction system was placed in a nitrogen atmosphere by vacuum pump replacement, and the reaction was heated to 65°C and stirred for 24 hours. After cooling to room temperature, the inorganic salt was filtered out, the solvent was removed by vacuum distillation with a high vacuum pump, and then dichloromethane was added to dissolve, followed by washing with water and saturated brine, respectively, dried over anhydrous sodium sulfate, filtered, vacuum distilled and concentrated, and purified by silica gel flash column chromatography (dichloromethane / methanol = 100:1-10:1 gradient elution) to obtain 7.14 g of TZ-20 with a yield of 85%. The product was identified by LC-MS: MS m / z: C 49 H 72 N4O6Si, [M+1] + , theoretical: 841.52, measured: 841.51.

[0261] 1.21 Synthesis of TZ-21

[0262]

[0263] TZ-20 (5.01 g, 5.96 mmol) was dissolved in anhydrous methanol (15 ml), and then p-toluenesulfonic acid (100 mg, 0.60 mmol, 0.1 equivalent) was added under ice bath stirring, and the reaction was stirred for 0.5 hours under ice bath, and saturated sodium bicarbonate solution was added to quench the reaction, and the mixture was extracted three times with dichloromethane. The combined organic phase was washed with water and saturated brine once each, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure and concentrated, and purified by silica gel flash column chromatography (dichloromethane / methanol = 100:1-10:1 gradient elution) to obtain 3.16 g of TZ-21 with a yield of 70%. The product was identified by LC-MS: MS m / z: C 44 H 64 N4O5Si, [M+1] + , theoretical: 757.46, measured: 757.45.

[0264] 1.22 Synthesis of TZ-22

[0265]

[0266] TZ-21 (3.10 g, 4.10 mmol) was dissolved in anhydrous pyridine (10 ml), and the reaction system was placed in a nitrogen atmosphere by vacuum pump replacement. Then, 4,4′-bismethoxytrityl chloride (1.53 g, 4.51 mmol, 1.1 equivalent) dissolved in anhydrous pyridine (10 ml) was added dropwise to the reaction through a syringe under ice bath stirring, and then the temperature was raised to room temperature and stirred overnight. Pyridine was then removed by vacuum distillation, and then dichloromethane was added to dissolve, and then washed once with saturated sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, filtered, vacuum distilled and concentrated, and purified by silica gel flash column chromatography (dichloromethane / methanol = 100:1-10:1, 0.5% triethylamine was added to each of mobile phases A / B, gradient elution) to obtain TZ-22 as a light yellow oil with a mass of 4.16 g and a yield of 96%. The product was identified by LC-MS: MS m / z: C 65 H 83 N4O7Si, [M+1] + , theoretical: 1059.60, measured: 1059.57.

[0267] 1.23 Synthesis of TZ-23

[0268]

[0269] TZ-22 (4.11 g, 3.88 mmol) and palladium / carbon (411 mg, 10% palladium loading) were added to anhydrous methanol (15 ml), and the reaction system was placed in a hydrogen atmosphere by vacuum pump replacement. Then, the mixture was stirred overnight at room temperature, and the palladium / carbon was removed by diatomaceous earth filtration, and the mixture was distilled under reduced pressure and concentrated. After being dried by vacuum pump, compound TZ-23 was obtained as a white foamy solid with a mass of 3.58 g and a yield of about 100%. This product can be directly used in the next step without purification. The product was identified by LC-MS: MS m / z: C 57 H 77 N4O5Si, [M+1] + , theoretical: 925.56, measured: 925.54.

[0270] 1.24 Synthetic TZ-24

[0271]

[0272] TZ-9 (6.30 g, 3.80 mmol) and TZ-23 (3.51 g, 3.80 mmol, 1 eq) were dissolved in anhydrous N,N-dimethylformamide (15 ml), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.88 g, 4.94 mmol, 1.3 eq) was added, and the reaction system was placed in a nitrogen atmosphere by vacuum pump replacement, and then N,N-diisopropylethylamine (1.98 ml, 11.40 mmol, 3.0 eq) was added while stirring in an ice bath, and the mixture was stirred at room temperature overnight, and then the reaction was quenched by adding water. The reaction solution was extracted three times with dichloromethane, and the combined organic phase was washed once with saturated sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure and concentrated, and then purified by silica gel flash column chromatography (dichloromethane / methanol = 100:1-10:1, 0.5% triethylamine was added to each of the mobile phases A / B, gradient elution). After vacuum pump drying, TZ-24 was obtained as a light yellow foamy solid with a mass of 6.33 g and a yield of 65%. The product was identified by LC-MS: MS m / z: C 131 H 189 N 13 O 37 Si, [M / 2+1] + , theoretical: 1283.15, measured: 1283.12.

[0273] 1.25 Synthetic TZ-25

[0274]

[0275] TZ-24 (5.00 g, 1.95 mmol) was dissolved in anhydrous tetrahydrofuran (15 ml), and the reaction system was placed in a nitrogen atmosphere by vacuum pump replacement. Tetrabutylammonium fluoride solution (1 M) was added under stirring in an ice bath. -1 The reaction mixture was heated to room temperature and stirred overnight, and then quenched with water. The reaction solution was extracted three times with dichloromethane, and the combined organic phase was washed once with saturated sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product TZ-25 (i.e., compound L1) as a light yellow foam solid with a mass of 4.52 g and a yield of about 100%. The product can be directly used in the next step without purification. The product was identified by LC-MS: MS m / z: C 115 H 171 N 13 O 37 , [(M-302) / 2+1] + , theoretical: 1013.03, measured: 1013.01.

[0276] 1.26 Synthetic TZ-26

[0277]

[0278] TZ-25 (233 mg, 0.10 mmol), succinic anhydride (20 mg, 0.20 mmol, 2.0 equiv) and 4-dimethylaminopyridine (36 mg, 0.30 mmol, 3.0 equiv) were dissolved in anhydrous dichloromethane (4 ml), and the reaction system was placed in a nitrogen atmosphere by vacuum pump replacement, and N,N-diisopropylethylamine (88 μL, 0.50 mmol, 5.0 equiv) was added with stirring in an ice bath. After the reaction was heated to room temperature, it was stirred overnight. After LC-MS detected that the reaction was complete, water was added to quench the reaction. The reaction system was extracted three times with dichloromethane. The combined organic phase was washed once with a saturated ammonium chloride solution and a saturated saline solution, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure and concentrated, and purified by C18 reverse phase column chromatography (methanol / water = 0-100% gradient elution). After vacuum pump drying, TZ-26 was obtained as a white foamy solid with a mass of 200 mg and a yield of about 82%. The product was identified by LC-MS: MS m / z: C 119 H 175 N 13 O 40 , [M / 2+1] + , theoretical: 1214.10, measured: 1214.05.

[0279] 1.27 Synthetic TZ-27

[0280]

[0281]

[0282] TZ-26 (200 mg, 0.08 mmol) and HBTU (37 mg, 0.10 mmol, 1.2 eq) were dissolved in anhydrous N,N-dimethylformamide (5 ml), and then N,N-diisopropylethylamine (56 μl, 0.32 mmol, 4.0 eq) was added. After shaking for 15 minutes, aminosilyl CPG (purchased from Hebei Dinaxingke Biotechnology Co., Ltd., 80 micromol / g, 1.0 g), and then shake the reaction for 24 hours. After the reaction is completed, filter and wash CPG three times with N, N-dimethylformamide, dichloromethane / methanol (v / v, 9:1) and dichloromethane, and then dry it with a vacuum pump. Then add acetic anhydride / pyridine (v / v, 1:3, 8 ml) to the CPG filter cake, shake and react for 30 minutes, so that the unconnected amino group is capped by the acetyl group, filter again, wash the CPG filter cake with dichloromethane / methanol (v / v, 9:1) and dichloromethane three times, and then completely dry it with a vacuum pump. The product TZ-27 is obtained, which is the CPG connected to the L1 ligand. The yield is 1.0 g, and the sample loading is measured to be 38.8 micromol / g by detecting the absorption of the 4,4′-dimethoxytrityl group (DMTr).

[0283] Preparation Example 2: Preparation of Compound L2

[0284] 2.1 Synthesis of TZ-28

[0285]

[0286] TZ-23 (0.92 g, 1.00 mmol), 5-(benzyloxycarbonylamino)pentanoic acid (0.25 g, 1.00 mmol, 1.0 equiv) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (0.49 g, 1.30 mmol, 1.3 equiv) were dissolved in anhydrous N,N-dimethylformamide (5 ml), and the reaction system was placed in a nitrogen atmosphere by vacuum pump replacement, and N,N-diisopropylethylamine (0.52 ml, 3.00 mmol, 3.0 equiv) was added with stirring in an ice bath. The reaction mixture was heated to room temperature and stirred overnight, and then quenched with water. The reaction solution was extracted three times with dichloromethane, and the combined organic phase was washed once with saturated sodium bicarbonate, water, and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. After purification, it was purified by silica gel flash column chromatography (dichloromethane / methanol = 100:1-10:1, 0.5% triethylamine was added to each mobile phase A / B, and gradient elution). After vacuum pump drying, the product TZ-28 was obtained as a light brown oily substance with a mass of 0.91 g and a yield of 78%. The product was identified by LC-MS: MS m / z: C 70 H 91 N5O8Si, [M+Na] + , theoretical: 1180.65, measured: 1180.62.

[0287] 2.2 Synthesis of TZ-29

[0288]

[0289] TZ-28 (0.91 g, 0.78 mmol) and palladium / carbon (92 mg, 10% palladium loading) were added to anhydrous methanol (5 ml), and the reaction system was placed in a hydrogen atmosphere by vacuum pump replacement. Then, the mixture was stirred overnight at room temperature, and the palladium / carbon was removed by filtration through diatomaceous earth. After vacuum distillation and concentration, the compound TZ-29 was dried by vacuum pump to obtain a white foamy solid with a mass of 0.80 g and a yield of 100%. This product can be directly used in the next step without chemical treatment. The product was identified by LC-MS: MS m / z: C 62 H 85 N5O6Si, [M+1] + , theoretical: 1024.63, measured: 1024.59.

[0290] 2.3 Synthesis of TZ-30

[0291]

[0292] TZ-9 (1.29 g, 0.78 mmol, 1.0 eq.) and TZ-29 (0.91 g, 0.78 mmol, 1.0 eq.) were dissolved in anhydrous N,N-dimethylformamide (15 ml), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (0.38 g, 1.01 mmol, 1.3 eq.) was added, and the reaction system was placed in a nitrogen atmosphere by vacuum pump replacement, and then N,N-diisopropylethylamine (0.41 ml, 2.34 mmol, 3.0 eq.) was added under stirring in an ice bath, and the mixture was stirred at room temperature overnight, and then the reaction was quenched by adding water. The reaction solution was extracted three times with dichloromethane, and the combined organic phase was washed once with saturated sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure and concentrated, and then purified by silica gel flash column chromatography (dichloromethane / methanol = 100:1-10:1, 0.5% triethylamine was added to each mobile phase A / B, gradient elution), and after vacuum pump drying, TZ-30 was obtained as a light yellow foamy solid with a mass of 1.10 g and a yield of 53%. The product was identified by LC-MS: MS m / z: C 136 H 198 N 14 O 38 Si, [M / 2+1] + , theoretical: 1332.69, measured: 1332.69.

[0293] 2.4 Synthesis of TZ-31

[0294]

[0295] TZ-30 (1.1 g, 0.41 mmol) was dissolved in anhydrous tetrahydrofuran (15 ml), and the reaction system was placed in a nitrogen atmosphere by vacuum pump replacement. Tetrabutylammonium fluoride solution (1 M) was added under stirring in an ice bath. -1 The reaction mixture was heated to room temperature and stirred overnight, and then quenched with water. The reaction solution was extracted three times with dichloromethane, and the combined organic phase was washed once with saturated sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product TZ-31 (i.e., compound L2) as a light yellow foamy solid with a mass of 1.00 g and a yield of about 100%. The product can be directly used in the next step without chemical treatment. The product was identified by LC-MS: MS m / z: C 120 H 180 N 14 O 38 , [(M-302) / 2+1] + , theoretical: 1062.63, measured: 1062.63.

[0296] 2.5 Synthesis of TZ-32

[0297]

[0298] TZ-31 (242 mg, 0.10 mmol), succinic anhydride (20 mg, 0.20 mmol, 2.0 equiv.) and 4-dimethylaminopyridine (37 mg, 0.30 mmol, 3.0 equiv.) were dissolved in anhydrous dichloromethane (4 ml). The reaction system was then placed in a nitrogen atmosphere by vacuum pump replacement. N,N-diisopropylethylamine (87 μL, 0.50 mmol, 5 equiv.) was added with stirring in an ice bath. The reaction was heated to room temperature and stirred overnight. After LC-MS detected that the reaction was complete, water was added to quench the reaction. The reaction system was extracted three times with dichloromethane. The combined organic phase was washed once with a saturated ammonium chloride solution and a saturated saline solution, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure and concentrated, and purified by C18 reverse phase column chromatography (methanol / water = 0-100% gradient elution). After vacuum pump drying, TZ-32 was obtained as a white foamy solid with a mass of 191 mg and a yield of about 76%. The product was identified by LC-MS: MS m / z: C 124 H 184 N 14 O 41 , [M / 2+1] + , theoretical: 1313.63, measured: 1313.60.

[0299] 2.6 Synthesis of TZ-33

[0300]

[0301]

[0302] TZ-32 (191 mg, 0.07 mmol) and HBTU (29 mg, 0.08 mmol, 1.2 eq) were dissolved in anhydrous N,N-dimethylformamide (5 ml), and then N,N-diisopropylethylamine (52 μl, 0.30 mmol, 4.0 eq) was added. After shaking for 15 minutes, aminosilyl CPG (purchased from Hebei Dinaxingke Biotechnology Co., Ltd., 80 micromol / g, 0.87 g), and then shake the reaction for 24 hours. After the reaction is completed, filter; CPG is washed three times with N, N-dimethylformamide, dichloromethane / methanol (v / v, 9:1) and dichloromethane, and then dried by a vacuum pump. Then acetic anhydride / pyridine (v / v, 1:3, 8 ml) is added to the CPG filter cake, and shake the reaction for 30 minutes to cap the unconnected amino group with the acetyl group, and then filter again. The CPG filter cake is washed three times with dichloromethane / methanol (v / v, 9:1) and dichloromethane, and then completely dried by a vacuum pump. The product TZ-33 is obtained, which is the CPG connected to the L2 ligand. The yield is 0.87 g, and the sample loading is measured to be 29.1 micromol / g by detecting the absorption of the 4,4′-dimethoxytrityl group (DMTr).

[0303] Preparation Example 3: Preparation of Compound L3

[0304] 3.1 Synthesis of TZ-34

[0305]

[0306] 8-aminooctanoic acid (purchased from Shanghai Haohong Biopharmaceutical Technology Co., Ltd., 15.90 g, 100 mmol, 1.0 equivalent) was dissolved in tetrahydrofuran (100 ml), potassium carbonate (41.40 g, 300 mmol, 3.0 equivalent) was added, and then benzyl chloroformate (20.40 g, 120 mmol, 1.2 equivalent) was added dropwise through a syringe under ice bath stirring. After the reaction was warmed to room temperature, it was stirred overnight, and water was added under ice bath to quench the reaction, and then 1M dilute hydrochloric acid was added to adjust the reaction to pH = 5. The reaction solution was extracted three times with ethyl acetate, and the combined organic phase was washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure and concentrated, and then purified by silica gel flash column chromatography (petroleum ether / ethyl acetate = 20:1-1:1 gradient elution), and the product TZ-34 was obtained as a white solid after vacuum pump drying, with a mass of 20.52 g and a yield of 70%. The product was identified by LC-MS: MS m / z: C 16 H 23 NO4, [M+1] + , theoretical: 294.16, measured: 294.15.

[0307] 3.2 Synthesis of TZ-35

[0308]

[0309] (3R, 5S)-5-{[bis(4-methoxyphenyl)(phenyl)methoxy]methyl}pyrrolidin-3-ol (purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd., 839 mg, 2.0 mmol, 1.0 eq) and TZ-34 (586 mg, 2.0 mmol, 1.0 eq) were dissolved in anhydrous N,N-dimethylformamide (5 ml), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (988 mg, 2.6 mmol, 1.3 eq) was added, and the reaction system was placed in a nitrogen atmosphere by vacuum pump replacement, and then N,N-diisopropylethylamine (1.0 ml, 6.0 mmol, 3.0 eq) was added while stirring in an ice bath, and the mixture was stirred at room temperature overnight, and then the reaction was quenched by adding water. The reaction solution was extracted three times with dichloromethane, and the combined organic phase was washed once with saturated sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure and concentrated, and then purified by silica gel flash column chromatography (dichloromethane / methanol = 100:1-10:1, 0.5% triethylamine was added to each mobile phase A / B, gradient elution), and after vacuum pump drying, TZ-35 was obtained as a light yellow foamy solid with a mass of 1.23 g and a yield of 89%. The product was identified by LC-MS: MS m / z: C 42 H 50 N2O7, [M+1] + , theoretical: 695.36, measured: 695.34.

[0310] 3.3 Synthesis of TZ-36

[0311]

[0312] TZ-35 (1.23 g, 1.77 mmol) and palladium / carbon (123 mg, 10% palladium loading) were added to anhydrous methanol (10 ml), and the reaction system was placed in a hydrogen atmosphere by vacuum pump replacement. Then, the mixture was stirred overnight at room temperature, and the palladium / carbon was removed by diatomaceous earth filtration. After vacuum distillation and concentration, the compound TZ-36 was obtained by vacuum pump drying as a light brown oil with a mass of 1.0 g and a yield of about 100%. This product can be directly used in the next step without purification. The product was identified by LC-MS: MS m / z: C 34 H 44 N2O5, [M+1] + , theoretical: 561.33, measured: 561.32.

[0313] 3.4 Synthesis of TZ-37

[0314]

[0315] TZ-9 (2.93 g, 1.77 mmol, 1.0 eq.) and TZ-36 (1.00 g, 1.77 mmol, 1.0 eq.) were dissolved in anhydrous N,N-dimethylformamide (15 ml), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (874 mg, 2.30 mmol, 1.3 eq.) was added, and the reaction system was placed in a nitrogen atmosphere by vacuum pump replacement, and then N,N-diisopropylethylamine (923 μL, 5.31 mmol, 3.0 eq.) was added under stirring in an ice bath, and the mixture was stirred at room temperature overnight, and then the reaction was quenched by adding water. The reaction solution was extracted three times with dichloromethane, and the combined organic phase was washed once with saturated sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure and concentrated, and then purified by silica gel flash column chromatography (dichloromethane / methanol = 100:1-10:1, 0.5% triethylamine was added to mobile phase A / B, gradient elution), and vacuum pumped to obtain TZ-37 (i.e., L3 compound) as a light yellow foamy solid with a mass of 2.80 g and a yield of 72%. The product was identified by LC-MS: m / z: C 108 H 157 N 11 O 37 , [M / 2+1] + , theoretical: 1101.03, measured: 1101.03.

[0316] 3.5 Synthesis of TZ-38

[0317]

[0318] TZ-37 (220 mg, 0.10 mmol, 1.0 equiv), succinic anhydride (20 mg, 0.20 mmol, 2.0 equiv) and 4-dimethylaminopyridine (37 mg, 0.3 mmol, 3.0 equiv) were dissolved in anhydrous dichloromethane (4 ml), and the reaction system was placed in a nitrogen atmosphere by vacuum pump replacement, and N,N-diisopropylethylamine (87 ml, 0.5 mmol, 3.0 equiv) was added with stirring in an ice bath. The reaction was heated to room temperature and stirred overnight. After the reaction was complete by LC-MS, water was added to quench the reaction. The reaction system was extracted three times with dichloromethane. The combined organic phase was washed once with a saturated ammonium chloride solution and a saturated saline solution, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure and concentrated, and purified by C18 reverse phase column chromatography (methanol / water = 0-100% gradient elution). After vacuum pump drying, TZ-38 was obtained as a white foamy solid with a mass of 165 mg and a yield of 72%. The product was identified by LC-MS: MS m / z: C 112 H 161 N 11 O 40 , [M / 2+1] + , theoretical: 1151.04, measured: 1151.04.

[0319] 3.6 Synthesis of TZ-39

[0320]

[0321]

[0322] TZ-38 (165 mg, 0.072 mmol) and HBTU (33 mg, 0.086 mmol, 1.2 eq) were dissolved in anhydrous N,N-dimethylformamide (5 ml), and then N,N-diisopropylethylamine (50 μl, 0.29 mmol, 4.0 eq) was added. After shaking for 15 minutes, aminosilyl CPG (purchased from Hebei Dinaxingke Biotechnology Co., Ltd., 80 micromol / g, 0.90 g), and then shake the reaction for 24 hours. After the reaction is completed, filter; CPG is washed three times with N, N-dimethylformamide, dichloromethane / methanol (v / v, 9:1) and dichloromethane, and then dried by a vacuum pump. Then acetic anhydride / pyridine (v / v, 1:3, 8 ml) is added to the CPG filter cake, and shake the reaction for 30 minutes to cap the unconnected amino group with the acetyl group, and then filter again. The CPG filter cake is washed three times with dichloromethane / methanol (v / v, 9:1) and dichloromethane, and then completely dried by a vacuum pump. The product TZ-39 is obtained, which is CPG connected to the L3 ligand. The yield is 0.90 g, and the sample loading is measured to be 29.6 micromol / g by detecting the absorption of the 4,4′-dimethoxytrityl group (DMTr).

[0323] Preparation Example 4: Preparation of Compound L4

[0324] 4.1 Synthesis of TZ-40

[0325]

[0326] N-Benzyloxycarbonyl-O-tert-butyl-L-serine (4.78 g, 20 mmol, 1.0 eq.) and N,N-diisopropylethylamine (10.4 ml, 60 mmol, 3.0 eq.) were dissolved in anhydrous dichloromethane (40 ml). A dichloromethane solution of 4,4'-dimethoxytriphenylmethane (4.73 g, 24 mmol, 1.2 eq.) was added dropwise under stirring in an ice bath. After the addition was completed, the reaction was heated to room temperature and stirred overnight, and water was added to quench the reaction. The reaction system was extracted three times with dichloromethane, and the combined organic phase was washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure and concentrated, and purified by silica gel flash column chromatography (dichloromethane / methanol = 100:1-10:1, 0.5% triethylamine was added to each mobile phase A / B, and gradient elution). After vacuum pump drying, TZ-40 was obtained as a brown oil with a mass of 5.33 g and a yield of 49%. The product was identified by LC-MS: m / z: C 32 H 31 NO7, [M+1] + , theoretical: 541.21, measured: 541.20. 1 HNMR (500MHz, DMSO) δ7.42–7.16 (m, 14H), 6.97 (brs, 1H), 6.90–6.76 (m, 4H), 5.05 (q, J=12.7Hz, 2H), 4.03 (brs, 1H), 3.72 (s, 6H), 3.27–3.15 (m, 2H).

[0327] 4.2 Synthesis of TZ-41

[0328]

[0329] TZ-40 (4.51 g, 8.34 mmol, 1.0 eq.) and 4-hydroxypiperidine hydrochloride (1.15 g, 8.34 mmol, 1.0 eq.) were dissolved in anhydrous N,N-dimethylformamide (16 ml), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (4.12 g, 10.84 mmol, 1.3 eq.) was added, and the reaction system was placed in a nitrogen atmosphere by vacuum pump replacement, and then N,N-diisopropylethylamine (5.8 ml, 33.34 mmol, 3.0 eq.) was added while stirring in an ice bath, and the mixture was stirred at room temperature overnight, and then the reaction was quenched by adding water. The reaction solution was extracted three times with dichloromethane, and the combined organic phase was washed once with saturated sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure and concentrated, and then purified by silica gel flash column chromatography (dichloromethane / methanol = 100:1-10:1, 0.5% triethylamine was added to mobile phase A / B, gradient elution), and vacuum pumped to obtain TZ-41 as a light yellow foamy solid with a mass of 3.95 g and a yield of 76%. The product was identified by LC-MS: m / z: C 37 H 40 N2O7, [M+1] + , theoretical: 625.28, measured: 625.28. 1 H NMR (500MHz, DMSO) δ7.42–7.10 (m, 15H), 6.95–6.75 (m, 4H), 5.07 (td, J=14.4, 7.5Hz, 2H), 4.73 (dd, J=15 .3, 5.7Hz, 2H), 3.73(s, 6H), 3.70–3.54(m, 5H), 3.27–3.15(m, 2H), 2.50–2.45(m, 2H), 1.73–1.51(m, 2H).

[0330] 4.3 Synthesis of TZ-42

[0331]

[0332] TZ-41 (2.24 g, 3.59 mmol) and palladium / carbon (224 mg, 10% palladium loading) were added to anhydrous methanol (10 ml), and the reaction system was placed in a hydrogen atmosphere by vacuum pump replacement. Then, the mixture was stirred overnight at room temperature, and the palladium / carbon was removed by diatomaceous earth filtration, and the mixture was distilled under reduced pressure and concentrated. The compound TZ-42 was obtained by vacuum pump drying as a light brown oil with a mass of 1.76 g and a yield of about 100%. The product can be directly used in the next step without purification. The product was identified by LC-MS: MS m / z: C 29 H 34 N2O5, [M+1] +, theoretical: 491.25, measured: 491.24.

[0333] 4.4 Synthesis of TZ-43

[0334]

[0335] TZ-42 (980 mg, 2.0 mmol, 1.0 eq.) and TZ-34 (528 mg, 2.0 mmol, 1.0 eq.) were dissolved in anhydrous N,N-dimethylformamide (5 ml), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (988 mg, 2.6 mmol, 1.3 eq.) was added, and the reaction system was placed in a nitrogen atmosphere by vacuum pump replacement, and then N,N-diisopropylethylamine (1.04 ml, 6.0 mmol, 3.0 eq.) was added under stirring in an ice bath, and the mixture was stirred at room temperature overnight, and then the reaction was quenched by adding water. The reaction solution was extracted three times with dichloromethane, and the combined organic phase was washed once with saturated sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure and concentrated, and then purified by silica gel flash column chromatography (dichloromethane / methanol = 100:1-10:1, 0.5% triethylamine was added to mobile phase A / B, gradient elution). After vacuum pump drying, TZ-43 was obtained as a white foamy solid with a mass of 830 mg and a yield of 54%. The product was identified by LC-MS: MS m / z: C 45 H 55 N3O8, [M+1] + , theoretical: 766.40, measured: 766.38.

[0336] 4.5 Synthesis of TZ-44

[0337]

[0338] TZ-43 (592 mg, 0.77 mmol) and palladium / carbon (60 mg, 10% palladium loading) were added to anhydrous methanol (10 ml), and the reaction system was placed in a hydrogen atmosphere by vacuum pump replacement. Then, the mixture was stirred overnight at room temperature, and the palladium / carbon was removed by diatomaceous earth filtration. Compound TZ-44 was obtained after reduced pressure distillation and concentration. This product can be directly used in the next step without further purification. Compound TZ-44 was dried by vacuum pump to obtain a white foamy solid with a mass of 490 mg and a yield of about 100%. The product was identified by LC-MS: MS m / z: C 37 H 49 N3O6, [M+1] + , theoretical: 632.36, measured: 632.35.

[0339] 4.6 Synthesis of TZ-45

[0340]

[0341] TZ-9 (664 mg, 0.40 mmol, 1.0 eq.) and TZ-44 (252 mg, 0.40 mmol, 1.0 eq.) were dissolved in anhydrous N,N-dimethylformamide (10 ml), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (198 mg, 0.52 mmol, 1.3 eq.) was added, and the reaction system was placed in a nitrogen atmosphere by vacuum pump replacement, and then N,N-diisopropylethylamine (208 μL, 0.60 mmol, 3.0 eq.) was added under stirring in an ice bath, and the mixture was stirred at room temperature overnight, and then the reaction was quenched by adding water. The reaction solution was extracted three times with dichloromethane, and the combined organic phase was washed once with saturated sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure and concentrated, and then purified by silica gel flash column chromatography (dichloromethane / methanol = 100:1-10:1, 0.5% triethylamine was added to each of mobile phases A / B, gradient elution), and vacuum pumped to obtain TZ-45 (i.e., L4 compound) as a white foamy solid with a mass of 552 mg and a yield of 61%. The product was identified by LC-MS: m / z: C 111 H 162 N 12 O 38 , [M / 2+1] + , theoretical: 1136.55, measured: 1136.56.

[0342] 4.7 Synthesis of TZ-46

[0343]

[0344] TZ-45 (500 mg, 0.22 mmol, 1.0 equiv), succinic anhydride (44 mg, 0.44 mmol, 2.0 equiv) and 4-dimethylaminopyridine (80 mg, 0.66 mmol, 3.0 equiv) were dissolved in anhydrous dichloromethane (4 ml), and the reaction system was placed in a nitrogen atmosphere by vacuum pump replacement, and N,N-diisopropylethylamine (191 μL, 1.10 mmol, 5.0 equiv) was added with stirring in an ice bath. The reaction was heated to room temperature and stirred overnight. After the reaction was complete by LC-MS, water was added to quench the reaction. The reaction system was extracted three times with dichloromethane. The combined organic phase was washed once with a saturated ammonium chloride solution and a saturated saline solution, dried over anhydrous sodium sulfate, filtered, distilled and concentrated under reduced pressure, and purified by C18 reverse phase column chromatography (methanol / water = 0-100% gradient elution). After vacuum pump drying, TZ-46 was obtained as a white foamy solid with a mass of 460 mg and a yield of about 88%. The product was identified by LC-MS: MS m / z: C 115 H166 N 12 O 41 , [M / 2+Na] + , theoretical: 1208.55, measured: 1208.56.

[0345] 4.8 Synthesis of TZ-47

[0346]

[0347]

[0348] TZ-46 (98 mg, 0.04 mmol) and HBTU (19 mg, 0.05 mmol, 1.2 eq) were dissolved in anhydrous N,N-dimethylformamide (5 ml), and then N,N-diisopropylethylamine (28 μl, 0.16 mmol, 4.0 eq) was added. After shaking for 15 minutes, aminosilyl CPG (purchased from Hebei Dinaxingke Biotechnology Co., Ltd., 80 micromol / g, 0.5 g), and then shake the reaction for 24 hours. After the reaction is completed, filter; CPG is washed three times with N, N-dimethylformamide, dichloromethane / methanol (v / v, 9:1) and dichloromethane, and then dried by a vacuum pump. Then acetic anhydride / pyridine (v / v, 1:3, 8 ml) is added to the CPG filter cake, and shake the reaction for 30 minutes to cap the unconnected amino group with the acetyl group, and then filter again. The CPG filter cake is washed three times with dichloromethane / methanol (v / v, 9:1) and dichloromethane, and then completely dried by a vacuum pump. The product TZ-47 is obtained, which is the CPG connected to the L4 ligand. The yield is 0.5 g, and the sample loading is measured to be 40.2 micromol / g by detecting the absorption of the 4,4′-dimethoxytrityl group (DMTr).

[0349] Preparation Example 5: Preparation of Compound L5

[0350] 5.1 Synthesis of TZ-48

[0351]

[0352] 4-Hydroxypiperidine hydrochloride (5.5 g, 40.00 mmol, 1.0 equivalent) was dissolved in acetonitrile (40 ml), and then triethylamine (19.6 ml, 180.00 mmol, 4.5 equivalent) and ethyl trifluoroacetate (15.2 ml, 100.00 mmol, 2.5 equivalent) were added under ice bath stirring, and then the mixture was heated to room temperature and stirred overnight, filtered, and the filter cake was washed with ethyl acetate. The combined filtrate was concentrated to obtain trifluoroacetic acid protected 4-hydroxypiperidine, which can be used for the next step without purification. The intermediate of the previous step was dissolved in anhydrous pyridine (100 ml), and then 4,4'-dimethoxytriphenylmethane (9.06 g, 43 mmol, 1.15 equivalent) was added under ice bath stirring, and the reaction was stirred overnight. Pyridine was removed by vacuum distillation with a high vacuum pump, and the obtained oil was dissolved in ethyl acetate, then washed once with water and saturated brine, dried over anhydrous sodium sulfate, filtered, vacuum distilled and concentrated, and used directly in the next step without purification. The DMTr-protected intermediate from the previous step was dissolved in ethanol (160 ml), and then 1 M HCl was added under stirring in an ice bath. -1 Aqueous solution of sodium hydroxide (80 ml, 80 mmol, 2.0 equivalents) was heated to room temperature and stirred for 1 hour. The solvent methanol was removed by vacuum distillation, and then the reaction solution was extracted with dichloromethane three times. The organic phases were combined and concentrated by vacuum distillation, and then purified by silica gel flash column chromatography (dichloromethane / methanol = 100:1-10:1, 0.5% triethylamine was added to each mobile phase A / B, gradient elution). After vacuum pump drying, TZ-48 was obtained as a colorless oil with a mass of 14.20 g and a yield of 88%. The product was identified by LC-MS: m / z: C 26 H 29 NO3, [M+1] + , theoretical: 404.21, measured: 404.21. 1 H NMR (500MHz, DMSO) δ7.47–7.40 (m, 2H), 7.35–7.27 (m, 7H), 6.92–6.87 (m, 4H), 3.73 (s, 6H), 3.38 (d, J= 6.7Hz, 1H), 3.06 (ddd, J=12.2, 7.7, 3.8Hz, 2H), 2.77 (ddt, J=12.2, 7.7, 3.8Hz, 2H), 1.44–1.21 (m, 4H).

[0353] 5.2 Synthesis of TZ-49

[0354]

[0355] TZ-48 (14.00 g, 34.72 mmol, 1.0 eq.) and N-benzyloxycarbonyl-O-tert-butyl-L-serine (8.30 g, 34.72 mmol, 1.0 eq.) were dissolved in anhydrous N,N-dimethylformamide (70 ml), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (17.15 g, 45.14 mmol, 1.3 eq.) was added, and the reaction system was placed in a nitrogen atmosphere by vacuum pump replacement, and then N,N-diisopropylethylamine (18 ml, 104.16 mmol, 3.0 eq.) was added while stirring in an ice bath, and the mixture was stirred at room temperature overnight, and then the reaction was quenched by adding water. The reaction solution was extracted three times with dichloromethane, and the combined organic phase was washed once with saturated sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure and concentrated, and then purified by silica gel flash column chromatography (dichloromethane / methanol = 100:1-10:1, 0.5% triethylamine was added to each mobile phase A / B, gradient elution), and after vacuum pumping, TZ-49 was obtained as a light brown oil with a mass of 16.47 g and a yield of 76%. The product was identified by LC-MS: m / z: C 37 H 40 N2O7, [M+1] + , theoretical: 625.28, measured: 625.28. 1 H NMR (500MHz, DMSO) δ7.47 (d, J=7.8Hz, 2H), 7.42–7.19 (m, 12H), 6.90 (d, J=8.7Hz, 2H), 4.99 (s, 2H), 4.80 (dt, J=37.7, 5 .9Hz, 1H), 4.54–4.46(m, 1H), 3.74(s, 6H), 3.68–3.51(m, 2H), 3.50–3.42(m, 1H), 3.22–2.97(m, 2H), 1.38–1.03(m, 4H).

[0356] 5.3 Synthesis of TZ-50

[0357]

[0358] TZ-49 (2.65 g, 4.24 mmol) and palladium / carbon (265 mg, 10% palladium loading) were added to anhydrous methanol (20 ml), and the reaction system was placed in a hydrogen atmosphere by vacuum pump replacement. Then, the mixture was stirred overnight at room temperature, and the palladium / carbon was removed by diatomaceous earth filtration. After vacuum distillation and concentration, compound TZ-50 was obtained. After drying by vacuum pump, the mixture was brown oily with a mass of 2.08 g and a yield of about 100%. The product can be directly used in the next step without further purification. The product was identified by LC-MS: MS m / z: C 29 H34 N2O5, [M+1] + , theoretical: 491.25, measured: 491.24.

[0359] 5.4 Synthesis of TZ-51

[0360]

[0361] TZ-50 (1.00 g, 2.04 mmol, 1.0 eq.) and TZ-34 (0.60 g, 2.04 mmol, 1.0 eq.) were dissolved in anhydrous N,N-dimethylformamide (10 ml), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.01 g, 2.65 mmol, 1.3 eq.) was added, and the reaction system was placed in a nitrogen atmosphere by vacuum pump replacement, and then N,N-diisopropylethylamine (1.06 ml, 6.12 mmol, 3.0 eq.) was added while stirring in an ice bath, and the mixture was stirred at room temperature overnight, and then the reaction was quenched by adding water. The reaction solution was extracted three times with dichloromethane, and the combined organic phase was washed once with saturated sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure and concentrated, and then purified by silica gel flash column chromatography (dichloromethane / methanol = 100:1-10:1, 0.5% triethylamine was added to mobile phase A / B, gradient elution), and after vacuum pump drying, TZ-51 was obtained as a brown oil with a mass of 1.22 g and a yield of 78%. The product was identified by LC-MS: MS m / z: C 45 H 55 N3O8, [M+1] + , theoretical: 766.40, measured: 766.38. 1 H NMR (500MHz, DMSO) δ7.91 (dd, J=35.4, 8.4Hz, 1H), 7.47–7.40 (m, 2H), 7.39–7.17 (m, 12H), 6.94–6.83 (m, 4H), 4.99 (d, J=4.5Hz , 2H), 4.80–4.63 (m, 2H), 3.73 (s, 6H), 3.71–3.45 (m, 4H), 3.18–2.90 (m, 4H), 2.06 (dt, J=25.4, 7.7Hz, 2H), 1.51–0.98 (m, 14H).

[0362] 5.5 Synthesis of TZ-52

[0363]

[0364] TZ-51 (610 mg, 0.80 mmol) and palladium / carbon (61 mg, 10% palladium loading) were added to anhydrous methanol (5 ml), and the reaction system was placed in a hydrogen atmosphere by vacuum pump replacement. Then, the mixture was stirred overnight at room temperature, and the palladium / carbon was removed by diatomaceous earth filtration. After vacuum distillation and concentration, compound TZ-52 was obtained. After vacuum pump drying, it was a white foamy solid with a mass of 500 mg and a yield of about 100%. This product can be directly used in the next step without purification. The product was identified by LC-MS: MS m / z: C 37 H 49 N3O6, [M+1] + , theoretical: 632.36, measured: 632.35. 1 H NMR (500MHz, DMSO) δ7.95 (dd, J=36.4, 8.2Hz, 1H), 7.50–7.41 (m, 2H), 7.41–7.15 (m, 7H), 6.90 (d, J=8.5Hz, 4H), 4.71 (q, J=7.4H z, 1H), 3.74 (s, 6H), 3.72–3.48 (m, 3H), 3.35 (dp, J=17.1, 5.8Hz, 1H), 3.27–2.94 (m, 4H), 2.13–1.98 (m, 2H), 1.55–1.03 (m, 14H).

[0365] 5.6 Synthesis of TZ-53

[0366]

[0367]

[0368] TZ-9 (1.31 g, 0.79 mmol, 1.0 eq.) and TZ-52 (0.50 g, 0.79 mmol, 1.0 eq.) were dissolved in anhydrous N,N-dimethylformamide (10 ml), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (0.39 g, 1.03 mmol, 1.3 eq.) was added, and the reaction system was placed in a nitrogen atmosphere by vacuum pump replacement, and then N,N-diisopropylethylamine (412 μL, 2.37 mmol, 3.0 eq.) was added under stirring in an ice bath, and the mixture was stirred at room temperature overnight, and then the reaction was quenched by adding water. The reaction solution was extracted three times with dichloromethane, and the combined organic phase was washed once with saturated sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure and concentrated, and then purified by silica gel flash column chromatography (dichloromethane / methanol = 100:1-10:1, 0.5% triethylamine was added to mobile phase A / B, gradient elution), and vacuum pumped to obtain TZ-53 (i.e., L5 compound) as a white foamy solid with a mass of 1.10 g and a yield of 61%. The product was identified by LC-MS: m / z: C 111 H 162 N 12 O 38 , [M / 2+1] + , theoretical: 1136.55, measured: 1136.55.

[0369] 5.7 Synthesis of TZ-54

[0370]

[0371]

[0372] TZ-53 (550 mg, 0.24 mmol, 1.0 equiv), succinic anhydride (48 mg, 0.48 mmol, 2.0 equiv) and 4-dimethylaminopyridine (88 mg, 0.72 mmol, 3.0 equiv) were dissolved in anhydrous dichloromethane (5 ml), and the reaction system was placed in a nitrogen atmosphere by vacuum pump replacement, and N,N-diisopropylethylamine (208 μL, 1.20 mmol, 5.0 equiv) was added with stirring in an ice bath. The reaction was heated to room temperature and stirred overnight. After LC-MS detected that the reaction was complete, water was added to quench the reaction. The reaction system was extracted three times with dichloromethane. The combined organic phase was washed once with a saturated ammonium chloride solution and a saturated saline solution, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure and concentrated, and purified by C18 reverse phase column chromatography (methanol / water = 0-100% gradient elution). After vacuum pump drying, TZ-54 was obtained as a colorless oil with a mass of 430 mg and a yield of about 75%. The product was identified by LC-MS: MS m / z: C 115 H 166 N12 O 41 , [M / 2+1] + , theoretical: 1186.56, measured: 1186.54.

[0373] 5.8 Synthesis of TZ-55

[0374]

[0375] TZ-54 (430 mg, 0.18 mmol, 1.0 eq.) and HBTU (82 mg, 0.22 mmol, 1.2 eq.) were dissolved in anhydrous N,N-dimethylformamide (10 ml), and then N,N-diisopropylethylamine (125 μl, 0.72 mmol, 4.0 eq.) was added. After shaking for 15 minutes, aminosilylated CPG (purchased from Hebei Dinaxingke Biotechnology Co., Ltd., 80 micromol / g, 2.25 g), and then shake the reaction for 24 hours. After the reaction is completed, filter; CPG is washed three times with N, N-dimethylformamide, dichloromethane / methanol (v / v, 9:1) and dichloromethane, and then dried by a vacuum pump. Then acetic anhydride / pyridine (v / v, 1:3, 12 ml) is added to the CPG filter cake, and shake the reaction for 30 minutes to cap the unconnected amino group with the acetyl group, and then filter again. The CPG filter cake is washed three times with dichloromethane / methanol (v / v, 9:1) and dichloromethane, and then completely dried by a vacuum pump. The product TZ-55 is obtained, which is CPG connected to the L5 ligand. The yield is 2.26 grams, and the sample loading is measured to be 31.3 micromol / g by detecting the absorption of the 4,4′-dimethoxytrityl group (DMTr).

[0376] Preparation Example 6: Preparation of L6-oligonucleotide conjugate (liquid phase coupling)

[0377] 6.1 Synthesis of TZ-56

[0378]

[0379] TZ-9 (300 mg, 0.18 mmol, 1.0 eq) was dissolved in anhydrous dichloromethane (6 ml), and then the reaction system was placed in a nitrogen atmosphere by vacuum pump replacement, and N,N-diisopropylethylamine (251 μL, 1.45 mmol, 8.0 eq) was added. Under ice bath stirring, pentafluorophenol trifluoroacetate (123 μL, 0.72 mmol, 4.0 eq) was added to the reaction system through a micro syringe, and then the mixture was heated to room temperature and stirred for 2 hours. After LC-MS detection of complete reaction, the mixture was concentrated under reduced pressure and purified by silica gel flash column chromatography (dichloromethane / methanol = 100:1-10:1, gradient elution) to obtain TZ-56 as a white solid with a mass of 320 mg and a yield of 97%. The product was identified by LC-MS: MS m / z: C 80 H 114 F5N9O 33 , [M+1] + , theoretical: 1823.74, measured: 1824.29.

[0380] 6.2 Synthesis of TZ-57 (coupling of TZ-56 and RNA modified with 3′-terminal C6 amino group)

[0381]

[0382] In a 1 ml centrifuge tube, dissolve the amino-modified RNA (235 nanomoles, 1.0 equivalents) in a sodium tetraborate (100 micromoles, pH = 8.8) buffer (470 microliters). At the same time, dissolve the pentafluorophenol active ester TZ-56 synthesized in the previous step in anhydrous dimethyl sulfoxide to prepare a 100 micromolar stock solution. Take 50 equivalents of the stock solution and add it to the amino-modified RNA buffer. After vortexing, place the reaction on a cyclotron, heat to 30 degrees, and react at a rate of 500rmp for 2 hours. After LC-MS detection of complete reaction, add 0.5 ml of water and 1 ml of ethyl acetate respectively, retain the aqueous phase after extraction, and then purify by high-performance liquid C18 reverse phase to obtain TZ-57 (200 nanomoles, 85%). The product was identified by LC-MS: MS m / z: [M-1] -1 , theoretical: 8620.2, measured: 8619.8.

[0383] 6.3 Synthesis of TZ-58

[0384]

[0385] Concentrated ammonia (500 μL) was added to the storage tube containing TZ-57 (200 nmol), and the tube was placed in a cyclotron shaker with the shaking rate set to 500 rpm and shaken at room temperature for 1 hour. The deacetylated protected product TZ-58 was then obtained after concentration by a concentrator, and the product could be used for the next annealing without further purification.

[0386] Preparation Example 7: Preparation of Compound L7

[0387] 7.1 Synthesis of TZ-59

[0388]

[0389] Benzyl piperazine-1-carboxylate (2.31 g, 10.50 mmol, 1.05 eq) and potassium carbonate (1.52 g, 11.00 mmol, 1.1 eq) were added to a mixed solvent of N,N-dimethylformamide and water (v / v=1:1, 10 ml), and 5-chloromethyl-2,4-dihydro[1,2,4]triazol-3-one (1.34 g, 10 mmol, 1.0 eq) was dissolved in N,N-dimethylformamide (5 ml) and added dropwise to the reaction. After stirring at room temperature for 2 hours, the mixture was diluted with water to precipitate a white solid, which was filtered. The precipitate was washed with water and a small amount of methyl tert-butyl ether to obtain the target product TZ-59 as a white solid with a mass of 1.96 g and a yield of 62%. The product was identified by LC-MS: MS m / z: C 15 H 19 N5O3, [M+1] -1 , theoretical: 318.15, measured: 318.16.

[0390] 7.2 Synthesis of TZ-60

[0391]

[0392] Compound TZ-59 (957 mg, 3.00 mmol, 1.0 eq) was dissolved in anhydrous N,N-dimethylformamide (5 ml), and added dropwise to a two-necked bottle containing NaH (60%, 360 mg, 9.00 mmol, 3.0 eq) by syringe under ice bath stirring and nitrogen atmosphere protection. After the addition, the mixture was heated to room temperature and stirred for 0.5 hours, and then cooled by ice bath, and a solution of 3-(tert-butyloxycarbonylamino)propyl bromide (2.14 g, 9.00 mmol, 3.0 eq) dissolved in N,N-dimethylformamide (5 ml) was added dropwise to the reaction by syringe, and the mixture was heated to room temperature and stirred for 6 hours. After the reaction was completed by TLC detection, water was added to quench the reaction, and ethyl acetate was added for extraction three times. The combined organic phase was washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure and concentrated. The residue was purified by silica gel flash column chromatography (dichloromethane / methanol = 100:1-10:1, gradient elution). After vacuum pump drying, TZ-60 was obtained as a white solid with a mass of 1.26 g and a yield of 67%. The product was identified by LC-MS: m / z: C 31 H 49 N7O7, [M+1] + , theoretical: 632.37, measured: 632.26.

[0393] 7.3 Synthesis of TZ-61

[0394]

[0395] Compound TZ-60 (300 mg, 0.50 mmol, 1.0 eq) was dissolved in methanol (5 mL) and stirred in an ice bath with a 1,4-dioxane solution of HCl (1 M -1 , 3 ml, 3.00 mmol, 6.0 equiv) was added to the reaction, the reaction was stirred overnight, and the compound TZ-61 was concentrated by distillation under reduced pressure to obtain a white solid, which could be directly used in the next step without purification.

[0396] 7.4 Synthesis of TZ-62

[0397]

[0398] (S)-2,6-bis((tert-butoxycarbonyl)amino)hexanoic acid (433 mg, 1.25 mmol, 2.5 eq), benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU, 569 mg, 1.50 mmol, 3.0 eq) and 1-hydroxybenzotriazole (HOBT, 203 mg, 1.50 mmol, 3.0 eq) were dissolved in anhydrous N,N-dimethylformamide (5 ml), and the reaction system was placed in a nitrogen atmosphere by vacuum pump replacement. N,N-diisopropylethylamine (784 μL, 4.5 mmol, 9.0 eq) was added dropwise to the reaction system while stirring in an ice bath. Then the mixture was heated to room temperature and stirred for 30 minutes. Compound TZ-61 (0.50 mmol, 1.0 equivalent) was dissolved in N,N-dimethylformamide (2.5 ml) and then added dropwise to the reaction mixture. The mixture was stirred overnight at room temperature. After TLC detection, dichloromethane was added to dilute the reaction mixture, and the mixture was washed once with saturated NaHCO3 solution, water and saturated brine, dried over anhydrous sodium sulfate, filtered, distilled and concentrated under reduced pressure. The residue was purified by silica gel flash column chromatography (dichloromethane / methanol = 100:1-10:1, gradient elution). After vacuum pump drying, TZ-62 was obtained as a light yellow solid with a mass of 700 mg and a yield of 52%. The product was identified by LC-MS: m / z: C 53 H 89 N 11 O 13 , [M+Na] + , theoretical: 1110.65, measured: 1110.65.

[0399] 7.5 Synthesis of TZ-63

[0400]

[0401] Compound TZ-62 (350 mg, 0.32 mmol, 1.0 eq) was dissolved in methanol (3 mL) and stirred in an ice bath with a 1,4-dioxane solution of HCl (1 M -1 , 2.5 ml, 2.50 mmol, 8.0 equiv) was added to the reaction, the reaction was stirred overnight, and the compound TZ-63 was concentrated by distillation under reduced pressure to obtain a light yellow solid, which can be directly used in the next step without purification.

[0402] 7.6 Synthesis of TZ-64

[0403]

[0404] Gal-5 (716 mg, 1.60 mmol, 5.0 equiv), benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU, 728 mg, 1.92 mmol, 6.0 equiv) and 1-hydroxybenzotriazole (HOBT, 259 mg, 1.92 mmol, 6.0 equiv) were dissolved in anhydrous N,N-dimethylformamide (10 ml), and the reaction system was placed in a nitrogen atmosphere by vacuum pump replacement. N,N-diisopropylethylamine (670 μL, 3.84 mmol, 12.0 equiv) was added dropwise to the reaction system while stirring in an ice bath. Then warm to room temperature and stir for 30 minutes, then dissolve compound TZ-63 (0.32 mmol, 1.0 equivalent) and N, N-diisopropylethylamine (670 μL, 3.84 mmol, 12.0 equivalent) in N, N-dimethylformamide solution (2.5 ml) and add dropwise to the reaction, and continue stirring overnight. After TLC detection, dichloromethane is added to dilute the reaction, and then washed once with saturated NaHCO3 solution, water and saturated brine, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure and concentrated, and the residue is purified by silica gel flash column chromatography (dichloromethane / methanol = 100:1-10:1, gradient elution), and after vacuum pump drying, TZ-64 is obtained as a white solid with a mass of 592 mg and a yield of 77%. The product is identified by LC-MS: m / z: C 109 H 165 N 15 O 45 , [M / 2+H] + , theoretical: 1203.06, measured: 1203.07.

[0405] 7.7 Synthesis of TZ-65

[0406]

[0407] TZ-64 (592 mg, 0.25 mmol) and palladium / carbon (60 mg, 10% palladium loading) were added to anhydrous methanol (10 ml), and the reaction system was placed in a hydrogen atmosphere by vacuum pump replacement. Then, the mixture was stirred overnight at room temperature, and the palladium / carbon was removed by diatomaceous earth filtration. Compound TZ-65 was obtained after reduced pressure distillation and concentration. This product can be directly used in the next step without further purification. Compound TZ-65 was dried by vacuum pump to obtain a white solid with a mass of 540 mg and a yield of about 97%. The product was identified by LC-MS: MS m / z: C 101 H 159 N 15 O 43 , [M / 2+1] + , theoretical: 1136.04, measured: 1136.05.

[0408] 7.8 Synthesis of TZ-66

[0409]

[0410] Monomethyl dodecanedioate (12.90 g, 52.60 mmol, 1.2 eq) was dissolved in anhydrous N,N-dimethylformamide (100 ml), and benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU, 24.90 g, 65.70 mmol, 1.5 eq) was added. The reaction system was placed in a nitrogen atmosphere by vacuum pump replacement, and then N,N-diisopropylethylamine (30 ml, 131.40 mmol, 3.0 eq) was slowly added under stirring in an ice bath. After stirring at room temperature for 30 minutes, (3R, 5S)-5-{[bis(4-methoxyphenyl)(phenyl)methoxy]methyl}pyrrolidin-3-ol (purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd., 18.38 g, 43.80 mmol, 1.0 eq) was added to the reaction, and the mixture was stirred at room temperature overnight. Add water to quench the reaction. The reaction solution was extracted three times with dichloromethane, and the combined organic phase was washed once with saturated sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure and concentrated, and then purified by silica gel flash column chromatography (dichloromethane / methanol = 100:1-10:1, 0.5% triethylamine was added to mobile phase A / B, gradient elution). After vacuum pump drying, TZ-66 was obtained as a light yellow foamy solid with a mass of 22.33 g and a yield of 79%. The product was identified by LC-MS: MS m / z: C 39 H 51 NO7, [M+1] + , theoretical: 646.37, measured: 646.38.

[0411] 7.9 Synthesis of TZ-67

[0412]

[0413] Compound TZ-66 (6.60 g, 10.23 mmol, 1.0 eq.) was dissolved in methanol (60 mL) and slowly added dropwise to a LiOH aqueous solution (1 M -1, 1.30 g, 30.70 mmol, 3.0 equivalents), then warmed to room temperature and stirred overnight. Under ice bath stirring, 10% acetic acid solution was added dropwise to adjust the pH to neutral, then dichloromethane was added for extraction three times, the combined organic phase was washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure and concentrated, the residue was purified by silica gel flash column chromatography (dichloromethane / methanol = 100:1-10:1, 0.5% triethylamine was added to each mobile phase A / B, gradient elution), and TZ-67 was obtained as a light yellow foamy solid after vacuum pump drying, with a mass of 6.10 g and a yield of 95%. The product was identified by LC-MS: MS m / z: C 38 H 49 NO7, [M+1] + , theoretical: 632.35, measured: 632.34.

[0414] 7.10 Synthesis of TZ-68

[0415]

[0416] Compound TZ-65 (227 mg, 0.10 mmol, 1.0 equiv), TZ-67 (76 mg, 0.12 mmol, 1.2 equiv) and benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU, 49 mg, 0.13 mmol, 1.3 equiv) were dissolved in anhydrous N,N-dimethylformamide (5 ml), and the reaction system was placed in a nitrogen atmosphere by vacuum pump replacement, and then N,N-diisopropylethylamine (52 μL, 0.30 mmol, 3.0 equiv) was slowly added with stirring in an ice bath. The mixture was heated to room temperature and stirred overnight, and dichloromethane was added to dilute the reaction. The mixture was washed with saturated sodium bicarbonate, water and saturated saline, dried over anhydrous sodium sulfate, filtered, evaporated and concentrated under reduced pressure, and purified by C18 reverse phase column chromatography (methanol / water = 0-100% gradient elution). After drying by vacuum pump, TZ-68 (i.e., compound L7) was obtained as a colorless oil with a mass of 228 mg and a yield of about 79%. The product was identified by LC-MS: MS m / z: C 139 H 206 N 16 O 49 , [M / 2+1] + , theoretical: 1442.71, measured: 1442.69.

[0417] 7.11 Synthesis of TZ-69

[0418]

[0419] TZ-68 (228 mg, 0.08 mmol, 1.0 equiv), succinic anhydride (237 mg, 0.24 mmol, 3.0 equiv) and 4-dimethylaminopyridine (29 mg, 0.24 mmol, 3.0 equiv) were dissolved in anhydrous dichloromethane (5 ml), and the reaction system was placed in a nitrogen atmosphere by vacuum pump replacement, and N,N-diisopropylethylamine (70 μL, 0.40 mmol, 5.0 equiv) was added with stirring in an ice bath. Then the reaction was heated to room temperature and stirred overnight. After LC-MS detected that the reaction was complete, water was added to quench the reaction. The reaction system was extracted three times with dichloromethane. The combined organic phase was washed once with a saturated ammonium chloride solution and a saturated saline solution, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure and concentrated, and purified by C18 reverse phase column chromatography (methanol / water = 0-100% gradient elution). After vacuum pump drying, TZ-69 was obtained as a colorless oil with a mass of 160 mg and a yield of about 68%. The product was identified by LC-MS: MS m / z: C 143 H 210 N 16 O 52 , [M / 2+1] + , theoretical: 1492.72, measured: 1492.71.

[0420] 7.12 Synthesis of TZ-70

[0421]

[0422]

[0423] TZ-69 (160 mg, 0.05 mmol, 1.0 eq.) and HBTU (24 mg, 0.06 mmol, 1.2 eq.) were dissolved in anhydrous N,N-dimethylformamide (4 ml), and then N,N-diisopropylethylamine (37 μl, 0.21 mmol, 4.0 eq.) was added. After shaking for 15 minutes, aminosilylated CPG (purchased from Hebei Dinaxingke Biotechnology Co., Ltd., 80 μmol / g, 625 mg), and then shake the reaction for 24 hours. After the reaction is completed, filter; CPG is washed three times with N, N-dimethylformamide, dichloromethane / methanol (v / v, 9:1) and dichloromethane, and then dried by vacuum pump. Then acetic anhydride / pyridine (v / v, 1:3, 12 ml) is added to the CPG filter cake, and shake the reaction for 30 minutes to cap the unconnected amino group with acetyl group, and then filter again. The CPG filter cake is washed three times with dichloromethane / methanol (v / v, 9:1) and dichloromethane, and then completely dried by vacuum pump. The product TZ-70 is obtained, which is CPG connected with L7 ligand. The yield is 620 mg, and the sample loading is measured as 26.5 μmol / g by detecting the absorption of 4,4′-dimethoxytrityl group (DMTr).

[0424] Preparation Example 8: Preparation of Compound L8

[0425] 8.1 Synthesis of TZ-71

[0426]

[0427] TZ-42 (980 mg, 2.00 mmol, 1.0 eq) and 8-(benzyloxy)-8-oxooctanoic acid (528 mg, 2.00 mmol, 1.0 eq) were dissolved in anhydrous N,N-dimethylformamide (5 ml), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (988 mg, 2.60 mmol, 1.3 eq) was added, and the reaction system was placed in a nitrogen atmosphere by vacuum pump replacement, and then N,N-diisopropylethylamine (1.04 ml, 6.00 mmol, 3.0 eq) was added while stirring in an ice bath, and the mixture was stirred at room temperature overnight, and then the reaction was quenched by adding water. The reaction solution was extracted three times with dichloromethane, and the combined organic phase was washed once with saturated sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure and concentrated, and then purified by silica gel flash column chromatography (dichloromethane / methanol = 100:1-10:1, 0.5% triethylamine was added to mobile phase A / B, gradient elution), and TZ-71 was obtained as a brown oil after vacuum pump drying, with a mass of 1.15 g and a yield of 78%. The product was identified by LC-MS: MS m / z: C 45 H 52 N2O8, [M+1] + , theoretical: 736.37, measured: 736.38.

[0428] 8.2 Synthesis of TZ-72

[0429]

[0430] TZ-71 (567 mg, 0.77 mmol) and palladium / carbon (57 mg, 10% palladium loading) were added to anhydrous methanol (5 ml), and the reaction system was placed in a hydrogen atmosphere by vacuum pump replacement. Then, the mixture was stirred overnight at room temperature, and the palladium / carbon was removed by diatomaceous earth filtration. After vacuum distillation and concentration, compound TZ-72 was obtained. After drying by vacuum pump, it was a white foamy solid with a mass of 498 mg and a yield of about 100%. This product can be directly used in the next step without further purification. The product was identified by LC-MS: MS m / z: C 37 H 46 N2O8, [M+1] + , theoretical: 647.33, measured: 647.32.

[0431] 8.3 Synthesis of TZ-73

[0432]

[0433] Compound TZ-65 (190 mg, 0.084 mmol, 1.0 equiv), TZ-72 (54 mg, 0.084 mmol, 1.0 equiv) and benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU, 41 mg, 0.109 mmol, 1.3 equiv) were dissolved in anhydrous N,N-dimethylformamide (5 ml), and the reaction system was placed in a nitrogen atmosphere by vacuum pump replacement, and then N,N-diisopropylethylamine (44 μL, 0.251 mmol, 3.0 equiv) was slowly added with stirring in an ice bath. The mixture was heated to room temperature and stirred overnight, and dichloromethane was added to dilute the reaction, and washed with saturated sodium bicarbonate, water and saturated brine respectively, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure and concentrated, and purified by C18 reverse phase column chromatography (methanol / water = 0-100% gradient elution). After vacuum pump drying, TZ-73 (i.e., compound L8) was obtained as a colorless oil with a mass of 184 mg and a yield of about 76%. The product was identified by LC-MS: MS m / z: C 138 H 203 N 17 O 50 , [M / 2+1] + , theoretical: 1450.19, measured: 1450.17.

[0434] 8.4 Synthesis of TZ-74

[0435]

[0436] TZ-73 (120 mg, 0.041 mmol, 1.0 equiv), succinic anhydride (12 mg, 0.124 mmol, 3.0 equiv) and 4-dimethylaminopyridine (15 mg, 0.124 mmol, 3.0 equiv) were dissolved in anhydrous dichloromethane (5 ml), and the reaction system was placed in a nitrogen atmosphere by vacuum pump replacement, and N,N-diisopropylethylamine (36 μL, 0.207 mmol, 5.0 equiv) was added with stirring in an ice bath. The reaction was heated to room temperature and stirred overnight. After the reaction was complete by LC-MS, water was added to quench the reaction. The reaction system was extracted three times with dichloromethane. The combined organic phase was washed once with a saturated ammonium chloride solution and a saturated saline solution, dried over anhydrous sodium sulfate, filtered, distilled and concentrated under reduced pressure, and purified by C18 reverse phase column chromatography (methanol / water = 0-100% gradient elution). After vacuum pump drying, TZ-74 was obtained as a colorless oil with a mass of 93 mg and a yield of about 76%. The product was identified by LC-MS: MS m / z: C 142 H 207 N 17 O 53 , [M / 2+1] + , theoretical: 1500.20, measured: 1500.22.

[0437] 8.5 Synthetic TZ-75

[0438]

[0439] TZ-74 (93 mg, 0.031 mmol, 1.0 eq.) and HBTU (14 mg, 0.037 mmol, 1.2 eq.) were dissolved in anhydrous N,N-dimethylformamide (4 ml), and then N,N-diisopropylethylamine (21 μl, 0.124 mmol, 4.0 eq.) was added. After shaking for 15 minutes, aminosilyl CPG (purchased from Hebei Dinaxingke Biotechnology Co., Ltd., 80 μmol / g, 387 mg, 1.0 equivalent), and then shake the reaction for 24 hours. After the reaction is completed, filter; CPG is washed three times with N, N-dimethylformamide, dichloromethane / methanol (v / v, 9:1) and dichloromethane, and then dried by a vacuum pump. Then acetic anhydride / pyridine (v / v, 1:3, 12 ml) is added to the CPG filter cake, and shake the reaction for 30 minutes to cap the unconnected amino group with the acetyl group, and then filter again. The CPG filter cake is washed three times with dichloromethane / methanol (v / v, 9:1) and dichloromethane, and then completely dried by a vacuum pump. The product TZ-75 is obtained, which is CPG connected to the L8 ligand. The yield is 390 mg, and the sample loading is measured to be 22.3 μmol / g by detecting the absorption of the 4,4′-dimethoxytrityl group (DMTr).

[0440] Preparation Example 9: Preparation of Compound L9

[0441] 9.1 Synthesis of TZ-76

[0442]

[0443] N′-tert-Butyloxycarbonyl-L-lysine benzyl ester hydrochloride (3.73 g, 10.00 mmol, 1.0 eq) and Boc-4-aminobutyric acid (2.44 g, 12.00 mmol, 1.2 eq) were dissolved in anhydrous N,N-dimethylformamide (10 ml), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (5.70 g, 15.00 mmol, 1.5 eq) was added, and the reaction system was placed in a nitrogen atmosphere by vacuum pump replacement, and then N,N-diisopropylethylamine (5.23 ml, 30.00 mmol, 3.0 eq) was added while stirring in an ice bath, and the mixture was stirred at room temperature overnight, and then the reaction was quenched by adding water. The reaction solution was extracted three times with dichloromethane, and the combined organic phase was washed once with saturated sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure and concentrated, and purified by silica gel flash column chromatography (petroleum ether / ethyl acetate = 10:1-1:1 gradient elution). After vacuum pump drying, TZ-76 was obtained as a white foamy solid with a mass of 4.70 g and a yield of 90%. The product was identified by LC-MS: MS m / z: C 27 H 43 N3O7, [M+1] + , theoretical: 522.32 measured: 522.33.

[0444] 9.2 Synthesis of TZ-77

[0445]

[0446] TZ-76 (4.65 g, 8.92 mmol) and palladium / carbon (465 mg, 10% palladium loading) were added to anhydrous methanol (25 ml), and the reaction system was placed in a hydrogen atmosphere by vacuum pump replacement. Then, the mixture was stirred overnight at room temperature, and the palladium / carbon was removed by diatomaceous earth filtration. After vacuum distillation and concentration, compound TZ-77 was obtained. After drying by vacuum pump, it was a white foamy solid with a mass of 3.85 g and a yield of about 100%. This product can be directly used in the next step without further purification. The product was identified by LC-MS: MS m / z: C 20 H 37 N3O7, [M+1] + , theoretical: 432.27, measured: 432.26.

[0447] 9.3 Synthesis of TZ-78

[0448]

[0449] TZ-77 (4.38 g, 10.15 mmol, 2.5 eq.), benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU, 4.61 mg, 12.18 mmol, 3.0 eq.) and 1-hydroxybenzotriazole (HOBT, 1.64 g, 12.18 mmol, 3.0 eq.) were dissolved in anhydrous N,N-dimethylformamide (25 ml). The reaction system was then placed in a nitrogen atmosphere by vacuum pump replacement. N,N-diisopropylethylamine (5.20 ml, 36.54 mmol, 9.0 eq.) was added dropwise to the reaction system while stirring in an ice bath. Then the temperature was raised to room temperature and stirred for 30 minutes. Compound TZ-61 (2.20 g, 4.06 mmol, 1.0 eq.) and N,N-diisopropylethylamine (2.60 ml, 18.27 mmol, 4.5 eq.) were dissolved in N,N-dimethylformamide (10 ml) and then added dropwise to the reaction mixture. The mixture was stirred overnight at room temperature. After TLC detection, dichloromethane was added to dilute the reaction mixture, and the mixture was washed once with saturated NaHCO3 solution, water and saturated saline solution, dried over anhydrous sodium sulfate, filtered, distilled and concentrated under reduced pressure. The residue was purified by silica gel flash column chromatography (dichloromethane / methanol = 100:1-10:1, gradient elution). After vacuum pump drying, TZ-78 was obtained as a light yellow solid with a mass of 2.26 g and a yield of 44%. The product was identified by LC-MS: m / z: C 61 H 103 N 13 O 15 , [M+H] + , theoretical: 1258.78, measured: 1258.77.

[0450] 9.4 Synthesis of TZ-79

[0451]

[0452] Compound TZ-78 (1.13 g, 0.90 mmol, 1.0 eq.) was dissolved in methanol (5 mL) and stirred in an ice bath with a 1,4-dioxane solution of HCl (1 M -1 , 3 ml, 3.00 mmol, 6.0 equiv) was added to the reaction, the reaction was stirred overnight, and the compound TZ-79 was concentrated by distillation under reduced pressure to obtain a white solid, which could be directly used in the next step without purification.

[0453] 9.5 Synthetic TZ-80

[0454]

[0455] Gal-5 (2.01 g, 4.49 mmol, 5.0 equiv), benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU, 2.04 g, 5.39 mmol, 6.0 equiv) and 1-hydroxybenzotriazole (HOBT, 0.73 g, 5.39 mmol, 6.0 equiv) were dissolved in anhydrous N,N-dimethylformamide (10 ml), and the reaction system was placed in a nitrogen atmosphere by vacuum pump replacement. N,N-diisopropylethylamine (1.89 ml, 10.78 mmol, 12.0 equiv) was added dropwise to the reaction system while stirring in an ice bath. Then warm to room temperature and stir for 30 minutes, then dissolve compound TZ-79 (0.90 mmol, 1.0 equivalent) and N, N-diisopropylethylamine (1.89 ml, 10.78 mmol, 12.0 equivalent) in N, N-dimethylformamide solution (10 ml) and add dropwise to the reaction, and continue stirring overnight. After TLC detection, dichloromethane is added to dilute the reaction, and then washed once with saturated NaHCO3 solution, water and saturated brine, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure and concentrated, and the residue is purified by silica gel flash column chromatography (dichloromethane / methanol = 100:1-10:1, gradient elution), and after vacuum pump drying, TZ-80 is obtained as a yellow foamy solid with a mass of 1.46 g and a yield of 63%. The product is identified by LC-MS: m / z: C 117 H 179 N 17 O 47 , [M / 2+H] + , theoretical: 1288.11, measured: 1288.09.

[0456] 9.6 Synthesis of TZ-81

[0457]

[0458] TZ-80 (1.46 g, 0.57 mmol) and palladium / carbon (146 mg, 10% palladium loading) were added to anhydrous methanol (10 ml), and the reaction system was placed in a hydrogen atmosphere by vacuum pump replacement. Then, the mixture was stirred overnight at room temperature, and the palladium / carbon was removed by diatomaceous earth filtration. After vacuum distillation and concentration, compound TZ-81 was obtained. After drying by vacuum pump, it was a light gray foamy solid with a mass of 1.38 g and a yield of about 100%. This product can be directly used in the next step without further purification. The product was identified by LC-MS: MS m / z: C 109 H 173 N 17 O 45 , [M / 2+1] + , theoretical: 1221.10, measured: 1221.08.

[0459] 9.6 Synthesis of TZ-82

[0460]

[0461] Compound TZ-81 (1.35 g, 0.59 mmol, 1.0 eq), TZ-72 (384 mg, 0.59 mmol, 1.0 eq) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 291 mg, 0.77 mmol, 1.3 eq) were dissolved in anhydrous N,N-dimethylformamide (15 ml), and the reaction system was placed in a nitrogen atmosphere by vacuum pump replacement, and then N,N-diisopropylethylamine (307 ml, 1.77 mmol, 3.0 eq) was slowly added with stirring in an ice bath. The mixture was heated to room temperature and stirred overnight, and dichloromethane was added to dilute the reaction, and washed with saturated sodium bicarbonate, water and saturated brine respectively, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure and concentrated, and purified by C18 reverse phase column chromatography (methanol / water = 0-100% gradient elution). After vacuum pump drying, TZ-82 (i.e., compound L9) was obtained as a white foamy solid with a mass of 1.28 g and a yield of about 75%. The product was identified by LC-MS: MS m / z: C 138 H 203 N 17 O 50 , [M / 2+1] + , theoretical: 1450.20, measured: 1450.17.

[0462] 9.7 Synthesis of TZ-83

[0463]

[0464] TZ-82 (1.25 g, 0.41 mmol, 1.0 equiv), succinic anhydride (122 mg, 1.22 mmol, 3.0 equiv) and 4-dimethylaminopyridine (149 mg, 1.22 mmol, 3.0 equiv) were dissolved in anhydrous dichloromethane (10 ml), and the reaction system was placed in a nitrogen atmosphere by vacuum pump replacement, and N,N-diisopropylethylamine (354 μL, 2.04 mmol, 5.0 equiv) was added with stirring in an ice bath. The reaction was heated to room temperature and stirred overnight. After LC-MS detected that the reaction was complete, water was added to quench the reaction. The reaction system was extracted three times with dichloromethane. The combined organic phase was washed once with a saturated ammonium chloride solution and a saturated saline solution, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure and concentrated, and purified by C18 reverse phase column chromatography (methanol / water = 0-100% gradient elution). After vacuum pump drying, TZ-83 was obtained as a white foamy solid with a mass of 1.05 g and a yield of about 81%. The product was identified by LC-MS: MS m / z: C 150 H221 N 19 O 55 , [M / 2+1] + , theoretical: 1585.26, measured: 1585.25.

[0465] 9.8 Synthesis of TZ-84

[0466]

[0467] TZ-83 (317 mg, 0.10 mmol, 1.0 eq.) and HBTU (45 mg, 0.12 mmol, 1.2 eq.) were dissolved in anhydrous N,N-dimethylformamide (8 ml), and then N,N-diisopropylethylamine (70 μl, 0.40 mmol, 4.0 eq.) was added. After shaking for 15 minutes, aminosilylated CPG (purchased from Hebei Dinaxingke Biotechnology Co., Ltd., 80 micromol / g, 1.25 g, 1.0 equivalent), and then shake the reaction for 24 hours. After the reaction is completed, filter; CPG is washed three times with N, N-dimethylformamide, dichloromethane / methanol (v / v, 9:1) and dichloromethane, and then dried by a vacuum pump. Then acetic anhydride / pyridine (v / v, 1:3, 12 ml) is added to the CPG filter cake, and shake the reaction for 30 minutes to cap the unconnected amino group with the acetyl group, and then filter again. The CPG filter cake is washed three times with dichloromethane / methanol (v / v, 9:1) and dichloromethane, and then completely dried by a vacuum pump. The product TZ-75 is obtained, which is CPG connected to the L9 ligand. The yield is 1.27 grams, and the sample loading is measured to be 38.3 micromol / g by detecting the absorption of the 4,4′-dimethoxytrityl group (DMTr).

[0468] Preparation Example 10: Preparation of Compound L10

[0469] 10.1 Synthesis of TZ-85

[0470]

[0471] Dissolve 3,5-dihydroxybenzoic acid methyl ester (0.84 g, 5.00 mmol, 1.0 eq) and 3-(tert-butyloxycarbonylamino)propyl bromide (3.57 g, 15.00 mmol, 3.0 eq) in anhydrous N,N-dimethylformamide (10 ml), then add anhydrous potassium carbonate (2.07 g, 15.00 mmol, 3.0 eq), and then replace the reaction system with nitrogen atmosphere by vacuum pump. The reaction was heated to 60 degrees and stirred overnight, then water was added to dilute the reaction, and the precipitated white solid was filtered and dried to obtain the product TZ-85, with a mass of 2.40 g and a yield of about 99%. The product was identified by LC-MS: MS m / z: C24 H 38 N2O8, [M+1] + , theoretical: 483.27, measured: 483.26.

[0472] 10.2 Synthesis of TZ-86

[0473]

[0474] Compound TZ-85 (2.40 g, 5.00 mmol, 1.0 eq) and potassium hydroxide (393 mg, 7.00 mmol, 1.4 eq) were dissolved in a mixed solvent of methanol and water (50 ml, v / v 4:1) and heated under reflux for 2 hours. After the reaction was cooled to room temperature, the reaction was adjusted to pH = 5 by acetic acid. The reaction was extracted three times with ethyl acetate, and the combined organic phase was washed three times with water, dried over anhydrous sodium sulfate, filtered, and distilled under reduced pressure and concentrated to obtain compound TZ-86 as a white solid with a mass of 2.04 g and a yield of about 87%.

[0475] 10.3 Synthesis of TZ-87

[0476]

[0477] TZ-86 (1.55 g, 3.31 mmol, 2.5 equivalents), TZ-61 (0.72 g, 1.67 mmol, 1.0 equivalents) and N,N-diisopropylethylamine (2.62 ml, 15.03 mmol, 9.0 equivalents) were dissolved in anhydrous N,N-dimethylformamide (25 ml), and the reaction system was placed in a nitrogen atmosphere by vacuum pump replacement. Benzotriazol-1-yloxy tris (dimethylamino) phosphonium hexafluorophosphate (Carter condensation agent, 1.75 g, 9.93 mmol, 3.0 equivalents) was added to the reaction system under ice bath stirring. Stirring was continued for 30 minutes under ice bath, and then heated to room temperature and stirred overnight. After the reaction was completed by TLC detection, dichloromethane was added to dilute the reaction, and then washed once with saturated NaHCO3 solution, water and saturated brine, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure and concentrated. The residue was purified by silica gel flash column chromatography (dichloromethane / methanol = 100:1-10:1, gradient elution), and after vacuum pumping, TZ-87 was obtained as a light yellow oil with a mass of 1.43 g and a yield of 64%. The product was identified by LC-MS: m / z: C 67 H 101 N 11 O 17 , [M+H] + , theoretical: 1332.74, measured: 1332.73.

[0478] 10.4 Synthesis of TZ-88

[0479]

[0480] Compound TZ-87 (1.43 g, 1.07 mmol, 1.0 eq) was dissolved in methanol (5 mL) and stirred in an ice bath with a 1,4-dioxane solution of HCl (1 M -1 , 6.5 ml, 6.44 mmol, 6.0 equiv) was added to the reaction, the reaction was stirred overnight, and the mixture was concentrated by distillation under reduced pressure to obtain compound TZ-88 as a light yellow oil, which could be directly used in the next step without purification.

[0481] 10.5 Synthesis of TZ-89

[0482]

[0483] Gal-5 (2.43 g, 5.44 mmol, 5.0 equiv), benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU, 2.47 g, 6.48 mmol, 6.0 equiv) and 1-hydroxybenzotriazole (HOBT, 0.88 g, 6.48 mmol, 6.0 equiv) were dissolved in anhydrous N,N-dimethylformamide (10 ml), and the reaction system was placed in a nitrogen atmosphere by vacuum pump replacement. N,N-diisopropylethylamine (2.2 ml, 12.96 mmol, 12.0 equiv) was added dropwise to the reaction system while stirring in an ice bath. Then the mixture was heated to room temperature and stirred for 30 minutes. Then compound TZ-79 (1.00 g, 1.08 mmol, 1.0 equivalent) and N,N-diisopropylethylamine (2.2 ml, 12.96 mmol, 12.0 equivalent) were dissolved in N,N-dimethylformamide solution (10 ml) and added dropwise to the reaction mixture. The mixture was stirred overnight. After the reaction was completed by TLC detection, dichloromethane was added to dilute the reaction mixture, and the mixture was washed once with saturated NaHCO3 solution, water and saturated saline solution, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure and concentrated. The residue was purified by silica gel flash column chromatography (dichloromethane / methanol = 100:1-10:1, gradient elution). After drying with a vacuum pump, TZ-64 was obtained as a yellow foamy solid with a mass of 2.09 g and a yield of 73%. The product was identified by LC-MS: m / z: C 123 H 177 N 15 O 49 , [M / 2+H] + , theoretical: 1325.10, measured: 1325.09.

[0484] 10.6 Synthesis of TZ-90

[0485]

[0486] TZ-89 (2.09 g, 0.79 mmol) and palladium / carbon (209 mg, 10% palladium loading) were added to anhydrous methanol (14 ml), and the reaction system was placed in a hydrogen atmosphere by vacuum pump replacement. Then, the mixture was stirred overnight at room temperature, and the palladium / carbon was removed by diatomaceous earth filtration. After vacuum distillation and concentration, compound TZ-90 was obtained. After drying by vacuum pump, it was a light gray foamy solid with a mass of 1.98 g and a yield of about 100%. This product can be directly used in the next step without further purification. The product was identified by LC-MS: MS m / z: C 115 H 171 N 15 O 47 , [M / 2+1] + , theoretical: 1258.08, measured: 1258.05.

[0487] 10.7 Synthesis of TZ-91

[0488]

[0489] Compound TZ-90 (1.35 g, 0.59 mmol, 1.0 eq), TZ-72 (384 mg, 0.59 mmol, 1.0 eq) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 291 mg, 0.77 mmol, 1.3 eq) were dissolved in anhydrous N,N-dimethylformamide (15 ml), and the reaction system was placed in a nitrogen atmosphere by vacuum pump replacement, and then N,N-diisopropylethylamine (307 ml, 1.77 mmol, 3.0 eq) was slowly added with stirring in an ice bath. The mixture was heated to room temperature and stirred overnight, and dichloromethane was added to dilute the reaction, and washed with saturated sodium bicarbonate, water and saturated brine respectively, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure and concentrated, and purified by C18 reverse phase column chromatography (methanol / water = 0-100% gradient elution). After vacuum pump drying, TZ-82 (i.e., compound L10) was obtained as a white foamy solid with a mass of 1.28 g and a yield of about 75%. The product was identified by LC-MS: MS m / z: C 138 H 203 N 17 O 50 , [M / 2+1] + , theoretical: 1450.20, measured: 1450.17.

[0490] 10.8 Synthesis of TZ-92

[0491]

[0492]

[0493] TZ-91 (1.25 g, 0.40 mmol, 1.0 equiv), succinic anhydride (119 mg, 1.19 mmol, 3.0 equiv) and 4-dimethylaminopyridine (146 mg, 1.19 mmol, 3.0 equiv) were dissolved in anhydrous dichloromethane (8 ml), and the reaction system was placed in a nitrogen atmosphere by vacuum pump replacement, and N,N-diisopropylethylamine (348 μL, 2.00 mmol, 5.0 equiv) was added with stirring in an ice bath. The reaction was heated to room temperature and stirred overnight. After the reaction was complete by LC-MS, water was added to quench the reaction. The reaction system was extracted three times with dichloromethane. The combined organic phase was washed once with a saturated ammonium chloride solution and a saturated saline solution, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure and concentrated, and purified by C18 reverse phase column chromatography (methanol / water = 0-100% gradient elution). After vacuum pump drying, TZ-92 was obtained as a white foamy solid with a mass of 1.02 g and a yield of about 79%. The product was identified by LC-MS: MS m / z: C 156 H 219 N 17 O 57 , [M / 2+1] + , theoretical: 1622.25, measured: 1622.23.

[0494] 10.9 Synthesis of TZ-93

[0495]

[0496]

[0497] TZ-92 (324 mg, 0.10 mmol, 1.0 eq.) and HBTU (45 mg, 0.12 mmol, 1.2 eq.) were dissolved in anhydrous N,N-dimethylformamide (8 ml), and then N,N-diisopropylethylamine (70 μl, 0.40 mmol, 4.0 eq.) was added. After shaking for 15 minutes, aminosilylated CPG (purchased from Hebei Dinaxingke Biotechnology Co., Ltd., 80 micromol / g, 1.25 g, 1.0 equivalent), and then shake the reaction for 24 hours. After the reaction is completed, filter; CPG is washed three times with N, N-dimethylformamide, dichloromethane / methanol (v / v, 9:1) and dichloromethane, and then dried by a vacuum pump. Then acetic anhydride / pyridine (v / v, 1:3, 12 ml) is added to the CPG filter cake, and shake the reaction for 30 minutes to cap the unconnected amino group with the acetyl group, and then filter again. The CPG filter cake is washed three times with dichloromethane / methanol (v / v, 9:1) and dichloromethane, and then completely dried by a vacuum pump. The product TZ-75 is obtained, which is CPG connected to the L10 ligand. The yield is 1.26 grams, and the sample loading is measured to be 40.2 micromol / g by detecting the absorption of the 4,4′-dimethoxytrityl group (DMTr).

[0498] Preparation Example 11: Synthesis of oligonucleotides and oligonucleotide conjugates

[0499] The oligonucleotides and oligonucleotide conjugates of the present invention are synthesized by the phosphoramidite solid phase synthesis method well known in the art. According to the nucleotide types and sequence of the oligonucleotides, the nucleoside monomers are sequentially connected from 3' to 5', and the connection of each nucleoside monomer must go through an identical cycle, including four steps of deprotection, coupling, capping, oxidation or sulfurization.

[0500] 1. Synthesis of sense strand (SS strand) and antisense strand (AS strand)

[0501] By solid phase phosphoramidite synthesis, using a blank CPG solid phase support or the CPG solid phase support connected with the GalNAc ligand (L1, L2, L3, L4, L5, L7, L8, L9, L10) of the present invention as the starting cycle, the nucleoside monomers are sequentially connected from 3' to 5' according to the nucleotide sequence, and the connection of each nucleoside monomer must go through an identical cycle, including four steps of deprotection, coupling, capping, oxidation or sulfurization. The synthesis conditions of the oligonucleotide with a synthesis scale of 500nmol are as follows:

[0502] Nucleoside monomers were prepared into 0.04 mol / L acetonitrile solution and Molecular sieve drying, the reaction conditions for each step are the same. The deprotection reagent is a 3% dichloromethane solution, the deprotection is three times, and the reaction time is 30 seconds. The coupling agent is a 0.25 mol / L acetonitrile solution of 5-ethylthio 1H-tetrazole, the coupling is three times, and the reaction time is 3 minutes. The coupling reaction is carried out in anhydrous acetonitrile. Capping uses 20% acetic anhydride-acetonitrile solution and N-methylimidazole / pyridine / acetonitrile solution (20 / 30 / 50, v / v / v), capping twice; oxidation uses 0.05 mol / L iodine pyridine / water solution (90 / 10, v / v); thiolation uses 0.20 mol / L 5-imino-1,2,4-dithiazolidine-3-thione / pyridine solution, thiolation twice.

[0503] 2. Oligonucleotide Purification and Annealing

[0504] 2.1 Aminolysis

[0505] The CPG carrier that has completed the synthesis step is added to a 5 mL centrifuge tube, and 1 mL of 25% to 28% concentrated ammonia water is added. The mixture is heated at 80 degrees in a blast oven for 2 hours or at 60 degrees for 6 hours. The mixture is then filtered, and the solid carrier is washed three times with water. The filtrate is concentrated by a concentrator to obtain a crude product for the next step of purification.

[0506] 2.2 Deprotection

[0507] When there is at least one 2′-TBDMS protection on the synthesized nucleotide sequence, the method further comprises contacting the crude product after the aminolysis step with triethylamine trihydrofluoride to remove the 2′-TBDMS protection. The deprotection reagent uses N-methylimidazole / triethylamine hydrofluoride / triethylamine solution (6 / 3 / 4, v / v / v) and reacts at 65 degrees for 2.5 hours.

[0508] 2.3 Purification

[0509] The methods of purification and desalting are well known to those skilled in the art. For example, a preparative ion chromatography purification column can be used to elute the nucleic acid by gradient elution with NaBr or NaCl; after the products are collected and combined, a reverse phase chromatography purification column can be used for desalting. The fractions with a purity greater than 95% are collected, concentrated and dried, and prepared into a PBS solution with a concentration of 100 μM.

[0510] 2.4 Annealing

[0511] The PBS solutions of the sense chain (SS chain) and the antisense chain (AS chain) were mixed in a centrifuge tube at a molar ratio (SS chain / AS chain = 1.1 / 1), heated to 85°C, maintained for 3-5 minutes, naturally cooled to room temperature, and the system was freeze-dried to obtain the product.

[0512] According to the above method, oligonucleotide conjugates 1-10 shown in Table 1 (the table shows that the compound is covalently linked to siRNA by forming a phosphate group) and the positive control group conjugate 11 shown in Table 2 were synthesized. The sequence of mTTR-siRNA is shown in Table 3. The information of the siRNA conjugate formed by the compound of the present invention and siRNA is shown in Table 4.

[0513] Table 1: Conjugates of the compounds of the present invention coupled with siRNA

[0514]

[0515]

[0516]

[0517] Table 2: Conjugates in the control group

[0518]

[0519] Table 3: Sequences of mTTR-siRNA (SEQ ID NO: 1 and 2)

[0520]

[0521] Note: The sequence table only shows the base sequence of RNA and does not show modifications.

[0522] Table 4: siRNA conjugates

[0523]

[0524]

[0525] Note: S: positive strand; AS: antisense strand; capital letters A, C, G, and U represent the base composition of nucleotides; 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′-fluorine-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; L indicates a conjugate.

[0526] Activity test of the oligonucleotide conjugate of the present invention

[0527] The inhibitory efficiency of the oligonucleotide conjugates of the present invention (1#, 2#, 3#, 4#, 5#, 6#, 7#, 8#, 9#, 10#), the positive control group conjugate 11# and the negative control 1×PBS on the expression of transthyretin (TTR) mRNA in C57B / 6j mice was investigated.

[0528] The C57B / 6j mice used in this example were purchased from Vital River, about 8 to 10 weeks old, male.

[0529] First, C57B / 6j mice were divided into 11 groups, namely, vehicle control group, positive control group, experimental groups 1-9 (using siRNA conjugates numbered 1-9 in Table 4, respectively), 3 mice in each group, injected at a dose of 5 mg / kg (in terms of siRNA content), injection volume 5 ml / kg, single subcutaneous administration, and shaved at the injection site. After 2 days, 7 days and 28 days of administration, the mice were killed, the livers were collected, the livers were ground into homogenate with Trizol at 4°C by a freezing grinder, and the total RNA of the liver tissue was extracted according to standard procedures.

[0530] Real-time fluorescence quantitative PCR was used to detect the expression level of TTR mRNA in mouse liver tissue. TM The extracted total RNA was reverse transcribed into cDNA according to the instructions of II 1st Strand cDNA Synthesis Kit: 6210A, and then the fluorescent quantitative PCR kit (Thermo TaqMan TM Fast Advanced Master Mix: 4444964) was used to detect the inhibition rate of siRNA on TTR mRNA expression in liver tissue. In this fluorescent quantitative PCR method, the mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an internal reference gene, and primers for mouse TTR and primers for mouse glyceraldehyde-3-phosphate dehydrogenase were used to detect mTTR and glyceraldehyde-3-phosphate dehydrogenase, respectively. The sequences of the primers are shown in Table 5.

[0531] Table 5: Sequences of detection primers (SEQ ID NO: 3-8)

[0532]

[0533] In this fluorescence quantitative PCR method, siRNA inhibitory activity was measured by the residual amount of TTR gene expression. -△△Ct It means that △△Ct=[(target gene in Ct experimental group-internal reference in Ct experimental group)-(target gene in Ct negative control group-internal reference in Ct negative control group)]. Then, the mRNA inhibition rate was calculated according to the following formula: mRNA inhibition rate=(1-remaining amount of mTTR gene expression)×100%. The inhibition rate experimental results of each experimental group are shown in Table 6.

[0534] Table 6: Inhibition of mTTR mRNA expression in mouse liver by siRNA conjugates

[0535]

[0536]

[0537] From the results in Table 6, it can be seen that the oligonucleotide conjugates of the present invention showed a high inhibition rate on the expression of TTR mRNA; in particular, experimental groups 3 and 4 showed an inhibitory activity superior to that of the positive control siRNA conjugate. At the same time, from the trend from 2 days to 28 days, experimental groups 3 and 4 also showed excellent long-term effects.

[0538] The ELISA method was used to detect the content of transthyretin (TTR) in mouse serum. Specifically, the mouse serum of each experimental group was detected using a mouse Prealbumin ELISA kit (abcam, ab282297) according to the instructions, and the OD450nm absorbance was read.

[0539] In the TTR protein detection, the protein inhibition activity is expressed by the relative inhibition rate of TTR protein expression, TTR protein inhibition rate % = [(absorbance of negative control group - absorbance of blank) - (absorbance of experimental group - absorbance of blank)] / (absorbance of negative control group - absorbance of blank) * 100%. The experimental results of the relative inhibition rate of protein expression of each experimental group are shown in Table 7.

[0540] Table 7: Relative inhibition of TTR protein expression in mouse liver by siRNA conjugates

[0541]

[0542]

[0543] It can be seen from the results in Table 7 that the oligonucleotide conjugates of the present invention exhibit a high inhibition rate on the expression of TTR protein; in particular, experimental group 4 showed an inhibitory activity superior to that of the positive control siRNA conjugate. At the same time, from the trend from 2 days to 28 days, experimental group 4 also showed excellent long-term effect.

Claims

1. A conjugate or a pharmaceutically acceptable salt thereof, characterized in that: The conjugate contains a liver cell-targeting structure shown in the following formula II or an isomer or isotopic variant thereof: In the formula, two of R1', R2' and R3' are monovalent groups, each of which independently contains a ligand having affinity for the asialoglycoprotein receptor on the surface of mammalian liver cells, and the remaining one is a divalent group, which contains a structure for covalently linking to a nucleic acid molecule; wherein: The monovalent groups are each independently selected from the following structures: -L1-X1-L1'-E, -CH(L2-X2-L2'-E)(X3-L3-E), -L4-X4-CH(X4-L4'-E)(L4'-X4-L4"-E), and -L5-X5-CH(X5-L5'-E)(L5'-X5-L5"-E); The divalent group is -L6-X6-L7-X7-L8'-R4'-; L1, L1', L2, L2', L3, L4, L4', L4", L5, L5' and L5" are each independently C1-C 20 Alkylene; X1, X2, X3, X4 and X5 are each independently selected from -CO-, -NH-, -CO-NH-, -NH-CO-, -CH=N-, -S(O)2-, C2-C 10 Alkenylene, C2~C 10 Alkynylidene, C6~C 14 Arylene, C3~C 18 Heterocyclylene and C5~C 10 heteroarylene; Each E is independently the ligand having affinity for the asialoglycoprotein receptor on the surface of mammalian liver cells; L6 is C1-C 20 Alkylene; X6 is selected from -CO-, -NH-, -CO-NH-, -NH-CO-, -CH=N-, -S(O)2-, C2~C 10 Olefin Base, C2~C 10 Alkynylidene, C6~C 14 Arylene, C3~C 18 Heterocyclylene and C5~C 10 heteroarylene; L7 is a bond, C1~C 20 Alkylene or carbonyl C1~C 20 Alkylene; X7 is a bond, -CO-, -NH-, -CO-NH-, -NH-CO-, -CH=N-, -S(O)2-, C2~C 10 Asia Alkenyl, C2~C 10 Alkynylidene, C6~C 14 Arylene, C3~C 18 Heterocyclylene or C5~C 10 heteroarylene; L8' is a bond or an optionally substituted C1-C 20 Alkylenecarbonyl; R4' is hydroxy, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl or optionally substituted heterocyclic, and at least one of L8' and R4' contains an -O- group for covalently linking to the phosphate group of the nucleic acid molecule via this group.

2. The conjugate according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: The L1, L1', L2, L2', L3, L4, L4', L4", L5, L5' and L5" are each independently C1-C 12 The alkylene groups are either independently C1-C8 alkylene groups, or independently C1-C6 alkylene groups, or independently C3-C8 alkylene groups, or independently C3-C6 alkylene groups.

3. The conjugate according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: In the definitions of X1, X2, X3, X4, X5, X6 and X7: the arylene group is a phenylene group, a naphthylene group or a biphenylene group; the heterocyclylene group is a divalent group containing an O and / or N heterocyclyl group, preferably selected from an oxetanediyl group, an azetidinyl group, a pyrrolidinyl group, a piperidinyl group, a piperazinyl group, a tetrahydropyranyl group or a morpholinyl group; the heteroarylene group is a divalent group containing a nitrogen heteroaryl group, preferably an imidazolyl group, a triazolyl group, a pyridinyl group or a pyrazinyl group; Preferably, X1, X2, X3, X4 and X5 are each independently -CO-, -NH-, -CO-NH-, -NH-CO-, -CH=N- or -S(O)2-; More preferably, X1, X2, X3, X4 and X5 are each independently -CO-, -NH-, -CO-NH- or -NH-CO-; More preferably, X1, X2, X3, X4 and X5 are each independently -CO-NH- or -NH-CO-; Preferably, X6 is -CO-, -NH-, -CO-NH-, -NH-CO-, -CH=N-, -S(O)2- or C3~C 18 More preferably, X6 is -CO-, -NH-, -CO-NH-, -NH-CO- or C4~C 10 More preferably, X6 is -CO-NH-, -NH-CO- or C4~C 10 Heterocyclylene; preferably, the heterocyclylene is a divalent group of a nitrogen-containing heterocyclic group, preferably a pyrrolidinylene, a piperazinylene or a piperidinylene; Preferably, X7 is a bond, -CO-, -NH-, -CO-NH-, -NH-CO-, -CH=N- or -S(O)2-; more preferably, X7 is a bond, -CO-, -NH-, -CO-NH- or -NH-CO-; more preferably, X7 is a bond, -CO-NH- or -NH-CO-.

4. The conjugate according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: The E is selected from a monosaccharide group, a monosaccharide derivative, a polysaccharide group or a polysaccharide derivative; Preferably, E is selected from the group consisting of mannosyl, L-mannopyranosyl, L-galactosyl, D-galactosyl, alpha-D-galactopyranosyl, beta-D-galactopyranosyl, alpha-D-galactofuranosyl, beta-D-galactofuranosyl, D-arabinosyl, D-glucosyl, L-glucosyl, alpha-D-glucopyranosyl, beta-D-glucopyranosyl, alpha-D-glucopyranosyl, beta a-D-glucofuranosyl, fructosyl, alpha-D-fructofuranosyl, alpha-D-fructopyranosyl, xylosyl, L-xylofuranosyl, D-xylofuranosyl, alpha-D-mannofuranosyl, beta-D-mannofuranosyl, alpha-D-mannopyranosyl, beta-D-mannopyranosyl, glucosamine, sialyl, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine Glucosamine, N-butyrylgalactosamine, N-isobutyrylgalactosamine, 2-amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-beta-D-glucopyranosyl, 2-deoxy-2-methylamino-L-glucopyranosyl, 2-deoxy-2-sulfoamino-D-glucopyranosyl, 4,6-dideoxy-4-carboxamido-2,3-di-O-methyl-D-mannopyranosyl, N-acetylneuraminic acid, 5-thio-beta-D-pyranosyl glucopyranosyl, 2,3,4-tri-O-acetyl-1-thio-6-O-trityl-alpha-D-glucopyranosyl methyl ester, 4-thio-beta-D-galactopyranosyl, 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-alpha-D-glucopyranosyl heptanoyl ethyl ester, 2,5-anhydro-D-allosenitrile, D-ribosyl, D-4-thioribosyl, L-ribosyl and L-4-thioribosyl; More preferably, the E has the following structure: In the formula, the wavy line indicates the position where E is connected to the rest of Formula II.

5. The conjugate according to claim 1 or a pharmaceutically acceptable salt thereof, wherein: The L6 is C1~C 12 The alkylene group may be independently C1-C8 alkylene group, C1-C6 alkylene group, C3-C8 alkylene group or C3-C6 alkylene group.

6. The conjugate according to claim 1 or a pharmaceutically acceptable salt thereof, wherein: The L7 is a bond, C1~C 12 Alkylene or carbonyl C1~C 12 The alkylene group is preferably a bond, a C1-C8 alkylene group or a carbonyl C1-C8 alkylene group, and more preferably a bond, a C3-C8 alkylene group or a carbonyl C3-C8 alkylene group.

7. The conjugate according to claim 1 or a pharmaceutically acceptable salt thereof, wherein: The L8' is a bond or an optionally substituted C1-C 12 The alkylenecarbonyl group is preferably a bond or an optionally substituted C1-C8 alkylenecarbonyl group, more preferably a bond or an optionally substituted C1-C6 alkylenecarbonyl group, more preferably a bond or an optionally substituted C1-C4 alkylenecarbonyl group; preferably, the alkylene group is substituted with a hydroxyl group, and the hydroxyl group is protected by a protecting group.

8. The conjugate according to claim 1 or a pharmaceutically acceptable salt thereof, wherein: The R4' is a hydroxyl group, an optionally substituted C3-C8 cycloalkyl group, an optionally substituted C6-C 14 Aryl, optionally substituted C5~C 10 Heteroaryl or optionally substituted C4~C 10 Heterocyclic group; Preferably, the cycloalkyl, aryl, heterocyclic and heteroaryl groups are optionally substituted by 1 to 3 substituents selected from carboxyl, C1-C8 alkyl, hydroxyl, hydroxyl-substituted C1-C8 alkyl and carboxyl and / or amino-substituted C1-C8 alkyl; Preferably, the hydroxyl group present in said R4' is protected by a protecting group.

9. The conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 8, characterized in that: The R1' and R3' are the monovalent groups, and the R2' is the divalent group; or The R1' and R2' are monovalent groups, and the R3' is a divalent group.

10. The conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 9, characterized in that: The two monovalent groups are: -L1-X1-L1'-E and -CH(L2-X2-L2'-E)(X3-L3-E); -L1-X1-L1'-E and -L4-X4-CH(X4-L4'-E)(L4'-X4-L4”-E); -L1-X1-L1'-E and -L5-X5-CH(X5-L5'-E)(L5'-X5-L5”-E); -CH(L2-X2-L2'-E)(X3-L3-E) and -L4-X4-CH(X4-L4'-E)(L4'-X4-L4”-E); -CH(L2-X2-L2'-E)(X3-L3-E) and -L5-X5-CH(X5-L5'-E)(L5'-X5-L5”-E); or -L4-X4-CH(X4-L4'-E)(L4'-X4-L4”-E) and -L5-X5-CH(X5-L5’-E)(L5’-X5-L5”-E); Wherein, L1, L1', L2, L2', L3, L4, L4', L4", L5, L5' and L5" are each as described in claim 1 or 2, preferably each independently represents a C3-C8 alkylene group; X1, X2, X3, X4 and X5 are each as described in claim 3, preferably each independently represents -CO-NH- or -NH-CO-; E is the ligand as described in claim 4, preferably an acetylgalactosamine group with a protected hydroxyl group; Preferably, R1' is -L1-X1-L1'-E, R3' is -CH(L2-X2-L2'-E)(X3-L3-E), -L4-X4-CH(X4-L4'-E)(L4'-X4-L4"-E) or -L5-X5-CH(X5-L5'-E)(L5'-X5-L5"-E); Preferably, R1' and R2' are each independently -CH(L2-X2-L2'-E)(X3-L3-E), -L4-X4-CH(X4-L4'-E)(L4'-X4-L4"-E) or -L5-X5-CH(X5-L5'-E)(L5'-X5-L5"-E).

11. The conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 10, characterized in that: The divalent group is: -L6-X6-L7-R4', wherein L6 is C1-C6 alkylene, X6 is -CO-NH- or -NH-CO-, L7 is C3-C8 alkylene, and R4' is a 5-10 membered nitrogen-containing heteroaryl group optionally substituted with a C1-C8 alkyl group substituted with 1 or 2 hydroxyl groups; the hydroxyl group is optionally protected by a protecting group; -L6-X6-L7-X7-L8'-R4', wherein L6, L7 and L8' are each independently C1-C6 alkyl, X6 and X7 are each independently -CO-NH- or -NH-CO-, and R4' is a 5-10 membered nitrogen-containing heteroaryl group optionally substituted with a C1-C8 alkyl group substituted with 1 or 2 hydroxyl groups; the hydroxyl group is optionally protected by a protecting group; -L6-X6-L7-X7-R4', wherein L6 is C1-C6 alkylene, X6 is -CO-NH- or -NH-CO-, L7 is C3-C8 alkylene, X7 is -CO-, R4' is a 4-10 membered nitrogen-containing heterocyclic group optionally substituted by 1 or 2 substituents selected from hydroxyl and C1-C8 alkyl substituted by hydroxyl, and the hydroxyl group is optionally protected by a protecting group; -L6-X6-L7-X7-L8'-R4', wherein L6 is C1-C6 alkylene, X6 and X7 are each independently -CO-NH- or -NH-CO-, L7 is C3-C8 alkylene, L8' is a C1-C4 alkylene carbonyl group optionally substituted with 1 to 3 hydroxyl groups, and R4' is a 4-10 membered nitrogen-containing heterocyclic group optionally substituted with 1 or 2 hydroxyl groups, and the hydroxyl groups are optionally protected by a protecting group; -L6-X6-R4', wherein L6 is C1-C6 alkylene, X6 is -CO-, and R4' is hydroxyl; the hydroxyl is optionally protected by a protecting group; or -L6-X6-L7-X7-L8'-R4', wherein L6 is a C1-C6 alkylene group; X6 is the C3-C 18 The heterocyclic group is preferably a divalent group containing a nitrogen-containing heterocyclic group, more preferably selected from a pyrrolidinyl group, a piperazinyl group or a piperidinyl group, preferably covalently linked to L6 through its nitrogen-containing atom; L7 is a carbonyl group C3-C 12 Alkylene; X7 is -CO-, -CO-NH- or -NH-CO-; L8' is a bond, or a C1-C4 alkylene carbonyl group optionally substituted by 1 to 3 hydroxyl groups; R4' is a 4-10 membered nitrogen-containing heterocyclic group optionally substituted by 1 or 2 substituents selected from hydroxyl and C1-C8 alkyl substituted by hydroxyl; wherein the hydroxyl group is optionally protected by a protecting group.

12. The conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 10, characterized in that: L8' is C1~C substituted by -O- 20 Alkylenecarbonyl, preferably -O-substituted C1-C 12 Alkylenecarbonyl, more preferably -O-substituted C1-C8 alkylenecarbonyl, more preferably -O-substituted C1-C6 alkylenecarbonyl; R4' is a 5- to 10-membered nitrogen-containing heteroarylene group optionally substituted by 1 or 2 C1-C8 alkyl groups substituted by hydroxy groups, or a 4- to 10-membered nitrogen-containing heterocyclylene group optionally substituted by 1 or 2 substituents selected from hydroxy groups and C1-C8 alkyl groups substituted by hydroxy groups, and wherein the hydrogen of one of the hydroxy groups is removed to form an -O- group.

13. The conjugate according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: The structure of the liver targeting structure is selected from: In the formula, the wavy line indicates the position where the structure is connected to the nucleic acid molecule.

14. The conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 13, characterized in that: The nucleic acid molecule has an oligonucleotide and an analog thereof having therapeutic or preventive activity against a disease; preferably, the length of the oligonucleotide and an analog thereof is less than 100 nucleotides; preferably, the oligonucleotide or an analog thereof is selected from small interfering RNA, microRNA, short hairpin RNA, dicer substrate interfering RNA, antisense oligonucleotide, short siRNA or single-stranded siRNA; Preferably, the nucleic acid molecule is a siRNA that specifically inhibits the expression of TTR mRNA gene.

15. A pharmaceutical composition, characterized in that The pharmaceutical composition contains the conjugate according to any one of claims 1 to 14 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

16. A compound represented by the following formula I or an isomer or isotopic variant thereof: In the formula, two of R1, R2 and R3 are groups containing ligands having affinity for asialoglycoprotein receptors on the surface of mammalian liver cells, and the remaining one is a group containing a structure for covalently linking to an oligonucleotide; wherein: The groups containing ligands having affinity for asialoglycoprotein receptors on the surface of mammalian liver cells have structures independently selected from the following groups: -L1-X1-L1'-E, -CH(L2-X2-L2'-E)(X3-L3-E), -L4-X4-CH(X4-L4'-E)(L4'-X4-L4"-E) and -L5-X5-CH(X5-L5'-E)(L5'-X5-L5"-E); The group comprising a structure for covalently linking to an oligonucleotide is -L6-X6-L7-X7-L8-R4; The L1, L1', L2, L2', L3, L4, L4', L4", L5, L5' and L5" are each independently C1-C 20 Alkylene; The X1, X2, X3, X4 and X5 are each independently selected from -CO-, -NH-, -CO-NH-, -NH-CO-, -CH=N-, -S(O)2-, C2-C 10 Alkenylene, C2~C 10 Alkynylidene, C6~C 14 Arylene, C3~C 18 Heterocyclylene and C5~C 10 Heteroarylene, each E is independently a ligand having affinity for the asialoglycoprotein receptor on the surface of mammalian liver cells; The L6 is C1-C 20 Alkylene; The X6 is selected from -CO-, -NH-, -CO-NH-, -NH-CO-, -CH=N-, -S(O)2-, C2~C 10 Alkenylene, C2~C 10 Alkynylidene, C6~C 14 Arylene, C3~C 18 Heterocyclylene and C5~C 10 heteroarylene; The L7 is a bond, C1~C 20 Alkylene or carbonyl C1~C 20 Alkylene; The X7 is a bond, -CO-, -NH-, -CO-NH-, -NH-CO-, -CH=N-, -S(O)2-, C2~C 10 Alkenylene, C2~C 10 Alkynylidene, C6~C 14 Arylene, C3~C 18 Heterocyclylene or C5~C 10 heteroarylene; The L8 is a bond or an optionally substituted C1-C 20 Alkylenecarbonyl; The R4 is hydroxy, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl or optionally substituted heterocyclyl.

17. The compound according to claim 16 or its isomer or isotopic variant, characterized in that: Said L1, L1', L2, L2', L3, L4, L4', L4", L5, L5' and L5" are as defined in claim 2; and / or Said X1, X2, X3, X4, X5, X6 and X7 are as defined in claim 3; and / or Said E is as defined in claim 4; and / or Said L6 is as defined in claim 5; and / or Said L7 is as defined in claim 6; and / or The L8 is a bond or an optionally substituted C1-C 12 Alkylenecarbonyl, preferably a bond or an optionally substituted C1-C8 alkylenecarbonyl, more preferably a bond or an optionally substituted C1-C6 alkylenecarbonyl, more preferably a bond or an optionally substituted C1-C4 alkylenecarbonyl; preferably, the alkylene is substituted with a hydroxyl group, and the hydroxyl group is protected by a protecting group; and / or The R4 is a hydroxyl group, an optionally substituted C3-C8 cycloalkyl group, an optionally substituted C6-C 14 Aryl, optionally substituted C5~C 10 Heteroaryl or optionally substituted C4~C 10 Heterocyclic group; preferably, the cycloalkyl, aryl, heterocyclic group and heteroaryl group are optionally substituted by 1 to 3 substituents selected from carboxyl, C1 to C8 alkyl, hydroxyl, C1 to C8 alkyl substituted by hydroxyl and C1 to C8 alkyl substituted by carboxyl and / or amino; preferably, the hydroxyl group present in R4 is optionally protected by a protecting group.

18. The compound according to claim 16 or its isomer or isotopic variant, characterized in that: Said R1 and R3 are said groups containing ligands having affinity for the asialoglycoprotein receptors on the surface of mammalian liver cells, and R2 is said group containing a structure for covalently linking to an oligonucleotide; or said R1 and R2 are said groups containing ligands having affinity for the asialoglycoprotein receptors on the surface of mammalian liver cells, and R3 is said group containing a structure for covalently linking to an oligonucleotide.

19. The compound according to claim 16 or its isomer or isotopic variant, characterized in that: The two groups containing the ligand having affinity for the asialoglycoprotein receptor on the surface of mammalian liver cells are: -L1-X1-L1'-E and -CH(L2-X2-L2'-E)(X3-L3-E), -L1-X1-L1'-E and -L4-X4-CH(X4-L4'-E)(L4'-X4-L4"-E), -L1-X1-L1'-E and -L5-X5-CH(X5-L5'-E)(L5'-X5-L5"-E), -CH(L2-X2 -L2'-E)(X3-L3-E) and -L4-X4-CH(X4-L4'-E)(L4'-X4-L4”-E), -CH(L2-X2-L2'-E)(X3-L3-E) and -L5-X5-CH(X5-L5 '-E)(L5'-X5-L5"-E), or -L4-X4-CH(X4-L4'-E)(L4'-X4-L4"-E) and -L5-X5-CH(X5-L5'-E)(L5'-X5-L5"-E), where, L1 L1', L2, L2', L3, L4, L4', L4", L5, L5' and L5" are each as described in claim 1 or 2, preferably each independently is a C3-C8 alkylene group; X1, X2, X3, X4 and X5 are each as described in claim 3, preferably each independently is -CO-NH- or -NH-CO-; E is the ligand according to claim 4, preferably a hydroxyl-protected acetylgalactosamine group; preferably, R1 is -L1-X1-L1'-E, R3 is -CH(L2-X2-L2'-E)(X3 -L3-E), -L4-X4-CH(X4-L4'-E)(L4'-X4-L4"-E) or -L5-X5-CH(X5-L5'-E)(L5'-X5-L5"-E); preferably, R1 and R2 are each independently -CH(L2-X2-L2'-E)(X3-L3-E), -L4-X4-CH(X4-L4'-E)(L4'-X4-L4"-E) or -L5-X5-CH(X5-L5'-E)(L5'-X5-L5"-E); and / or The group comprising a structure for covalently linking to an oligonucleotide is: -L6-X6-L7-R4, wherein L6 is C1-C6 alkylene, X6 is -CO-NH- or -NH-CO-, L7 is C3-C8 alkylene, and R4 is a 5-10 membered nitrogen-containing heteroaryl group optionally substituted with 1 or 2 hydroxyl groups and substituted with a C1-C8 alkyl group; the hydroxyl group is optionally protected by a protecting group; -L6-X6-L7-X7-L8-R4, wherein L6, L7 and L8 are each independently a C1-C6 alkyl group, X6 and X7 are each independently -CO-NH- or -NH-CO-, and R4 is a 5-10 membered nitrogen-containing heteroaryl group optionally substituted with a C1-C8 alkyl group which is optionally substituted with 1 or 2 hydroxyl groups; the hydroxyl group is optionally protected by a protecting group; -L6-X6-L7-X7-R4, wherein L6 is C1-C6 alkylene, X6 is -CO-NH- or -NH-CO-, L7 is C3-C8 alkylene, X7 is -CO-, R4 is a 4-10 membered nitrogen-containing heterocyclic group optionally substituted by 1 or 2 substituents selected from hydroxyl and C1-C8 alkyl substituted by hydroxyl, and the hydroxyl group is optionally protected by a protecting group; -L6-X6-L7-X7-L8-R4, wherein L6 is C1-C6 alkylene, X6 and X7 are each independently -CO-NH- or -NH-CO-, L7 is C3-C8 alkylene, L8 is a C1-C4 alkylene carbonyl group optionally substituted with 1 to 3 hydroxyl groups, and R4 is a 4-10 membered nitrogen-containing heterocyclic group optionally substituted with 1 or 2 hydroxyl groups, and the hydroxyl groups are optionally protected by a protecting group; -L6-X6-R4, wherein L6 is a C1-C6 alkylene group, X6 is -CO-, and R4 is a hydroxyl group; the hydroxyl group is optionally protected by a protecting group; -L6-X6-L7-X7-L8-R4, wherein L6 is a C1-C6 alkylene group; X6 is the C3-C 18 The heterocyclic group is preferably a divalent group containing a nitrogen-containing heterocyclic group, more preferably selected from a pyrrolidinyl group, a piperazinyl group or a piperidinyl group, preferably covalently linked to L6 through its nitrogen-containing atom; L7 is a carbonyl group C3-C 12 Alkylene; X7 is -CO-, -CO-NH- or -NH-CO-; L8 is a bond, or is a C1-C4 alkylene carbonyl group optionally substituted by 1 to 3 hydroxyl groups; R4 is a 4-10 membered nitrogen-containing heterocyclic group optionally substituted by 1 or 2 substituents selected from hydroxyl and C1-C8 alkyl substituted by hydroxyl; wherein the hydroxyl group is optionally protected by a protecting group.

20. The compound according to claim 16 or its isomer or isotopic variant, characterized in that The compound of formula I has the structure shown below: wherein L4, L4', L4", L5, L5', L5", L6, L7, L8, X4, X5, X6 and E are as defined in claim 16 or 17; preferably: L4, L4', L4", L5, L5' and L5" are each independently C1-C 12 Alkylene, preferably C3-C8 alkylene; X4 and X5 are each independently -CO-, -NH-, -CO-NH-, -NH-CO-, -CH=N- or -S(O)2-; preferably, X4 and X5 are each independently -CO-, -NH-, -CO-NH- or -NH-CO-; more preferably, X4 and X5 are each independently -CO-NH- or -NH-CO-; E is the ligand having affinity for the asialoglycoprotein receptor (ASGPR) on the surface of mammalian liver cells, preferably an acetylgalactosamine group with a protected hydroxyl group, more preferably: L6 is a C1-C6 alkylene group; X6 is C3~C 18 a heterocyclylene group, preferably a divalent group of a nitrogen-containing heterocyclyl group, more preferably selected from a pyrrolidinylene group, a piperazinylene group or a piperidinylene group, preferably covalently linked to L6 via its nitrogen-containing atom; L7 is a carbonyl group C3~C 12 Alkylene; X7 is -CO-, -CO-NH- or -NH-CO-; L8 is a bond, or a C1-C4 alkylene carbonyl group optionally substituted by 1-3 hydroxyl groups; the hydroxyl groups are optionally protected by a protecting group R4 is a 4- to 10-membered nitrogen-containing heterocyclic group optionally substituted by 1 or 2 substituents selected from hydroxyl and C1-C8 alkyl substituted by hydroxyl; wherein the hydroxyl group is optionally protected by a protecting group.

21. The compound according to any one of claims 16 to 20 or an isomer or isotopic variant thereof, characterized in that The -X7-L8-R4 is selected from the following groups: Wherein, the wavy line indicates the position where the group is connected to the rest of the compound, R5 is a C1-C6 alkyl group optionally substituted with 1, 2 or 3 hydroxyl groups; preferably, the hydroxyl group on the pyrrolidyl group is located at the meta position, and the hydroxyl group on the piperidinyl group is located at the para position or meta position; preferably, the hydroxyl group is protected by a hydroxyl protecting group; Preferably, the -X7-L8-R4 is selected from the following groups: The wavy line indicates the position where the group is connected to the rest of the compound, and n is an integer from 1 to 6.

22. The compound according to claim 16 or an isomer or isotopic variant thereof, characterized in that The compound of formula I is selected from: 23.Select from the following applications: (1) Use of the structure of formula (II) or its isomer or isotopic variant according to any one of claims 1 to 13 or the compound represented by formula (I) or its isomer or isotopic variant according to any one of claims 16 to 22 in the preparation of a conjugate targeting hepatocytes; (2) Use of the conjugate according to any one of claims 1 to 15 or a pharmaceutically acceptable salt thereof in the preparation of a drug, especially a drug for inhibiting the expression of a target nucleic acid; and (3) Use of the conjugate according to any one of claims 1 to 15 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating or preventing a TTR-mediated disease.

24. The use according to claim 23, characterized in that The TTR-mediated diseases include hATTR amyloidosis, polyneuropathy and cardiomyopathy associated with hATTR amyloidosis, and cell damage and organ dysfunction caused by the formation of amyloid fibrils and plaques by misfolded TTR monomers in the extracellular space of tissues.

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