Conjugation groups and conjugates thereof

Abstract

The present disclosure relates to novel conjugation groups that can be linked to compounds, such as therapeutic compounds, which can be used to direct the compounds to targets in vivo. The conjugation groups disclosed herein can target expression-inhibiting oligomeric nucleotides, such as RNAi agents, to liver cells to modulate gene expression. The conjugation groups disclosed herein, when linked to expression-inhibiting oligomeric nucleotides, can be used for a variety of uses, including uses for therapy, diagnosis, target validation, and genome development. Compositions comprising the conjugation groups disclosed herein, when linked to expression-inhibiting oligomeric nucleotides, are capable of mediating expression of a target nucleic acid sequence in liver cells, such as hepatocytes, which can be used to treat diseases or conditions responsive to gene expression or activity in cells, tissues, or organisms.

Description

[0001] This application claims priority to:

[0002] CN202010522407.6, filed June 10, 2020;

[0003] CN202011524307.3, filed December 21, 2020. TECHNICAL FIELD

[0004] The present disclosure relates to a novel conjugation group and uses thereof. The conjugation groups disclosed herein can be linked to a compound, such as a therapeutic agent, to direct the compound to a target in vivo. BACKGROUND

[0005] Many compounds need to be delivered to a specific location (e.g., a desired cell) to have a therapeutic effect or to be useful for diagnostic purposes, particularly when attempting to deliver a therapeutic compound in vivo. In addition, the ability to efficiently deliver a compound to a specific location can limit or potentially eliminate unwanted consequences (e.g., off-target effects) that can result from the administration of the compound. One method to facilitate the delivery of a compound, such as a therapeutic agent, to a desired location in vivo is by linking or attaching the compound to a conjugation group.

[0006] One class of therapeutic agents that can be targeted using a conjugation group is an oligonucleotide. Oligonucleotides comprising a nucleotide sequence that is at least partially complementary to a target nucleic acid have been shown to alter the function and activity of the target in vitro and in vivo. Oligonucleotides have been shown to modulate the expression of a target when delivered to a cell containing the target nucleic acid (e.g., mRNA), resulting in an alteration in the transcription or translation of the target nucleic acid. In certain examples, oligonucleotides can reduce gene expression by inhibiting the nucleic acid target and / or triggering degradation of the target nucleic acid.

[0007] If the target nucleic acid is mRNA, one mechanism by which an expression-inhibiting oligonucleotide can modulate mRNA target expression is through RNA interference. RNA interference is a biological process that causes RNA or RNA-like molecules, such as chemically modified RNA molecules, to be degraded, silencing gene expression. This post-transcriptional gene silencing process is thought to be an evolutionarily conserved cellular defense mechanism against foreign gene expression.

[0008] Synthetic RNA and RNA-like molecules have been shown to trigger RNA interference in vivo. For example, Elbashir et al. (Nature 2000, 411, 494-98) describe the induction of RNAi by double-stranded body of synthetic 21-nucleotide RNA molecules introduced into cultured mammalian cells. The types of synthetic RNA or RNA-like molecules that can trigger the RNAi response mechanism can include modified nucleotides and / or one or more non-phosphodiester linkages.

[0009] Meier et al. (J. Mol. Biol. 2000, 300, 857-65) reported an acetylgalactosamine (GalNAc) group that can bind tightly to the asialoglycoprotein receptor (ASGPR) highly expressed in liver cells and the co-crystal structure upon binding. Khorev et al. (Bioorg. Med. Chem. 2008, 16, 5216-31) reported the use of this group to achieve targeted delivery of a fluorescent chromophore to liver cells. Prakash et al. (J. Med. Chem. 2016, 59, 2718-33) and WO2009 / 073809 reported the use of the GalNAc delivery platform to deliver antisense nucleotides and siRNA to the liver and achieve corresponding gene silencing, respectively. Thus, the structural unit of GalNAc has a broad application prospect in the delivery of macromolecules to liver cells. SUMMARY

[0010] The present disclosure relates to a novel conjugation group that can be linked to a compound, such as a therapeutic agent, which can be used to direct the compound to a target in vivo. The conjugation groups disclosed herein can target an expression-inhibiting oligomeric nucleotide, such as an RNAi agent, to liver cells to modulate gene expression.

[0011] The conjugation groups disclosed herein can be used for various purposes when linked to an expression-inhibiting oligomeric nucleotide, including therapeutic, diagnostic, target validation, and genome development purposes. Compositions comprising the conjugation groups disclosed herein can mediate expression of a target nucleic acid sequence in liver cells, such as hepatocytes, when linked to an expression-inhibiting oligomeric nucleotide, which can be used to treat a disease or condition responsive to gene expression or activity in the cells, tissues, or organisms.

[0012] Thus, in a first aspect, the present disclosure provides a conjugation group having the structure of Formula (I)

[0013]

[0014] wherein n is selected from an integer from 8 to 12.

[0015] In some embodiments of the first aspect, the conjugation group can have the following structure:

[0016]

[0017] In a second aspect, the present disclosure provides a conjugate comprising a conjugation group according to the first aspect of the present disclosure and a therapeutic agent linked to the conjugation group.

[0018] In some embodiments of the second aspect, the therapeutic agent in the conjugate described above is an expression-inhibiting oligomeric nucleotide.

[0019] In some embodiments of the second aspect, the expression-inhibiting oligonucleotide in the above conjugate is an RNAi agent.

[0020] In some embodiments of the second aspect, the RNAi agent in the above conjugate comprises one or more modified nucleotides.

[0021] In some embodiments of the second aspect, the RNAi agent in the above conjugate is a double-stranded siRNA comprising a sense strand and an antisense strand.

[0022] In some embodiments of the second aspect, the double-stranded siRNA in the above conjugate is linked to the conjugate group at the 5' end of its sense strand.

[0023] In some embodiments of the second aspect, the expression-inhibiting oligonucleotide in the above conjugate is linked to the conjugate group via a phosphate group, a thiophosphate group, or a phosphonate group.

[0024] In some embodiments of the second aspect, the thiophosphate moiety of the above conjugate or expression-inhibiting oligonucleotide comprises (R)- and (S)-enantiomers, diastereomers, and / or racemic mixtures thereof.

[0025] In some embodiments of the second aspect, the above disclosure further provides a salt of the conjugate.

[0026] In some embodiments of the second aspect, the salt as described above is selected from the group consisting of a base addition salt, an acid addition salt, and a combination thereof.

[0027] In some embodiments of the second aspect, the base addition salt as described above is selected from the group consisting of sodium, potassium, calcium, ammonium, organic amine, magnesium salts, and combinations thereof, and the acid addition salt is selected from the group consisting of inorganic acid salts, organic acid salts, and combinations thereof.

[0028] In some embodiments of the second aspect, the inorganic acid as described above is selected from the group consisting of hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, and combinations thereof, and the organic acid is selected from the group consisting of acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzene sulfonic acid, p-toluene sulfonic acid, citric acid, tartaric acid, methanesulfonic acid, and combinations thereof.

[0029] In a third aspect, the disclosure provides a compound having the structure of Formula (II) or Formula (III)

[0030]

[0031] wherein m is selected from an integer from 8 to 12.

[0032] In some embodiments of the third aspect, the compound can have the following structure

[0033]

[0034]

[0035] In a fourth aspect, the disclosure provides a pharmaceutical composition comprising a conjugate according to the second aspect of the disclosure and a pharmaceutically acceptable carrier or excipient.

[0036] In a fifth aspect, the disclosure provides a method for inhibiting expression of a target nucleic acid in a subject in need thereof, comprising the step of administering to the subject a conjugate according to the second aspect of the disclosure or a pharmaceutical composition according to the fourth aspect of the disclosure.

[0037] In some embodiments of the fifth aspect, the target nucleic acid in the method is a nucleic acid from a virus. The virus can be, for example, a virus that causes liver disease, such as hepatitis B virus.

[0038] In a sixth aspect, the disclosure provides a method of treating a disease, comprising the step of administering to a subject a conjugate according to the second aspect of the disclosure or a pharmaceutical composition according to the fourth aspect of the disclosure.

[0039] In a seventh aspect, the disclosure provides use of a conjugate according to the second aspect of the disclosure or a pharmaceutical composition according to the fourth aspect of the disclosure in the manufacture of a medicament for treating a disease.

[0040] In an eighth aspect, the disclosure provides a conjugate according to the second aspect of the disclosure or a pharmaceutical composition according to the fourth aspect of the disclosure for use in treating a disease.

[0041] In some embodiments of the seventh and eighth aspects, the disease is a viral infection.

[0042] In some embodiments of the seventh and eighth aspects, the disease is a liver disease.

[0043] In some embodiments of the seventh and eighth aspects, the disease is hepatitis B.

[0044] Definitions and Descriptions

[0045] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be construed as undefined or unclear unless specifically defined, but should be interpreted as understood by one of ordinary skill in the art. When a trade name appears herein, it is intended to refer to its corresponding product or active ingredient thereof.

[0046] The conjugate groups described herein can enhance delivery of a therapeutic agent to a particular target location (e.g., a particular organ or tissue) within a subject, such as a human or an animal. In some embodiments of the disclosure, the conjugate groups can enhance targeted delivery of an expression-inhibiting oligonucleotide. In some embodiments of the disclosure, the conjugate groups can enhance delivery of an expression-inhibiting oligonucleotide to the liver.

[0047] The conjugate groups described herein can be linked, directly or indirectly, to a compound, such as a therapeutic agent, e.g., an expression-inhibiting oligonucleotide, e.g., to the 3' or 5' end of an expression-inhibiting oligonucleotide. In some embodiments of the disclosure, the expression-inhibiting oligonucleotide comprises one or more modified nucleotides. In some embodiments of the disclosure, the expression-inhibiting oligonucleotide is an RNAi agent, such as a double-stranded RNAi agent comprising a sense strand and an antisense strand. In some embodiments of the disclosure, the conjugate groups disclosed herein are linked to the 5' end of the sense strand of a double-stranded RNAi agent. In some embodiments, the conjugate groups disclosed herein are linked to the expression-inhibiting oligonucleotide agent at the 5' end of the sense strand of a double-stranded RNAi agent via a phosphate, phosphorothioate, or phosphonate group.

[0048] The term "linked" as described herein, when referring to the association between two molecules, means that the two molecules are connected by a covalent bond or that the two molecules are associated via a non-covalent bond (e.g., a hydrogen bond or an ionic bond).

[0049] An "oligonucleotide" as described herein is a nucleotide sequence containing 10 to 50 nucleotides or nucleotide base pairs. In some embodiments of the disclosure, the oligonucleotide has a nucleobase sequence that is at least partially complementary to a coding sequence in a target nucleic acid or target gene expressed in a cell. The nucleotides can optionally be modified. In some embodiments of the disclosure, upon delivery of the oligonucleotide to a cell expressing a gene, the oligonucleotide is capable of inhibiting expression of the underlying gene and is referred to in the disclosure as an "expression-inhibiting oligonucleotide" that can inhibit gene expression in vitro or in vivo. "Oligonucleotide" includes, but is not limited to, a single-stranded oligonucleotide, a single-stranded antisense oligonucleotide, a short interfering RNA (siRNA), a double-stranded RNA (dsRNA), a microRNA (miRNA), a short hairpin RNA (shRNA), a ribozyme, an interfering RNA molecule, and a Dicer enzyme substrate.

[0050] The term "RNAi agent" as used herein refers to an agent comprising an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule capable of degrading or inhibiting translation of a messenger RNA (mRNA) transcript of a target mRNA in a sequence-specific manner. The RNAi agents described herein can operate through the RNA interference mechanism (i.e., by interacting with components of the RNA interference pathway of a mammalian cell (the RNA-induced silencing complex or RISC) to induce RNA interference) or through any other mechanism or pathway. Although the RNAi agents described herein primarily operate through the RNA interference mechanism, the disclosed RNAi agents are not limited to or constrained by any particular mechanism or pathway of action. RNAi agents include, but are not limited to, single-stranded oligonucleotides, single-stranded antisense oligonucleotides, short interfering RNAs (siRNAs), double-stranded RNAs (dsRNAs), microRNAs (miRNAs), short hairpin RNAs (shRNAs), and Dicer substrates. The RNAi agents described herein include oligonucleotides having a strand that is at least partially complementary to a targeted mRNA. In some embodiments of the disclosure, the RNAi agents described herein are double-stranded and include an antisense strand and a sense strand that is at least partially complementary to the antisense strand. The RNAi agents can include modified nucleotides and / or one or more non-phosphodiester linkages. In some embodiments, the RNAi agents described herein are single-stranded.

[0051] The term "single-stranded oligonucleotide" as used herein refers to a single-stranded oligonucleotide having a sequence that is at least partially complementary to a target mRNA, which is capable of hybridizing to the target mRNA through hydrogen bonding under mammalian physiological conditions (or equivalent in vitro conditions). In some embodiments of the disclosure, the single-stranded oligonucleotide is a single-stranded antisense oligonucleotide.

[0052] Short interfering RNA (siRNA) as used herein refers to a class of RNA molecules, 20-25 base pairs in length, similar to miRNAs, and operating within the RNA interference (RNAi) pathway, which interferes with the translation of mRNA of a specific gene complementary to the nucleotide sequence, resulting in mRNA degradation. The short interfering RNA (siRNA) described herein includes double-stranded siRNA (including a sense strand and an antisense strand) and single-stranded siRNA (only an antisense strand).

[0053] The term "silencing," "reducing," "inhibiting," "down-regulating," or "knocking down," when referring to expression of a given gene, means that the expression of the gene is reduced when the cell, cell population, or tissue is treated with an oligonucleotide linked to a conjugate group as described herein, as compared to a second cell, cell population, or tissue that has not been so treated, as measured by the level of RNA transcribed from the gene or the level of polypeptide, protein, or protein subunit translated from mRNA in the cell, cell population, tissue, or subject from which the transcribed gene.

[0054] The term "sequence" or "nucleotide sequence" as used herein refers to the order or succession of nucleobases or nucleotides described by a sequence of letters using standard nucleotide nomenclature.

[0055] The term "HBV gene" as used herein refers to a DNA sequence as shown in Genbank accession number NC_003977.1. The gene as shown in Genbank accession number NC_003977.1 is the complete genome of HBV.

[0056] In some embodiments, the double-stranded siRNA analogs can target the X open reading frame (X ORF) of HBV.

[0057] In further embodiments, the double-stranded siRNA analogs can target the S ORF of HBV.

[0058] In further embodiments, the double-stranded siRNA analogs can target the P ORF of HBV.

[0059] The term "modification" of a nucleotide as used herein includes, but is not limited to, methoxy modification, fluoro modification, phosphorothioate linkage, etc. The sequences as used herein can include those listed in Table 1 as "further modified sequences".

[0060] In the present disclosure, capital letters C, G, U, A represent the base composition of a nucleotide, unless otherwise specified. Lowercase letters c, g, u, a represent the corresponding nucleotide represented by its capital letter is modified by methoxy; underlined represents the nucleotide represented by the capital letter is modified by fluoro; and the dash "·" represents a phosphorothioate linkage between the two nucleotide residues adjacent to the left and right of the dash "·". For example, "a·g" represents a phosphorothioate linkage between the a and g residues.

[0061] The term "fluoro modified nucleotide" as used herein refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose group is replaced by fluorine. The term "methoxy modified nucleotide" as used herein refers to a nucleotide in which the 2'-hydroxyl group of the ribose group is replaced by methoxy.

[0062] In the present disclosure, "complementary" has the meaning well known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one strand pair with the bases on the other strand in a complementary manner. The purine base adenine (A) always pairs with the pyrimidine base uracil (U); the purine base guanine (G) always pairs with the pyrimidine base cytosine (C). Each base pair includes one purine and one pyrimidine. When the adenine on one strand always pairs with the uracil on the other strand, and the guanine always pairs with the cytosine, the two strands are considered to be complementary to each other, and the sequence of one strand can be inferred from the sequence of its complementary strand.

[0063] The compounds of the present disclosure can exist in particular geometric or stereoisomeric forms. The present disclosure contemplates all such compounds, including (R)- and (S)-enantiomers, diastereomers, as well as the racemic mixtures and other mixtures of those, for example, atropisomers, enantiomeric or diastereomeric mixtures, all falling within the scope of the present disclosure. Additional asymmetric carbon atoms can be present in a substituent group. All such isomers, as well as mixtures thereof, are included within the scope of the present disclosure.

[0064] The term "enantiomeric" or "optical isomer" means a stereoisomer which is a mirror image of the other and which is not superimposable on the other.

[0065] The term "diastereomeric" means a stereoisomer which has two or more chiral centers and which is not a mirror image of the other.

[0066] Unless otherwise indicated, the absolute configuration of a stereogenic center is indicated by a wedged and dashed wedge The relative configuration of a stereogenic center is indicated by a straight and dashed straight line The relative configuration of a stereogenic center is indicated by a straight and dashed straight line or dashed wedge or by a wavy line The relative configuration of a stereogenic center is indicated by a straight and / or dashed straight line

[0067] The terms "enantiomeric excess" or "enantiomeric excess" refer to the difference between the relative percentages of two enantiomers, unless otherwise indicated. For example, where one enantiomer is present in an amount of 90% and the other enantiomer is present in an amount of 10%, the enantiomeric excess (ee value) is 80%.

[0068] The terms "enantiomeric excess" or "enantiomeric excess" refer to the difference between the relative percentages of two enantiomers, unless otherwise indicated. For example, where one enantiomer is present in an amount of 90% and the other enantiomer is present in an amount of 10%, the enantiomeric excess (ee value) is 80%.

[0069] Optically active (R)- and (S)-isomers and D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the disclosure is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting mixture of diastereomers is separated and the auxiliary group cleaved to provide the pure desired enantiomeric isomer. Alternatively, when a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group) is present in the molecule, diastereomeric salts with appropriate optically active acids or bases can be formed and the desired enantiomer recovered by conventional means known in the art. Further, separation of enantiomeric and diastereomeric mixtures can be achieved by the formation of salts with chiral bases or acids, or by chromatographic techniques using chiral stationary phases. The compounds of the disclosure can contain unnatural proportions of atomic isotopes at one or more atoms, for example, deuterium ( 3 H), iodine-125 ( 125 I) or carbon-14 ( 14 C). For example, deuterium substituted drugs can be formed by replacing hydrogen with deuterium. The bond between deuterium and carbon is stronger than the bond between ordinary hydrogen and carbon. Deuterium substituted drugs have advantages over non-deuterated drugs, such as reduced toxicity, increased drug stability, enhanced efficacy, and increased biological half-life. All isotopic variations of the compounds of the disclosure, whether radioactive or not, are included within the scope of the disclosure.

[0070] The term "salt" refers to salts of the compounds of the present disclosure, which are prepared from compounds of the present disclosure having acidic or basic groups with relatively non-toxic acids or bases. When the compounds of the present disclosure contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compounds in solution or suspension with a sufficient amount of the base to provide the desired salt. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or similar salts. When the compounds of the present disclosure contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds in solution or suspension with a sufficient amount of the acid to provide the desired salt. Examples of pharmaceutically acceptable acid addition salts include inorganic acids, such as hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, hydrogen sulfate, hydroiodic, phosphorous, and the like; and organic acids, such as acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like; also salts of amino acids, such as arginate, and salts of organic acids not

[0071] The salts of the present disclosure can be synthesized from the parent compound that contains an acidic or basic moiety by conventional chemical methods. Generally, such salts are prepared either by contacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent or in a mixture of the two, in the presence of a suitable solvent.

[0072] The compounds of the present disclosure can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments set forth below, embodiments formed by a combination of the specific embodiments set forth below with other chemical synthetic methods known to those skilled in the art, and equivalents thereof known to those skilled in the art, preferred embodiments including but not limited to the examples of the present disclosure.

[0073] The solvents used in the present disclosure are commercially available.

[0074] The solvent ratios used in the column chromatography and preparative thin layer silica gel chromatography of the present disclosure are all volume ratios, unless otherwise specified.

[0075] List of Abbreviations

[0076] Ac acetyl Boc tert-butyloxycarbonyl DMSO dimethyl sulfoxide DMT / DMTr 4,4'-dimethoxytrityl dsRNA double-stranded ribonucleic acid EC 50 ]]> EC50 half maximal effective concentration EDTA ethylenediaminetetraacetic acid i-Pr isopropyl Me methyl Ms methanesulfonyl Ph phenyl p-HPLC preparative high performance liquid chromatography, used for purification of compounds RNA ribonucleic acid RNAi ribonucleic acid interference technology siRNA small interfering ribonucleic acid t-Bu tert-butyl Tris

[0077] Compounds are named according to the principles of nomenclature conventionally used in the art or using software nomenclature, and commercially available compounds are named using the supplier's catalog name. Software nomenclature, and commercially available compounds are named using the supplier's catalog name. DETAILED DESCRIPTION

[0078] The present disclosure is described in detail by the following examples, but it does not mean any unfavorable limitation to the present disclosure. The compounds of the present disclosure can be prepared by various synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by the combination of the specific embodiments with other chemical synthetic methods, and the equivalent replacement methods well known to those skilled in the art, and the preferred embodiments include but are not limited to the examples of the present disclosure. It will be obvious to those skilled in the art that various changes and improvements can be made to the specific embodiments of the present disclosure without departing from the spirit and scope of the present disclosure.

[0079] Example 1: Synthesis of DOl

[0080]

[0081]

[0082]

[0083] Step A: 11-Dodecyn-1-ol (25 g, 137.14 mmol) and triethylamine (16.65 g, 164.56 mmol) were dissolved in dichloromethane (250 mL) and methanesulfonyl chloride (18.85 g, 164.56 mmol) was added at 0 °C. The mixture was stirred at 0 °C for 2 h. The reaction solution was diluted with water (400 mL) and extracted with dichloromethane (800 mL, 400 mL x 2). The combined organic phase was washed with water (400 mL, 200 mL x 2) and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give 2-2.

[0084] Step B: The compound represented by formula 2-3 (20 g, 67.26 mmol) was dissolved in N,N-dimethylformamide (200 mL) and sodium hydride (60% purity, 4.04 g, 100.89 mmol) was added at 0 °C, followed by the compound represented by formula 2-2 (19.27 g, 73.99 mmol). The mixture was stirred at 25 °C for 16 h. The reaction solution was quenched with water (1 L) and extracted with dichloromethane (1.6 L, 800 mL x 2). The combined organic phase was washed with saturated brine (800 mL, 800 mL x 1), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give 2-4. 1 H NMR (400 MHz, DMSO-d6): δ 7.63-6.89 (m, 10H), 5.64-5.52 (m, 2H), 4.27-4.01 (m, 2H), 3.98-3.77 (m, 2H), 3.72-3.18 (m, 4H), 2.23-2.14 (m, 2H), 1.98-1.92 (m, 1H), 1.54-1.23 (m, 16H).

[0085] Step C: To a solution of the compound of formula 2-4 (48 g, 103.98 mmol) in methanol (870 mL) was added hydrogen chloride in methanol (4 mol / L, 400 mL, 1.6 mol). The mixture was stirred at 30 °C for 2 h. To the reaction mixture was added hydrogen chloride in methanol (4 mol / L, 350 mL, 1.4 mol). The mixture was stirred at 30 °C for 16 h. The reaction mixture was concentrated under reduced pressure. To the residue was added chloroform (200 mL, 100 mL x 2) and concentrated under reduced pressure until white solid appeared. To the residue was added toluene (130 mL) and petroleum ether (130 mL). The mixture was stirred at 15 °C for 16 h. The reaction mixture was filtered through a Buchner funnel. The collected solid was dried under vacuum to give a white solid. The white solid was dissolved in dichloromethane (50 mL). To the solution was added a solution of sodium hydroxide (6.59 g, 164.66 mmol) in water (50 mL). The mixture was stirred at 20 °C for 1 h. The reaction mixture was diluted with water (500 mL) and extracted with dichloromethane (1 L, 500 mL x 2). The combined organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give 2-5.

[0086] Step D: To a mixture of the compound of formula 2-5 (23 g, 80.58 mmol) and sodium hydroxide (322.31 mg, 8.06 mmol) in dimethyl sulfoxide (70 mL) and water (6 mL) was added tert-butyl acrylate (22.72 g, 177.28 mmol). The mixture was stirred at 25 °C for 16 h under nitrogen. The reaction mixture was diluted with water (500 mL) and extracted with ethyl acetate (1 L, 500 mL x 2). The combined organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a crude product. The crude product was purified by column chromatography (Si02, petroleum ether / ethyl acetate / ethanol (containing 0.1% ammonia water) = 36 / 3 / 1 to 16 / 3 / 1) to give 2-6. 1 H NMR (400 MHz, DMSO-d6): δ 3.60-3.54 (m, 4H), 3.32 (br s, 5H), 3.15 (s, 5H), 2.74-2.66 (m, 1H), 2.40 (t, J = 6.0 Hz, 4H), 2.18-2.11 (m, 2H), 1.58-1.38 (m, 22H), 1.34-1.23 (m, 12H).

[0087] Step E: To a solution of the compound of formula 2-6 (24.5 g, 45.22 mmol) in dichloromethane (250 mL) was added triethylamine (9.15 g, 90.45 mmol) and succinic anhydride (6.79 g, 67.83 mmol). The mixture was stirred at 20 °C for 16 h. To the reaction mixture was added dichloromethane (1 L) and hydrochloric acid (1 mol / L, 1 L). The organic phase was separated and dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give 2-7. 1H NMR (400 MHz, CDC13): δ 6.49-6.37 (m, 1H), 3.72 (s, 2H), 3.70-3.57 (m, 8H), 3.37 (t, J = 6.7 Hz, 2H), 2.69-2.51 (m, 4H), 2.50-2.36 (m, 4H), 2.22-2.13 (m, 2H), 1.96-1.90 (m, 1H), 1.57-1.47 (m, 4H), 1.46-1.40 (m, 18H), 1.40-1.31 (m, 2H), 1.30-1.21 (m, 10H).

[0088] Step F: The compound of formula 2-7 (27.4 g, 42.69 mmol) was dissolved in formic acid (140 mL), and the mixture was stirred at 20 °C for 16 h under nitrogen. The reaction mixture was concentrated under reduced pressure, and toluene (300 mL, 150 mL x 2) was added. The mixture was concentrated under reduced pressure to give 2-8. 1 H NMR (400 MHz, CDC13): δ 6.49-6.37 (m, 1H), 3.72 (s, 2H), 3.70-3.57 (m, 8H), 3.37 (t, J = 6.7 Hz, 2H), 2.69-2.51 (m, 4H), 2.50-2.36 (m, 4H), 2.22-2.13 (m, 2H), 1.96-1.90 (m, 1H), 1.57-1.47 (m, 4H), 1.46-1.40 (m, 18H), 1.40-1.31 (m, 2H), 1.30-1.21 (m, 10H).

[0089] Step G: The compound of formula 2-8 (22.6 g, 42.67 mmol), N,N- diisopropylethylamine (33.09 g, 256.03 mmol), and O-(7-azabenzotriazol-1-yl)- N,N,N,N-tetramethyluronium hexafluorophosphate (51.92 g, 136.55 mmol) were dissolved in N,N-dimethylformamide (250 mL), and tert-butyl N-(3- aminopropyl)carbamate (29.74 g, 170.69 mmol) was added. The mixture was stirred at 20 °C for 16 h. The reaction mixture was added to dichloromethane (1 L) and hydrochloric acid (1 mol / L, 1 L), and the organic phase was washed with water (1 L, 1 L x 1), aqueous sodium bicarbonate (1 L, 1 L x 1), and saturated brine (1 L, 1 L x 1) in sequence, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product. Purification by column chromatography (Si02, petroleum ether / ethyl acetate / ethanol = 40 / 3 / 1 to 10 / 3 / 1) gave 2-9. 1H NMR (400 MHz, CDC13): δ 7.22-6.79 (m, 3H), 6.77-6.44 (m, IH), 5.45-5.00 (m, 3H), 3.86-3.73 (m, 2H), 3.72-3.63 (m, 4H), 3.62-3.45 (m, 4H), 3.41-3.32 (m, 2H), 3.32-3.20 (m, 6H), 3.19-3.03 (m, 6H), 2.56-2.47 (m, 4H), 2.47-2.39 (m, 4H), 2.21-2.12 (m, 2H), 1.95-1.90 (m, IH), 1.70-1.57 (m, 6H), 1.56-1.47 (m, 4H), 1.46-1.38 (m, 29H), 1.30-1.25 (m, 10H).

[0090] Step H: The compound of formula 2-9 (15 g, 15.03 mmol) was dissolved in dichloromethane (114 ml) and trifluoroacetic acid (38 ml) was added. The mixture was stirred at 20 °C for 16 hours. The reaction solution was concentrated under reduced pressure, and toluene / acetonitrile = 3 / 1 mixture 750 ml (250 ml x 3) was added and concentrated under reduced pressure to obtain 2-10.

[0091] Step I: The compound of formula 2-11 (22.15 g, 49.50 mmol), N,N- diisopropylethylamine (7.75 g, 60.00 mmol), 1-hydroxy-7-azabenzotriazole (6.12 g, 45.00 mmol), and O-(7-azabenzotriazol-1-yl)-N,N,N,N- tetramethyluronium hexafluorophosphate (20.53 g, 54.00 mmol) were dissolved in N,N-dimethylformamide (90 ml), and to this mixture was added a solution of the compound of formula 2-10 (tris(trifluoroacetate), 15.6 g, 15.00 mmol) and N,N- diisopropylethylamine (21.32 g, 165.00 mmol) in N,N-dimethylformamide (120 ml). The mixture was stirred at 20 °C for 16 hours. To the reaction solution was added dichloromethane (1.2 L) and hydrochloric acid (1 mol / L, 1 L), and the organic phase was washed successively with water (1 L x 1), aqueous sodium bicarbonate (1 L x 1), and saturated brine (1 L x 1), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. Purification by column chromatography (Si02, dichloromethane / methanol = 100 / 1 to 10 / 1 to dichloromethane / ethanol = 1 / 1) gave 2-12. 1H NMR (400 MHz, DMSO-d6): δ 7.87-7.66 (m, 9H), 7.09 (s, IH), 5.21 (d, J = 3.4 Hz, 3H), 4.96 (dd, J = 3.4, 11.3 Hz, 3H), 4.48 (d, J = 8.5 Hz, 3H), 4.06-3.98 (m, 9H), 3.91-3.82 (m, 3H), 3.74-3.66 (m, 3H), 3.58-3.46 (m, 12H), 3.31 (br s, 3H), 3.07-2.98 (m, 12H), 2.71 (t, J = 2.6 Hz, IH), 2.33-2.22 (m, 8H), 2.16-2.12 (m, 2H), 2.10 (s, 9H), 2.04 (br t, J = 7.1 Hz, 6H), 1.99 (s, 9H), 1.89 (s, 9H), 1.81-1.74 (m, 9H), 1.54-1.39 (m, 22H), 1.32 (br dd, J = 4.5, 6.7 Hz, 2H), 1.24 (s, 10H).

[0092] Step J: To a solution of the compound of formula 2-12 (1.00 g, 0.50 mmol) and N-methyl-N,N,N-trinormal octyl ammonium chloride (20.35 mg, 50.35 μmol) in a mixture of acetic acid (2.7 mL) and n-pentane (6.3 mL) at 0 °C was added dropwise a solution of potassium permanganate (0.40 g, 2.52 mmol) in water (9 mL). The mixture was stirred at 0 to 15 °C for 2 hours. The reaction was quenched with sodium bisulfite (1.27 g), hydrochloric acid (2 M, 5 mL) and water (30 mL) were added, and the mixture was extracted with a mixture of chloroform / isopropanol = 3 / 1 (120 mL, 40 mL x 3). The combined organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Toluene / acetonitrile = 1 / 1 (180 mL, 30 mL x 6) was added and concentrated under reduced pressure to give 2-13. 1HNMR (400 MHz, CD3OD): δ 5.34 (d, J = 2.9 Hz, 3H), 5.06 (dd, J = 3.3, 11.2 Hz, 3H), 4.56 (d, J = 8.4 Hz, 3H), 4.19-4.06 (m, 9H), 4.04-3.98 (m, 3H), 3.87 (td, J = 5.7, 9.9 Hz, 4H), 3.72-3.64 (m, 9H), 3.57-3.50 (m, 3H), 3.39 (br t, J = 6.4 Hz, 2H), 3.22 (q, J = 6.4 Hz, 12H), 2.51-2.40 (m, 9H), 2.21 (br t, J = 7.3 Hz, 6H), 2.14 (s, 9H), 2.03 (s, 9H), 1.94 (d, J = 7.9 Hz, 18H), 1.72-1.57 (m, 22H), 1.39 (br s, 12H).

[0093] Step K: To a solution of the compound represented by formula 2-13 (1.00 g, 0.50 mmol) in N,N-dimethylformamide (10 ml) was added N,N-diisopropylethylamine (0.26 g, 1.99 mmol) and O-(7-azabenzotriazol-l-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (0.23 g, 0.60 mmol). After the mixture was stirred, the compound represented by formula 2-14 (0.23 g, 0.55 mmol) was added. The mixture was stirred at 15 °C for 16 hours. The reaction solution was added to dichloromethane (50 ml) and water (50 ml), and the organic phase was washed with saturated aqueous sodium bicarbonate solution 50 ml (50 ml x 1), water 50 ml (50 ml x 1), and saturated brine 50 ml (50 ml x 1) in this order, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. Purification by column chromatography (SiO2, dichloromethane / methanol (containing 0.1% triethylamine) = 20 / 1 to 10 / 1) gave 2-15. 1H NMR (400 MHz, DMSO-d6): δ 7.90-7.82 (m, 6H), 7.78 (br d, J = 4.8 Hz, 3H), 7.40-7.26 (m, 10H), 6.91 (br dd, J = 3.1, 9.0 Hz, 4H), 5.26 (d, J = 3.4 Hz, 3H), 5.03-4.99 (m, 3H), 4.53 (d, J = 8.4 Hz, 3H), 4.43 (br d, J = 3.8 Hz, 1H), 4.23-4.14 (m, 1H), 4.12-4.02 (m, 9H), 3.92 (td, J = 9.0, 11.0 Hz, 3H), 3.78 (s, 6H), 3.77-3.71 (m, 3H), 3.66-3.51 (m, 13H), 3.49-3.41 (m, 4H), 3.11-3.01 (m, 16H), 2.38-2.37 (m, 1H), 2.32 (br s, 9H), 2.14 (s, 9H), 2.08 (br t, J = 6.9 Hz, 7H), 2.04 (s, 9H), 1.93 (s, 9H), 1.82 (s, 9H), 1.57-1.46 (m, 22H), 1.31-1.26 (m, 12H).

[0094] Step L: To a solution of the compound of formula 2-15 (0.80 g, 0.33 mmol) in dichloromethane (8 mL) was added triethylamine (67.24 mg, 0.64 mmol), 4-N,N-dimethylaminopyridine (0.12 g, 1.00 mmol) and succinic anhydride (83.13 mg, 0.83 mmol) successively. The mixture was stirred at 10 °C for 16 hours. The reaction solution was added dichloromethane (50 mL), water (30 mL) and saturated brine (30 mL) successively. The organic phase was washed with water (30 mL x 1) and saturated brine (30 mL x 1) successively, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a crude product. Purification by p-HPLC (separation column: Waters Xbridge C18 (specification: 150 mm x 50 mm, particle size: 10 μm); mobile phase: [water (10 mM ammonium bicarbonate) - acetonitrile]; elution gradient: 27% - 57%, 11 min) gave D01. 1H NMR (400 MHz, DMSO-d6): δ 7.96-7.69 (m, 9H), 7.33-7.09 (m, 10H), 6.90-6.78 (m, 4H), 5.21 (d, J = 3.3 Hz, 3H), 4.97 (dd, J = 3.3, 11.2 Hz, 3H), 4.49 (d, J = 8.4 Hz, 3H), 4.06-3.97 (m, 9H), 3.91-3.83 (m, 3H), 3.79-3.66 (m, 11H), 3.63-3.45 (m, 18H), 3.02 (br d, J = 4.6 Hz, 14H), 2.46-2.37 (m, 4H), 2.35-2.14 (m, 12H), 2.10 (s, 9H), 2.04 (br t, J = 7.0 Hz, 6H), 1.99 (s, 9H), 1.88 (s, 9H), 1.77 (s, 9H), 1.57-1.37 (m, 22H), 1.22 (br s, 12H).

[0095] D is a residue of small molecule fragment D01 after chemical reaction, which is covalently bound to nucleic acid, and its structure is shown in the following formula:

[0096]

[0097] Example 2: Synthesis of D02

[0098]

[0099]

[0100] Step A: Preparation of compound 3-2 Reference to the preparation of compound D01 in Example 1, replace 2-1 with 3-1.

[0101] Step B: Compound represented by formula 2-3 (35.70 g, 120.06 mmol) was dissolved in 2-methyltetrahydrofuran (285 mL), and potassium tert-butoxide (17.51 g, 156.07 mmol) was added. The mixture was stirred at 85°C for 2 hours. Then, compound represented by formula 3-2 (28.40 g, 114.34 mmol) was added. The mixture was stirred at 85°C for 12 hours. The reaction solution was added with water (400 mL), and extracted with dichloromethane (800 mL, 400 mL x 2). The combined organic phase was dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 3-3.

[0102] Step C: The compound of formula 3-3 (35 g, 77.84 mmol) was dissolved in methanol (525 mL) and hydrogen chloride in methanol (4 mole per liter, 175 mL, 750 mmol) was added. The mixture was stirred at 50 °C for 12 hours. The reaction solution was poured into a mixture of potassium carbonate (80 g) and methanol (500 mL), filtered through a Buchner funnel and concentrated under reduced pressure. The resulting crude product was dissolved in methanol (240 mL), sodium acetate (12.77 g, 155.68 mmol) and hydroxylamine hydrochloride (5.41 g, 77.84 mmol) were added. The mixture was stirred at 25 °C for 0.5 hours. The reaction solution was filtered through a Buchner funnel and concentrated under reduced pressure. The resulting crude product was added to aqueous sodium hydroxide (1 mole per liter, 500 mL) and extracted with dichloromethane 500 mL (500 mL x 1). The combined organic phase was washed with aqueous sodium hydroxide (1 mole per liter, 500 mL) and saturated brine 500 mL (500 mL x 1) successively, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give 3-4. 1 H NMR (400 MHz, CDC13): δ 5.81-5.66 (m, 1H), 4.98-4.79 (m, 2H), 3.50-3.37 (m, 4H), 3.36-3.30 (m, 2H), 3.24 (br s, 2H), 2.03-1.91 (m, 2H), 1.53-1.42 (m, 2H), 1.35-1.27 (m, 2H), 1.25-1.13 (m, 10H).

[0103] Steps D-I: Refer to the respective steps of Example 1, Compound D01 for the preparation.

[0104] Step J: The compound of formula 3-10 (20 g, 10.13 mmol) and trichlororuthenium hydride (52.98 mg, 202.61 μmol) were dissolved in a mixture of dichloromethane (60 mL), acetonitrile (60 mL) and water (90 mL), and sodium periodate (10.83 g, 50.65 mmol) was slowly added to the mixture. The mixture was stirred at 25 °C for 3.5 hours. The reaction solution was added to water (500 mL) and extracted with a mixture of dichloromethane / isopropanol = 3 / 1 1 L (500 mL x 2). The combined organic phase was washed with saturated aqueous sodium sulfite 150 mL (150 mL x 1) and concentrated under reduced pressure. The crude product was added to saturated aqueous sodium bicarbonate (500 mL), washed with dichloromethane 1 L (500 mL x 2), added with hydrochloric acid (1 mole per liter) to pH = 3, and extracted with a mixture of dichloromethane / isopropanol = 3 / 1 1.5 L (500 mL x 3). The combined organic phase was washed with saturated brine 500 mL (500 mL x 1), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give 3-11. 1H NMR (400 MHz, CD3OD): δ 5.37-5.29 (m, 3H), 5.06 (dd, J = 3.4, 11.3 Hz, 3H), 4.61-4.51 (m, 3H), 4.20-3.96 (m, 12H), 3.92-3.82 (m, 3H), 3.70-3.63 (m, 9H), 3.57-3.49 (m, 3H), 3.43-3.36 (m, 2H), 3.28-3.14 (m, 12H), 2.50-2.38 (m, 8H), 2.33-2.24 (m, 3H), 2.24-2.17 (m, 6H), 2.17-2.11 (m, 9H), 2.03-1.99 (m, 9H), 1.98-1.90 (m, 18H), 1.75-1.51 (m, 22H), 1.38-1.27 (m, 10H).

[0105] Step K: To a solution of the compound represented by formula 3-11 (11 g, 5.52 mmol) in dichloromethane (110 mL) was added N, N-diisopropylethylamine (0.71 g, 5.52 mmol), 1-hydroxy-7-azabenzotriazole (1.50 g, 11.04 mmol), O-(7-azabenzotriazol-1-yl)-N, N, N, N-tetramethyluronium hexafluorophosphate (2.52 g, 6.63 mmol) and the compound represented by formula 2-14 (2.43 g, 5.80 mmol). After the mixture was stirred, N, N-diisopropylethylamine (2.14 g, 16.56 mmol) was added. The mixture was stirred at 25 °C for 12 hours. The reaction solution was added to dichloromethane (1 L) and water (500 mL), and the organic phase was washed with water 500 mL (500 mL x 1) and saturated brine 500 mL (500 mL x 1) successively, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product. Purification by column chromatography (SiO2, dichloromethane / methanol (containing 0.5% triethylamine) = 30 / 1 to 10 / 1) gave 3-12. 1H NMR (400 MHz, DMSO-d6) δ 7.92 - 7.73 (m, 9H), 7.38 - 7.16 (m, 10H), 6.94 - 6.83 (m, 4H), 5.22 (d, J = 3.4 Hz, 3H), 4.97 (dd, J = 3.4, 11.3 Hz, 3H), 4.53 - 4.44 (m, 3H), 4.44 - 4.35 (m, 1H), 4.21 - 4.09 (m, 1H), 4.03 (s, 8H), 3.94 - 3.83 (m, 3H), 3.77 - 3.68 (m, 9H), 3.58 - 3.48 (m, 10H), 3.43 (br s, 3H), 3.36 - 3.28 (m, 4H), 3.03 (br s, 14H), 2.33 - 2.19 (m, 10H), 2.11 (s, 9H), 2.04 (br t, J = 6.9 Hz, 7H), 2.00 (s, 9H), 1.89 (s, 9H), 1.81 - 1.74 (m, 9H), 1.57 - 1.38 (m, 22H), 1.37 - 1.17 (m, 10H).

[0106] Step L: Referring to the preparation of compound D01 of Example 1 to obtain D02. 1 H NMR (400 MHz, DMSO-d6) δ 7.92 - 7.73 (m, 9H), 7.38 - 7.16 (m, 10H), 6.94 - 6.83 (m, 4H), 5.22 (d, J = 3.4 Hz, 3H), 4.97 (dd, J = 3.4, 11.3 Hz, 3H), 4.53 - 4.44 (m, 3H), 4.44 - 4.35 (m, 1H), 4.21 - 4.09 (m, 1H), 4.03 (s, 8H), 3.94 - 3.83 (m, 3H), 3.77 - 3.68 (m, 9H), 3.58 - 3.48 (m, 10H), 3.43 (br s, 3H), 3.36 - 3.28 (m, 4H), 3.03 (br s, 14H), 2.33 - 2.19 (m, 10H), 2.11 (s, 9H), 2.04 (br t, J = 6.9 Hz, 7H), 2.00 (s, 9H), 1.89 (s, 9H), 1.81 - 1.74 (m, 9H), 1.57 - 1.38 (m, 22H), 1.37 - 1.17 (m, 10H).

[0107] Example 3: Synthesis of D-01-M

[0108]

[0109] Step A: To a solution of D-01 (100 mg, 39.88 μmol) in acetonitrile (0.7 mL) was added ammonia water (30%, 1.4 mL). The mixture was stirred at 50 °C for 12 h. The reaction mixture was filtered and purified by p-HPLC (separation column: Phenomenex Gemini-NX C18 (size: 75 mm x 30 mm, particle size: 3 μm); mobile phase: [water (0.05% ammonia water) - acetonitrile]; elution gradient: 14% - 42%, 7 min) to give D-01-M. 1 H NMR (400 MHz, DMSO-d6): δ 7.90-7.57 (m, 9H), 7.38-7.10 (m, 10H), 6.93-6.80 (m, 4H), 4.67-4.52 (m, 6H), 4.52-4.44 (m, 3H), 4.22-4.19 (m, 3H), 3.77-3.62 (m, 15H), 3.58-3.47 (m, 16H), 3.46-3.43 (m, 2H), 3.32-3.25 (m, 6H), 3.12-2.91 (m, 14H), 2.36-2.16 (m, 10H), 2.11-1.99 (m, 7H), 1.85-1.73 (m, 9H), 1.59-1.35 (m, 22H), 1.32-1.13 (m, 12H).

[0110] Test of the binding ability of the compound to human asialoglycoprotein receptor

[0111] 1. Purpose of the experiment:

[0112] The binding ability of the compound to human asialoglycoprotein receptor (ASGPR) was detected by surface plasmon resonance technology (SPR), and the kinetic K D value of the compound was used as an index to evaluate the binding ability of the compound to ASGPR, so as to reflect the ability of the compound to specifically target liver cells to deliver nucleic acid molecules.

[0113] 2. Experimental materials:

[0114] 2.1 Protein:

[0115] Asialoglycoprotein Receptor Protein, Mouse, Recombinant (His Tag), Sinobiological-50083-M07H-50 μg; Asialoglycoprotein Receptor Protein, Human, Recombinant (His Tag), Sino biological-10773-H07H-50 μg

[0116] 2.2 Reagents:

[0117] NiHC 1500M Chip (Xan Tec-SCN ihc1500m0720); HEPES (SIGMA-V900477); NaCl (SIGMA-71376); Tween-20 (Aladin-T104863); CaCl2(SIGMA-C3306-250G); EDTA (SIGMA-3609); NiCl2(Energy-chemical-V830089); 10x PBS (Sangon-E607016-0500)

[0118] 2.3 Consumables and instruments:

[0119] Biacore 8k; Series S CM5 chip (GE Healthcare - BR100530) 96-well plate - 250 μL (Greiner-650201); 384-well plate - 200 μL (Greiner-781270); 96-well plate - 1 mL (Greiner-780201); 96 Microplate foils (GE Healthcare-28975816); 384 Microplate foils (GE Healthcare-BR100577)

[0120] 3. Experimental steps and methods:

[0121] 3.1 Dissolve two proteins to 0.25 mg / mL with 1x PBS, dissolve the test compound with DMSO, prepare running buffer (10 mM HEPES, 150 mM NaCl, 0.05% Tween 20, 50 mM CaCl2, 50 μM EDTA) and filter with 0.22 μm membrane.

[0122] 3.2 Dock the NiHC 1500M Chip into the biacore, switch the system to running buffer.

[0123] 3.3 Protein labeling: The chip is first treated with 350 mM EDTA for 5 min, 30 μl / min, washed with running buffer for 2 min, then bound with 40 mM NiCl2for 2 min, 30 μl / min, then label the protein diluted to 5 μg / mL with running buffer onto the chip, the flow rate is 10 μl / min, and the final labeling amount is 1500-2000 RU

[0124] 3.4 Instrument system switched to running buffer containing 2% DMSO.

[0125] 3.5 Compound was diluted 9 concentrations with running buffer 2% DMSO, starting concentration was 2 μM, final concentration of DMSO was 2%.

[0126] 3.6 Test: each cycle binding 60 s, dissociation 180 s, flow rate was 50 μl / min, compound concentration was tested from low to high, and 4 cycles of blank (2% DMSO running buffer) were added before compound gradient test, and 1.5%-3.5% DMSO (6 concentrations) was used for solvent correction.

[0127] 3.7 Data analysis:

[0128] Data was analyzed by Biacore Insight Evaluation Software, kinetics was analyzed by 1:1 model, and affinity was analyzed by steady state affinity model.

[0129] tris(hydroxymethyl)aminomethane K D (mol / L)]]> K a (L / (mol·s)) K d (1 / s) sample D-01-M 1.56 x 10 -8 ]]> 1.08 x 10 6 ]]> 1.68 x 10 -2 ]]>

[0130] Experimental conclusion:

[0131] In this experiment, the test substance D-01-M showed good binding force in the SPR experiment.

[0132] The present application shows an efficient oligonucleic acid molecule delivery platform with high hepatocyte targeting: it can bind to the specific and highly expressed ASGPR protein on the surface of hepatocytes, enter the endosome through endocytosis and release into the cytoplasm to take effect. The binding constant of the delivery platform to ASGPR is better than that of the prior art. It shows good tissue distribution and metabolic stability in vivo, and is expected to achieve more efficient liver-targeted molecular delivery and drug efficacy. The related conjugates using the present application show good activity for reducing HBsAg, and show long-term inhibition of HBsAg efficacy.

Claims

1. A conjugating group having the structure of Formula (I) ###0001### Formula (I) wherein n is selected from an integer from 8 to 12. ， 2. The conjugating group according to claim 1 having the structure: ###0002### 3. A conjugate comprising the conjugating group according to claim 1 or 2 and a therapeutic agent linked to the conjugating group. 。 4. The conjugate according to claim 3, wherein the therapeutic agent is an expression- inhibiting oligonucleotide.

5. The conjugate according to claim 4, wherein the expression-inhibiting oligonucleotide is an RNAi agent.

6. The conjugate according to claim 5, wherein the RNAi agent comprises one or more modified nucleotides.

7. The conjugate according to claim 5 or 6, wherein the RNAi agent is a double- stranded siRNA comprising a sense strand and an antisense strand.

8. The conjugate according to claim 7, wherein the double-stranded siRNA is linked to the conjugating group at the 5' end of its sense strand.

9. The conjugate according to any one of claims 4-8, wherein the expression-inhibiting oligonucleotide is linked to the conjugating group via a phosphate group, a thiophosphate group, or a phosphonate group.

10. A compound having the structure of Formula (II) or Formula (III) ###0003### Formula (II) Formula (III) wherein m is independently selected from an integer from 8 to 12.

11. The compound according to claim 10 having the structure: ###0004### 、 ， 12. A pharmaceutical composition comprising the conjugate according to any one of claims 3-9 and a pharmaceutically acceptable carrier or excipient.

13. Use of the conjugate according to any one of claims 3-9 or the pharmaceutical composition according to claim 12 in the manufacture of a medicament for the treatment of a liver disease. 、 or 。 14. The use according to claim 13, wherein the liver disease is a viral infection that causes liver disease.

15. The use according to claim 13, wherein the liver disease is hepatitis B. ​ ​

Images