Galnac compound comprising ribose ring or derivative structure thereof, and oligonucleotide conjugate thereof

Abstract

Provided in the present invention are a GalNAc compound comprising a ribose ring or a derivative structure thereof, and an oligonucleotide conjugate thereof. The oligonucleotide conjugate provided in the present invention enables efficient liver-targeted delivery, thereby improving the efficacy of a drug.

Description

A GalNAc compound containing a ribose ring or its derivative structure and its oligonucleotide conjugate

[0001] This application claims priority to Chinese patent application No. 202311118366.4, entitled “A GalNAc compound containing a ribose ring or its derivative structure and its oligonucleotide conjugate”, and the contents disclosed in the above-mentioned Chinese patent application are cited in full as part of this application. Technical Field

[0002] The present invention relates to the field of biomedicine, and in particular to a GalNAc compound having a ribose ring structure and a GalNAc oligonucleotide conjugate prepared therefrom. Background Art

[0003] Nucleic acid drugs, especially oligonucleotide drugs, are widely used due to their simple synthesis and high activity. Oligonucleotide drugs generally include antisense oligonucleotides (ASOs), small interfering RNA (siRNA), microRNA (miRNA), and nucleic acid aptamers.

[0004] Oligonucleotides are a class of short DNA or RNA molecules or oligomers that readily bind to their respective complementary oligonucleotides, DNA, or RNA in a sequence-specific manner to form duplexes or, less commonly, higher-order hybrids. This fundamental property has led to their widespread application in genetic testing, targeted gene therapy research, and medicine. These small fragments of nucleic acid can be manufactured as single-stranded molecules with any specified sequence. In nature, oligonucleotides are often small RNA molecules that play a role in regulating gene expression, or degradation intermediates derived from the breakdown of larger nucleic acid molecules.

[0005] RNA interference is a natural defense mechanism against foreign genes. siRNA can knock out target genes by recognizing specific sequences and breaking down target mRNA.

[0006] N-acetylgalactosamine (GalNAc) is a ligand that binds to the asialoglycoprotein receptor (ASGPR) on the surface of the liver. The asialoglycoprotein receptor is an endocytic receptor specifically expressed on the surface of hepatocytes. In recent years, certain progress has been made in the liver-targeted delivery of nucleic acid drugs using the high-affinity ligand GalNAc of ASGPR as a targeting molecule. For example, Alnylam Pharmaceuticals, Inc. reported that siRNA based on GalNAc conjugation technology exerted gene silencing activity in mice (Nair JK, et al. J. Am. Chem. Soc. 2014, 136, 16958). The article reported that the conjugate of GalNAc and siRNA showed good delivery activity in both in vivo and in vitro experiments. Through in vivo experiments in mice administered subcutaneously, a single dose of ED 50 The dose was determined to be 1 mg / kg, with a single injection dose of less than 1 mL. In long-term dosing experiments, weekly subcutaneous injections achieved stable interfering activity for up to nine months. Studies have shown that tetra- and tri-antennary GalNAc compounds have significantly higher affinity for ASGPR than bi- and mono-antennary GalNAc compounds.

[0007] Proprotein convertase subtilisin / kexin type 9 (PCSK9) is a 692-amino acid glycoprotein and the ninth member of the proprotein convertase (PCs) family. It is a secreted serine protease primarily expressed in tissues such as the liver and intestines and then secreted into the blood. Once in the circulation, PCSK9 specifically binds to the epidermal growth factor-like domain of the low-density lipoprotein receptor (LDL-R) on the surface of hepatocytes, directing it to the lysosomes where it is degraded. This leads to a decrease in LDL-R on the hepatocyte surface, which in turn reduces the liver's ability to bind and clear LDL-C, ultimately leading to elevated LDL-C levels in the blood. Therefore, inhibiting PCSK9 could be a promising approach for treating hypercholesterolemia. Furthermore, recent studies have shown that elevated PCSK9 levels are closely associated with obesity and type 2 diabetes, as well as chronic kidney diseases such as nephrotic syndrome and proteinuria. Therefore, inhibiting PCSK9 could be an important approach for preventing and treating these and other related conditions.

[0008] Different GalNAc ligand structures have very different effects on nucleic acid delivery. In order to improve the delivery effect of liver-targeted drugs, such as PCSK9 inhibitors, there is still a need in the art to develop new GalNAc ligand compounds.

[0009] Summary of the Invention

[0010] The present invention designs a series of novel GalNAc compounds. Through solid-phase synthesis, the novel GalNAc compounds of the present invention can be efficiently conjugated with oligonucleotides. The GalNAc oligonucleotide conjugates prepared therefrom significantly improve the liver-targeted delivery efficiency compared with GalNAc compounds with similar structures in the prior art.

[0011] In a first aspect, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0012] in,

[0013] R1 is oxygen or sulfur, preferably oxygen;

[0014] R2 is hydrogen, C 1-4 Alkyl, C 1-4 Alkoxy or halogen;

[0015] R3 is hydrogen, hydroxyl protecting group, phosphorus-containing active reactive group, or -CO(CH2) x COOH, wherein x is an integer from 1 to 10, It is controlled pore glass or polystyrene;

[0016] R4 is hydrogen or a hydroxyl protecting group;

[0017] A is -(CH2) a -、-(CH2CH2O) b -、-((CH2) c NHCO) d -or-((CH2) c CONH) d -, wherein a is an integer of 1-15, preferably an integer of 3-13, b is an integer of 1-7, preferably an integer of 2-5, c is an integer of 1-7, preferably an integer of 2-6, and d is an integer of 1-5, preferably an integer of 1-3;

[0018] B is -(CH2) e -, wherein e is an integer of 0-7, preferably an integer of 1-5.

[0019] L is -CONH- or -NHCO-;

[0020] G is

[0021] in,

[0022] T is N-acetyl-galactosamine in which the hydroxyl groups are fully protected by acyl groups, galactose in which the hydroxyl groups are fully protected by acyl groups, galactosamine in which the hydroxyl groups are fully protected by acyl groups, N-formyl-galactosamine in which the hydroxyl groups are fully protected by acyl groups, N-propionyl-galactosamine in which the hydroxyl groups are fully protected by acyl groups, N-n-butanoyl-galactosamine in which the hydroxyl groups are fully protected by acyl groups, or N-isobutanoyl-galactosamine in which the hydroxyl groups are fully protected by acyl groups, preferably N-acetyl-galactosamine in which the hydroxyl groups are fully protected by acyl groups, wherein the acyl groups are, for example, acetyl or benzoyl, preferably acetyl;

[0023] X1 is -(CH2) f -or-(CH2CH2O) f CH2-, where f is an integer from 1 to 5;

[0024] X2 is -(CH2) g -, where g is an integer from 1 to 6;

[0025] Y1 is 0 or 1;

[0026] Y2 is 0, 1, or 2;

[0027] Y3 is 1, 2 or 3;

[0028] m is an integer from 0 to 4, preferably 0, 1 or 2;

[0029] n is an integer of 0-4, preferably 0, 1 or 2.

[0030] In one embodiment, A is -(CH2) 10 -, -(CH2)7-, -(CH2)8-, -(CH2)9-, -(CH2) 11 -、-(CH2) 12 -, -(CH2CH2O)3-, -(CH2)4NHCO- or -(CH2)6NHCO-.

[0031] In one embodiment, B is -(CH2)0-, -CH2-, -(CH2)2-, -(CH2)4-, or -(CH2)3-.

[0032] In one embodiment, R1 is oxygen.

[0033] In one embodiment, R1 is in the α configuration or the β configuration.

[0034] In one embodiment, R2 is hydrogen or -OCH3.

[0035] In one embodiment, R3 is a hydroxy protecting group or a phosphorus-containing reactive group. Preferably, R3 is an acyl group, a silyl group, a trityl group, a monomethoxytrityl group, a 4,4'-dimethoxytrityl group or a More preferably, R3 is acetyl, 1,1,3,3-tetraisopropyldisiloxanylidene (TIPDS, 1,1,3,3-tetraisopropyldisiloxanylidene), t-butyldimethylsilyl, phenyldimethylsilyl, 4,4'-dimethoxytrityl or

[0036] In another embodiment, R3 is In yet another embodiment, R3 is -CO(CH2)2COOH.

[0037] In one embodiment, R4 is a hydroxy protecting group, preferably trityl, monomethoxytrityl or 4,4'-dimethoxytrityl, more preferably 4,4'-dimethoxytrityl.

[0038] In one embodiment, m is 1.

[0039] In one embodiment, n is 0.

[0040] In one embodiment, G is

[0041] In one embodiment, the compound of formula (I) is a compound YK-GAL-301, YK-GAL-302, YK-GAL-303, YK-GAL-304, YK-GAL-305, YK-GAL-306, YK-GAL-307, YK-GAL-308, YK-GAL-309, YK-GAL-310, YK-GAL-311, YK-GAL-312 or YK-GAL-313 having the following structure:

[0042] in It is controlled pore glass.

[0043] In another embodiment, the compound of formula (I) is a compound YK-GAL-314, YK-GAL-315, YK-GAL-316, YK-GAL-317, YK-GAL-318, YK-GAL-319, YK-GAL-320, YK-GAL-321, YK-GAL-322, YK-GAL-323, YK-GAL-324, YK-GAL-325 or YK-GAL-326 having the following structure:

[0044] Optionally, the compound of formula (I) or a pharmaceutically acceptable salt thereof can bind to asialoglycoprotein receptor (ASGPR).

[0045] In a second aspect, the present invention provides a conjugate comprising an oligonucleotide and a GalNAc moiety, having the structure shown below:

[0046] Wherein, Oligo represents oligonucleotide, and R1, R2, R3, R4, A, B, L, G, m and n are as described above.

[0047] Optionally, the hydroxyl protecting group acetyl in the N-acetyl-galactosamine of G in the conjugate is removed.

[0048] Alternatively, the oligonucleotide comprises a non-thio oligonucleotide and a thio oligonucleotide. In one embodiment, the non-thio oligonucleotide and the GalNAc moiety are connected by a phosphate bond. In another embodiment, the thio oligonucleotide and the GalNAc moiety are connected by a thiophosphate bond.

[0049] Alternatively, the oligonucleotide comprises a small interfering nucleotide (siRNA), DNA, microRNA (miRNA), small activating RNA (saRNA), small guide RNA (sgRNA), transfer RNA (tRNA), antisense nucleotide (ASO) or aptamer (Aptamer), preferably an antisense nucleotide (ASO) or a small interfering nucleotide (siRNA). In one embodiment, each nucleotide in the antisense nucleotide (ASO) or small interfering nucleotide (siRNA) is independently a modified or unmodified nucleotide.

[0050] Optionally, the oligonucleotide modulates expression of a target gene.

[0051] In a third aspect, the present invention provides a pharmaceutical composition comprising the conjugate according to the second aspect and at least one pharmaceutically acceptable excipient.

[0052] In a fourth aspect, the present invention provides use of the conjugate of the second aspect or the pharmaceutical composition of the third aspect in the preparation of a medicament for treating and / or preventing pathological conditions or diseases caused by the expression of specific genes in hepatocytes, optionally, the specific gene is selected from the hepatitis B virus gene, angiopoietin 3 gene or apolipoprotein C3 gene.

[0053] In one embodiment, the disease is selected from chronic liver disease, hepatitis, liver fibrosis, liver proliferative disease and dyslipidemia; optionally, the dyslipidemia is hypercholesterolemia, hypertriglyceridemia or atherosclerosis.

[0054] In a fifth aspect, the present invention provides a method for inhibiting the expression of a specific gene in a hepatocyte, comprising contacting an effective amount of the conjugate of the second aspect or the pharmaceutical composition of the third aspect with the hepatocyte;

[0055] Optionally, the specific gene is selected from one of the following genes: proprotein convertase subtilisin / kexin type 9 gene (PCSK9), ApoB, ApoC, ANGPTL3, SCD1, FVII, p53, HBV, HCV;

[0056] Optionally, the specific gene is selected from the group consisting of proprotein convertase subtilisin / kexin type 9 gene (PCSK9), hepatitis B virus gene, angiopoietin-like protein 3 gene or apolipoprotein C3 gene.

[0057] In a sixth aspect, the present invention provides a kit comprising the conjugate according to the second aspect. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings of the embodiments will be briefly introduced below. Obviously, the drawings in the following description only relate to some embodiments of the present invention, but are not intended to limit the present invention.

[0059] Figure 1 shows the inhibition rate of PCSK9 protein expression in mouse serum by GalNAc-conjugated siRNA sequences inc-G4, inc-G5, inc-G6, inc-G7, inc-G11, inc-G12, inc-G13, G18-inc, inc-L96, inc-NAG0052, inc-GalNAc 1b, and inc-GalNAc 2 on days 7 and 14.

[0060] Figure 2 shows the inhibition rate of PCSK9 protein expression in mouse serum by GalNAc-conjugated siRNA sequences inc-G1, inc-G2, inc-G3, inc-G8, inc-G9, inc-G10, inc-G25, G26-inc, inc-L96, inc-NAG0052, inc-GalNAc 1b and inc-GalNAc 2 on days 7 and 14.

[0061] Figure 3 shows the inhibition rate of PCSK9 gene expression in mouse liver by GalNAc-conjugated siRNA sequences inc-G4, inc-G5, inc-G6, inc-G7, inc-G11, inc-G12, inc-G13, G18-inc, inc-L96, inc-NAG0052, inc-GalNAc 1b and inc-GalNAc 2 on day 14.

[0062] Figure 4 shows the inhibition rate of PCSK9 gene expression in mouse liver by GalNAc-conjugated siRNA sequences inc-G1, inc-G2, inc-G3, inc-G8, inc-G9, inc-G10, inc-G25, G26-inc, inc-L96, inc-NAG0052, inc-GalNAc 1b and inc-GalNAc 2 on day 14.

[0063] Figure 5 shows the effects of GalNAc-conjugated siRNA sequences inc-G4, inc-G5, inc-G6, inc-G7, inc-G11, inc-G12, inc-G13, G18-inc, inc-L96, inc-NAG0052, inc-GalNAc 1b, and inc-GalNAc 2 on LDL-C levels in mouse serum on days 7 and 14.

[0064] Figure 6 shows the effects of GalNAc-conjugated siRNA sequences inc-G1, inc-G2, inc-G3, inc-G8, inc-G9, inc-G10, inc-G25, G26-inc, inc-L96, inc-NAG0052, inc-GalNAc 1b, and inc-GalNAc 2 on LDL-C levels in mouse serum on days 7 and 14.

[0065] Figure 7 shows the activity imaging of mice 8 hours after administration of the GalNAc-conjugated siRNA sequences inc-G5, inc-G7, inc-L96, and inc-GalNAc 1b. (a: inc-G5; b: inc-G7; c: inc-L96; d: inc-GalNAc 1b)

[0066] Figure 8 shows the half-lives of GalNAc-conjugated siRNA sequences inc-G4, inc-G5, inc-G6, inc-G7, inc-G11, inc-G12, inc-G13, G18-inc, inc-L96, inc-NAG0052, inc-GalNAc 1b, and inc-GalNAc 2 in mouse liver.

[0067] Figure 9 shows the half-lives of GalNAc-conjugated siRNA sequences inc-G1, inc-G2, inc-G3, inc-G8, inc-G9, inc-G10, inc-G25, G26-inc, inc-L96, inc-NAG0052, inc-GalNAc 1b, and inc-GalNAc 2 in mouse liver. DETAILED DESCRIPTION

[0068] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the described embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0069] The present invention may be implemented in other specific forms without departing from the essential attributes of the present invention. It should be understood that, without conflict, any and all embodiments of the present invention may be combined with the technical features of any other embodiment or multiple other embodiments to produce additional embodiments. The present invention includes additional embodiments resulting from such combinations.

[0070] All publications and patents mentioned in the present invention are hereby incorporated into the present invention in their entirety by reference. If the purposes or terms used in any publications and patents incorporated by reference conflict with the purposes or terms used in the present invention, then the purposes and terms of the present invention shall prevail.

[0071] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0072] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly used in the art to which the claimed subject matter belongs. If there are multiple definitions for a term, the definition herein shall prevail.

[0073] Except in the working examples or otherwise indicated, all numbers stating quantitative properties such as dosage in the specification and claims should be understood to be modified by the term "about" in all cases. It should also be understood that any numerical range recited herein is intended to include all subranges within the range and any combination of the various endpoints of the range or subrange.

[0074] As used herein, words such as "include," "comprising," or "including" mean that the elements preceding the word include the elements listed after the word and their equivalents, without excluding unrecited elements. The terms "comprising" or "including" as used herein may be open, semi-closed, or closed. In other words, the terms also encompass "consisting essentially of" or "consisting of."

[0075] The term "pharmaceutically acceptable" as used herein means that the compound or composition is chemically and / or toxicologically compatible with the other ingredients constituting the formulation and / or with humans or mammals for the prevention or treatment of a disease or condition.

[0076] The term "pharmaceutically acceptable salt" refers to a relatively non-toxic, inorganic or organic acid addition salt of a compound of the present invention. For example, see SM Berge et al. "Pharmaceutical Salts", J. Pharm. Sci. 1977, 66, 1-19. Among them, inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid or nitric acid; organic acids such as formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, hexanoic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxybenzoyl)-benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, digluconic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, pamoic acid, pectinic acid, 3-phenylpropionic acid, picric acid, Pivalic acid, 2-hydroxyethanesulfonic acid, itaconic acid, amidosulfonic acid, trifluoromethanesulfonic acid, dodecylsulfuric acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, glucoheptonic acid, glycerophosphoric acid, aspartic acid, sulfosalicylic acid, etc. For example, HCl (or hydrochloric acid), HBr (or hydrobromic acid solution), methanesulfonic acid, sulfuric acid, tartaric acid or fumaric acid can be used to form a pharmaceutically acceptable salt with the compound shown in formula (I).

[0077] The term "alkyl" in this application refers to a monovalent hydrocarbon group including branched and straight chain saturated aliphatic groups having a specified number of carbon atoms. 1-4 "Alkyl" includes methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl.

[0078] The term "alkoxy" refers to the formula -OR, where R is an alkyl group as defined herein. A non-limiting list of alkoxy groups is methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, phenoxy, and benzoyloxy. In some cases, the alkoxy group can be -OR, where R is an unsubstituted C1-4 alkyl group. The alkoxy group can be substituted or unsubstituted.

[0079] "Halogen" refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I), preferably fluorine (F) and chlorine (Cl). In one embodiment, halogen is fluorine.

[0080] As used herein, the term "protecting group" refers to any atom or group of atoms introduced into a molecule to prevent existing groups in the molecule from undergoing undesired chemical reactions, which can be removed to leave an unprotected group.

[0081] The hydroxyl protecting group can be a protecting group commonly used to protect the hydroxyl group of the ribose structure in the synthesis of RNA or its derivatives, and can also refer to Green et al. in Protective Groups in Organic Synthesis, 3rd Edition, 1999, John Protecting groups described in the literature of Wiley & Sons, Inc., for example, acetyl, phenoxyacetyl, pivaloyl, benzyl, 4-methoxybenzyl, benzoyl, triphenylmethyl, 4,4'-dimethoxytrityl (DMTr), monomethoxytrityl (MMTr, monomethoxytrityl), 9-phenyl-xanthen-9-yl, 9-(p-tolyl)-xanthen-9-yl, trimethylsilyl, tert-butyldimethylsilyl (TBDMS), cyanomethoxymethyl, 2-(cyanoethoxy)ethyl, cyanoethoxymethyl, etc., preferably 4,4'-dimethoxytrityl (DMTr, 4,4'-dimethoxytrityl).

[0082] As used herein, the term "phosphorus-containing reactive group" refers to a phosphorus-containing group that is capable of reacting with a hydroxyl or amine group contained in another molecule, particularly another nucleotide unit or another nucleotide analog, by a nucleophilic attack reaction. Typically, such a reaction produces an ester-type internucleoside bond connecting a nucleotide unit or nucleotide analog unit to another nucleotide unit or nucleotide analog unit. These phosphorus-containing reactive groups are known in the art and include P III or P VPhosphorus atoms with a valence state, the phosphorus-containing reactive groups include but are not limited to phosphoramidites, H-phosphonates, phosphotriesters and phosphorus-containing chiral auxiliary agents. Phosphorus-containing reactive groups are for example:

[0083] 2-cyanoethoxy-N, N-diisopropylaminophosphinoyl,

[0084] 2-Propyleneoxy--N, N-diisopropylaminophosphine,

[0085] Methoxy-N, N-diisopropylaminophosphino,

[0086] In one embodiment, the phosphorus-containing reactive group is

[0087] Controlled pore glass (CPG) and polystyrene (highly cross-linked polystyrene microbeads) described herein are solid supports for oligonucleotide synthesis. They are insoluble particles that bind to oligonucleotides during the synthesis process and are commercially available.

[0088] The term "nucleotide" as used herein includes naturally occurring nucleotides and chemically modified nucleotides. Chemically modified nucleotides are non-naturally occurring nucleotides, also referred to herein as "nucleotide analogs."

[0089] The “hydroxyl groups are fully protected with acyl groups” described herein for “T” means that all hydroxyl groups in the galactose structure except the hydroxyl group used for connection to X1 are protected by acyl groups, wherein the acyl group is, for example, acetyl, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, isobutyryl or benzoyl.

[0090] Compound of formula (I) or a pharmaceutically acceptable salt thereof

[0091] The present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0092] in,

[0093] R1 is oxygen or sulfur, preferably oxygen;

[0094] R2 is hydrogen, C 1-4 Alkyl, C 1-4 Alkoxy or halogen;

[0095] R3 is hydrogen, hydroxyl protecting group, phosphorus-containing active reactive group, or -CO(CH2) x COOH, wherein x is an integer from 1 to 10, It is controlled pore glass or polystyrene;

[0096] R4 is hydrogen or a hydroxyl protecting group;

[0097] A is -(CH2) a -、-(CH2CH2O) b -、-((CH2) c NHCO) d -or-((CH2) c CONH) d -, wherein a is an integer of 1-15, preferably an integer of 3-13, b is an integer of 1-7, preferably an integer of 2-5, c is an integer of 1-7, preferably an integer of 2-6, and d is an integer of 1-5, preferably an integer of 1-3;

[0098] B is -(CH2) e -, wherein e is an integer of 0-7, preferably an integer of 1-5.

[0099] L is -CONH- or -NHCO-;

[0100] G is

[0101] in,

[0102] T is N-acetyl-galactosamine with fully protected hydroxyl groups, galactose with fully protected hydroxyl groups, galactosamine with fully protected hydroxyl groups, N-formyl-galactosamine with fully protected hydroxyl groups, N-propionyl-galactosamine with fully protected hydroxyl groups, N-n-butanoyl-galactosamine with fully protected hydroxyl groups, or N-isobutanoyl-galactosamine with fully protected hydroxyl groups. For example, T is N-acetyl-galactosamine with fully protected hydroxyl groups, wherein the acyl group is acetyl or benzoyl, preferably acetyl;

[0103] X1 is -(CH2) f -or-(CH2CH2O) f CH2-, where f is an integer from 1 to 5;

[0104] X2 is -(CH2) g -, where g is an integer from 1 to 6;

[0105] Y1 is 0 or 1;

[0106] Y2 is 0, 1, or 2;

[0107] Y3 is 1, 2 or 3;

[0108] m is an integer from 0 to 4, preferably 0, 1 or 2;

[0109] n is an integer of 0-4, preferably 0, 1 or 2.

[0110] In one embodiment, A is -(CH2) 10 -, -(CH2)7-, -(CH2)8-, -(CH2)9-, -(CH2) 11 -、-(CH2) 12 -, -(CH2CH2O)3-, -(CH2)4NHCO- or -(CH2)6NHCO-.

[0111] In one embodiment, B is -(CH2)0-, -CH2-, -(CH2)2-, -(CH2)4-, or -(CH2)3-.

[0112] In one embodiment, R1 is oxygen.

[0113] In one embodiment, R1 is in the α configuration or the β configuration.

[0114] In one embodiment, R2 is hydrogen or -OCH3.

[0115] In one embodiment, R3 is a hydroxyl protecting group or a phosphorus-containing reactive group. The hydroxyl protecting group can be, for example, an acyl group (e.g., acetyl, phenoxyacetyl, 4-isopropylphenoxyacetyl), a silyl group (silyl), a trityl group (trityl), a monomethoxytrityl group (MMTr) or a 4,4'-dimethoxytrityl group (DMTr), and the phosphorus-containing reactive group can be, for example, Preferably, R3 is acetyl, 1,1,3,3-tetraisopropyldisiloxanylidene (TIPDS, 1,1,3,3-tetraisopropyldisiloxanylidene), t-butyldimethylsilyl, phenyldimethylsilyl, 4,4'-dimethoxytrityl or More preferably, R3 is

[0116] In another embodiment, R3 is In yet another embodiment, R3 is -CO(CH2)2COOH.

[0117] In one embodiment, R4 is a hydroxy protecting group, preferably trityl, monomethoxytrityl or 4,4'-dimethoxytrityl, more preferably 4,4'-dimethoxytrityl.

[0118] In one embodiment, m is 1.

[0119] In one embodiment, n is 0.

[0120] In one embodiment, G is

[0121] In one embodiment, the compound of formula (I) is a compound having the following structure: YK-GAL-301, YK-GAL-302, YK-GAL-303, YK-GAL-304, YK-GAL-305, YK-GAL-306, YK-GAL-307, YK-GAL-308, YK-GAL-309, YK-GAL-310, YK-GAL-311, YK-GAL-312 or YK-GAL-313:

[0122] in It is controlled pore glass.

[0123] In another embodiment, the compound of formula (I) is a compound YK-GAL-314, YK-GAL-315, YK-GAL-316, YK-GAL-317, YK-GAL-318, YK-GAL-319, YK-GAL-320, YK-GAL-321, YK-GAL-322, YK-GAL-323, YK-GAL-324, YK-GAL-325 or YK-GAL-326 having the following structure:

[0124] Optionally, the compound of formula (I) or a pharmaceutically acceptable salt thereof can bind to asialoglycoprotein receptor (ASGPR).

[0125] Conjugate

[0126] The present invention provides a conjugate comprising an oligonucleotide and a GalNAc moiety having the structure shown below:

[0127] Wherein, Oligo represents oligonucleotide, and R1, R2, R3, R4, A, B, L, G, m and n are as described above.

[0128] The conjugates of the present invention can be prepared by solid-phase synthesis reaction of the compound of formula (I) or a pharmaceutically acceptable salt thereof with an oligonucleotide. The conventional method for conjugating GalNAc to oligonucleotides is solid-phase synthesis. One method is to connect the GalNAc compound to a CPG (Controlled Pore Glass) column and achieve the connection of the GalNAc compound to the oligonucleotide by solid-phase synthesis. Another method is to first prepare the GalNAc compound into a phosphoramidite monomer and achieve the connection of the GalNAc compound to any position of the oligonucleotide by solid-phase synthesis.

[0129] The oligonucleotides in this application include single-stranded oligonucleotides (e.g., antisense nucleotides, referred to as ASOs) and double-stranded oligonucleotides (e.g., small interfering nucleotides, referred to as siRNAs). In a preferred embodiment, the oligonucleotide comprises 7-30 nucleotides. Conjugates prepared by such oligonucleotides will have greater therapeutic value.

[0130] In one embodiment, the oligonucleotide is selected from a small interfering nucleotide, DNA, microRNA (miRNA), small activating RNA (saRNA), small guide RNA (sgRNA), transfer RNA (tRNA), antisense nucleotide or aptamer, preferably the oligonucleotide is an antisense nucleotide or a small interfering nucleotide.

[0131] Oligonucleotides in this application include natural oligonucleotides and chemically modified oligonucleotides. Chemical modifications herein include nucleoside modifications (including sugar moiety modifications and core base modifications) and internucleoside linkage modifications. Chemical modifications of oligonucleotides do not include situations where there are differences only in the core base sequence. Natural herein refers to situations corresponding to naturally occurring RNA or DNA.

[0132] Natural oligonucleotides have difficulty entering cells and are easily degraded by intracellular nucleases, resulting in poor efficacy. Chemical modification of oligonucleotides can improve their properties and increase their bioavailability. Among the many modified oligonucleotides, the most representative are thiolated oligonucleotides. One type of thiolation is that a non-bridging oxygen atom in the phosphodiester bond is replaced by a sulfur atom, for example

[0133] Thio-oligonucleotides can be commercially available or prepared by conventional solid phase synthesis methods. Hydroxanthin can be used as a sulfurizing agent. For example, reference can be made to CN1479745A and CN113150041A for synthesizing thio-oligonucleotides.

[0134] Optionally, the hydroxyl protecting group acetyl in the N-acetyl-galactosamine of G in the conjugate is removed, for example, by hydrolysis in an alkaline solution to obtain a hydroxyl group.

[0135] In one embodiment, the GalNAc portion of the conjugate has the structure

[0136] R1, R2, A, B, L, X1, X2, Y1, Y2, Y3, m and n are as described above.

[0137] For example, the oligonucleotide and the GalNAc moiety are connected via a bond or a cleavable linker. The bond herein may include, but is not limited to, a phosphate bond and a phosphorothioate bond.

[0138] As used herein, a cleavable linker refers to a linker that is cleaved by intracellular metabolism after internalization, for example, by hydrolysis, reduction, or enzymatic reaction. Suitable linkers include, but are not limited to, acid-labile linkers, hydrolysis-labile linkers, enzymatically cleavable linkers, and reduction-labile linkers. Acid-labile linkers can refer to the acid-labile linkers in ADC drugs (Mylotarg, Besponsas, Trodelvys).

[0139] Alternatively, the oligonucleotide comprises a non-thio oligonucleotide and a thio oligonucleotide. In one embodiment, the non-thio oligonucleotide and the GalNAc moiety are connected by a phosphate bond. In another embodiment, the thio oligonucleotide and the GalNAc moiety are connected by a thiophosphate bond.

[0140] Alternatively, the oligonucleotide comprises a small interfering nucleotide (siRNA), DNA, microRNA (miRNA), small activating RNA (saRNA), small guide RNA (sgRNA), transfer RNA (tRNA), antisense nucleotide (ASO) or aptamer (Aptamer), preferably an antisense nucleotide (ASO) or a small interfering nucleotide (siRNA). In one embodiment, each nucleotide in the antisense nucleotide (ASO) or small interfering nucleotide (siRNA) is independently a modified or unmodified nucleotide.

[0141] Optionally, the oligonucleotide modulates expression of a target gene.

[0142] Pharmaceutical composition

[0143] In addition to the conjugate, the composition provided herein may include any substance that can be used in a pharmaceutical composition. For example, the composition may include one or more pharmaceutically acceptable excipients or auxiliary ingredients, such as but not limited to one or more solvents, dispersion media, diluents, dispersing aids, suspension aids, granulation aids, disintegrants, fillers, glidants, liquid vehicles, adhesives, surfactants, isotonic agents, thickeners or emulsifiers, buffers, lubricants, oils, preservatives, flavorings, coloring agents, etc. Excipients such as starch, lactose or dextrin. Pharmaceutically acceptable excipients are well known in the art (see, for example, Remington's The Science and Practice of Pharmacy, 21st edition, A.R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, MD, 2006).

[0144] Pharmaceutically acceptable diluents include phosphate buffered saline (PBS), for example, sterile phosphate buffered saline. In some embodiments, the conjugate is used in a pharmaceutically acceptable diluent at a concentration of 50-500 μM solution. WO 2007 / 031091 provides suitable pharmaceutically acceptable diluents, carriers, and excipients, as well as suitable dosages, formulations, routes of administration, compositions, dosage forms, combinations with other therapeutic agents, and prodrug formulations (incorporated by reference).

[0145] The oligonucleotide conjugates of the present invention can be mixed with pharmaceutically acceptable active substances or inert substances to prepare pharmaceutical compositions or preparations, and can be sterilized by conventional sterilization techniques or can be sterile filtered.

[0146] Reagent test kit

[0147] The present application also provides a kit comprising the conjugate as described above. In some embodiments, the kit provided herein comprises a container comprising the conjugate as described above. In some embodiments, the kit provided herein further comprises a pharmaceutically acceptable excipient, such as a stabilizer or preservative. In some embodiments, the kit further comprises instructions for mixing the conjugate with a pharmaceutically acceptable excipient or other ingredients (if present).

[0148] Example

[0149] The present invention will be further described below with reference to the following examples. However, the present invention is not limited to the following examples. The implementation conditions used in the examples can be further adjusted according to the specific requirements of the application. The implementation conditions not specified are conventional conditions in the industry. In the specific examples of the present invention, the raw materials used can be obtained commercially. Unless otherwise stated, all temperatures are given in degrees Celsius. The technical features involved in the various embodiments of the present invention may be combined with each other as long as they do not conflict with each other.

[0150] Example 1: Synthesis of GalNAc-CPG Compounds

[0151] The following abbreviations represent the following reagents: DCM: dichloromethane; PE: petroleum ether; EA: ethyl acetate; THF: tetrahydrofuran; DMF: N,N-dimethylformamide; ACN: acetonitrile; BF3·Et2O: boron trifluoride etherate; LiOH·H2O: lithium hydroxide monohydrate; DMTrCl: 4,4′-dimethoxytriphenylmethane chloride; TEAB: aqueous triethylamine ammonium bicarbonate solution; HBTU: O-benzotriazole-tetramethyluronium hexafluorophosphate; DIPEA: N,N-diisopropylethylamine; DMAP: 4-dimethylaminopyridine; TMSOTf: trimethylsilyl trifluoromethanesulfonate; TIPDSCl2: 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane; TEA·3HF: triethylamine trihydrofluoride; Proton sponge: 1,8-bis(dimethylaminonaphthalene).

[0152] 1. Synthesis of YK-GAL-301

[0153] The synthetic route is as follows:

[0154] Step 1: Synthesis of G1-3

[0155] To dried compound G1-1 (781 mg, 3.00 mmol), dried compound G1-2 (0.50 g, 2.31 mmol), and 3A molecular sieves (2.0 g) was added dichloromethane (20 mL). After cooling to 0°C, boron trifluoride etherate (656 mg, 4.62 mmol) was added and stirring continued for 2 h. The reaction was quenched by the addition of saturated aqueous sodium bicarbonate solution (20 mL), and the mixture was extracted with dichloromethane (10 mL × 2). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to remove the solvent to obtain a crude product. Purification by column chromatography (PE / EA) gave a yellow oily substance G1-3 (1.17 g, 2.81 mmol, α, β mixed isomers, 93.7%), which was then separated by high pressure preparation to give G8-1 (533.2 mg, 1.28 mmol, α configuration, 42.7%) and G1-3 (625.6 mg, 1.5 mmol, β configuration, 50.1%). 1HNMR (400MHz, CDCl3) δppm 5.23-5.13 (m, 2H), 4.27 (dd, J=11.0, 5.6Hz, 1H), 4.23-4.15 (m, 1H), 4.09 (dd, J=11.0, 6.4Hz, 1H), 3.69-3.63 (m, 4H), 3.37-3.28 (m, 1H), 2.39-2.33 (m, 1H), 2.29 (t, J=7.6Hz, 2H), 2.19-1.94 (m, 8H), 1.65-1.56 (m, 2H), 1.53-1.48 (m, 2H), 1.32-1.21 (m, 11H). MS(ESI)m / z[M+Na] + =439.1.

[0156] Step 2: Synthesis of G1-4

[0157] G1-3 (0.12 g, 288 μmol) was dissolved in tetrahydrofuran (3.0 mL), and 1.5 mL of an aqueous solution of lithium hydroxide monohydrate (38.7 mg, 921 μmol) was added. The mixture was stirred at 15°C for 12 h. The reaction mixture was concentrated under reduced pressure to remove the solvent, yielding a yellow solid. The crude product G1-4 (92 mg) was used directly in the next step. MS (ESI) m / z [M+Na] + =341.2.

[0158] Step 3: Synthesis of G1-5

[0159] The crude product G1-4 (92 mg, 288 μmol) and anhydrous pyridine (20 mL × 3) were co-rotated to remove water. The dried G1-4 and 4,4′-dimethoxytriphenylmethane (195 mg, 576 μmol) were dissolved in pyridine (3.0 mL) and stirred at 15°C for 28 h under nitrogen. Methanol (10 mL) was added to quench the reaction, and the solvent was removed by concentration under reduced pressure to obtain the crude product. Preparative chromatography (H2O (10 mM NH4HCO3)-ACN) gave G1-5 (109.5 mg, 176.5 μmol) as a white solid in a yield of 61.3%. 1HNMR (400MHz, CDCl3) δppm 7.48-7.41(m, 2H), 7.37-7.27(m, 6H), 7.21(dd, J=8.2, 6.2Hz, 1H), 6.88-6.78(m, 4H), 5 .15(dd, J=5.3, 2.2Hz, 1H), 4.43-4.39(m, 1H), 3.98-3.91(m, 1H), 3.79(s, 6H), 3.62-3.5 6 (m, 1H), 3.35-3.23 (m, 2H), 3.15 (dd, J=9.4, 6.8Hz, 1H), 2.33 (t, J=7.4Hz, 5H), 2.21-2. 15(m, 2H), 2.07-2.00(m, 1H), 1.66-1.59(m, 2H), 1.44-1.41(m, 2H), 1.29-1.22(m, 10H). MS(ESI)m / z[M+Na] + =643.4.

[0160] Step 4: Synthesis of G1-6

[0161] G1-5 (40 mg, 64.4 μmol), diisopropylethylamine (16.7 mg, 129 μmol), and O-benzotriazole-tetramethyluronium hexafluorophosphate (36.7 mg, 96.6 μmol) were dissolved in N,N-dimethylformamide (0.5 mL). GalNAc-NH2.TFA (123 mg, 64.4 μmol, TFA) was added and stirred at 15°C under nitrogen for 1 h. The reaction mixture was used directly in the next step. MS (ESI) m / z [M-2H] 2- =1197.4.

[0162] Step 5: Synthesis of G1-7

[0163] G1-7 (154 mg, 64.2 μmol), 4-dimethylaminopyridine (15.7 mg, 128 μmol), diisopropylethylamine (16.6 mg, 128 μmol), and succinic anhydride (64.4 mg, 642 μmol) were dissolved in N,N-dimethylformamide (2.0 mL) and stirred at 30°C under nitrogen for 48 h. Preparative chromatography (HO(10 mM TEAB)-ACN) afforded G1-7 (59 mg, 26.7 μmol) as a white solid in a 41.5% yield. 1HNMR (400MHz, CDCl3) δppm 7.44 (d, J=7.6Hz, 2H), 7.32 (d, J=8.5Hz, 3H), 7.25-7.14 (m, 5H), 6.97 (t, J=6.3Hz, 3H), 6.78 (dd, J=11.8, 8.6Hz, 6H) , 6.48 (s, 1H), 5.33 (d, J=3.2Hz, 3H), 5.19-5.15 (m, 5H), 4.59 (d, J=8.3Hz, 3H), 4.18-4.03 (m, 10H), 3.95-3.88 (m, 6H ), 3.77 (s, 6H), 3.68-3.61 (m, 13H), 3.52-3.46 (m, 3H), 3.39-3.09 (m, 15H), 2.61-2.48 (m, 4H), 2.42 (t, J=5.8Hz, 5H) , 2.31-2.09(m, 20H), 2.03-1.99(m, 16H), 1.98-1.93(m, 21H), 1.72-1.54(m, 16H), 1.41(s, 2H), 1.24-1.21(m, 10H). MS(ESI)m / z[M-2H] 2- =1247.4.

[0164] Step 6: Synthesis of YK-GAL-301

[0165] G1-7 (59 mg, 22.7 μmol), 4-dimethylaminopyridine (2.77 mg, 22.7 μmol), diisopropylethylamine (23.5 mg, 182 μmol), and O-benzotriazole-tetramethyluronium hexafluorophosphate (43.1 mg, 113 μmol) were dissolved in N,N-dimethylformamide (4.0 mL). CPG-NH2 (425 mg) was then added and stirred at 40°C for 12 h. The reaction mixture was filtered, and the filtrate was washed sequentially with methanol and dichloromethane and dried under vacuum. The filtrate was then added to 4 mL of acetic anhydride / pyridine (1:5) solution and stirred at 40°C for 0.5 h. The mixture was filtered, washed sequentially with dichloromethane and methanol, and dried under vacuum for 12 h to obtain compound YK-GAL-301 (338 mg, loading 27.0 μmol / g) as a white solid.

[0166] 2. Synthesis of YK-GAL-302

[0167] The synthetic route is as follows:

[0168] Step 1: Synthesis of G2-1

[0169] Compound G2 (800 mg, 3.84 mmol) was dissolved in methanol (16 mL), sulfuric acid (1.18 g, 11.7 mmol) was added, and the mixture was stirred at 70°C for 16 h. The reaction was quenched by the addition of 10 mL of sodium sulfate, and the solvent was removed by concentration under reduced pressure to obtain the crude product. Purification by column chromatography (DCM / MeOH) afforded G2-1 (470 mg, 2.11 mmol) as a colorless oil in a 55.0% yield. 1 HNMR (400MHz, CDCl3) δppm 4.17 (s, 2H), 3.78-3.65 (m, 13H), 3.61 (dd, J=5.4, 3.7Hz, 2H), 2.30 (s, 1H)

[0170] Step 2: Synthesis of G2-2

[0171] To compound G1-1 (660 mg, 2.54 mmol), compound G2-1 (470 mg, 2.11 mmol), and 4A molecular sieves (900 mg) was added dichloromethane (9.0 mL). The temperature was cooled to 0°C, and boron trifluoride etherate (600 mg, 4.23 mmol) was added. Stirring was continued for 2 h. Stirring was then continued at 15°C for 3 h. The reaction was quenched by the addition of saturated aqueous sodium bicarbonate (30 mL), and the mixture was extracted with dichloromethane (30 mL x 3). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to remove the solvent to obtain the crude product. Purification by column chromatography (PE / EA) afforded G2-2 (443.3 mg, 1.05 mmol) as a yellow oil in a 49.8% yield. 1 HNMR (400MHz, CDCl3) δppm 5.24(d, J=5.4Hz, 1H), 5.04-5.00(m, 1H), 4.36-4.23(m, 2H), 4.19-4.14(m, 3H), 3.87-3.80(m, 1H), 3.75(s, 3H), 3.73-3.68(m, 5H), 3.67-3.64(m, 6H), 2.45-2.38(m, 1H), 2.11-2.01(m, 7H). MS(ESI)m / z[M+Na] + =445.0.

[0172] Step 3: Synthesis of G2-3

[0173] G2-2 (320 mg, 757 μmol) was dissolved in tetrahydrofuran (2.5 mL), and 2.5 mL of an aqueous solution of lithium hydroxide monohydrate (95.3 mg, 2.27 mmol) was added. The mixture was stirred at 15°C for 16 h. The reaction mixture was concentrated under reduced pressure to remove the solvent, yielding a white solid. The crude product G2-3 (245 mg) was used directly in the next step. MS (ESI) m / z [M+Na] + =346.9.

[0174] Step 4: Synthesis of G2-4

[0175] The dried crude product G2-3 (245 mg, 755 μmol) and 4,4′-dimethoxytriphenylmethane (511 mg, 1.51 mmol) were dissolved in pyridine (3.0 mL) and stirred at 15°C under nitrogen for 6 h. Methanol (2 mL) was added to quench the reaction, and the solvent was removed by concentration under reduced pressure to obtain the crude product. Purification by preparative chromatography (H2O(10 mM NH4HCO3)-ACN) afforded G2-4 as a white solid (309.1 mg, 494 μmol, 65.2% yield). 1 HNMR (400MHz, CDCl3) δppm 7.47 (d, J=7.6Hz, 1H), 7.39-7.33 (m, 4H), 7.20-7.16 (m, 1H), 6.82-6.80 (m, 4H), 5.13 (d, J=4.8Hz, 1H), 4.43-4.38 (m, 2 H), 4.05-4.38(m, 5H), 3.79-3.78(m, 8H), 3.67-3.52(m, 10H), 3.46-3.13(m, 4H), 2.13-2.16(m, 1H), 2.03-1.98(m, 1H). MS(ESI)m / z[MH] - =625.4.

[0176] Step 5: Synthesis of G2-5

[0177] G2-4 (80.0 mg, 127 μmol), diisopropylethylamine (33.0 mg, 255 μmol, 44.4 μL), and O-benzotriazole-tetramethyluronium hexafluorophosphate (72.6 mg, 191 μmol) were dissolved in N,N-dimethylformamide (2.0 mL). GalNAc-NH2.TFA (206 mg, 108 μmol, TFA) was added and stirred at 15°C under nitrogen for 4 h. The reaction mixture was used directly in the next step. MS (ESI) m / z [M-2H] 2- =1200.3.

[0178] Step 6: Synthesis of G2-6

[0179] G2-5 (305 mg, 126 μmol), 4-dimethylaminopyridine (62.0 mg, 507 μmol), diisopropylethylamine (49.2 mg, 380 μmol), and succinic anhydride (114 mg, 1.14 mmol) were dissolved in N,N-dimethylformamide (3.0 mL) and stirred at 30°C under nitrogen for 48 h. Preparative chromatography (HO(10 mM TEAB)-ACN) afforded G2-6 (130 mg, 51.9 μmol) as a white solid in a 40.9% yield. 1 HNMR (400MHz, CDCl3) δppm 7.45 (s, 1H), 7.43 (s, 1H), 7.32 (d, J=8.5Hz, 4H), 7.25-7.16 (m, 4H), 6.95 (dd, J=36.7, 13.2Hz, 4H), 6.85-6.72 (m, 6H), 5.35 (d, J =3.4Hz, 2H), 5.22 (td, J = 11.0, 10.3, 4.0Hz, 4H), 4.63 (dd, J = 8.3, 2.6Hz, 2H), 4.12 (dtd, J = 27.6, 11.1, 4.6Hz, 9H), 3.96-3.85 (m, 7H), 3.78 (d, J=2.6Hz, 5H), 3.71-3.62 (m, 14H), 3.54 (dd, J=25.9, 6.2Hz, 7H), 3.33-3.16 (m, 12H), 2.83 (s, 10H), 2.67-2.53 (m, 4 H), 2.42 (t, J=5.9Hz, 4H), 2.28-2.14 (m, 14H), 2.11-1.93 (m, 26H), 1.67 (ddd, J=39.3, 13.2, 7.3Hz, 16H), 1.23 (d, J=7.4Hz, 15H). MS(ESI)m / z[M-2H] 2- =1250.3.

[0180] Step 7: Synthesis of YK-GAL-302

[0181] G2-6 (130 mg, 51.9 μmol), 4-dimethylaminopyridine (6.35 mg, 51.9 μmol), diisopropylethylamine (53.7 mg, 415 μmol), and O-benzotriazole-tetramethyluronium hexafluorophosphate (98.7 mg, 259.5 μmol) were dissolved in N,N-dimethylformamide (6.0 mL). CPG-NH2 (830 mg, 1.41 mmol) was then added and stirred at 30°C for 16 h. The reaction mixture was filtered, and the filtrate was washed sequentially with methanol and dichloromethane and dried under vacuum. The filtrate was then added to 12 mL of acetic anhydride / pyridine (1:5) solution and stirred at 40°C for 0.5 h. The mixture was filtered, washed sequentially with dichloromethane and methanol, and dried under vacuum for 12 h to obtain compound YK-GAL-302 (840 mg, loading 34.9 μmol / g) as a white solid.

[0182] 3. Synthesis of YK-GAL-303

[0183] The synthetic route is as follows:

[0184] Step 1: Synthesis of G3-2

[0185] To dried compound G1-1 (1.19 g, 4.58 mmol), dried compound G3-1 (663.2 mg, 3.52 mmol), and 3A molecular sieves (2.0 g) was added dichloromethane (26 mL). The mixture was cooled to 0°C and then added with boron trifluoride etherate (999.95 mg, 7.05 mmol). The mixture was then stirred at 25°C for 2 h. The reaction was quenched with saturated aqueous sodium bicarbonate (30 mL) and extracted with dichloromethane (30 mL x 3). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to remove the solvent to obtain the crude product. Purification by column chromatography (PE / EA) afforded G3-2 (612.2 mg, 1.58 mmol) as a yellow oil in a yield of 44.8%. MS (ESI) m / z [M+Na] + =411.0.

[0186] Step 2: Synthesis of G3-3

[0187] G3-2 (130 mg, 335 μmol) was dissolved in tetrahydrofuran (2.0 mL), and 1.0 mL of an aqueous solution of lithium hydroxide monohydrate (44.9 mg, 1.07 mmol) was added. The mixture was stirred at room temperature for 16 h. The reaction mixture was concentrated under reduced pressure to remove the solvent, yielding a yellow solid. The crude product G3-3 (97 mg) was used directly in the next step. MS (ESI) m / z [MH] - =289.3.

[0188] Step 3: Synthesis of G3-4

[0189] Dried G3-3 (97 mg, 335 μmol) and 4,4′-dimethoxytriphenylmethane (406 mg, 1.2 mmol) were dissolved in pyridine (4.0 mL) and stirred at room temperature under nitrogen for 16 h. Methanol (10 mL) was added to quench the reaction, and the solvent was removed by concentration under reduced pressure to obtain the crude product. Column chromatography (DCM / MeOH) afforded G3-4 (126.7 mg, 213.9 μmol) as a white solid in a yield of 63.86%. MS (ESI) m / z [MH] - =591.5.

[0190] Step 4: Synthesis of G3-5

[0191] G3-4 (85 mg, 143 μmol), diisopropylethylamine (37.1 mg, 287 μmol, 49.9 μL), and O-benzotriazole-tetramethyluronium hexafluorophosphate (81.6 mg, 215 μmol) were dissolved in N,N-dimethylformamide (3.0 mL). GalNAc-NH2.TFA (274 mg, 143 μmol, TFA) was added and stirred at room temperature under nitrogen for 2 h. The reaction mixture was used directly in the next step. MS (ESI) m / z [M-2H] 2- =1183.2.

[0192] Step 5: Synthesis of G3-6

[0193] G3-5 (339 mg, 143 μmol), 4-dimethylaminopyridine (17.5 mg, 143 μmol), diisopropylethylamine (74.1 mg, 573 μmol), and succinic anhydride (71.7 mg, 717 μmol) were dissolved in N,N-dimethylformamide (3.0 mL) and stirred at 30°C under nitrogen for 16 h. Preparative chromatography (HO(10 mM TEAB)-ACN) afforded G3-6 (187 mg, 75.7 μmol) as a white solid in a 52.8% yield. 1HNMR (400 MHz, CDCl3) δ ppm 7.46-7.41(m, 2H), 7.34-7.27(m, 5H), 7.25-7.21(m, 2H), 7.19-7.14(m, 1 H), 7.05 (t, J=6.1Hz, 2H), 6.96 (d, J=9.0Hz, 2H), 6.83-6.75 (m, 4H), 6.50 (d, J=5.4Hz, 1H), 5.34-5.31 (m, 2H), 5.18 (dt, J=11.3, 3.9Hz, 4H), 4.60 ( dd, J=8.4, 2.6Hz, 3H), 4.50 (d, J=8.0Hz, 1H), 4.18-4.04 (m, 9H), 3.95-3.8 8(m, 5H), 3.76(s, 5H), 3.69-3.58(m, 12H), 3.52-3.46(m, 3H), 3.32-3.11 (m, 14H), 2.80 (q, J=7.2Hz, 8H), 2.57-2.54 (m, 1H), 2.48 (t, J=6.5Hz, 2H) , 2.41 (t, J=5.8Hz, 5H), 2.29-2.06 (m, 18H), 2.05-1.91 (m, 24H), 1.76-1. 51 (m, 18H), 1.40 (t, J=6.7Hz, 2H), 1.23-1.19 (m, 7H), 1.16-1.13 (m, 12H). MS (ESI) m / z [M-2H] 2- =1233.3.

[0194] Step 6: Synthesis of YK-GAL-303

[0195] G3-6 (50 mg, 19.1 μmol), 4-dimethylaminopyridine (2.34 mg, 19.1 μmol), diisopropylethylamine (19.8 mg, 153 μmol), and O-benzotriazole-tetramethyluronium hexafluorophosphate (36.3 mg, 95.7 μmol) were dissolved in N,N-dimethylformamide (4.0 mL). CPG-NH2 (332 mg) was then added and stirred at 40°C for 16 h. The reaction mixture was filtered, and the filtrate was washed sequentially with methanol and dichloromethane and dried under vacuum. The filtrate was then added to 4 mL of acetic anhydride / pyridine (1:4) solution and stirred at 40°C for 0.5 h. The mixture was filtered, washed sequentially with dichloromethane and methanol, and dried under vacuum for 12 h to obtain compound YK-GAL-303 (292 mg, loading 31.7 μmol / g) as a white solid.

[0196] 4. Synthesis of YK-GAL-304

[0197] The synthetic route is as follows:

[0198] Step 1: Synthesis of G4-1

[0199] G4A (3.00 g, 18.7 mmol), diisopropylethylamine (4.84 g, 37.4 mmol), and O-benzotriazole-tetramethyluronium hexafluorophosphate (7.81 g, 20.6 mmol) were dissolved in N,N-dimethylformamide (30.0 mL), and G4A-1 (1.50 g, 16.9 mmol) was added. The mixture was stirred at 15° C. for 2 h. Preparative chromatography (H O (0.1% TFA)-ACN) gave G4-1 (1.70 g) as a white solid. 1 HNMR (400MHz, DMSO-d6) δppm 7.73-7.75(m, 1H), 4.69(s, 1H), 3.62(s, 3H), 3.35-3.57(m, 2H), 2.99- 3.02(m, 2H), 2.27-2.30(m, 2H), 2.04-2.05(m, 2H), 1.38-1.48(s, 8H). MS(ESI)m / z[M+H] + =231.9.

[0200] Step 2: Synthesis of G4-2

[0201] To compound G1-1 (900 mg, 3.46 mmol), compound G4-1 (880 mg, 3.80 mmol), and 4A molecular sieves (1.60 g) was added dichloromethane (16.0 mL). After cooling to 0°C, trimethylsilyl trifluoromethanesulfonate (1.54 g, 6.92 mmol) was added and stirring continued for 2 h. The reaction was quenched by adding saturated aqueous sodium bicarbonate (20.0 mL) and extracted with dichloromethane (20.0 mL x 3). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to remove the solvent to obtain the crude product. Purification by column chromatography (PE / EA) afforded G4-2 (617.7 mg, 1.43 mmol) as a colorless oil in a 41.4% yield. 1 HNMR (400MHz, CDCl3) δppm 5.69-5.54(m, 1H), 5.30-5.17(m, 1H), 4.31-4.04(m, 3H), 3.75-3.62(m, 4H), 3.43-3.34(m, 1H), 3.31-3.19(m, 2H), 2.41-2.29(m, 3H), 2.21-2.16(m, 3H), 2.13-2.00(m, 5H), 1.72-1.51(m, 10H). MS(ESI)m / z[M+Na] + =454.0.

[0202] Step 3: Synthesis of G4-3

[0203] Dissolve G4-2 (160 mg, 371 μmol) in tetrahydrofuran (2.2 mL), add 1.0 mL of an aqueous solution of lithium hydroxide monohydrate (49.8 mg, 1.19 mmol), and stir at 20°C for 16 h. The reaction mixture is concentrated under reduced pressure to remove the solvent, yielding a white solid. The crude product, G4-3 (123 mg), is used directly in the next step.

[0204] Step 4: Synthesis of G4-4

[0205] Dried G4-3 (123 mg, 369 μmol) and 4,4′-dimethoxytriphenylmethane (463 mg, 1.37 mmol) were dissolved in pyridine (1.50 mL) and stirred at 25°C under nitrogen for 48 h. Methanol (1.00 mL) was added to quench the reaction, and the solvent was removed by concentration under reduced pressure to obtain the crude product. Preparative chromatography (HO (10 mM TEAB)-ACN) afforded G4-4 (148 mg, 233 μmol) as a white solid in a 62.8% yield. 1 HNMR (400 MHz, CDCl3) δ ppm 7.19-7.41(m, 9H), 6.73-6.77(m, 4H), 5.81-5.65(m, 2H), 5.07-5.08(s, 1H), 4.31-4.34(m, 1H), 3.91-3.92(m, 1H), 3.54-3.71(m, 6H), 3.42-3.5 4(m, 1H), 3.16-3.17(m, 1H), 3.10-3.14(m, 1H), 3.06-3.09(m, 4H), 2.22 -2.25 (m, 2H), 1.92-2.10 (m, 4H), 1.54-1.57 (m, 4H), 1.12-1.16 (m, 4H). MS (ESI) m / z [MH] - =634.4.

[0206] Step 5: Synthesis of G4-5

[0207] G4-4 (30.0 mg, 47.2 μmol), diisopropylethylamine (12.2 mg, 94.4 μmol), and O-benzotriazole-tetramethyluronium hexafluorophosphate (26.8 mg, 70.8 μmol) were dissolved in N,N-dimethylformamide (1.0 mL). GalNAc-NH2.TFA (45.0 mg, 23.6 μmol, TFA) was added and stirred at 25°C under nitrogen for 2 h. The reaction mixture was used directly in the next step. MS (ESI) m / z [M-2H] 2- =1204.8.

[0208] Step 6: Synthesis of G4-6

[0209] G4-5 (113 mg, 46.9 μmol), 4-dimethylaminopyridine (23.1 mg, 187 μmol), diisopropylethylamine (66.7 mg, 515 μmol), and succinic anhydride (42.2 mg, 421 μmol) were dissolved in N,N-dimethylformamide (1.0 mL) and stirred at 30°C under nitrogen for 48 h. Purification by preparative chromatography (H2O(10 mM TEAB)-ACN) afforded G4-6 (55.3 mg, 22 μmol) as a white solid in a 47.0% yield. MS (ESI) m / z [M-2H] 2- =1254.1.

[0210] Step 7: Synthesis of YK-GAL-304

[0211] G4-6 (20 mg, 7.57 μmol), 4-dimethylaminopyridine (1.00 mg, 8.20 μmol), diisopropylethylamine (7.82 mg, 60.5 μmol), and O-benzotriazole-tetramethyluronium hexafluorophosphate (14.4 mg, 37.8 μmol) were dissolved in N,N-dimethylformamide (1.5 mL). CPG-NH2 (131 mg) was then added and stirred at 40°C for 16 h. The reaction mixture was filtered, and the filtrate was washed sequentially with methanol and dichloromethane and dried under vacuum. The filtrate was then added to 1.20 mL of acetic anhydride / pyridine (1:5) solution and stirred at 40°C for 0.5 h. The mixture was filtered, washed sequentially with dichloromethane and methanol, and dried under vacuum for 12 h to obtain compound YK-GAL-304 (100 mg, loading 35.57 μmol / g) as a white solid. 5. Synthesis of YK-GAL-305

[0212] The synthetic route is as follows:

[0213] Step 1: Synthesis of G5-2

[0214] To dried compound G5-1 (3.53 g, 11.1 mmol), dried compound G1-2 (2.0 g, 9.25 mmol), and 3A molecular sieves (10.0 g) was added dichloromethane (100.0 mL). After cooling to 0°C, boron trifluoride etherate (2.62 g, 18.5 mmol) was added and stirring continued for 2 h. The reaction was quenched by the addition of triethylamine (2.7 mL), and the mixture was washed with saturated aqueous sodium bicarbonate (50 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to remove the solvent to obtain the crude product. Purification by column chromatography (PE / EA) afforded G5-2 (2.11 g, 4.46 mmol) as a colorless oil in a 48.2% yield. 1HNMR (400MHz, CDCl3) δppm 5.32(dd, J=6.7, 4.8Hz, 1H), 5.25-5.18(m, 1H), 4.97(s, 1H), 4.39-4.22(m, 2H), 4.14-4.00 (m, 1H), 3.73-3.60 (m, 4H), 3.36 (dt, J=9.3, 6.7Hz, 1H), 2.29 (t, J=7.5 Hz, 2H), 2.11-2.04(m, 8H), 1.64-1.52(m, 5H), 1.30-1.24(m, 12H). MS(ESI)m / z[M+Na] + =497.1.

[0215] Step 2: Synthesis of G5-3

[0216] Dissolve G5-2 (1.80 g, 3.79 mmol) in methanol (10.0 mL), add sodium methoxide (68.3 mg, 379 μmol), and stir at 15°C for 2 h. Quench the reaction by adding hydrogen ion exchange resin, filter, and concentrate the filtrate under reduced pressure to remove the solvent to obtain a yellow solid. The crude product G5-3 (1.32 g) was used directly in the next step. 1 HNMR (400MHz, CDCl3) δppm 4.91 (s, 1H), 4.32 (t, J=5.5Hz, 1H), 4.08-3.97 (m, 2H), 3.76 (dd, J=11.8, 3.3Hz, 1H), 3.71-3.57 (m, 5H), 3.41 (dt, J=9.3, 6.5Hz, 1H), 2.74 (s, 2H), 2.27 (t, J=7.5Hz, 2H), 1.55 (dq, J=20.6, 7.0Hz, 5H), 1.28-1.25 (m, 12H). MS(ESI)m / z[M+Na] + =371.2.

[0217] Step 3: Synthesis of G5-4

[0218] G5-3 (1.32 g, 3.79 mmol) was dissolved in N,N-dimethylformamide (10.0 mL), and 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (1.31 g, 4.17 mmol) and imidazole (645 mg, 9.47 mmol) were added. The mixture was stirred at 15°C for 2 h under nitrogen. Saturated aqueous sodium bicarbonate (10.0 mL) was added to quench the mixture, and the mixture was extracted with dichloromethane (10 mL x 2). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to remove the solvent to obtain the crude product. Purification by column chromatography (PE / EA) afforded G5-4 (1.20 g, 2.03 mmol) as a colorless oil in a yield of 53.6%. 1HNMR (400MHz, CDCl3) δppm 4.91 (s, 1H), 4.51 (t, J=5.3Hz, 1H), 4.06-3.97 (m, 3H), 3.76 (dd, J=10.4, 8.7Hz, 1H), 3.66 (s, 3H), 3.61 (dt, J=9.6, 6.8Hz, 1H), 3.34 (dt, J= 9.5, 6.4Hz, 1H), 2.97 (s, 1H), 2.30 (t, J=7.5Hz, 2H), 1.61 (t, J=7.4Hz, 3H), 1.51 (q, J=6.8Hz, 3H), 1.27-1.32 (m, 12H), 1.11-1.02 (m, 26H). MS (ESI) m / z [M+Na] + =613.4.

[0219] Step 4: Synthesis of G5-5

[0220] G5-4 (1.20 g, 2.03 mmol) was dissolved in dichloromethane (12.0 mL), and 1,8-bis(dimethylamino)naphthalene (1.18 g, 5.48 mmol) and trimethyloxonium tetrafluoroborate (751 mg, 5.08 mmol) were added. The mixture was stirred at 15°C for 16 h under nitrogen. Aqueous ammonium chloride (10.0 mL) was added to quench the mixture, and the mixture was extracted with dichloromethane (10 mL x 2). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to remove the solvent to obtain the crude product. Purification by column chromatography (PE / EA) afforded G5-5 (1.05 g, 1.74 mmol, yield 85.5%) as a colorless oil. 1 HNMR (400MHz, CDCl3) δppm 4.84 (s, 1H), 4.49 (dd, J=7.6, 4.3Hz, 1H), 3.98 (dq, J=9.0, 3.1Hz, 2H), 3.86 (dd, J=12.6, 6.6Hz, 1H), 3.69-3.54 (m, 8H), 3.40-3.27 (m, 1H), 2.30 (t, J=7.5Hz, 2H), 1.65-1.57 (m, 2H), 1.53-1.48 (m, 2H), 1.33-1.24 (m, 12H), 1.11-0.97 (m, 28H). MS(ESI)m / z [M+Na] + =627.4.

[0221] Step 5: Synthesis of G5-6

[0222] G5-5 (1.05 g, 1.74 mmol) was dissolved in tetrahydrofuran (10.0 mL), and triethylamine trihydrofluoride (839 mg, 5.20 mmol) was added. The mixture was stirred at 15°C for 16 h. Saturated aqueous sodium bicarbonate solution (10.0 mL) was added to quench the mixture, and the mixture was extracted with dichloromethane (10 mL × 2). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to remove the solvent to obtain the crude product G5-6 (0.63 g), which was used directly in the next step. 1 HNMR (400MHz, CDCl3) δppm 4.93 (d, J=1.3Hz, 1H), 4.25 (t, J=5.2Hz, 1H), 4.02-3.94 (m, 1H), 3.75-3.54 (m, 6H), 3.48-3.31 (m, 4H), 2.55 (brs, 1H), 2.23 (t, J=7.5Hz, 2H), 1.53 (dp, J=14.1, 7.0Hz, 4H), 1.22 (d, J=5.8Hz, 12H), 0.99 (dq, J=5.8, 3.1, 2.6Hz, 2H). MS(ESI)m / z[M+Na] + =385.2.

[0223] Step 6: Synthesis of G5-7

[0224] Dried G5-6 (0.63 g, 1.74 mmol) and 4,4'-dimethoxytriphenylmethane (618 mg, 1.83 mmol) were dissolved in pyridine (10.0 mL) and stirred at 15°C for 16 h under nitrogen. The reaction was quenched by the addition of saturated aqueous sodium bicarbonate (10.00 mL) and extracted with dichloromethane (15.0 mL x 2). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to remove the solvent to obtain the crude product. Purification by column chromatography (PE / EA) afforded G5-7 (1.01 g, 1.52 mmol) as a white solid in an 87.4% yield. 1HNMR (400MHz, CDCl3) δppm 7.52-7.46(m, 2H), 7.40-7.34(m, 4H), 7.29-7.24(m, 2H), 7.22-7.16(m, 1H), 6.83-6.78(m, 4H), 5.0 2 (d, J=1.4Hz, 1H), 4.19 (dt, J=8.3, 5.5Hz, 1H), 4.04 (td, J=5.8, 3.8Hz, 1H), 3.78 (brs, 6H), 3.76-3. 63 (m, 6H), 3.50 (s, 3H), 3.38 (dt, J=9.4, 6.8Hz, 1H), 3.27 (dd, J=9.9, 3.8Hz, 1H), 3.14 (dd, J=9.9, 5 .6Hz, 1H), 2.48 (d, J=8.4Hz, 1H), 2.29 (t, J=7.6Hz, 2H), 1.60 (d, J=14.7Hz, 2H), 1.28-1.23 (m, 11H). MS(ESI)m / z[MH] - =663.6.

[0225] Step 7: Synthesis of G5-8

[0226] Dissolve G5-7 (1.00 g, 1.50 mmol) in tetrahydrofuran (12.0 mL). Add 6.0 mL of an aqueous solution of lithium hydroxide monohydrate (94.7 mg, 2.26 mmol). Stir at 15°C for 12 h. The reaction mixture is concentrated under reduced pressure to remove the solvent, yielding a yellow solid. The crude product, G5-8 (0.98 g), is used directly in the next step. 1 HNMR (400MHz, CDCl3) δppm 7.53-7.45 (m, 2H), 7.35 (d, J=8.50Hz, 4H), 7.23 (t, J=7.19Hz, 2H), 7.19-7.14 (m, 1H), 6.84-6.73 (m, 4H), 5.04 (s, 1H), 4.26-4.00 (m, 2H), 3.80-3.63 (m, 8H), 3.45 (s, 3H), 3.39 (s, 1H), 3.30-3.20 (m,1H),3.12(br s, 1H), 2.12-2.05 (m, 1H), 2.04-1.98 (m, 3H), 1.58-1.41 (m, 4H), 1.19 (br s, 12H),. MS(ESI)m / z[MH] - =649.6.

[0227] Step 8: Synthesis of G5-9

[0228] G5-8 (0.97 g, 1.49 mmol), diisopropylethylamine (385 mg, 2.98 mmol), and O-benzotriazole-tetramethyluronium hexafluorophosphate (848 mg, 2.24 mmol) were dissolved in N,N-dimethylformamide (35.0 mL). GalNAc-NH2.TFA (2.84 g, 1.49 mmol, TFA) was added and stirred at 15°C under nitrogen for 1 h. The reaction mixture was used directly in the next step. MS (ESI) m / z [M-2H] 2- =1212.4.

[0229] Step 9: Synthesis of G5-10

[0230] G5-9 (3.62 g, 1.49 mmol), 4-dimethylaminopyridine (364 mg, 2.98 mmol), diisopropylethylamine (771 mg, 5.97 mmol), and succinic anhydride (896 mg, 8.95 mmol) were dissolved in N,N-dimethylformamide (35.0 mL) and stirred at 30°C under nitrogen for 48 h. Preparative chromatography (HO (50 mM TEAB)-ACN) afforded G5-10 (2.10 g, 937 μmol) as a white solid in a 62.8% yield. 1 HNMR (400 MHz, CDCl3) δ ppm 7.48-7.41(m, 2H), 7.33(d, J=8.6Hz, 3H), 7.28-7.21(m, 5H), 7.17(t, J=7 .2Hz, 1H), 6.99 (t, J=6.3Hz, 2H), 6.87-6.77 (m, 5H), 6.49 (d, J=6.0Hz, 1H ), 5.34 (d, J = 3.3Hz, 3H), 5.21-5.13 (m, 3H), 4.98 (d, J = 2.3Hz, 1H), 4.61 ( d, J=8.5Hz, 2H), 4.23 (q, J=4.9Hz, 1H), 4.19-4.01 (m, 8H), 3.91 (dt, J=13 .1, 5.6Hz, 6H), 3.77 (s, 5H), 3.68 (d, J=7.6Hz, 11H), 3.55-3.44 (m, 3H), 3 .39-3.19 (m, 14H), 3.11 (dd, J=9.9, 5.0Hz, 1H), 2.80-2.59 (m, 13H), 2.49 (t, J=7.4Hz, 2H), 2.42 (t, J=5.7Hz, 5H), 2.30-2.06 (m, 16H), 2.06-1.91 ( m, 22H), 1.81-1.44 (m, 19H), 1.31-1.19 (m, 10H), 1.11 (t, J=7.2Hz, 16H). MS (ESI) m / z [M-2H]2- =1262.5.

[0231] Step 10: Synthesis of YK-GAL-305

[0232] G5-10 (1.00 g, 380 μmol), 4-dimethylaminopyridine (46.5 mg, 381 μmol), diisopropylethylamine (393 mg, 3.04 mmol), and O-benzotriazole-tetramethyluronium hexafluorophosphate (722 mg, 1.90 mmol) were dissolved in N,N-dimethylformamide (65.0 mL). CPG-NH2 (6.60 g) was then added and stirred at 40°C for 16 h. The reaction mixture was filtered, and the filtrate was washed sequentially with methanol and dichloromethane and dried under vacuum. The filtrate was then added to 65 mL of acetic anhydride / pyridine (1:5) solution and stirred at 40°C for 0.5 h. The mixture was filtered, washed sequentially with dichloromethane and methanol, and dried under vacuum for 12 h to obtain compound YK-GAL-305 (7.05 g, loading 34.4 μmol / g) as a white solid.

[0233] 6. Synthesis of YK-GAL-306

[0234] The synthetic route is as follows:

[0235] Step 1: Synthesis of G6-1

[0236] To dried compound G5-1 (1.58 g, 4.95 mmol), dried compound G2-1 (1.00 g, 4.50 mmol), and 3A molecular sieves (1.50 g, 4.50 mmol) was added dichloromethane (15.0 mL). The temperature was cooled to 0°C, and boron trifluoride etherate (1.28 g, 9.00 mmol) was added, and stirring was continued for 2 h. The temperature was then raised to 15°C and stirring was continued for 2 h. Triethylamine (1.32 mL) was added to quench the reaction, and the mixture was washed with saturated aqueous sodium bicarbonate (30.0 mL x 2). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to remove the solvent to obtain the crude product. Purification by column chromatography (PE / EA) afforded G6-1 (1.60 g, 3.33 mmol) as a yellow oil in a yield of 74.0%. 1 HNMR (400MHz, CDCl3) δppm 5.34 (dd, J=6.7, 4.8Hz, 1H), 5.26 (d, J=4.8Hz, 1H), 4.37-4.25 (m, 2H), 4.12 (dd, J=11.3, 5 .9Hz, 2H), 3.89-3.77(m, 1H), 3.75-3.56(m, 16H), 2.10(s, 3H), 2.08(s, 3H), 2.04(s, 3H). MS(ESI)m / z[M+Na] +=503.0.

[0237] Step 2: Synthesis of G6-2

[0238] Dissolve G6-1 (1.60 g, 3.33 mmol) in methanol (16.0 mL), add sodium methoxide (60.0 mg, 333 μmol), and stir at 15°C for 2 h. Quench the reaction by adding hydrogen ion exchange resin, filter, and concentrate the filtrate under reduced pressure to remove the solvent to obtain a white solid. The crude product G6-2 (1.18 g) was used directly in the next step. 1 H NMR (400MHz, MeOH-d4) δ4.89 (s, 1H), 4.13 (dd, J=7.1, 4.7Hz, 1H), 3.99-3.89 (m, 2H), 3.88-3.80 (m, 1H), 3.78-3.53 (m, 18H). MS(ESI)m / z[M+Na] + =376.9.

[0239] Step 3: Synthesis of G6-3

[0240] G6-2 (1.18 g, 3.33 mmol) was dissolved in N,N-dimethylformamide (12.0 mL), and 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (1.16 g, 3.66 mmol) and imidazole (567 mg, 8.33 mmol) were added. The mixture was stirred at 15°C for 2 h under nitrogen. Saturated aqueous sodium bicarbonate (10.0 mL) was added to quench the mixture, and the mixture was extracted with dichloromethane (10 mL x 2). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to remove the solvent to obtain the crude product. Purification by column chromatography (PE / EA) afforded G6-3 (1.38 g, 2.27 mmol) as a colorless oil in a 68.0% yield. 1 HNMR (400MHz, MeOH-d4) δppm 4.52 (t, J = 5.4Hz, 1H), 4.09-3.97 (m, 3H), 3.80-3.54 (m, 20H), 1.09-1.02 (m, 28H).

[0241] Step 4: Synthesis of G6-4

[0242] G6-3 (1.25 g, 2.09 mmol) was dissolved in dichloromethane (17.0 mL), and 1,8-bis(dimethylamino)naphthalene (1.48 g, 6.91 mmol) and trimethyloxonium tetrafluoroborate (929 mg, 6.28 mmol) were added. The mixture was stirred at 15°C for 6 h under nitrogen. Aqueous ammonium chloride (30.0 mL) was added to quench the mixture, and the mixture was extracted with dichloromethane (15 mL x 2). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to remove the solvent to obtain the crude product. Purification by column chromatography (PE / EA) afforded G6-4 (1.11 g, 1.82 mmol) as a colorless oil in an 86% yield. 1 HNMR (400MHz, MeOH-d4) δppm 4.43 (dd, J=8.1, 4.3Hz, 1H), 3.95-3.44 (m, 26H), 1.03-0.95 (m, 28H).

[0243] Step 5: Synthesis of G6-5

[0244] G6-4 (1.11 g, 1.82 mmol) was dissolved in tetrahydrofuran (20.0 mL), and triethylamine trihydrofluoride (1.46 g, 9.09 mmol) was added. The mixture was stirred at 20°C for 2 h. Saturated aqueous sodium bicarbonate (10.0 mL) was added to quench the mixture, and the mixture was extracted with dichloromethane (25 mL x 2). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to remove the solvent, yielding a colorless oil. The crude product, G6-5 (0.67 g), was used directly in the next step.

[0245] Step 6: Synthesis of G6-6

[0246] Dried G6-5 (0.67 g, 1.82 mmol) and 4,4′-dimethoxytriphenylmethane (616 mg, 1.82 mmol) were dissolved in pyridine (10.0 mL) and stirred at 25°C for 2 h under nitrogen. Methanol (1.00 mL) was added to quench the reaction, and the solvent was removed by concentration under reduced pressure to obtain the crude product. Preparative chromatography (H2O(10 mM NH4HCO3)-ACN) afforded G6-6 (1.05 g, 1.57 mmol) as a white solid in an 86.0% yield. 1HNMR (400MHz, CD3CN) δppm 7.46-7.36(m, 2H), 7.31-7.25(m, 4H), 7.22-7.16(m, 2H), 7.15-7.06(m, 1H), 6.79-6.71(m, 4H), 4.15(s, 1H), 4.08 (s, 2H), 3.97 (td, J=6.1, 3.8Hz, 1H), 3.77-3.42 (m, 27H), 3.21 (dd, J=9.9, 3.7Hz, 1H), 3.08 (dd, J=9.9, 5.8Hz, 1H). MS(ESI)m / z[MH] - =669.4.

[0247] Step 7: Synthesis of G6-7

[0248] Dissolve G6-6 (195 mg, 291 μmol) in tetrahydrofuran (1.0 mL). Add 1.0 mL of an aqueous solution of lithium hydroxide monohydrate (60.0 mg, 333 μmol). Stir at 25°C for 12 h. The reaction mixture is concentrated under reduced pressure to remove the solvent, yielding a white solid. The crude product, G6-7 (190 mg), is used directly in the next step. 1 HNMR (400MHz, MeOH-d4) δppm 7.52-7.45 (m, 2H), 7.39-7.31 (m, 4H), 7.31-7.24 (m, 2H), 7.24-7.15 (m, 1H), 6.92-6.80 (m, 4H), 4.22 (dd, J=7.2, 4.7Hz, 1H), 4.0 7-4.03 (m, 1H), 3.86-3.68 (m, 10H), 3.65-3.51 (m, 12H), 3.49 (s, 3H), 3.27 (dd, J=10.0, 3.1Hz, 1H), 3.12 (dd, J=10.0, 5.8Hz, 1H). MS(ESI)m / z[MH] - =655.4.

[0249] Step 8: Synthesis of G6-8

[0250] G6-7 (190 mg, 293 μmol), diisopropylethylamine (112 mg, 868 μmol), and O-benzotriazole-tetramethyluronium hexafluorophosphate (165 mg, 433 μmol) were dissolved in N,N-dimethylformamide (5.0 mL). GalNAc-NH2.TFA (442 mg, 232 μmol, 0.8 eq, TFA) was added and stirred at 15°C for 2 h under nitrogen. The reaction mixture was used directly in the next step. MS (ESI) m / z [M-2H] 2- =1215.0.

[0251] Step 9: Synthesis of G6-9

[0252] G6-8 (703 mg, 289 μmol), 4-dimethylaminopyridine (70.6 mg, 578 μmol), diisopropylethylamine (261 mg, 2.02 mmol), and succinic anhydride (173 mg, 1.73 mmol) were dissolved in N,N-dimethylformamide (1.0 mL) and stirred at 30°C under nitrogen for 48 h. Preparative chromatography (H2O(50 mM TEAB)-ACN) afforded G6-9 (300 mg, 118 mol) as a white solid in a 50.0% yield. MS (ESI) m / z [M-2H] 2- =1265.3.

[0253] Step 10: Synthesis of YK-GAL-306

[0254] G6-9 (300 mg, 110 μmol), 4-dimethylaminopyridine (13.4 mg, 109 μmol), diisopropylethylamine (113 mg, 877 μmol), and O-benzotriazole-tetramethyluronium hexafluorophosphate (208 mg, 548 μmol) were dissolved in N,N-dimethylformamide (16.0 mL). CPG-NH2 (1.9 g) was then added and stirred at 40°C for 12 h. The reaction mixture was filtered, and the filtrate was washed sequentially with methanol and dichloromethane and dried under vacuum. The filtrate was then added to 20 mL of acetic anhydride / pyridine (1:5) solution and stirred at 40°C for 0.5 h. The mixture was filtered, washed sequentially with dichloromethane and methanol, and dried under vacuum for 12 h to obtain compound YK-GAL-306 (1.94 g, loading 37.43 μmol / g) as a white solid.

[0255] 7. Synthesis of YK-GAL-307

[0256] The synthetic route is as follows:

[0257] Step 1: Synthesis of G7-1

[0258] To dried compound G5-1 (5.00 g, 15.7 mmol), dried compound G4-1 (3.50 g, 15.1 mmol), and 4A molecular sieves (1.0 g) was added dichloromethane (70.0 mL). After cooling to 0°C, boron trifluoride etherate (4.30 g, 30.2 mmol) was added and stirring continued for 2 h. Triethylamine (3.06 g, 30.2 mmol) was added to quench the reaction. The mixture was washed with saturated aqueous sodium bicarbonate (20 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to remove the solvent to obtain the crude product. Purification by column chromatography (PE / EA) afforded G7-1 (3.70 g, 7.56 mmol) as a colorless oil in a 50.1% yield. 1 HNMR (400MHz, CDCl3) δppm 5.74 (d, J=31.1Hz, 1H), 5.33-5.14 (m, 1H), 4.98 (d, J=13.6Hz, 1H), 4.30-4.26(m, 1H), 4.19-4.03(m, 2H), 3.87-3.61(m, 4H), 3.43-3.37(m1H), 3 .33-3.18(m, 2H), 2.35-2.31(m, 2H), 2.23-1.98(m, 11H), 1.71-1.50(m, 9H). MS(ESI)m / z[M+Na] + =512.1.

[0259] Step 2: Synthesis of G7-2

[0260] G7-1 (3.10 g, 6.33 mmol) was dissolved in methanol (30.0 mL), and sodium methoxide (114 mg, 633 μmol) was added. The mixture was stirred at 25°C for 2 h. The reaction was quenched by adding hydrogen ion exchange resin, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent to obtain a yellow oil. The crude product G7-2 (2.20 g) was used directly in the next step. MS (ESI) m / z [MH] - =362.2.

[0261] Step 3: Synthesis of G7-3

[0262] G7-2 (2.20 g, 6.05 mmol) was dissolved in N,N-dimethylformamide (20.0 mL), and 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (2.10 g, 6.66 mmol) and imidazole (1.03 g, 15.1 mmol) were added. The mixture was stirred at 25°C for 2 h under nitrogen. Saturated aqueous sodium bicarbonate (10.0 mL) was added to quench the mixture, and the mixture was extracted with dichloromethane (10 mL x 2). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to remove the solvent to obtain the crude product. Purification by column chromatography (PE / EA) afforded G7-3 (2.15 g, 3.56 mmol) as a yellow oil in a 58.8% yield. 1 HNMR (400MHz, CDCl3) δppm 5.59 (s, 1H), 4.90 (s, 1H), 4.49 (t, J=5.4Hz, 1H), 4.08-3.91 (m, 3H), 3.80-3.60 (m, 6H), 3.40-3.35 (m, 1H), 3.26 (q, J= 6.4Hz, 2H), 2.35-2.32(m, 2H), 2.22-2.15(m, 2H), 1.68-1.64(p, J=3.7Hz, 4H), 1.59-1.54(m, 4H), 1.10-1.02(m, 28H). MS(ESI)m / z[M+H] + =606.4.

[0263] Step 4: Synthesis of G7-4

[0264] G7-3 (1.0 g, 1.65 mmol) was dissolved in dichloromethane (15.0 mL), and 1,8-bis(dimethylamino)naphthalene (955 mg, 4.46 mmol) and trimethyloxonium tetrafluoroborate (610 mg, 4.13 mmol) were added. The mixture was stirred at 15°C under nitrogen for 6 h. The mixture was quenched by the addition of aqueous ammonium chloride (10.0 mL) and extracted with dichloromethane (10 mL x 2). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to remove the solvent to obtain the crude product. Purification by column chromatography (PE / EA) afforded G7-4 (890.2 mg, 1.43 mmol) as a colorless oil in an 87.1% yield. 1HNMR (400MHz, CDCl3) δppm 5.59 (d, J=6.1Hz, 1H), 4.53-4.42 (m, 1H), 4.08-3.85 (m, 3H), 3.80-3.60 (m, 5H), 3.58 (d, J=7.2Hz, 2H), 3.47-3.31 (m, 3H), 3. 27-3.20 (m, 2H), 2.38-2.29 (m, 2H), 2.18 (q, J=6.2, 5.0Hz, 2H), 1.66 (h, J=3.8Hz, 4H), 1.60-1.51 (m, 4H), 1.17-0.89 (m, 28H). MS(ESI)m / z[M+H] + =620.5.

[0265] Step 5: Synthesis of G7-5

[0266] G7-4 (300 mg, 484 μmol) was dissolved in tetrahydrofuran (10.0 mL), and triethylamine trihydrofluoride (390 mg, 2.42 mmol) was added. The mixture was stirred at 25°C for 3 h. Saturated aqueous sodium bicarbonate solution (10.0 mL) was added to quench the mixture, and the mixture was extracted with dichloromethane (20 mL × 3). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to remove the solvent to obtain a yellow oil. The crude product G7-6 (182 mg) was used directly in the next step. MS (ESI) m / z [M+H] + =378.2.

[0267] Step 6: Synthesis of G7-6

[0268] Dried G7-5 (180 mg, 477 μmol) and 4,4′-dimethoxytriphenylmethane (161 mg, 477 μmol) were dissolved in pyridine (2.0 mL) and stirred at 25°C for 3 h under nitrogen. Methanol (2.00 mL) was added to quench the reaction, and the solvent was removed by concentration under reduced pressure to obtain the crude product. Preparative chromatography (H2O(10 mM NH4HCO3)-ACN) afforded G7-6 (262 mg, 385.7 μmol) as a white solid in an 80.85% yield. 1HNMR (400MHz, CDCl3) δppm 7.52-7.45(m, 2H), 7.40-7.32(m, 4H), 7.30-7.26(m, 2H), 7.19(dd, J=14.1, 8.0Hz, 1H), 6.87-6.78(m , 4H), 5.38 (s, 1H), 5.01 (d, J=1.5Hz, 1H), 4.26-4.17 (m, 1H), 4.07-4.03 (m, 1H), 3.82-3.71 (m, 7H), 3 .66 (d, J=6.2Hz, 4H), 3.51 (d, J=2.2Hz, 3H), 3.41 (dd, J=9.7, 5.9Hz, 1H), 3.30 (dd, J=10.0, 3.7Hz, 1H ), 3.22-3.07 (m, 3H), 2.48 (d, J=8.3Hz, 1H), 2.34-2.29 (m, 2H), 2.07-1.98 (m, 2H), 1.60-1.47 (m, 8H). MS(ESI)m / z[MH] - =678.5.

[0269] Step 7: Synthesis of G7-7

[0270] G7-6 (100 mg, 147 μmol) was dissolved in tetrahydrofuran (2.5 mL), and 2.5 mL of an aqueous solution of lithium hydroxide monohydrate (9.26 mg, 220 μmol) was added. The mixture was stirred at 15°C for 16 h. The reaction mixture was concentrated under reduced pressure to remove the solvent, yielding a yellow solid. The crude product G7-7 (0.95 g) was used directly in the next step. MS (ESI) m / z [MH] - =664.5.

[0271] Step 8: Synthesis of G7-8

[0272] G7-7 (90.0 mg, 135 μmol), diisopropylethylamine (34.9 mg, 270 μmol), and O-benzotriazole-tetramethyluronium hexafluorophosphate (76.9 mg, 202 μmol) were dissolved in N,N-dimethylformamide (2.0 mL). GalNAc-NH2.TFA (206 mg, 108 μmol, TFA) was added and stirred at 15°C for 4 h under nitrogen. The reaction solution was used directly in the next step. MS (ESI) m / z [M-2H] 2- =1219.9.

[0273] Step 9: Synthesis of G7-9

[0274] G7-8 (280 mg, 114 μmol), 4-dimethylaminopyridine (28.0 mg, 229 μmol), diisopropylethylamine (44.4 mg, 344 μmol), and succinic anhydride (91.8 mg, 917 μmol) were dissolved in N,N-dimethylformamide (2.0 mL) and stirred at 30°C under nitrogen for 48 h. Preparative chromatography (HO (50 mM TEAB)-ACN) gave G7-9 (172 mg, 68.1 μmol) as a yellow solid in a 59.7% yield. 1 HNMR (400MHz, CDCl3) δppm 7.54-7.45 (m, 4H), 7.38-7.24 (m, 7H), 7.23-7.20 (m, 1H), 7.15-7.07 (m, 5H), 6.83 (dd, J=8.8, 3.8Hz, 4H), 5.37 (d, J=3.8Hz, 2H) , 5.24-5.16 (m, 3H), 5.01 (t, J=3.1Hz, 1H), 4.65 (dd, J=8.4, 3.9Hz, 2H), 4.26 (t, J=4.7Hz, 1H), 4.21-4.07 (m, 8H), 3.99-3.91 (m, 6H), 3.81-3.75(m, 8H), 3.70-3.65(m, 11H), 3.55-3.50(m, 2H), 3.40(d, J=3.9Hz, 3H), 3.29-3.25(m, 12H), 2.81-2.64(m, 16H), 2 .52(dd, J=7.8, 4.0Hz, 2H), 2.46-2.42(m, 5H), 2.26-2.16(m, 16H), 2.09-1.94(m, 26H), 1.75-1.54(m, 14H), 1.16-1.11(m, 18H). MS(ESI)m / z[M-2H] 2- =1269.9.

[0275] Step 10: Synthesis of YK-GAL-307

[0276] G7-9 (120 mg, 47.2 μmol), 4-dimethylaminopyridine (5.77 mg, 47.2 μmol), diisopropylethylamine (48.8 mg, 377 μmol), and O-benzotriazole-tetramethyluronium hexafluorophosphate (89.5 mg, 236 μmol) were dissolved in N,N-dimethylformamide (10.0 mL). CPG-NH2 (790 mg) was then added and stirred at 40°C for 16 h. The reaction mixture was filtered, and the filtrate was washed sequentially with methanol and dichloromethane and dried under vacuum. The filtrate was then added to 12 mL of acetic anhydride / pyridine (1:5) solution and stirred at 40°C for 0.5 h. The mixture was filtered, washed sequentially with dichloromethane and methanol, and dried under vacuum for 12 h to obtain compound YK-GAL-307 (810 mg, loading 38.9 μmol / g) as a white solid.

[0277] 8. Synthesis of YK-GAL-308

[0278] The synthetic route is as follows:

[0279] Step 1: Synthesis of G8-2

[0280] G8-1 (0.34 g, 816 μmol) was dissolved in tetrahydrofuran (8.0 mL), and 4.0 mL of an aqueous solution of lithium hydroxide monohydrate (109 mg, 2.61 mmol) was added. The mixture was stirred at 15°C for 12 h. The reaction mixture was concentrated under reduced pressure to remove the solvent, yielding a yellow solid. The crude product, G8-2 (0.26 g), was used directly in the next step. MS (ESI) m / z [M+Na] + =341.2.

[0281] Step 2: Synthesis of G8-3

[0282] The crude product G8-2 (0.26 g, 816 μmol) and anhydrous pyridine (20 mL x 3) were co-rotatingly evaporated to remove water. The dried G8-2 and 4,4′-dimethoxytriphenylmethane (580 mg, 1714 μmol) were dissolved in pyridine (6.0 mL) and stirred at 15°C for 36 h under nitrogen. Methanol (20 mL) was added to quench the reaction, and the solvent was removed by concentration under reduced pressure to obtain the crude product. Preparative chromatography (H2O(10 mM NH4HCO3)-ACN) gave G8-3 (401.9 mg, 647.9 μmol) as a white solid in a yield of 79.4%. MS (ESI) m / z [M+Na] + =643.4.

[0283] Step 3: Synthesis of G8-4

[0284] G8-3 (90 mg, 145 μmol), diisopropylethylamine (37.5 mg, 290 μmol), and O-benzotriazole-tetramethyluronium hexafluorophosphate (82.5 mg, 217 μmol) were dissolved in N,N-dimethylformamide (3.0 mL). GalNAc-NH2.TFA (331.3 mg, 174 μmol, 1.2 eq, TFA) was added and stirred at 15°C for 2 h under nitrogen. The reaction mixture was used directly in the next step. MS (ESI) m / z [M-2H] 2- =1197.3.

[0285] Step 4: Synthesis of G8-5

[0286] G8-4,4-Dimethylaminopyridine (35.4 mg, 289 μmol), diisopropylethylamine (149.8 mg, 1158 μmol), and succinic anhydride (159.4 mg, 1592 μmol) were dissolved in N,N-dimethylformamide (3.0 mL) and stirred at 30°C under nitrogen for 48 h. Preparative chromatography (HO(10 mM TEAB)-ACN) afforded G8-5 (190 mg, 85.9 μmol) as a white solid in a 59.4% yield. 1 HNMR (400MHz, CDCl3) δppm 7.44-7.37 (m, 2H), 7.34-7.26 (m, 8H), 7.20 (d, J=7.1Hz, 2H), 6.99 (d, J=5.7Hz, 4H), 6.81 (d, J=8.7Hz, 4H), 6.72 (d, J=8.9Hz, 2H) , 6.57 (s, 1H), 5.35 (d, J = 3.3Hz, 2H), 5.26 (d, J = 5.1Hz, 1H), 5.23-5.12 (m, 4H), 4.61 (d, J = 8.4Hz, 3H), 4.23-4.04 (m, 10H), 3.95-3 .89 (m, 6H), 3.78 (s, 6H), 3.73-3.62 (m, 14H), 3.53-3.47 (m, 4H), 3.43-3.35 (m, 2H), 3.30-3.24 (m, 12H), 3.17 (dd, J=9.9, 4.0Hz, 2H), 2.62-2.57(m, 4H), 2.43(d, J=5.7Hz, 6H), 2.28-2.15(m, 13H), 2.10-1.92(m, 30H), 1.74-1.51(m, 22H), 1.238-1.26(m, 14H). MS(ESI)m / z[M-2H] 2- =1247.4.

[0287] Step 5: Synthesis of YK-GAL-308

[0288] G8-5 (190 mg, 76.1 μmol), 4-dimethylaminopyridine (9.30 mg, 76.1 μmol), diisopropylethylamine (78.7 mg, 609 μmol), and O-benzotriazole-tetramethyluronium hexafluorophosphate (144 mg, 381 μmol) were dissolved in N,N-dimethylformamide (12 mL). CPG-NH2 (1.27 g) was then added and stirred at 40°C for 16 h. The reaction mixture was filtered, and the filtrate was washed sequentially with methanol and dichloromethane and dried under vacuum. The filtrate was then added to 12 mL of acetic anhydride / pyridine (1:5) solution and stirred at 40°C for 0.5 h. The mixture was filtered, washed sequentially with dichloromethane and methanol, and dried under vacuum for 12 h to obtain compound YK-GAL-308 (1.24 g, loading 29.7 μmol / g) as a white solid.

[0289] 9. Synthesis of YK-GAL-309

[0290] The synthetic route is as follows:

[0291] Step 1: Synthesis of G9-2

[0292] To dried compound G1-1 (734 mg, 2.82 mmol), dried compound G9-1 (0.50 g, 2.17 mmol), and 3A molecular sieves (2.0 g) was added dichloromethane (20 mL). After cooling to 0°C, boron trifluoride etherate (616 mg, 4.34 mmol) was added and stirring continued for 2 h. The reaction was quenched by adding saturated aqueous sodium bicarbonate (20 mL) and extracted with dichloromethane (10 mL x 2). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to remove the solvent to obtain the crude product. Column chromatography (PE / EA) gave G9-2 (589 mg, 1.37 mmol) as a yellow oil in a yield of 48.5%. MS (ESI) m / z [M+Na] + =453.1.

[0293] Step 2: Synthesis of G9-3

[0294] G9-2 (0.30 g, 696 μmol) was dissolved in tetrahydrofuran (4.0 mL), and 2.0 mL of an aqueous solution of lithium hydroxide monohydrate (93.6 mg, 2.23 mmol) was added. The mixture was stirred at 15°C for 12 h. The reaction mixture was concentrated under reduced pressure to remove the solvent, yielding a yellow solid. The crude product, G9-3 (0.23 g), was used directly in the next step. MS (ESI) m / z [M+Na] + =355.0.

[0295] Step 3: Synthesis of G9-4

[0296] The crude product G9-3 (0.23 g, 692 μmol) and anhydrous pyridine (20 mL x 3) were co-rotated to remove water. The dried G9-3 and 4,4′-dimethoxytriphenylmethane (534.58 mg, 1579.8 μmol) were dissolved in pyridine (5.0 mL) and stirred at 15°C for 28 h under nitrogen. Methanol (10 mL) was added to quench the reaction, and the solvent was removed by concentration under reduced pressure to obtain the crude product. Preparative chromatography (H2O(10 mM NH4HCO3)-ACN) gave G9-4 (337.3 mg, 531.7 μmol) as a white solid in a yield of 76.84%. MS (ESI) m / z [MH] - =633.5.

[0297] Step 4: Synthesis of G9-5

[0298] G9-4 (30 mg, 47.3 μmol), diisopropylethylamine (12.2 mg, 94.5 μmol), and O-benzotriazole-tetramethyluronium hexafluorophosphate (26.9 mg, 70.9 μmol) were dissolved in N,N-dimethylformamide (0.5 mL). GalNAc-NH2.TFA (90.2 mg, 47.3 μmol, TFA) was added and stirred at 15°C under nitrogen for 1 h. The reaction solution was used directly in the next step. MS (ESI) m / z [M-2H] 2- =1204.3.

[0299] Step 5: Synthesis of G9-6

[0300] G9-5 (114 mg, 47.3 μmol), 4-dimethylaminopyridine (11.55 mg, 94.58 μmol), diisopropylethylamine (55.1 mg, 425.45 μmol), and succinic anhydride (52.1 mg, 519 μmol) were dissolved in N,N-dimethylformamide (2.0 mL) and stirred at 30°C under nitrogen for 36 h. Preparative chromatography (HO(10 mM TEAB)-ACN) gave G9-6 (56.7 mg, 22.6 μmol) as a white solid in a 47.8% yield. 1HNMR (400MHz, CDCl3) δppm 7.44-7.38(m, 2H), 7.33-7.26(m, 6H), 7.24-7.17(m, 1H), 6.86-6.79(m, 4H), 5.24(d, J=4. 3Hz, 1H), 4.27-4.21 (m, 1H), 4.19 (d, J=5.6Hz, 1H), 3.79 (s, 6H), 3.74 (dd, J=9.6, 6.8Hz, 1 H), 3.40 (dt, J=9.6, 6.5Hz, 2H), 3.27 (s, 1H), 3.16-3.07 (m, 3H), 2.33 (t, J=7.4Hz, 2H), 2. 21-2.15 (m, 1H), 2.02 (d, J=13.3Hz, 1H), 1.67-1.52 (m, 4H), 1.23-1.30 (d, J=13.9Hz, 13H). MS(ESI)m / z[M-2H] 2- =1254.4.

[0301] Step 6: Synthesis of YK-GAL-309

[0302] G9-6 (32 mg, 12.7 μmol), 4-dimethylaminopyridine (1.56 mg, 12.7 μmol), diisopropylethylamine (13.2 mg, 102 μmol), and O-benzotriazole-tetramethyluronium hexafluorophosphate (24.2 mg, 63.7 μmol) were dissolved in N,N-dimethylformamide (4.0 mL). CPG-NH2 (221 mg) was then added and stirred at 40°C for 12 h. The reaction mixture was filtered, and the filtrate was washed sequentially with methanol and dichloromethane and dried under vacuum. The filtrate was then added to 4 mL of acetic anhydride / pyridine (1:5) solution and stirred at 40°C for 0.5 h. The mixture was filtered, washed sequentially with dichloromethane and methanol, and dried under vacuum for 12 h to obtain compound YK-GAL-309 (103 mg, loading 24.5 μmol / g) as a white solid.

[0303] 10. Synthesis of YK-GAL-310

[0304] The synthetic route is as follows:

[0305] Step 1: Synthesis of G10-2

[0306] To dried compound G1-1 (692 mg, 2.66 mmol), dried compound G10-1 (500 mg, 2.05 mmol), and 3A molecular sieves (2.0 g) was added dichloromethane (20 mL). The temperature was cooled to 0°C, and boron trifluoride etherate (581 mg, 4.09 mmol) was added. The temperature was then raised to 25°C and stirred for 2 h. The reaction was quenched by adding saturated aqueous sodium bicarbonate (20 mL), and the mixture was extracted with dichloromethane (10 mL x 2). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to remove the solvent to obtain the crude product. Purification by column chromatography (PE / EA) afforded G10-2 (576.6 mg, 1.30 mmol) as a colorless oil in a yield of 48.8%. MS (ESI) m / z [M+Na] + =467.1.

[0307] Step 2: Synthesis of G10-3

[0308] G10-2 (69 mg, 155 μmol) was dissolved in tetrahydrofuran (2.0 mL), and 1.0 mL of an aqueous solution of lithium hydroxide monohydrate (20.8 mg, 497 μmol) was added. The mixture was stirred at room temperature for 16 h. The reaction mixture was concentrated under reduced pressure to remove the solvent, yielding a yellow oil. The crude product, G10-3 (53.8 mg), was used directly in the next step. MS (ESI) m / z [M+Na] + =369.0.

[0309] Step 3: Synthesis of G10-4

[0310] The crude product G10-3 (53.8 mg, 155 μmol) and anhydrous pyridine (20 mL x 3) were co-rotated to remove water. The dried G10-3 and 4,4′-dimethoxytriphenylmethane (189 mg, 559 μmol) were dissolved in pyridine (3.0 mL) and stirred at room temperature under nitrogen for 16 h. Methanol (10 mL) was added to quench the reaction, and the solvent was removed by concentration under reduced pressure to obtain the crude product. Column chromatography (DCM / MeOH) was used to obtain G10-4 (79.1 mg, 122 μmol) as a pale yellow solid in a yield of 78.7%. MS (ESI) m / z [MH] - =647.5.

[0311] Step 4: Synthesis of G10-5

[0312] G10-4 (39 mg, 60.1 μmol), diisopropylethylamine (15.5 mg, 120 μmol), and O-benzotriazole-tetramethyluronium hexafluorophosphate (34.2 mg, 90.2 μmol) were dissolved in N,N-dimethylformamide (1.5 mL). GalNAc-NH2.TFA (115 mg, 60.1 μmol) was added and stirred at room temperature under nitrogen for 1 h. The reaction mixture was used directly in the next step. MS (ESI) m / z [M-2H] 2- =1211.3.

[0313] Step 5: Synthesis of G10-6

[0314] G10-5 (146 mg, 60.1 μmol), 4-dimethylaminopyridine (7.34 mg, 60.1 μmol), diisopropylethylamine (31.1 mg, 240 μmol), and succinic anhydride (30.1 mg, 301 μmol) were dissolved in N,N-dimethylformamide (1.5 mL) and stirred at 30°C under nitrogen for 16 h. Preparative chromatography (HO(10 mM TEAB)-ACN) afforded G10-6 (74.2 mg, 49.4 μmol) as a white solid in a 48.9% yield. 1 HNMR (400MHz, CDCl3) δppm 7.49-7.42 (m, 2H), 7.36-7.31 (m, 4H), 7.28-7.23 (m, 8H), 7.21-7.16 (m, 1H), 6.99 (t, J=6.0Hz, 3H), 6.79 (dd, J=11.8, 8.8Hz, 6H), 6.47 (d, J=9.6Hz, 1H), 5.35 (d, J=3.3Hz, 2H), 5.23-5.17 (dt, J=11.2, 3.8Hz, 4H), 4.61 (d, J=8.5Hz, 2H), 4.19-4.04 (m, 9H), 3.99-3.86 (m, 6H), 3.7 8 (d, J=2.1Hz, 6H), 3.69-3.60 (m, 11H), 3.53-3.48 (m, 3H), 3.35-3.14 (m, 15H), 2.60-2.50 (m, 4H), 2.43 (t, J=5.7Hz, 7H), 2.29-2.13 (m, 24 H), 2.10 (d, J=5.0Hz, 3H), 2.04 (s, 9H), 1.99 (s, 9H), 1.94 (d, J=2.5Hz, 8H), 1.75-1.55 (m, 18H), 1.42 (s, 2H), 1.26-1.21 (d, J=3.6Hz, 15H). MS(ESI)m / z[M-2H] 2- =1261.1.

[0315] Step 6: Synthesis of YK-GAL-310

[0316] G10-6 (59 mg, 22.1 μmol), 4-dimethylaminopyridine (2.70 mg, 22.13 μmol), diisopropylethylamine (22.88 mg, 177.01 μmol), and O-benzotriazole-tetramethyluronium hexafluorophosphate (41.96 mg, 110.63 μmol) were dissolved in N,N-dimethylformamide (4.0 mL). CPG-NH2 (383 mg) was then added and stirred at 40°C for 16 h. The reaction mixture was filtered, and the filtrate was washed sequentially with methanol and dichloromethane and dried under vacuum. The filtrate was then added to 4 mL of acetic anhydride / pyridine (1:4) solution and stirred at 40°C for 0.5 h. The mixture was filtered, washed sequentially with dichloromethane and methanol, and dried under vacuum for 12 h to obtain compound YK-GAL-310 (362 mg, loading 30.2 μmol / g) as a white solid.

[0317] 11. Synthesis of YK-GAL-311

[0318] The synthetic route is as follows:

[0319] Step 1: Synthesis of G11-1

[0320] G11A (2.00 g, 13.7 mmol, 1.72 mL), diisopropylethylamine (3.54 g, 27.3 mmol), and O-benzotriazole-tetramethyluronium hexafluorophosphate (5.71 g, 15.0 mmol) were dissolved in N,N-dimethylformamide (20.0 mL), and G11A-1 (1.92 g, 16.4 mmol) was added. The mixture was stirred at 15°C for 2 h. Preparative chromatography (HO (0.1% TFA)-ACN) gave G11-1 (2.00 g, 8.15 mmol) as a white solid in a yield of 59.5%. 1 HNMR (400MHz, DMSO-d6) δppm 7.76-7.73 (m, 1H), 4.38-4.30 (m, 1H), 3.57 (s, 3H), 3.36 (t, J=6.4Hz, 2H), 3.02-2.97 (m, 2H), 2.28 (t, J=7.6Hz, 2H), 2.69 (t, J=7.2Hz, 2H), 1.76-1.68 (m, 2H), 1.42-1.18 (m, 8H). MS(ESI)m / z[M+H] + =245.9.

[0321] Step 2: Synthesis of G11-2

[0322] To compound G1-1 (500 mg, 1.92 mmol), compound G11-1 (565 mg, 2.31 mmol), and 4A molecular sieves (1.0 g) was added dichloromethane (10.0 mL). After cooling to 0°C, trimethylsilyl trifluoromethanesulfonate (854 mg, 3.84 mmol) was added and stirring continued for 2 h. The reaction was quenched by adding saturated aqueous sodium bicarbonate (30.0 mL) and extracted with dichloromethane (15.0 mL x 3). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to remove the solvent to obtain the crude product. Purification by column chromatography (PE / EA) afforded G11-2 (400 mg, 899 μmol) as a colorless oil in a 46.8% yield. 1 HNMR (400MHz, CDCl3) δppm 5.61-5.54 (m, 1H), 5.24-5.20 (m, 1H), 4.32-4.00 (m, 3H), 3.67 (d, J=1.0Hz, 4H), 3.34 (dt, J=9.4, 6.6Hz, 1H), 3. 27-3.19 (m, 2H), 2.42-2.30 (m, 3H), 2.27-2.02 (m, 9H), 2.02-1.90 (m, 3H), 1.57-1.45 (m, 4H), 1.37-1.31 (m, 4H). MS(ESI)m / z[M+H] + =446.0.

[0323] Step 3: Synthesis of G11-3

[0324] G11-2 (180 mg, 404 μmol, 1.0 eq) was dissolved in tetrahydrofuran (1.8 mL). A 0.9 mL aqueous solution of lithium hydroxide monohydrate (67.8 mg, 1.62 mmol, 4.0 eq) was added, and the mixture was stirred at 35°C for 16 h. The reaction mixture was concentrated under reduced pressure to remove the solvent, yielding a brown oil. The crude product, G11-3 (140.36 mg), was used directly in the next step.

[0325] Step 4: Synthesis of G11-4

[0326] Dried G11-3 (40.0 mg, 115 μmol) and 4,4′-dimethoxytriphenylmethane (65.0 mg, 191 μmol) were dissolved in pyridine (1.00 mL) and stirred at 15°C under nitrogen for 8 h. Methanol (2.00 mL) was added to quench the reaction, and the solvent was removed by concentration under reduced pressure to obtain the crude product. Preparative chromatography (HO (10 mM TEAB)-ACN) afforded G11-4 (54.7 mg, 84.3 μmol) as a white solid in a yield of 73.3%. 1HNMR (400MHz, CDCl3) δppm 7.38 (d, J=7.7Hz, 2H), 7.29-7.20 (m, 6H), 7.13 (t, J=7.3Hz, 1H), 6.75 (d, J=8.6Hz, 4H), 5.60 (s, 1H), 5.08 (dd, J=5.5, 2.3Hz, 1H), 4.34 (q, J=6.2Hz, 1H), 3.89 (q, J=5.6Hz, 1H), 3.72 (s, 6H ), 3.56-3.50 (m, 1H), 3.29-3.17 (m, 3H), 3.16-3.06 (m, 3H), 2.33 (d, J=7.0Hz, 3H), 2.18 (t, J =7.2Hz, 3H), 2.01-1.95 (m, 1H), 1.88 (t, J = 7.0Hz, 2H), 1.40-1.31 (m, 4H), 1.21-1.15 (m, 4H). MS(ESI)m / z[MH] - =648.2.

[0327] Step 5: Synthesis of G11-5

[0328] G11-4 (17.0 mg, 26.1 μmol), diisopropylethylamine (6.76 mg, 52.3 μmol), and O-benzotriazole-tetramethyluronium hexafluorophosphate (14.8 mg, 39.2 μmol) were dissolved in N,N-dimethylformamide (0.3 mL). GalNAc-NH2.TFA (62.4 mg, 27.4 μmol, TFA) was added and stirred at 15°C under nitrogen for 6 h. The reaction mixture was used directly in the next step. MS (ESI) m / z [M-2H] 2- =1211.6.

[0329] Step 6: Synthesis of G11-6

[0330] G11-5 (63.0 mg, 25.9 μmol), 4-dimethylaminopyridine (6.35 mg, 51.9 μmol), diisopropylethylamine (6.71 mg, 51.9 μmol), and succinic anhydride (13.0 mg, 129 μmol) were dissolved in N,N-dimethylformamide (0.5 mL) and stirred at 30°C under nitrogen for 16 h. Preparative chromatography (HO(10 mM TEAB)-ACN) afforded G11-6 (35.0 mg, 13.8 μmol) as a white solid in a 53.3% yield. 1HNMR (400MHz, CDCl3) δppm 7.49-7.44(m, 5H), 7.35-7.31(m, 4H), 7.28(s, 1H), 7.24(s, 1H), 7.17(d, J=7.5Hz, 1H), 7.01-7.00(m, 3H), 6.91(d, J= 9.0Hz, 2H), 6.83-6.72 (m, 6H), 5.35 (d, J=3.3Hz, 2H), 5.19 (dd, J=10.9, 3.3Hz, 5H), 4.62 (d, J=8.3Hz, 3H), 4.18-4.05 ( m, 10H), 3.95-3.89 (m, 6H), 3.78 (s, 7H), 3.67-3.61 (m, 12H), 3.50 (s, 4H), 3.31-3.15 (m, 17H), 2.57 (d, J=6.3Hz, 2H), 2 .51 (d, J=6.7Hz, 2H), 2.41 (t, J=5.8Hz, 6H), 2.28-2.12 (m, 21H), 2.12-1.84 (m, 34H), 1.75-1.61 (m, 20H), 1.25 (s, 4H). MS(ESI)m / z[M-2H] 2- =1261.9.

[0331] Step 7: Synthesis of YK-GAL-311

[0332] G11-7 (35.0 mg, 13.8 μmol), 4-dimethylaminopyridine (1.69 mg, 13.8 μmol), diisopropylethylamine (14.3 mg, 110 μmol), and O-benzotriazole-tetramethyluronium hexafluorophosphate (26.2 mg, 69.3 μmol) were dissolved in N,N-dimethylformamide (2.0 mL). CPG-NH2 (220 mg) was then added and stirred at 40°C for 16 h. The reaction mixture was filtered, and the filtrate was washed sequentially with methanol and dichloromethane and dried in vacuo. The filtrate was then added to 12.0 mL of acetic anhydride / pyridine (1:5) solution and stirred at 40°C for 0.5 h. The mixture was filtered, washed sequentially with dichloromethane and methanol, and dried in vacuo for 12 h to obtain compound YK-GAL-311 (205 mg, loading 32.4 μmol / g) as a pale yellow solid.

[0333] 12. Synthesis of YK-GAL-312

[0334] The synthetic route is as follows:

[0335] Step 1: Synthesis of G12-1

[0336] G5-8 (1.3 g, 2.0 mmol), diisopropylethylamine (517 mg, 4.0 mmol), and O-benzotriazole-tetramethyluronium hexafluorophosphate (1.14 g, 3.0 mmol) were dissolved in N,N-dimethylformamide (100.0 mL). GalNAc-NH2 (synthesized according to the method of patent CN115315263A, total yield 20.18%) (1.24 g, 2.0 mmol) was added and stirred at 15°C under nitrogen for 1 h. The reaction solution was used directly in the next step. MS (ESI) m / z [MH] - =1249.9.

[0337] Step 2: Synthesis of G12-2

[0338] G12-1 (2.0 mmol), 4-dimethylaminopyridine (488.7 mg, 4.0 mmol), diisopropylethylamine (1.03 g, 8.0 mmol), and succinic anhydride (1.2 g, 12.0 mmol) were dissolved in N,N-dimethylformamide (100.0 mL) and stirred at 30°C under nitrogen for 48 h. Preparative chromatography (H2O(50 mM TEAB)-ACN) afforded G12-2 (1.9 g, 1.31 mmol) as a white solid in a 65.5% yield. MS (ESI) m / z [MH] - =1350.1.

[0339] Step 3: Synthesis of YK-GAL-312

[0340] G12-2 (1.9 g, 1.31 mmol), 4-dimethylaminopyridine (160 mg, 1.31 mmol), diisopropylethylamine (1.35 g, 10.48 mmol), and O-benzotriazole-tetramethyluronium hexafluorophosphate (2.48 g, 6.55 mmol) were dissolved in N,N-dimethylformamide (210 mL). CPG-NH2 (13.6 g) was then added and stirred at 40°C for 16 h. The reaction mixture was filtered, and the filtrate was washed sequentially with methanol and dichloromethane and dried under vacuum. The filtrate was then added to 124 mL of acetic anhydride / pyridine (1:5) solution and stirred at 40°C for 0.5 h. The mixture was filtered, washed sequentially with dichloromethane and methanol, and dried under vacuum for 12 h to obtain compound YK-GAL-312 (24.3 g, loading 36.2 μmol / g) as a white solid.

[0341] 13. Synthesis of YK-GAL-313

[0342] The synthetic route is as follows:

[0343] Step 1: Synthesis of G13-1

[0344] G5-8 (1.3 g, 2.0 mmol), diisopropylethylamine (517 mg, 4.0 mmol), and O-benzotriazole-tetramethyluronium hexafluorophosphate (1.14 g, 3.0 mmol) were dissolved in N,N-dimethylformamide (100.0 mL). GalNAc-NH2 (synthesized according to the method of patent WO2023288033A1, total yield 2.1%) (2.41 g, 2.0 mmol) was added and stirred at 15°C under nitrogen for 1 h. The reaction solution was used directly in the next step. MS (ESI) m / z [MH] - =1837.5.

[0345] Step 2: Synthesis of G13-2

[0346] G13-1 (2.0 mmol), 4-dimethylaminopyridine (488.7 mg, 4.0 mmol), diisopropylethylamine (1.03 g, 8.0 mmol), and succinic anhydride (1.2 g, 12.0 mmol) were dissolved in N,N-dimethylformamide (100.0 mL) and stirred at 30°C under nitrogen for 48 h. Preparative chromatography (H2O(50 mM TEAB)-ACN) afforded G13-2 (2.66 g, 1.29 mmol) as a white solid in a 64.5% yield. MS (ESI) m / z [MH] - =1937.2.

[0347] Step 3: Synthesis of YK-GAL-313

[0348] G13-2 (2.66 g, 1.29 mmol), 4-dimethylaminopyridine (157.6 mg, 1.29 mmol), diisopropylethylamine (1.33 g, 10.32 mmol), and O-benzotriazole-tetramethyluronium hexafluorophosphate (2.45 g, 6.45 mmol) were dissolved in N,N-dimethylformamide (210.0 mL). CPG-NH2 (18.9 g) was then added and stirred at 40°C for 16 h. The reaction mixture was filtered, and the filtrate was washed sequentially with methanol and dichloromethane and dried under vacuum. The filtrate was then added to 173 mL of acetic anhydride / pyridine (1:5) solution and stirred at 40°C for 0.5 h. The mixture was filtered, washed sequentially with dichloromethane and methanol, and dried under vacuum for 12 h to obtain compound YK-GAL-313 (26.3 g, loading 37.1 μmol / g) as a white solid.

[0349] 14. Synthesis of NAG0052

[0350] According to the synthesis method of NAG0052 on page 100 of WO2023109938A1, 288 mg of product was obtained with a total yield of 1.26% and a loading of 31.5 μmol / g.

[0351] Example 2: Synthesis of GalNAc phosphoramidite compounds

[0352] 1. Synthesis of YK-GAL-318

[0353] Compound G5-9 (2.43 g, 1.0 mmol) and DCM (25 mL) were added to a reaction flask and stirred until dissolved. 3-bis(diisopropylamino)phosphinoyloxypropionitrile (0.75 g, 2.5 mmol) and 4,5-dicyanoimidazole (0.24 g, 2.0 mmol) were added sequentially and stirred at 10°C for 2 h. The reaction solution was diluted with DCM (100 mL) and washed twice with NaHCO3 (100 mL). The organic phase was dried over anhydrous Na2SO4 and concentrated to remove the solvent to obtain a residue. The residue was dissolved in DCM (30 mL) and slurried with n-heptane / methyl tert-butyl ether (5 / 1) at 10-15°C for 10 min, slurried three times in total, to obtain 2.39 g of a white solid, with a yield of 91%. MS (ESI) m / z [M+H] + =2626.8.

[0354] 2. Synthesis of other GalNAc phosphoramidite compounds

[0355] Other GalNAc phosphoramidite compounds listed in Table 1 were synthesized using G1-6, G2-5, G3-5, G4-5, G6-8, G7-8, G8-4, G9-5, G10-5, G11-5, G12-1 and G13-1 as starting materials, respectively, according to the method for synthesizing YK-GAL-318.

[0356] Table 1: GalNAc phosphoramidite compounds

[0357] 3. Synthesis of GalNAc 1b

[0358] According to the synthesis method of GalNAc 1b in WO2023014938A1, page 145, 377.2 mg of product was obtained with a total yield of 0.58%. MS (ESI) m / z [M+H] + =1137.9.

[0359] 4. Synthesis of GalNAc 2

[0360] According to the synthesis method of GalNAc 2 on page 216 of WO2023049258A1, 468.5 mg of the product was obtained with a total yield of 1.75%. MS (ESI) m / z [M+H] + =1217.7.

[0361] Example 3: Coupling of GalNAc Compounds to Oligonucleotides

[0362] In this example, the same siRNA sequence was used for synthesis. The conjugated siRNA sequence was the sequence numbered inc. The inc sequence is as follows:

[0363] Justice chain (inc-SS):

[0364] 5'-Cms-Ums-Am-Gm-Am-Cm-Cf-Um-Gf-Um-dT-Um-Um-Gm-Cm-Um-Um-Um-Um-Gm-Um-3' (SEQ ID NO: 1),

[0365] Antisense strand (inc-AS):

[0366] 5'-Ams-Cfs-Am-Af-Af-Af-Gm-Cf-Am-Af-Am-Af-Cm-Af-Gm-Gf-Um-Cf-Um-Am-Gms-Ams-Am-3' (SEQ ID NO: 2).

[0367] Among them, A, U, C, and G represent the base composition of the nucleotide; dT represents deoxythymidine nucleotide; m represents that the nucleotide adjacent to the left of m is 2'-OMe modified; f represents that the nucleotide adjacent to the left of m is 2'-F modified; s represents that the two nucleotides adjacent to the left and right of s are connected by phosphorothioate groups.

[0368] The sequence comes from a public sequence and is the oligonucleotide sequence of the siRNA drug inclisiran that has been marketed overseas.

[0369] The inclisiran sequence is a synthetic, chemically modified double-stranded small interfering RNA (siRNA) that targets and binds to the mRNA encoding the PCSK9 protein, inhibiting the production of the PCSK9 protein through the RNA interference mechanism, thereby regulating the recycling and reuse of the LDL receptor and enhancing its binding to LDL, thereby achieving the purpose of lowering LDL in the blood.

[0370] 1. Preparation of GalNAc-conjugated siRNA sense strand

[0371] Oligonucleotide GalNAc conjugates were synthesized on solid supports according to phosphoramidite chemistry.

[0372] When synthesizing conjugates 1-11 and 18 (sequence numbers inc-G1, inc-G2, inc-G3, inc-G4, inc-G5, inc-G6, inc-G7, inc-G8, inc-G9, inc-G10, inc-G11 and inc-NAG0052), the GalNAc-CPG compounds synthesized in Example 1 (including YK-GAL-301, YK-GAL-302, YK-GAL-303, YK-GAL-304, YK-GAL The solid phase support was constructed using the following methods: YK-GAL-305, YK-GAL-306, YK-GAL-307, YK-GAL-308, YK-GAL-309, YK-GAL-310, YK-GAL-311, and NAG0052) as solid phase supports; when synthesizing conjugate 17 (sequence numbered inc-L96), CPG-L96 (purchased from Tianjin WuXi AppTec New Drug Development Co., Ltd., L96 is disclosed in US10465194B2, claim 10) was used as the solid phase support. The solid phase support was synthesized on a 1 μmol scale. These GalNAc compounds were conjugated to the 3' end of the oligonucleotide.

[0373] When synthesizing conjugates 12-16, 19, and 20 (sequence numbers: inc-G12, inc-G13, G18-inc, inc-G25, G26-inc, inc-GalNAc 1b, and inc-GalNAc 2), a universal CPG solid phase support was used, and the GalNAc phosphoramidite compounds synthesized in Example 2 (YK-GAL-318, YK-GAL-325, YK-GAL-326, inc-GalNAc 1b, and inc-GalNAc 2) were used as the first or last monomer for solid phase synthesis, where the solid phase support synthesis scale was 1 μmol. Among them, conjugates 12 (sequence numbered inc-G12), 13 (sequence numbered inc-G13), 15 (sequence numbered inc-G25), 19 (sequence numbered inc-GalNAc 1b) and 20 (sequence numbered inc-GalNAc 2) are GalNAc ligand compounds conjugated to the 3' end of the oligonucleotide, and conjugates 14 (sequence numbered G18-inc) and 16 (sequence numbered G26-inc) are GalNAc ligand compounds conjugated to the 5' end of the oligonucleotide.

[0374] (1) Preparation of reagents and monomers

[0375] The monomer solution in acetonitrile (1 / 20, w / v), 0.25M 5-benzylthiotetrazole solution in acetonitrile as an activating agent, 0.2M hydroxanthin solution in acetonitrile / pyridine (1 / 4, v / v) as a thiolation reagent, 0.05M iodine solution in water / pyridine (1 / 9, v / v) as an oxidizing reagent, 20% acetic anhydride in acetonitrile (v / v) as a capping agent A, 20 / 30 / 50 (1-methylimidazole / pyridine / acetonitrile, v / v / v) as a capping agent B, 20% diethylamine in acetonitrile (v / v) as a decyanoethylation reagent, and 3% dichloroacetic acid in toluene (v / v) as a de-DMT reagent were loaded into the designated reagent position of a 192 P model DNA / RNA automatic synthesizer.

[0376] (2) Crude product synthesis

[0377] Enter the designated oligonucleotide sequence, set up the synthesis program, and after checking for errors, begin the cyclic oligonucleotide synthesis. The monomer coupling time is approximately 1 minute, with oxo-substitution taking approximately 30-45 seconds and thio-substitution taking approximately 2 minutes. After the cycle is complete, the solid-phase synthesis of the oligonucleotide is complete.

[0378] (3) Deprotection

[0379] After the synthesis is completed, the solid phase support is transferred to a reactor and the oligonucleotide is cleaved from the solid phase support with concentrated ammonia (25-28%) at 50-60°C for 16-24 hours. The system is cooled to room temperature and then filtered and rinsed with a mixed solution of purified water and ethanol. The filtrates are combined and concentrated at low temperature to obtain a crude residue.

[0380] (4) Purification

[0381] The crude residue after deprotection was dissolved in purified water and subjected to HPLC purification. The product peak solution was collected and the content was measured using an enzyme marker, and the molecular weight was confirmed by ESI MS.

[0382] In this step, the GalNAc-CPG compound synthesized in Example 1 and the GalNAc phosphoramidite compound synthesized in Example 2 are conjugated to the 3' or 5' end of the sense strand of the siRNA.

[0383] YK-GAL-325 was used as the phosphoramidite monomer in the synthesis of conjugate 12. Since YK-GAL-325 has a monoantennary structure, it was repeated three times in the synthesis sequence, resulting in the synthesized conjugate 12 having three GalNAc modification groups.

[0384] When synthesizing conjugate 13, YK-GAL-326 was used as the phosphoramidite monomer. Since YK-GAL-326 is biantennary, it was repeated twice in the synthetic sequence, so that the synthesized conjugate 13 has four GalNAc modification groups.

[0385] 2. Preparation of siRNA antisense strand without GalNAc conjugation

[0386] The siRNA antisense chain was synthesized according to the method for synthesizing the siRNA sense chain, wherein a universal CPG solid phase carrier was used, and the synthesis scale of each antisense chain complementary to the sense chain was 1 μmol.

[0387] 3. Preparation of Conjugated Double-stranded siRNA

[0388] The siRNA sense strand and the complementary antisense strand were mixed at a ratio of 1:1 based on UV absorption, heated to 95°C for 3 minutes, and cooled to room temperature to form a double-stranded product. The resulting double-stranded solution was characterized by HPLC for purity, and the content was determined using a microplate reader. The resulting solid powder was lyophilized and stored for future use. The sequence and molecular weight of the resulting GalNAc-conjugated siRNA double-strands are shown in Table 2.

[0389] Table 2: GalNAc-conjugated double-stranded siRNA

[0390] Wherein: SS is the sense strand, AS is the antisense strand, and the resulting conjugated siRNA structure is as follows:

[0391] (GalNAc compound is L96, disclosed in US10465194B2, compound of claim 10),

[0392] (The GalNAc compound is NAG0052, WO2023109938A1, page 100),

[0393] (GalNAc compound is GalNAc 1b, WO2023014938A1, page 145),

[0394] (The GalNAc compound is GalNAc 2, WO2023049258A1, page 216).

[0395] Example 4: Inhibitory Effect of GalNAc-Conjugated siRNA on PCSK9 Expression in Mouse Serum and Liver and Its Effect on LDL-C Levels

[0396] This example examines the inhibitory effect of the siRNA conjugates listed in Table 2 on PCSK9 expression in mouse serum and liver, as well as their effects on LDL-C levels. After entering the blood, the GalNAc-conjugated inclisiran siRNA sequence first specifically binds to the asialoglycoprotein receptor (ASGPR) on the hepatocyte membrane via GalNAc, thereby entering the hepatocyte. Once inside the hepatocyte, the inclisiran siRNA sequence binds to the RNA-induced silencing complex (RISC) and, mediated by the antisense strand, binds to the mRNA encoding the PCSK9 protein, inhibiting PCSK9 protein production. Reduction in PCSK9 protein in the liver promotes LDL-R circulation, thereby increasing the number of LDL-R receptors on the hepatocyte surface, increasing the uptake and degradation of plasma LDL-C, and further lowering plasma LDL-C levels. Therefore, the higher the amount of inclisiran siRNA sequence delivered to the liver, i.e., the higher the efficiency of GalNAc delivery, the lower the amount of PCSK9 protein in the liver and serum, and the lower the LDL-C level in the liver.

[0397] The results showed that compared with the existing GalNAc oligonucleotide conjugates, the PCSK9 expression inhibition rate and LDL-C reduction levels in mouse serum and liver in the inc-G4, inc-G5, inc-G6, inc-G7, inc-G11, inc-G12, inc-G13 and G18-inc experimental groups were significantly improved. For example, compared with inc-L96, inc-NAG0052, inc-GalNAc 1b and inc-GalNAc 2, the PCSK9 inhibition rate of inc-G5 in serum increased by 14.2%, 13.1%, 14.5% and 51.2% on the 7th day, and increased by 10.2%, 10.7%, 10.4% and 43.0% on the 14th day, respectively; the PCSK9 inhibition rate in mouse liver increased by 11.1%, 13.2%, 11.0% and 44.0%, respectively; the LDL-C reduction level increased by 20.4%, 17.1%, 15.8% and 29.8% on the 7th day, and increased by 23.5%, 20.5%, 20.0% and 33.3% on the 14th day, respectively.

[0398] Animal preparation:

[0399] First, hPCSK9 transgenic mice (SPF grade, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were adaptively bred and then randomly divided into a negative control group (no drug administration) and an siRNA test drug group (inc-G1 to inc-G13, G18-inc, inc-G25, G26-inc, inc-L96, inc-NAG0052, inc-GalNAc 1b, and inc-GalNAc 2 in Table 2) according to the serum PSCK9 protein content, with 5 mice in each group, all of whom were male.

[0400] Administration and dosage:

[0401] The drug was administered by a single subcutaneous injection at a dose of 6 mg / kg, a volume of 1 mL / kg, and a concentration of 6 mg / mL. The day of administration was designated as day 0.

[0402] 1. Inhibitory efficiency of different GalNAc-conjugated siRNAs on PCSK9 in hPCSK9 mouse serum

[0403] Results of PCSK9 inhibition in hPCSK9 mouse serum showed that the oligonucleotide conjugates prepared with the GalNAc compound of the present invention significantly improved the inhibition rate of PCSK9 protein expression in mouse serum compared to the prior art GalNAc. For example, compared with inc-L96, inc-NAG0052, inc-GalNAc 1b, and inc-GalNAc 2, the inhibition rate of inc-G5 increased by 14.2%, 13.1%, 14.5%, and 51.2% on day 7, and by 10.2%, 10.7%, 10.4%, and 43.0% on day 14, respectively.

[0404] Experimental process:

[0405] Approximately 200 μL of blood (without anticoagulation) was collected from the orbital venous plexus of the experimental animals before administration (D0), 7 days after administration (D7), and 14 days after administration (D14). Whole blood samples were temporarily stored in an ice box before centrifugation. Serum was separated by centrifugation at 4000 rpm for 10 minutes at 4°C. Serum PCSK9 protein levels were measured using an ELISA kit (Sino Biological).

[0406] Experimental results:

[0407] The inhibition rates of PCSK9 protein in serum of the negative control group and each test drug group 7 days and 14 days after administration are shown in Table 4. Data were statistically analyzed using GraphPad Prism 9 software.

[0408] 1) Structural differences

[0409] The structural differences of GalNAc oligonucleotide conjugates are shown in Table 3:

[0410] Table 3: Comparison of GalNAc oligonucleotide conjugate structures

[0411] 2) PCSK9 inhibition efficiency in hPCSK9 mouse serum

[0412] Table 4: PCSK9 protein expression inhibition rate in mouse serum

[0413] 1) Inc-G4, Inc-G5, Inc-G6, Inc-G7, Inc-G11, Inc-G12, Inc-G13, and G18-inc showed the highest inhibition rates for PCSK9 protein expression in mouse serum. For example, Inc-G5 achieved inhibition rates of 94.4% and 95.6% on days 7 and 14, respectively. This demonstrates that a cleverly designed linker structure can significantly enhance drug bioavailability and enhance efficacy.

[0414] As can be seen in Table 4, the conjugates prepared from the GalNAc compounds designed in this invention showed significant differences in their inhibitory effects on PCSK9 protein expression in mouse serum. Inc-G4, Inc-G5, Inc-G6, Inc-G7, Inc-G11, Inc-G12, Inc-G13, and G18-inc showed significantly higher inhibition rates than the other groups, exceeding 90% on both days 7 and 14. Inc-G5 achieved the highest inhibition rate, reaching 94.4% and 95.6% on days 7 and 14, respectively. (Figure 1)

[0415] Inc-G2, Inc-G3, Inc-G8, and Inc-G9 also had high inhibition rates, around 85%. Inc-G1, Inc-G10, and G26-inc had inhibition rates between 70-80%. Inc-G25 had the lowest inhibition rate, at 55.9%. (Figure 2)

[0416] The inhibition rate of inc-G5, which had the highest inhibition rate, increased by 38.5% and 35.5% on the 7th and 14th days, respectively, compared with that of inc-G25, which had the lowest inhibition rate, showing significant differences.

[0417] 2) The GalNAc compounds designed by the present invention with three, one, or two antennae are used to prepare conjugates with three or four GalNAc groups, all of which have very high inhibition rates. Moreover, the GalNAc compounds can effectively inhibit PCSK9 protein expression regardless of whether they are conjugated to the 3' end or the 5' end of the oligonucleotide.

[0418] The GalNAc compound YK-GAL-325 designed in the present invention has one antennae, and YK-GAL-326 has two antennae (see Table 1 in Example 2). Inc-G12 is a conjugate with three GalNAc groups obtained by linking YK-GAL-325 via two phosphodiester bonds. Inc-G13 is a conjugate with four GalNAc groups obtained by linking YK-GAL-326 via one phosphodiester bond. Activity assay results showed that inc-G12 and inc-G13 also had significant inhibitory effects on PCSK9 protein expression in mouse serum, with inhibition rates reaching 93.8% and 94.2% on day 7, and 95.3% and 95.5% on day 14, respectively, comparable to inc-G5.

[0419] The GalNAc ligand of G18-inc is conjugated to the 5' end of the oligonucleotide, while the GalNAc ligand of inc-G5 is conjugated to the 3' end of the oligonucleotide. Activity assay results showed that G18-inc also significantly inhibited PCSK9 protein expression in mouse serum, with inhibition rates reaching 93.5% and 95.2% on days 7 and 14, respectively, comparable to those of inc-G5.

[0420] It can be seen from this that the oligonucleotide conjugates with 3 or 4 GalNAc groups prepared by the GalNAc compounds with 3 antennae, 1 antennae or 2 antennae designed by the present invention have very high delivery efficiency. Moreover, regardless of whether they are conjugated to the 3' end or the 5' end of the oligonucleotide, they can efficiently deliver the oligonucleotide inclisiran siRNA sequence to the liver, achieving a significant inhibitory effect on PCSK9 protein expression in mouse serum.

[0421] 3) Oligonucleotide conjugates prepared from the GalNAc compounds designed in this invention significantly improved the inhibition rate of PCSK9 protein expression in mouse serum compared to inc-L96, inc-NAG0052, inc-GalNAc 1b, and inc-GalNAc 2, all prepared from existing GalNAc compounds. For example, inc-G5 showed a 14.2% improvement over inc-L96.

[0422] Conjugates prepared from prior art GalNAc compounds, including inc-L96, inc-NAG0052, inc-GalNAc 1b and inc-GalNAc 2, inhibited PCSK9 protein expression in mouse serum by 80.2%, 81.3%, 79.9% and 43.2% on day 7, and by 85.4%, 84.9%, 85.2% and 52.6% on day 14, respectively.

[0423] The conjugates prepared from the GalNAc compounds of the present invention, including inc-G4, inc-G5, inc-G6, inc-G7, inc-G11, inc-G12, inc-G13 and G18-inc, significantly improve the inhibition rate of PCSK9 protein expression in mouse serum compared with the prior art GalNAc.

[0424] For example, the inhibition rate of inc-G5 on day 7 was 14.2%, 13.1%, 14.5%, and 51.2% higher than that of inc-L96, inc-NAG0052, inc-GalNAc 1b, and inc-GalNAc 2, respectively, and on day 14, it was 10.2%, 10.7%, 10.4%, and 43.0%, respectively. Both inc-G25 and inc-GalNAc 2 are oligonucleotide conjugates with one GalNAc group, but the inhibition rate of inc-G25 was significantly higher than that of inc-GalNAc 2, reaching 12.7%.

[0425] 4) Oligonucleotide conjugates prepared from GalNAc compounds with similar structures are likely to have very different inhibition rates on PCSK9 protein expression in mouse serum; oligonucleotide conjugates prepared from GalNAc compounds with large structural differences are also likely to have very similar inhibition rates on PCSK9 protein expression in mouse serum.

[0426] A comparison of the GalNAc oligonucleotide conjugate structures (Example 3) and Table 3 demonstrates that the structures of this series of GalNAc compounds designed by the present invention are very similar, with only slight differences in individual groups. They are also very similar to the structures of the prior art GalNAc compounds L-96, NAG0052, GalNAc 1b, and GalNAc 2. However, the inhibitory rates of PCSK9 protein expression in mouse serum achieved by oligonucleotide conjugates prepared from these structurally similar GalNAc compounds ranged from similar to significant differences.

[0427] For example, compared with inc-L96, inc-G5 only replaces the prolinol structure in the main chain of inc-L96. Change to ribose ring The other structures are exactly the same, but the inhibition rate of PCSK9 protein expression of inc-G5 can be increased by 14.2% compared with inc-L96, and the inhibitory activity is significantly improved.

[0428] Compared with GalNAc 1b, inc-G12 differs only in the atom connected to the 1' position of the ribose ring. GalNAc 1b is carbon, while inc-G12 is oxygen. The connecting arm of inc-G12 is longer than that of GalNAc 1b. The other structures are exactly the same, but the inhibition rate of inc-G12 is significantly improved by 13.9% compared with GalNAc 1b.

[0429] The only difference between inc-G5 and inc-G1 is the 2' position of the ribose ring, which is a methoxy group while inc-G1 is a hydrogen group. However, the inhibition rate of inc-G5 is 21.4% higher than that of inc-G1.

[0430] Therefore, GalNAc compounds with similar chemical structures do not necessarily have similar oligonucleotide delivery efficiencies. The GalNAc oligonucleotide conjugates prepared from them have different inhibitory effects on the PCSK9 gene in mouse serum and are likely to have very large differences.

[0431] 2. Inhibitory Efficiency of Different GalNAc-Conjugated siRNAs on PCSK9 in the Liver of hPCSK9 Mice

[0432] Experimental process:

[0433] On the 14th day after administration (D14), the experimental animals were anesthetized and killed, and then perfused to collect the liver.

[0434] The tissue was quickly placed in a 1.5 mL RNase-free EP tube containing 1 mL of RNA lysis solution (trizol) at a ratio of 100 mg tissue weight to 1 mL RNA lysis solution (trizol). Three 3 mm steel balls (RNA-free treated) were added to the tube, which was then placed in a tissue homogenizer. The tube was run at 50 Hz for 30 seconds, followed by a 10-second pause, for three cycles to prepare a tissue homogenate.

[0435] Centrifuge at 12,000 × g for 3 minutes at 4°C. Transfer 400 μL of the homogenate supernatant to a 1.5 mL RNase-free EP tube and place on ice. Add 80 μL of chloroform to each tube, shake vigorously for 15 seconds, and let stand at room temperature for 5 minutes. Centrifuge at 12,000 × g for 15 minutes at 4°C. Transfer 150 μL of the supernatant to a new EP tube.

[0436] An equal volume of isopropanol was added, and the liquid in the tube was gently mixed by inverting. The tube was allowed to stand at -20°C for 10 minutes, and then centrifuged at 4°C, 12,000 × g for 15 minutes, and the supernatant was discarded.

[0437] Add 1 mL of 75% ethanol and gently wash the RNA pellet. Centrifuge at 7,500 × g for 5 minutes at 4°C and aspirate the supernatant. Repeat the rinse cycle once more, centrifuging at 7,500 × g for 5 minutes at 4°C. Remove any remaining ethanol using a micropipette.

[0438] After drying the remaining ethanol at room temperature for 10 minutes, 150 μL of RNase-free ddH₂O was added for dissolution. RNA concentration was measured using a micro-UV spectrophotometer. PCSK9 gene expression was assessed by qPCR, measuring mRNA. The results are shown in Table 5.

[0439] Experimental results:

[0440] The qPCR results (Mean ± SD) were statistically analyzed and plotted using GraphPad Prism 9 software. The specific results are shown in Table 5.

[0441] Table 5 PCSK9 gene expression inhibition rate in mouse liver on day 14 after administration

[0442] 1) Inc-G4, Inc-G5, Inc-G6, Inc-G7, Inc-G11, Inc-G12, Inc-G13, and G18-inc exhibited the highest inhibition rates for PCSK9 in mouse liver. For example, Inc-G5, Inc-G6, and Inc-G12 all achieved inhibition rates of 90%. This suggests that cleverly designed linker structures can significantly enhance drug bioavailability and enhance efficacy.

[0443] As shown in Table 5, the conjugates prepared using the GalNAc compounds designed in this invention exhibited widely varying inhibition rates on PCSK9 gene expression in mouse liver. Inc-G4, Inc-G5, Inc-G6, Inc-G7, Inc-G11, Inc-G12, Inc-G13, and G18-inc achieved the highest inhibition rates, all exceeding 85%. Inc-G5, Inc-G6, and Inc-G12 achieved inhibition rates exceeding 90%. (Figure 3)

[0444] Inc-G2 and Inc-G3 also had high inhibition rates, ranging from 80-85%. Inc-G8, Inc-G9, and G26-inc had inhibition rates between 70-80%. Inc-G1, Inc-G10, and Inc-G25 had inhibition rates between 60-70%. (Figure 4)

[0445] The inhibition rate of inc-G5, which had the highest inhibition rate, was 30.3% higher than that of inc-G10, which had the lowest inhibition rate, showing a significant difference.

[0446] 2) The present invention designs three-antennary, one-antennary, or two-antennary GalNAc compounds, and prepares oligonucleotide conjugates with three or four GalNAc groups, all of which have very high inhibition rates. Moreover, these GalNAc compounds, whether conjugated to the 3' end or the 5' end of the oligonucleotide, can effectively inhibit PCSK9 gene expression.

[0447] The GalNAc compounds YK-GAL-325 designed in the present invention have one antennae, and YK-GAL-326 has two antennae (see Table 1 in Example 2). Inc-G12 is a conjugate with three GalNAc groups, obtained by linking YK-GAL-325 via two phosphodiester bonds. Inc-G13 is a conjugate with four GalNAc groups, obtained by linking YK-GAL-326 via one phosphodiester bond. Activity assays showed that inc-G12 and inc-G13 also significantly inhibited PCSK9 gene expression in mouse liver, with inhibition rates of 90.1% and 89.9%, respectively, comparable to those of inc-G5.

[0448] The GalNAc ligand of G18-inc is conjugated to the 5' end of the oligonucleotide, while the GalNAc ligand of inc-G5 is conjugated to the 3' end of the oligonucleotide. Activity assay results showed that G18-inc also significantly inhibited PCSK9 gene expression in mouse liver, with an inhibition rate of 89.7%, comparable to that of inc-G5.

[0449] It can be seen from this that the oligonucleotide conjugates with 3 or 4 GalNAc groups prepared by the GalNAc compounds with 3 antennae, 1 antennae or 2 antennae designed by the present invention have very high delivery efficiency. Moreover, regardless of whether they are conjugated to the 3' end or the 5' end of the oligonucleotide, they can efficiently deliver the oligonucleotide Inclisiran siRNA sequence to the liver and have a significant inhibitory effect on PCSK9 gene expression in mouse liver.

[0450] 3) Oligonucleotide conjugates prepared using the GalNAc compounds designed in this invention showed significantly improved inhibition rates compared to inc-L96, inc-NAG0052, inc-GalNAc 1b, and inc-GalNAc 2, all prepared using prior art GalNAc compounds. For example, inc-G5 showed an 11.1% improvement over inc-L96.

[0451] Conjugates prepared from prior art GalNAc compounds, including inc-L96, inc-NAG0052, inc-GalNAc 1b and inc-GalNAc 2, inhibited PCSK9 gene expression in mouse liver by 80.1%, 78.0%, 80.2% and 47.2%, respectively.

[0452] The conjugates prepared from the GalNAc compounds of the present invention, including inc-G4, inc-G5, inc-G6, inc-G7, inc-G11, inc-G12, inc-G13 and G18-inc, significantly improve the inhibition rate of PCSK9 gene expression in mouse liver compared with the prior art GalNAc.

[0453] For example, the inhibition rate of inc-G5 was 11.1%, 13.2%, 11.0%, and 44.0% higher than that of inc-L96, inc-NAG0052, inc-GalNAc 1b, and inc-GalNAc 2, respectively. Both inc-G25 and inc-GalNAc 2 are oligonucleotide conjugates with one GalNAc group, but the inhibition rate of inc-G25 was significantly higher than that of inc-GalNAc 2, reaching 14.4%.

[0454] 4) Oligonucleotide conjugates prepared from GalNAc compounds with similar structures are likely to have very different inhibition rates on PCSK9 gene expression in mouse liver; oligonucleotide conjugates prepared from GalNAc compounds with large structural differences are also likely to have very similar inhibition rates on PCSK9 gene expression in mouse liver.

[0455] A comparison of the GalNAc oligonucleotide conjugate structures (Example 3) and Table 3 reveals that the structures of this series of GalNAc compounds designed by the present invention are very similar, with only slight differences in individual groups. They are also very similar in structure to the prior art GalNAc compounds L-96, NAG0052, GalNAc 1b, and GalNAc 2. However, the inhibitory rates of PCSK9 gene expression in mouse liver by oligonucleotide conjugates prepared from these structurally similar GalNAc compounds range from similar to significant differences in some cases.

[0456] For example, compared with inc-L96, inc-G5 only changes the prolinol structure in the inc-L96 main chain to a ribose ring structure, and the other structures are exactly the same. However, the inhibition rate of PCSK9 gene expression of inc-G5 can be increased by 11.1% compared with inc-L96, and the inhibitory activity is significantly improved.

[0457] Compared with GalNAc 1b, inc-G12 differs only in the atom connected to the 1' position of the ribose ring. GalNAc 1b is carbon, while inc-G12 is oxygen. The connecting arm of inc-G12 is longer than that of GalNAc 1b. The other structures are exactly the same, but the inhibition rate of inc-G12 is significantly improved by 9.9% compared with GalNAc 1b.

[0458] The only difference between inc-G5 and inc-G1 is the 2' position of the ribose ring, which is a methoxy group while inc-G1 is a hydrogen group. However, the inhibition rate of inc-G5 is 21.3% higher than that of inc-G1.

[0459] Both inc-GalNAc 2 and inc-G25 are oligonucleotide conjugates with one GalNAc group, but the inhibition rate of inc-G25 is significantly increased by 14.4% compared with inc-GalNAc 2.

[0460] Therefore, GalNAc compounds with similar chemical structures do not necessarily have similar oligonucleotide delivery efficiencies. The GalNAc oligonucleotide conjugates prepared from them have different inhibitory effects on the PCSK9 gene in mouse liver and are likely to have very large differences.

[0461] 3. Effects of different GalNAC-conjugated siRNAs on LDL-C levels in hPCSK9 mice

[0462] Experimental process:

[0463] Approximately 200 μL of blood (without anticoagulation) was collected from the orbital venous plexus of the experimental animals before administration (D0), 7 days after administration (D7), and 14 days after administration (D14). Whole blood samples were temporarily stored in an ice box before centrifugation. Serum was separated by centrifugation at 4000 rpm for 10 minutes at 4°C, and serum LDL-C levels were measured.

[0464] Experimental results:

[0465] Serum LDL-C (Mean±SD) was statistically analyzed and graphed using GraphPad Prism 9 software. The specific results are shown in Table 6.

[0466] Table 6: Reduction levels of serum LDL-C in each experimental group

[0467] 1) Inc-G4, Inc-G5, Inc-G6, Inc-G7, Inc-G11, Inc-G12, Inc-G13, and G18-inc significantly reduced LDL-C compared to the other groups. For example, Inc-G5 achieved 49.6% and 54.1% reductions on days 7 and 14, respectively. This demonstrates that a cleverly designed linker structure can significantly enhance drug bioavailability and enhance efficacy.

[0468] As shown in Table 6, the conjugates prepared from the GalNAc compounds designed in this invention, inc-G4, inc-G5, inc-G6, inc-G7, inc-G11, inc-G12, inc-G13, and G18-inc, showed the highest reductions in mouse serum LDL-C, exceeding 35% on both days 7 and 14, significantly higher than those in the other experimental groups. The highest reduction was achieved with inc-G5, reaching 49.6% and 54.1% on days 7 and 14, respectively (Figure 5).

[0469] The reduction levels for inc-G2, inc-G3, and inc-G8 ranged from 30% to 35%. The reduction levels for inc-G9, inc-G25, and G26-inc ranged from 20% to 30%. The reduction levels for inc-G1 and inc-G10 were less than 20%, with inc-G1 showing only 15.9% and 17.0% on days 7 and 14, respectively. (Figure 6)

[0470] The highest reduction level of inc-G5 was more than 30% higher than that of inc-G1, the lowest, and the difference was significant.

[0471] 2) The present invention designs tri-antennary, uni-antennary, or bi-antennary GalNAc compounds, and oligonucleotide conjugates with three or four GalNAc groups, all exhibit very high LDL-C reduction levels. Furthermore, these GalNAc compounds, whether conjugated to the 3' or 5' end of the oligonucleotide, can effectively reduce LDL-C levels.

[0472] The GalNAc compounds YK-GAL-325 designed in the present invention have one antennae, and YK-GAL-326 has two antennae (see Table 1 in Example 2). Inc-G12 is a conjugate with three GalNAc groups, formed by linking YK-GAL-325 via two phosphodiester bonds. Inc-G13 is a conjugate with four GalNAc groups, formed by linking YK-GAL-326 via one phosphodiester bond. Activity assays showed that inc-G12 and inc-G13 also significantly reduced LDL-C levels in mouse serum, with reductions of 48.5% and 47.3% on day 7 and 51.9% and 49.5% on day 14, respectively, comparable to those of inc-G5.

[0473] The GalNAc ligand of G18-inc was conjugated to the 5' end of the oligonucleotide, while the GalNAc ligand of inc-G5 was conjugated to the 3' end of the oligonucleotide. Activity assay results showed that G18-inc also significantly reduced LDL-C levels in mouse serum, with reductions of 45.6% and 47.9% on days 7 and 14, respectively, comparable to those of inc-G5.

[0474] It can be seen from this that the oligonucleotide conjugates with 3 or 4 GalNAc groups prepared by the GalNAc compounds with 3 antennae, 1 antennae or 2 antennae designed by the present invention have a significant inhibitory effect on PCSK9 gene expression. Moreover, regardless of whether conjugated to the 3' end or 5' end of the oligonucleotide, the oligonucleotide Inclisiran siRNA sequence can be efficiently delivered to the liver, achieving a significant inhibitory effect on PCSK9 gene expression in mouse serum, thereby reducing LDL-C levels.

[0475] 3) Oligonucleotide conjugates prepared using the GalNAc compounds designed in this invention significantly reduced LDL-C in mouse serum compared to inc-L96, inc-NAG0052, inc-GalNAc 1b, and inc-GalNAc 2, all prepared using existing GalNAc compounds. For example, inc-G5 achieved 20.4% and 23.5% higher LDL-C levels than inc-L96 on days 7 and 14, respectively.

[0476] Among the conjugates prepared from prior art GalNAc compounds, the reduction levels of inc-L96, inc-NAG0052, and inc-GalNAc 1b were between 25-35%, and the reduction level of inc-GalNAc 2 was around 20%.

[0477] The conjugates prepared from the GalNAc compounds of the present invention, including inc-G4, inc-G5, inc-G6, inc-G7, inc-G11, inc-G12, inc-G13 and G18-inc, significantly improve the level of LDL-C reduction in mouse serum compared with the prior art GalNAc.

[0478] For example, the reduction level of inc-G5 on day 7 was 20.4%, 17.1%, 15.8% and 29.8% higher than that of inc-L96, inc-NAG0052, inc-GalNAc 1b and inc-GalNAc 2, respectively, and on day 14 it was 23.5%, 20.5%, 20.0% and 33.3%, respectively.

[0479] 4) Oligonucleotide conjugates prepared from structurally similar GalNAc compounds are likely to have very different effects on the reduction of LDL-C in mouse serum; oligonucleotide conjugates prepared from structurally different GalNAc compounds are also likely to have very similar effects on the reduction of LDL-C in mouse serum.

[0480] A comparison of the GalNAc oligonucleotide conjugate structures (Example 3) and Table 3 demonstrates that the structures of this series of GalNAc compounds designed by the present invention are very similar, with only minor differences in individual groups. They are also very similar to the existing GalNAc compounds L-96, NAG0052, GalNAc 1b, and GalNAc 2. However, the effects of oligonucleotide conjugates prepared from these structurally similar GalNAc compounds on mouse serum LDL-C levels range from similar to significant differences.

[0481] For example, compared to inc-L96, inc-G5 only changes the prolinol structure in the inc-L96 backbone to a ribose ring structure; the rest of the structure is identical. However, inc-G5 significantly improves serum LDL-C lowering by over 20% compared to inc-L96. Compared to GalNAc 1b, inc-G12 differs only in the atom attached to the 1' position of the ribose ring—GalNAc 1b is a carbon atom, while inc-G12 is an oxygen atom. The linker arm of inc-G12 is longer than that of GalNAc 1b. While the rest of the structure is identical, inc-G12 significantly improves serum LDL-C lowering by over 15%. Compared with inc-G1, inc-G5 differs only in the 2' position of the ribose ring. Inc-G5 is a methoxy group, while inc-G1 is a hydrogen group. However, in terms of its effect on LDL-C levels in mouse serum, inc-G5 can reduce it by 50%, while inc-G1 only reduces it by about 15%. The reduction level of inc-G5 is more than 30% higher than that of inc-G1, which is a significant difference.

[0482] Therefore, GalNAc compounds with similar chemical structures do not necessarily have similar oligonucleotide delivery efficiencies. The effects of GalNAc oligonucleotide conjugates prepared from them on LDL-C levels in mouse serum are not consistent and are likely to have very large differences.

[0483] Example 5: Distribution of different GalNAc-conjugated siRNAs in different tissues and organs of mice

[0484] After entering the bloodstream, the GalNAc-liganded inclisiran siRNA sequence initially enters liver cells, where it inhibits the target gene. The remaining siRNA in the serum is cleared through the kidneys. Since plasma siRNA is primarily cleared through the kidneys, the siRNA sequence is also distributed in the kidneys in addition to the liver.

[0485] Experimental process:

[0486] Six- to eight-week-old wild-type C57BL / 6 mice were used, with six mice per group. Each group was administered with Cy5-fluorophore-containing siRNA conjugates: inc-G1, inc-G2, inc-G3, inc-G4, inc-G5, inc-G6, inc-G7, inc-G8, inc-G9, inc-G10, inc-G11, inc-G12, inc-G13, G18-inc, inc-G25, G26-inc, inc-L96, inc-NAG0052, inc-GalNAc 1b, and inc-GalNAc 2. A negative control group received no treatment. Mice were weighed and administered subcutaneously at a dose of 6 mg / kg. The injection volume should not exceed 0.1-0.2 mL, depending on the dosing volume.

[0487] After drug administration, the mice were shaved clean of abdominal hair and placed in a supine position under isoflurane anesthesia in a small animal living imaging system. Images of the mice were observed using the Cy5 channel 4 and 8 hours after drug administration. Luminescence intensity was calculated using the Living Image software, and differences between the different test groups were compared.

[0488] Experimental results:

[0489] The results of the fluorescence intensity test in mouse liver and kidney (which can represent the relative content of oligonucleic acid) are shown in Table 7.

[0490] Table 7: Fluorescence intensity in mouse liver and kidney

[0491]

[0492] 1) The liver fluorescence intensities of inc-G4, inc-G5, inc-G6, inc-G7, inc-G11, inc-G12, inc-G13, and G18-inc were significantly higher than those of the other groups. For example, inc-G5, which showed the highest fluorescence intensity, reached 9.12E+09 and 9.16E+09 at 4 and 8 hours, respectively. This demonstrates that cleverly designed linker structures can significantly improve the delivery of oligonucleotide drugs.

[0493] As can be seen in Table 7, the Cy5 fluorescently labeled oligonucleotides were distributed throughout the mouse liver and kidneys. The fluorescence intensities of the conjugates prepared using the GalNAc compound designed by the present invention varied significantly. The fluorescence intensities of inc-G4, inc-G5, inc-G6, inc-G7, inc-G12, inc-G13, and G18-inc in the liver were significantly higher than those of the other groups, indicating that the relative oligonucleotide content was significantly higher than in the other groups. Among these, inc-G5 had the highest fluorescence intensity, reaching 9.12E+09 at 4 hours and 9.16E+09 at 8 hours. (Figure 7)

[0494] The fluorescence intensities of inc-G2, inc-G3, and inc-G8 were also high, ranging from 8.00E+09 to 8.50E+09 at both 4 and 8 hours. The fluorescence intensities of inc-G9, inc-G10, and G26-inc ranged from 7.00E+09 to 8.00E+09 at 4 hours and from 6.50E+09 to 8.00E+09 at 8 hours. The fluorescence intensities of inc-G1 and inc-G25 ranged from 5.00E+09 to 7.50E+09.

[0495] Inc-G5, which had the highest fluorescence intensity, showed a significant improvement of 52.0% and 23.5% at 4 hours and 8 hours, respectively, compared with inc-G1, which had the lowest fluorescence intensity.

[0496] 2) The present invention designs GalNAc compounds with three, one, or two antennae to prepare oligonucleotide conjugates with three or four GalNAc groups, all of which have very high fluorescence intensity in the liver. Moreover, these GalNAc compounds, whether conjugated to the 3' end or the 5' end of the oligonucleotide, can efficiently deliver the oligonucleotide to the liver.

[0497] The GalNAc compounds designed in this invention, YK-GAL-325, have one antennae, while YK-GAL-326 has two antennae (see Table 1 in Example 2). Inc-G12 is a conjugate containing three GalNAc groups by linking YK-GAL-325 via two phosphodiester bonds. Inc-G13 is a conjugate containing four GalNAc groups by linking YK-GAL-326 via one phosphodiester bond. The fluorescence intensities of inc-G12 and inc-G13 at 4 hours were 9.01E+09 and 8.98E+09, respectively, and at 8 hours, 9.09E+09 and 9.01E+09, respectively, comparable to those of inc-G5.

[0498] The GalNAc ligand of G18-inc was conjugated to the 5' end of the oligonucleotide, while the GalNAc ligand of inc-G5 was conjugated to the 3' end of the oligonucleotide. The fluorescence intensities of G18-inc at 4 and 8 hours were 8.95E+09 and 9.13E+09, respectively, comparable to those of inc-G5.

[0499] It can be seen from this that the oligonucleotide conjugates with 3 or 4 GalNAc groups prepared by the GalNAc compounds with 3 antennae, 1 antennae or 2 antennae designed by the present invention have very high delivery efficiency, and regardless of whether they are conjugated to the 3' end or 5' end of the oligonucleotide, they can efficiently deliver the oligonucleotide inclisiran siRNA sequence to the liver.

[0500] 3) Oligonucleotide conjugates prepared using the GalNAc compounds designed in this invention showed significantly improved fluorescence absorption intensity in mouse liver compared to inc-L96, inc-NAG0052, inc-GalNAc 1b, and inc-GalNAc 2, all prepared using existing GalNAc compounds. For example, inc-G5 showed a greater than 50% improvement compared to inc-L96.

[0501] As can be seen from Table 7 and Figure 7, oligonucleotides labeled with Cy5 fluorescence are distributed in the liver and kidney of mice. The fluorescence intensity of inc-G4, inc-G5, inc-G6, inc-G7, inc-G12, inc-G13 and G18-inc in the liver is significantly higher than that of inc-L96, inc-NAG0052, inc-GalNAc1b and inc-GalNAc 2 at 4 hours and 8 hours. For example, the liver fluorescence intensity of inc-G5 at 4 hours and 8 hours was 9.12E+09 and 9.16E+09, respectively, which was 10.9% and 52.9% higher than inc-L96, 13.9% and 40.5% higher than inc-NAG0052, 17.7% and 44.3% higher than inc-GalNAc 1b, and 110.6% and 127.3% higher than inc-GalNAc 2, which was a significant improvement. The fluorescence intensity of inc-G6 at 4 hours and 8 hours was 8.92E+09 and 9.03E+09, respectively, which was 8.5% and 50.8% higher than inc-L96, 11.4% and 38.5% higher than inc-NAG0052, 15.1% and 42.2% higher than inc-GalNAc 1b, and 106.0% and 124.1% higher than inc-GalNAc 2, which was a significant improvement.

[0502] 4) Oligonucleotide conjugates prepared from GalNAc compounds with similar structures are likely to have very different efficiencies in delivering oligonucleotides to the liver; oligonucleotide conjugates prepared from GalNAc compounds with very different structures are also likely to have very similar efficiencies in delivering oligonucleotides to the liver.

[0503] A comparison of the GalNAc oligonucleotide conjugate structures (Example 3) and Table 3 demonstrates that the structures of this series of GalNAc compounds designed by the present invention are very similar, with only slight differences in individual groups. They are also very similar to the structures of the prior art GalNAc compounds L-96, NAG0052, GalNAc 1b, and GalNAc 2. However, the inhibitory rates of PCSK9 protein expression in mouse serum achieved by oligonucleotide conjugates prepared from these structurally similar GalNAc compounds ranged from similar to significant differences.

[0504] For example, compared with inc-L96, inc-G5 only changes the prolinol structure in the inc-L96 main chain to a ribose ring structure, and the other structures are exactly the same. However, the liver fluorescence intensity of inc-G5 can be increased by more than 50% compared with inc-L96, and the delivery efficiency is significantly improved.

[0505] Compared with GalNAc 1b, inc-G12 differs only in the atom connected to the 1' position of the ribose ring. GalNAc 1b is carbon, while inc-G12 is oxygen. The connecting arm of inc-G12 is longer than that of GalNAc 1b. The other structures are exactly the same, but the liver fluorescence intensity of inc-G12 is more than 40% higher than that of GalNAc 1b.

[0506] The only difference between inc-G5 and inc-G1 is the 2' position of the ribose ring, which is a methoxy group while inc-G1 is a hydrogen group. However, the liver fluorescence intensity of inc-G5 is more than 50% higher than that of inc-G1.

[0507] Therefore, GalNAc compounds with similar chemical structures do not necessarily have similar oligonucleotide delivery efficiencies. On the contrary, they are likely to have very large differences.

[0508] Example 6: Pharmacokinetics of different GalNAc-conjugated siRNAs in animal liver

[0509] Experimental process:

[0510] The experimental animals were 6-9 week old male Sprague-Dawley rats, with 30 rats per group. Each group was administered with siRNA conjugates inc-G1, inc-G2, inc-G3, inc-G4, inc-G5, inc-G6, inc-G7, inc-G8, inc-G9, inc-G10, inc-G11, inc-G12, inc-G13, G18-inc, inc-G25, G26-inc, inc-L96, inc-NAG0052, inc-GalNAc 1b, and inc-GalNAc 2. Rats were weighed and administered subcutaneously at a dose of 5 mg / kg at a concentration of 1 mg / mL in a 5 mL volume. Tissue samples were collected at 9 time points, namely 6, 24, 72, 168, 336, 504, 672, 1008 and 1344 h after administration: the experimental animals were killed with carbon dioxide, the liver was removed and rinsed with pre-cooled saline, then dried with filter paper, weighed and transferred to a labeled tube, and homogenized under ice-cold conditions at a ratio of 1:9 (1 g of tissue added to 9 mL of homogenate) (homogenate: 100 mM Tris, 10 mM EDTA, pH 8.0). Approximately 800 μL of the homogenized sample was stored at -80°C, and the drug concentration in the liver was then detected by LC-MS / MS.

[0511] Experimental results: The half-life, peak drug concentration and area under the drug-time curve data are shown in Table 8.

[0512] Table 8: Half-life, peak concentration and area under the drug-time curve data

[0513]

[0514] 1) The half-lives of inc-G4, inc-G5, inc-G6, inc-G7, inc-G11, inc-G12, inc-G13, and G18-inc were significantly improved compared to other groups. For example, the half-lives of inc-G5, inc-G6, inc-G7, inc-G12, and G18-inc reached 110 hours, 103 hours, 103 hours, 101 hours, and 102 hours, respectively. This demonstrates that cleverly designed linker structures can significantly improve the in vivo pharmacokinetic properties of drugs.

[0515] As can be seen from Table 8, the half-lives of the conjugates prepared using the GalNAc compounds designed according to the present invention varied greatly. The half-lives of inc-G4, inc-G5, inc-G6, inc-G7, inc-G11, inc-G12, inc-G13, and G18-inc were significantly improved compared to the other groups. For example, the half-lives of inc-G5, inc-G6, inc-G7, and G18-inc reached 110 hours, 103 hours, 103 hours, and 102 hours, respectively, while inc-G4, inc-G11, and inc-G13 all reached 85 hours. (Figure 8)

[0516] The half-lives of inc-G2, inc-G3, inc-G9, inc-G25, and G26-inc ranged from 70 to 85 hours. The half-lives of inc-G8 and inc-G10 ranged from 60 to 70 hours. The shortest half-life was inc-G1, at 56 hours. (Figure 9)

[0517] The half-life of inc-G5 with the longest half-life and inc-G1 with the shortest half-life differ by 54 hours. The half-life of inc-G5 is twice that of inc-G1, which is a very significant difference.

[0518] The increasing or decreasing trends of the peak concentration and area under the drug-time curve of each test drug group were also consistent with the half-life.

[0519] 2) Conjugates with three or four GalNAc groups prepared from the GalNAc compounds designed by the present invention, which have three antennae, one antennae, or two antennae, all have long half-lives in mice. Moreover, the half-lives are long regardless of whether the GalNAc compounds are conjugated to the 3' end or the 5' end of the oligonucleotide.

[0520] The GalNAc compounds designed in this invention, YK-GAL-325, have one antennae, while YK-GAL-326 has two antennae (see Table 1 in Example 2). Inc-G12 is a conjugate of YK-GAL-325 linked via two phosphodiester bonds, resulting in three GalNAc groups. Inc-G13 is a conjugate of YK-GAL-326 linked via one phosphodiester bond, resulting in four GalNAc groups. The in vivo half-lives of inc-G12 and inc-G13 in mice are 101 hours and 95 hours, respectively, comparable to those of inc-G5.

[0521] The GalNAc ligand of G18-inc is conjugated to the 5' end of the oligonucleotide, while the GalNAc ligand of inc-G5 is conjugated to the 3' end of the oligonucleotide. The half-life of G18-inc in mice reached 102 hours, comparable to that of inc-G5.

[0522] It can be seen from this that the oligonucleotide conjugates with three or four GalNAc groups prepared by the GalNAc compounds with three, one or two antennae designed by the present invention are very stable in mice and have a long half-life regardless of whether they are conjugated to the 3' end or the 5' end of the oligonucleotide.

[0523] The increasing or decreasing trends of the peak concentration and area under the drug-time curve of each test drug group were also consistent with the half-life.

[0524] 3) Oligonucleotide conjugates prepared using the GalNAc compounds designed in this invention exhibit significantly improved in vivo half-lives in mice compared to inc-L96, inc-NAG0052, inc-GalNAc 1b, and inc-GalNAc 2, all prepared using existing GalNAc compounds. For example, inc-G5 exhibits a 48.6% improvement over inc-L96.

[0525] Conjugates prepared from prior art GalNAc compounds, including inc-L96, inc-NAG0052, inc-GalNAc 1b, and inc-GalNAc 2, had in vivo half-lives of 74 hours, 75 hours, 79 hours, and 65 hours in mice, respectively. Conjugates prepared from the present invention's GalNAc compounds, including inc-G4, inc-G5, inc-G6, inc-G7, inc-G11, inc-G12, inc-G13, and G18-inc, showed significantly improved in vivo half-lives in mice compared to prior art GalNAc.

[0526] For example, the half-life of inc-G5 was significantly improved by 48.6%, 46.7%, 39.2% and 69.2% compared with inc-L96, inc-NAG0052, inc-GalNAc 1b and inc-GalNAc 2, respectively.

[0527] Both inc-G25 and inc-GalNAc 2 are oligonucleotide conjugates with one GalNAc group, but the half-life of inc-G25 is significantly increased by 15.4% compared to inc-GalNAc 2.

[0528] The increasing or decreasing trends of the peak concentration and area under the drug-time curve of each test drug group were also consistent with the half-life.

[0529] 4) Oligonucleotide conjugates prepared from structurally similar GalNAc compounds are likely to have very different half-lives in mice; oligonucleotide conjugates prepared from structurally different GalNAc compounds are also likely to have very similar half-lives in mice.

[0530] As can be seen from the structural diagram of the GalNAc oligonucleotide conjugate (Example 3) and the structural comparison in Table 3, the structures of this series of GalNAc compounds designed by the present invention are very similar, with only slight differences in individual groups. They are also very similar to the existing GalNAc compounds L-96, NAG0052, GalNAc 1b, and GalNAc 2. However, the half-lives of oligonucleotide conjugates prepared from these structurally similar GalNAc compounds in mice vary significantly.

[0531] For example, compared with inc-L96, inc-G5 only changes the prolinol structure in the inc-L96 main chain to a ribose ring structure, while the other structures are exactly the same. However, the half-life of inc-G5 can be increased by 48.6% compared with inc-L96, which is a significant improvement.

[0532] Compared with GalNAc 1b, inc-G12 differs only in the atom connected to the 1' position of the ribose ring. GalNAc1b is carbon, while inc-G12 is oxygen. The connecting arm of inc-G12 is longer than that of GalNAc 1b. The other structures are exactly the same, but the half-life of inc-G12 is significantly improved by 27.8% compared with GalNAc 1b.

[0533] Compared with inc-G1, inc-G5 differs only in the 2' position of the ribose ring, which is a methoxy group while inc-G1 is a hydrogen group. However, the half-life of inc-G5 is significantly improved by 96.4% compared with inc-G1.

[0534] Therefore, it is not necessarily the case that oligonucleotide conjugates prepared from GalNAc compounds with similar chemical structures will have similar half-lives in mice. On the contrary, they are likely to have very large differences.

[0535] The increasing or decreasing trends of the peak concentration and area under the drug-time curve of each test drug group were also consistent with the half-life.

[0536] The present invention designs a series of novel GalNAc compounds, such as YK-GAL-304, YK-GAL-305, YK-GAL-306, YK-GAL-307, YK-GAL-311, YK-GAL-318, YK-GAL-325 and YK-GAL-326. The GalNAc oligonucleotide conjugates prepared from them can achieve efficient liver-targeted delivery, and the activity and half-life are significantly improved compared with the representative GalNAc compounds in the prior art.

[0537] 1. The designed GalNAc compound has a significantly different chemical structure compared to the prior art GalNAc compound. The designed GalNAc compound of the present invention introduces a ribose ring structure into the linker arm and introduces an oxygen or sulfur atom at the 1' position of the ribose ring.

[0538] 2. The oligonucleotide conjugates inc-G4, inc-G5, inc-G6, inc-G7, inc-G11, inc-G12, inc-G13 and G18-inc prepared by the GalNAc compounds designed by the present invention have significantly improved delivery efficiency and half-life compared with other groups. This shows that the cleverly designed connection structure can significantly improve the bioavailability of the drug and improve the pharmacokinetic properties of the drug, thereby exerting better drug efficacy. The GalNAc compounds with 3 antennae, 1 antennae or 2 antennae designed by the present invention are used to prepare conjugates with 3 or 4 GalNAc groups, all of which have high activity and long half-life. Moreover, the activity and half-life of oligonucleotide conjugates prepared from GalNAc compounds with similar structures are very likely to be very different; the activity and half-life of oligonucleotide conjugates prepared from GalNAc compounds with large structural differences are also very likely to be very close.

[0539] The details are as follows:

[0540] 1) Inhibition rate of PCSK9 protein expression in mouse serum

[0541] I. inc-G4, inc-G5, inc-G6, inc-G7, inc-G11, inc-G12, inc-G13, and G18-inc significantly inhibited PCSK9 protein expression in mouse serum compared to other groups. For example, inc-G5 achieved inhibition rates of 94.4% and 95.6% on days 7 and 14, respectively. Furthermore, compared to inc-L96, inc-NAG0052, inc-GalNAc 1b, and inc-GalNAc 2, representative GalNAc compounds prepared from existing technologies, the inhibition rates of PCSK9 protein expression in mouse serum were significantly improved. For example, inc-G5 achieved a 14.2% improvement over inc-L96. This demonstrates that cleverly designed linker structures can significantly enhance drug bioavailability and achieve greater efficacy.

[0542] II. The GalNAc compounds designed by the present invention with three, one, or two antennae are used to prepare conjugates with three or four GalNAc groups, all of which have very high inhibition rates. Moreover, regardless of whether the GalNAc compound is conjugated to the 3' end or the 5' end of the oligonucleotide, it can effectively inhibit PCSK9 protein expression in serum.

[0543] III. Oligonucleotide conjugates prepared from GalNAc compounds with similar structures are likely to have very different inhibition rates on PCSK9 protein expression in mouse serum; oligonucleotide conjugates prepared from GalNAc compounds with large structural differences are also likely to have very similar inhibition rates on PCSK9 protein expression in mouse serum.

[0544] 2) Inhibition rate of PCSK9 gene expression in mouse liver

[0545] I. Incc-G4, inc-G5, inc-G6, inc-G7, inc-G11, inc-G12, inc-G13, and G18-inc significantly inhibited PCSK9 gene expression in mouse livers compared to other groups. For example, inc-G5, inc-G6, and inc-G12 all achieved inhibition rates of 90%. Furthermore, compared to inc-L96, inc-NAG0052, inc-GalNAc 1b, and inc-GalNAc 2, prepared using existing GalNAc compounds, the inhibition rates were significantly improved. For example, inc-G5 showed an 11.1% improvement over inc-L96. This demonstrates that cleverly designed linker structures can significantly enhance drug bioavailability and achieve greater efficacy.

[0546] II. The present invention designs three-antennary, one-antennary, or two-antennary GalNAc compounds, and prepares oligonucleotide conjugates with three or four GalNAc groups, all of which have very high inhibition rates. Moreover, these GalNAc compounds, regardless of whether they are conjugated to the 3' end or the 5' end of the oligonucleotide, can effectively inhibit PCSK9 gene expression in the liver.

[0547] III. Oligonucleotide conjugates prepared from GalNAc compounds with similar structures are likely to have very different inhibition rates on PCSK9 protein expression in mouse liver; oligonucleotide conjugates prepared from GalNAc compounds with large structural differences are also likely to have very similar inhibition rates on PCSK9 gene expression in mouse liver.

[0548] 3) Effects on LDL-C levels in mice

[0549] I. inc-G4, inc-G5, inc-G6, inc-G7, inc-G11, inc-G12, inc-G13, and G18-inc resulted in significantly higher LDL-C reductions than the other groups. For example, inc-G5 achieved reductions of 49.6% and 54.1% on days 7 and 14, respectively. Furthermore, compared to inc-L96, inc-NAG0052, inc-GalNAc 1b, and inc-GalNAc 2, all prepared using prior art GalNAc compounds, the reduction in LDL-C in mouse serum was significantly enhanced. For example, inc-G5 achieved 20.4% and 23.5% improvements over inc-L96 on days 7 and 14, respectively. This demonstrates that a cleverly designed linker structure can significantly improve drug bioavailability and enhance efficacy.

[0550] II. The present invention designs three-antennary, one-antennary, or two-antennary GalNAc compounds, and prepares oligonucleotide conjugates with three or four GalNAc groups, all of which have very high reduction levels. Moreover, these GalNAc compounds, whether conjugated to the 3' end or the 5' end of the oligonucleotide, can effectively reduce LDL-C levels.

[0551] III. Oligonucleotide conjugates prepared from structurally similar GalNAc compounds are likely to have very different effects on the reduction of LDL-C in mouse serum; oligonucleotide conjugates prepared from structurally different GalNAc compounds are also likely to have very similar effects on the reduction of LDL-C in mouse serum.

[0552] 4) Distribution in mouse liver

[0553] I. The fluorescence intensities of inc-G4, inc-G5, inc-G6, inc-G7, inc-G11, inc-G12, inc-G13, and G18-inc were significantly higher than those of the other groups. For example, the fluorescence intensity of inc-G5 reached 9.12E+09 and 9.16E+09 at 4 and 8 hours, respectively. Furthermore, compared to inc-L96, inc-NAG0052, inc-GalNAc 1b, and inc-GalNAc 2, prepared using existing GalNAc compounds, the fluorescence absorption intensity in mouse liver was significantly improved. For example, inc-G5 showed an improvement of over 50% compared to inc-L96. This demonstrates that a cleverly designed linker structure can significantly enhance drug bioavailability and achieve greater efficacy.

[0554] II. The present invention designs tri-antennary, uni-antennary, or bi-antennary GalNAc compounds to prepare oligonucleotide conjugates with three or four GalNAc groups, all of which have very high fluorescence intensity. Moreover, these GalNAc compounds, whether conjugated to the 3' or 5' end of the oligonucleotide, can efficiently deliver the oligonucleotide to the liver.

[0555] III. Oligonucleotide conjugates prepared from structurally similar GalNAc compounds are likely to have vastly different efficiencies in oligonucleotide delivery to the liver; oligonucleotide conjugates prepared from structurally distinct GalNAc compounds are also likely to have very similar efficiencies in oligonucleotide delivery to the liver.

[0556] 5) Half-life in mice

[0557] I. The half-lives of inc-G4, inc-G5, inc-G6, inc-G7, inc-G11, inc-G12, inc-G13, and G18-inc were significantly improved compared to other groups. For example, the half-lives of inc-G5, inc-G6, inc-G7, inc-G12, and G18-inc reached 110 hours, 103 hours, 103 hours, 101 hours, and 102 hours, respectively. Furthermore, compared to inc-L96, inc-NAG0052, inc-GalNAc1b, and inc-GalNAc 2, prepared using prior art GalNAc compounds, the in vivo half-life of inc-G5 was significantly prolonged in mice. For example, the half-life of inc-G5 was 48.6% higher than that of inc-L96. This demonstrates that cleverly designed linker structures can significantly improve the in vivo pharmacokinetic properties of drugs.

[0558] II. Conjugates with three or four GalNAc groups prepared using the tri-antennary, uni-antennary, or bi-antennary GalNAc compounds designed by the present invention all had long half-lives in mice. Furthermore, the half-lives of the GalNAc compounds were long regardless of whether they were conjugated to the 3' or 5' end of the oligonucleotide.

[0559] III. Conjugates prepared from structurally similar GalNAc compounds are likely to have vastly different half-lives in mice; oligonucleotide conjugates prepared from structurally distinct GalNAc compounds are also likely to have very similar half-lives in mice.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof: in, R1 is oxygen or sulfur; R2 is hydrogen, C 1-4 Alkyl, C 1-4 Alkoxy or halogen; R3 is hydrogen, a hydroxyl protecting group, a phosphorus-containing reactive group, or -CO(CH2) x COOH, wherein x is an integer from 1 to 10, It is controlled pore glass or polystyrene; R4 is hydrogen or a hydroxyl protecting group; A is -(CH2) a -、-(CH2CH2O) b -、-((CH2) c NHCO) d -or-((CH2) c CONH) d -, wherein a is an integer from 1 to 15, b is an integer from 1 to 7, c is an integer from 1 to 7, and d is an integer from 1 to 5; B is -(CH2) e -, where e is an integer from 0 to 7; L is -CONH- or -NHCO-; G is in, T is N-acetyl-galactosamine in which the hydroxyl groups are fully protected by acyl groups, galactose in which the hydroxyl groups are fully protected by acyl groups, galactosamine in which the hydroxyl groups are fully protected by acyl groups, N-formyl-galactosamine in which the hydroxyl groups are fully protected by acyl groups, N-propionyl-galactosamine in which the hydroxyl groups are fully protected by acyl groups, N-n-butyryl-galactosamine in which the hydroxyl groups are fully protected by acyl groups, or N-isobutyryl-galactosamine in which the hydroxyl groups are fully protected by acyl groups, wherein the acyl groups are acetyl or benzoyl groups; X1 is -(CH2) f -or-(CH2CH2O) f CH2-, where f is an integer from 1 to 5; X2 is -(CH2) g -, where g is an integer from 1 to 6; Y1 is 0 or 1; Y2 is 0, 1 or 2; Y3 is 1, 2 or 3; m is an integer from 0 to 4; n is an integer from 0 to 4.

2. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein A is -(CH2) 10 -, -(CH2)7-, -(CH2)8-, -(CH2)9-, -(CH2) 11 -、-(CH2) 12 -, -(CH2CH2O)3-, -(CH2)4NHCO- or -(CH2)6NHCO-.

3. A compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein B is -(CH2)0-, -CH2-, -(CH2)2-, -(CH2)4- or -(CH2)3-.

4. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein R1 is oxygen. 5 . The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1 , wherein R 1 is an α configuration or a β configuration.

6. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein R2 is hydrogen or -OCH3.

7. A compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6, wherein R3 is a hydroxyl protecting group or a phosphorus-containing active reactive group.

8. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 7, wherein R3 is acetyl, 1,1,3,3-tetraisopropyldisiloxanyl, tert-butyldimethylsilyl, phenyldimethylsilyl, 4,4′-dimethoxytrityl or 9. A compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6, wherein R3 is 10. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6, wherein R3 is -CO(CH2)2COOH.

11. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6, wherein R4 is a hydroxyl protecting group.

12. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 11, wherein R4 is trityl, monomethoxytrityl or 4,4'-dimethoxytrityl.

13. A compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6, wherein m is 1.

14. A compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6, wherein n is 0.

15. A compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6, wherein G is 16. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein the compound of formula (I) is a compound YK-GAL-301, YK-GAL-302, YK-GAL-303, YK-GAL-304, YK-GAL-305, YK-GAL-306, YK-GAL-307, YK-GAL-308, YK-GAL-309, YK-GAL-310, YK-GAL-311, YK-GAL-312 or YK-GAL-313 having the following structure: in It is controlled pore glass.

17. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein the compound of formula (I) is a compound YK-GAL-314, YK-GAL-315, YK-GAL-316, YK-GAL-317, YK-GAL-318, YK-GAL-319, YK-GAL-320, YK-GAL-321, YK-GAL-322, YK-GAL-323, YK-GAL-324, YK-GAL-325 or YK-GAL-326 having the following structure:

18. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6, which is capable of binding to asialoglycoprotein receptor (ASGPR).

19. A conjugate comprising an oligonucleotide and a GalNAc moiety having the structure shown below: Among them, Ol igo represents oligonucleotide, and R1, R2, R3, R4, A, B, L, G, m and n are the same as those in claim 1.

20. The conjugate according to claim 19, wherein the hydroxyl protecting group acetyl group in N-acetyl-galactosamine of G is removed.

21. The conjugate of claim 19 or 20, wherein the oligonucleotide comprises a non-thio oligonucleotide and a thio oligonucleotide.

22. The conjugate of claim 21, wherein the non-thio oligonucleotide and the GalNAc moiety are linked by a phosphate bond.

23. The conjugate of claim 21, wherein the thio-oligonucleotide and the GalNAc moiety are linked by a phosphorothioate bond.

24. The conjugate of claim 21, wherein the oligonucleotide comprises a small interfering nucleotide (siRNA), a DNA, a micro RNA (miRNA), a small activating RNA (saRNA), a small guide RNA (sgRNA), a transfer RNA (tRNA), an antisense nucleotide (ASO), or an aptamer.

25. The conjugate according to claim 24, wherein each nucleotide in the antisense nucleotide (ASO) or small interfering nucleotide (siRNA) is independently a modified or unmodified nucleotide.

26. The conjugate of claim 21, wherein the oligonucleotide modulates expression of a target gene.

27. A pharmaceutical composition comprising the conjugate of any one of claims 19-26 and at least one pharmaceutically acceptable excipient.

28. Use of the conjugate according to any one of claims 19 to 26 or the pharmaceutical composition according to claim 27 in the preparation of a medicament for treating and / or preventing a pathological condition or disease caused by the expression of a specific gene in hepatocytes, optionally, the specific gene is selected from the hepatitis B virus gene, angiopoietin 3 gene or apolipoprotein C3 gene.

29. The use according to claim 28, wherein The disease is selected from chronic liver disease, hepatitis, liver fibrosis, liver proliferative disease and dyslipidemia; optionally, the dyslipidemia is hypercholesterolemia, hypertriglyceridemia or atherosclerosis.

30. A method for inhibiting the expression of a specific gene in a hepatocyte, comprising contacting an effective amount of the conjugate according to any one of claims 19 to 26 or the pharmaceutical composition according to claim 27 with the hepatocyte; Optionally, the specific gene is selected from one of the following genes: proprotein convertase subtilisin / kexin type 9 gene (PCSK9), ApoB, ApoC, ANGPTL3, SCD1, FVII, p53, HBV, HCV; Optionally, the specific gene is selected from the group consisting of proprotein convertase subtilisin / kexin type 9 gene (PCSK9), hepatitis B virus gene, angiopoietin-like protein 3 gene or apolipoprotein C3 gene.

31. A kit comprising the conjugate according to any one of claims 19-26.

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