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

Abstract

The present invention provides a GalNAc compound containing a ribose ring or its derivative structure and an oligonucleotide conjugate thereof. With the oligonucleotide conjugate provided by the present invention, efficient liver-targeted delivery can be achieved, improving the drug efficacy.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 202311118366.4, with the invention title of "A GalNAc Compound Containing a Ribose Ring or Its Derivative Structure and Its Oligonucleotide Conjugate". Technical Field

[0002] The present invention relates to the field of biomedicine, and specifically relates 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, have been widely used due to their simple synthesis and high activity. Oligonucleotide drugs usually include antisense oligonucleotides (ASO), small interfering RNAs (siRNA), microRNAs (miRNA), and aptamers, etc.

[0004] Oligonucleotides are a class of short DNA or RNA molecules, oligomers. Oligonucleotides can easily bind to their respective complementary oligonucleotides, DNA, or RNA in a sequence-specific manner to form double-stranded bodies, or more rarely, higher-order hybrids. This basic property makes oligonucleotides widely used in gene detection, research on targeted gene therapy, and medicine. These small nucleic acid fragments can be fabricated into single-stranded molecules with any specified sequence. In nature, oligonucleotides are usually small RNA molecules that play a role in gene expression regulation, or degradation intermediates derived from the decomposition 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 decomposing target mRNA.

[0006] N-acetylgalactosamine (GalNAc) is a ligand that binds to the asialoglycoprotein receptor (ASGPR) on the liver surface. 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 by 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 exhibited gene silencing activity in mice (Nair JK, et al. J. Am. Chem. Soc. 2014, 136, 16958). The conjugate of GalNAc and siRNA reported in the article showed good delivery activity in both in vivo and in vitro experiments. Through in vivo experiments on mice by subcutaneous administration, a single dose of ED 50It is determined to be 1 mg / kg, and the single injection dose is less than 1 mL. In the long-term administration experiment, subcutaneous injection is performed once a week, and stable interference activity can be obtained for up to 9 months. It is found that the tetra-antennary and tri-antennary GalNAc compounds have much higher affinity for ASGPR than the di-antennary and mono-antennary GalNAc compounds.

[0007] Proprotein convertase subtilisin / kexin type 9 (PCSK9) is a glycoprotein composed of 692 amino acids, which is the ninth member of the proprotein convertase (PCs) family. It is a secreted serine protease, mainly expressed in tissues such as the liver and intestine, and then secreted into the blood. After entering the blood circulation, PCSK9 can specifically bind to the epidermal growth factor-like domain of the low-density lipoprotein receptor (LDL-R) on the surface of hepatocytes, guiding it into hepatocytes to reach lysosomes, causing LDL-R to be degraded in lysosomes, thereby reducing the LDL-R on the surface of hepatocytes, and further reducing the ability of the liver to bind and clear LDL-C, ultimately leading to an increase in the level of LDL-C in the blood. Therefore, hypercholesterolemia can be treated by inhibiting PCSK9. In addition, the latest research shows that the elevation of PCSK9 is closely related to obesity and type 2 diabetes, and is also closely related to chronic kidney diseases such as nephrotic syndrome and proteinuria. Therefore, inhibiting PCSK9 can become an important means for preventing and treating these related diseases.

[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 inhibitor drugs, there is still a need in the art to develop new GalNAc ligand compounds. Summary of the Invention

[0009] The present invention designs a series of GalNAc compounds. Through solid-phase synthesis methods, the 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.

[0010] In a first aspect, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0011]

[0012] Wherein,

[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, a hydroxyl protecting group, a phosphorus-containing reactive group, or -CO(CH2) x COOH, where x is an integer from 1 to 10, 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 -, where a is an integer from 1 to 15, preferably an integer from 3 to 13, b is an integer from 1 to 7, preferably an integer from 2 to 5, c is an integer from 1 to 7, preferably an integer from 2 to 6, and d is an integer from 1 to 5, preferably an integer from 1 to 3;

[0018] B is -(CH2) e -, where e is an integer from 0 to 7, preferably an integer from 1 to 5.

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

[0020] G is

[0021] where,

[0022] T is N-acetyl-galactosamine with all hydroxyl groups fully protected by acyl groups, galactose with all hydroxyl groups fully protected by acyl groups, galactosamine with all hydroxyl groups fully protected by acyl groups, N-formyl-galactosamine with all hydroxyl groups fully protected by acyl groups, N-propionyl-galactosamine with all hydroxyl groups fully protected by acyl groups, N-n-butanoyl-galactosamine with all hydroxyl groups fully protected by acyl groups, or N-isobutanoyl-galactosamine with all hydroxyl groups fully protected by acyl groups, preferably N-acetyl-galactosamine with all hydroxyl groups fully protected by acyl groups, where the acyl group is, 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 from 0 to 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 β-configuration.

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

[0035] In one embodiment, R3 is a hydroxyl protecting group or a phosphorus-containing reactive group. Preferably, R3 is an acyl group, silyl, trityl, monomethoxytrityl, 4,4'-dimethoxytrityl or More preferably, R3 is acetyl, 1,1,3,3-tetraisopropyldisiloxanylidene (TIPDS), 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 hydroxyl 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]

[0042] 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:

[0043]

[0044]

[0045]

[0046] wherein is controlled pore glass.

[0047] 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:

[0048]

[0049]

[0050]

[0051] Optionally, the compound of formula (I) or a pharmaceutically acceptable salt thereof can bind to the asialoglycoprotein receptor (ASGPR).

[0052] In a second aspect, the present invention provides a conjugate comprising an oligonucleotide and a GalNAc moiety, having the structure shown below:

[0053]

[0054] Wherein, Oligo represents an oligonucleotide, and R1, R2, R3, R4, A, B, L, G, m and n are as described above.

[0055] Optionally, the acetyl group, which is a hydroxyl protecting group in N-acetyl-galactosamine of G in the conjugate, is removed.

[0056] Optionally, the oligonucleotide includes a non-thiolated oligonucleotide and a thiolated oligonucleotide. In one embodiment, the non-thiolated oligonucleotide and the GalNAc moiety are linked by a phosphodiester bond. In another embodiment, the thiolated oligonucleotide and the GalNAc moiety are linked by a phosphorothioate bond.

[0057] Optionally, the oligonucleotide includes small interfering ribonucleic acid (siRNA), deoxyribonucleic acid (DNA), micro ribonucleic acid (miRNA), small activating RNA (saRNA), small guide RNA (sgRNA), transfer ribonucleic acid (tRNA), antisense oligonucleotide (ASO) or aptamer, preferably antisense oligonucleotide (ASO) or small interfering ribonucleic acid (siRNA). In one embodiment, each nucleotide in the antisense oligonucleotide (ASO) or small interfering ribonucleic acid (siRNA) is independently a modified or unmodified nucleotide.

[0058] Optionally, the oligonucleotide regulates the expression of a target gene.

[0059] In a third aspect, the present invention provides a pharmaceutical composition comprising the conjugate of the second aspect and at least one pharmaceutically acceptable excipient.

[0060] In a fourth aspect, the present invention provides the use of the conjugate of the second aspect or the pharmaceutical composition of the third aspect in the preparation of a drug 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 hepatitis B virus gene, angiopoietin-3 gene or apolipoprotein C3 gene.

[0061] In one embodiment, the disease is selected from chronic liver disease, hepatitis, liver fibrosis disease, liver hyperplastic disease and dyslipidemia; optionally, the dyslipidemia is hypercholesterolemia, hypertriglyceridemia or atherosclerosis.

[0062] Fifth aspect, the present invention provides a method for inhibiting the expression of a specific gene in hepatocytes, comprising contacting the hepatocytes with an effective amount of the conjugate of the second aspect or the pharmaceutical composition of the third aspect;

[0063] 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;

[0064] Optionally, the specific gene is selected from the proprotein convertase subtilisin / kexin type 9 gene (PCSK9), hepatitis B virus gene, angiopoietin-like protein 3 gene or apolipoprotein C3 gene.

[0065] Sixth aspect, the present invention provides a kit, which comprises the conjugate of the second aspect. Description of the Drawings

[0066] 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 and do not limit the present invention.

[0067] Figure 1 Showing the inhibition rates 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 the expression of PCSK9 protein in mouse serum on the 7th day and the 14th day.

[0068] Figure 2 Showing the inhibition rates of GalNAc-conjugated siRNA sequences inc-G1, inc-G2, inc-G3, inc-G8, inc-G9, inc-G10, inc-G25, G-inc26, inc-L96, inc-NAG0052, inc-GalNAc 1b and inc-GalNAc 2 on the expression of PCSK9 protein in mouse serum on the 7th day and the 14th day.

[0069] Figure 3Show the inhibition rates 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 PCSK9 gene expression in mouse liver on the 14th day.

[0070] Figure 4 Show the inhibition rates of GalNAc-conjugated siRNA sequences inc-G1, inc-G2, inc-G3, inc-G8, inc-G9, inc-G10, inc-G25, G-inc26, inc-L96, inc-NAG0052, inc-GalNAc 1b, and inc-GalNAc 2 on PCSK9 gene expression in mouse liver on the 14th day.

[0071] Figure 5 Show 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 the 7th and 14th days.

[0072] Figure 6 Show the effects of GalNAc-conjugated siRNA sequences inc-G1, inc-G2, inc-G3, inc-G8, inc-G9, inc-G10, inc-G25, G-inc26, inc-L96, inc-NAG0052, inc-GalNAc 1b, and inc-GalNAc 2 on LDL-C levels in mouse serum on the 7th and 14th days.

[0073] Figure 7 Show the in vivo imaging of mice after 8 hours of administration of GalNAc-conjugated siRNA sequences inc-G5, inc-G7, inc-L96, and inc-GalNAc 1b groups. (a: inc-G5; b: inc-G7; c: inc-L96; d: inc-GalNAc 1b)

[0074] Figure 8To show 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 the mouse liver.

[0075] Figure 9 To show the half-lives of GalNAc-conjugated siRNA sequences inc-G1, inc-G2, inc-G3, inc-G8, inc-G9, inc-G10, inc-G25, G-inc26, inc-L96, inc-NAG0052, inc-GalNAc 1b, and inc-GalNAc 2 in the mouse liver. Detailed implementation manners

[0076] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the described embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0077] The present invention can be implemented in other specific forms without departing from its basic attributes. It should be understood that, on the premise of no conflict, any and all implementation manners of the present invention can be combined with the technical features in any other implementation manner or multiple other implementation manners to obtain additional implementation manners. The present invention includes such additional implementation manners obtained by such combinations.

[0078] All publications and patents mentioned in the present invention are hereby incorporated by reference in their entirety into the present invention. If there are conflicts between the uses or terms used in any incorporated publications and patents and those used in the present invention, then the uses and terms of the present invention shall prevail.

[0079] The section titles used herein are only for the purpose of organizing the article and should not be construed as limiting the subject matter described.

[0080] Unless otherwise specified, all technical terms and scientific terms used herein have the ordinary meanings in the field to which the claimed subject matter belongs. If there are multiple definitions for a certain term, the definition in this article shall prevail.

[0081] Except where otherwise indicated in the working examples or elsewhere, all numbers expressing quantitative properties such as dosages stated in the specification and claims are to be understood as being modified in all instances by the term "about". It should also be understood that any numerical range recited in this application is intended to include all sub-ranges within that range and any combination of the individual endpoints of that range or sub-ranges.

[0082] As used herein, words such as "comprising", "containing" or "including" and the like are intended to mean that the elements appearing before the word encompass the elements recited after the word and their equivalents, without excluding elements not recited. The term "containing" or "including (comprising)" used herein may be open-ended, semi-closed and closed. In other words, the said term also includes "consisting essentially of...", or "consisting of...".

[0083] The term "pharmaceutically acceptable" as used in this application means that a compound or composition is chemically and / or toxicologically compatible with the other components of the formulation and / or with the human or mammalian subject to which it is administered for the prevention or treatment of a disease or disorder.

[0084] The term "pharmaceutically acceptable salt" refers to relatively non-toxic, inorganic or organic acid addition salts of the compounds of the present invention. For example, see S.M. 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, etc.; 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, aminosulfonic 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 pharmaceutically acceptable salts with the compounds of formula (I).

[0085] The term "alkyl" as used in this application refers to branched and straight-chain saturated aliphatic monovalent hydrocarbon groups having a specified number of carbon atoms. For example, "C 1-4 alkyl" includes methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl.

[0086] The term "alkoxy" refers to -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.

[0087] "Halogen" refers to fluorine (F), chlorine (Cl), bromine (Br), and iodine (I), preferably fluorine (F) and chlorine (Cl). In one embodiment, the halogen is fluorine.

[0088] The term "protecting group" as used herein refers to any atom or group of atoms that is introduced into a molecule to prevent an existing group in the molecule from undergoing an undesired chemical reaction and that can be removed to leave the unprotected group.

[0089] Hydroxy protecting groups can be those protecting groups that are commonly used to protect the hydroxyl groups of ribose structures in the synthesis of RNA or its derivatives, and reference can also be made to the protecting groups described in the literature of Green et al. in Protective Groups in Organic Synthesis, 3rd Edition, 1999, John Wiley & Sons, Inc., such as: acetyl, phenoxyacetyl, pivaloyl, benzyl, 4-methoxybenzyl, benzoyl, triphenylmethyl, 4,4'-dimethoxytriphenylmethyl (DMTr), monomethoxytriphenylmethyl (MMTr), 9-phenyl-xanthen-9-yl, 9-(p-tolyl)-xanthen-9-yl, trimethylsilyl, tert-butyldimethylsilyl (TBDMS), cyanomethoxymethyl, 2-(cyanoethoxy)ethyl, cyanomethoxymethyl, etc., preferably 4,4'-dimethoxytriphenylmethyl (DMTr, 4,4'-dimethoxytrityl).

[0090] The term "phosphorus-containing reactive group" as used herein refers to a phosphorus-containing group that is capable of reacting, through a nucleophilic attack reaction, with a hydroxyl group or an amino group contained in another molecule, especially another nucleotide unit or another nucleotide analogue. Generally, such a reaction produces an ester-type internucleoside bond that links a nucleotide unit or a nucleotide analogue unit to another nucleotide unit or another nucleotide analogue unit. These phosphorus-containing reactive groups are known in the art and include P III or P VPhosphorus atoms in a valence state, and the phosphorus-containing active reaction groups include, but are not limited to, phosphoramidites, H-phosphonates, triphosphates, and phosphorus-containing chiral auxiliaries. Examples of phosphorus-containing active reaction groups are:

[0091] 2-cyanoethoxy-N,N-diisopropylaminophosphino,

[0092] 2-propenyloxy--N,N-diisopropylaminophosphino,

[0093] methoxy-N,N-diisopropylaminophosphino,

[0094] bis(diisopropylamino)phosphino, etc. In one embodiment, the phosphorus-containing active reaction group is

[0095] The controlled pore glass (CPG) and polystyrene (highly cross-linked polystyrene microbeads) described herein are solid supports for oligonucleotide synthesis, which are insoluble particles that bind to oligonucleotides during synthesis and are commercially available.

[0096] The term "nucleotide" as used herein includes naturally occurring nucleotides and chemically modified nucleotides. Chemically modified nucleotides are non-naturally occurring nucleotides and are also referred to as "nucleotide analogs" herein.

[0097] "The hydroxyl group is fully protected with an acyl group" described for "T" herein means that in the galactose structure, other hydroxyl groups except the hydroxyl group used for connection with X1 are protected with an acyl group, where the acyl group is, for example, acetyl, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, isobutyryl, or benzoyl.

[0098] Compound of formula (I) or a pharmaceutically acceptable salt thereof

[0099] The present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0100]

[0101] Wherein,

[0102] R1 is oxygen or sulfur, preferably oxygen;

[0103] R2 is hydrogen, C 1-4 alkyl, C 1-4 alkoxy, or halogen;

[0104] R3 is hydrogen, a hydroxyl-protecting group, a phosphorus-containing active reaction group, or -CO(CH2) x COOH, where x is an integer from 1 to 10, is controlled pore glass or polystyrene;

[0105] R4 is hydrogen or a hydroxyl protecting group;

[0106] A is -(CH2) a -, -(CH2CH2O) b -,-((CH2) c NHCO) d - or -((CH2) c CONH) d -, where a is an integer from 1 to 15, preferably an integer from 3 to 13, b is an integer from 1 to 7, preferably an integer from 2 to 5, c is an integer from 1 to 7, preferably an integer from 2 to 6, and d is an integer from 1 to 5, preferably an integer from 1 to 3;

[0107] B is -(CH2) e -, where e is an integer from 0 to 7, preferably an integer from 1 to 5.

[0108] L is -CONH- or -NHCO-;

[0109] G is

[0110] where

[0111] T is N-acetyl-galactosamine with all hydroxyl groups fully protected by acyl groups, galactose with all hydroxyl groups fully protected by acyl groups, galactosamine with all hydroxyl groups fully protected by acyl groups, N-formyl-galactosamine with all hydroxyl groups fully protected by acyl groups, N-propionyl-galactosamine with all hydroxyl groups fully protected by acyl groups, N-n-butanoyl-galactosamine with all hydroxyl groups fully protected by acyl groups, or N-isobutanoyl-galactosamine with all hydroxyl groups fully protected by acyl groups. For example, T is N-acetyl-galactosamine with all hydroxyl groups fully protected by acyl groups, where the acyl group is acetyl or benzoyl, preferably acetyl;

[0112] X1 is -(CH2) f - or -(CH2CH2O) f CH2-, where f is an integer from 1 to 5;

[0113] X2 is -(CH2) g -, where g is an integer from 1 to 6;

[0114] Y1 is 0 or 1;

[0115] Y2 is 0, 1 or 2;

[0116] Y3 is 1, 2 or 3;

[0117] m is an integer from 0 to 4, preferably 0, 1 or 2;

[0118] n is an integer from 0 to 4, preferably 0, 1 or 2.

[0119] In one embodiment, A is -(CH2) 10 -, -(CH2)7-, -(CH2)8-, -(CH2)9-, -(CH2) 11 -, -(CH2) 12 -, -(CH2CH2O)3-, -(CH2)4NHCO- or -(CH2)6NHCO-.

[0120] In one embodiment, B is -(CH2)0-, -CH2-, -(CH2)2-, -(CH2)4- or -(CH2)3-.

[0121] In one embodiment, R1 is oxygen.

[0122] In one embodiment, R1 is in the α configuration or the β configuration.

[0123] In one embodiment, R2 is hydrogen or -OCH3.

[0124] 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), silyl, trityl, monomethoxytrityl (MMTr) or 4,4'-dimethoxytrityl (DMTr). 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

[0125] In another embodiment, R3 is In yet another embodiment, R3 is -CO(CH2)2COOH.

[0126] In one embodiment, R4 is a hydroxyl protecting group, preferably trityl, monomethoxytrityl or 4,4'-dimethoxytrityl, more preferably 4,4'-dimethoxytrityl.

[0127] In one embodiment, m is 1.

[0128] In one embodiment, n is 0.

[0129] In one embodiment, G is

[0130]

[0131] 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:

[0132]

[0133]

[0134]

[0135] wherein is controlled pore glass.

[0136] 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:

[0137]

[0138]

[0139]

[0140] Optionally, the compound of formula (I) or a pharmaceutically acceptable salt thereof can bind to the asialoglycoprotein receptor (ASGPR).

[0141] Conjugate

[0142] The present invention provides a conjugate comprising an oligonucleotide and a GalNAc moiety, having the structure shown below:

[0143]

[0144] Wherein, Oligo represents an oligonucleotide, and R1, R2, R3, R4, A, B, L, G, m and n are as described above.

[0145] The conjugate of the present invention can be prepared by a 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 with an oligonucleotide is the solid-phase synthesis method. One method is to connect the GalNAc compound to a CPG (Controlled Pore Glass) column, and realize the connection of the GalNAc compound with the oligonucleotide through solid-phase synthesis. Another method is to first prepare the GalNAc compound into a phosphoramidite monomer, and realize the connection of the GalNAc compound with any position of the oligonucleotide through solid-phase synthesis.

[0146] The oligonucleotides in the present application include single-stranded oligonucleotides (such as antisense nucleotides, abbreviated as ASO) and double-stranded oligonucleotides (such as small interfering nucleotides, abbreviated as siRNA). In a preferred embodiment, the oligonucleotide comprises 7-30 nucleotides. The conjugate prepared by such an oligonucleotide will have greater therapeutic value.

[0147] In one embodiment, the oligonucleotide is selected from small interfering nucleotides, DNA, microRNA (miRNA), small activating RNA (saRNA), small guide RNA (sgRNA), transfer RNA (tRNA), antisense nucleotides or aptamers, and preferably the oligonucleotide is an antisense nucleotide or a small interfering nucleotide.

[0148] The oligonucleotides in the present application include natural oligonucleotides and chemically modified oligonucleotides. The chemical modifications herein include nucleoside modifications (including sugar moiety modifications and nucleobase modifications) and internucleoside linkage modifications. The chemical modification of the oligonucleotide does not include the case where there are only differences in the nucleobase sequence. The natural herein refers to the corresponding case of naturally occurring RNA or DNA.

[0149] Natural oligonucleotides have difficulty entering cells and are easily degraded by intracellular nucleases, resulting in poor effects. By chemically modifying oligonucleotides, their properties can be improved and their bioavailability increased. Among the many modified oligonucleotides, the most representative is phosphorothioate oligonucleotides, in which one non-bridging oxygen atom in the phosphodiester bond is replaced by a sulfur atom. For example

[0150] Phosphorothioate oligonucleotides can be commercially available or prepared by conventional solid-phase synthesis methods. Hydrogen xanthate can be used as a sulfurizing agent. For example, reference can be made to CN1479745A and CN113150041A for synthesizing phosphorothioate oligonucleotides.

[0151] Optionally, the acetyl protecting group on the hydroxyl group in N-acetyl-galactosamine of G in the conjugate is removed, for example, by hydrolysis in an alkaline solution to obtain a hydroxyl group.

[0152] In one embodiment, the GalNAc moiety of the conjugate has the structure

[0153]

[0154] R1, R2, A, B, L, X1, X2, Y1, Y2, Y3, m and n are as described above.

[0155] For example, the oligonucleotide and the GalNAc moiety are linked by a bond or a cleavable linker. The bond here may include but is not limited to a phosphoester bond and a phosphorothioate bond.

[0156] The cleavable linker used in this application 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. The acid-labile linker can refer to the acid-labile linker in ADC drugs (Mylotarg, Besponsas, Trodelvys).

[0157] Optionally, the oligonucleotide includes non-phosphorothioate oligonucleotides and phosphorothioate oligonucleotides. In one embodiment, the non-phosphorothioate oligonucleotide and the GalNAc moiety are linked by a phosphoester bond. In another embodiment, the phosphorothioate oligonucleotide and the GalNAc moiety are linked by a phosphorothioate bond.

[0158] Optionally, the oligonucleotide includes small interfering nucleotide (siRNA), DNA, microRNA (miRNA), small activating RNA (saRNA), small guide RNA (sgRNA), transfer RNA (tRNA), antisense oligonucleotide (ASO), or aptamer, preferably antisense oligonucleotide (ASO) or small interfering nucleotide (siRNA). In one embodiment, each nucleotide in the antisense oligonucleotide (ASO) or small interfering nucleotide (siRNA) is independently a modified or unmodified nucleotide.

[0159] Optionally, the oligonucleotide regulates the expression of a target gene.

[0160] Pharmaceutical composition

[0161] Except for the conjugate, the compositions provided in the present application may include any substances that can be used in pharmaceutical compositions. For example, the composition may include one or more pharmaceutically acceptable excipients or adjuvants, such as but not limited to one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, granulation aids, disintegrants, fillers, glidants, liquid vehicles, binders, surfactants, isotonic agents, thickening agents or emulsifiers, buffers, lubricants, oils, preservatives, flavoring agents, 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).

[0162] 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, and also provides suitable dosages, formulations, routes of administration, compositions, dosage forms, combinations with other therapeutic agents, prodrug formulations (this document is incorporated by reference).

[0163] The oligonucleotide conjugate of the present invention can be mixed with pharmaceutically active or inert substances to prepare pharmaceutical compositions or formulations, can be sterilized by conventional sterilization techniques, or can be sterile filtered.

[0164] Kit

[0165] The present application also provides a kit comprising the conjugate as described above. In some embodiments, the kit provided by the present application comprises a container, which contains the conjugate as described above. In some embodiments, the kit provided by the present application further includes pharmaceutically acceptable excipients, such as stabilizers or preservatives. In some embodiments, the kit further comprises instructions for mixing the conjugate with pharmaceutically acceptable excipients or other components (if any).

[0166] Examples

[0167] The present invention will be further described below in conjunction with 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 different requirements of specific use, and 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 specified, all temperatures are given in degrees Celsius. The technical features involved in each embodiment of the present invention can be combined with each other as long as they do not conflict with each other.

[0168] Example 1: Synthesis of GalNAc-CPG compound

[0169] The following abbreviated letters represent the following reagents respectively: DCM: dichloromethane; PE: petroleum ether; EA: ethyl acetate; THF: tetrahydrofuran; DMF: N,N-dimethylformamide; ACN: acetonitrile; BF3·Et2O: boron trifluoride diethyl etherate; LiOH·H2O: lithium hydroxide monohydrate; DMTrCl: 4,4'-dimethoxytriphenyl chloride; TEAB: triethylammonium bicarbonate aqueous solution; HBTU: O-benzotriazole-N,N,N',N'-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(dimethylamino)naphthalene.

[0170] 1. Synthesis of YK-GAL-301

[0171] The synthesis route is as follows:

[0172]

[0173] Step 1: Synthesis of G1-3

[0174] To dry compound G1-1 (781 mg, 3.00 mmol), dry compound G1-2 (0.50 g, 2.31 mmol) and 3A molecular sieve (2.0 g), add dichloromethane (20 mL). After cooling to 0 °C, add boron trifluoride diethyl etherate (656 mg, 4.62 mmol), and continue stirring for 2 h. Quench the reaction by adding saturated sodium bicarbonate aqueous solution (20 mL), extract with dichloromethane (10 mL × 2), dry the organic phase with anhydrous sodium sulfate, and concentrate under reduced pressure to remove the solvent to obtain the crude product. Purify by column chromatography (PE / EA) to obtain yellow oil G1-3 (1.17 g, 2.81 mmol, α, β mixed isomers, 93.7%). After preparative separation by high pressure, obtain G8-1 (533.2 mg, 1.28 mmol, α configuration, 42.7%), G1-3 (625.6 mg, 1.5 mmol, β configuration, 50.1%). 1 HNMR (400 MHz, CDCl3) δ ppm 5.23–5.13 (m, 2H), 4.27 (dd, J = 11.0, 5.6 Hz, 1H), 4.23–4.15 (m, 1H), 4.09 (dd, J = 11.0, 6.4 Hz, 1H), 3.69–3.63 (m, 4H), 3.37–3.28 (m, 1H), 2.39–2.33 (m, 1H), 2.29 (t, J = 7.6 Hz, 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.

[0175] Step 2: Synthesis of G1-4

[0176] Dissolve G1-3 (0.12 g, 288 μmol) in tetrahydrofuran (3.0 mL), add 1.5 mL of an aqueous solution of lithium hydroxide monohydrate (38.7 mg, 921 μmol), and stir at 15 °C for 12 h. Concentrate the reaction solution under reduced pressure to remove the solvent to obtain a yellow solid. The crude product G1-4 (92 mg) is directly used for the next step. MS (ESI) m / z [M+Na] + = 341.2.

[0177] Step 3: Synthesis of G1-5

[0178] The crude product G1-4 (92 mg, 288 μmol) and anhydrous pyridine (20 mL × 3) were co-evaporated to remove water. The dried G1-4 and 4,4'-dimethoxytriphenyl chloromethane (195 mg, 576 μmol) were dissolved in pyridine (3.0 mL), and stirred at 15 °C for 28 h under nitrogen protection. Methanol (10 mL) was added to quench the reaction, and the solvent was removed by concentration under reduced pressure to obtain a crude product. Purification by preparative chromatography (H2O (10 mM NH4HCO3)-ACN) gave a white solid G1-5 (109.5 mg, 176.5 μmol) with a yield of 61.3%. 1 HNMR (400 MHz, CDCl3) δ ppm 7.48–7.41 (m, 2H), 7.37–7.27 (m, 6H), 7.21 (dd, J = 8.2, 6.2 Hz, 1H), 6.88–6.78 (m, 4H), 5.15 (dd, J = 5.3, 2.2 Hz, 1H), 4.43–4.39 (m, 1H), 3.98–3.91 (m, 1H), 3.79 (s, 6H), 3.62–3.56 (m, 1H), 3.35–3.23 (m, 2H), 3.15 (dd, J = 9.4, 6.8 Hz, 1H), 2.33 (t, J = 7.4 Hz, 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.

[0179] Step 4: Synthesis of G1-6

[0180] G1-5 (40 mg, 64.4 μmol), diisopropylethylamine (16.7 mg, 129 μmol) and O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (36.7 mg, 96.6 μmol) were dissolved in N,N-dimethylformamide (0.5 mL), and GalNAc-NH2.TFA (123 mg, 64.4 μmol, TFA) was added. The mixture was stirred at 15 °C for 1 h under nitrogen protection. The reaction solution was directly used for the next step. MS (ESI) m / z [M-2H] 2- = 1197.4.

[0181] Step 5: Synthesis of G1-7

[0182] Dissolve 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) in N,N-dimethylformamide (2.0 mL), and stir at 30 °C for 48 h under nitrogen protection. Purify by preparative chromatography (H2O (10 mM TEAB)-ACN) to obtain white solid G1-7 (59 mg, 26.7 μmol) with a yield of 41.5%. 1 HNMR (400 MHz, CDCl3) δ ppm 7.44 (d, J = 7.6 Hz, 2H), 7.32 (d, J = 8.5 Hz, 3H), 7.25–7.14 (m, 5H), 6.97 (t, J = 6.3 Hz, 3H), 6.78 (dd, J = 11.8, 8.6 Hz, 6H), 6.48 (s, 1H), 5.33 (d, J = 3.2 Hz, 3H), 5.19–5.15 (m, 5H), 4.59 (d, J = 8.3 Hz, 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 = 5.8 Hz, 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.

[0183] Step 6: Synthesis of YK-GAL-301

[0184] Dissolve G1-7 (59 mg, 22.7 μmol), 4-dimethylaminopyridine (2.77 mg, 22.7 μmol), diisopropylethylamine (23.5 mg, 182 μmol), and O-benzotriazole-tetramethylurea hexafluorophosphate (43.1 mg, 113 μmol) in N,N-dimethylformamide (4.0 mL), then add CPG-NH2 (425 mg), and stir at 40 °C for 12 h. Filter the reaction solution, wash the filtrate successively with methanol and dichloromethane, and dry in vacuo. Then add the filtrate to a 4 mL acetic anhydride / pyridine (1:5) solution, and stir at 40 °C for 0.5 h. Filter, wash successively with dichloromethane and methanol, and dry the filtrate in vacuo for 12 h to obtain white solid compound YK-GAL-301 (338 mg, loading 27.0 μmol / g).

[0185] 2. Synthesis of YK-GAL-302

[0186] The synthesis route is as follows:

[0187]

[0188] Step 1: Synthesis of G2-1

[0189] Dissolve compound G2 (800 mg, 3.84 mmol) in methanol (16 mL), add sulfuric acid (1.18 g, 11.7 mmol), and stir at 70 °C for 16 h. Add 10 mL of sodium sulfate to quench the reaction, and concentrate under reduced pressure to remove the solvent to obtain a crude product. Purify by column chromatography (DCM / MeOH) to obtain colorless oily substance G2-1 (470 mg, 2.11 mmol) with a yield of 55.0%. 1 HNMR (400 MHz, CDCl3) δ ppm 4.17 (s, 2H), 3.78–3.65 (m, 13H), 3.61 (dd, J = 5.4, 3.7 Hz, 2H), 2.30 (s, 1H)

[0190] Step 2: Synthesis of G2-2

[0191] Add dichloromethane (9.0 mL) to compound G1-1 (660 mg, 2.54 mmol), compound G2-1 (470 mg, 2.11 mmol) and 4A molecular sieve (900 mg). After cooling to 0 °C, add boron trifluoride diethyl etherate (600 mg, 4.23 mmol), and continue to stir for 2 h. Then stir at 15 °C for 3 h. Add saturated sodium bicarbonate aqueous solution (30 mL) to quench the reaction, extract with dichloromethane (30 mL × 3), dry the organic phase with anhydrous sodium sulfate, and concentrate under reduced pressure to remove the solvent to obtain a crude product. Purify by column chromatography (PE / EA) to obtain yellow oily substance G2-2 (443.3 mg, 1.05 mmol) with a yield of 49.8%. 1 HNMR (400 MHz, CDCl3) δ ppm 5.24 (d, J = 5.4 Hz, 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.

[0192] Step 3: Synthesis of G2-3

[0193] Dissolve G2-2 (320 mg, 757 μmol) in tetrahydrofuran (2.5 mL), add 2.5 mL of an aqueous solution of lithium hydroxide monohydrate (95.3 mg, 2.27 mmol), and stir at 15 °C for 16 h. Concentrate the reaction mixture under reduced pressure to remove the solvent to obtain a white solid. The crude product G2-3 (245 mg) was directly used in the next step. MS (ESI) m / z [M+Na] + = 346.9.

[0194] Step 4: Synthesis of G2-4

[0195] Dissolve the dried crude product G2-3 (245 mg, 755 μmol) and 4,4'-dimethoxytrityl chloride (511 mg, 1.51 mmol) in pyridine (3.0 mL), and stir at 15 °C for 6 h under nitrogen protection. Quench the reaction by adding methanol (2 mL), and concentrate the reaction mixture under reduced pressure to remove the solvent to obtain a crude product. Purify by preparative chromatography (H2O (10 mM NH4HCO3)-ACN) to obtain a white solid G2-4 (309.1 mg, 494 μmol, 65.2% yield). 1 1H NMR (400 MHz, CDCl3) δ ppm 7.47 (d, J = 7.6 Hz, 1H), 7.39–7.33 (m, 4H), 7.20–7.16 (m, 1H), 6.82–6.80 (m, 4H), 5.13 (d, J = 4.8 Hz, 1H), 4.43–4.38 (m, 2H), 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 [M-H] - = 625.4.

[0196] Step 5: Synthesis of G2-5

[0197] Dissolve G2-4 (80.0 mg, 127 μmol), diisopropylethylamine (33.0 mg, 255 μmol, 44.4 μL), and O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (72.6 mg, 191 μmol) in N,N-dimethylformamide (2.0 mL), add GalNAc-NH2.TFA (206 mg, 108 μmol, TFA), and stir at 15 °C for 4 h under nitrogen protection. The reaction mixture was directly used in the next step. MS (ESI) m / z [M-2H] 2- = 1200.3.

[0198] Step 6: Synthesis of G2-6

[0199] Dissolve 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) in N,N-dimethylformamide (3.0 mL), and stir at 30 °C for 48 h under nitrogen protection. Purify by preparative chromatography (H2O (10 mM TEAB)-ACN) to obtain white solid G2-6 (130 mg, 51.9 μmol), with a yield of 40.9%. 1 HNMR (400 MHz, CDCl3) δ ppm 7.45 (s, 1H), 7.43 (s, 1H), 7.32 (d, J = 8.5 Hz, 4H), 7.25–7.16 (m, 4H), 6.95 (dd, J = 36.7, 13.2 Hz, 4H), 6.85–6.72 (m, 6H), 5.35 (d, J = 3.4 Hz, 2H), 5.22 (td, J = 11.0, 10.3, 4.0 Hz, 4H), 4.63 (dd, J = 8.3, 2.6 Hz, 2H), 4.12 (dtd, J = 27.6, 11.1, 4.6 Hz, 9H), 3.96–3.85 (m, 7H), 3.78 (d, J = 2.6 Hz, 5H), 3.71–3.62 (m, 14H), 3.54 (dd, J = 25.9, 6.2 Hz, 7H), 3.33–3.16 (m, 12H), 2.83 (s, 10H), 2.67–2.53 (m, 4H), 2.42 (t, J = 5.9 Hz, 4H), 2.28–2.14 (m, 14H), 2.11–1.93 (m, 26H), 1.67 (ddd, J = 39.3, 13.2, 7.3 Hz, 16H), 1.23 (d, J = 7.4 Hz, 15H). MS (ESI) m / z [M-2H] 2- = 1250.3

[0200] Step 7: Synthesis of YK-GAL-302

[0201] Dissolve 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) in N,N-dimethylformamide (6.0 mL), then add CPG-NH2 (830 mg, 1.41 mmol), and stir at 30 °C for 16 h. Filter the reaction solution, wash the filtrate with methanol and dichloromethane successively, and dry it under vacuum. Then add the filtrate to a 12 mL acetic anhydride / pyridine (1:5) solution, and stir at 40 °C for 0.5 h. Filter, wash with dichloromethane and methanol successively, and dry the filtrate under vacuum for 12 h to obtain the white solid compound YK-GAL-302 (840 mg, loading amount 34.9 μmol / g).

[0202] 3. Synthesis of YK-GAL-303

[0203] The synthesis route is as follows:

[0204]

[0205] Step 1: Synthesis of G3-2

[0206] Add dichloromethane (26 mL) to dry compound G1-1 (1.19 g, 4.58 mmol), dry compound G3-1 (663.2 mg, 3.52 mmol) and 3A molecular sieve (2.0 g). After cooling to 0 °C, add boron trifluoride diethyl etherate (999.95 mg, 7.05 mmol), and then stir at 25 °C for 2 h. Quench the reaction by adding saturated sodium bicarbonate aqueous solution (30 mL), extract with dichloromethane (30 mL × 3), dry the organic phase with anhydrous sodium sulfate, and concentrate under reduced pressure to remove the solvent to obtain the crude product. Purify by column chromatography (PE / EA) to obtain the yellow oil G3-2 (612.2 mg, 1.58 mmol), with a yield of 44.8%. MS (ESI) m / z [M+Na] + = 411.0.

[0207] Step 2: Synthesis of G3-3

[0208] Dissolve G3-2 (130 mg, 335 μmol) in tetrahydrofuran (2.0 mL), add 1.0 mL of an aqueous solution of lithium hydroxide monohydrate (44.9 mg, 1.07 mmol), and stir at room temperature for 16 h. Concentrate the reaction solution under reduced pressure to remove the solvent to obtain a yellow solid. The crude product G3-3 (97 mg) is directly used for the next step. MS (ESI) m / z [M-H] - = 289.3.

[0209] Step 3: Synthesis of G3-4

[0210] The dried G3-3 (97 mg, 335 μmol) and 4,4'-dimethoxytriphenylmethyl chloride (406 mg, 1.2 mmol) were dissolved in pyridine (4.0 mL), and the mixture was stirred at room temperature for 16 h under nitrogen protection. Methanol (10 mL) was added to quench the reaction, and the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (DCM / MeOH) to give white solid G3-4 (126.7 mg, 213.9 μmol) with a yield of 63.86%. MS (ESI) m / z [M-H] - = 591.5.

[0211] Step 4: Synthesis of G3-5

[0212] G3-4 (85 mg, 143 μmol), diisopropylethylamine (37.1 mg, 287 μmol, 49.9 μL), and O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (81.6 mg, 215 μmol) were dissolved in N,N-dimethylformamide (3.0 mL), and GalNAc-NH2·TFA (274 mg, 143 μmol, TFA) was added. The mixture was stirred at room temperature for 2 h under nitrogen protection. The reaction solution was directly used for the next step. MS (ESI) m / z [M-2H] 2- = 1183.2.

[0213] Step 5: Synthesis of G3-6

[0214] 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 the mixture was stirred at 30 °C for 16 h under nitrogen protection. The product was purified by preparative chromatography (H2O (10 mM TEAB)-ACN) to give white solid G3-6 (187 mg, 75.7 μmol) with a yield of 52.8%. 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, 1H), 7.05 (t, J = 6.1 Hz, 2H), 6.96 (d, J = 9.0 Hz, 2H), 6.83–6.75 (m, 4H), 6.50 (d, J = 5.4 Hz, 1H), 5.34–5.31 (m, 2H), 5.18 (dt, J = 11.3, 3.9 Hz, 4H), 4.60 (dd, J = 8.4, 2.6 Hz, 3H), 4.50 (d, J = 8.0 Hz, 1H), 4.18–4.04 (m, 9H), 3.95–3.88 (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 = 7.2 Hz, 8H), 2.57–2.54 (m, 1H), 2.48 (t, J = 6.5 Hz, 2H), 2.41 (t, J = 5.8 Hz, 5H), 2.29–2.06 (m, 18H), 2.05–1.91 (m, 24H), 1.76–1.51 (m, 18H), 1.40 (t, J = 6.7 Hz, 2H), 1.23–1.19 (m, 7H), 1.16–1.13 (m, 12H). MS (ESI) m / z [M-2H] 2- = 1233.3.

[0215] Step 6: Synthesis of YK-GAL-303

[0216] Dissolve G3-6 (50 mg, 19.1 μmol), 4-dimethylaminopyridine (2.34 mg, 19.1 μmol), diisopropylethylamine (19.8 mg, 153 μmol), and O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (36.3 mg, 95.7 μmol) in N,N-dimethylformamide (4.0 mL), then add CPG-NH2 (332 mg) and stir at 40 °C for 16 h. Filter the reaction mixture, wash the filtrate successively with methanol and dichloromethane, and dry it under vacuum. Then add the filtrate to a 4 mL solution of acetic anhydride / pyridine (1:4), stir at 40 °C for 0.5 h. Filter, wash successively with dichloromethane and methanol, and dry the filtrate under vacuum for 12 h to obtain the white solid compound YK-GAL-303 (292 mg, loading 31.7 μmol / g).

[0217] 4. Synthesis of YK-GAL-304

[0218] The synthesis route is as follows:

[0219]

[0220] Step 1: Synthesis of G4-1

[0221] Dissolve G4A (3.00 g, 18.7 mmol), diisopropylethylamine (4.84 g, 37.4 mmol) and O-benzotriazole-tetramethylurea hexafluorophosphate (7.81 g, 20.6 mmol) in N,N-dimethylformamide (30.0 mL), add G4A-1 (1.50 g, 16.9 mmol), and stir at 15 °C for 2 h. Purify by preparative chromatography (H2O (0.1% TFA)-ACN) to obtain white solid G4-1 (1.70 g). 1 1H NMR (400 MHz, DMSO-d6) δ ppm 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.

[0222] Step 2: Synthesis of G4-2

[0223] Add dichloromethane (16.0 mL) to compound G1-1 (900 mg, 3.46 mmol), compound G4-1 (880 mg, 3.80 mmol) and 4A molecular sieve (1.60 g). After cooling to 0 °C, add trimethylsilyl trifluoromethanesulfonate (1.54 g, 6.92 mmol), and continue stirring for 2 h. Quench the reaction by adding saturated aqueous sodium bicarbonate solution (20.0 mL), extract with dichloromethane (20.0 mL × 3), dry the organic phase with anhydrous sodium sulfate, and concentrate under reduced pressure to remove the solvent to obtain the crude product. Purify by column chromatography (PE / EA) to obtain colorless oil G4-2 (617.7 mg, 1.43 mmol), with a yield of 41.4%. 1 1H NMR (400 MHz, CDCl3) δ ppm 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.

[0224] Step 3: Synthesis of G4-3

[0225] Dissolve G4-2 (160 mg, 371 μmol) in tetrahydrofuran (2.2 mL), add an aqueous solution (1.0 mL) of lithium hydroxide monohydrate (49.8 mg, 1.19 mmol), and stir at 20 °C for 16 h. Concentrate the reaction mixture under reduced pressure to remove the solvent to obtain a white solid. The crude product G4-3 (123 mg) is directly used in the next step.

[0226] Step 4: Synthesis of G4-4

[0227] Dissolve the dried G4-3 (123 mg, 369 μmol) and 4,4'-dimethoxytrityl chloride (463 mg, 1.37 mmol) in pyridine (1.50 mL), and stir at 25 °C for 48 h under nitrogen protection. Quench the reaction by adding methanol (1.00 mL), and concentrate the reaction mixture under reduced pressure to remove the solvent to obtain a crude product. Purify by preparative chromatography (H2O (10 mM TEAB)-ACN) to obtain a white solid G4-4 (148 mg, 233 μmol) with a yield of 62.8%. 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), 3.42–3.54 (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 [M-H] - = 634.4.

[0228] Step 5: Synthesis of G4-5

[0229] Dissolve G4-4 (30.0 mg, 47.2 μmol), diisopropylethylamine (12.2 mg, 94.4 μmol), and O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (26.8 mg, 70.8 μmol) in N,N-dimethylformamide (1.0 mL), add GalNAc-NH2.TFA (45.0 mg, 23.6 μmol, TFA), and stir at 25 °C for 2 h under nitrogen protection. The reaction mixture is directly used in the next step. MS (ESI) m / z [M-2H] 2- = 1204.8.

[0230] Step 6: Synthesis of G4-6

[0231] Dissolve 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) in N,N-dimethylformamide (1.0 mL), and stir at 30 °C for 48 h under nitrogen protection. Purify by preparative chromatography (H2O (10 mM TEAB)-ACN) to obtain white solid G4-6 (55.3 mg, 22 μmol) with a yield of 47.0%. MS (ESI) m / z [M-2H] 2- = 1254.1.

[0232] Step 7: Synthesis of YK-GAL-304

[0233] Dissolve 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-tetramethylurea hexafluorophosphate (14.4 mg, 37.8 μmol) in N,N-dimethylformamide (1.5 mL), then add CPG-NH2 (131 mg), and stir at 40 °C for 16 h. Filter the reaction solution, wash the filtrate with methanol and dichloromethane in sequence, and dry under vacuum. Then add the filtrate to a 1.20 mL acetic anhydride / pyridine (1:5) solution, and stir at 40 °C for 0.5 h. Filter, wash with dichloromethane and methanol in sequence, and dry the filtrate under vacuum for 12 h to obtain white solid compound YK-GAL-304 (100 mg, loading amount 35.57 μmol / g). 5. Synthesis of YK-GAL-305

[0234] The synthesis route is as follows:

[0235]

[0236] Step 1: Synthesis of G5-2

[0237] Add dichloromethane (100.0 mL) to dry compound G5-1 (3.53 g, 11.1 mmol), dry compound G1-2 (2.0 g, 9.25 mmol) and 3A molecular sieve (10.0 g), cool to 0 °C, then add boron trifluoride diethyl etherate (2.62 g, 18.5 mmol), and continue stirring for 2 h. Quench the reaction by adding triethylamine (2.7 mL), wash with saturated sodium bicarbonate aqueous solution (50 mL), dry the organic phase with anhydrous sodium sulfate, and concentrate under reduced pressure to remove the solvent to obtain a crude product. Purify by column chromatography (PE / EA) to obtain colorless oily substance G5-2 (2.11 g, 4.46 mmol) with a yield of 48.2%.1 HNMR (400 MHz, CDCl3) δ ppm 5.32 (dd, J = 6.7, 4.8 Hz, 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, 6.7 Hz, 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。

[0238] Step 2: Synthesis of G5-3

[0239] 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 with 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) is directly used in the next step. 1 HNMR (400 MHz, CDCl3) δ ppm 4.91 (s, 1H), 4.32 (t, J = 5.5 Hz, 1H), 4.08–3.97 (m, 2H), 3.76 (dd, J = 11.8, 3.3 Hz, 1H), 3.71–3.57 (m, 5H), 3.41 (dt, J = 9.3, 6.5 Hz, 1H), 2.74 (s, 2H), 2.27 (t, J = 7.5 Hz, 2H), 1.55 (dq, J = 20.6, 7.0 Hz, 5H), 1.28–1.25 (m, 12H). MS (ESI) m / z [M+Na] + = 371.2。

[0240] Step 3: Synthesis of G5-4

[0241] Dissolve G5-3 (1.32 g, 3.79 mmol) in N,N-dimethylformamide (10.0 mL), add 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (1.31 g, 4.17 mmol) and imidazole (645 mg, 9.47 mmol), and stir at 15 °C for 2 h under nitrogen protection. Quench with saturated aqueous sodium bicarbonate solution (10.0 mL), extract with dichloromethane (10 mL × 2), dry the organic phase over anhydrous sodium sulfate, and concentrate under reduced pressure to remove the solvent to obtain a crude product. Purify by column chromatography (PE / EA) to obtain a colorless oil G5-4 (1.20 g, 2.03 mmol), with a yield of 53.6%.1 HNMR (400 MHz, CDCl3) δ ppm 4.91 (s, 1H), 4.51 (t, J = 5.3 Hz, 1H), 4.06–3.97 (m, 3H), 3.76 (dd, J = 10.4, 8.7 Hz, 1H), 3.66 (s, 3H), 3.61 (dt, J = 9.6, 6.8 Hz, 1H), 3.34 (dt, J = 9.5, 6.4 Hz, 1H), 2.97 (s, 1H), 2.30 (t, J = 7.5 Hz, 2H), 1.61 (t, J = 7.4 Hz, 3H), 1.51 (q, J = 6.8 Hz, 3H), 1.27–1.32 (m, 12H), 1.11–1.02 (m, 26H). MS (ESI) m / z [M+Na] + = 613.4。

[0242] Step 4: Synthesis of G5-5

[0243] Dissolve G5-4 (1.20 g, 2.03 mmol) in dichloromethane (12.0 mL), add 1,8-bis(dimethylamino)naphthalene (1.18 g, 5.48 mmol) and trimethyloxonium tetrafluoroborate (751 mg, 5.08 mmol), stir at 15 °C for 16 h under nitrogen protection. Quench with aqueous ammonium chloride solution (10.0 mL), extract with dichloromethane (10 mL × 2), dry the organic phase over anhydrous sodium sulfate, and concentrate under reduced pressure to remove the solvent to obtain the crude product. Purify by column chromatography (PE / EA) to obtain colorless oily G5-5 (1.05 g, 1.74 mmol), yield 85.5%). 1 HNMR (400 MHz, CDCl3) δ ppm 4.84 (s, 1H), 4.49 (dd, J = 7.6, 4.3 Hz, 1H), 3.98 (dq, J = 9.0, 3.1 Hz, 2H), 3.86 (dd, J = 12.6, 6.6 Hz, 1H), 3.69–3.54 (m, 8H), 3.40–3.27 (m, 1H), 2.30 (t, J = 7.5 Hz, 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。

[0244] Step 5: Synthesis of G5-6

[0245] 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. The reaction was quenched by adding saturated aqueous sodium bicarbonate solution (10.0 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, obtaining the crude product G5-6 (0.63 g), which was directly used in the next step. 1 HNMR (400 MHz, CDCl3) δ ppm 4.93 (d, J = 1.3 Hz, 1H), 4.25 (t, J = 5.2 Hz, 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 = 7.5 Hz, 2H), 1.53 (dp, J = 14.1, 7.0 Hz, 4H), 1.22 (d, J = 5.8 Hz, 12H), 0.99 (dq, J = 5.8, 3.1, 2.6 Hz, 2H). MS (ESI) m / z + = 385.2.

[0246] Step 6: Synthesis of G5-7

[0247] The dried G5-6 (0.63 g, 1.74 mmol) and 4,4'-dimethoxytriphenyl chloromethane (618 mg, 1.83 mmol) were dissolved in pyridine (10.0 mL), and the mixture was stirred at 15 °C for 16 h under nitrogen protection. The reaction was quenched by adding saturated aqueous sodium bicarbonate solution (10.00 mL), and the mixture was extracted with dichloromethane (15.0 mL × 2). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to remove the solvent, obtaining the crude product. The crude product was purified by column chromatography (PE / EA) to obtain the white solid G5-7 (1.01 g, 1.52 mmol) with a yield of 87.4%. 1HNMR (400 MHz, CDCl3) δ ppm 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), 5.02 (d, J = 1.4 Hz, 1H), 4.19 (dt, J = 8.3, 5.5 Hz, 1H), 4.04 (td, J = 5.8, 3.8 Hz, 1H), 3.78 (brs, 6H), 3.76–3.63 (m, 6H), 3.50 (s, 3H), 3.38 (dt, J = 9.4, 6.8 Hz, 1H), 3.27 (dd, J = 9.9, 3.8 Hz, 1H), 3.14 (dd, J = 9.9, 5.6 Hz, 1H), 2.48 (d, J = 8.4 Hz, 1H), 2.29 (t, J = 7.6 Hz, 2H), 1.60 (d, J = 14.7 Hz, 2H), 1.28–1.23 (m, 11H). MS (ESI) m / z [M-H] - = 663.6。

[0248] Step 7: Synthesis of G5-8

[0249] Dissolve G5-7 (1.00 g, 1.50 mmol) in tetrahydrofuran (12.0 mL), add an aqueous solution (6.0 mL) of lithium hydroxide monohydrate (94.7 mg, 2.26 mmol), and stir at 15 °C for 12 h. Concentrate the reaction mixture under reduced pressure to remove the solvent, and a yellow solid is obtained. The crude product G5-8 (0.98 g) is directly used in the next step. 1 HNMR (400 MHz, CDCl3) δ ppm 7.53–7.45 (m, 2H), 7.35 (d, J = 8.50 Hz, 4H), 7.23 (t, J = 7.19 Hz, 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 [M-H] - = 649.6。

[0250] Step 8: Synthesis of G5-9

[0251] Dissolve G5-8 (0.97 g, 1.49 mmol), diisopropylethylamine (385 mg, 2.98 mmol) and O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (848 mg, 2.24 mmol) in N,N-dimethylformamide (35.0 mL), add GalNAc-NH2.TFA (2.84 g, 1.49 mmol, TFA), and stir at 15 °C for 1 h under nitrogen protection. The reaction solution is directly used for the next step. MS (ESI) m / z [M-2H] 2- = 1212.4

[0252] Step 9: Synthesis of G5-10

[0253] Dissolve 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) in N,N-dimethylformamide (35.0 mL), and stir at 30 °C for 48 h under nitrogen protection. Purify by preparative chromatography (H2O (50 mM TEAB)-ACN) to obtain white solid G5-10 (2.10 g, 937 μmol) with a yield of 62.8%. 1 1H NMR (400 MHz, CDCl3) δ ppm 7.48–7.41 (m, 2H), 7.33 (d, J = 8.6 Hz, 3H), 7.28–7.21 (m, 5H), 7.17 (t, J = 7.2 Hz, 1H), 6.99 (t, J = 6.3 Hz, 2H), 6.87–6.77 (m, 5H), 6.49 (d, J = 6.0 Hz, 1H), 5.34 (d, J = 3.3 Hz, 3H), 5.21–5.13 (m, 3H), 4.98 (d, J = 2.3 Hz, 1H), 4.61 (d, J = 8.5 Hz, 2H), 4.23 (q, J = 4.9 Hz, 1H), 4.19–4.01 (m, 8H), 3.91 (dt, J = 13.1, 5.6 Hz, 6H), 3.77 (s, 5H), 3.68 (d, J = 7.6 Hz, 11H), 3.55–3.44 (m, 3H), 3.39–3.19 (m, 14H), 3.11 (dd, J = 9.9, 5.0 Hz, 1H), 2.80–2.59 (m, 13H), 2.49 (t, J = 7.4 Hz, 2H), 2.42 (t, J = 5.7 Hz, 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 = 7.2 Hz, 16H). MS (ESI) m / z [M-2H]2- = 1262.5.

[0254] Step 10: Synthesis of YK-GAL-305

[0255] Dissolve G5-1 (1.00 g, 380 μmol), 4-dimethylaminopyridine (46.5 mg, 381 μmol), diisopropylethylamine (393 mg, 3.04 mmol), and O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (722 mg, 1.90 mmol) in N,N-dimethylformamide (65.0 mL), then add CPG-NH2 (6.60 g) and stir at 40 °C for 16 h. Filter the reaction solution, wash the filtrate successively with methanol and dichloromethane, and dry it under vacuum. Then add the filtrate to a 65 mL acetic anhydride / pyridine (1:5) solution and stir at 40 °C for 0.5 h. Filter, wash successively with dichloromethane and methanol, and dry the filtrate under vacuum for 12 h to obtain the white solid compound YK-GAL-305 (7.05 g, loading 34.4 μmol / g).

[0256] 6. Synthesis of YK-GAL-306

[0257] The synthesis route is as follows:

[0258]

[0259] Step 1: Synthesis of G6-1

[0260] Add dichloromethane (15.0 mL) to dry compound G5-1 (1.58 g, 4.95 mmol), dry compound G2-1 (1.00 g, 4.50 mmol), and 3A molecular sieve (1.50 g, 4.50 mmol). After cooling to 0 °C, add boron trifluoride diethyl etherate (1.28 g, 9.00 mmol) and continue stirring for 2 h. Then warm to 15 °C and continue stirring for 2 h. Add triethylamine (1.32 mL) to quench the reaction, wash with saturated sodium bicarbonate aqueous solution (30.0 mL × 2), dry the organic phase with anhydrous sodium sulfate, and concentrate under reduced pressure to remove the solvent to obtain the crude product. Purify by column chromatography (PE / EA) to obtain the yellow oil G6-1 (1.60 g, 3.33 mmol) with a yield of 74.0%. 1HNMR(400MHz, CDCl3) δ ppm 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

[0261] Step 2: Synthesis of G6-2

[0262] 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 with 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) is directly used for the next step. 1 H NMR(400MHz, MeOH-d4) δ 4.89(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

[0263] Step 3: Synthesis of G6-3

[0264] Dissolve G6-2 (1.18 g, 3.33 mmol) in N,N-dimethylformamide (12.0 mL), add 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (1.16 g, 3.66 mmol) and imidazole (567 mg, 8.33 mmol), and stir at 15 °C for 2 h under nitrogen protection. Quench with saturated aqueous sodium bicarbonate solution (10.0 mL), extract with dichloromethane (10 mL × 2), dry the organic phase with anhydrous sodium sulfate, and concentrate under reduced pressure to remove the solvent to obtain a crude product. Purify by column chromatography (PE / EA) to obtain a colorless oil G6-3 (1.38 g, 2.27 mmol) with a yield of 68.0%. 1 HNMR(400MHz, MeOH-d4) δ ppm 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).

[0265] Step 4: Synthesis of G6-4

[0266] Dissolve G6-3 (1.25 g, 2.09 mmol) in dichloromethane (17.0 mL), add 1,8-bis(dimethylamino)naphthalene (1.48 g, 6.91 mmol) and trimethyloxonium tetrafluoroborate (929 mg, 6.28 mmol), and stir at 15 °C for 6 h under nitrogen protection. Quench with aqueous ammonium chloride solution (30.0 mL), extract with dichloromethane (15 mL×2), dry the organic phase over anhydrous sodium sulfate, and concentrate under reduced pressure to remove the solvent to obtain the crude product. Purify by column chromatography (PE / EA) to obtain colorless oil G6-4 (1.11 g, 1.82 mmol) with a yield of 86%. 1 HNMR (400 MHz, MeOH-d4) δ ppm 4.43 (dd, J = 8.1, 4.3 Hz, 1H), 3.95–3.44 (m, 26H), 1.03–0.95 (m, 28H).

[0267] Step 5: Synthesis of G6-5

[0268] Dissolve G6-4 (1.11 g, 1.82 mmol) in tetrahydrofuran (20.0 mL), add triethylamine trihydrofluoride (1.46 g, 9.09 mmol), and stir at 20 °C for 2 h. Quench with saturated aqueous sodium bicarbonate solution (10.0 mL), extract with dichloromethane (25 mL×2), dry the organic phase over anhydrous sodium sulfate, and concentrate under reduced pressure to remove the solvent to obtain a colorless oil. The crude product G6-5 (0.67 g) is directly used in the next step.

[0269] Step 6: Synthesis of G6-6

[0270] Dissolve dry G6-5 (0.67 g, 1.82 mmol) and 4,4'-dimethoxytrityl chloride (616 mg, 1.82 mmol) in pyridine (10.0 mL), and stir at 25 °C for 2 h under nitrogen protection. Quench the reaction with methanol (1.00 mL), concentrate under reduced pressure to remove the solvent to obtain the crude product. Purify by preparative chromatography (H2O (10 mM NH4HCO3)-ACN) to obtain white solid G6-6 (1.05 g, 1.57 mmol) with a yield of 86.0%. 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[M-H] - = 669.4.

[0271] Step 7: Synthesis of G6-7

[0272] Dissolve G6-6 (195 mg, 291 μmol) in tetrahydrofuran (1.0 mL), add an aqueous solution (1.0 mL) of lithium hydroxide monohydrate (60.0 mg, 333 μmol), and stir at 25 °C for 12 h. Concentrate the reaction solution under reduced pressure to remove the solvent to obtain a white solid. The crude product G6-7 (190 mg) is directly used 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),4.07–4.03(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[M-H] - = 655.4.

[0273] Step 8: Synthesis of G6-8

[0274] Dissolve G6-7 (190 mg, 293 μmol), diisopropylethylamine (112 mg, 868 μmol) and O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (165 mg, 433 μmol) in N,N-dimethylformamide (5.0 mL), add GalNAc-NH2.TFA (442 mg, 232 μmol, 0.8 eq, TFA), and stir at 15 °C for 2 h under nitrogen protection. The reaction solution is directly used in the next step. MS(ESI)m / z[M-2H] 2- = 1215.0.

[0275] Step 9: Synthesis of G6-9

[0276] Dissolve 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) in N,N-dimethylformamide (1.0 mL), and stir at 30 °C for 48 h under nitrogen protection. Purify by preparative chromatography (H2O (50 mM TEAB)-ACN) to obtain white solid G6-9 (300 mg, 118 μmol), with a yield of 50.0%. MS (ESI) m / z [M-2H] 2- = 1265.3.

[0277] Step 10: Synthesis of YK-GAL-306

[0278] Dissolve G6-9 (300 mg, 110 μmol), 4-dimethylaminopyridine (13.4 mg, 109 μmol), diisopropylethylamine (113 mg, 877 μmol), and O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (208 mg, 548 μmol) in N,N-dimethylformamide (16.0 mL), then add CPG-NH2 (1.9 g), and stir at 40 °C for 12 h. Filter the reaction solution, wash the filtrate successively with methanol and dichloromethane, and dry in vacuo. Then add the filtrate to a 20 mL acetic anhydride / pyridine (1:5) solution, and stir at 40 °C for 0.5 h. Filter, wash successively with dichloromethane and methanol, and dry the filtrate in vacuo for 12 h to obtain white solid compound YK-GAL-306 (1.94 g, loading amount 37.43 μmol / g).

[0279] 7. Synthesis of YK-GAL-307

[0280] The synthesis route is as follows:

[0281]

[0282] Step 1: Synthesis of G7-1

[0283] To dry compound G5-1 (5.00 g, 15.7 mmol), dry compound G4-1 (3.50 g, 15.1 mmol) and 4A molecular sieve (1.0 g), add dichloromethane (70.0 mL). After cooling to 0 °C, add boron trifluoride etherate (4.30 g, 30.2 mmol), and continue stirring for 2 h. Add triethylamine (3.06 g, 30.2 mmol) to quench the reaction, wash with saturated aqueous sodium bicarbonate solution (20 mL), dry the organic phase with anhydrous sodium sulfate, and concentrate under reduced pressure to remove the solvent to obtain the crude product. Purify by column chromatography (PE / EA) to obtain colorless oil G7-1 (3.70 g, 7.56 mmol), with a yield of 50.1%. 1 HNMR (400 MHz, CDCl3) δ ppm 5.74 (d, J = 31.1 Hz, 1H), 5.33–5.14 (m, 1H), 4.98 (d, J = 13.6 Hz, 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.

[0284] Step 2: Synthesis of G7-2

[0285] Dissolve G7-1 (3.10 g, 6.33 mmol) in methanol (30.0 mL), add sodium methoxide (114 mg, 633 μmol), and stir at 25 °C for 2 h. Add hydrogen ion exchange resin to quench the reaction, filter and concentrate the filtrate under reduced pressure to remove the solvent to obtain a yellow oil. The crude product G7-2 (2.20 g) is directly used for the next step. MS (ESI) m / z [M-H] - = 362.2.

[0286] Step 3: Synthesis of G7-3

[0287] Dissolve G7-2 (2.20 g, 6.05 mmol) in N,N-dimethylformamide (20.0 mL), add 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (2.10 g, 6.66 mmol) and imidazole (1.03 g, 15.1 mmol), and stir at 25 °C for 2 h under nitrogen protection. Quench with saturated aqueous sodium bicarbonate solution (10.0 mL), extract with dichloromethane (10 mL × 2), dry the organic phase over anhydrous sodium sulfate, and concentrate under reduced pressure to remove the solvent to obtain the crude product. Purify by column chromatography (PE / EA) to obtain a yellow oil G7-3 (2.15 g, 3.56 mmol), with a yield of 58.8%. 1 HNMR (400 MHz, CDCl3) δ ppm 5.59 (s, 1H), 4.90 (s, 1H), 4.49 (t, J = 5.4 Hz, 1H), 4.08–3.91 (m, 3H), 3.80–3.60 (m, 6H), 3.40–3.35 (m, 1H), 3.26 (q, J = 6.4 Hz, 2H), 2.35–2.32 (m, 2H), 2.22–2.15 (m, 2H), 1.68–1.64 (p, J = 3.7 Hz, 4H), 1.59–1.54 (m, 4H), 1.10–1.02 (m, 28H). MS (ESI) m / z + = 606.4.

[0288] Step 4: Synthesis of G7-4

[0289] Dissolve G7-3 (1.0 g, 1.65 mmol) in dichloromethane (15.0 mL), add 1,8-bis(dimethylamino)naphthalene (955 mg, 4.46 mmol) and trimethyloxonium tetrafluoroborate (610 mg, 4.13 mmol), and stir at 15 °C for 6 h under nitrogen protection. Quench with aqueous ammonium chloride solution (10.0 mL), extract with dichloromethane (10 mL × 2), dry the organic phase over anhydrous sodium sulfate, and concentrate under reduced pressure to remove the solvent to obtain the crude product. Purify by column chromatography (PE / EA) to obtain a colorless oil G7-4 (890.2 mg, 1.43 mmol), with a yield of 87.1%. 1HNMR (400 MHz, CDCl3) δ ppm 5.59 (d, J = 6.1 Hz, 1H), 4.53–4.42 (m, 1H), 4.08–3.85 (m, 3H), 3.80–3.60 (m, 5H), 3.58 (d, J = 7.2 Hz, 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, 5.0 Hz, 2H), 1.66 (h, J = 3.8 Hz, 4H), 1.60–1.51 (m, 4H), 1.17–0.89 (m, 28H). MS (ESI) m / z [M+H] + = 620.5.

[0290] Step 5: Synthesis of G7-5

[0291] Dissolve G7-4 (300 mg, 484 μmol) in tetrahydrofuran (10.0 mL), add triethylamine trihydrofluoride (390 mg, 2.42 mmol), and stir at 25 °C for 3 h. Quench with saturated aqueous sodium bicarbonate (10.0 mL), extract with dichloromethane (20 mL × 3), dry the organic phase over anhydrous sodium sulfate, and concentrate under reduced pressure to remove the solvent to obtain a yellow oil. The crude product G7-6 (182 mg) was directly used in the next step. MS (ESI) m / z [M+H] + = 378.2.

[0292] Step 6: Synthesis of G7-6

[0293] Dissolve dry G7-5 (180 mg, 477 μmol) and 4,4'-dimethoxytriphenylmethyl chloride (161 mg, 477 μmol) in pyridine (2.0 mL), stir at 25 °C for 3 h under nitrogen protection. Quench the reaction with methanol (2.00 mL), concentrate under reduced pressure to remove the solvent to obtain a crude product. Purify by preparative chromatography (H2O (10 mM NH4HCO3)-ACN) to obtain white solid G7-6 (262 mg, 385.7 μmol), with a yield of 80.85%. 1HNMR (400 MHz, CDCl3) δ ppm 7.52–7.45 (m, 2H), 7.40–7.32 (m, 4H), 7.30–7.26 (m, 2H), 7.19 (dd, J=14.1, 8.0 Hz, 1H), 6.87–6.78 (m, 4H), 5.38 (s, 1H), 5.01 (d, J=1.5 Hz, 1H), 4.26–4.17 (m, 1H), 4.07–4.03 (m, 1H), 3.82–3.71 (m, 7H), 3.66 (d, J=6.2 Hz, 4H), 3.51 (d, J=2.2 Hz, 3H), 3.41 (dd, J=9.7, 5.9 Hz, 1H), 3.30 (dd, J=10.0, 3.7 Hz, 1H), 3.22–3.07 (m, 3H), 2.48 (d, J=8.3 Hz, 1H), 2.34–2.29 (m, 2H), 2.07–1.98 (m, 2H), 1.60–1.47 (m, 8H). MS (ESI) m / z [M-H] - = 678.5。

[0294] Step 7: Synthesis of G7-7

[0295] Dissolve G7-6 (100 mg, 147 μmol) in tetrahydrofuran (2.5 mL), add 2.5 mL of an aqueous solution of lithium hydroxide monohydrate (9.26 mg, 220 μmol), and stir at 15 °C for 16 h. Concentrate the reaction solution under reduced pressure to remove the solvent to obtain a yellow solid. The crude product G7-7 (0.95 g) is directly used in the next step. MS (ESI) m / z [M-H] - = 664.5。

[0296] Step 8: Synthesis of G7-8

[0297] Dissolve G7-7 (90.0 mg, 135 μmol), diisopropylethylamine (34.9 mg, 270 μmol) and O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (76.9 mg, 202 μmol) in N,N-dimethylformamide (2.0 mL), add GalNAc-NH2.TFA (206 mg, 108 μmol, TFA), and stir at 15 °C for 4 h under nitrogen protection. The reaction solution is directly used in the next step. MS (ESI) m / z [M-2H] 2- = 1219.9。

[0298] Step 9: Synthesis of G7-9

[0299] Dissolve 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) in N,N-dimethylformamide (2.0 mL), and stir at 30 °C for 48 h under nitrogen protection. Purify by preparative chromatography (H2O (50 mM TEAB)-ACN) to obtain yellow solid G7-9 (172 mg, 68.1 μmol) with a yield of 59.7%. 1 HNMR (400 MHz, CDCl3) δ ppm 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, 3.8 Hz, 4H), 5.37 (d, J = 3.8 Hz, 2H), 5.24–5.16 (m, 3H), 5.01 (t, J = 3.1 Hz, 1H), 4.65 (dd, J = 8.4, 3.9 Hz, 2H), 4.26 (t, J = 4.7 Hz, 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 = 3.9 Hz, 3H), 3.29–3.25 (m, 12H), 2.81–2.64 (m, 16H), 2.52 (dd, J = 7.8, 4.0 Hz, 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 2- = 1269.9.

[0300] Step 10: Synthesis of YK-GAL-307

[0301] Dissolve G7-9 (120 mg, 47.2 μmol), 4-dimethylaminopyridine (5.77 mg, 47.2 μmol), diisopropylethylamine (48.8 mg, 377 μmol), and O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (89.5 mg, 236 μmol) in N,N-dimethylformamide (10.0 mL). Subsequently, add CPG-NH2 (790 mg) and stir at 40 °C for 16 h. Filter the reaction mixture, wash the filtrate successively with methanol and dichloromethane, and dry it under vacuum. Then add the filtrate to a 12 mL acetic anhydride / pyridine (1:5) solution and stir at 40 °C for 0.5 h. Filter, wash successively with dichloromethane and methanol, and dry the filtrate under vacuum for 12 h to obtain the white solid compound YK-GAL-307 (810 mg, loading 38.9 μmol / g).

[0302] 8. Synthesis of YK-GAL-308

[0303] The synthesis route is as follows:

[0304]

[0305] Step 1: Synthesis of G8-2

[0306] Dissolve G8-1 (0.34 g, 816 μmol) in tetrahydrofuran (8.0 mL), add an aqueous solution (4.0 mL) of lithium hydroxide monohydrate (109 mg, 2.61 mmol), and stir at 15 °C for 12 h. Concentrate the reaction mixture under reduced pressure to remove the solvent to obtain a yellow solid. The crude product G8-2 (0.26 g) is directly used in the next step. MS (ESI) m / z [M+Na] + = 341.2.

[0307] Step 2: Synthesis of G8-3

[0308] Co-evaporate the crude product G8-2 (0.26 g, 816 μmol) and anhydrous pyridine (20 mL × 3) to remove water. Dissolve the dried G8-2 and 4,4'-dimethoxytrityl chloride (580 mg, 1714 μmol) in pyridine (6.0 mL) and stir at 15 °C for 36 h under nitrogen protection. Quench the reaction by adding methanol (20 mL), concentrate the reaction mixture under reduced pressure to remove the solvent to obtain a crude product. Purify it by preparative chromatography (H2O (10 mM NH4HCO3)-ACN) to obtain the white solid G8-3 (401.9 mg, 647.9 μmol) with a yield of 79.4%. MS (ESI) m / z [M+Na] + = 643.4.

[0309] Step 3: Synthesis of G8-4

[0310] Dissolve G8-3 (90 mg, 145 μmol), diisopropylethylamine (37.5 mg, 290 μmol), and O-benzotriazole-tetramethyluronium hexafluorophosphate (82.5 mg, 217 μmol) in N,N-dimethylformamide (3.0 mL), add GalNAc-NH2.TFA (331.3 mg, 174 μmol, 1.2 eq, TFA), and stir at 15 °C for 2 h under nitrogen protection. The reaction solution is directly used for the next step. MS (ESI) m / z [M-2H] 2- = 1197.3.

[0311] Step 4: Synthesis of G8-5

[0312] Dissolve G8-4, 4-dimethylaminopyridine (35.4 mg, 289 μmol), diisopropylethylamine (149.8 mg, 1158 μmol), and succinic anhydride (159.4 mg, 1592 μmol) in N,N-dimethylformamide (3.0 mL), and stir at 30 °C for 48 h under nitrogen protection. Purify by preparative chromatography (H2O (10 mM TEAB)-ACN) to obtain white solid G8-5 (190 mg, 85.9 μmol) with a yield of 59.4%. 1 1H NMR (400 MHz, CDCl3) δ ppm 7.44–7.37 (m, 2H), 7.34–7.26 (m, 8H), 7.20 (d, J = 7.1 Hz, 2H), 6.99 (d, J = 5.7 Hz, 4H), 6.81 (d, J = 8.7 Hz, 4H), 6.72 (d, J = 8.9 Hz, 2H), 6.57 (s, 1H), 5.35 (d, J = 3.3 Hz, 2H), 5.26 (d, J = 5.1 Hz, 1H), 5.23–5.12 (m, 4H), 4.61 (d, J = 8.4 Hz, 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, 4.0 Hz, 2H), 2.62–2.57 (m, 4H), 2.43 (d, J = 5.7 Hz, 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.

[0313] Step 5: Synthesis of YK-GAL-308

[0314] G8-5 (190 mg, 76.1 μmol), 4-dimethylaminopyridine (9.30 mg, 76.1 μmol), diisopropylethylamine (78.7 mg, 609 μmol), and O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (144 mg, 381 μmol) were dissolved in N,N-dimethylformamide (12 mL). Subsequently, CPG-NH2 (1.27 g) was added, and the mixture was stirred at 40 °C for 16 h. The reaction solution was filtered, and the filtrate was washed successively with methanol and dichloromethane and dried under vacuum. The filtrate was then added to a 12 mL solution of acetic anhydride / pyridine (1:5), and the mixture was stirred at 40 °C for 0.5 h. After filtration, the residue was washed successively with dichloromethane and methanol and dried under vacuum for 12 h to obtain a white solid compound YK-GAL-308 (1.24 g, loading amount 29.7 μmol / g).

[0315] 9. Synthesis of YK-GAL-309

[0316] The synthesis route is as follows:

[0317]

[0318] Step 1: Synthesis of G9-2

[0319] Dry dichloromethane (20 mL) was added to dry compound G1-1 (734 mg, 2.82 mmol), dry compound G9-1 (0.50 g, 2.17 mmol), and 3A molecular sieve (2.0 g). After cooling to 0 °C, boron trifluoride diethyl etherate (616 mg, 4.34 mmol) was added, and the mixture was stirred for an additional 2 h. The reaction was quenched by adding 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, yielding a crude product. The crude product was purified by column chromatography (PE / EA) to obtain a yellow oil, G9-2 (589 mg, 1.37 mmol), with a yield of 48.5%. MS (ESI) m / z [M+Na] + = 453.1.

[0320] Step 2: Synthesis of G9-3

[0321] 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 solution was concentrated under reduced pressure to remove the solvent, yielding a yellow solid. The crude product G9-3 (0.23 g) was directly used in the next step. MS (ESI) m / z [M+Na] + = 355.0.

[0322] Step 3: Synthesis of G9-4

[0323] The crude product G9-3 (0.23 g, 692 μmol) and anhydrous pyridine (20 mL × 3) were co-evaporated to remove water. The dried G9-3 and 4,4'-dimethoxytriphenylmethyl chloride (534.58 mg, 1579.8 μmol) were dissolved in pyridine (5.0 mL), and stirred at 15 °C for 28 h under nitrogen protection. Methanol (10 mL) was added to quench the reaction, and the solvent was removed by concentration under reduced pressure to obtain a crude product. Purification by preparative chromatography (H2O (10 mM NH4HCO3)-ACN) gave the white solid G9-4 (337.3 mg, 531.7 μmol) with a yield of 76.84%. MS (ESI) m / z [M-H] - = 633.5

[0324] Step 4: Synthesis of G9-5

[0325] 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), and GalNAc-NH2.TFA (90.2 mg, 47.3 μmol, TFA) was added. The mixture was stirred at 15 °C for 1 h under nitrogen protection. The reaction solution was directly used for the next step. MS (ESI) m / z [M-2H] 2- = 1204.3

[0326] Step 5: Synthesis of G9-6

[0327] 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 for 36 h under nitrogen protection. Purification by preparative chromatography (H2O (10 mM TEAB)-ACN) gave the white solid G9-6 (56.7 mg, 22.6 μmol) with a yield of 47.8%. 1HNMR (400 MHz, CDCl3) δ ppm 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 = 4.3 Hz, 1H), 4.27–4.21 (m, 1H), 4.19 (d, J = 5.6 Hz, 1H), 3.79 (s, 6H), 3.74 (dd, J = 9.6, 6.8 Hz, 1H), 3.40 (dt, J = 9.6, 6.5 Hz, 2H), 3.27 (s, 1H), 3.16–3.07 (m, 3H), 2.33 (t, J = 7.4 Hz, 2H), 2.21–2.15 (m, 1H), 2.02 (d, J = 13.3 Hz, 1H), 1.67–1.52 (m, 4H), 1.23–1.30 (d, J = 13.9 Hz, 13H). MS (ESI) m / z [M-2H] 2- = 1254.4。

[0328] Step 6: Synthesis of YK-GAL-309

[0329] Dissolve G9-6 (32 mg, 12.7 μmol), 4-dimethylaminopyridine (1.56 mg, 12.7 μmol), diisopropylethylamine (13.2 mg, 102 μmol), and O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (24.2 mg, 63.7 μmol) in N,N-dimethylformamide (4.0 mL), then add CPG-NH2 (221 mg) and stir at 40 °C for 12 h. Filter the reaction mixture, wash the filtrate successively with methanol and dichloromethane, and dry it under vacuum. Then add the filtrate to a 4 mL acetic anhydride / pyridine (1:5) solution and stir at 40 °C for 0.5 h. Filter, wash successively with dichloromethane and methanol, and dry the filtrate under vacuum for 12 h to obtain the white solid compound YK-GAL-309 (103 mg, loading 24.5 μmol / g).

[0330] 10. Synthesis of YK-GAL-310

[0331] The synthesis route is as follows:

[0332]

[0333] Step 1: Synthesis of G10-2

[0334] To dry compound G1-1 (692 mg, 2.66 mmol), dry compound G10-1 (500 mg, 2.05 mmol) and 3A molecular sieve (2.0 g), add dichloromethane (20 mL). After cooling to 0 °C, add boron trifluoride diethyl etherate (581 mg, 4.09 mmol). Then warm to 25 °C and continue stirring for 2 h. Quench the reaction by adding saturated aqueous sodium bicarbonate solution (20 mL), extract with dichloromethane (10 mL × 2), dry the organic phase over anhydrous sodium sulfate, and concentrate under reduced pressure to remove the solvent to obtain the crude product. Purify by column chromatography (PE / EA) to obtain colorless oil G10-2 (576.6 mg, 1.30 mmol), with a yield of 48.8%. MS (ESI) m / z + = 467.1.

[0335] Step 2: Synthesis of G10-3

[0336] Dissolve G10-2 (69 mg, 155 μmol) in tetrahydrofuran (2.0 mL), add 1.0 mL of an aqueous solution of lithium hydroxide monohydrate (20.8 mg, 497 μmol), and stir at room temperature for 16 h. Concentrate the reaction mixture under reduced pressure to remove the solvent to obtain a yellow oil. The crude product G10-3 (53.8 mg) is directly used in the next step. MS (ESI) m / z + = 369.0.

[0337] Step 3: Synthesis of G10-4

[0338] Co-evaporate the crude product G10-3 (53.8 mg, 155 μmol) and anhydrous pyridine (20 mL × 3) to remove water. Dissolve the dried G10-3 and 4,4'-dimethoxytriphenylmethyl chloride (189 mg, 559 μmol) in pyridine (3.0 mL), and stir at room temperature for 16 h under nitrogen protection. Quench the reaction by adding methanol (10 mL), and concentrate under reduced pressure to remove the solvent to obtain the crude product. Purify by column chromatography (DCM / MeOH) to obtain pale yellow solid G10-4 (79.1 mg, 122 μmol), with a yield of 78.7%. MS (ESI) m / z - = 647.5.

[0339] Step 4: Synthesis of G10-5

[0340] Dissolve G10-4 (39 mg, 60.1 μmol), diisopropylethylamine (15.5 mg, 120 μmol), and O-benzotriazole-tetramethyluronium hexafluorophosphate (34.2 mg, 90.2 μmol) in N,N-dimethylformamide (1.5 mL), add GalNAc-NH2.TFA (115 mg, 60.1 μmol), and stir at room temperature for 1 h under nitrogen protection. The reaction solution is directly used for the next step. MS (ESI) m / z [M-2H] 2- = 1211.3.

[0341] Step 5: Synthesis of G10-6

[0342] Dissolve 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) in N,N-dimethylformamide (1.5 mL), and stir at 30 °C for 16 h under nitrogen protection. Purify by preparative chromatography (H2O (10 mM TEAB)-ACN) to obtain white solid G10-6 (74.2 mg, 49.4 μmol), with a yield of 48.9%. 1 1H NMR (400 MHz, CDCl3) δ ppm 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 = 6.0 Hz, 3H), 6.79 (dd, J = 11.8, 8.8 Hz, 6H), 6.47 (d, J = 9.6 Hz, 1H), 5.35 (d, J = 3.3 Hz, 2H), 5.23–5.17 (dt, J = 11.2, 3.8 Hz, 4H), 4.61 (d, J = 8.5 Hz, 2H), 4.19–4.04 (m, 9H), 3.99–3.86 (m, 6H), 3.78 (d, J = 2.1 Hz, 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 = 5.7 Hz, 7H), 2.29–2.13 (m, 24H), 2.10 (d, J = 5.0 Hz, 3H), 2.04 (s, 9H), 1.99 (s, 9H), 1.94 (d, J = 2.5 Hz, 8H), 1.75–1.55 (m, 18H), 1.42 (s, 2H), 1.26–1.21 (d, J = 3.6 Hz, 15H). MS (ESI) m / z [M-2H] 2- = 1261.1.

[0343] Step 6: Synthesis of YK-GAL-310

[0344] Dissolve 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) in N,N-dimethylformamide (4.0 mL). Subsequently, add CPG-NH2 (383 mg) and stir at 40 °C for 16 h. Filter the reaction solution, and wash the filtrate successively with methanol and dichloromethane, then dry it under vacuum. Then add the filtrate to a 4 mL acetic anhydride / pyridine (1:4) solution and stir at 40 °C for 0.5 h. Filter, wash successively with dichloromethane and methanol, and dry the filtrate under vacuum for 12 h to obtain the white solid compound YK-GAL-310 (362 mg, loading 30.2 μmol / g).

[0345] 11. Synthesis of YK-GAL-311

[0346] The synthesis route is as follows:

[0347]

[0348] Step 1: Synthesis of G11-1

[0349] Dissolve 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) in N,N-dimethylformamide (20.0 mL). Add G11A-1 (1.92 g, 16.4 mmol) and stir at 15 °C for 2 h. Purify by preparative chromatography (H2O (0.1% TFA)-ACN) to obtain the white solid G11-1 (2.00 g, 8.15 mmol) with a yield of 59.5%. 1 HNMR (400 MHz, DMSO-d6) δ ppm 7.76–7.73 (m, 1H), 4.38–4.30 (m, 1H), 3.57 (s, 3H), 3.36 (t, J = 6.4 Hz, 2H), 3.02–2.97 (m, 2H), 2.28 (t, J = 7.6 Hz, 2H), 2.69 (t, J = 7.2 Hz, 2H), 1.76–1.68 (m, 2H), 1.42–1.18 (m, 8H). MS (ESI) m / z [M+H] + = 245.9.

[0350] Step 2: Synthesis of G11-2

[0351] To compound G1-1 (500 mg, 1.92 mmol), compound G11-1 (565 mg, 2.31 mmol) and 4A molecular sieve (1.0 g), dichloromethane (10.0 mL) was added. After cooling to 0 °C, trimethylsilyl trifluoromethanesulfonate (854 mg, 3.84 mmol) was added, and stirring was continued for 2 h. The reaction was quenched by adding saturated aqueous sodium bicarbonate solution (30.0 mL), and the mixture was extracted with dichloromethane (15.0 mL×3). The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by concentration under reduced pressure to obtain a crude product. Purification by column chromatography (PE / EA) gave colorless oil G11-2 (400 mg, 899 μmol) with a yield of 46.8%. 1 HNMR (400 MHz, CDCl3) δ ppm 5.61–5.54 (m, 1H), 5.24–5.20 (m, 1H), 4.32–4.00 (m, 3H), 3.67 (d, J = 1.0 Hz, 4H), 3.34 (dt, J = 9.4, 6.6 Hz, 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.

[0352] Step 3: Synthesis of G11-3

[0353] G11-2 (180 mg, 404 μmol, 1.0 eq) was dissolved in tetrahydrofuran (1.8 mL), and an aqueous solution (0.9 mL) of lithium hydroxide monohydrate (67.8 mg, 1.62 mmol, 4.0 eq) was added. The mixture was stirred at 35 °C for 16 h. The reaction solution was concentrated under reduced pressure to remove the solvent, and a brown oil was obtained. The crude product G11-3 (140.36 mg) was directly used in the next step.

[0354] Step 4: Synthesis of G11-4

[0355] The dried G11-3 (40.0 mg, 115 μmol) and 4,4'-dimethoxytriphenylmethyl chloride (65.0 mg, 191 μmol) were dissolved in pyridine (1.00 mL), and the mixture was stirred at 15 °C for 8 h under nitrogen protection. The reaction was quenched by adding methanol (2.00 mL), and the solvent was removed by concentration under reduced pressure to obtain a crude product. Purification by preparative chromatography (H2O (10 mM TEAB)-ACN) gave white solid G11-4 (54.7 mg, 84.3 μmol) with a yield of 73.3%. 1HNMR (400 MHz, CDCl3) δ ppm 7.38 (d, J = 7.7 Hz, 2H), 7.29–7.20 (m, 6H), 7.13 (t, J = 7.3 Hz, 1H), 6.75 (d, J = 8.6 Hz, 4H), 5.60 (s, 1H), 5.08 (dd, J = 5.5, 2.3 Hz, 1H), 4.34 (q, J = 6.2 Hz, 1H), 3.89 (q, J = 5.6 Hz, 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 = 7.0 Hz, 3H), 2.18 (t, J = 7.2 Hz, 3H), 2.01–1.95 (m, 1H), 1.88 (t, J = 7.0 Hz, 2H), 1.40–1.31 (m, 4H), 1.21–1.15 (m, 4H). MS (ESI) m / z [M-H] - = 648.2。

[0356] Step 5: Synthesis of G11-5

[0357] Dissolve G11-4 (17.0 mg, 26.1 μmol), diisopropylethylamine (6.76 mg, 52.3 μmol), and O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (14.8 mg, 39.2 μmol) in N,N-dimethylformamide (0.3 mL), add GalNAc-NH2·TFA (62.4 mg, 27.4 μmol, TFA), and stir at 15 °C for 6 h under nitrogen protection. The reaction solution is directly used for the next step. MS (ESI) m / z [M-2H] 2- = 1211.6。

[0358] Step 6: Synthesis of G11-6

[0359] Dissolve 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) in N,N-dimethylformamide (0.5 mL), and stir at 30 °C for 16 h under nitrogen protection. Purify by preparative chromatography (H2O (10 mM TEAB)-ACN) to obtain white solid G11-6 (35.0 mg, 13.8 μmol) with a yield of 53.3%. 1HNMR (400 MHz, CDCl3) δ ppm 7.49–7.44 (m, 5H), 7.35–7.31 (m, 4H), 7.28 (s, 1H), 7.24 (s, 1H), 7.17 (d, J = 7.5 Hz, 1H), 7.01–7.00 (m, 3H), 6.91 (d, J = 9.0 Hz, 2H), 6.83–6.72 (m, 6H), 5.35 (d, J = 3.3 Hz, 2H), 5.19 (dd, J = 10.9, 3.3 Hz, 5H), 4.62 (d, J = 8.3 Hz, 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 = 6.3 Hz, 2H), 2.51 (d, J = 6.7 Hz, 2H), 2.41 (t, J = 5.8 Hz, 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。

[0360] Step 7: Synthesis of YK-GAL-311

[0361] Dissolve G11-7 (35.0 mg, 13.8 μmol), 4-dimethylaminopyridine (1.69 mg, 13.8 μmol), diisopropylethylamine (14.3 mg, 110 μmol), and O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (26.2 mg, 69.3 μmol) in N,N-dimethylformamide (2.0 mL), then add CPG-NH2 (220 mg) and stir at 40 °C for 16 h. Filter the reaction mixture, wash the filtrate successively with methanol and dichloromethane, and dry it under vacuum. Then add the filtrate to a 12.0 mL acetic anhydride / pyridine (1:5) solution and stir at 40 °C for 0.5 h. Filter, wash successively with dichloromethane and methanol, and dry the filtrate under vacuum for 12 h to obtain a pale yellow solid compound YK-GAL-311 (205 mg, loading 32.4 μmol / g).

[0362] 12. Synthesis of YK-GAL-312

[0363] The synthesis route is as follows:

[0364]

[0365] Step 1: Synthesis of G12-1

[0366] Dissolve G5-8 (1.3 g, 2.0 mmol), diisopropylethylamine (517 mg, 4.0 mmol) and O-benzotriazole-tetramethylurea hexafluorophosphate (1.14 g, 3.0 mmol) in N,N-dimethylformamide (100.0 mL), add GalNAc-NH2 (this compound was synthesized according to the synthesis method of patent CN115315263A, with a total yield of 20.18%) (1.24 g, 2.0 mmol), and stir at 15 °C for 1 h under nitrogen protection. The reaction solution was directly used for the next step. MS(ESI) m / z [M-H] - = 1249.9.

[0367] Step 2: Synthesis of G12-2

[0368] Dissolve 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) in N,N-dimethylformamide (100.0 mL), and stir at 30 °C for 48 h under nitrogen protection. Purify by preparative chromatography (H2O (50 mM TEAB)-ACN) to obtain white solid G12-2 (1.9 g, 1.31 mmol) with a yield of 65.5%. MS(ESI) m / z [M-H] - = 1350.1.

[0369] Step 3: Synthesis of YK-GAL-312

[0370] Dissolve 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-tetramethylurea hexafluorophosphate (2.48 g, 6.55 mmol) in N,N-dimethylformamide (210 mL), then add CPG-NH2 (13.6 g), and stir at 40 °C for 16 h. Filter the reaction solution, wash the filtrate successively with methanol and dichloromethane, and dry in vacuo. Then add the filtrate to 124 mL of acetic anhydride / pyridine (1:5) solution, and stir at 40 °C for 0.5 h. Filter, wash successively with dichloromethane and methanol, and dry the filtrate in vacuo for 12 h to obtain white solid compound YK-GAL-312 (24.3 g, loading amount 36.2 μmol / g).

[0371] 13. Synthesis of YK-GAL-313

[0372] The synthesis route is as follows:

[0373]

[0374] Step 1: Synthesis of G13-1

[0375] Dissolve G5-8 (1.3 g, 2.0 mmol), diisopropylethylamine (517 mg, 4.0 mmol) and O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.14 g, 3.0 mmol) in N,N-dimethylformamide (100.0 mL), add GalNAc-NH2 (synthesized according to the synthesis method of patent WO2023288033A1, total yield 2.1%) (2.41 g, 2.0 mmol), and stir at 15 °C for 1 h under nitrogen protection. The reaction solution is directly used for the next step. MS(ESI) m / z [M-H] - = 1837.5.

[0376] Step 2: Synthesis of G13-2

[0377] Dissolve 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) in N,N-dimethylformamide (100.0 mL), and stir at 30 °C for 48 h under nitrogen protection. Purify by preparative chromatography (H2O (50 mM TEAB)-ACN) to obtain white solid G13-2 (2.66 g, 1.29 mmol), with a yield of 64.5%. MS(ESI) m / z [M-H] - = 1937.2.

[0378] Step 3: Synthesis of YK-GAL-313

[0379] Dissolve 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-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (2.45 g, 6.45 mmol) in N,N-dimethylformamide (210.0 mL), then add CPG-NH2 (18.9 g), and stir at 40 °C for 16 h. Filter the reaction solution, wash the filtrate with methanol and dichloromethane in sequence, and dry in vacuo. Then add the filtrate to 173 mL of acetic anhydride / pyridine (1:5) solution, and stir at 40 °C for 0.5 h. Filter, wash with dichloromethane and methanol in sequence, and dry the filtrate in vacuo for 12 h to obtain white solid compound YK-GAL-313 (26.3 g, loading amount 37.1 μmol / g).

[0380] 14. Synthesis of NAG0052

[0381]

[0382] According to the synthesis method of NAG0052 on page 100 of WO2023109938A1, 288 mg of the product was obtained, with an overall yield of 1.26% and a loading of 31.5 μmol / g.

[0383] Example 2: Synthesis of GalNAc phosphoramidite compounds

[0384] 1. Synthesis of YK-GAL-318

[0385]

[0386] Compound G5-9 (2.43 g, 1.0 mmol) and DCM (25 mL) were added to a reaction flask and stirred until dissolved. Then, 3-bis(diisopropylamino)phosphoryloxypropionitrile (0.75 g, 2.5 mmol) and 4,5-dicyanoimidazole (0.24 g, 2.0 mmol) were added successively, and the mixture was 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, and this was repeated three times to obtain 2.39 g of a white solid with a yield of 91%. MS(ESI) m / z [M+H] + = 2626.8.

[0387] 2. Synthesis of other GalNAc phosphoramidite compounds

[0388] 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, other GalNAc phosphoramidite compounds listed in Table 1 were synthesized according to the method for synthesizing YK-GAL-318.

[0389] Table 1: GalNAc phosphoramidite compounds

[0390]

[0391]

[0392] 3. Synthesis of GalNAc 1b

[0393]

[0394] According to the synthesis method of GalNAc 1b on page 145 of WO2023014938A1, 377.2 mg of the product was obtained with an overall yield of 0.58%, MS(ESI) m / z + = 1137.9.

[0395] 4. Synthesis of GalNAc 2

[0396]

[0397] According to the synthesis method of GalNAc 2 on page 216 of WO2023049258A1, 468.5 mg of the product was obtained with an overall yield of 1.75%, MS(ESI) m / z + = 1217.7.

[0398] Example 3: Conjugation of GalNAc compound with oligonucleotide

[0399] In this example, the same siRNA sequence was used for synthesis. The conjugated siRNA sequence was the sequence numbered inc, and the inc sequence was as follows:

[0400] Sense strand (inc-SS):

[0401] 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),

[0402] Antisense strand (inc-AS):

[0403] 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).

[0404] Among them, A, U, C, and G represent the base composition of nucleotides; 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 there is a phosphorothioate linkage between the two nucleotides adjacent to the left and right of s.

[0405] The sequence is from a publicly available sequence and is the oligonucleotide sequence of the foreign marketed siRNA drug inclisiran.

[0406] The inclisiran sequence is a synthetic chemically modified double-stranded small interfering ribonucleic acid (siRNA). By targeting and binding to the mRNA encoding the PCSK9 protein, it inhibits the production of the PCSK9 protein through the RNA interference mechanism, thereby regulating the recycling and reuse of LDL receptors, enhancing their binding to LDL, and achieving the goal of reducing LDL in the blood.

[0407] 1. Preparation of GalNAc-conjugated siRNA sense strand

[0408] Synthesize the oligonucleotide GalNAc conjugate on a solid-phase support according to the phosphoramidite chemistry method.

[0409] 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), use the GalNAc-CPG compounds synthesized in Example 1 (including 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, and NAG0052) as the solid-phase support; when synthesizing conjugate 17 (sequence number inc-L96), use the purchased CPG-L96 (purchased from WuXi AppTec Co., Ltd., Tianjin, L96 is disclosed in US 10465194B2, claim 10) as the solid-phase support, where the solid-phase support synthesis scale is 1 μmol. All these GalNAc compounds are conjugated to the 3'-end of the oligonucleotide.

[0410] When synthesizing conjugates 12-16, 19 and 20 (SEQ ID NO: inc-G12, inc-G13, G18-inc, inc-G25, G26-inc, inc-GalNAc 1b and inc-GalNAc 2), a general 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 monomer or the last monomer for solid-phase synthesis, with a solid-phase support synthesis scale of 1 μmol. Among them, conjugates 12 (SEQ ID NO: inc-G12), 13 (SEQ ID NO: inc-G13), 15 (SEQ ID NO: inc-G25), 19 (SEQ ID NO: inc-GalNAc 1b) and 20 (SEQ ID NO: inc-GalNAc 2) have the GalNAc ligand compound conjugated to the 3'-end of the oligonucleotide, and conjugates 14 (SEQ ID NO: G18-inc) and 16 (SEQ ID NO: G26-inc) have the GalNAc ligand compound conjugated to the 5'-end of the oligonucleotide.

[0411] (1) Reagent and monomer preparation

[0412] By using an acetonitrile solution of the monomer (1 / 20, w / v), a 0.25 M acetonitrile solution of 5-benzylthiotetrazole as the activator, a 0.2 M acetonitrile / pyridine (1 / 4, v / v) solution of hydrogen xanthate as the sulfurizing reagent, a 0.05 M water / pyridine (1 / 9, v / v) solution of iodine as the oxidizing reagent, 20% acetic anhydride in acetonitrile (v / v) as capping agent A, 20 / 30 / 50 (1-methylimidazole / pyridine / acetonitrile, v / v / v) as capping agent B, 20% diethylamine in acetonitrile (v / v) as the cyanoethyl removal reagent, and 3% dichloroacetic acid in toluene (v / v) as the DMT removal reagent. And they were loaded into the designated reagent positions in a 192P model DNA / RNA automatic synthesizer.

[0413] (2) Crude product synthesis

[0414] Input the specified oligonucleotide sequence, set the synthesis program, and after checking for errors, start the cyclic synthesis of the oligonucleotide. The monomer coupling time is about 1 minute, among which the oxygenation time is about 30-45 seconds and the sulfurization time is about 2 minutes. After the cycle ends, the solid-phase synthesis of the oligonucleotide is completed.

[0415] (3) Deprotection

[0416] After the synthesis is completed, transfer the solid support to a reactor and cleave the oligonucleotide from the solid support with concentrated ammonia water (25-28%) at 50-60 °C for 16-24 hours. Let the system cool to room temperature, then filter, wash with a mixed solution of purified water and ethanol, combine the filtrates, and concentrate the filtrates at low temperature to obtain a crude residue.

[0417] (4) Purification

[0418] Dissolve the deprotected crude residue in purified water, perform HPLC purification, collect the product peak solution, measure the content with a microplate reader, and confirm the molecular weight by ESIMS.

[0419] This step conjugates the GalNAc-CPG compound synthesized in Example 1 and the GalNAc phosphoramidite compound synthesized in Example 2 to the 3' or 5' end of the sense strand of siRNA.

[0420] YK-GAL-325 is used as the phosphoramidite monomer when synthesizing conjugate 12. Since YK-GAL-325 has a single arm, it is repeated 3 times in the synthesis sequence, so that the synthesized conjugate 12 has 3 GalNAc modification groups.

[0421] YK-GAL-326 is used as the phosphoramidite monomer when synthesizing conjugate 13. Since YK-GAL-326 has a double arm, it is repeated 2 times in the synthesis sequence, so that the synthesized conjugate 13 has 4 GalNAc modification groups.

[0422] 2. Preparation of GalNAc-unconjugated siRNA antisense strand

[0423] Synthesize the siRNA antisense strand according to the method for synthesizing the siRNA sense strand, using a universal CPG solid support, and the synthesis scale of each antisense strand complementary to the sense strand is 1 μmol.

[0424] 3. Preparation of conjugated double-stranded siRNA

[0425] Mix the siRNA sense strand and the complementary antisense strand in a ratio of 1:1 according to the UV absorption content, heat to 95 °C, and cool to room temperature after 3 minutes to form a double strand. After characterizing the product purity of the obtained double-strand solution by HPLC and measuring the content with a microplate reader, lyophilize to obtain a solid powder for storage and standby. The sequences and molecular weights of the GalNAc-conjugated siRNA double strands are shown in Table 2.

[0426] Table 2: GalNAc-conjugated double-stranded siRNA

[0427]

[0428] Wherein: SS is the sense strand, AS is the antisense strand, and the conjugated siRNA structure obtained is as follows:

[0429]

[0430]

[0431]

[0432]

[0433] (The GalNAc compound is L96, disclosed in US10465194B2, claim 10 compound),

[0434]

[0435] (The GalNAc compound is NAG0052, WO2023109938A1, page 100),

[0436]

[0437] (The GalNAc compound is GalNAc 1b, WO2023014938A1, page 145),

[0438]

[0439] (The GalNAc compound is GalNAc 2, WO2023049258A1, page 216).

[0440] Example 4: Inhibitory effect of GalNAc-conjugated siRNA on PCSK9 expression in mouse serum and liver and its effect on LDL-C level

[0441] In this example, the inhibitory rates of the siRNA conjugates in Table 2 on PCSK9 expression in mouse serum and liver and their effects on LDL-C levels were investigated. After the GalNAc-conjugated Inclisiran siRNA sequence enters the bloodstream, it first specifically binds to the asialoglycoprotein receptor (ASGPR) on the liver cell membrane through GalNAc, and then enters the hepatocytes. After the Inclisiran siRNA sequence enters the hepatocytes, it binds to the RNA-induced silencing complex (RISC), and under the mediation of the antisense strand, binds to the mRNA encoding the PCSK9 protein, inhibiting the production of the PCSK9 protein. The reduction of the PCSK9 protein in the liver promotes the recycling of LDL-R, thereby increasing the number of LDL-R receptors on the surface of hepatocytes, increasing the uptake and degradation of plasma LDL-C, and further reducing the plasma LDL-C level. Therefore, the higher the amount of the Inclisiran siRNA sequence delivered to the liver, that is, the higher the GalNAc delivery efficiency, the lower the amount of the PCSK9 protein in the liver and serum, and the lower the level of LDL-C in the liver.

[0442] The results showed that compared with the existing technology GalNAc oligonucleotide conjugates, in the inc-G4, inc-G5, inc-G6, inc-G7, inc-G11, inc-G12, inc-G13 and G18-inc experimental groups, the inhibitory rates of PCSK9 expression and the reduction levels of LDL-C in mouse serum and liver were significantly improved. For example, compared with inc-L96, inc-NAG0052, inc-GalNAc 1b and inc-GalNAc2, the inhibitory rates of inc-G5 on PCSK9 in serum increased by 14.2%, 13.1%, 14.5% and 51.2% respectively on the 7th day, and by 10.2%, 10.7%, 10.4% and 43.0% respectively on the 14th day; the inhibitory rates on PCSK9 in mouse liver increased by 11.1%, 13.2%, 11.0% and 44.0% respectively; the reduction levels of LDL-C increased by 20.4%, 17.1%, 15.8% and 29.8% respectively on the 7th day, and by 23.5%, 20.5%, 20.0% and 33.3% respectively on the 14th day.

[0443] Animal preparation:

[0444] First, after adaptively raising hPCSK9 transgenic mice (SPF grade, purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd.), they were randomly divided into a negative control group (without drug administration) and siRNA test drug groups (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. Each group had 5 male mice.

[0445] Route and dose of drug administration:

[0446] Administer the drug by single subcutaneous injection. The dose is 6 mg / kg, the volume is 1 mL / kg, and the concentration is 6 mg / mL. The day of administration is recorded as day 0.

[0447] 1. Inhibitory efficiency of different GalNAc-conjugated siRNAs on PCSK9 in the serum of hPCSK9 mice

[0448] The results of the inhibitory efficiency of PCSK9 in the serum of hPCSK9 mice showed that, compared with the existing technology GalNAc, the oligonucleotide conjugate prepared from the GalNAc compound of the present invention significantly improved the inhibitory rate of PCSK9 protein expression in the serum of mice. For example, compared with inc-L96, inc-NAG0052, inc-GalNAc 1b, and inc-GalNAc 2, the inhibitory rates of inc-G5 on the 7th day increased by 14.2%, 13.1%, 14.5%, and 51.2% respectively, and on the 14th day, they increased by 10.2%, 10.7%, 10.4%, and 43.0% respectively.

[0449] Experimental procedure:

[0450] Before drug administration (D0), 7 days after drug administration (D7), and 14 days after drug administration (D14) of the experimental animals, approximately 200 μL of blood was collected from the orbital venous plexus (without anticoagulation). The whole blood samples were temporarily stored in an ice box before centrifugation and centrifuged at 4000 r / min for 10 min at 4°C to separate the serum. The PCSK9 protein level in the serum was detected using an ELISA kit (Sino Biological).

[0451] Experimental results:

[0452] The inhibitory rates of the negative control group and each test drug group on the PCSK9 protein in the serum 7 days and 14 days after drug administration are shown in Table 4. Data statistics and analysis were performed using GraphPad Prism 9 software.

[0453] 1) Structural differences

[0454] The structural differences of GalNAc oligonucleotide conjugates are shown in Table 3:

[0455] Table 3: Structural comparison of GalNAc oligonucleotide conjugates

[0456]

[0457] 2) Inhibitory efficiency of PCSK9 in the serum of hPCSK9 mice

[0458] Table 4: Inhibitory rate of PCSK9 protein expression in mouse serum

[0459]

[0460] 1) inc-G4, inc-G5, inc-G6, inc-G7, inc-G11, inc-G12, inc-G13 and G18-inc have the highest inhibitory rates on PCSK9 protein expression in mouse serum. For example, the inhibitory rates of inc-G5 reached 94.4% and 95.6% on the 7th and 14th days respectively. This shows that the ingeniously designed linker structure can significantly improve the bioavailability of the drug and exert better drug efficacy.

[0461] As can be seen from Table 4, the conjugates prepared from the GalNAc compounds designed by the present invention have very different inhibitory effects on PCSK9 protein expression in mouse serum. The inhibitory rates of inc-G4, inc-G5, inc-G6, inc-G7, inc-G11, inc-G12, inc-G13 and G18-inc are significantly higher than those of other groups, and the inhibitory rates both exceed 90% on the 7th and 14th days. The highest inhibitory rate is inc-G5, reaching 94.4% and 95.6% on the 7th and 14th days respectively. ( Figure 1 )

[0462] The inhibitory rates of inc-G2, inc-G3, inc-G8 and inc-G9 are also relatively high, about 85%. The inhibitory rates of inc-G1, inc-G10 and G26-inc are between 70 - 80%. The lowest inhibitory rate is inc-G25, which is 55.9%.

[0463] ( Figure 2 )

[0464] The inc-G5 with the highest inhibitory rate is 38.5% and 35.5% higher than the inc-G25 with the lowest inhibitory rate on the 7th and 14th days respectively, showing a significant difference.

[0465] 2) The GalNAc compounds designed by the present invention with 3 antennae, 1 antenna or 2 antennae are used to prepare conjugates with 3 or 4 GalNAc groups, all of which have very high inhibition rates. Moreover, regardless of whether the GalNAc compound is conjugated to the 3'-end or 5'-end of the oligonucleotide, it can efficiently inhibit the expression of PCSK9 protein.

[0466] The GalNAc compound YK-GAL-325 designed by the present invention has 1 antenna, and YK-GAL-326 has 2 antennae (see Table 1 in Example 2). inc-G12 is obtained by connecting YK-GAL-325 through 2 phosphodiester bonds to form a conjugate with 3 GalNAc groups. inc-G13 is obtained by connecting YK-GAL-326 through 1 phosphodiester bond to form a conjugate with 4 GalNAc groups. The results of activity detection show that inc-G12 and inc-G13 also have significant inhibitory effects on the expression of PCSK9 protein in mouse serum. The inhibition rates reached 93.8% and 94.2% respectively on the 7th day, and 95.3% and 95.5% respectively on the 14th day, which are comparable to inc-G5.

[0467] 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 results of activity detection show that G18-inc also has a significant inhibitory effect on the expression of PCSK9 protein in mouse serum. The inhibition rates reached 93.5% and 95.2% respectively on the 7th day and the 14th day, and the inhibition rate is comparable to inc-G5.

[0468] Therefore, it can be seen that the oligonucleotide conjugates prepared from the GalNAc compounds designed by the present invention with 3 antennae, 1 antenna or 2 antennae all have very high delivery efficiencies. Moreover, 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 and achieve a significant inhibitory effect on the expression of PCSK9 protein in mouse serum.

[0469] 3) Compared with inc-L96, inc-NAG0052, inc-GalNAc 1b and inc-GalNAc2 prepared from GalNAc compounds in the prior art, the oligonucleotide conjugates prepared from the GalNAc compounds designed by the present invention have a significantly increased inhibition rate of PCSK9 protein expression in mouse serum. For example, inc-G5 is 14.2% higher than inc-L96.

[0470] Conjugates prepared from prior art GalNAc compounds, including inc-L96, inc-NAG0052, inc-GalNAc1b, and inc-GalNAc 2, had inhibition rates of PCSK9 protein expression in mouse serum of 80.2%, 81.3%, 79.9%, and 43.2% on the 7th day, and 85.4%, 84.9%, 85.2%, and 52.6% on the 14th day, respectively.

[0471] 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, showed a significant increase in the inhibition rate of PCSK9 protein expression in mouse serum compared to prior art GalNAc.

[0472] For example, the inhibition rate of inc-G5 on the 7th day was increased by 14.2%, 13.1%, 14.5%, and 51.2% compared to inc-L96, inc-NAG0052, inc-GalNAc 1b, and inc-GalNAc 2, respectively, and on the 14th day was increased by 10.2%, 10.7%, 10.4%, and 43.0%, respectively. Both inc-G25 and inc-GalNAc 2 are oligonucleotide conjugates with 1 GalNAc group, but the inhibition rate of inc-G25 was increased by 12.7% compared to inc-GalNAc 2, showing a significant increase.

[0473] 4) Oligonucleotide conjugates prepared from GalNAc compounds with similar structures are very likely to have extremely different inhibition rates of PCSK9 protein expression in mouse serum; oligonucleotide conjugates prepared from GalNAc compounds with large structural differences are also very likely to have very close inhibition rates of PCSK9 protein expression in mouse serum.

[0474] From the structural comparison in the GalNAc oligonucleotide conjugate structure diagram (Example 3) and Table 3, it can be seen that the structures of this series of GalNAc compounds designed in the present invention are very similar, with only slight differences in individual groups, and are also very similar to the structures of prior art GalNAc compounds L-96, NAG0052, GalNAc 1b, and GalNAc 2. However, for oligonucleotide conjugates prepared from these GalNAc compounds with similar structures, the inhibition rates of PCSK9 protein expression in mouse serum are some close and some very different.

[0475] For example, compared with inc-L96, inc-G5 only changes the prolinol structure in the backbone of inc-L96 to a ribose ring The other structures are exactly the same, but for the inhibitory rate of PCSK9 protein expression, inc-G5 can increase by 14.2% compared to inc-L96, and the inhibitory activity is significantly improved.

[0476] Compared with GalNAc 1b, inc-G12 only differs in the atom connected to the 1'-position of the ribose ring. In GalNAc 1b, it is carbon, while in inc-G12, it is oxygen. The linker arm of inc-G12 is longer than that of GalNAc 1b. The other structures are exactly the same, but the inhibitory rate of inc-G12 is 13.9% higher than that of GalNAc 1b, showing a significant improvement.

[0477] inc-G5 only differs from inc-G1 in the 2'-position of the ribose ring. inc-G5 has a methoxy group, while inc-G1 has a hydrogen atom, but the inhibitory rate of inc-G5 is 21.4% higher than that of inc-G1.

[0478] Therefore, GalNAc compounds with similar chemical structures do not necessarily have similar oligonucleotide delivery efficiencies. The inhibitory effects of GalNAc oligonucleotide conjugates prepared from them on the PCSK9 gene in mouse serum are not consistent and are very likely to have significant differences.

[0479] 2. Inhibitory efficiency of siRNAs conjugated with different GalNAcs on PCSK9 in the livers of hPCSK9 mice

[0480] Experimental procedure:

[0481] On the 14th day (D14) after drug administration to the experimental animals, the animals were anesthetized and sacrificed, followed by perfusion, and the livers were collected.

[0482] According to the tissue weight: RNA lysis buffer (trizol, Ambion) = 100 mg: 1 mL, quickly place it into a 1.5 mL RNase-free EP tube containing 1 mL of RNA lysis buffer (trizol). Add 3 3-mm steel beads (treated without RNA) to the tube, put it into a tissue homogenizer, run it at 50 Hz for 30 seconds, pause for 10 seconds, and run it 3 times to prepare tissue homogenate.

[0483] Centrifuge at 4°C at 12,000×g for 3 minutes. Transfer 400 μL of the homogenate supernatant to a 1.5 mL RNase-free EP tube and place it on ice. Add 80 μL of chloroform to each tube, shake vigorously for 15 seconds, and let it stand at room temperature for 5 minutes. Centrifuge at 4°C at 12,000×g for 15 minutes, and transfer 150 μL of the supernatant to a new EP tube.

[0484] Add an equal volume of isopropanol, gently mix the liquid in the tube by inverting it up and down, let it stand at -20°C for 10 minutes, centrifuge at 4°C at 12,000×g for 15 minutes, and discard the supernatant.

[0485] Add 1 mL of 75% ethanol, gently wash the RNA pellet, centrifuge at 7,500×g for 5 minutes at 4°C, and aspirate the supernatant. Repeat the rinsing once, centrifuge at 7,500×g for 5 minutes at 4°C, and remove the residual ethanol completely with a micropipette tip.

[0486] Air-dry the residual ethanol at room temperature for 10 minutes, add 150 μL of RNase-free ddH2O, and dissolve. Detect the RNA concentration using a micro UV spectrophotometer. Detect the expression of the PCSK9 gene by qPCR, measuring the mRNA, and the results are shown in Table 5.

[0487] Experimental results:

[0488] By statistically analyzing the qPCR results (Mean±SD) and using GraphPad Prism 9 software for plotting and data analysis, the specific results are shown in Table 5.

[0489] Table 5 Inhibition rate of PCSK9 gene expression in mouse liver on the 14th day after administration

[0490]

[0491] 1) inc-G4, inc-G5, inc-G6, inc-G7, inc-G11, inc-G12, inc-G13, and G18-inc have the highest inhibition rates on PCSK9 in mouse liver. For example, the inhibition rates of inc-G5, inc-G6, and inc-G12 all reached 90%. This shows that a cleverly designed linking structure can significantly improve the bioavailability of the drug and exert better pharmacological effects.

[0492] As can be seen from Table 5, the conjugates prepared from the GalNAc compounds designed by the present invention have very different inhibition rates on the expression of the PCSK9 gene in mouse liver. inc-G4, inc-G5, inc-G6, inc-G7, inc-G11, inc-G12, inc-G13, and G18-inc have the highest inhibition rates, all above 85%, and among them, inc-G5, inc-G6, and inc-G12 reached 90%. ( Figure 3 )

[0493] The inhibition rates of inc-G2 and inc-G3 are also relatively high, between 80 - 85%. The inhibition rates of inc-G8, inc-G9, and G26-inc are between 70 - 80%. The inhibition rates of inc-G1, inc-G10, and inc-G25 are between 60 - 70%. ( Figure 4 )

[0494] The inc-G5 with the highest inhibition rate is 30.3% higher than inc-G10 with the lowest inhibition rate, showing a significant difference.

[0495] 2) The GalNAc compounds designed in the present invention with 3 antennas, 1 antenna or 2 antennas are used to prepare oligonucleotide conjugates with 3 or 4 GalNAc groups, all of which have very high inhibition rates. Moreover, these GalNAc compounds can efficiently inhibit the expression of the PCSK9 gene whether conjugated to the 3'-end or 5'-end of the oligonucleotide.

[0496] The GalNAc compound YK-GAL-325 designed in the present invention has 1 antenna, and YK-GAL-326 has 2 antennas (see Table 1 in Example 2). inc-G12 is obtained by connecting YK-GAL-325 through 2 phosphodiester bonds to get a conjugate with 3 GalNAc groups. inc-G13 is obtained by connecting YK-GAL-326 through 1 phosphodiester bond to get a conjugate with 4 GalNAc groups. The results of activity detection show that inc-G12 and inc-G13 also have significant inhibitory effects on the expression of the PCSK9 gene in mouse liver, and the inhibition rates are 90.1% and 89.9% respectively, which are comparable to inc-G5.

[0497] 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 results of activity detection show that G18-inc also has a significant inhibitory effect on the expression of the PCSK9 gene in mouse liver, and the inhibition rate is 89.7%, which is comparable to inc-G5.

[0498] Therefore, it can be seen that the oligonucleotide conjugates prepared from the GalNAc compounds designed in the present invention with 3 antennas, 1 antenna or 2 antennas, which have 3 or 4 GalNAc groups, all have very high delivery efficiencies. Moreover, whether conjugated to the 3'-end or 5'-end of the oligonucleotide, they can efficiently deliver the oligonucleotide Inclisiran siRNA sequence to the liver and have a significant inhibitory effect on the expression of the PCSK9 gene in mouse liver.

[0499] 3) Compared with inc-L96, inc-NAG0052, inc-GalNAc 1b and inc-GalNAc2 prepared from GalNAc compounds in the prior art, the inhibition rates of the oligonucleotide conjugates prepared from the GalNAc compounds designed in the present invention are significantly improved. For example, inc-G5 is 11.1% higher than inc-L96.

[0500] Conjugates prepared from prior art GalNAc compounds, including inc-L96, inc-NAG0052, inc-GalNAc1b, and inc-GalNAc 2, had inhibition rates of PCSK9 gene expression in mouse liver of 80.1%, 78.0%, 80.2%, and 47.2%, respectively.

[0501] 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, showed a significant increase in the inhibition rate of PCSK9 gene expression in mouse liver compared to prior art GalNAc.

[0502] For example, the inhibition rate of inc-G5 was increased by 11.1%, 13.2%, 11.0%, and 44.0% compared to inc-L96, inc-NAG0052, inc-GalNAc 1b, and inc-GalNAc2, respectively. Both inc-G25 and inc-GalNAc 2 are oligonucleotide conjugates with 1 GalNAc group, but the inhibition rate of inc-G25 was increased by 14.4% compared to inc-GalNAc2, showing a significant improvement.

[0503] 4) Oligonucleotide conjugates prepared from GalNAc compounds with similar structures are very likely to have greatly different inhibition rates of PCSK9 gene expression in mouse liver; oligonucleotide conjugates prepared from GalNAc compounds with large structural differences are also very likely to have very similar inhibition rates of PCSK9 gene expression in mouse liver.

[0504] From the structural comparison in the GalNAc oligonucleotide conjugate structure diagram (Example 3) and Table 3, it can be seen that the structures of this series of GalNAc compounds designed in the present invention are very close to each other, with only slight differences in individual groups, and are also very close to the structures of prior art GalNAc compounds L-96, NAG0052, GalNAc 1b, and GalNAc 2. However, for the oligonucleotide conjugates prepared from these GalNAc compounds with similar structures, the inhibition rates of PCSK9 gene expression in mouse liver are some close and some very different.

[0505] For example, compared with inc-L96, inc-G5 only changes the prolinol structure in the backbone of inc-L96 to a ribose ring structure, and the other structures are exactly the same. However, for the inhibition rate of PCSK9 gene expression, inc-G5 can be increased by 11.1% compared to inc-L96, showing a significant improvement in inhibitory activity.

[0506] Compared with GalNAc 1b, inc-G12 only differs in the atom connected to the 1'-position of the ribose ring. In GalNAc 1b, it is carbon, while in inc-G12, it is oxygen. The linker arm of inc-G12 is longer than that of GalNAc 1b, and the other structures are exactly the same. However, the inhibition rate of inc-G12 is 9.9% higher than that of GalNAc 1b, showing a significant increase.

[0507] inc-G5 and inc-G1 only differ in the 2'-position of the ribose ring. In inc-G5, it is a methoxy group, while in inc-G1, it is hydrogen. However, the inhibition rate of inc-G5 is 21.3% higher than that of inc-G1.

[0508] Both inc-GalNAc 2 and inc-G25 are oligonucleotide conjugates with one GalNAc group, but the inhibition rate of inc-G25 is 14.4% higher than that of inc-GalNAc 2, showing a significant increase.

[0509] Therefore, it is not necessarily the case that GalNAc compounds with similar chemical structures have similar oligonucleotide delivery efficiencies. The inhibitory effects of GalNAc oligonucleotide conjugates prepared from them on the PCSK9 gene in mouse liver are not consistent and are very likely to have extremely large differences.

[0510] 3. Effects of siRNAs conjugated with different GalNACs on LDL-C levels in hPCSK9 mice

[0511] Experimental procedure:

[0512] Approximately 200 μL of blood (non-anticoagulated) was collected from the orbital venous plexus of the experimental animals before drug administration (D0), on the 7th day after drug administration (D7), and on the 14th day after drug administration (D14). The whole blood samples were temporarily stored in an ice box before centrifugation and centrifuged at 4000 r / min for 10 minutes at 4 °C to separate the serum, and the level of serum LDL-C was detected.

[0513] Experimental results:

[0514] Through the statistics of serum LDL-C (Mean±SD), GraphPad Prism 9 software was used for plotting and data analysis. The specific results are shown in Table 6.

[0515] Table 6: Reduction levels of LDL-C in the serum of each experimental group

[0516]

[0517] 1) inc-G4, inc-G5, inc-G6, inc-G7, inc-G11, inc-G12, inc-G13 and G18-inc showed significantly higher LDL-C reduction levels than other groups. For example, inc-G5 reached 49.6% and 54.1% on the 7th and 14th days respectively. This indicates that a cleverly designed linkage structure can significantly improve the bioavailability of the drug and exert better drug efficacy.

[0518] As can be seen from Table 6, for the conjugates prepared from the GalNAc compounds designed by the present invention, inc-G4, inc-G5, inc-G6, inc-G7, inc-G11, inc-G12, inc-G13 and G18-inc had the highest LDL-C reduction levels in mouse serum, exceeding 35% on both the 7th and 14th days, which was significantly higher than other experimental groups. Among them, the highest reduction level was inc-G5, reaching 49.6% and 54.1% on the 7th and 14th days respectively. ( Figure 5 )

[0519] The reduction levels of inc-G2, inc-G3 and inc-G8 were between 30 - 35%. The reduction levels of inc-G9, inc-G25 and G26-inc were all between 20 - 30%. The reduction levels of inc-G1 and inc-G10 were lower than 20%, and inc-G1 was only 15.9% and 17.0% on the 7th and 14th days respectively. ( Figure 6 )

[0520] The inc-G5 with the highest reduction level was more than 30% higher than inc-G1 with the lowest reduction level, and the difference was significant.

[0521] 2) The GalNAc compounds with 3 arms, 1 arm or 2 arms designed by the present invention were used to prepare oligonucleotide conjugates with 3 or 4 GalNAc groups, all of which had very high reduction levels. And these GalNAc compounds could efficiently reduce the LDL-C level whether conjugated to the 3'-end or 5'-end of the oligonucleotide.

[0522] The GalNAc compound YK-GAL-325 designed by the present invention has 1 arm, and YK-GAL-326 has 2 arms (see Table 1 in Example 2). inc-G12 is a conjugate with 3 GalNAc groups obtained by connecting YK-GAL-325 through 2 phosphodiester bonds. inc-G13 is a conjugate with 4 GalNAc groups obtained by connecting YK-GAL-326 through 1 phosphodiester bond. The results of activity detection show that inc-G12 and inc-G13 can also significantly reduce the LDL-C level in the serum of mice. The reduction levels on the 7th day are 48.5% and 47.3% respectively, and on the 14th day are 51.9% and 49.5% respectively, which are comparable to inc-G5.

[0523] 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 results of activity detection show that G18-inc can also significantly reduce the LDL-C level in the serum of mice. The reduction levels on the 7th day and the 14th day are 45.6% and 47.9% respectively, which are comparable to inc-G5.

[0524] It can be seen from this that the oligonucleotide conjugates prepared from the GalNAc compounds with 3 arms, 1 arm or 2 arms designed by the present invention all have a significant inhibitory effect on the expression of PCSK9 gene. And whether conjugated to the 3'-end or the 5'-end of the oligonucleotide, they can efficiently deliver the oligonucleotide Inclisiran siRNA sequence to the liver, achieve a significant inhibitory effect on the expression of PCSK9 gene in the serum of mice, and further reduce the LDL-C level.

[0525] 3) Compared with inc-L96, inc-NAG0052, inc-GalNAc 1b and inc-GalNAc2 prepared from GalNAc compounds in the prior art, the oligonucleotide conjugates prepared from the GalNAc compounds designed by the present invention have a significantly improved reduction level of LDL-C in the serum of mice. For example, inc-G5 is 20.4% and 23.5% higher than inc-L96 on the 7th day and the 14th day respectively.

[0526] Among the conjugates prepared from GalNAc compounds in the prior art, the reduction levels of inc-L96, inc-NAG0052 and inc-GalNAc1b are between 25% and 35%, and the reduction level of inc-GalNAc 2 is about 20%.

[0527] 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, show a significantly improved reduction level of LDL-C in mouse serum compared with the prior art GalNAc.

[0528] For example, on the 7th day, the reduction levels of inc-G5 were increased by 20.4%, 17.1%, 15.8% and 29.8% respectively compared with inc-L96, inc-NAG0052, inc-GalNAc1b and inc-GalNAc 2, and on the 14th day, they were increased by 23.5%, 20.5%, 20.0% and 33.3% respectively.

[0529] 4) The oligonucleotide conjugates prepared from GalNAc compounds with similar structures are very likely to have a huge difference in the reduction level of LDL-C in mouse serum; the oligonucleotide conjugates prepared from GalNAc compounds with large structural differences are very likely to be very close in the reduction level of LDL-C in mouse serum.

[0530] It can be seen from the structural comparison in the GalNAc oligonucleotide conjugate structure diagram (Example 3) and Table 3 that the structures of this series of GalNAc compounds designed in the present invention are very close, with only slight differences in individual groups, and are also very similar to the structures of the prior art GalNAc compounds L-96, NAG0052, GalNAc 1b and GalNAc 2. However, the effects of the oligonucleotide conjugates prepared from these GalNAc compounds with similar structures on the LDL-C level in mouse serum are some close and some very different.

[0531] For example, compared with inc-L96, inc-G5 only changes the prolinol structure in the backbone of inc-L96 to a ribose ring structure, and the other structures are exactly the same. However, for the reduction level of LDL-C in serum, inc-G5 can be increased by more than 20% compared with inc-L96, showing a significant improvement. Compared with GalNAc 1b, inc-G12 only has a different atom connected to the 1'-position of the ribose ring. GalNAc 1b is carbon, while inc-G12 is oxygen. The linker arm of inc-G12 is longer than that of GalNAc 1b, and the other structures are exactly the same. However, the reduction level of inc-G12 is increased by more than 15% compared with GalNAc 1b, showing a significant improvement. The only difference between inc-G5 and inc-G1 is at the 2'-position of the ribose ring. inc-G5 has a methoxy group, while inc-G1 has a hydrogen atom. However, for the effect on the LDL-C level 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 increased by more than 30% compared with inc-G1, showing a significant difference.

[0532] Therefore, GalNAc compounds with similar chemical structures do not necessarily have similar oligonucleotide delivery efficiencies. The effects of GalNAc oligonucleotide conjugates prepared therefrom on LDL-C levels in mouse serum are not consistent and are very likely to have extremely large differences.

[0533] Example 5: Distribution of Different GalNAc-Conjugated siRNAs in Different Tissues and Organs of Mice

[0534] After the Inclisiran siRNA sequence conjugated with the GalNAc ligand enters the blood, it first partially enters hepatocytes with the help of GalNAc and inhibits the target gene. The remaining siRNAs in the serum are cleared by the kidneys. Since siRNAs in plasma are mainly cleared by the kidneys, siRNA sequences are also distributed in the kidneys in addition to the liver.

[0535] Experimental procedure:

[0536] Six- to eight-week-old wild-type C57BL / 6 mice, six mice in each group. Each group was respectively administered 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 with a Cy5 fluorophore. The negative control group was not administered. After weighing the mice, they were administered at a dose of 6 mg / kg by subcutaneous injection, and the injection volume was determined according to the dosing volume, with the injection amount not exceeding 0.1 - 0.2 mL.

[0537] After administration, the abdominal hair of the mice was shaved off, and after anesthesia with isoflurane, the mice were placed in a small animal in vivo imaging system in a supine position, and the imaging of the mice was observed 4 hours and 8 hours after administration under the Cy5 channel. The Living Image in vivo imaging software was used to statistically analyze the luminescence intensity of the mice and compare the differences between different test groups.

[0538] Experimental results:

[0539] The detection results of the fluorescence intensity (which can represent the relative content of oligonucleic acid) in the mouse liver and kidneys are shown in Table 7.

[0540] Table 7: Fluorescence Intensity in Mouse Liver and Kidneys

[0541] Experimental group 4 h - Liver 4 h - Kidney 8 h - Liver 8 h - Kidney Negative control group 6.14E+07 5.54E+07 6.35E+07 5.75E+07 inc - G4 8.50E+09 6.39E+09 9.03E+09 6.35E+09 inc - G5 9.12E+09 4.31E+09 9.16E+09 3.44E+09 inc - G6 8.92E+09 5.70E+09 9.03E+09 4.11E+09 inc - G7 8.84E+09 6.67E+09 9.09E+09 5.90E+09 inc - G11 8.58E+09 6.45E+09 9.03E+09 5.09E+09 inc - G12 9.01E+09 4.15E+09 9.09E+09 3.02E+09 inc - G13 8.98E+09 4.20E+09 9.01E+09 3.22E+09 G18 - inc 8.95E+09 3.99E+09 9.13E+09 3.10E+09 inc - G1 6.00E+09 7.32E+09 7.42E+09 9.19E+09 inc - G2 8.17E+09 7.97E+09 8.45E+09 7.07E+09 inc - G3 8.13E+09 7.67E+09 8.01E+09 7.01E+09 inc - G8 8.05E+09 7.91E+09 8.10E-+09 8.35E+09 inc - G9 7.99E+09 8.86E+09 7.69E+09 8.92E+09 inc - G10 7.83E-+09 8.88E+09 7.64E+09 8.62E+09 inc - G25 5.89E+09 6.40E+09 5.08E+09 6.33E+09 G26 - inc 7.02E+09 5.52E+09 6.65E+09 5.87E+09 inc - L96 8.22E+09 9.01E+09 5.99E+09 9.15E+09 inc - NAG0052 8.01E+09 8.83E+09 6.52E+09 8.94E+09 inc - GalNAc1b 7.75E+09 8.42E+09 6.35E+09 8.77E+09 inc - GalNAc2 4.33E+09 4.01E+09 4.03E+09 5.24E+09

[0542] 1) The fluorescence intensities of inc-G4, inc-G5, inc-G6, inc-G7, inc-G11, inc-G12, inc-G13, and G18-inc in the liver were significantly higher than those of other groups. For example, the highest inc-G5 had fluorescence intensities of 9.12E+09 and 9.16E+09 at 4 hours and 8 hours, respectively, indicating that the ingeniously designed linking structure can significantly improve the delivery effect of oligonucleotide drugs.

[0543] As can be seen from Table 7, the oligonucleotides labeled with Cy5 fluorescence were distributed in both the mouse liver and kidney. The fluorescence intensities of the conjugates prepared from the GalNAc compounds designed in the present invention varied greatly. 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 other groups, that is, the relative content of oligonucleotides was significantly higher than that of other groups. Among them, the highest fluorescence intensity was inc-G5, reaching 9.12E+09 at 4 hours and 9.16E+09 at 8 hours. ( Figure 7 )

[0544] The fluorescence intensities of inc-G2, inc-G3, and inc-G8 were also relatively high, both between 8.00E+09 and 8.50E+09 at 4 hours and 8 hours. The fluorescence intensities of inc-G9, inc-G10, and G26-inc were between 7.00E+09 and 8.00E+09 at 4 hours and between 6.50E+09 and 8.00E+09 at 8 hours. The fluorescence intensities of inc-G1 and inc-G25 were between 5.00E+09 and 7.50E+09.

[0545] The inc-G5 with the highest fluorescence intensity could increase by 52.0% and 23.5% compared with the inc-G1 with the lowest fluorescence intensity at 4 hours and 8 hours, respectively, showing a significant improvement.

[0546] 2) The GalNAc compounds with 3 arms, 1 arm, or 2 arms designed in the present invention were used to prepare oligonucleotide conjugates with 3 or 4 GalNAc groups, and all of them had very high fluorescence intensities in the liver. Moreover, these GalNAc compounds could efficiently deliver oligonucleotides to the liver whether conjugated to the 3'-end or 5'-end of the oligonucleotide.

[0547] The GalNAc compound YK-GAL-325 designed in the present invention has 1 antenna, and YK-GAL-326 has 2 antennas (see Table 1 in Example 2). inc-G12 is a conjugate with 3 GalNAc groups obtained by connecting YK-GAL-325 through 2 phosphodiester bonds. inc-G13 is a conjugate with 4 GalNAc groups obtained by connecting YK-GAL-326 through 1 phosphodiester bond. The fluorescence intensities of inc-G12 and inc-G13 at 4 hours are 9.01E+09 and 8.98E+09 respectively, and at 8 hours are 9.09E+09 and 9.01E+09 respectively, which are comparable to inc-G5.

[0548] 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 fluorescence intensities of G18-inc at 4 hours and 8 hours are 8.95E+09 and 9.13E+09 respectively, which are comparable to inc-G5.

[0549] It can be seen from this that the oligonucleotide conjugates prepared from the GalNAc compounds with 3 antennas, 1 antenna or 2 antennas designed in the present invention all have very high delivery efficiency, and whether conjugated to the 3'-end or 5'-end of the oligonucleotide, they can efficiently deliver the oligonucleotide Inclisiran siRNA sequence to the liver.

[0550] 3) The oligonucleotide conjugates prepared from the GalNAc compounds designed in the present invention show a significant increase in the fluorescence absorption intensity in the mouse liver compared with inc-L96, inc-NAG0052, inc-GalNAc 1b and inc-GalNAc2 prepared from the GalNAc compounds in the prior art. For example, inc-G5 is more than 50% higher than inc-L96.

[0551] From Table 7 and Figure 7It can be seen that the oligonucleotides labeled with Cy5 are distributed in the liver and kidneys of mice. The fluorescence intensities of inc-G4, inc-G5, inc-G6, inc-G7, inc-G12, inc-G13, and G18-inc in the liver are significantly higher than those of inc-L96, inc-NAG0052, inc-GalNAc1b, and inc-GalNAc 2 at 4 hours and 8 hours. For example, the fluorescence intensities of inc-G5 in the liver at 4 hours and 8 hours are 9.12E+09 and 9.16E+09 respectively, which are 10.9% and 52.9% higher than those of inc-L96, 13.9% and 40.5% higher than those of inc-NAG0052, 17.7% and 44.3% higher than those of inc-GalNAc 1b, and 110.6% and 127.3% higher than those of inc-GalNAc 2, showing a significant increase. The fluorescence intensities of inc-G6 at 4 hours and 8 hours are 8.92E+09 and 9.03E+09 respectively, which are 8.5% and 50.8% higher than those of inc-L96, 11.4% and 38.5% higher than those of inc-NAG0052, 15.1% and 42.2% higher than those of inc-GalNAc 1b, and 106.0% and 124.1% higher than those of inc-GalNAc 2, showing a significant increase.

[0552] 4) For the oligonucleotide conjugates prepared from GalNAc compounds with similar structures, it is very likely that there are huge differences in the efficiency of delivering oligonucleotides to the liver; for the oligonucleotide conjugates prepared from GalNAc compounds with large structural differences, it is also very likely that the efficiency of delivering oligonucleotides to the liver is very close.

[0553] As can be seen from the structural comparison in the GalNAc oligonucleotide conjugate structure diagram (Example 3) and Table 3, the structures of this series of GalNAc compounds designed in the present invention are very similar, with only slight differences in individual groups, and are also very similar to the structures of the prior art GalNAc compounds L-96, NAG0052, GalNAc 1b, and GalNAc 2. However, for the oligonucleotide conjugates prepared from these GalNAc compounds with similar structures, the inhibition rates of PCSK9 protein expression in mouse serum are some close and some very different.

[0554] For example, when inc-G5 is compared with inc-L96, only the prolinol structure in the backbone of inc-L96 is changed to a ribose ring structure, and the other structures are exactly the same. However, the fluorescence intensity of inc-G5 in the liver can be increased by more than 50% compared with inc-L96, and the delivery efficiency is significantly improved.

[0555] Compared with GalNAc 1b, inc-G12 only differs in the atom connected to the 1'-position of the ribose ring. For GalNAc 1b, it is carbon, while for inc-G12, it is oxygen. The linker arm of inc-G12 is longer than that of GalNAc 1b, and the other structures are exactly the same. However, the fluorescence intensity of inc-G12 in the liver is more than 40% higher than that of GalNAc 1b.

[0556] inc-G5 and inc-G1 only differ in the 2'-position of the ribose ring. For inc-G5, it is methoxy, while for inc-G1, it is hydrogen. However, the fluorescence intensity of inc-G5 in the liver is more than 50% higher than that of inc-G1.

[0557] Therefore, GalNAc compounds with similar chemical structures do not necessarily have similar oligonucleotide delivery efficiencies. On the contrary, there is a very high probability that there are huge differences.

[0558] Example 6: Pharmacokinetics of Different GalNAc-Conjugated siRNAs in Animal Livers

[0559] Experimental Procedure:

[0560] Male Sprague-Dawley rats aged 6 - 9 weeks were used as experimental animals, with 30 rats in each group. Each group was respectively administered 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. After weighing the rats, they were administered at a dose of 5 mg / kg by subcutaneous injection. The administration concentration was 1 mg / mL, and the administration volume was 5 mL. Tissue samples were collected at 9 time points: 6, 24, 72, 168, 336, 504, 672, 1008, and 1344 h after administration. After sacrificing the experimental animals with carbon dioxide, the livers were removed, rinsed with pre-cooled physiological saline, dried with filter paper, weighed, and transferred to a labeled tube. They were homogenized under ice-cold conditions at a ratio of 1:9 (1 g of tissue was added with 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 then the drug concentration in the liver was detected by LC-MS / MS.

[0561] Experimental Results: The data of half-life, peak drug concentration, and area under the drug-time curve are shown in Table 8.

[0562] Table 8: Data of Half-Life, Peak Drug Concentration, and Area under the Drug-Time Curve

[0563] Experimental group Half - life (h) Peak drug concentration (ng / mL) Area under the drug - time curve (h * ng / mL) inc - G4 94 4057 292836 inc - G5 110 4450 339466 inc - G6 103 4200 343186 inc - G7 103 4077 329675 inc - G11 85 4249 292469 inc - G12 101 4351 330245 inc - G13 95 4280 329874 G18 - inc 102 4179 304785 inc - G1 56 3210 218457 inc - G2 84 4020 274861 inc - G3 78 3920 248637 inc - G8 60 3408 249106 inc - G9 73 3426 245634 inc - G10 64 3358 221649 inc - G25 75 3504 253012 G26 - inc 82 3980 301254 inc - L96 74 3537 250520 inc - NAG0052 75 3612 257841 inc - GalNAc 1b 79 3705 261218 inc - GalNAc 2 65 3401 231250

[0564] 1) The half-lives of inc-G4, inc-G5, inc-G6, inc-G7, inc-G11, inc-G12, inc-G13, and G18-inc were significantly increased compared with 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 indicates that the ingeniously designed linking structure can significantly improve the in vivo pharmacokinetic properties of drugs.

[0565] As can be seen from Table 8, the half-lives of the conjugates prepared from the GalNAc compounds designed by the present invention vary greatly. The half-lives of inc-G4, inc-G5, inc-G6, inc-G7, inc-G11, inc-G12, inc-G13, and G18-inc were significantly increased compared with 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, and those of inc-G4, inc-G11, and inc-G13 also reached 85 hours.( Figure 8 )

[0566] The half-lives of inc-G2, inc-G3, inc-G9, inc-G25, and G26-inc were between 70 and 85 hours. The half-lives of inc-G8 and inc-G10 were between 60 and 70 hours. The shortest half-life was that of inc-G1, which was 56 hours.

[0567] ( Figure 9 )

[0568] The difference between the longest half-life of inc-G5 and the shortest half-life of inc-G1 was 54 hours, and inc-G5 reached twice that of inc-G1, with a very significant difference.

[0569] The trends of increase or decrease in the peak concentration and the area under the concentration-time curve of each tested drug group were also consistent with the half-life.

[0570] 2) The conjugates with 3, 1, or 2 antennae GalNAc compounds designed by the present invention, which were prepared to have conjugates with 3 or 4 GalNAc groups, all had relatively long half-lives in mice, and regardless of whether the GalNAc compound was conjugated to the 3'-end or 5'-end of the oligonucleotide, the half-life was relatively long.

[0571] The GalNAc compound YK-GAL-325 designed by the present invention has 1 arm, and YK-GAL-326 has 2 arms (see Table 1 in Example 2). inc-G12 is a conjugate with 3 GalNAc groups obtained by connecting YK-GAL-325 through 2 phosphodiester bonds. inc-G13 is a conjugate with 4 GalNAc groups obtained by connecting YK-GAL-326 through 1 phosphodiester bond. The half-lives of inc-G12 and inc-G13 in mice are 101 hours and 95 hours respectively, which are comparable to that of inc-G5.

[0572] 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 reaches 102 hours, which is comparable to that of inc-G5.

[0573] It can be seen therefrom that the oligonucleotide conjugates with 3, 1 or 2 arms of GalNAc compounds designed by the present invention are all very stable in mice, and regardless of whether they are conjugated to the 3'-end or 5'-end of the oligonucleotide, they have a relatively long half-life.

[0574] The trends of increase or decrease in the peak concentration and the area under the drug-time curve of each test drug group are also consistent with the half-life.

[0575] 3) The oligonucleotide conjugates prepared from the GalNAc compounds designed by the present invention have a significantly improved half-life in mice compared with inc-L96, inc-NAG0052, inc-GalNAc 1b and inc-GalNAc2 prepared from GalNAc compounds of the prior art. For example, inc-G5 is 48.6% higher than inc-L96.

[0576] The conjugates prepared from GalNAc compounds of the prior art, including inc-L96, inc-NAG0052, inc-GalNAc1b and inc-GalNAc 2, have half-lives of 74 hours, 75 hours, 79 hours and 65 hours in mice respectively. 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, have a significantly improved half-life in mice compared with GalNAc of the prior art.

[0577] For example, the half-life of inc-G5 was increased by 48.6%, 46.7%, 39.2% and 69.2% respectively compared with inc-L96, inc-NAG0052, inc-GalNAc 1b and inc-GalNAc2, showing a significant improvement.

[0578] Both inc-G25 and inc-GalNAc 2 are oligonucleotide conjugates with one GalNAc group, but the half-life of inc-G25 was increased by 15.4% compared with inc-GalNAc 2, showing a significant improvement.

[0579] The trends of increase or decrease in the peak concentration and the area under the concentration-time curve of each tested drug group were also consistent with the half-life.

[0580] 4) Oligonucleotide conjugates prepared from GalNAc compounds with similar structures are very likely to have very different half-lives in mice; oligonucleotide conjugates prepared from GalNAc compounds with large structural differences are also very likely to be very close in half-life in mice.

[0581] It can be seen from the structural comparison in the GalNAc oligonucleotide conjugate structure diagram (Example 3) and Table 3 that the structures of this series of GalNAc compounds designed in the present invention are very similar, with only slight differences in individual groups, and are also very similar to the structures of the prior art GalNAc compounds L-96, NAG0052, GalNAc 1b and GalNAc 2. However, the half-lives of oligonucleotide conjugates prepared from these GalNAc compounds with similar structures are very different in mice.

[0582] For example, compared with inc-L96, inc-G5 only changes the prolinol structure in the backbone of inc-L96 to a ribose ring structure, and the other structures are exactly the same. However, the half-life of inc-G5 can be increased by 48.6% compared with inc-L96, showing a significant improvement.

[0583] Compared with GalNAc 1b, inc-G12 only differs in the atom connected to the 1'-position of the ribose ring. GalNAc1b is carbon, while inc-G12 is oxygen. The linker arm of inc-G12 is longer than that of GalNAc 1b, and the other structures are exactly the same. However, the half-life of inc-G12 was increased by 27.8% compared with GalNAc 1b, showing a significant improvement.

[0584] inc-G5 and inc-G1 only differ in the 2'-position of the ribose ring. inc-G5 is methoxy, while inc-G1 is hydrogen. However, the half-life of inc-G5 was increased by 96.4% compared with inc-G1, showing a significant improvement.

[0585] Therefore, oligonucleotide conjugates prepared from GalNAc compounds with similar chemical structures do not necessarily have similar half-lives in mice. On the contrary, there may be very significant differences.

[0586] The trends of increase or decrease in the peak drug concentration and the area under the drug-time curve in each test drug group were also consistent with the half-life.

[0587] The present invention designed a series of 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. GalNAc oligonucleotide conjugates prepared therefrom can achieve efficient liver-targeted delivery, and have significantly improved activities and half-lives compared with representative GalNAc compounds in the prior art.

[0588] 1. The designed GalNAc compounds are significantly different in chemical structure from the GalNAc compounds in the prior art. In the designed GalNAc compounds of the present invention, a ribose ring structure is introduced into the linker arm, and an oxygen or sulfur atom is introduced at the 1'-position of the ribose ring.

[0589] 2. The oligonucleotide conjugates inc-G4, inc-G5, inc-G6, inc-G7, inc-G11, inc-G12, inc-G13, and G18-inc prepared from the designed GalNAc compounds of the present invention have significantly improved delivery efficiency and half-lives compared with other groups. This shows that a cleverly designed linker structure can significantly improve the bioavailability of drugs and improve the pharmacokinetic properties of drugs, thereby exerting better drug effects. Conjugates with 3 or 4 GalNAc groups prepared from the designed GalNAc compounds with 3 arms, 1 arm, or 2 arms of the present invention all have high activities and long half-lives. Moreover, oligonucleotide conjugates prepared from GalNAc compounds with similar structures are very likely to have huge differences in activity and half-life; oligonucleotide conjugates prepared from GalNAc compounds with large structural differences are also very likely to be very close in activity and half-life.

[0590] Specifically as follows:

[0591] 1) Inhibitory rate of PCSK9 protein expression in mouse serum

[0592] I. inc-G4, inc-G5, inc-G6, inc-G7, inc-G11, inc-G12, inc-G13 and G18-inc showed significantly higher inhibitory rates on the expression of PCSK9 protein in mouse serum than other groups. For example, the inhibitory rates of inc-G5 on the 7th day and the 14th day reached 94.4% and 95.6% respectively. Moreover, compared with inc-L96, inc-NAG0052, inc-GalNAc 1b and inc-GalNAc 2 prepared from representative GalNAc compounds of the prior art, the inhibitory rates on the expression of PCSK9 protein in mouse serum were significantly increased. For example, inc-G5 was 14.2% higher than inc-L96. This indicates that the ingeniously designed linkage structure can significantly improve the bioavailability of the drug and exert better drug efficacy.

[0593] II. GalNAc compounds designed by the present invention with 3 arms, 1 arm or 2 arms can be used to prepare conjugates with 3 or 4 GalNAc groups, all of which have very high inhibitory rates. Moreover, whether the GalNAc compound is conjugated to the 3'-end or 5'-end of the oligonucleotide, it can efficiently inhibit the expression of PCSK9 protein in serum.

[0594] III. The inhibitory rates of oligonucleotide conjugates prepared from GalNAc compounds with similar structures on the expression of PCSK9 protein in mouse serum are very likely to vary greatly; the inhibitory rates of oligonucleotide conjugates prepared from GalNAc compounds with large structural differences on the expression of PCSK9 protein in mouse serum are also very likely to be very close.

[0595] 2) Inhibitory rate on the expression of PCSK9 gene in mouse liver

[0596] I. inc-G4, inc-G5, inc-G6, inc-G7, inc-G11, inc-G12, inc-G13 and G18-inc showed significantly higher inhibitory rates on the expression of PCSK9 gene in mouse liver than other groups. For example, the inhibitory rates of inc-G5, inc-G6 and inc-G12 all reached 90%. Moreover, compared with inc-L96, inc-NAG0052, inc-GalNAc 1b and inc-GalNAc 2 prepared from GalNAc compounds of the prior art, the inhibitory rates were significantly increased. For example, inc-G5 was 11.1% higher than inc-L96. This indicates that the ingeniously designed linkage structure can significantly improve the bioavailability of the drug and exert better drug efficacy.

[0597] II. The GalNAc compounds designed in the present invention with 3 antennae, 1 antenna or 2 antennae, when used to prepare oligonucleotide conjugates with 3 or 4 GalNAc groups, all have very high inhibition rates. Moreover, these GalNAc compounds, whether conjugated to the 3'-end or 5'-end of the oligonucleotide, can efficiently inhibit the expression of PCSK9 gene in the liver.

[0598] III. The oligonucleotide conjugates prepared from GalNAc compounds with similar structures are very likely to have extremely large differences in the inhibition rate of PCSK9 protein expression in the mouse liver; the oligonucleotide conjugates prepared from GalNAc compounds with large structural differences are also very likely to have very close inhibition rates of PCSK9 gene expression in the mouse liver.

[0599] 3) Effects on the LDL-C level in mice

[0600] I. inc-G4, inc-G5, inc-G6, inc-G7, inc-G11, inc-G12, inc-G13 and G18-inc led to significantly higher decreases in LDL-C levels than other groups. For example, the decreases on the 7th day and 14th day of inc-G5 reached 49.6% and 54.1% respectively. Moreover, compared with inc-L96, inc-NAG0052, inc-GalNAc 1b and inc-GalNAc 2 prepared from the prior art GalNAc compounds, the decreases in LDL-C levels in the mouse serum were significantly increased. For example, inc-G5 was 20.4% and 23.5% higher than inc-L96 on the 7th day and 14th day respectively. This shows that the ingeniously designed linking structure can significantly improve the bioavailability of the drug and exert better drug effects.

[0601] II. The GalNAc compounds designed in the present invention with 3 antennae, 1 antenna or 2 antennae, when used to prepare oligonucleotide conjugates with 3 or 4 GalNAc groups, all have very high reduction levels. Moreover, these GalNAc compounds, whether conjugated to the 3'-end or 5'-end of the oligonucleotide, can efficiently reduce the LDL-C level.

[0602] III. The oligonucleotide conjugates prepared from GalNAc compounds with similar structures are very likely to have extremely large differences in the reduction level of LDL-C in the mouse serum; the oligonucleotide conjugates prepared from GalNAc compounds with large structural differences are also very likely to have very close reduction levels of LDL-C in the mouse serum.

[0603] 4) Distribution in the mouse liver

[0604] 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 other groups. For example, the fluorescence intensities of inc-G5 at 4 hours and 8 hours reached 9.12E+09 and 9.16E+09 respectively. Moreover, compared with inc-L96, inc-NAG0052, inc-GalNAc 1b and inc-GalNAc 2 prepared from the prior art GalNAc compounds, the fluorescence absorption intensity in the mouse liver was significantly increased. For example, inc-G5 was more than 50% higher than inc-L96. This shows that the ingeniously designed linking structure can significantly improve the bioavailability of the drug and exert better pharmacodynamic effects.

[0605] II. The GalNAc compounds designed in the present invention with 3 arms, 1 arm or 2 arms are used to prepare oligonucleotide conjugates with 3 or 4 GalNAc groups, all of which have very high fluorescence intensities. Moreover, these GalNAc compounds can efficiently deliver oligonucleotides to the liver whether conjugated to the 3'-end or 5'-end of the oligonucleotide.

[0606] III. For the oligonucleotide conjugates prepared from GalNAc compounds with similar structures, it is very likely that there are huge differences in the efficiency of delivering oligonucleotides to the liver; for the oligonucleotide conjugates prepared from GalNAc compounds with large structural differences, it is also very likely that the efficiency of delivering oligonucleotides to the liver is very close.

[0607] 5) Half-life in mice

[0608] I. The half-lives of inc-G4, inc-G5, inc-G6, inc-G7, inc-G11, inc-G12, inc-G13 and G18-inc were significantly increased compared with 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. Moreover, compared with inc-L96, inc-NAG0052, inc-GalNAc1b and inc-GalNAc 2 prepared from the prior art GalNAc compounds, the half-life in mice was significantly prolonged. For example, inc-G5 was 48.6% higher than inc-L96. This shows that the ingeniously designed linking structure can significantly improve the in vivo pharmacokinetic properties of the drug.

[0609] II. The GalNAc compounds designed by the present invention with 3 antennas, 1 antenna or 2 antennas, when prepared into conjugates with 3 or 4 GalNAc groups, all have a long half-life in mice, and regardless of whether the GalNAc compound is conjugated to the 3'-end or 5'-end of the oligonucleotide, the half-life is long.

[0610] III. The conjugates prepared from GalNAc compounds with similar structures are very likely to have a huge difference in half-life in mice; the oligonucleotide conjugates prepared from GalNAc compounds with large structural differences are also very likely to be very close in half-life in mice.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof: ； Wherein, R1 is oxygen; R1 is in the α-configuration or β-configuration; R2 is hydrogen or C 1-4 alkoxy; R3 is hydrogen, a hydroxyl protecting group, a phosphorus-containing reactive group, or -CO(CH2) x COOH, where x is an integer from 1 to 10, 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 -, where 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 , Wherein, T is N-acetyl-galactosamine with all hydroxyl groups fully protected by acyl 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 1; n is 0.

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. The 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 R2 is hydrogen or -OCH3.

5. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1-4, wherein R3 is a hydroxyl-protecting group or a phosphorus-containing reactive group.

6. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 5, wherein R3 is acetyl, 1,1,3,3-tetraisopropyldisiloxanyl, tert-butyldimethylsilyl, phenyldimethylsilyl, 4,4'-dimethoxytriphenylmethyl or .

7. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1-4, wherein R3 is .

8. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1-4, wherein R3 is -CO(CH2)2COOH.

9. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1-4, wherein R4 is a hydroxyl-protecting group.

10. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 9, wherein R4 is trityl, monomethoxytrityl or 4,4'-dimethoxytrityl.

11. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1-4, wherein G is ， , or 。 12. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1-4, which is capable of binding to the asialoglycoprotein receptor.

13. A conjugate comprising an oligonucleotide and an N-acetyl-galactosamine GalNAc moiety, having the structure shown below: or ； Among them, Oligo represents an oligonucleotide, and R1, R2, R3, R4, A, B, L, G, m and n are the same as in claim 1.

14. The conjugate according to claim 13, wherein the acyl group of the hydroxyl-protecting group in the N-acetyl-galactosamine of G is removed.

15. The conjugate according to claim 13 or 14, wherein the oligonucleotide comprises a non-thiophosphorothioate oligonucleotide and a phosphorothioate oligonucleotide.

16. The conjugate according to claim 15, wherein the non-thiophosphorothioate oligonucleotide and the GalNAc moiety are linked by a phosphodiester bond.

17. The conjugate according to claim 15, wherein the phosphorothioate oligonucleotide and the GalNAc moiety are linked by a phosphorothioate bond.

18. The conjugate according to claim 15, wherein the oligonucleotide comprises a small interfering nucleotide, DNA, microRNA, small activating RNA, small guide RNA, transfer RNA, antisense nucleotide or aptamer.

19. The conjugate according to claim 18, wherein each nucleotide in the antisense nucleotide or small interfering nucleotide is independently a modified or unmodified nucleotide.

20. The conjugate according to claim 15, wherein the oligonucleotide regulates the expression of a target gene.

21. A pharmaceutical composition comprising the conjugate according to any one of claims 13-20 and at least one pharmaceutically acceptable excipient.

22. Use of the conjugate according to any one of claims 13-20 or the pharmaceutical composition according to claim 21 in the preparation of a medicament for the treatment and / or prevention of a disease caused by the expression of a specific gene in hepatocytes, wherein the specific gene is selected from hepatitis B virus gene, angiopoietin-3 gene or apolipoprotein C-3 gene; and the disease is selected from chronic liver disease, hepatitis, liver fibrosis disease, liver hyperplastic disease and dyslipidemia.

23. The use according to claim 22, wherein The dyslipidemia is hypercholesterolemia, hypertriglyceridemia or atherosclerosis.

24. A kit comprising the conjugate according to any one of claims 13-20.

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