Compounds, conjugates and uses thereof

Abstract

The present invention provides a compound having the structure shown in formula (I) and (II). The present invention also provides a conjugate of the compound linked to a drug molecule, and uses of the compound and conjugate, such as uses in detection and treatment.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, specifically, the present invention relates to compounds, conjugates and uses thereof, more specifically, the present invention relates to compounds, conjugates, methods for preparing compounds, compositions and uses thereof. Background Art

[0002] The asialoglycoprotein receptor (ASGPR) is an abundant, endocytic, heterologous oligomeric receptor primarily found on the sinusoidal surface of liver parenchymal cells. It specifically recognizes sugars. Because the terminal sialic acid residues of various glycoproteins are removed by enzymatic hydrolysis or acidolysis, the exposed secondary termini are galactose residues. Therefore, the sugar-binding specificity of ASGPR lies in the galactose moiety, earning it the name galactose-specific receptor. ASGPR is primarily found in liver parenchymal cells, with low levels in other cells, making it an optimal receptor for targeted transport to the liver.

[0003] Glycoproteins with non-reducing galactose (Gal) or N-acetylgalactosamine (GalNAc) residues at the end can be recognized by ASGPR, and the affinity of GalNAc for binding to ASGPR is approximately 50 times higher than that of Gal (Lobst ST et al, J Biol Chem, 1996, 271(12):6686-6693). In vitro experiments have shown that clustered sugar residues can occupy the binding sites of the receptor simultaneously, making their affinity much higher than that of non-clustered sugar residues. The order of affinity is: tetraantennary > triantennary >> biantennary >> monoantennary galactosides (Lee YC, et al, J Biol Chem, 1983, 258(1):199-202).

[0004] ASGPR receptor-mediated liver-targeting oligonucleotides represent a breakthrough in the field of innovative nucleic acid drug research. In 2012, Alnylam Pharmaceuticals (USA) covalently linked a previously developed triantennary GalNAc structure to small interfering RNA (siRNA), achieving liver-targeted siRNA delivery in vivo. Using this technology, researchers have developed drugs for diseases such as amyloidosis, hemophilia, hypercholesterolemia, hepatic porphyria, and hepatitis B. In 2019, the first GalNAc-siRNA drug was marketed, two are under development, and more than a dozen candidates have entered clinical trials (http: / / www.alnylam.com / product-pipeline / ). In 2014, ISIS Pharmaceuticals (USA) covalently linked a triantennary GalNAc structure to an antisense nucleic acid, achieving liver-targeted delivery in animals. The activity of the antisense nucleic acid increased tenfold after this link (Prakash TP et al., Nucleic Acids Res. 42, 8796-807).

[0005] However, ASGPR receptor-mediated liver-targeting oligonucleotide technology still needs further development and improvement. Summary of the Invention

[0006] The applicants of this application use pentaerythritol (PET) as the main structure to form a multi-antennary ASGPR ligand. One of the four hydroxyl groups of pentaerythritol can be converted into a variety of different functional groups, such as amino, azide, carboxyl, carbonyl, etc., which can react with a variety of chemical groups to form a variety of connected GalNAc receptors. Compared with Alnylam's product, which uses trishydroxymethylaminomethane (TRIS) as the main structure to form a multi-antennary ASGPR ligand, the multi-antennary ASGPR ligand of this application can obtain GalNAc-coupled oligonucleotides with more diverse structures.

[0007] Based on this, in the first aspect of the present invention, the present invention proposes a compound. According to an embodiment of the present invention, the compound has a structure shown in formula (I),

[0008]

[0009] Among them, R1 is the part that binds to ASGPR; R2 is -CH2-,

[0010] C 6-14 Aromatic group, C 2-6 Alkenyl or C 2-6 Alkynyl; R3 is -O-、C 6-14 aromatic group or 3 to 10 membered heterocyclic group; R4 is -CH2-,

[0011] C 6-14 Aryl or 3 to 10 membered heterocyclic group; R5 is -OH, -COOH, PG is a carboxyl protecting group; PG2 is a hydroxyl protecting group; a, b, c, and d are each independently an integer from 1 to 10; and n is an integer from 1 to 3. The compound represented by formula (I) according to the embodiments of the present invention can be linked to a variety of oligonucleotide chains via a phosphodiester bond, and the linking position can be at the 5' end, 3' end, or between the 5' end and the 3' end of the oligonucleotide chain. The types of oligonucleotides that can be linked are greatly expanded compared to the prior art, and the non-restricted linking position provides more options for oligonucleotide modification, which is beneficial for improving the pharmacokinetic properties of nucleic acid drugs.

[0012] According to an embodiment of the present invention, the above compound may further include at least one of the following additional technical features:

[0013] According to an embodiment of the present invention, the R1 moiety terminates in galactose or N-acetylgalactosamine;

[0014] According to an embodiment of the present invention, the R1 moiety is selected from the following structures:

[0015]

[0016] wherein q and q' are independently any integer from 1 to 10.

[0017] According to an embodiment of the present invention, the carboxyl protecting group is selected from succinate, Wherein h is any integer from 1 to 5.

[0018] According to an embodiment of the present invention, the hydroxy protecting group is selected from silyl, monomethoxytrityl (MMTr), 4,4'-dimethoxytrityl (DMTr) and trityl.

[0019] According to an embodiment of the present invention, the silyl group is selected from tert-butyldimethylsilyl ether (TBMDS), tert-butyldiphenylsilyl (TBDPS) and triisopropylsilyl ether (TIPS).

[0020] According to an embodiment of the present invention, the compound has the structure shown below:

[0021]

[0022]

[0023]

[0024] In the second aspect of the present invention, the present invention provides a compound. According to an embodiment of the present invention, the compound has a structure shown in formula (II),

[0025]

[0026] Among them, R1 is the part that binds to asialoglycoprotein receptor (ASGPR); R2 is -CH2-, C 6-14 Aromatic group, C 2-6 Alkenyl or C 2-6 Alkynyl; a and b are each independently an integer from 1 to 10; and n is an integer from 1 to 3.

[0027] The compound represented by formula (II) according to the embodiment of the present invention can be used as a coupling reagent to be directly coupled with an oligonucleotide containing an alkyne group, or can be used as a precursor of a coupling reagent to react mildly and efficiently with a compound having an active group such as an alkyne-containing pentafluorophenol ester to obtain a Gal or GalNAc coupling reagent of the pentafluorophenol ester, which can then be coupled with oligonucleotides having various functional groups to obtain Gal or GalNAc-coupled oligonucleotides with more diverse structures.

[0028] According to an embodiment of the present invention, the above compound may further include at least one of the following additional technical features:

[0029] According to an embodiment of the present invention, the R1 moiety terminates in galactose or N-acetylgalactosamine.

[0030] According to an embodiment of the present invention, the R1 moiety is selected from the following structures:

[0031]

[0032]

[0033] wherein q and q' are each independently any integer from 1 to 10.

[0034] According to an embodiment of the present invention, the compound has the structure shown below:

[0035]

[0036] In the third aspect of the present invention, the present invention provides a conjugate. According to an embodiment of the present invention, the conjugate has a structure shown in formula (III),

[0037]

[0038] Among them, R1 is the part that binds to ASGPR; R2 is -CH2-,

[0039] C 6-14 Aromatic group, C 2-6 Alkenyl or C 2-6 Alkynyl; R3 is -O-、C 6-14 aromatic group or 3 to 10 membered heterocyclic group; R4 is -CH2-,

[0040] C 6-14 an aryl group or a 3- to 10-membered heterocyclic group; R is O or S; Rx is a drug molecule selected from a cytotoxic agent, a chemotherapeutic agent, a growth inhibitory agent, a toxin, a radioactive isotope, and an oligonucleotide chain; a, b, c, and d are each independently an integer from 1 to 10; and n is an integer from 1 to 3.

[0041] According to an embodiment of the present invention, the above conjugate may further include at least one of the following additional technical features:

[0042] According to an embodiment of the present invention, the conjugate has a structure shown in formula (IV),

[0043] Wherein oligo is an oligonucleotide chain, optionally, the oligonucleotide chain is a single-stranded oligonucleotide or a double-stranded oligonucleotide or a combination thereof.

[0044] In a fourth aspect, the present invention provides a conjugate. According to an embodiment of the present invention, the conjugate is formed by linking the compound of the first or second aspect of the present invention to a drug molecule, wherein the drug molecule is selected from a cytotoxic agent, a chemotherapeutic agent, a growth inhibitory agent, a toxin, a radioisotope, and an oligonucleotide chain.

[0045] The conjugates according to the embodiments of the third aspect or fourth aspect of the present invention can specifically target the liver, and the coupled drug molecules, such as oligonucleotide chains, are specifically introduced into hepatocytes to achieve intervention or detection of specific genes in hepatocytes, or to treat or prevent pathological conditions or diseases caused by the expression of specific genes in hepatocytes.

[0046] According to an embodiment of the present invention, the drug molecule is an oligonucleotide chain, and optionally, the oligonucleotide chain is a single-stranded oligonucleotide, a double-stranded oligonucleotide, or a combination thereof.

[0047] According to the embodiment of the conjugate of the third aspect or the fourth aspect of the present invention, the conjugate may further include at least one of the following additional technical features:

[0048] According to an embodiment of the present invention, the oligonucleotide chain comprises unmodified nucleotides and / or modified nucleotides.

[0049] According to an embodiment of the present invention, the modified nucleotides are each independently selected from 2′-methoxyethyl modified nucleotides, 2′-O-alkyl modified nucleotides, 2′-O-allyl modified nucleotides, 2′-C-allyl modified nucleotides, 2′-fluoro modified nucleotides, 2′-deoxy modified nucleotides, 2′-hydroxy modified nucleotides, locked nucleotide modified nucleotides, glycerol nucleic acid (glycolnucleic acid, GNA) modified nucleotides and unlocked nucleic acid modified nucleotides (unlocked nucleic acid, UNA).

[0050] According to an embodiment of the present invention, the 2'-O-alkyl modification is a 2'-O-methyl modification.

[0051] According to an embodiment of the present invention, the oligonucleotide chain has a terminal modification.

[0052] According to an embodiment of the present invention, the terminal modification is selected from cholesterol, polyethylene glycol, a fluorescent probe, biotin, a polypeptide, a vitamin, a tissue-targeting molecule, or a combination thereof.

[0053] According to an embodiment of the present invention, the length of the oligonucleotide chain is 5-100 bp.

[0054] According to an embodiment of the present invention, the compound is linked to the 5' end, 3' end, or any nucleotide between the 5' end and the 3' end of at least one oligonucleotide chain via a phosphate bond. Compared to existing technologies, the types of oligonucleotides that can be linked are greatly expanded, and the non-restricted linking position provides more options for oligonucleotide modification, which is beneficial for improving the pharmacokinetic properties of nucleic acid drugs.

[0055] According to an embodiment of the present invention, the phosphate bond is a phosphodiester bond or a modified phosphate bond.

[0056] According to an embodiment of the present invention, the modified phosphate bond is selected from a thio-modified phosphate bond and an amino-modified phosphate bond.

[0057] According to an embodiment of the present invention, the ribose-phosphate backbone of the oligonucleotide chain is replaced by polypeptide nucleic acid (PNA) or morpholino antisense nucleotide (PMO).

[0058] According to an embodiment of the present invention, the conjugate is obtained by solid phase synthesis or liquid phase synthesis.

[0059] In a fifth aspect of the present invention, the present invention provides a method for preparing a compound represented by formula (II). According to an embodiment of the present invention, the method comprises subjecting pentaerythritol, NaN3 and galactose or a galactose derivative to a synthesis reaction to obtain the compound.

[0060] The method according to the embodiment of the present invention uses pentaerythritol (PET) as the main structure for the first time to prepare a multi-antennary ASGPR ligand. One of the four hydroxyl groups is converted into azide. The prepared product can be directly coupled with an oligonucleotide containing an alkyne group, or can be subjected to a mild and efficient reaction with a compound with an active group such as an alkyne-containing pentafluorophenol ester to obtain a Gal or GalNAc coupling reagent of the pentafluorophenol ester, which can then be coupled with oligonucleotides with various functional groups to obtain Gal or GalNAc-coupled oligonucleotides with diverse structures.

[0061] In a sixth aspect of the present invention, the present invention provides a method for preparing a compound of formula (I). According to an embodiment of the present invention, the method comprises contacting a first compound with a second compound to obtain the compound, wherein the first compound is as defined in the second aspect or obtained according to the method described in the fifth aspect.

[0062] According to a specific embodiment of the present invention, the second compound has the structure shown below:

[0063]

[0064] wherein c and d are independently any integer from 1 to 10.

[0065] According to the above method of the embodiment of the present invention, the compound represented by formula (II) reacts with a compound containing an alkyne group and carrying an active group in a mild and efficient manner to obtain a Gal or GalNAc coupling reagent with an active group; or the compound represented by formula (II) is converted from an azide group to an amino group in the presence of H2, and then reacts with a second compound to form a Gal or GalNAc coupling reagent with an active group. The compound prepared by the above method according to the embodiment of the present invention can be connected to a variety of oligonucleotide chains through a phosphodiester bond, and the connection position can be at the 5' end, 3' end, or between the 5' end and the 3' end of the oligonucleotide chain. The types of oligonucleotides that can be connected are greatly expanded compared to the prior art, and the non-limited connection position provides more options for the modification of oligonucleotides, which is beneficial to improving the pharmacokinetic properties of nucleic acid drugs.

[0066] In its seventh aspect, the present invention provides a composition. According to an embodiment of the present invention, the composition comprises the conjugate described in the third or fourth aspect of the present invention. The composition according to an embodiment of the present invention can specifically target hepatocytes, allowing the conjugated drug molecule, such as an oligonucleotide, to enter the hepatocytes, thereby achieving gene intervention or detection within the hepatocytes.

[0067] According to an embodiment of the present invention, the above composition may further include at least one of the following additional technical features:

[0068] According to an embodiment of the present invention, the composition further comprises a pharmaceutically acceptable carrier or excipient.

[0069] According to an embodiment of the present invention, the dosage form of the composition is powder, tablet, granule, capsule, solution, emulsion, suspension, injection, spray, aerosol, powder spray or microneedle patch.

[0070] According to an embodiment of the present invention, the composition is administered to the subject by intravenous injection, intramuscular injection, or subcutaneous injection.

[0071] According to an embodiment of the present invention, the subject is a mammal.

[0072] According to a particular embodiment of the present invention, the mammal is selected from the group consisting of bovines, equines, ovines, porcines, canines, felines, rodents and primates.

[0073] In an eighth aspect of the present invention, the present invention provides the use of the conjugate described in the third aspect or the fourth aspect in the preparation of a medicament for treating and / or preventing pathological conditions or diseases associated with the expression or overexpression of genes in hepatocytes. The composition according to an embodiment of the present invention can specifically target hepatocytes, allowing the drug molecules to which it is coupled, such as oligonucleotides, to enter the hepatocytes, thereby achieving gene intervention in the hepatocytes. The medicament prepared from the composition according to an embodiment of the present invention can be used for the effective treatment and / or prevention of pathological conditions or diseases associated with the expression or overexpression of genes in hepatocytes.

[0074] According to a specific embodiment of the present invention, the gene is selected from HBV genome, HCV genome, PCSK9, xanthine oxidase, URAT1, APOB, liver fibrosis-related genes (AP3S2, AQP2, AZIN1, DEGS1, STXBP5L, TLR4, TRPM5, etc.), non-alcoholic fatty liver disease (PNPLA3, FDFT1), primary biliary cirrhosis (HLA-DQB1, IL-12, IL-12RB2, etc.), or a combination thereof.

[0075] According to a specific embodiment of the present invention, the disease is selected from hereditary angioedema, familial tyrosinemia type I, Alagille syndrome, alpha-1-antitrypsin deficiency, bile acid synthesis and metabolism defects, biliary atresia, cystic fibrosis liver disease, idiopathic neonatal hepatitis, mitochondrial liver disease, progressive familial intrahepatic cholestasis, primary sclerosing cholangitis, transthyretin amyloidosis, hemophilia, homozygous familial hypercholesterolemia, hyperlipidemia, hepatitis B (HBV), hepatitis C (HCV), steatohepatitis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), hyperglycemia or diseases involving abnormally increased hepatic glucose production similar to type 2 diabetes, hepatitis and hepatic porphyrin.

[0076] In the ninth aspect of the present invention, the present invention provides use of the compound described in the first or second aspect or the conjugate described in the third or fourth aspect in preparing a composition for liver targeting.

[0077] In the tenth aspect of the present invention, the present invention provides use of the conjugate described in the third aspect or the fourth aspect in preparing a composition for liver-targeted RNA detection or localization.

[0078] In the eleventh aspect of the present invention, the present invention provides the conjugate according to the third aspect or the fourth aspect for use in treating and / or preventing pathological conditions or diseases associated with gene expression or overexpression in hepatocytes.

[0079] According to an embodiment of the present invention, the gene is selected from HBV genome, HCV genome, PCSK9, xanthine oxidase, URAT1, APOB, liver fibrosis-related genes (AP3S2, AQP2, AZIN1, DEGS1, STXBP5L, TLR4, TRPM5, etc.), non-alcoholic fatty liver disease (PNPLA3, FDFT1), primary biliary cirrhosis (HLA-DQB1, IL-12, IL-12RB2, etc.), or a combination thereof.

[0080] According to an embodiment of the present invention, the disease is selected from hereditary angioedema, familial tyrosinemia type I, Alagille syndrome, alpha-1-antitrypsin deficiency, bile acid synthesis and metabolism defects, biliary atresia, cystic fibrosis liver disease, idiopathic neonatal hepatitis, mitochondrial liver disease, progressive familial intrahepatic cholestasis, primary sclerosing cholangitis, transthyretin amyloidosis, hemophilia, homozygous familial hypercholesterolemia, hyperlipidemia, hepatitis B (HBV), hepatitis C (HCV), steatohepatitis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), hyperglycemia or diseases involving abnormally increased hepatic glucose production similar to type II diabetes, hepatitis and hepatic porphyrin.

[0081] In the twelfth aspect of the present invention, the present invention provides the compound described in the first or second aspect or the conjugate described in the third or fourth aspect for liver targeting.

[0082] In the thirteenth aspect of the present invention, the present invention provides the conjugate according to the third aspect or the fourth aspect, for use in liver-targeted RNA detection or localization.

[0083] In a fourteenth aspect, the present invention provides a method for treating and / or preventing a pathological condition or disease associated with the expression or overexpression of a gene in hepatocytes. According to an embodiment of the present invention, the method comprises administering to a subject a pharmaceutically acceptable amount of the conjugate of the third or fourth aspect.

[0084] In the fifteenth aspect of the present invention, the present invention proposes a method for intervening or detecting a predetermined gene in a liver cell. According to an embodiment of the present invention, the method comprises: contacting a cell population with the conjugate described in the third or fourth aspect, wherein the cell population contains the liver cell, and the oligonucleotide chain in the conjugate interacts with the predetermined gene. According to the method of the embodiment of the present invention, the conjugate specifically targets the liver cell, and the oligonucleotide chain coupled to the conjugate enters the liver cell, thereby achieving intervention or detection of the gene in the liver cell. It should be noted that the manner in which "the oligonucleotide chain in the conjugate interacts with the predetermined gene" described in this application is not particularly limited, and the mode of action may be direct or indirect, and the mode of interaction includes but is not limited to binding and complementary pairing. The effect produced after the interaction is adapted to the mode of interaction between the two. For example, when the conjugated oligonucleotide chain is an siRNA targeting a predetermined gene, after the interaction, the silencing of the predetermined gene can be achieved. For example, when the conjugated oligonucleotide chain is a probe targeting a predetermined gene, after the interaction, the positioning or detection of the predetermined gene can be achieved.

[0085] Compared with the prior art, the present invention has the following significant differences and technical advances:

[0086] 1. Different chemical structures. The prior art uses tris(hydroxymethyl)aminomethane (TRIS) as the main structure to form a multi-antennary ASGPR ligand. The present invention uses pentaerythritol (PET) as the main structure to form a multi-antennary ASGPR ligand. One of the four hydroxyl groups can be converted into a variety of different functional groups, such as amino, azide, carboxyl, carbonyl, etc., which can be linked with a variety of chemical groups to form a diversely linked GalNAc receptor. For example, azide-PETGalNAc can be coupled with oligonucleotide chains containing alkyne groups and can react mildly and efficiently with compounds with active groups such as alkyne-containing pentafluorophenol esters to obtain pentafluorophenol ester GalNAc coupling reagents. This can be directly coupled with oligonucleotides containing a variety of different functional groups to obtain GalNAc-coupled oligonucleotides with diverse structures.

[0087] 2. The synthesis method is simpler and more efficient. The azide compound obtained in the present invention can obtain the target molecule through a one-step click reaction with a branched alkyne group, which reduces the number of reaction steps and improves the synthesis efficiency. The reaction route, experimental process, and subsequent purification are all simpler and easier to operate.

[0088] 3. The covalent attachment method of the ligand to the oligonucleotide chain is different. The prior art uses a method of attaching a triantennary GalNAc ligand to a solid phase support, and the modified solid phase support is used for solid phase synthesis of oligonucleotides, so that the ligand can only be attached to the 3' end of the oligonucleotide. The present invention can not only attach the phosphoramidite of the triantennary GalNAc ligand to the 5' end oligonucleotide by solid phase synthesis, but also can, after completing solid phase synthesis, (1) attach the triantennary PETGalNAc ligand to the end (3' or 5') or any middle position of the oligonucleotide by liquid phase ligation of an activated ester such as pentafluorophenol ester of the triantennary GalNAc ligand with an amino-modified oligonucleotide; (2) attach the triantennary GalNAc ligand to the end or any middle position of an acetylene-modified oligonucleotide by liquid phase ligation. This method has a mild reaction, high coupling yield, and simple operation; (3) can be coupled to oligonucleotides with multiple amino or acetylene groups to achieve multi-ligand modification. The present invention expands the types of oligonucleic acid modifications, is conducive to finding drug-forming molecules with higher activity, and is more suitable for large-scale production.

[0089] 4. Different oligonucleotide sites and combinations can be modified. The terminal sites in oligonucleotide drug molecules are usually modified with cholesterol, polyethylene glycol (PEG), etc. to improve pharmacokinetic properties. The triantennary GalNAc ligands designed in the prior art are only used for 3'-terminal modification of oligonucleotide chains, occupying the terminal modification site and reducing the types of oligonucleotide modifications that can be applied. The new compounds of the present invention can be modified at any position of the oligonucleotide, and other modifications are not affected by the end. The preparation of a new compound mixed with terminal cholesterol to modify an oligonucleotide chain is described in the embodiment of the present invention. DETAILED DESCRIPTION

[0090] The following examples are provided to further illustrate the present invention. 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.

[0091] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0092] Terminology used in this article

[0093] In the structural formula herein, n represents the number of adjacent atoms to which the group in the square brackets is connected. For example, in the structural formula (I), n means there are n Attached to adjacent carbon atoms.

[0094] As used herein, the term "oligonucleotide chain" refers to an oligomeric compound containing multiple or all chemically modified or unmodified nucleotides, which has a length of less than about 100 nucleotides (e.g., 1-20 nucleotides or 1-50 nucleotides). In certain embodiments, a non-nucleic acid conjugated group may be included in the oligonucleotide chain. In certain embodiments, the oligonucleotide comprises ribonucleic acid (RNA), deoxyribonucleic acid (DNA), or oligopeptide nucleotides (PNA). In certain embodiments, the oligonucleotide chain is double-stranded or single-stranded. In certain embodiments, the oligonucleotide chain is siRNA, nucleic acid aptamer, antisense nucleic acid, sgRNA, tractRNA, or crRNA. The m before the nucleotide in the sequence of the present application represents 2'OMe modification, and f represents 2'F modification. For example, mG represents that glycine has 2'OMe modification, and fU represents that uracil has 2'F modification.

[0095] As used herein, "conjugate group" means an atom or group of atoms that is bound to an oligonucleotide chain. In some cases, conjugate groups alter one or more properties of the oligonucleotide to which they are attached, including but not limited to pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge and / or clearance properties.

[0096] As used herein, the term "conjugate" refers to a coupled molecule of a compound of the present invention and an oligonucleotide chain, for example, the compound of formula (III) of the present application, or Z1001-Z1013 of the present application (wherein n, m, and m' are each independently selected from integers between 1 and 10).

[0097]

[0098]

[0099]

[0100] As used herein, the term "coupling reagent" means a compound or substance that can couple other compound molecules, for example, the compound represented by formula (I) or formula (II) of the present application, and for example, GalNAc-1 to GalNAc-14 of the present application.

[0101] GalNAc-1

[0102]

[0103] GalNAc-2

[0104]

[0105] GalNAc-3

[0106]

[0107] GalNAc-4

[0108]

[0109] GalNAc-5

[0110]

[0111] GalNAc-6

[0112]

[0113] GalNAc-7:

[0114] GalNAc-8:

[0115] GalNAc-9:

[0116] GalNAc-10:

[0117]

[0118] GalNAc-11:

[0119]

[0120] GalNAc-12:

[0121]

[0122] GalNAc-13

[0123]

[0124] GalNAc-14

[0125]

[0126] As used herein, the term "receptor" refers to a biological macromolecule composed of glycoproteins or lipoproteins, which is present in the cell membrane, cytoplasm or nucleus. Different receptors have specific structures and configurations. As used herein, the term "ligand" refers to a substance or compound that has the ability to recognize and bind to a receptor. In certain embodiments, the ligand is a ligand that binds to an asialoglycoprotein receptor (ASGPR). In certain embodiments, the ligand is a carbohydrate, such as a monosaccharide and a polysaccharide, including but not limited to: galactose, N-acetylgalactosamine, mannose, glucose, glucosamine and fucose.

[0127] As used herein, the term "polysaccharide" refers to a polymer composed of multiple monosaccharide groups linked by glycosidic bonds. In the present invention, polysaccharides include oligosaccharides and oligosaccharides. Generally, "oligosaccharides" refer to polymers composed of 2-10 monosaccharide groups linked by glycosidic bonds, and "oligosaccharides" refer to polymers composed of 20 or fewer monosaccharide groups linked by glycosidic bonds.

[0128] As used herein, the term "about" should be understood by those skilled in the art and will vary to some extent depending on the context in which it is used. If the meaning is not clear to one skilled in the art based on the context in which the term is used, then "about" means a deviation of no more than plus or minus 10% from the specified value or range.

[0129] As used herein, the term "prevent" or "prevent" means to stop or delay the occurrence of a disease.

[0130] As used herein, the term "treating" refers to curing or at least partially arresting the progression of a disease, or alleviating the symptoms of a disease.

[0131] As used herein, the term "effective amount" refers to an amount effective to achieve the intended purpose. For example, a disease-preventive effective amount refers to an amount effective to prevent, inhibit, or delay the onset of a disease. Determining such an effective amount is within the capabilities of those skilled in the art.

[0132] When a numerical range is listed, it is intended to include every value and sub-range within the stated range. For example, "C 1-6 "Alkyl" includes C1, C2, C3, C4, C5, C6, C 1-6 、C 1-5 、C 1-4 、C 1-3 、C 1-2 、C 2-6 、C 2-5 、C 2-4 、C 2-3 、C 3-6 、C 3-5 、C 3-4 、C 4-6 、C 4-5 and C 5-6 alkyl.

[0133] "Alkyl" refers to a straight or branched chain saturated hydrocarbon group having carbon atoms. In some embodiments, C 1-6 Alkyl groups are preferred, C 1-4 Alkyl is particularly preferred. Examples of the alkyl include, but are not limited to, methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), isobutyl (C4), n-pentyl (C5), 3-pentyl (C5), pentyl (C5), neopentyl (C5), 3-methyl-2-butyl (C5), tert-pentyl (C5) and n-hexyl (C6). Regardless of whether the alkyl group is modified with "substituted", each of the alkyl groups is independently optionally substituted, for example, 1 to 5 substituents, 1 to 3 substituents or 1 substituent.

[0134] "Alkenyl" refers to a straight or branched chain hydrocarbon group having at least one carbon-carbon double bond. 2-6 Alkenyl is preferred, C 2-4 Alkenyl is more preferred. 2-6 Examples of alkenyl groups include ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like. The term "C 2-6"Alkenyl" also includes heteroalkenyl groups in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). Regardless of whether the alkenyl group is preceded by "substituted," each substituent of the alkenyl group is independently optionally substituted, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0135] "Alkynyl" refers to a straight or branched chain hydrocarbon group having at least one carbon-carbon triple bond and optionally one or more carbon-carbon double bonds. 2-6 Alkynyl is preferred, C 2-4 Alkynyl is more preferred. 2-6 Examples of alkynyl groups include, but are not limited to, ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), pentynyl (C5), hexynyl (C6), and the like. The term "C 2-6 "Alkynyl" also includes heteroalkynyl groups in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). Regardless of whether the alkynyl group is preceded by "substituted," each substituent of the alkynyl group is independently optionally substituted, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. Suitable substituents are defined below.

[0136] “C 6-14 "Aryl" refers to a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in a cyclic arrangement) having 6-14 ring carbon atoms and zero heteroatoms. In some embodiments, an aryl group has six ring carbon atoms ("C6 aryl"; e.g., phenyl). In some embodiments, an aryl group has ten ring carbon atoms ("C 10 In some embodiments, an aryl group has fourteen ring carbon atoms ("C 14 In some embodiments, C 6-10 Aryl is particularly preferred, and C6 aryl is more preferred. Aryl also includes ring systems in which the aforementioned aryl ring is fused to one or more cycloalkyl or heterocyclic groups, and the point of attachment is on the aryl ring. In this case, the number of carbon atoms continues to represent the number of carbon atoms in the aryl ring system. Regardless of whether the aryl group is preceded by "substituted," each aryl group is independently optionally substituted, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0137] "3- to 10-membered heterocyclyl" refers to a radical of a 3- to 10-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon. In heterocyclyl groups containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom, as valence permits. In some embodiments, a 3- to 7-membered heterocyclyl is preferred, which is a 3- to 7-membered non-aromatic ring system having ring carbon atoms and 1 to 3 ring heteroatoms; in some embodiments, a 3- to 6-membered heterocyclyl is particularly preferred, which is a 3- to 6-membered non-aromatic ring system having ring carbon atoms and 1 to 3 ring heteroatoms; more preferably, a 5- to 6-membered heterocyclyl is a 5- to 6-membered non-aromatic ring system having ring carbon atoms and 1 to 3 ring heteroatoms. Heterocyclyl also includes ring systems in which the above-mentioned heterocyclyl ring is fused to one or more cycloalkyl, aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring; and in such cases, the number of ring members continues to represent the number of ring members in the heterocyclyl ring system. Regardless of whether the heterocyclyl group is preceded by "substituted", each of the heterocyclyl groups is independently optionally substituted, for example, with 1 to 5 substituents, 1 to 3 substituents or 1 substituent.

[0138] Exemplary 3-membered heterocyclic groups containing one heteroatom include, but are not limited to, aziridine, oxirane, and thiorenyl. Exemplary 4-membered heterocyclic groups containing one heteroatom include, but are not limited to, azetidinyl, oxetane, and thietanyl. Exemplary 5-membered heterocyclic groups containing one heteroatom include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclic groups containing two heteroatoms include, but are not limited to, dioxolanyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-membered heterocyclic groups containing three heteroatoms include, but are not limited to, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyls containing one heteroatom include, but are not limited to, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyls containing two heteroatoms include, but are not limited to, piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary 6-membered heterocyclyls containing three heteroatoms include, but are not limited to, hexahydrotriazinyl. Exemplary 7-membered heterocyclyls containing one heteroatom include, but are not limited to, azepanyl, oxepanyl, and thiepanyl. Exemplary 8-membered heterocyclyls containing one heteroatom include, but are not limited to, azocanyl, oxepanyl, and thiocanyl. Exemplary 5-membered heterocyclyls fused to a C6 aryl ring (also referred to herein as 5,6-bicyclic heterocyclyls) include, but are not limited to, dihydroindolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, benzoxazolinyl, and the like. Exemplary 6-membered heterocyclyl groups (also referred to herein as 6,6-bicyclic heterocyclyl groups) fused to a C6 aryl ring include, but are not limited to, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like.

[0139] Exemplary substituents include, but are not limited to, halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, alkyl, haloalkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl.

[0140] The term "stereoisomers" refers to compounds that have identical chemical constitution but differ in the way the atoms or groups are arranged in space. Stereoisomers include enantiomers, diastereomers, conformers (rotamers), geometric isomers (cis / trans) isomers, atropisomers, and the like.

[0141] The stereochemical definitions and conventions used herein generally follow those of SP Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S, "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc, New York, 1994. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. In describing optically active compounds, the prefixes D and L or R and S are used to denote the absolute configuration of the molecule about one or more of its chiral centers. The prefixes d and l or (+) and (-) are the symbols used to designate the rotation of plane-polarized light caused by the compound, where (-) or l indicates that the compound is levorotatory. A compound prefixed with (+) or d is dextrorotatory. A specific stereoisomer is an enantiomer, and a mixture of such isomers is called an enantiomeric mixture. A 50:50 mixture of enantiomers is called a racemic mixture or racemate and can occur when there is no stereoselectivity or stereospecificity in a chemical reaction or process.

[0142] Any resulting mixture of stereoisomers can be separated into the pure or substantially pure geometric isomers, enantiomers, and diastereomers on the basis of the differences in the constituent physicochemical properties, for example, by chromatography and / or fractional crystallization.

[0143] The term "tautomer" or "tautomeric form" refers to structural isomers with different energies that are interconvertible across a low energy barrier. If tautomerism is possible (e.g., in solution), a chemical equilibrium of the tautomers can be achieved. For example, proton tautomers (also known as prototropic tautomers) include interconversions via proton migration, such as keto-enol isomerization and imine-enamine isomerization.

[0144] The term "pharmaceutically acceptable salt" refers to salts that are suitable for contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic reaction, etc., and are commensurate with a reasonable benefit / risk ratio, within the scope of sound medical judgment. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66: 1-19. Pharmaceutically acceptable salts of the compounds of the present invention include salts derived from suitable inorganic and organic acids and inorganic and organic bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or salts formed with organic acids, such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid. Salts formed using conventional methods in the art, such as ion exchange methods, are also included. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, gluconate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. Pharmaceutically acceptable salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N + (C 1-4 Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Other pharmaceutically acceptable salts include non-toxic ammonium salts, quaternary ammonium salts, and amine cations formed with counterions such as halides, hydroxides, formates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates, if appropriate.

[0145] treat

[0146] "Subjects" to be administered include, but are not limited to, humans (i.e., males or females of any age group, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged adults, or older adults)) and / or non-human animals, e.g., mammals, e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys), cattle, pigs, horses, sheep, goats, rodents, cats, and / or dogs. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal. The terms "human," "patient," and "subject" are used interchangeably herein.

[0147] In the therapeutic method of the present invention, " effective amount " refers to the amount or dosage that is enough to produce the desired therapeutic benefit in the individuality of the treatment in need.The effective amount or dosage of the compounds of this invention can be determined by conventional methods (such as modeling, dose escalation or clinical trials) and conventional factors (such as the mode or approach of drug delivery, the pharmacokinetics of medicament, the severity and process of infection, the health status and body weight of the individual and the judgment of the treating physician).Exemplary dosage is in the range of about 0.1mg to 1g per day or about 1mg to 50mg per day or about 50mg to 250mg per day or about 250mg to 1g per day.Total dose can be administered by single dose or separate dosage units (for example, BID, TID, QID).

[0148] After the patient's disease improves, the dosage can be adjusted for preventive or maintenance treatment. For example, the dosage or frequency of administration or both can be reduced to the amount required to maintain the desired treatment or preventive effect, depending on the symptoms. Of course, if the symptoms have been alleviated to an appropriate degree, treatment can be stopped. However, when any symptom recurs, the patient may need long-term intermittent treatment. The patient may also need long-term slow treatment.

[0149] Pharmaceutical compositions, preparations and kits

[0150] The present invention provides pharmaceutical compositions comprising a compound or conjugate of the present invention (also referred to as an "active ingredient") and a pharmaceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical composition comprises an effective amount of the active ingredient. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the active ingredient. In some embodiments, the pharmaceutical composition comprises a prophylactically effective amount of the active ingredient.

[0151] The pharmaceutically acceptable excipients used in the present invention refer to non-toxic carriers, adjuvants or vehicles that do not destroy the pharmacological activity of the compound or conjugate formulated together. Pharmaceutically acceptable carriers, adjuvants or vehicles that can be used in the compositions of the present invention include, but are not limited to, ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffer substances (such as phosphates), glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (such as protamine sulfate), disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, silica gel, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and lanolin.

[0152] The present invention also includes kits (e.g., pharmaceutical packaging). The kits provided may include a compound of the invention, other therapeutic agents, and first and second containers (e.g., vials, ampoules, bottles, syringes, and / or dispersible packaging or other suitable containers) containing the compound of the invention and other therapeutic agents. In some embodiments, the kit provided may also optionally include a third container containing a pharmaceutical excipient for diluting or suspending the compound of the invention and / or other therapeutic agents. In some embodiments, the compound of the invention and other therapeutic agents provided in the first and second containers are combined to form a unit dosage form.

[0153] Pharmaceutical compositions provided by the invention can be administered by many routes, including but not limited to: oral administration, parenteral administration, inhalation administration, topical administration, rectal administration, nasal administration, oral administration, vaginal administration, administration by implant or other modes of administration. For example, parenteral administration used herein includes subcutaneous administration, intradermal administration, intravenous administration, intramuscular administration, intraarticular administration, intraarterial administration, intrasynovial administration, intrasternal administration, intrathecal administration, intralesional administration, and intracranial injection or infusion technology.

[0154] Typically, an effective amount of the compounds provided herein is administered. The amount of compound actually administered can be determined by a physician based on the relevant circumstances, including the condition being treated, the route of administration selected, the compound actually administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.

[0155] When used to prevent the conditions described herein, the compounds provided herein are administered to a subject at risk of developing the condition, typically based on the advice and under the supervision of a physician, at dosage levels as described above. Subjects at risk of developing a particular condition typically include those with a family history of the condition, or those identified by genetic testing or screening as being particularly susceptible to developing the condition.

[0156] The pharmaceutical compositions provided herein can also be administered long-term ("chronic administration"). Long-term administration refers to administration of a compound or pharmaceutical composition thereof over an extended period of time, e.g., 3 months, 6 months, 1 year, 2 years, 3 years, 5 years, etc., or administration can continue indefinitely, e.g., for the remainder of the subject's life. In some embodiments, long-term administration is intended to provide a constant level of the compound in the blood over an extended period of time, e.g., within the therapeutic window.

[0157] Various methods of administration can be used to further deliver the pharmaceutical composition of the present invention. For example, in some embodiments, the pharmaceutical composition can be administered by push injection, for example, in order to rapidly increase the concentration of the compound in the blood to an effective level. The push dose depends on the target systemic level of the active ingredient, for example, an intramuscular or subcutaneous push dose slowly releases the active ingredient, and a push (for example, by IV intravenous drip) delivered directly to the vein can be delivered more rapidly so that the concentration of the active ingredient in the blood is rapidly increased to an effective level. In other embodiments, the pharmaceutical composition can be given in a continuous infusion form, for example, by IV intravenous drip, so as to provide a steady-state concentration of the active ingredient in the subject's body. In addition, in other embodiments, the pharmaceutical composition of the push dose can be first given, and then continuous infusion.

[0158] To provide blood levels similar to, or lower than, those obtained with an injectable dose, a transdermal dose is typically selected in an amount of about 0.01 to about 20% by weight, preferably about 0.1 to about 20% by weight, preferably about 0.1 to about 10% by weight, and more preferably about 0.5 to about 15% by weight.

[0159] From about 1 to about 120 hours, and particularly from 24 to 96 hours, the injected dose level is in the range of about 0.1 mg / kg / hour to at least 10 mg / kg / hour. To achieve adequate steady-state levels, a preload bolus of about 0.1 mg / kg to about 10 mg / kg or more may also be administered. For a 40 to 80 kg human patient, the maximum total dose may not exceed about 2 g / day.

[0160] Injectable compositions are typically based on sterile saline or phosphate buffered saline for injection, or other injectable excipients known in the art. As previously mentioned, in such compositions, the active compound is typically a minor component, often about 0.05 to 10% by weight, with the remainder being injectable excipients and the like.

[0161] The above components of the composition for injection or topical administration are merely representative. Other materials and processing techniques are described in Section 8 of Remington's Pharmaceutical Sciences, 17th edition, 1985, Mack Publishing Company, Easton, Pennsylvania, which is incorporated herein by reference.

[0162] The compounds of the invention can also be administered in sustained release form or from a sustained release delivery system. Descriptions of representative sustained release materials can be found in Remington's Pharmaceutical Sciences.

[0163] The present invention also relates to pharmaceutically acceptable formulations of the compounds of the present invention. In one embodiment, the formulation comprises water.

[0164] Pharmaceutical composition

[0165] The compounds of the present invention or conjugates described herein can be used in combination with one or more other active ingredients in pharmaceutical compositions or methods to treat the diseases and conditions described herein. Other additional active ingredients include other therapeutic agents or agents that mitigate adverse effects of the intended disease target. The combination can be used to increase efficacy, improve other disease symptoms, reduce one or more side effects, or reduce the required dose of the compounds of the present invention. The additional active ingredients can be formulated into separate pharmaceutical compositions from the compounds of the present invention or can be included in a single pharmaceutical composition with the compounds of the present invention. The additional active ingredients can be administered simultaneously with, before, or after the administration of the compounds of the present invention.

[0166] Combination agents include those active ingredients known or observed to be effective in treating the diseases and conditions described herein, including those that are effective against another target associated with the disease. For example, the compositions and formulations of the present invention, as well as the methods of treatment, may further comprise other drugs, such as other agents that can be used to treat or alleviate the target disease or related symptoms or conditions. The pharmaceutical compositions of the present invention may further comprise one or more of the aforementioned active agents, and the methods of treatment may further comprise administering an effective amount of one or more of the aforementioned active agents.

[0167] The embodiments of the present invention will be further described in detail below with reference to the examples, but those skilled in the art will appreciate that the following examples are intended only to illustrate the present invention and are not intended to limit the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all commercially available conventional products.

[0168] Example 1 Preparation of GalNAc-1

[0169] 1. Synthesis route

[0170]

[0171] 2. Synthesis method

[0172] (1) 50 g of pentaerythritol was dissolved in 150 g of tert-butyl acrylate, 6 ml of 50% sodium hydroxide solution was added, and then 9.5 g of tetrabutylammonium hydroxide was added. After the reaction was completed at room temperature, ethyl acetate was added for extraction to obtain an organic phase, which was concentrated to dryness and purified by column separation to obtain compound 1 (PE (petroleum ether): EA (ethyl acetate) = 20:1--7:1); (2) 10 g of compound 1 and 6.5 g of TsCl were dissolved in 100 ml of Py (pyridine) solution and stirred at 70°C for reaction. After the reaction is complete, pyridine is removed by concentration, ethyl acetate is added for dilution, the mixture is dried and concentrated, and the mixture is separated and purified by column chromatography to obtain compound 2 (PE: EA = 20: 1-10: 1); (3) 4.2 g of compound 2 is dissolved in 50 ml of anhydrous DMF, 0.53 g of sodium azide is added, and the mixture is stirred at 100 ° C for complete reaction, the DMF is removed by concentration, ethyl acetate is added for separation and extraction, the mixture is concentrated to dryness, and the mixture is separated and purified by column chromatography to obtain compound 3 (PE: EA = 20: 1-10: 1); (4) 2 g of compound 3 is dissolved in 10 ml of formic acid, the mixture is stirred for complete reaction, and the mixture is concentrated to dryness to obtain compound 4; (5) 1.6 g of compound 4 is dissolved in 50 ml of dichloromethane, 2.23 g of HOBt, 3.0 g of EDCI, and DIEA are added. 3.3g, mono-Boc-propylenediamine 2.7g, after stirring at room temperature for complete reaction, dichloromethane was added for extraction, dried and concentrated, and purified by column separation to obtain compound 5 (dichloromethane: methanol = 2%--5%); (6) 2.5g of compound 5 was dissolved in 50ml of dichloromethane, 20ml of 2N trifluoroacetic acid was added, and after complete reaction at room temperature, the product was concentrated and dried to obtain compound 6; (7) 11g of galactovalerate was dissolved in 100ml of dichloromethane, 6.2g of DCC and 5.5g of pentafluorophenol were added, and after complete reaction at room temperature, the product was filtered and the organic phase was washed with water, dried and concentrated to dryness, and purified by column separation to obtain compound 7 (PE: EA = 3:1-1:1); (8) 2.46g of compound 6 and 10g of compound 7 were dissolved in 50ml of dichloromethane solution, 8ml of DIEA, stirred at room temperature for complete reaction, extracted with dichloromethane, dried and concentrated the organic phase, separated and purified by column to obtain compound 8 (dichloromethane: methanol = 10% - 20%); (9) 10g of glutaric anhydride and 4.82g of propargylamine were dissolved in 50ml of tetrahydrofuran, stirred at room temperature for complete reaction, and concentrated to dryness to obtain compound 12; (10) 3.7g of compound 12 was dissolved in 50ml of dichloromethane, 5.41g of DCC and 4.83g of pentafluorophenol were added, stirred at room temperature for complete reaction, filtered, and the organic phase was washed with water, dried and concentrated to dryness, separated and purified by column to obtain the product compound 13 (PE: EA = 3:1-1:1); (11) 6.5g of compound 13, 2.54 g of aminocaproic acid was dissolved in 50 ml of tetrahydrofuran, stirred at room temperature until the reaction was complete, concentrated to dryness, and the organic phase was extracted with dichloromethane, dried and concentrated to dryness, and purified by column separation to obtain compound 14 (dichloromethane: methanol = 3%-8%); (12) 4.5 g of compound 14 was dissolved in 100 ml of dichloromethane, 3.94 g of DCC and 3.52 g of pentafluorophenol were added, stirred at room temperature until the reaction was complete, filtered, and the organic phase was washed with water, dried and concentrated to dryness, and purified by column separation to obtain the product compound. 15 (PE:EA=3:1-1:1); (13) 0.34g of compound 8 was dissolved in 5ml of THF, and 0.09g of compound 15, 0.092g of anhydrous copper sulfate, and 5ml of an aqueous solution of 0.146g of sodium ascorbate were added. After stirring at room temperature and the reaction was complete, the THF was removed by concentration, the mixture was diluted with dichloromethane, and the mixture was dried and decolorized with activated carbon and anhydrous sodium sulfate. The insoluble matter was removed by filtration, and the mixture was concentrated to dryness. The mixture was purified by column chromatography (dichloromethane:methanol=10%-15%) to obtain GalNAc-1.

[0173] 3. Experimental Results

[0174] About 0.4 g of GalNAc-1 was obtained as a white foamy solid.

[0175] 1 HNMR(400MHz, CDCl3)δ:ppm.7.69(s,1H),7.36,dd,4H),6.97(t,3H),6.81( d,1H),6.74(d,2H),6.40(s,1H),5.35(t,3H),5.19(dd,3H),4.61(d,3H),4. 51(d,2H),4.32(s,2H),4.13(m,9H),3.92(dd,6H),3.65(d,6H),3.50(m,3H ),3.27(m,18H),2.68(t,2H),2.44(t,6H),2.33(t,2H),2.21-1.26(m,61H). MS(ESI-TOF):m / z(M+H) + 2283.44,(M+Na) + 2305.41.

[0176] Example 2 Preparation of GalNAc-2

[0177] 1. Synthesis route

[0178]

[0179] The synthesis methods of Compound 8 and Compound 13 are the same as those in Example 1.

[0180] 2. Synthesis method: Prepared according to the synthesis method of Example 1.

[0181] 3. Experimental Results

[0182] About 0.2 g of GalNAc-2 was obtained as a white foamy solid.

[0183] 1HNMR(400MHz, CDCl3)δ:ppm 7.73(s,1H),6.90-6.40(m,10H),5.36(d,3H),5.08(m,3H),4.60(s,3H),4.50(d,2H),4.35(s,2H),4.14(m,9H),3.9 2(s,6H),3.66(s,6H),3.51(m,3H),3.27(m,18H),2.79(t,2H),2.45(m,6H),2.25-1.80(m,44H),1.80-1.26(m,20H)

[0184] MS (ESI-TOF): m / z (M+Na) + 2191.38.

[0185] Example 3 Preparation of GalNAc-3

[0186] 1 Synthesis route

[0187]

[0188] The synthesis methods of Compound 4 and Compound 15 are the same as those in Example 1.

[0189] 2. Synthesis method

[0190] (1) Referring to the synthesis method of compound 7, compound 8, and GalNAc-1 in Example 1, compound 9, compound 11, and GalNAc-3 were obtained;

[0191] (2) 4.5 g of compound Cbz-hexylamine-galactose (purchased from Alading) was dissolved in 100 ml of ethyl acetate, and 1 g of 10% palladium carbon was added. After stirring at room temperature and the hydrogenation reaction was complete, the mixture was filtered, dried, and concentrated to dryness to obtain the product compound 10.

[0192] 3. Experimental Results

[0193] About 0.2 g of GalNAc-3 was obtained as a white foamy solid.

[0194] 1HNMR(400MHz, CDCl3)δ:ppm.7.47-6.32(m,9H),5.36(s,3H),5.28(m,3H),4.69(s,3H),4.54(s,2H),4.36(m,2H),4.15(m,6H),4.0 5(m,3H),3.94(dd,6H),3.66(s,6H),3.47(m,3H),3.25(m,12H),2.68(t,2H),2.44(m,6H),2.30-1.80(m,42H),1.80-1.26(m,32H).

[0195] MS (ESI-TOF): m / z (M+Na) + 2133.43.

[0196] Example 4 Preparation of GalNAc-4

[0197] 1. Synthesis route

[0198]

[0199] The synthesis method of compound 13 is the same as that of Example 1, and the synthesis method of compound 11 is the same as that of Example 3.

[0200] 2. Synthesis method: Prepared according to the synthesis method of Example 1.

[0201] 3. Experimental Results

[0202] About 0.2 g of GalNAc-4 was obtained as a white foamy solid.

[0203] 1 HNMR(400MHz, CDCl3)δ:ppm.7.74-6.43(m,8H),5.36(d,3H),5.28(m,3H),4.68(d,3H),4.53(d,2H),4.31(s,2H),4.14(ddd,6H),4.05 (dd,3H),3.89(ddd,6H),3.67(m,6H),3.46(dd,3H),3.24(s,12H),2.78(t,2H),2.43(m,6H),2.30-1.80(m,38H),1.80-1.26(m,26H).

[0204] MS (ESI-TOF): m / z (M+H) + 1998.43,(M+Na) + 2020.42.

[0205] Example 5 Preparation of GalNAc-5

[0206] 1. Synthesis route

[0207]

[0208] The synthesis method of compound 11 is the same as that of Example 3.

[0209] 2. Synthesis Method

[0210] (1) 1.6 g of compound 16 was dissolved in 50 ml of dichloromethane, and 2.23 g of HOBt, 3.0 g of EDCI, 3.3g DIEA, 0.3g monopropargylamine, after stirring at room temperature for complete reaction, dichloromethane was added to extract the organic phase, dried and concentrated, and purified by column separation to obtain compound 17 (dichloromethane: methanol = 2%--5%); (2) 2g of the raw material was dissolved in 10ml of methanol, 0.5g of palladium carbon (10%) was added, and after the hydrogenation reaction was complete, it was filtered, the filtrate was collected and concentrated to dryness to obtain compound 18; (3) 4.5g of PFP-dodecanoate was dissolved in 100ml of dichloromethane, 3.94g of DCC and 3.52g of pentafluorophenol were added, after stirring at room temperature for complete reaction, it was filtered, dried and concentrated to dryness, and purified by column separation (PE: EA = 3:1-1:1) to obtain compound 19; (4) Referring to the synthesis method of GalNAc-1 in Example 1, GalNAc-5 was obtained.

[0211] 3. Experimental Results

[0212] About 0.4 g of GalNAc-5 was obtained as a white foamy solid.

[0213] 1 HNMR (400MHz, CDCl3) δ: ppm. 7.73-6.40(m,8H),5.35(d,3H),5.26(m,3H),4.67(d,3H),4.51(s,2H),4.33(s,2H),4.11(dd,6H),4.07(d,3H),3.89 (dt,6H),3.66(m,6H),3.46(dd,3H),3.24(s,12H),2.75(t,2H),2.41(m,6H),2.25-1.80(m,38H),1.80-1.26(m,40H).

[0214] MS (ESI-TOF): m / z (M+H) + 2096.19,(M+Na) + 2119.20.

[0215] Example 6 Preparation of GalNAc-6

[0216] 1. Synthesis route

[0217]

[0218] The synthesis method of compound 8 is the same as that of Example 1.

[0219] 2. Synthesis method

[0220] (1) 47.9 g of aminocaproic acid was dissolved in 700 ml of toluene, and 57 ml of benzyl alcohol and 74 g of 4-toluenesulfonic acid were added. After stirring at 115° C. and the reaction was complete, the mixture was cooled to room temperature and stirred. A large amount of solid precipitated. 500 ml of methyl tert-butyl ether was added, the mixture was filtered and dried to obtain compound 22. (2) 19 g of compound 22 was dissolved in 100 ml of dichloromethane, and 11 g of glutaric anhydride in 100 ml of dichloromethane solution was added. After stirring at room temperature and the reaction was complete, the mixture was dried and concentrated to obtain compound 23. (3) 5 g of compound 23 was dissolved in 3.69 g of DCC and 3.3 g of pentafluorophenol were added to 100 ml of dichloromethane. After the reaction was complete at room temperature, the mixture was filtered and concentrated to dryness. Compound 24 (PE:EA=3:1-2:1) was separated and purified by column chromatography to obtain compound 24; (4) 0.13 g of compound 8 was dissolved in 10 ml of ethyl acetate and 10 ml of anhydrous methanol, 0.2 g of palladium carbon and 3 drops of acetic acid were added, and after the displacement reaction was complete in hydrogen, the mixture was filtered and the filtrate was concentrated to dryness to obtain compound 25; (5) 2.8 g of compound 25 was dissolved in 50 ml of dichloromethane, 0.5 g of DIEA and 0.78g of compound 24 were reacted at room temperature, dried and concentrated to dryness, and purified by column separation to obtain compound 26 (dichloromethane: methanol 15%--25%); (6) 2.5g of compound 26 was dissolved in 30ml of methanol and 30ml of ethyl acetate, 1.3g of palladium carbon was added to cause hydrogen displacement reaction, and then filtered and the filtrate was dried and concentrated to dryness to obtain compound 27; (7) 0.8g of compound 27 was dissolved in dichloromethane, 0.12g of DCC and 0.11g of pentafluorophenol were added, and the reaction was completed at room temperature, dichloromethane was added to dilute, dried and concentrated to dryness, and purified by column separation to obtain GalNAc-6 (dichloromethane: methanol = 10%--15%).

[0221] 3. Experimental Results

[0222] About 0.7 g of GalNAc-6 was obtained as a white foamy solid.

[0223] 1HNMR(400MHz,d-DMSO)δ:ppm.7.75-7.26(m,11H),5.85(d,3H),5.59(d,3H),5.11(dd,3H),4.63(m,6H),4.50(d,6H),4.37 (s,3H),4.17(d,6H),4.07(s,3H),3.90-3.70(m,18H),3.28(m,2H),2.92(d,6H),2.80-2.25(m,52H),2.25-1.26(m,24H).

[0224] MS (ESI-TOF): m / z (M+H) + 2201.82, (M+Na) + 2223.86.

[0225] Example 7 Preparation of GalNAc-7

[0226] 1. Synthesis route

[0227]

[0228] The synthesis method of compound 25 is the same as that of Example 6.

[0229] 2. Synthesis method

[0230] (1) Referring to the synthesis method of compound 13 in Example 1, compound 21 was obtained; (2) Referring to the synthesis method of compound 26, compound 27, and GalNAc-6 in Example 6, compound 29, compound 30, and GalNAc-7 were obtained.

[0231] 3. Experimental Results

[0232] About 0.5 g of GalNAc-7 was obtained as a white foamy solid.

[0233] 1 HNMR(400MHz,CDCl3)δ:ppm 7.32-6.63(m,10H),5.35(s,3H),5.20(s,3H),4.62(d,3H),4.13(ddd,9H),3.92(d,6H),3.66(dd, 6H),3.52(d,3H),3.33(m,20H),2.77(t,2H),2.44(s,6H),2.25-1.80(m,44H),1.80-1.26(m,20H).

[0234] MS (ESI-TOF): m / z (M+H) + 2088.08, (M+Na) +2109.36.

[0235] Example 8 Preparation of GalNAc-8

[0236] 1. Synthesis route

[0237]

[0238] The synthesis method of compound 11 is the same as that of Example 3, and the synthesis method of compound 24 is the same as that of Example 6.

[0239] 2. Synthesis method: Compound 32, Compound 36, Compound 37, and GalNAc-8 were obtained by referring to the synthesis method of Compound 25, Compound 26, Compound 27, and GalNAc-6 in Example 6;

[0240] 3. Experimental results

[0241] About 0.45 g of GalNAc-8 was obtained.

[0242] 1HNMR(400MHz, CDCl3)δ:ppm.7.7.06-6.37(m,8H),5.36(d,3H),5.270(m,3H),4.69(t,3H),4.15(m,6H),4.00(m,3H),3.90(m, 6H),3.65(t,6H),3.47(dt,3H),3.28(m,16H),2.68(t,2H),2.42(m,6H),2.29(t,4H),2.15-1.80(s,36H),1.80-1.26(m,32H).

[0243] MS (ESI-TOF): m / z (M+H) + 2030.38, (M+Na) + 2052.41.

[0244] Example 9 Preparation of GalNAc-9

[0245] 1. Synthesis route

[0246]

[0247] The synthesis method of compound 11 is the same as that of Example 3.

[0248] 2. Synthesis method

[0249] (1) 1.3 g of azide compound 11 was dissolved in 15 ml of THF, and 0.3 g of palladium carbon and 0.11 g of glutaric anhydride were added. The reaction was replaced by hydrogen. After the reaction was complete at room temperature, the palladium carbon was removed by filtration. The filtrate was concentrated to dryness, and the organic phase was diluted with dichloromethane and extracted. The organic phase was concentrated and purified by column separation (dichloromethane:methanol = 10%-15%) to obtain about 0.8 g of the product compound 34.

[0250] (2) Referring to the synthesis method of GalNAc-6 in Example 6, GalNAc-9 was obtained.

[0251] 3. Experimental results

[0252] About 0.8 g of GalNAc-9 was obtained as a foamy solid.

[0253] 1HNMR(400MHz,d-DMSO)δ:ppm.7.63(s,1H),7.42(s,3H),7.17(s,3H),5.85(d,3H),5.60(t,3H),5.10(dd,3H),4.63(td,6H),4.49(m,6 H),4.34(d,3H),4.16(m,6H),4.03(d,3H),3.84(d,6H),3.73(d,6H),3.35(m,2H),2.91(d,6H),2.70-2.40(m,42H),2.21-1.76(m,24H).

[0254] MS (ESI-TOF): m / z (M+H) + 1917.93, (M+Na) + 1938.69.

[0255] Example 10 Preparation of GalNAc-10

[0256] 1 Synthesis route

[0257]

[0258] The synthesis method of compound 28 refers to the synthesis of compound 24 in Example 6.

[0259] 2. Synthesis method

[0260] (1) 3 g of the amino compound was dissolved in 30 ml of dichloromethane, and DIEA (1 ml) and 1.31 g of monobenzyl dodecanoate-pentafluorophenol ester were added. After the reaction was complete at room temperature, dichloromethane was added to dilute the mixture, dried and concentrated to dryness, and purified by column chromatography (dichloromethane:methanol 10%-15%) to obtain compound 38; (2) Referring to the synthesis method of compound 27 and GalNAc-6 in Example 6, compound 39 and GalNAc-10 were obtained.

[0261] 3. Experimental results

[0262] About 0.45 g of GalNAc-10 was obtained.

[0263] 1 HNMR(400MHz,CDCl3)δ:ppm 7.7.06-6.40(m,7H),5.35(d,3H),5.28(m,3H),4.67(t,3H),4.14(m,6H),4.02(m,3H),3.93(m,6H),3.61(t,6H ),3.48(dt,3H),3.29(m,14H),2.67(t,2H),2.43(m,6H),2.28(t,2H),2.15-1.80(s,36H),1.80-1.26(m,40H).

[0264] MS (ESI-TOF): m / z (M+H) + 2015.11,(M+Na) + 2038.47.

[0265] Example 11 Preparation of GalNAc-11

[0266] 1. Synthesis route

[0267]

[0268] The synthesis method of compound GalNAc-4 is the same as that in Example 4.

[0269] 2. Synthesis method

[0270] (1) 3 g of GalNAc-4 was dissolved in 30 ml of dichloromethane, and DIEA (0.3 ml) and 0.18 g of 1-hydroxyhexanoic acid were added. After the reaction was complete at room temperature, the mixture was diluted with dichloromethane, dried and concentrated to dryness, and purified by column chromatography (dichloromethane: methanol 10%-15%) to obtain compound 40; (2) 1 g of compound 40 was dissolved in 10 ml of anhydrous dichloromethane, and DIEA (0.2 ml) and phosphoryl chloride (0.14 g) were added. After the reaction was complete at 0°C, the mixture was diluted with dichloromethane, dried and concentrated, and purified by column chromatography (dichloromethane: methanol 10%-15%) to obtain GalNAc-11.

[0271] 3. Experimental results

[0272] The spectral data of compound 40 are:

[0273] 1HNMR(400MHz, CDCl3)δ:ppm.7.71-6.38(m,9H),5.36(d,3H),5.27(m,3H),4.68(d,3H),4.53(s,2H),4.31(s,2H),4.15(m,6H),4.01(dd,3H) )),3.89(ddd,6H),3.65(dd,8H),3.46(m,3H),3.24(m,14H),2.46(d,6H),2.31(d,2H),2.23(s,2H),2.16-1.80(m,36H),1.80-1.26(m,34H).

[0274] MS (ESI-TOF): m / z (MH) - 1929.81.

[0275] Approximately 0.5 g of GalNAc-11 was obtained. A white foamy solid. MS (ESI-TOF): m / z (M+Na) + 2153.62.

[0276] Example 12 Preparation of GalNAc-12

[0277] 1. Synthesis route

[0278]

[0279] Compound 42 (purchased from Alading) and compound GalNAc-5 were the same as those in Example 5.

[0280] 2. Synthesis method

[0281] (1) 2 g of GalNAc-5 was dissolved in 20 ml of dichloromethane, and DIEA (0.3 ml) and 0.44 g of DMTr-hydroxyproline (compound 42) were added. After the reaction was complete at room temperature, the mixture was diluted with dichloromethane, dried, concentrated to dryness, and separated and purified by column chromatography (dichloromethane: methanol 10%-15%) to obtain compound 41; (2) 1 g of compound 41 was dissolved in 10 ml of anhydrous dichloromethane, and DIEA (0.2 ml) and phosphoryl chloride (0.12 g) were added. After the reaction was complete at 0°C, the mixture was diluted with dichloromethane, dried, concentrated, and separated and purified by column chromatography (dichloromethane: methanol 10%-15%) to obtain GalNAc-12.

[0282] 3. Experimental results

[0283] About 0.6 g of GalNAc-12 was obtained as a white foamy solid.

[0284] Example 13 Preparation of modified single-stranded oligonucleotide (antisense strand)

[0285] The exemplary antisense strand sequences involved in Examples 13, 16-18 are as follows:

[0286] Sequence 1 (SEQ ID NO:1):

[0287] 5'-Cy5-mUmGmAfCmAmAmAfCmGmGmGfCmAmAfCmAfUmAfC-3'

[0288] In this example, the modified oligonucleotide was synthesized according to a theoretical yield of 1 μmol using the following process:

[0289] (1) Weigh 1 μmol of universal solid support CPG (controlled pore glass) or 3'-cholesterol-modified CPG (purchased from Chemgenes), or 3'-amino-modified solid support (purchased from Kinovite), 2'-O-TBDMS-protected RNA phosphoramidite monomers, DNA monomers, 2'-methoxy monomers, 2'-fluoro monomers (purchased from Sigma-Aldrich), and phosphoramidites used to synthesize 5'-modified amino-C16-12-phosphoramidites or fluorescent compounds and dissolve them in anhydrous acetonitrile solution to a concentration of 0.2 M. For oligonucleotides with phosphate backbone thiolation modifications, 0.2 M PADS solution was used as the thiolation reagent. A 5-ethylthio-1H-tetrazole (purchased from Chemgenes) acetonitrile solution was prepared as an activating agent (0.25 M), a 0.02 M iodine pyridine / water solution was prepared as an oxidizing agent, and a 3% trichloroacetic acid dichloromethane solution was prepared as a deprotection reagent and placed in the corresponding reagent designated position of a DNA / RNA automatic synthesizer (GE AKTAOP100).

[0290] (2) Set up the synthesis program, input the specified oligonucleotide base sequence, and after checking that it is correct, start the cyclic oligonucleotide synthesis. The coupling time for each step is 6 minutes, and the coupling time for the galactose ligand corresponding monomer is 10-20 minutes. After the automatic cycle, the oligonucleotide solid phase synthesis is completed.

[0291] (3) Dry the CPG with dry nitrogen, transfer it to a 5mL EP tube, add 2mL of ammonia / ethanol solution (3 / 1), and heat at 55℃ for 16-18 hours. Centrifuge at 10000rpm for 10min to obtain the supernatant, drain the concentrated ammonia / ethanol to obtain a white colloidal solid. The solid was dissolved in 200μL 1M TBAF THF solution and shaken at room temperature for 20 hours. Add 0.5mL 1M Tris-HCl buffer (pH 7.4), shake at room temperature for 15 minutes, place in a centrifuge and pump down to 1 / 2 of the original volume to remove THF. The solution was extracted twice with 0.5mL chloroform, 1mL 0.1M TEAA loading solution was added, and the mixed solution was poured into a solid phase extraction column to remove excess salt in the solution.

[0292] (4) The concentration of the oligonucleotides was determined using a micro-UV spectrophotometer (KO5500). Mass spectrometry analysis was performed on an OligoHTCS LC-MS system (Novatia). The molecular weight of the nucleic acid was calculated using Promass software after normalization using the primary scan.

[0293] Example 14 Preparation of modified single-stranded oligonucleotide (sense strand)

[0294] The exemplary sense strand sequences involved in Examples 14-18 are as follows:

[0295] Sequence 2 (SEQ ID NO:2):

[0296] 5'-mGfUmAfUmGfUfUmGfCfCfCmGfUfUfUmGfUfCmA-3'

[0297] In this example, the modified oligonucleotide was synthesized according to the specification of a theoretical yield of 1 μmol, and the process was as follows:

[0298] (1) Weigh 1 μmol of a universal solid support CPG or 3'-cholesterol-modified CPG (purchased from Chemgenes), a 3'-amino-modified solid support (purchased from Kinovite), a DNA monomer, a 2'-methoxy monomer, a 2'-fluoro monomer (purchased from Sigma Aldrich), or a phosphoramidite for synthesizing a 5'-modified amino-C16-12-phosphoramidite or fluorescent compound and dissolve them in anhydrous acetonitrile solution to a concentration of 0.2 M. For oligonucleotides with phosphate backbone thiolation modification, a 0.2 M PADs solution is used as the thiolation reagent. Prepare an acetonitrile solution of 5-ethylthio-1H-tetrazole (purchased from Chemgenes) as an activator (0.25 M), a 0.02 M iodine-pyridine / water solution as an oxidant, and a 3% trichloroacetic acid in dichloromethane solution as a deprotection reagent, and place them in the designated reagent position of the DNA / RNA automatic synthesizer.

[0299] (2) Set up the synthesis program, input the designated oligonucleotide base sequence, and after checking that it is correct, begin cyclic oligonucleotide synthesis. Each coupling step takes 6 minutes, and the coupling time for the galactose ligand-corresponding monomer is 6-10 minutes. After the automatic cycle, the oligonucleotide solid phase synthesis is completed.

[0300] (3) Dry the CPG with dry nitrogen and transfer it to a 5 mL EP tube. Add 2 mL of ammonia solution and heat at 55°C for 16–18 hours. Centrifuge at 10,000 rpm for 10 minutes, remove the supernatant, and drain the concentrated ammonia / ethanol mixture to obtain a white or yellow colloidal solid. Add 1 mL of 0.1 M TEAA loading solution and pour the mixed solution into a solid phase extraction column to remove excess salt.

[0301] (4) The concentration of the oligonucleotides was determined using a micro-UV spectrophotometer (KO5500). Mass spectrometry analysis was performed on an OligoHTCS LC-MS system (Novatia). The molecular weight of the nucleic acid was calculated using Promass software after normalization using the primary scan.

[0302] Example 15 Preparation of GalNAc-modified oligonucleotides

[0303] An amino-modified oligonucleotide prepared according to the method of Example 14 was dissolved in buffer. Various GalNAc-pentafluorophenol esters (GalNAc selected from GalNAc-1 to GalNAc-10) dissolved in acetonitrile were added to the amino-modified oligonucleotide solution, mixed thoroughly, and allowed to react at room temperature for at least 3 hours. After completion of the reaction, the acetyl protecting group was removed. The GalNAc-modified oligonucleotide was purified by ion exchange chromatography (WATERS) using a DNAPAc PA-100 ion exchange column with a linear gradient. Mobile phase A: 20 mM NaOH; mobile phase B: a mixture of 20 mM NaOH and 2 M NaCl. The sequences of exemplary GalNAc-modified oligonucleotides and their corresponding molecular weight determination results are shown in Table 1.

[0304] Table 1 GalNAc-modified oligonucleotides (sense strand)

[0305]

[0306] Abbreviations: N = RNA; dN = DNA; mN = 2'OMe modification; fN = 2'F modification.

[0307] Example 16 Preparation of double-stranded oligonucleotides

[0308] The process is as follows: the 5'-Cy5-antisense oligonucleotide synthesized in Example 13 was mixed with the GalNAc-1-10 sense oligonucleotide in Example 15 at a ratio of 1:1 according to the UV absorption content, heated to 95°C in a water bath for three minutes, and then cooled to room temperature to form a GalNAc-duplex (as shown in Table 2).

[0309] Table 2 GalNAc-double-stranded RNA structure

[0310]

[0311]

[0312] Abbreviations: N = RNA; dN = DNA; mN = 2'OMe modification; fN = 2'F modification.

[0313] Example 17 Cell Targeting Detection of Modified Oligonucleotides

[0314] Modified oligonucleotides used in animal experiments were filtered through a 0.22 μm membrane before injection.

[0315] 1. Isolation of primary mouse hepatocytes

[0316] Mice (purchased from Beijing Weitonglihua Experimental Animal Co., Ltd.) were anesthetized, and the skin and muscle layers were cut open to expose the liver. A perfusion catheter was inserted into the portal vein, and a small incision was made in the inferior vena cava to prepare for liver perfusion. Perfusion Solution I (Hank's, 0.5 mM EGTA, pH 8) and Perfusion Solution II (Low-glucose DMEM, 100 U / mL Type IV, pH 7.4) were preheated at 40°C. Perfusion Solution I was perfused into the liver via the portal vein cannula at a flow rate of 7 mL / min for 5 minutes, until the liver turned grayish white. The liver was then perfused with Perfusion Solution II at a flow rate of 7 mL / min for 7 minutes at 37°C. Following perfusion, the liver was removed and placed in Solution III (10% FBS low-glucose DMEM, 4°C) to terminate digestion. The liver capsule was ruptured with forceps and gently shaken to release hepatocytes. The hepatocytes were filtered through a 70 μm cell strainer and centrifuged at 50 g for 2 minutes, after which the supernatant was discarded. Resuspend the cells in Solution IV (40% percoll low-glucose DMEM, 4°C), centrifuge at 100g for 2 minutes, and discard the supernatant. Add 2% FBS low-glucose DMEM and resuspend the cells for later use. Cell viability was assessed by trypan blue staining.

[0317] 2. Determination of GalNAc-siRNA binding curve and Kd value

[0318] Freshly isolated mouse primary hepatocytes were plated into 96-well plates at a rate of 2 × 10 4 / well, 100 μL / well. Different GalNAc-siRNAs were added to each well (see Table 2). The final concentration of each GalNAc-siRNA was set to 0.9 nM, 2.7 nM, 8.3 nM, 25 nM, 50 nM or 100 nM. After incubation at 4 ° C for 2 hours, centrifuge at 50g for 2 minutes and discard the supernatant. Resuspend the cells with 10 μg / mL PI, stain for 10 minutes, and then centrifuge at 50g for 2 minutes. Wash the cells with pre-cooled PBS, centrifuge at 50g for 2 minutes and discard the supernatant. Resuspend the cells with PBS. The mean fluorescence intensity MFI (Mean Fluorescence Intensity) of living cells was measured by flow cytometer (Beckman), and GraphPadPrism 5 software was used for nonlinear fitting and dissociation constant K. d Value calculation.

[0319] The results are shown in Table 3. The data demonstrate that GalNAc-siRNA can specifically target hepatocytes; the Kd values ​​of the GalNAc ligands for the cell receptor range from 1.5 to 27.6 nM, exhibiting higher affinity than prior art galactose ligands. The RB-101, RB-104, RB-108, and RB-109 structures exhibit relatively strong receptor affinity (the smaller the Kd value, the greater the affinity).

[0320] Table 3 K of each experimental group d and K d Value (nM)

[0321] Sample number RB-101 RB-102 RB-103 RB-104 RB-105 Kd 1.879 3.118 4.265 1.958 19.64 Ki 1.6 2.922 1.049 1.031 12.57 Sample number RB-106 RB-107 RB-108 RB-109 RB-110 Kd 4.632 2.066 1.874 1.982 6.371 Ki 2.984 1.161 1.018 1.057 2.708

[0322] Example 18 In vivo liver targeting test

[0323] Thirty male, 6- to 7-week-old, SPF-grade Balb / c-nu mice (purchased from Beijing Weitonglihua Laboratory Animal Co., Ltd.) were randomly divided into six groups: blank control, NC1 (unconjugated ligand), test group 1, test group 2, test group 3, and test group 4. Each group consisted of five animals, and the drug was administered via tail vein injection at a dose of approximately 10 mg / kg (see Table 4 for experimental design). All animals underwent in vivo imaging, including white light imaging, before drug administration and 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, and 6 hours after drug administration. Six hours after euthanasia, the brain, salivary glands, heart, spleen, lungs, liver, kidneys, and intestine were removed for ex vivo organ imaging (Bruker, XTREME).

[0324] Table 4 Liver-targeted experimental design

[0325]

[0326]

[0327] The in vitro imaging results are shown in Table 5.

[0328] Table 5 Statistical results of fluorescence intensity values ​​of isolated organs after background subtraction (average total photon number p / sec)

[0329]

[0330] The results showed that the test substances RB-100, RB-104, RB-107, RB-108 and RB-110 were mainly distributed in the liver, kidney and gastrointestinal tract, and were less distributed in tissues such as the brain, heart, lung and spleen.

[0331] Compared with the RB-100 group (negative control group), combined with the statistical results of the average total photon number, the RB-104, RB-107, RB-108, and RB-110 test substances had liver-targeting effects. Comparison of the fluorescence intensities of the test substances in different organs showed that the liver fluorescence intensities of the RB-107 and RB-108 test substances showed extremely significant statistical differences (P < 0.001), the liver fluorescence intensities of the RB-104 test substance showed statistically significant differences (P < 0.01), and the liver fluorescence intensities of the RB-110 test substance showed statistically significant differences (P < 0.05).

[0332] The contents of all references cited throughout this application (including literature references, issued patents, published patent applications and co-pending patent applications) are hereby expressly incorporated by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0333] All features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature having the same, equivalent, or similar purpose. Therefore, unless expressly stated otherwise, each feature disclosed is merely an example of a series of equivalent or similar features.

[0334] From the above description, those skilled in the art can easily determine the essential features of the present invention, and without departing from the spirit and scope of the present invention, various changes and modifications can be made to the present invention to adapt it to various uses and conditions. Therefore, other embodiments are also within the scope of the appended claims.

Claims

1. A compound of formula (I), or a tautomer, stereoisomer or pharmaceutically acceptable salt thereof, in, R1 is R2 is -CH2- or R3 is R4 is -CH2- or R5 is -OH, -COOH、 PG is a carboxyl protecting group selected from succinate and Where h is any integer from 1 to 5; PG2 is a hydroxy protecting group selected from tert-butyldimethylsilyl ether, tert-butyldiphenylsilyl, triisopropylsilyl ether, monomethoxytrityl, 4,4'-dimethoxytrityl and trityl; a, b, c and d are each independently any integer from 1 to 10; n is 3.

2. The compound according to claim 1, characterized in that The PG2 is selected from tert-butyldimethylsilyl ether, tert-butyldiphenylsilyl and triisopropylsilyl ether.

3. The compound according to claim 1 or 2, characterized in that The carboxyl protecting group is Wherein h is any integer from 1 to 5.

4. The compound according to claim 3, characterized in that The carboxyl protecting group is 5. The compound according to claim 1, characterized in that The hydroxyl protecting group is 4,4'-dimethoxytrityl (DMTr).

6. The compound according to claim 1, characterized in that The compound has the structure shown below:

7. A compound of formula (II), or a tautomer, stereoisomer or pharmaceutically acceptable salt thereof, in, R1 is R2 is -CH2- or a and b are each independently any integer from 1 to 10; n is 3.

8. The compound according to claim 7, characterized in that The compound has the structure shown below:

9. A conjugate, characterized in that Having the structure shown in formula (III), in, R1 is R2 is -CH2- or R3 is R4 is -CH2- or R is O; Rx is an oligonucleotide chain having an antisense chain sequence shown in SEQ ID NO: 1 and a sense chain sequence shown in SEQ ID NO: 2; a, b, c and d are each independently any integer from 1 to 10; n is 3.

10. The conjugate according to claim 9, characterized in that Having the structure shown in formula (IV), Where oligo is an oligonucleotide chain, The oligonucleotide chain has an antisense chain sequence shown in SEQ ID NO: 1 and a sense chain sequence shown in SEQ ID NO:

2.

11. The conjugate according to claim 9 or 10, characterized in that The compound is linked to the 5' end, 3' end or any nucleotide between the 5' end and the 3' end of at least one chain of the oligonucleotide chain through a phosphate bond.

12. A method for preparing a compound of formula (I), in, R1 is R2 is -CH2- or R3 is R4 is R5 is PG is a carboxyl protecting group selected from succinate and Where h is any integer from 1 to 5; PG2 is a hydroxy protecting group selected from the group consisting of silyl, monomethoxytrityl (MMTr), 4,4'-dimethoxytrityl (DMTr) and trityl; a, b, c and d are each independently any integer from 1 to 10; n is 3, It is characterized in that a first compound is contacted with a second compound to obtain the compound, wherein the first compound is as defined in claim 7 and the second compound has the structure shown below: wherein c and d are independently any integer from 1 to 10.

13. A composition, characterized in that include: The conjugate according to any one of claims 9 to 11, optionally, further comprising a pharmaceutically acceptable carrier or excipient.

14. The composition according to claim 13, characterized in that The dosage form of the composition is powder, tablet, granule, capsule, solution, emulsion, suspension, injection, spray, aerosol, powder spray or microneedle patch.

15. The composition according to claim 14, characterized in that The composition is administered to the subject by intravenous injection, intramuscular injection, or subcutaneous injection; Optionally, the subject is a mammal; Optionally, the mammal is selected from bovine, equine, ovine, porcine, canine, feline, rodent, and primate.

16. Use of the compound according to any one of claims 1 to 8 or the conjugate according to any one of claims 9 to 11 in preparing a composition for liver targeting.

17. Use of the conjugate according to any one of claims 9 to 11 in preparing a composition for liver-targeted RNA detection or localization.

18. An in vitro or ex vivo method for intervening in or detecting a predetermined gene in a hepatocyte, characterized in that: contacting a cell population with the conjugate according to any one of claims 9 to 11, wherein the cell population comprises the hepatocytes, and the oligonucleotide chain in the conjugate interacts with the predetermined gene; The method described herein is not a method for treating or diagnosing a disease.

19. Use of the conjugate according to any one of claims 9 to 11 in preparing a kit for intervening in or detecting a predetermined gene in hepatocytes.

20. A method for intervening in or detecting a predetermined gene in a liver cell, characterized in that: A cell population is contacted with the conjugate according to any one of claims 9 to 11, wherein the cell population comprises the hepatocytes, the oligonucleotide chain in the conjugate interacts with the predetermined gene, and the method is not a method for treating or diagnosing a disease.

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