Galnac compound containing aromatic ring skeleton and application thereof

Abstract

The application provides a GalNAc compound containing an aromatic ring skeleton and an application thereof, and particularly provides a GalNAc compound containing an aromatic ring skeleton shown in formula (I) and an oligonucleotide conjugate thereof. The oligonucleotide conjugate provided in the application can realize efficient liver-targeted delivery and improve drug efficacy.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and in particular to a GalNAc compound containing an aromatic ring skeleton, and a GalNAc conjugated oligonucleotide prepared therefrom can achieve efficient liver targeted delivery. BACKGROUND

[0002] In recent years, the breakthrough progress of small nucleic acid drugs is inseparable from the innovation of delivery technology. As a key factor determining whether the drug can effectively enter the target cell, the optimization of the delivery system has become the focus of research in this field. Among them, N-acetylgalactosamine (GalNAc) has attracted attention as a high-affinity ligand for asialoglycoprotein receptor (ASGPR). ASGPR is an endocytic receptor specifically expressed on the surface of hepatocyte membranes, and the GalNAc-ASGPR complex can be efficiently internalized through the clathrin-mediated endocytosis pathway. When GalNAc is covalently combined with therapeutic drugs, this delivery system can ensure that the drugs enter the liver cells simultaneously. With its advantages of significant liver targeting, low dosage requirement, good safety and long-lasting efficacy, the GalNAc delivery system has become the preferred strategy for liver-targeted therapy of small nucleic acid drugs.

[0003] Currently, the structural optimization of the GalNAc delivery system is in a critical development stage. A large number of studies have shown that the molecular structure of the GalNAc ligand is significantly correlated with its delivery efficiency and clinical efficacy. By modifying the structure of the GalNAc compound, the binding affinity to ASGPR can be effectively improved, the intracellular transport efficiency can be improved, and the targeting specificity can be enhanced. In order to break through the limitations of existing delivery technologies and meet the growing demand for precision medicine, there is a need in the art to develop new GalNAc compounds. These breakthroughs will strongly promote the clinical translation of the next generation of liver-targeted drugs and provide more effective solutions for the treatment of complex liver diseases. SUMMARY

[0004] The present application provides a GalNAc compound containing an aromatic ring skeleton and its application in liver-targeted delivery of small nucleic acid drugs. Specifically, the present application synthesizes a series of GalNAc compounds containing an aromatic ring skeleton, and the GalNAc compound containing an aromatic ring skeleton of the present application can efficiently bind to the 3' end of the oligonucleotide chain through solid-phase synthesis method. Compared with the prior art, the oligonucleotide GalNAc conjugate containing the GalNAc compound containing an aromatic ring skeleton of the present application has stronger inhibitory efficiency on the target gene.

[0005] The present application provides a compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0006]

[0007] wherein,

[0008] R1is oxygen or sulfur;

[0009] R2is hydrogen, halogen, C 1-6 alkyl or C 1-6 alkoxy;

[0010] R3and R4are each independently hydrogen, a hydroxyl protecting group, a phosphorus-containing reactive reactive group, -CO(CH2) z CONH-E or -CO(CH2) z COOH, wherein z is an integer from 1 to 10 and E is a solid support;

[0011] A and B are each independently oxygen, sulfur, -C(O)NH- or -NHC(O)-;

[0012] L is -(CH2) a - or -(CH2CH2O) b (CH2) a wherein a is independently an integer from 1 to 10 and b is an integer from 1 to 5;

[0013] G is -X1-O-T;

[0014] T is ; R5is -OH or -NHR 5a ; R 5a , R 6a , R 6b and R 6c are independently hydrogen, formyl, acetyl, propionyl, n-butyryl, isobutyryl or benzoyl;

[0015] X1is -(CH2) f - or -(CH2CH2O) f CH2-, f is independently an integer from 1 to 5;

[0016] m and n are each independently an integer from 0 to 4;

[0017] x and y are each independently an integer from 0 to 7.

[0018] In some embodiments, R1is in the alpha configuration or the beta configuration.

[0019] In some embodiments, R1is oxygen.

[0020] In some embodiments, R2is C 1-6 alkoxy.

[0021] In some embodiments, R2is -OCH3.

[0022] In some embodiments, R3is a phosphorous-containing active reactive group or -CO(CH2) z CONH-E; E is controlled pore glass (CPG).

[0023] In some embodiments, z is 1, 2, 3, or 4.

[0024] In some embodiments, R3is a phosphorous-containing active reactive group; the phosphorous-containing active reactive group can be a phosphoramidite group, a phosphotriester group, an H-phosphonate group, or a phosphorous chiral auxiliary group.

[0025] In some embodiments, R3is or -CO(CH2)2CONH-E; E is controlled pore glass.

[0026] In some embodiments, R4is a hydroxyl protecting group.

[0027] In some embodiments, R4is a hydroxyl protecting group that is 4, 4'- dimethoxytrityl, monomethoxytrityl, trityl, t-butyldimethylsilyl, t- butyldiphenylsilyl, triisopropylsilyl, or isopropyldimethylsilyl.

[0028] In some embodiments, R4is 4, 4'-dimethoxytrityl (DMTr).

[0029] In some embodiments, A and B are each independently -C(O)NH- or -NHC(O)-.

[0030] In some embodiments, A is -C(O)NH- a , the a end is attached to L.

[0031] In some embodiments, B is -NHC(O)- b , the b end is attached to G.

[0032] In some embodiments, L is -(CH2) a - or -(CH2CH2O) b (CH2) a -, where a is 1, 2, 3, 4, 5, or 6, and b is 1, 2, or 3.

[0033] In some embodiments, L is -(CH2)2-, -(CH2)3-, -(CH2)4-, -(CH2)5-, or -CH2CH2OCH2CH2-.

[0034] In some embodiments, X1is -(CH2) f -, and f is 1, 2, 3, 4, or 5.

[0035] In some embodiments, R5is -NHR 5a .

[0036] In some embodiments, R 5a , R 6a , R 6b , and R 6c are independently acetyl, propionyl, or benzoyl.

[0037] In some embodiments, R 5a is acetyl, propionyl, n-butyryl, or isobutyryl.

[0038] In some embodiments, R 6a , R 6b , and R 6c are the same and are independently acetyl or benzoyl.

[0039] In some embodiments, G is .

[0040] In some embodiments, m and n are each independently 1 or 2.

[0041] In some embodiments, m is 1.

[0042] In some embodiments, n is 1.

[0043] In some embodiments, x and y are each independently 1, 2, or 3.

[0044] In some embodiments, x is 1.

[0045] In some embodiments, y is 1.

[0046] In some embodiments, is or .

[0047] In some embodiments, the compound of Formula (I) is a compound of Formula (la):

[0048] ,

[0049] wherein R1, R2, R3, R4, A, B, L, G, x, y, m, and n are as defined above.

[0050] In some embodiments, R1is oxygen;

[0051] R2is C 1-6 alkoxy;

[0052] R3is a phosphorus-containing active reactive group or -CO(CH2)z CONH-E; z is 1, 2, 3, or 4; E is a controlled pore glass;

[0053] R4is a hydroxyl protecting group;

[0054] A and B are each independently -C(O)NH- or -NHC(O)-;

[0055] L is -(CH2) a - or -(CH2CH2O) b (CH2) a - wherein a is 1, 2, 3, 4, 5, or 6, and b is 1, 2, or 3;

[0056] G is -X1-O-T;

[0057] X1is -(CH2)4-;

[0058] T is ; R5is -NHR 5a ; R 5a is acetyl;

[0059] R 6a , R 6b , and R 6c are the same and are independently acetyl or benzoyl;

[0060] m and n are 1;

[0061] x and y are 1.

[0062] In some embodiments, the compound of Formula (I) is compound YK-GAL-601, YK-GAL-602, YK-GAL-603, YK-GAL-604, YK-GAL-605, YK-GAL-601-SP, YK-GAL-602-SP, YK-GAL-603-SP, YK-GAL-604-SP, or YK-GAL-605-SP, wherein E is a controlled pore glass:

[0063] ,

[0064] ,

[0065] ,

[0066] ,

[0067] ,

[0068] ,

[0069] 、

[0070] 、

[0071] or

[0072] .

[0073] In some embodiments, the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is capable of binding to the Asialoglycoprotein Receptor (ASGPR).

[0074] The present application provides a conjugate or a pharmaceutically acceptable salt thereof, comprising an oligonucleotide and a GalNAc moiety, having the structure as shown below:

[0075] or

[0076] wherein Oligo represents an oligonucleotide, X is oxygen or sulfur, G1 is -X1-O-T1; T1 is ; q is 1, 2, 3 or 4, and R1, R2, R5, A, B, L, X1, x, y, m and n are defined as above.

[0077] In some embodiments, the oligonucleotide comprises a non-thio oligonucleotide and a thio oligonucleotide.

[0078] In some embodiments, the oligonucleotide comprises a small interfering nucleotide (siRNA), DNA, microRNA (miRNA), small activating RNA (saRNA), small guide RNA (sgRNA), transfer RNA (tRNA), antisense nucleotide (ASO) or aptamer (Aptamer).

[0079] In some embodiments, each nucleotide in the antisense nucleotide (ASO) or small interfering nucleotide (siRNA) is independently a modified or unmodified nucleotide.

[0080] In some embodiments, the sense strand of the small interfering nucleotide has a sequence as set forth in SEQ ID NO. 1; the antisense strand of the small interfering nucleotide has a sequence as set forth in SEQ ID NO. 2.

[0081] In some embodiments, the conjugate has the structure as shown below:

[0082] or ;

[0083] wherein Oligo, X, R1, R2, A, B, L, G1, q, x, y, m and n are as defined above.

[0084] In some embodiments, the conjugate is any one of the following conjugates:

[0085] ,

[0086] ,

[0087] ,

[0088] or

[0089] ;

[0090] wherein the sense strand of the siRNA has a sequence as set forth in SEQ ID NO. 1; and the antisense strand of the siRNA has a sequence as set forth in SEQ ID NO. 2.

[0091] In some embodiments, the oligonucleotide modulates the expression of a target gene.

[0092] The present application provides a pharmaceutical composition comprising the conjugate as described above and at least one pharmaceutically acceptable excipient.

[0093] The present application provides use of the conjugate as described above or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition as described above in the manufacture of a medicament for treating and / or preventing a pathological condition or a disease caused by the expression of a specific gene in liver tissue or in a virus.

[0094] In some embodiments, the specific gene is selected from a hepatitis B virus gene, a preprotein convertase subtilisin 9 gene, a coagulation factor gene, a lipoprotein a gene or an angiopoietin-like protein 3 gene, an angiotensinogen gene or an apolipoprotein C3 gene; preferably an angiotensinogen gene.

[0095] In some embodiments, the disease is selected from a chronic liver disease, hepatitis, a liver fibrosis disease, a liver hyperplasia disease and a cardiovascular and cerebrovascular disease.

[0096] In some embodiments, the cardiovascular and cerebrovascular disease is hypercholesterolemia, hypertriglyceridemia, atherosclerosis or coagulation dysfunction.

[0097] The present application provides a kit comprising the conjugate as described above or a pharmaceutically acceptable salt thereof.

[0098] The present application provides a method for inhibiting expression of a specific gene in a hepatocyte, the method comprising contacting an effective amount of the conjugate as described above or the pharmaceutical composition as described above with the hepatocyte; optionally, the specific gene is selected from the group consisting of a proprotein convertase subtilisin 9 gene, a hepatitis B virus gene, an apolipoprotein a gene, a coagulation factor XI gene, an angiopoietin-like protein 3 gene, an angiotensinogen gene, or an apolipoprotein C3 gene.

[0099] It should be understood that, in the above technical solutions, the above uses provided by the present application can include therapeutic, diagnostic, non-therapeutic, and non-diagnostic uses. For example, the therapeutic use can include using the conjugate or the pharmaceutically acceptable salt thereof provided by the present application, or using the composition of the present application, to deliver the active ingredient contained therein to the target organ / tissue / cell, so as to achieve the effects of treating diseases, improving symptoms, regulating physiological activities in the body, etc.; the diagnostic use can include packaging the active ingredient for disease diagnosis in the conjugate or the pharmaceutically acceptable salt thereof provided by the present application or the composition, so as to deliver the active ingredient to the target organ / tissue / cell, thereby achieving the purpose of disease diagnosis; the non-therapeutic and non-diagnostic purpose can include using the conjugate or the pharmaceutically acceptable salt thereof provided by the present application, or using the composition provided by the present application to encapsulate the active ingredient, so as to deliver the active ingredient to the target organ / tissue / cell, for the purpose of scientific research, detection, etc. (such as disease mechanism research, drug mechanism research, new drug development, drug screening, etc.).

[0100] All publications and patents mentioned in the present application are hereby incorporated by reference in their entirety into the present application. To the extent that any publication or patent incorporated by reference contradicts any disclosure contained in the present application, the disclosure contained in the present application will control.

[0101] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this application belongs. In case of conflict between any reference incorporated by reference and the disclosure contained in the present application, the disclosure contained in the present application controls.

[0102] Except in the Examples, or where otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, dosages, and so forth used herein are to be understood as being modified in all instances by the term "about." Also, any numerical range recited herein is intended to include all sub-ranges of the same numerical precision subsumed therein. In other words, only single value is to be treated as an approximation.

[0103] As used in this application, the terms "comprise", "contain", or "include" and like terms are intended to be open-ended, non-limiting terms that specifically permit the inclusion of other elements notwithstanding the presence of the recited elements.

[0104] The term "pharmaceutically acceptable" means, as used in this application, that the compound or composition is chemically and / or toxicologically compatible with the other ingredients of the formulation and / or the subject to be treated therewith.

[0105] The term "pharmaceutically acceptable salt" refers to a relatively non-toxic, inorganic or organic acid addition salt of a compound of the present application. For example, see S. M. Berge et al. "Pharmaceutical Salts", J. Pharm. Sci. 1977, 66, 1-19. Inorganic acids such as, for example, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, or nitric acid and the like; and organic acids such as, for example, formic acid, acetic acid, acetoacetic acid, propionic 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, methanesulfonic acid, trifluoromethanesulfonic acid, dodecylsulfic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalene- disulfonic 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, glucoheptanoic acid, glycerylphosphoric acid, aspartic acid, sulfosalicylic acid, and the like. For example, HCl (or hydrochloric acid), HBr (or hydrobromic acid solution), methanesulfonic acid, sulfuric acid, tartaric acid, or fumaric acid can be used to form a pharmaceutically acceptable salt with a compound of Formula (I).

[0106] The term "C 1-6 " refers to a group having a total number of carbon atoms in the principal chain and branch chains of any integer value in the range of from 1 to 6, e.g., 1, 2, 3, 4, 5, 6 carbon atoms.

[0107] The term "alkyl" as used herein refers to both straight chain and branched chain saturated aliphatic monovalent hydrocarbon radicals having the indicated number of carbon atoms. For example, "C 1-6 alkyl" includes methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, and n-hexyl.

[0108] The term "alkoxy" refers to the group -OR, wherein R is alkyl as defined herein. A non-limiting list of alkoxy groups is methoxy, ethoxy, n-propyloxy, 1-methylethoxy (isopropoxy), n-butyloxy, isobutyloxy, sec-butyloxy, t-butyloxy. In some cases, the alkoxy group can be -OR, wherein R is unsubstituted C 1-6 alkyl.

[0109] "Halogen" refers to fluorine (F), chlorine (CI), bromine (Br), and iodine (I), preferably fluorine (F) and chlorine (CI).

[0110] The term "protecting group" as used herein refers to any atom or atom group that is introduced into a molecule to prevent an existing group in the molecule from undergoing an undesired chemical reaction, which can be removed to leave the unprotected group.

[0111] The term "hydroxyl protecting group" can be any suitable hydroxyl protecting group, i.e., a labile chemical moiety known in the art to protect a hydroxyl group from undesirable reactions during synthetic procedures. The protecting group as described herein can be selectively removed after one or more of the synthetic procedures. See, e.g., A. Isidro-Llobet et al., Amino Acid-Protecting Groups, Chem. Rev. 109:2455-2504 (2009) and T. Greene and P. Wuts, Protective Groups in Organic Synthesis (3d Ed. 1999). In some embodiments, the hydroxyl protecting group is an acid-stable hydroxyl protecting group. Examples of hydroxyl protecting groups include, but are not limited to, alkyl, cycloalkyl, arylalkyl, aryl, ether, ester, cyclic ether, cyclic ester, acetal, cyclic acetal, ketal, and cyclic ketal groups, among others, which can be removed under acidic or basic conditions, thereby removing the protecting group and replacing it with a hydrogen atom. Particular hydroxyl protecting groups include, but are not limited to, methyl, ethyl, acetate, ethyl acetate, propionate, ethylene glycol, propylene glycol, 4-methoxybenzyl, benzyl, trityl, trimethylsilyl, tetrahydropyranyl, and benzoyl. Other hydroxyl protecting groups include, but are not limited to, methoxymethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), silanyloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl (CTMP), 1,4-dioxanyl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl, t-butyl, allyl, p- chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl, p-methoxybenzyl, 3,4- dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p- cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido, diphenylmethyl, p,p''-dinitrosydiphenylmethyl, 5-dibenzosuberyl, triphenylmethyl, a- naphthyl diphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4''-bromobenzoylmethyloxyphenyl)diphenylmethyl, 4,4'',4''-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4'',4''-tris(acetoacetyloxyphenyl)methyl, 4,4'',4''-tris(benzoyloxyphenyl)methyl, 3-(imidazol-1-yl)bis(4'',4''-dimethoxyphenyl)methyl, 1,1-bis(4-methoxyphenyl)-1''- pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo))anthryl, 1,3- benzodithiol-2-yl, S,S-dioxobenzisothiazolyl, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylethylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-cymylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), t-butytmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxalylpentanoate (leucinol), 4,4-(ethylenedithio)pentanoate (acetoacetyl dithioacetal), pivaloate, adamantoate, crotonoate, 4-methoxycrotonoate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), alkylmethylcarbonate, 9-fluorenylmethyl carbonate (Fmoc), alkylethylcarbonate, alkyl 2,2,2-trichloroethyl (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2- (phenylsulfonyl)ethyl carbonate (Psec); 2-(triphenylphosphonio)ethyl carbonate (Peoc), alkyl isobutyl carbonate, alkyl vinyl carbonate, alkyl allyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate, 4-ethoxy-1-naphthyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylvalerate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4- (methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2,6- dichloro-4-methylphenoxyacetate, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)- phenoxyacetate, 2,4-bis(1,1 -dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinate, (E)-2-methyl-2-butenoate, o- (methoxycarbonyl)benzoate, a-naphthaleneacetate, nitrate, alkyl N,N,N",N"- tetramethylphosphoniodylamide, alkyl N-phenylcarbamate, boronate, dimethylthiophosphoniodyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methane sulfonate (mesylate), benzyl sulfonate, and tosylate (Ts). Methods for the protection and deprotection of hydroxyl groups are well known and can be found, for example, in Protective Groups in Organic Synthesis (T. Green and P. Wuts; 3rdEdition; John Wiley and Sons, 1999).

[0112] Hydroxyl protecting groups can be used, for example, protecting groups commonly used in the synthesis of RNA or derivatives thereof to protect the hydroxyl groups of the ribose structure, and also the protecting groups described in Green et al. in Protective Groups in Organic Synthesis, 3rdEdition, 1999, John Wiley & Sons, Inc., such as, for example: acetyl, phenoxyacetyl, pivaloyl, benzyl, 4-methoxybenzyl, benzoyl, triphenylmethyl, 4, 4'-dimethoxytrityl (DMTr), monomethoxytrityl (MMTr), 9-phenyl-xanthen-9-yl, 9-(p-tolyl)-xanthen-9-yl, trimethylsilyl, tert-butyldimethylsilyl (TBDMS), cyanomethoxymethyl, 2-(cyanoethoxy)ethyl, cyanoethoxymethyl, and the like, preferably 4, 4'-dimethoxytrityl (DMTr).

[0113] The term "phosphorus-containing reactive reactive group" as used herein refers to a phosphorus-containing group capable of reacting by nucleophilic attack with a hydroxyl or amine group contained in another molecule, especially another nucleotide unit or another nucleotide analogue. Typically, such a reaction results in an ester-type internucleosidic linkage connecting the nucleotide unit or nucleotide analogue unit to another nucleotide unit or nucleotide analogue unit. These phosphorus-containing reactive reactive groups are known in the art and comprise phosphorus atoms in the PIII or PV valence state, including but not limited to phosphoramidites, H-phosphonates, phosphotriesters, and phosphorus-containing chiral auxiliaries, for example:

[0114] 2-cyanoethoxy-N,N-diisopropylaminophosphorus;

[0115] 2-propenyloxy-N,N-diisopropylaminophosphorus;

[0116] methoxy-N,N-diisopropylaminophosphorus;

[0117] bis-diisopropylaminophosphorus, and the like.

[0118] In some embodiments, the phosphorus-containing reactive reactive group is .

[0119] Controllable Pore Glass (CPG) and polystyrene (highly cross-linked polystyrene microbeads) as described herein refer to solid supports useful for oligonucleotide synthesis, which are commercially available for linking signal entities, reactive groups, nucleobases or analogs thereof, or phosphates, etc. to oligonucleotides during synthesis.

[0120] The term "pharmaceutically acceptable excipient" in the present application refers to all substances contained in a pharmaceutical formulation other than the active ingredient.

[0121] The term "treatment" as used herein refers to the application of one or more pharmaceutical substances to a patient or subject suffering from a disease or having symptoms of a particular disease, in order to cure, alleviate, relieve, lessen, improve, or affect the disease or the symptoms of the disease. As used herein, the term includes preventing the worsening of the disease, disorder, or symptoms related thereto. It is understood that the term treatment as used herein can not be effective in all subjects to be treated. Preferably, however, the term shall require that a statistically significant proportion of subjects suffering from a disease or disorder described herein can be successfully treated. Statistical significance can be determined by a variety of well-known statistical evaluation tools, such as confidence interval determination, p-value determination, t-test, Mann-Whitney test, etc. In some embodiments, treatment comprises inhibiting the proliferation of cancer cells, preferably comprises killing cancer cells. Preferably, treating cancer is reducing the tumor and / or cancer cell burden in a subject. As will be appreciated by the skilled person, the effectiveness of e.g. cancer treatment depends on a variety of factors, including e.g. the cancer stage and the cancer type. Also preferably, cancer treatment further comprises at least one of chemotherapy, immunotherapy, surgery, and radiotherapy.

[0122] The term "prevention" refers to maintaining health in relation to a particular disease or disorder in a subject over a period of time. It is understood that the period of time can depend on the amount of drug already administered and the individual factors of the subject. It is understood that prevention can not be effective in all subjects treated. Preferably, however, the term shall require that a statistically significant proportion of subjects of a group or population effectively prevented from suffering from a disease or disorder described herein or a concomitant symptom thereof. Statistical significance can be determined by a variety of well-known statistical evaluation tools, such as confidence interval determination, p-value determination, t-test, Mann-Whitney test, etc. In the case of cancer treatment, prevention particularly relates to preventing cancer development, preventing metastasis formation, and / or preventing recurrence, preferably to preventing metastasis formation and / or preventing recurrence.

[0123] "Oligonucleotide" as used in the present application can be a single-stranded oligonucleotide (e.g. antisense oligonucleotide, ASO for short) or a double-stranded oligonucleotide (e.g. small interfering nucleotide, siRNA for short), the specific meaning being known to the skilled person depending on the specific context and technical scenario.

[0124] The oligonucleotides in the present application comprise, in most cases, any unmodified oligonucleotide and / or modified oligonucleotide. The modification of the oligonucleotide usually includes modification of ribose moiety, modification of base moiety, modification of phosphate backbone moiety and / or modification of terminal, etc. In addition, in the art, the linkage between adjacent nucleosides is also referred to as being modified in some cases, for example, the 3', 5'-phosphodiester bond between two adjacent nucleosides comprises a thio-modification, forming a chiral pure 3', 5'-thiophosphodiester bond.

[0125] The above-mentioned preferred conditions can be combined arbitrarily without departing from the common general knowledge in the art, thereby obtaining various preferred embodiments of the present application.

[0126] Unless otherwise specified, the reagents and raw materials used in the present application can be obtained through commercial channels.

[0127] The positive progress effect of the present application is that a series of novel GalNAc compounds containing aryl skeleton are synthesized, and the oligonucleotide-GalNAc conjugate containing the GalNAc compound of the present application can significantly improve the inhibitory activity of siRNA on target genes. For example, compared with the corresponding conjugate formed by conjugation of oligonucleotide with compounds such as L96 and YK-GAL-325 in the prior art, the oligonucleotide-GalNAc conjugate containing the GalNAc compound of the present application has significantly improved delivery efficiency, and in animal tests, a significant decrease in the content of AGT protein in serum and a significant improvement in the inhibition rate of AGT mRNA in the liver can be observed. In addition, in the synthesis method of the compound of the present application, commercially available 4-(hydroxymethyl) methyl benzoate is used as the starting material, and the intermediates generated in the synthesis method all contain an aryl ring, which is a UV-visible fluorescent group, facilitating the detection and purification of the reaction, and thus the synthesis method has economic advantages in terms of methodology. DETAILED DESCRIPTION

[0128] In order to make the purpose, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions of the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments, and thus the present application is not limited to the following embodiments. Based on the described embodiments of the present application, all other embodiments obtained by those skilled in the art through adjustment, change, etc. without departing from the spirit and main point of the present application, all belong to the scope of protection of the present application.

[0129] The experimental conditions used in the examples can be further adjusted according to different requirements of specific use, and the experimental conditions not specified are the conventional conditions in the industry. In the specific examples in the present application, the raw materials used can be obtained by market purchase or can be synthesized using conventional methods. Unless otherwise specified, all temperatures are given in degrees Celsius. The technical features involved in each embodiment of the present application can be combined with each other as long as they do not conflict with each other.

[0130] Example 1: Synthesis of GalNAc compound

[0131] The following abbreviations represent the following reagents, respectively: DCM: dichloromethane; PE: petroleum ether; EA: ethyl acetate; THF: tetrahydrofuran; DMF: N, N-dimethylformamide; ACN: acetonitrile; HBTU: O-benzotriazol- tetramethyluronium hexafluorophosphate; DIPEA: N, N-diisopropylethylamine; NaHCO3: sodium bicarbonate; NaCl: sodium chloride; Na2SO4: sodium sulfate; TFA: trifluoroacetic acid; BF3·Et2O: boron trifluoride ether; MeONa: sodium methoxide; TIPDSCl2: 1,3-dichloro-1,1,3,3-tetraisopropyl disiloxane; H2O: water; NH4Cl: ammonium chloride; Py: pyridine; DMTrCl: 4,4'-dimethoxytrityl chloride; TEA: triethylamine; MeOH: methanol; Heptane: n-heptane; MTBE: methyl tert-butyl ether; Me3O·BF4: trimethyloxonium tetrafluoroborate; LiOH·H2O: lithium hydroxide monohydrate; 3HF·TEA: triethylamine trihydrofluoride; SiO2: silicon dioxide; HCl: hydrochloric acid; DMAP: 4-dimethylaminopyridine.

[0132] 1. Synthesis of GalNAc phosphoramidite compound

[0133] (1) Synthesis of YK-GAL-601

[0134] The synthetic route is as follows:

[0135]

[0136]

[0137] Step 1: Synthesis of 1-3

[0138] Into a reaction flask was placed 1-1 (300.0 g, 94.2 mmol, 1.1 eq.), 1-2 (154.2 g, 85.6 mmol, 1.0 eq.), 4A molecular sieves (activated, 90 g) and super dry DCM (3 L) and stirred in an ice water bath for about 5-10 minutes. BF3Et20 (243.0 g, 171.2 mmol, 2.0 eq.) was added. The ice bath was removed and stirred at room temperature for about 20 hours. CAD indicated a clear product peak. The reaction was quenched with saturated aqueous NaHC03(600 mL) and extracted with DCM (600 mL x 3). The organic phase was dried over anhydrous Na2S04and concentrated under reduced pressure to remove the solvent to give a residue. Column purification (Si02, PE / EA = 4 / 1 ~ 7 / 3) gave about 139.3 g of a light yellow oil with a CAD purity of about 60.0% which was used directly in the next step. 1 H NMR (400 MHz, d6-DMSO): δ 7.26 - 7.32 (m, 4 H), 5.25 (t, J = 5.70 Hz, 1 H), 5.18 (d, J = 4.94 Hz, 1 H), 5.15 (s, 1 H), 4.68 (d, J = 12.22 Hz, 1 H), 4.50 (d, J = 12.22 Hz, 1 H), 4.29 - 4.35 (m, 2 H), 4.08 - 4.12 (m, 1 H), 3.69 (s, 2 H), 3.63 (s, 3 H), 2.19 (s, 3 H), 2.05 (s, 3H), 2.00 (s, 3H) ppm.

[0139] Step 2: Synthesis of 1-4

[0140] Into a reaction flask was placed 1-3 (139.3 g, 317.0 mmol, 1.0 eq., 60% purity) and super dry MeOH (1.4 L) and stirred to dissolve. MeONa (17.2 mL, 95.0 mmol, 0.3 eq.) was added and stirred at room temperature for about 2 hours. CAD indicated no starting material remained. Amberlite ion exchange resin IR120 was added to adjust the pH to neutral and filtered, washing with MeOH. The solvent was removed under reduced pressure to give a light brown oil. Column purification (Si02, MeOH / DCM = 3 / 97 ~ 1 / 19) gave 50.0 g of a white solid with a yield of about 18.7% (total yield for the first and second steps). 1H NMR (400 MHz, d6-DMSO): δ 7.22 - 7.33 (m, 4H), 4.85 (s, 1 H), 4.62-4.69 (m, 1 H), 4.49 (s, 1 H), 4.39-4.43 (m, 1 H),3.75-3.99 (m, 3 H), 3.68 (d, J = 7.22 Hz, 2 H), 3.63 (d, J = 1.90 Hz, 3 H),3.53 - 3.61 (m, 1 H), 3.39 - 3.50 (m, 1 H) ppm.

[0141] Step 3: Synthesis of 1-5

[0142] Into the reaction flask was added 1-4 (50.0 g, 161.0 mmol, 1.0 eq.) and concentrated with super dry acetonitrile once with water. Added super dry DCM (1 L) and imidazole (27.4 g, 402.0 mmol, 2.5 eq.) and stirred to dissolve. Added TIPDSCl2 (55.5 g, 176.0 mmol, 1.1 eq.). Stirred at room temperature for about 2.5 hours. No starting material was detected by CAD. Diluted with DCM (2 L) and washed sequentially with aqueous NH4Cl (2 L x 2) and saturated aqueous NaCl (2 L). Dried over anhydrous Na2SO4 and concentrated under reduced pressure to remove the solvent to give a residue. Purified by column (SiO2, PE / EA = 9 / 1 ~ 7 / 3) to give a white oil about 71.9 g with a yield of about 80.4%. 1 H NMR (400 MHz, d6-DMSO): δ 7.26 (s, 4 H), 5.12 (s, 1 H), 4.84 (s, 1 H), 4.58(d, J = 11.43 Hz, 1 H), 4.36-4.42 (m, 2 H), 3.89-3.96 (m, 3 H), 3.80-3.85 (m,1 H), 3.68 (s, 2 H), 3.62 (s, 3 H), 0.98-1.05 (m, 28 H) ppm.

[0143] Step 4: Synthesis of 1-6

[0144] Into a reaction flask was placed 1-5 (71.9 g, 129.0 mmol, 1.0 eq.) and super dry DCM (1.4 L) and stirred to dissolve. Added 1,8-bis(dimethylamino)naphthalene (150.2 g, 704.0 mmol, 5.4 eq.) and Me3OBF4(95.8 g, 647.0 mmol, 5.0 eq.). Stirred at room temperature for about 20 hours. The reaction was filtered, the filter cake was rinsed with DCM, and the filtrate was washed with H2O (2 L x 2) and saturated aqueous NaCl (2 L), dried over anhydrous Na2SO4, and concentrated under reduced pressure to remove the solvent to give a residue. Column purification (SiO2, PE / EA = 19 / 1 to 9 / 1) gave about 58.2 g of a white oil in about 78.7% yield. 1 H NMR (400 MHz, d6-DMSO): δ 7.26 (s, 4 H), 4.96 (s, 1 H), 4.60 (d, J = 11.70 Hz, 1H), 4.42-4.50 (m, 2 H), 3.82-3.94 (m, 3 H), 3.66-3.69 (m, 3 H), 3.63 (s, 3H), 3.48 (s, 3 H), 1.01-1.07 (m, 28 H) ppm.

[0145] Step 5: Synthesis of 1-7

[0146] Into a reaction flask was placed 1-6 (58.2 g, 102.0 mmol, 1.0 eq.) and super dry THF (1.2 L) and stirred to dissolve. Added 3HF·TEA (82.2 g, 510.0 mmol, 5.0 eq.). Stirred at room temperature for about 3 hours. To the reaction was added saturated aqueous NaHCO3solution to quench, extracted with DCM three times, the combined organic phase was washed with saturated aqueous NaCl, dried over anhydrous Na2SO4, and concentrated under reduced pressure to remove the solvent to give a residue. Column purification (SiO2, PE / EA = 2 / 3 to 1 / 4) gave about 15.0 g of a light blue oil in about 46.0% yield. 1H NMR (400 MHz, d6-DMSO): δ 7.45 (d, J = 7.60 Hz, 2 H), 7.27-7.33 (m, 6 H), 7.16-7.23 (m, 5 H),6.85-6.89 (m, 4 H), 5.08 (s, 1 H), 4.94 (d, J = 7.18 Hz, 1 H), 4.68 (d, J =11.64 Hz, 1 H), 4.47 (d, J = 11.80 Hz, 1 H), 4.06-4.11 (m, 1 H), 3.97-4.02(m, 1 H), 3.73 (s, 3 H), 3.72 (s, 3 H), 3.68 (s, 2 H), 3.63 (s, 3 H), 3.56(d, J = 4.52 Hz, 1 H), 3.41 (s, 3 H), 3.17 (dd, J = 9.95 Hz, 2.60 Hz, 1 H),3.00-3.04 (m, 1 H) ppm.

[0147] Step 6: Synthesis of 1-8

[0148] To the reaction bottle was added 1-7 (15.0 g, 46.1 mmol, 1.0 eq.) and concentrated with super dry acetonitrile once with water. Added super dry Py (310 mL) and stirred to dissolve. Added DMTrCl (23.4 g, 69.1 mmol, 1.5 eq.). Stirred at room temperature for about 3 hours. To the reaction was added CH3OH (60 mL) to quench and concentrated under reduced pressure to remove the solvent to give a residue. Column purification (Si02, PE / EA = 17 / 3 ~ 13 / 7) gave about 22.2 g of a light yellow oil with a yield of about 74.1%. 1 H NMR (400 MHz, d6-DMSO): δ 7.45 (d, J = 7.60 Hz, 2 H), 7.27-7.33 (m, 6 H), 7.16-7.23 (m, 5 H),6.85-6.89 (m, 4 H), 5.08 (s, 1 H), 4.94 (d, J = 7.18 Hz, 1 H), 4.68 (d, J =11.64 Hz, 1 H), 4.47 (d, J = 11.80 Hz, 1 H), 4.06-4.11 (m, 1 H), 3.97-4.02(m, 1 H), 3.73 (s, 3 H), 3.72 (s, 3 H), 3.68 (s, 2 H), 3.63 (s, 3 H), 3.56(d, J = 4.52 Hz, 1 H), 3.41 (s, 3 H), 3.17 (dd, J = 9.95 Hz, 2.60 Hz, 1 H),3.00-3.04 (m, 1 H) ppm.

[0149] Step 7: Synthesis of 1-9

[0150] A mixture solution of 1-8 (22.2 g, 35.4 mmol, 1.0 eq.) and THF / H2O (V / V, 1 / 1, 600 mL) was added into a reaction flask and stirred to dissolve. LiOH-H2O (2.2 g, 53.1 mmol, 1.5 eq.) was added. It was stirred at room temperature for about 4-5 hours. The pH was adjusted to neutral with concentrated HCl and the solvent was removed by concentration under reduced pressure to obtain a residue. H2O (300 mL) was added to dissolve and the pH was adjusted to about 4-5 with concentrated HCl (with obvious solid precipitation). The aqueous phase was extracted with EA for 3 times, the organic phase was combined and washed with saturated NaCl aqueous solution for 2 times, dried over anhydrous Na2SO4, and the solvent was removed by concentration under reduced pressure to obtain a residue of about 13.2 g with a yield of about 60.8%. 1 H NMR (400MHz, d6-DMSO): δ 7.45 (d, J = 7.21 Hz, 2 H), 7.27-7.33 (m, 6 H), 7.14-7.24(m, 5 H), 6.86-6.89 (m, 4 H), 5.08 (s, 1 H), 4.68 (d, J = 11.80 Hz, 1 H),4.47 (d, J = 11.75 Hz, 1 H),4.07-4.10 (m, 1 H), 3.97-4.01(m, 1 H), 3.73 (s, 3H), 3.72 (s, 3 H), 3.54 (s, 2 H), 3.41 (s, 3 H), 3.17 (dd, J = 10.17 Hz, 2.75Hz, 1 H), 3.01-3.06 (m, 1 H), 2.76 (s, 2 H) ppm.

[0151] Step 8: Synthesis of 1-12

[0152] A mixture of 1-10 (1.0 g, 2.2 mmol, 1.0 eq.), HBTU (1.0 g, 2.7 mmol, 1.2 eq.), DIPEA (0.6 g, 4.4 mmol, 2.0 eq.) and super dry DMF (10 mL) was added into a reaction flask and stirred for about 5-10 minutes. 1-11 (0.6 g, 3.3 mmol, 1.5 eq.) was added and stirred at room temperature for about 22 hours. It was diluted with EA and washed with saturated NaCl aqueous solution for 4 times, dried over anhydrous Na2SO4, and the solvent was removed by concentration under reduced pressure to obtain a residue. Column purification (MeOH / DCM = 4-6%) obtained white solid 1.1 g with a yield of about 85.0%. 1H NMR (400 Hz, d6-DMSO): δ 7.81 (d, J = 9.22 Hz, 1 H), 7.71(t, J = 5.70 Hz, 1 H), 6.74 (t, J = 5.00 Hz, 1 H), 5.22 (d, J = 3.71 Hz, 1H), 4.99 (dd, J = 11.10 Hz, 3.41 Hz, 1 H), 4.50 (d, J = 8.47 Hz, 1 H), 4.01-4.06 (m, 3 H), 3.85-3.92 (m, 1 H), 3.69-3.75 (m, 1 H), 3.40-3.45 (m, 1 H),3.00-3.05 (m, 2 H), 2.89-2.94 (m, 2 H), 2.12 (s, 3 H), 2.06 (t, J = 7.03 Hz,2 H), 2.01 (s, 3 H), 1.91 (s, 3 H), 1.79 (s, 3 H), 1.46-1.54 (m, 6 H), 1.39(s, 9 H) ppm. MS [M + H] + : 604.3.

[0153] Step 9: Synthesis of 1-13

[0154] Into a reaction vial was placed 1-12 (1.1 g, 1.8 mmol, 1.0 eq.) and super dry DCM (7.5 mL) and stirred to dissolve. TFA / DCM (2 / 3, 7.5 mL) was added. Stirring at room temperature for about 2 hours. LCMS check showed no starting material left. Concentrated under reduced pressure to remove solvent to give residue about 1.2 g, which was used directly for next step. 1H NMR (400 Hz, d6-DMSO): δ 12.82 (s, 2 H), 7.95 (t, J = 5.50 Hz, 1 H), 7.87 (d, J = 9.20 Hz, 1 H), 5.23 (d, J = 3.17 Hz, 1 H), 4.99 (dd, J = 11.24 Hz, 3.30 Hz, 1 H), 4.51 (d, J = 8.29 Hz, 1 H), 4.00-4.06 (m, 3 H), 3.86-3.93 (m, 1 H), 3.71-3.76 (m, 1 H), 3.40-3.46 (m, 1 H), 3.10-3.14 (m, 2 H), 2.91 (s, 1 H), 2.76-2.81 (m, 2 H), 2.11 (s, 3 H), 2.07-2.09 (m, 1 H), 2.01 (s, 3 H), 1.90 (s, 3 H), 1.79 (s, 3 H), 1.66-1.72 (m, 2 H), 1.47 - 1.52 (m, 4 H). MS [M + H] + : 504.2.

[0155] Step 10: Synthesis of 1-14

[0156] Into a reaction vial was placed 1-9 (1.1 g, 1.8 mmol, 1.0 eq.), HBTU (1.0 g, 2.6 mmol, 1.4 eq.), DIPEA (3.7 mL, 21.9 mmol, 12.0 eq.) and super dry DMF (45 mL), stirred at room temperature for about 5-10 min. Added 1-13 (0.3 g, 2.7 mmol, 1.5 eq.), stirred at room temperature for about 2-4 h. Diluted with appropriate amount of EA, washed with saturated aqueous NaCl solution for 3 times, dried over anhydrous Na2SO4, concentrated under reduced pressure to remove solvent to give residue. Purified by column (DCM / MeOH = 97 / 3 ~ 23 / 2) to give white solid 1.1 g, HPLC purity 93.7%, yield about 56.0%. 1H NMR (400 Hz, d6-DMSO): δ 8.04 (t, J = 5.50 Hz, 1 H), 7.85 (d, J = 9.17 Hz, 1 H), 7.76 (t, J = 5.50 Hz, 1 H), 7.46 (d, J = 7.68 Hz, 2 H), 7.28-7.33 (m, 6 H), 7.20-7.24 (m, 3 H), 7.15 (d, J = 8.00 Hz, 2 H), 6.86-6.89 (m, 4 H), 6.23 (d, J = 3.32 Hz, 1 H), 5.07 (s, 1 H), 4.99 (dd, J = 10.92 Hz, 3.32 Hz, 1 H), 4.94 (d, J = 7.26 Hz, 1 H), 4.67 (d, J = 11.60 Hz, 1 H), 4.44-4.52 (m, 2 H), 4.01-4.10 (m, 4 H), 3.86-3.93 (m, 1 H), 3.74 (s, 3 H), 3.73 (s, 3 H), 3.54 (d, J = 4.60 Hz, 1 H), 3.36-3.44 (m, 8 H), 3.17 (dd, J = 9.93 Hz, 2.37 Hz, 1 H), 3.00-3.06 (m, 5 H), 2.12 (s, 3 H), 2.06 (t, J = 6.92 Hz, 2 H), 2.01 (s, 3 H), 1.91 (s, 3 H), 1.79 (s, 3 H), 1.47-1.57 (m, 6 H) ppm. MS [M - H] - : 1098.3.

[0157] Step 11: Synthesis of YK-GAL-601

[0158] Into a reaction vial was placed 1-14 (1.0 g, 0.9 mmol, 1.0 eq.), bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.1 g, 3.6 mmol, 4.0 eq.) and super dry DCM (36 mL) and stirred to dissolve. Added tetrazole (127.4 mg, 1.8 mmol, 2.0 eq.) and stirred at room temperature for about 2 hours. Diluted with appropriate amount of DCM and washed with saturated aqueous NaHCO3 and NaCl twice, dried over anhydrous Na2SO4 and concentrated under reduced pressure to remove solvent to give a residue. Column purified (ACN / DCM (1% TEA) = 1 / 1 ~ 3 / 2) to give a white oil, which was dissolved in DCM (2 mL) and added Heptane / MTBE (V / V, 5 / 1, 10 mL) under stirring condition, stirred for about 20 minutes, removed the supernatant and repeated the above procedure (total of 6 times) to give a white solid about 0.6 g with HPLC purity about 96.0% and yield about 50.0%. 1H NMR (400 Hz, d6-DMSO): δ 8.03 (t, J = 5.50 Hz, 1 H), 7.84 (d, J = 9.15 Hz, 1 H), 7.75 (t, J = 5.50 Hz, 1 H), 7.46 (dd, J = 7.13 Hz, 2.89 Hz, 2 H), 7.26-7.33 (m, 6 H), 7.14-7.23 (m, 5 H), 6.84-6.88 (m, 4 H), 5.23 (d, J = 3.23 Hz, 1 H), 5.13 (d, J = 2.26 Hz, 1 H), 4.99 (dd, J = 11.41 Hz, 3.65 Hz, 1 H), 4.71 (dd, J = 11.91 Hz, 4.18 Hz, 1 H), 4.51 (d, J = 8.51 Hz, 2 H), 4.02-4.11 (m, 4 H), 3.86-3.93 (m, 1 H), 3.67-3.76 (m, 9 H), 3.47-3.58 (m, 3 H), 3.43 (t, J = 5.10 Hz, 2 H), 3.39 (s, 3 H), 3.36 (s, 3 H), 2.99-3.06 (m, 5 H), 2.77 (t, J = 6.12 Hz, 1 H), 2.58 (t, J = 5.90 Hz, 1 H), 2.12 (s, 3 H), 2.06 (t, J = 7.00 Hz, 2 H), 2.01 (s, 3 H), 1.91 (s, 3 H), 1.79 (s, 3 H), 1.46-1.53 (m, 6 H), 1.08-1.13 (m, 9 H), 0.92 (d, J = 6.72 Hz, 3 H) ppm. 31 P NMR (400 Hz, d6-DMSO): 149.18, 148.69 ppm. MS [M - H] - : 1299.3.

[0159] (2) Synthesis of YK-GAL-602

[0160] The synthetic route is as follows:

[0161]

[0162] Step 1: Synthesis of 2-2

[0163] Starting from 2-1 (5.0 g, 11.2 mmol, 1.0 eq.) and 1-11 (2.7 g, 13.4 mmol, 1.2 eq.), white solid 6.2 g, yield 85.2% was obtained according to the method for synthesis of compound 1-12. 1 H NMR (400 Hz, d6-DMSO): δ 7.79-7.84 (m, 2 H), 6.78 (t, J = 5.08 Hz, 1 H), 5.23 (d, J = 3.29 Hz, 1 H), 4.99 (dd, J = 11.35 Hz, 3.39 Hz, 1 H), 4.50 (d, J = 8.49 Hz, 1 H), 4.04 (s, 3 H), 3.85-3.92 (m, 1 H), 3.70-3.76 (m, 1 H), 3.36-3.45 (m, 5 H), 3.17-3.22 (m, 2 H), 3.06-3.10 (m, 2 H), 2.12 (s, 3 H), 2.08 (t, J = 7.01 Hz, 2 H), 2.02 (s, 3 H), 1.91 (s, 3 H), 1.79 (s, 3 H), 1.48-1.55 (m, 4 H), 1.39 (s, 9 H) ppm. MS [M + H] + : 634.3.

[0164] Step 2: synthesis of 2-3

[0165] Starting from 2-2 (1.0 g, 1.6 mmol, 1.0 eq.), oil 0.9 g was obtained according to the method for synthesis of compound 1-13, which was used directly in the next step. 1H NMR (400 Hz, d6-DMSO): δ 7.88 (d, J = 9.05 Hz,2 H), 5.23 (d, J = 3.36 Hz, 1 H), 4.99 (dd, J = 11.28 Hz, 3.35 Hz, 1 H), 4.50(d, J = 8.48 Hz, 1 H), 4.02-4.07 (m, 3 H), 3.86-3.94 (m, 1 H), 3.70-3.76 (m,1 H), 3.59 (t, J = 5.16 Hz, 2 H), 3.40-3.47 (m, 3 H), 3.23-3.28 (m, 2 H),3.92-3.04 (m, 3 H), 2.11 (s, 3 H), 2.07-2.09 (m, 2 H), 2.00 (s, 3 H), 1.90(s, 3 H), 1.80 (s, 3 H),1.46-1.54 (m, 5 H) ppm. MS [M + H] + 534.2.

[0166] Step 3: Synthesis of 2-4

[0167] Using 1-9 (1.0 g, 1.6 mmol, 1.0 eq.) and 2-3 (1.1 g, 2.0 mmol, 1.3 eq.) as starting material, following the procedure for synthesizing compound 1-14, afforded 0.8 g of white solid in about 45.3% yield with HPLC purity of about 90.8%. 1HNMR (400 Hz, d6-DMSO): δ 8.11 (t, J = 5.33 Hz, 1 H), 7.80-7.85 (m, 2 H), 7.46(d, J = 7.58 Hz, 2 H), 7.28-7.33 (m, 6 H), 7.20-7.24 (m, 3 H), 7.15 (d, J =8.07 Hz, 2 H), 6.86-6.89 (m, 4 H), 5.77 (s, 1 H), 5.24 (d, J = 3.21 Hz, 1 H),5.07 (s, 1 H), 4.94-5.01 (m, 2 H), 4.67 (d, J = 11.70 Hz, 1 H), 4.44-4.52 (m,2 H), 4.00-4.09 (m, 5 H), 3.87-3.94 (m, 1 H), 3.74 (s, 3 H), 3.73 (s, 3 H),3.54 (d, J = 4.34 Hz, 1 H), 3.41-3.42 (m, 10 H), 3.16-3.26 (m, 5 H), 3.01-3.04 (m, 1 H), 2.12 (s, 3 H), 2.07-2.09 (m, 2 H), 2.01 (s, 3 H), 1.91 (s, 3H), 1.80 (s, 3 H), 1.49-1.52 (m, 4 H) ppm. MS [M - H] - : 1128.5.

[0168] Step 4: Synthesis of YK-GAL-602

[0169] Starting from 2-4 (1.5 g, 1.3 mmol, 1.0 eq.) and bis(diisopropylamino)(2- cyanoethoxy)phosphine (2.00 g, 6.5 mmol, 5.0 eq.), following the procedure for the synthesis of YK-GAL-601, white solid 1.1 g was obtained with HPLC purity about 96.5%, yield about 64.2%. 1H NMR (400 Hz, d6-DMSO): δ 8.12 (t, J = 5.26 Hz, 1 H), 7.81-7.86 (m, 2 H), 7.45 (d, J = 7.49 Hz, 2 H), 7.26-7.33 (m, 6 H), 7.14-7.23 (m, 5 H), 6.86 ((t, J = 7.23 Hz, 4 H), 5.24 (d, J = 3.31 Hz, 1 H), 5.13 (s, 1 H), 4.99 (dd, J = 11.17 Hz, 3.31 Hz, 1 H), 4.71 (dd, J = 11.78 Hz, 4.27 Hz, 1 H), 4.50 (d, J = 8.51 Hz, 2 H), 4.23-4.46 (m, 1 H), 4.04-4.11 (m, 4 H), 3.86-3.98 (m, 1 H), 3.67-3.75 (m, 9 H), 3.50-3.57 (m, 3 H), 3.39-3.47 (m, 10 H), 3.21-3.23 (m, 4 H), 3.09 (s, 1 H), 2.98-3.02 (m, 1 H), 2.77 (t, J = 6.10 Hz, 1 H), 2.58 (t, J = 5.87 Hz, 1 H), 2.11 (s, 3 H), 2.07-2.08 (m, 2 H), 2.01 (s, 3 H), 1.91 (s, 3 H), 1.79 (s, 3 H), 1.46-1.52 (m, 4 H), 1.12 (s, 6 H), 1.09 (t, J = 6.35 Hz, 6 H) ppm. 31 P NMR (400 Hz, d6-DMSO): 149.11, 148.68 ppm. MS [M - H] - : 1329.3.

[0170] (3) Synthesis of YK-GAL-603

[0171] The synthetic route is as follows:

[0172]

[0173] Step 1: Synthesis of 3-2

[0174] Starting from 3-1 (6.0 g, 13.2 mmol, 1.0 eq.) and 1-11 (3.3 g, 16.2 mmol, 1.2 eq.), following the procedure for the synthesis of compound 1-12, white solid 8.4 g, yield about 99.0% was obtained. 1 H NMR (400 Hz, d6-DMSO): δ 7.83 (d, J = 9.02 Hz, 1 H), 7.72 (d, J = 5.92 Hz, 1 H), 6.77 (t, J =5.70 Hz, 1 H), 5.24 (d, J = 3.33 Hz, 1 H), 4.99 (dd, J = 11.20 Hz, 3.39 Hz, 1H), 4.50 (d, J = 8.36 Hz, 1 H), 4.02-4.09 (m, 3 H), 3.85-3.93 (m, 1 H), 3.70-3.75 (m, 1 H), 3.40-3.46 (m, 1 H), 2.99-3.04 (m, 2 H), 2.88-2.92 (m, 3 H),2.13 (s, 3 H), 2.05 (t, J = 7.07 Hz, 2 H), 2.02 (s, 3 H), 1.91 (s, 3 H), 1.79(s, 3 H), 1.44-1.55 (m, 4 H), 1.34-1.39 (m, 14 H) ppm. MS [M + H] + : 632.2.

[0175] Step 2: Synthesis of 3-3

[0176] Starting from 3-2 (8.4 g, 13.3 mmol, 1.0 eq.), following the procedure for the synthesis of compound 1-13, oil 16.4 g was obtained, which was used directly for the next step. 1H NMR (400 Hz, d6-DMSO): δ 7.88 (d, J = 9.39Hz, 1 H), 7.76 (d, J = 5.59 Hz, 1 H), 5.23 (d, J = 3.32 Hz, 1 H), 4.99 (dd, J= 11.26 Hz, 3.41 Hz, 1 H), 4.50 (d, J = 8.46 Hz, 1 H), 4.01-4.09 (m, 3 H),3.86-3.94 (m, 1 H), 3.70-3.75 (m, 1 H), 3.40-3.45 (m, 1 H), 3.02-3.06 (m, 2H), 2.91 (s, 1 H),2.74-2.82 (m, 2 H), 2.11 (s, 3 H), 2.06 (t, J = 6.95 Hz, 2H), 2.01 (s, 3 H), 1.90 (s, 3 H), 1.79 (s, 3 H), 1.39-1.56 (m, 9 H), 1.26-1.35 (m, 2 H) ppm. MS [M + H] + : 532.2.

[0177] Step 3: Synthesis of 3-4

[0178] Using 1-9 (2.5 g, 4.0 mmol, 1.0 eq.) and 3-3 (2.7 g, 5.0 mmol, 1.3 eq.) as starting material, following the procedure for synthesizing compound 1-14, gave 2.1 g of white solid in about 46.9% yield with HPLC purity of about 96.2%. 1HNMR (400 Hz, d6-DMSO): δ 8.01 (t, J = 5.64 Hz, 1 H), 7.54 (d, J = 9.34 Hz, 1H), 7.72 (t, J = 5.51 Hz, 1 H), 7.45 (d, J = 7.79 Hz, 2 H), 7.27-7.33 (m, 6H), 7.13-7.23 (m, 5 H), 6.86-6.89 (m, 4 H), 5.78 (s, 1 H), 5.23 (d, J = 3.50Hz, 1 H), 5.07 (s, 1 H), 4.93-5.00 (m, 2 H), 4.67 (d, J = 11.60 Hz, 1 H),4.44-4.51 (m, 2 H), 3.97-4.10 (m, 5 H), 3.56-3.93 (m, 1 H), 3.70-3.75 (m, 7H), 3.54 (d, J = 4.38 Hz, 1 H), 3.39-3.45 (m, 6 H), 3.16 (dd, J = 9.93 Hz,2.67 Hz, 1 H), 3.00-3.06 (m, 5 H), 2.12 (s, 3 H), 2.06 (t, J = 7.06 Hz, 2 H),2.01 (s, 3 H), 1.91 (s, 3 H), 1.79 (s, 3 H), 1.37-1.48 (m, 6 H), 1.23-1.29(m, 3 H) ppm. MS [M - H] - : 1127.3.

[0179] Step 4: Synthesis of YK-GAL-603

[0180] Starting from 3-4 (2.6 g, 2.3 mmol, 1.0 eq.) and bis(diisopropylamino)(2- cyanoethoxy)phosphine (4.2 g, 13.9 mmol, 6.0 eq.), following the procedure for the synthesis of YK-GAL-601, white solid 2.2 g was obtained with HPLC purity of about 96.0% and yield of about 69.8%. 1H NMR (400 Hz, d6-DMSO): δ 8.01 (t, J = 5.75 Hz, 1 H), 7.54 (d, J = 9.05 Hz, 1 H), 7.72 (t, J = 5.61 Hz, 1 H), 7.44-7.47 (m, 2H), 7.26-7.33 (m, 6 H), 7.14-7.23 (m, 5 H), 6.84-6.88 (m, 4 H), 5.23 (d, J = 3.40 Hz, 1 H), 5.13 (d, J = 2.38 Hz, 1 H), 4.99 (dd, J = 11.20 Hz, 3.47 Hz, 1H), 4.71 (dd, J = 11.76 Hz, 4.33 Hz, 1 H), 4.50 (d, J = 8.77 Hz, 2 H), 4.23-4.46 (m, 1 H), 4.09-4.11 (m, 1 H), 4.02-4.07 (m, 3 H), 3.86-3.93 (m, 1 H),3.67-3.77 (m, 9 H), 3.47-3.56 (m, 3 H), 3.38-3.45 (m, 6 H), 3.00-3.10 (m, 5H), 2.77 (t, J = 6.04 Hz, 1 H), 2.58 (t, J = 5.93 Hz, 1 H), 2.12 (s, 3 H),2.05 (t, J = 7.06 Hz, 2 H), 2.01 (s, 3 H), 1.91 (s, 3 H), 1.79 (s, 3 H),1.35-1.53 (m, 8 H), 1.23-1.30 (m, 5 H), 1.08-1.16 (m, 10 H) ppm. 31 P NMR (400Hz, d6-DMSO): δ 149.18, 148.69 ppm. MS [M - H] - : 1327.4.

[0181] (4) Synthesis of YK-GAL-604

[0182] The synthetic route is as follows:

[0183]

[0184] Step 1: Synthesis of 4-2

[0185] Starting from 4-1 (6.0 g, 13.4 mmol, 1.0 eq.) and 1-11 (2.6 g, 16.1 mmol, 1.2 eq.), white solid 4.3 g, yield 54.7% was obtained according to the procedure for synthesis of compound 1-12. 1 H NMR (400 Hz, d6-DMSO): δ 7.82 (d, J = 9.10 Hz, 1 H), 7.77 (t, J = 5.57 Hz, 1 H), 6.79 (t, J =5.59 Hz, 1 H), 6.23 (d, J = 3.36 Hz, 1 H), 4.99 (dd, J = 11.26 Hz, 3.40 Hz, 1H), 4.50 (d, J = 8.45 Hz, 1 H), 4.02-4.06 (m, 3 H), 3.86-3.92 (m, 1 H), 3.69-3.76 (m, 1 H), 3.40-3.49 (m, 1 H), 3.04-3.09 (m, 2 H), 2.94-2.99 (m, 2 H),2.12 (s, 3 H), 2.06 (t, J = 7.00 Hz, 2 H), 2.01 (s, 3 H), 1.91 (s, 3 H), 1.79(s, 3 H), 1.44-1.54 (m, 4 H),1.39 (s, 9 H) ppm。MS [M + Na] + : 612.2.

[0186] Step 2: Synthesis of 4-3

[0187] Starting from 4-2 (4.0 g, 7.1 mmol, 1.0 eq.), oil 8.7 g was obtained according to the procedure for synthesis of compound 1-13, which was used directly in the next step. 1H NMR (400 Hz, d6-DMSO): δ 7.97 (t, J = 5.62 Hz,1 H), 7.87 (d, J = 9.30 Hz, 1 H), 5.23 (d, J = 3.33 Hz, 1 H), 4.99 (dd, J =11.21 Hz, 3.48 Hz, 1 H), 4.51 (d, J = 8.46 Hz, 1 H), 4.01-4.07 (m, 3 H),3.86-3.93 (m, 1 H), 3.70 -3.76 (m, 1 H), 3.40-3.46 (m, 1 H), 3.26-3.31 (m, 2H), 2.82-2.92 (m, 3 H), 2.11 (s, 5 H), 2.01 (s, 3 H), 1.90 (s, 3 H), 1.79 (s,3 H), 1.46-1.57 (m, 5 H) ppm。MS [M + Na] + : 512.2.

[0188] Step 3: Synthesis of 4-4

[0189] Using 1-9 (3.5 g, 5.7 mmol, 1.0 eq.) and 4-3 (3.5 g, 7.1 mmol, 1.3 eq.) as starting material, following the procedure for synthesizing compound 1-14, afforded 3.2 g of white solid, yield about 52.1%, HPLC purity 87.0%. 1H NMR (400 Hz, d6-DMSO): δ 8.08 (s, 1 H), 7.82-7.86 (m, 2 H), 7.43-7.47 (m, 2 H), 7.27-7.33 (m, 6 H), 7.14-7.24 (m, 5 H), 6.85-6.89 (m, 4 H), 5.77 (s, 1 H), 5.23 (d, J = 3.43 Hz, 1 H), 5.07 (s, 1 H), 4.99 (dd, J = 11.33 Hz, 3.54 Hz, 1 H), 4.94 (d, J = 7.19 Hz, 1 H), 4.65 (d, J = 11.77 Hz, 1 H), 4.44-4.52 (m, 2 H), 3.97-4.10 (m, 5 H), 3.86 -3.94 (m, 1 H), 3.70-3.76 (m, 7 H), 3.55 (d, J = 4.51 Hz, 1 H), 3.40-3.42 (m, 5 H), 3.17 (dd, J = 10.00 Hz, 2.74 Hz, 1 H), 3.10-3.13 (m, 4 H), 3.00-3.04 (m, 1 H), 2.12 (s, 3 H), 2.06 (t, J = 7.08 Hz, 2 H), 2.01 (s, 3 H), 1.91 (s, 3 H), 1.80 (s, 3 H), 1.45-1.54 (m, 4 H) ppm. MS [M - H] - : 1085.3.

[0190] Step 4: Synthesis of YK-GAL-604

[0191] Starting from 4-4 (2.1 g, 2.0 mmol, 1.0 eq.) and bis(diisopropylamino)(2-cyanoethoxy) phosphine (2.9 g, 9.8 mmol, 5.0 eq.), following the procedure for synthesis of YK-GAL-601, about 1.8 g of white solid was obtained with HPLC purity 98.0%, yield about 71.4%. 1H NMR (400 Hz, d6-DMSO): δ 8.09 (s, 1 H), 7.81-7.86 (m, 2 H), 7.44-7.47 (m, 2 H), 7.26-7.33 (m, 6 H), 7.14-7.23 (m, 5 H), 6.84-6.88 (m, 4 H), 5.23 (d, J = 3.41 Hz, 1 H), 5.13 (s, 1 H), 4.99 (dd, J = 11.23 Hz, 3.45 Hz, 1 H), 4.72 (dd, J = 11.82 Hz, 4.20 Hz, 1 H), 4.51 (d, J = 8.52 Hz, 2 H), 4.23-4.45 (m, 1 H), 4.02-4.11 (m, 4 H), 3.86 -3.93 (m, 1 H), 3.67-3.76 (m, 9 H), 3.39-3.56 (m, 9 H), 3.09-3.11 (m, 4 H), 2.98-3.02 (m, 1 H), 2.77 (t, J = 6.02 Hz, 1 H), 2.58 (t, J = 6.00 Hz, 1 H), 2.12 (s, 3 H), 2.06 (t, J = 7.18 Hz, 2 H), 2.01 (s, 3 H), 1.91 (s, 3 H), 1.79 (s, 3 H), 1.46-1.54 (m, 4 H), 1.25-1.32 (m, 2 H), 1.08-1.16 (m, 11 H) ppm. 31 P NMR (400 Hz, d6-DMSO): δ 148.71, 149.20 ppm. MS [M - H] - : 1285.3.

[0192] (5) Synthesis of YK-GAL-605

[0193] The synthetic route is as follows:

[0194]

[0195] Step 1: Synthesis of 5-2

[0196] Using 5-1 (9.9 g, 22.1 mmol, 1.0 eq.) and 1-11 (5.0 g, 26.6 mmol, 1.2 eq.) as the starting material, following the procedure for synthesizing compound 1-12, about 11.9 g of white solid was obtained in 80.1% yield. 1 H NMR (400 Hz, d6-DMSO): δ 7.82 (d, J = 9.27 Hz, 1 H), 7.72 (t, J = 5.59 Hz, 1 H), 6.78 (t, J = 6.21 Hz, 1 H), 6.23 (d, J = 3.35 Hz, 1 H), 4.99 (dd, J = 11.21 Hz, 3.50 Hz, 1H), 4.50 (d, J = 8.49 Hz, 1 H), 4.01-4.06 (m, 3 H), 3.86-3.92 (m, 1 H), 3.69-3.76 (m, 1 H), 3.39-3.45 (m, 1 H), 2.89-3.04 (m, 5 H), 2.12 (s, 3 H), 2.06(t, J = 7.00 Hz, 2 H), 2.01 (s, 3 H), 1.91 (s, 3 H), 1.79 (s, 3 H), 1.44-1.54(m, 4 H),1.39 (s, 9 H), 1.33-1.36 (m, 3 H) ppm. MS [M + Na] + : 640.3.

[0197] Step 2: Synthesis of 5-3

[0198] Using 5-2 (11.9 g, 19.1 mmol, 1.0 eq.) as the starting material, following the procedure for synthesizing compound 1-13, 23.6 g of oil was obtained, which was used directly in the next step. 1H NMR (400 Hz, d6-DMSO): δ 7.88 (d, J = 9.44Hz, 1 H), 7.83 (d, J = 5.82 Hz, 1 H), 5.23 (d, J = 3.31 Hz, 1 H), 4.99 (dd, J= 11.02 Hz, 3.26 Hz, 1 H), 4.50 (d, J = 8.60 Hz, 1 H), 4.00-4.06 (m, 3 H),3.86-3.93 (m, 1 H), 3.70 -3.76 (m, 1 H), 3.40-3.45 (m, 1 H), 3.04-3.08 (m, 2H), 2.91 (s, 1 H), 2.76-2.84 (m, 2 H), 2.11 (s, 3 H), 2.06 (t, J = 7.07 Hz, 2H), 2.00 (s, 3 H), 1.89 (s, 3 H), 1.79 (s, 3 H), 1.42-1.68 (m, 9 H) ppm. MS [M+ Na] + : 540.2.

[0199] Step 3: Synthesis of 5-4

[0200] Using 1-9 (3.5 g, 5.7 mmol, 1.0 eq.) and 5-3 (4.4 g, 7.1 mmol, 1.3 eq.) as starting material, following the procedure for synthesizing compound 1-14, gave 1.9 g white solid in about 29.3% yield with 95.2% HPLC purity. 1H NMR (400 Hz, d6-DMSO): δ 8.02 (t, J = 5.44 Hz, 1 H), 7.83 (d, J = 9.09 Hz, 1 H), 7.74 (t, J = 5.43 Hz, 1 H), 7.45 (d, J = 7.39 Hz, 2 H), 7.26-7.32 (m, 6 H), 7.13-7.24 (m, 5 H), 6.86-6.89 (m, 4 H), 5.23 (d, J = 3.03 Hz, 1 H), 5.06 (s, 1 H), 4.87-5.00 (m, 3 H), 4.66 (d, J = 11.60 Hz, 1 H), 4.40-4.61 (m, 2 H), 3.86-4.10 (m, 8 H), 3.70-3.76 (m, 7 H), 3.53 (d, J = 4.45 Hz, 1 H), 3.38-3.43 (m, 7 H), 3.13-3.18 (m, 1 H), 3.00-3.06 (m, 5 H), 2.12 (s, 3 H), 2.06 (t, J = 6.85 Hz, 2 H), 2.01 (s, 3 H), 1.91 (s, 3 H), 1.79 (s, 3 H), 1.36-1.41 (m, 4 H) ppm. MS [M - H] - : 1113.3.

[0201] Step 4: Synthesis of YK-GAL-605

[0202] Using 5-4 (1.9 g, 1.7 mmol, 1.0 eq.) and bis(diisopropylamino)(2-cyanoethoxy) phosphine (2.8 g, 8.4 mmol, 5.0 eq.) as starting material, following the procedure for the synthesis of YK-GAL-601, about 1.6 g of white solid was obtained with 95.4% HPLC purity, about 71.3% yield. 1H NMR (400 Hz, d6-DMSO): δ 8.03 (t, J = 5.15 Hz, 1H), 7.84 (d, J = 9.36 Hz, 1 H), 7.74 (t, J = 5.73 Hz, 1 H), 7.44-7.48 (m, 2H), 7.26-7.33 (m, 6 H), 7.14-7.24 (m, 5 H), 6.84-6.89 (m, 4 H), 5.23 (d, J =3.46 Hz, 1 H), 5.13 (s, 1 H), 4.99 (dd, J = 11.29 Hz, 3.45 Hz, 1 H), 4.71(dd, J = 11.87 Hz, 4.20 Hz, 1 H), 4.51 (d, J = 8.56 Hz, 2 H), 4.23-4.46 (m, 1H), 4.02-4.11 (m, 4 H), 3.86 -3.93 (m, 1 H), 3.67-3.76 (m, 9 H), 3.46-3.56(m, 3 H), 3.39-3.43 (m, 6 H), 3.10(s, 1 H), 2.99-3.06 (m, 5 H), 2.77 (t, J =6.17 Hz, 1 H), 2.58 (t, J = 5.91 Hz, 1 H), 2.12 (s, 3 H), 2.06 (t, J = 7.00Hz, 2 H), 2.01 (s, 3 H), 1.91 (s, 3 H), 1.80 (s, 3 H), 1.25-1.32 (m, 5 H),1.08-1.16 (m, 15 H) ppm. 31 P NMR (400 Hz, d6-DMSO): δ 148.71, 149.20 ppm. MS [M- H] - : 1313.3.

[0203] (6) Synthesis of YK-GAL-325

[0204]

[0205] Following the synthesis of YK-GAL-325 in CN116854754B page 62, product 1.1 g, MS [M-H] - =1450.1.

[0206] 2. Synthesis of GalNAc solid support compounds

[0207] (1) Synthesis of YK-GAL-601-SP

[0208] The synthetic route is as follows:

[0209]

[0210] Step 1: Synthesis of 1-15

[0211] 1-14 (0.5 g, 0.5 mmol), 4-dimethylaminopyridine (54.7 mg, 0.5 mmol), diisopropylethylamine (231.4 mg, 1.8 mmol) and succinic anhydride (224.6 mg, 2.2 mmol) were dissolved in N,N-dimethylformamide (4 mL) and stirred at 30°C for 16 hours under nitrogen protection. Purification by preparative chromatography gave white solid 1-15 (0.3 g, 0.3 mmol) with a yield of 57.5%.

[0212] Step 2: Synthesis of YK-GAL-601-SP

[0213] 1-15 (0.3 g, 0.3 mmol), 4-dimethylaminopyridine (31.8 mg, 0.3 mmol), diisopropylethylamine (269.1 mg, 2.1 mmol) and O-benzotriazol- tetramethyluronium hexafluorophosphate (493.1 mg, 1.3 mmol) were dissolved in N,N-dimethylformamide (56 mL), followed by the addition of CPG-NH2 (4.50 g) and stirring at 40°C for 16 hours. The reaction solution was filtered, and the filter was washed with methanol and dichloromethane in turn and dried in vacuum. The filter was then added to a 56 mL solution of acetic anhydride / pyridine (1:4), and stirred at 40°C for 0.5 hours. Filtration was performed, and the filter was washed with dichloromethane and methanol in turn, and dried in vacuum for 12 hours to obtain white solid compound YK-GAL-601-SP (4.0 g, loading 31.7 μmol / g).

[0214] (2) Synthesis of other solid phase carrier compounds

[0215] YK-GAL-602-SP, YK-GAL-603-SP, YK-GAL-604-SP and YK-GAL-605-SP, other GalNAc solid phase carrier compounds listed in Table 1, were synthesized according to the method for synthesizing YK-GAL-601-SP, using 2-4, 3-4, 4-4 and 5-4 as starting materials, respectively.

[0216] Table 1 GalNAc solid phase carrier compounds

[0217]

[0218] Example 2: Coupling of GalNAc compound with oligonucleotide

[0219] Exemplarily, a double-stranded oligonucleotide siRNA was synthesized in this example, and the GalNAc compound of the present application was coupled with the oligonucleotide, thereby obtaining a GalNAc-conjugated oligonucleotide conjugate (which can also be referred to simply as conjugate herein), the siRNA being numbered as D579-DV25P, and the sequences of the sense and antisense strands thereof being as follows:

[0220] Sense strand (D579-DV25P-SS): 5'-Cms-Cms-Um-Um-Um-Um-Cf-Um-Uf-Cf-Uf-Am-Am-Um-Gm-Am-Gf-Um-Cm-Gm-Am-3' (SEQ ID NO. 1)

[0221] Antisense strand (D579-DV25P-AS): 5'-UmsEVP-Cfs-Gm-Am-Cm-Uf-Cm-Am-Um-Um-Am-Gm-Am-Af-Gm-Af-Am-Am-Am-Gm-Gms-Ums-Gm-3' (SEQ ID NO. 2)

[0222] Wherein, A, U, C, G represent nucleotides comprising adenine, uracil, cytosine or guanine as the base, respectively; m represents a nucleotide adjacent to the left of the letter as a 2'-OMe (2'-methoxy) modified nucleotide; f represents a nucleotide adjacent to the left of the letter as a 2'-F (2'-fluoro) modified nucleotide; s represents a 3', 5'-phosphorothioate group between the two nucleotides adjacent to the left and right of s; EVP represents a nucleotide adjacent to the left thereof as a vinyl-(E)-phosphonate modified nucleotide.

[0223] The nucleoside monomers without GalNAc structure used in the synthesis of the sense and antisense strands can be obtained through commercial channels.

[0224] 1. Synthesis of GalNAc-conjugated siRNA sense strand

[0225] Instruments and reagents: GenXpert 192 P model DNA / RNA automatic synthesizer, Primer support 5G Unylinker 350 (Cytiva) model.

[0226] The GalNAc-conjugated oligonucleotide conjugate was synthesized on a solid support according to the phosphoramidite chemical method.

[0227] For the synthesis of conjugates 1-5 (siRNA IDs: D579-DV25PG601, D579-DV25PG602, D579-DV25PG603, D579-DV25PG604, and D579-DV25PG605), the general CPG solid support or the GalNAc solid support compound (YK-GAL-601-SP, YK-GAL-602-SP, YK-GAL-603-SP, YK-GAL-604-SP, and YK-GAL-605-SP) synthesized in Example 1 was used, and the GalNAc phosphoramidite compound (YK-GAL-601, YK-GAL-602, YK-GAL-603, YK-GAL-604, and YK-GAL-605) synthesized in Example 1 was used as a phosphoramidite monomer linked to the 3' end of the oligonucleotide sense strand, respectively. For the synthesis of conjugate 6 (siRNA ID: D579-DV25PG325), the general CPG solid support was used, and the GalNAc phosphoramidite compound YK-GAL-325 synthesized in Example 1 was used as a phosphoramidite monomer linked to the 3' end of the oligonucleotide sense strand. For the synthesis of conjugate 7 (siRNA ID: D579-DV25PL96), the purchased CPG-L96 (Jiangsu Shenji Biological Technology Co., Ltd., Cat. No. DS004) was used as a solid support, and the solid support was synthesized in a scale of 1 μmol. These GalNAc compounds were conjugated to the 3' end of the oligonucleotide sense strand.

[0228] (1) Preparation of reagents and phosphoramidite monomer solution

[0229] An acetonitrile solution of 0.25 M 5-benzylthio tetrazole was used as an activator, an acetonitrile / pyridine (1 / 4, v / v) solution of 0.2 M hydrogenated xanthine was used as a thio reagent, an aqueous / pyridine (1 / 9, v / v) solution of 0.05 M iodine was used as an oxidizing agent, a 20% (v / v) acetic anhydride acetonitrile solution was used as a capping agent A, 20 / 30 / 50 (1-methylimidazole / pyridine / acetonitrile, v / v / v) was used as a capping agent B, a 20% (v / v) diethylamine acetonitrile solution was used as a de-cyanethyl reagent, a 3% (v / v) dichloroacetic acid toluene solution was used as a DMT-removing reagent, and an acetonitrile solution (1 / 20, w / v) of the phosphoramidite monomer was prepared. Then, the above reagents and phosphoramidite monomer solution were loaded into the designated reagent positions of the DNA / RNA automatic synthesizer of model 192P.

[0230] (2) Crude synthesis

[0231] The sequence was inputted into the synthesizer according to the nucleotide sequence of the oligonucleotide to be synthesized, and the synthesis program was set according to the manufacturer's instructions, and the synthesis of the oligonucleotide was performed. The monomer coupling time was about 1 minute, wherein the oxo time was about 30 to 45 seconds, and the thio time was about 2 minutes. After the cycle was completed, the solid-phase synthesis of the oligonucleotide was completed.

[0232] (3) Deprotection

[0233] After the synthesis was completed, the solid-phase carrier was transferred to the reactor, and the oligonucleotide was cleaved from the solid-phase carrier with concentrated ammonia water (25-28%) at 50-60°C for 16-24 hours, then reduced to room temperature, then filtered, rinsed with a mixture of purified water and ethanol, combined the filtrates, and concentrated the filtrates at low temperature to obtain a crude residue.

[0234] (4) Purification

[0235] The crude residue after deprotection was dissolved with purified water and subjected to HPLC purification, the product peak solution was collected and the content was measured with a microplate reader, and the molecular weight was verified by ESI-MS.

[0236] When synthesizing conjugates 1-6, the GalNAc phosphoramidite compounds (YK-GAL-601, YK-GAL-602, YK-GAL-603, YK-GAL-604, YK-GAL-605, and YK-GAL-325) synthesized in Example 1 were used as phosphoramidite monomers, and when a general CPG solid-phase carrier was used, the GalNAc phosphoramidite compound was repeated 3 times in the synthesis sequence; when the GalNAc solid-phase carrier synthesized in Example 1 was used, the GalNAc phosphoramidite compound was repeated 2 times in the synthesis sequence, finally making the synthesized conjugates 1-6 conjugate 3 GalNAc compounds.

[0237] 2. Synthesis of siRNA antisense strand without GalNAc conjugation

[0238] The siRNA antisense strand was synthesized according to the conventional solid-phase synthesis method in the art, and the monomers used were commercially available monomers, wherein the synthesis scale of each antisense strand complementary to the sense strand was 1 μmol using a general CPG solid-phase carrier.

[0239] 3. Synthesis of GalNAc conjugated oligonucleotide

[0240] The GalNAc conjugated siRNA sense strand and the corresponding antisense strand were mixed according to the ultraviolet absorption content 1:1, heated to 95℃, and cooled to room temperature after 3 minutes, to form a conjugate of the GalNAc conjugated siRNA double-stranded. The obtained double-stranded solution was characterized by HPLC to determine the product purity, and then the content was determined by a microplate reader, and the solid powder was obtained by freeze-drying for storage. The obtained conjugate and the corresponding molecular weight are shown in Table 2.

[0241] Table 2 Conjugate and corresponding molecular weight

[0242]

[0243] wherein: SS is the sense strand, AS is the antisense strand, and the structure of the obtained GalNAc conjugated oligonucleotide is as follows:

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250] The GalNAc compound in D579-DV25PG325 is YK-GAL-325 (CN116854754B, page 62),

[0251]

[0252] The GalNAc compound in D579-DV25PL96 is L96 (N-[tris(GalNAc-alkyl)-dodecanamido]-4-hydroxyprolinol, see US10465194B2, claim 10).

[0253] Example 3: Inhibition of AGT protein expression in mouse serum by GalNAc conjugated oligonucleotide and effect on liver AGT mRNA level

[0254] Angiotensinogen (AGT) protein is a secreted protein mainly expressed in the liver in the human body. As an upstream protein of the renin-angiotensin-aldosterone system (RAAS), AGT protein inhibits the expression of RAAS system, which fundamentally inhibits the role of RAAS system in raising blood pressure, thereby reducing blood pressure.

[0255] Example 2, the AGT protein level in serum at different time points was detected by ELISA method and the AGT mRNA level in liver was detected by PCR method.

[0256] It should be noted that the conjugate synthesized in Example 2 is used to illustrate the inhibition of the corresponding target gene AGT. Those skilled in the art can clearly know that for different target genes, oligonucleotides with different sequences will be conjugated with the corresponding GalNAc compound to produce the corresponding inhibitory effect. Therefore, any example in the present application does not mean that the GalNAc compound of the present application can only be applied to the oligonucleotide in Example 2. For oligonucleotides with other specific sequences, the corresponding conjugate generated after conjugation can also have the beneficial effects of the present application, such as but not limited to, inhibition of the corresponding target gene of the oligonucleotide.

[0257] 1. Experimental materials

[0258] Test drug:

[0259] GalNAc-conjugated oligonucleotides: D579-DV25PL96, D579-DV25PG325, D579-DV25PG601, D579-DV25PG602, D579-DV25PG603, D579-DV25PG604 and D579-DV25PG605.

[0260] Experimental animal information:

[0261] hAGT transgenic mice; SPF level; male; a total of 48; 6-8 weeks old; body weight about 18-28 g (Jiangsu Jizhuangkang Biotechnology Co., Ltd., Production License No.: SCXK(Su)2018-0008).

[0262] 2. Experimental methods

[0263] Dose design and grouping

[0264] Test date definition: the day when the test animals are administered with the solvent or the test drug is defined as day 0 (day 0).

[0265] Grouping and administration: after 3 days of adaptive feeding of the test animals, they were randomly divided into negative control group and test drug group according to the serum AGT protein content, 6 in each group. The administration was performed by subcutaneous single injection, the administration dose was 1 mg / kg, the administration volume was 5 mL / kg, the administration concentration was 0.2 mg / mL, and the administration day was recorded as day 0.

[0266] The animal grouping information is shown in Table 3:

[0267] Table 3 Information of animal grouping

[0268]

[0269] Detection index

[0270] (1) General observation

[0271] From 1 week before self-administration to the end of the experiment, the animals were observed once a day.

[0272] Observation content: Observe the death or near-death, mental state, behavior activity, feces shape, feed and water supply, etc. of the animals.

[0273] Animals for detection: All animals in the negative control group and the test drug group.

[0274] (2) Expression of AGT protein in serum

[0275] Detection time: Before administration on day-3 (day-3), 1 week after administration on day 7 (D7), 2 weeks after administration on day 14 (D14), and 3 weeks after administration on day 21 (D21).

[0276] AGT protein level detection method: ELISA kit was used for detection.

[0277] Animals for detection: All animals in the negative control group and the test drug group.

[0278] (3) Detection of liver mRNA level

[0279] Collection of liver tissue and preparation of homogenate: On day 21, fresh liver tissue was taken from all animals, and RNA lysis solution (Trizol) was added at a ratio of 100 mg of tissue: 1 mL of solution. The EP tube containing 1 mL of RNA lysis solution (TRIzol) was quickly placed in the prepared EP tube, and liver homogenate was prepared. The homogenate was immediately detected or stored at -80°C. The remaining liver tissue was quickly frozen and stored at -80°C.

[0280] RNA extraction:

[0281] a. The tissue was taken and placed in a 1.5 mL RNAase-free EP tube. 1 mL of TRIzol reagent was added to every 100 mg of tissue, and tissue homogenate was prepared by shaking and grinding.

[0282] b. Centrifugation at 4°C, 12000 g, 3 minutes, 400 μL of tissue homogenate supernatant was transferred to a 1.5 mL RNAase-free EP tube and placed on ice. 80 μL of chloroform was added to each tube, and it was vigorously shaken for 15 seconds and allowed to stand at room temperature for 5 minutes. Centrifugation at 4°C, 12000 g for 15 minutes, and 150 μL of supernatant was taken to a new EP tube.

[0283] c. Add equal volume of isopropanol, invert the tube to mix the liquid gently, stand at -20°C for 10 minutes, centrifuge at 4°C (white precipitate can be seen), 12000 g, 15 minutes, discard the supernatant.

[0284] d. Add 1 mL 75% ethanol, wash the RNA precipitate gently, centrifuge at 4°C 7500 g for 5 minutes, remove the supernatant. Repeat the rinse once at 4°C 7500 g for 5 minutes, remove the residual ethanol with a micropipette tip.

[0285] e. Dry the residual ethanol at room temperature for 10 minutes, add 100 μL RNase-free ddH2O, dissolve.

[0286] RNA concentration detection and reverse transcription: Use the UV-visible spectrophotometer to detect the concentration of RNA. Use 2 μL RNase-free ddH2O as a blank control, detect 2 μL of RNA sample each time. Record the sample concentration. Then use PrimeScript RT Master Mix to synthesize cDNA according to the instructions. Prepare the reverse transcription reaction system in a 0.2 mL multi-well plate according to the following Table 4. The volume of RNA and sterile enzyme-free water can be adjusted according to the concentration of RNA, 10 μL system can handle up to 500 ng of RNA, and the system can be proportionally expanded according to the required amount of RNA.

[0287] Table 4 Reverse transcription system

[0288]

[0289] Tap to mix the system. Use the centrifuge Short function for a short time, then use the PCR instrument for reverse transcription reaction, the program is as follows: 37°C for 15 minutes, 85°C for 5 seconds, 4°C for maintenance.

[0290] Realtime-qPCR: Use TB Green® Premix Ex Taq TMqPCR. The reaction system of 10 μL was prepared in a 96-well plate according to Table 4, and the cDNA of GAPDH and AGT was detected by qPCR, and GAPDH was used as an internal reference gene. qPCR was performed in a 96-well plate, and each well contained 10 μL of the reaction system. Each sample had 3 wells of GAPDH primers and 3 wells of each pair of target gene primers. When the number of samples was too large and the samples of one biological repeat needed to be dispersed for detection in multiple 96-well plates, it was ensured that each group of samples was amplified simultaneously on the same 96-well plate, and the control samples were included on each 96-well plate. The qPCR reaction program was as follows: heating at 95℃ for 30 seconds, then entering the cycle mode, heating at 95℃ for 5 seconds, then heating at 60℃ for 34 seconds, a total of 40 cycles; template heating at 95℃ for 15 seconds, heating at 60℃ for 1 minute, then heating at 95℃ for 15 seconds, and then melting curve analysis.

[0291] Table 5 qPCR reaction system

[0292]

[0293] (4) Data processing and statistical analysis

[0294] The data were analyzed using GraphPad Prism 8.3 analysis software. Statistical analysis was performed using two-way ANOVA and post-hoc test, LSD test for homogeneity of variance, and Dunnett's test for heterogeneity of variance.

[0295] 3. Experimental results

[0296] The specific experimental results are shown in Tables 6 and 7.

[0297] (1) Inhibition rate of AGT protein level in serum

[0298] Table 6 Inhibition rate of AGT protein level in serum by different GalNAc conjugated oligonucleotides

[0299]

[0300] 1) The siRNA sequences containing aromatic ring skeleton GalNAc modification in the present application can efficiently inhibit the expression of AGT protein in mouse serum.

[0301] As can be seen from Table 6, the siRNA sequences containing aromatic ring skeleton GalNAc modification in the present application can continuously and significantly inhibit the AGT protein level in mouse serum. For example, the inhibition rates of D579-DV25PG601 on day 7, day 14 and day 21 reached 72.82%, 83.07% and 81.36%, respectively.

[0302] 2) The siRNA sequence of the application containing an aromatic ring skeleton GalNAc modified siRNA sequence compared with prior art compound modified siRNA sequence, the inhibition rate of AGT protein expression in mouse serum is significantly improved.

[0303] Compared with siRNA conjugates synthesized by prior art GalNAc compounds YK-GAL-325 and L96, the siRNA conjugates synthesized by the aromatic skeleton GalNAc compound of the application have a significantly improved inhibition rate of AGT protein levels in serum. For example, the inhibition rate of the D579-DV25PG604 group on the 21st day was increased by 15.83% and 18.01% compared with the D579-DV25PG325 group and the D579-DV25PL96 group, respectively, and the inhibition rate of the D579-DV25PG601 group on the 21st day was increased by 16.93% and 19.11% compared with the D579-DV25PG325 group and the D579-DV25PL96 group, respectively.

[0304] From the structure, compared with YK-GAL-325, the connecting arm between the ribose ring and GalNAc of YK-GAL-601 is shorter and contains an aromatic ring, and the inhibition rate of the corresponding modified siRNA on AGT protein expression in mouse serum is significantly different.

[0305] (2) Inhibition rate of AGT mRNA level in liver

[0306] Table 7 Inhibition rate of AGT mRNA level in liver of hAGT transgenic mice after 21 days of administration

[0307]

[0308] As can be seen from Table 7, the aromatic ring skeleton GalNAc conjugated oligonucleotide of the application can significantly inhibit the AGT mRNA level in the liver of mice, and the inhibition rate of the D579-DV25PG601 group is the highest, reaching 82.60%. The experimental results show that the oligonucleotide sequence D579-DV25P coupled with the GalNAc compound of the application can be efficiently delivered to the liver of animals and significantly inhibit the AGT mRNA level in the liver.

[0309] Compared with siRNA conjugates synthesized by prior art GalNAc compounds YK-GAL-325 and L96, the GalNAc conjugated oligonucleotide synthesized by the GalNAc compound of the application has a significantly improved inhibition rate of AGT mRNA level in the liver of mice. For example, the inhibition rate of the D579-DV25P601 group was increased by 16.88% and 21.04% compared with the D579-DV25PG325 group and the D579-DV25PL96 group, respectively.

[0310] While the application has been illustrated by a description of the previous specific embodiments, it is not the intention to restrict or limit it by means of the specific embodiments described, but rather the intention is to cover all the alternatives, modifications, and equivalents falling within the spirit and scope of the application.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof: ； in, R1 is oxygen or sulfur; R2 is C 1-6 Alkoxy; R3 is or -CO(CH2) z CONH-E; z is 1, 2, 3 or 4; E is controllable aperture glass; R4 is a hydroxyl protecting group, wherein the hydroxyl protecting group is 4,4'-dimethoxytriphenylmethyl, monomethoxytriphenylmethyl, or triphenylmethyl; A and B are each independently -C(O)NH- or -NHC(O)-; L stands for -(CH2) a -or-(CH2CH2O) b (CH2) a -, where a is independently 1, 2, 3, 4, 5 or 6, and b is 1; G stands for -X1-OT; T is R5 is -NHR 5a ;R 5a R 6a R 6b and R 6c It can be acetyl, propionyl, n-butyryl, or isobutyryl independently; X1 is -(CH2) f -, f is 4; m and n are both 1; x and y are both 1.

2. The compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R 6a R 6b and R 6c They are identical and independently acetyl groups.

3. The compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof, wherein... for or .

4. The compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound of formula (I) satisfies any one or a combination of at least two of the following conditions (1) to (8): (1) R1 is oxygen; (2) R2 is -OCH3; (3) R3 is Or -CO(CH2)2CONH-E; E represents glass with controllable aperture; (4) R4 is 4,4'-dimethoxytriphenylmethyl; (5) A is -C(O)NH- a End a is connected to L; (6) B is -NHC(O)- b The b end is connected to the G end; (7) L is -(CH2)2-, -(CH2)3-, -(CH2)4-, -(CH2)5- or -CH2CH2OCH2CH2-; and (8) G is .

5. The compound of formula (I) according to any one of claims 1-4, or a pharmaceutically acceptable salt thereof, wherein, The compound of formula (I) is the compound shown in formula (Ia): 。 6. The compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound of formula (I) is compound YK-GAL-601, YK-GAL-602, YK-GAL-603, YK-GAL-604, YK-GAL-605, YK-GAL-601-SP, YK-GAL-602-SP, YK-GAL-603-SP, YK-GAL-604-SP or YK-GAL-605-SP, wherein E is a controllable aperture glass. 、 、 、 、 、 、 、 、 or 。 7. A conjugate or a pharmaceutically acceptable salt thereof, comprising an oligonucleotide and a GalNAc moiety, having the following structure: or ; in, Oligo stands for oligonucleotide, where X represents oxygen or sulfur, G1 stands for -X1-O-T1, and T1 represents... q is 1, 2, 3 or 4, and R1, R2, R5, A, B, L, X1, x, y, m and n are defined as in claim 1.

8. The conjugate of claim 7 or a pharmaceutically acceptable salt thereof, wherein the oligonucleotide comprises nonthio oligonucleotides and thio oligonucleotides.

9. The conjugate of claim 7 or a pharmaceutically acceptable salt thereof, wherein the oligonucleotide is a small interfering nucleotide, DNA, microRNA, small activating RNA, small guide RNA, transfer RNA, antisense nucleotide or aptamer.

10. The conjugate of claim 9 or a pharmaceutically acceptable salt thereof, wherein each nucleotide in the antisense nucleotide or small interfering nucleotide is independently an unmodified nucleotide.

11. The conjugate of claim 9 or a pharmaceutically acceptable salt thereof, wherein the sense strand of the small interfering nucleotide has the sequence shown in SEQ ID NO. 1; and the antisense strand of the small interfering nucleotide has the sequence shown in SEQ ID NO.

2.

12. The conjugate according to claim 7 or a pharmaceutically acceptable salt thereof, wherein the conjugate is any of the following: 、 、 、 or ； in, The sense strand of the siRNA has the sequence shown in SEQ ID NO. 1; the antisense strand of the siRNA has the sequence shown in SEQ ID NO.

2.

13. A pharmaceutical composition comprising the conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 7-12, and at least one pharmaceutically acceptable excipient.

14. Use of a conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 7-12, or the pharmaceutical composition of claim 13, in the preparation of a medicament for treating and / or preventing a pathological condition or disease caused by the expression of a specific gene in liver tissue or a virus, said specific gene being the angiotensinogen gene.

15. The use according to claim 14, wherein the disease is selected from chronic liver disease, hepatitis, liver fibrosis, liver proliferative disease, and cardiovascular and cerebrovascular diseases.

16. The use according to claim 15, wherein the cardiovascular disease is hypercholesterolemia, hypertriglyceridemia, atherosclerosis, or coagulation dysfunction.

17. A kit comprising the conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 7-12.

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