Galnac compounds containing a triazole structure and oligonucleotide conjugates thereof

Abstract

GalNAc compounds containing a triazole structure and oligonucleotide conjugates thereof are provided.SOLUTION: Provided is a GalNAc compound represented by the following formula, or a pharmaceutically acceptable salt thereof. The GalNAc compound in the present invention can form a conjugate with an oligonucleotide, and the conjugate can achieve highly efficient liver-targeted delivery of the oligonucleotide, control gene expression, and be used for the prevention and / or treatment of a disease caused by the expression of a specific gene in hepatocytes.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to the field of biomedicine, and specifically to GalNAc compounds having a triazole ring structure and GalNAc-oligonucleotide conjugates prepared therewith. [Background technology]

[0002] In recent years, following small molecule drugs and antibody drugs, oligonucleotide drugs are gradually becoming the third wave of new drug research and development. Oligonucleotide drugs mainly bind to the upstream mRNA of pathogenic proteins and can regulate them at the gene level, providing therapeutic potential for urgent clinical needs for which no drugs are available, and showing great potential in the treatment of neurological disorders and cardiovascular diseases.

[0003] Due to their unique properties, small molecule nucleic acid drugs (oligonucleotides) have gradually developed as an alternative to traditional small molecule drugs and are being used to control the function of disease-related proteins. Oligonucleotides can be used to silence or activate gene expression in specific diseases, thereby preventing or promoting the formation of specific proteins and treating the disease. Oligonucleotides include, but are not limited to, antisense oligonucleotides (ASOs), small interfering RNA (siRNA), small activating RNA (saRNA), and microRNA (miRNA). Due to their relatively good therapeutic effects and technological breakthroughs, oligonucleotide drugs have become the most popular technology, with several drugs approved for sale worldwide.

[0004] Currently available oligonucleotide drug delivery systems include lipid nanoparticle (LNP) delivery systems and N-acetylgalactosamine (GalNAc) delivery systems, of which the GalNAc delivery system is the most widely used. GalNAc can bind to the asialoglycoprotein receptor (ASGPR), an endocytic receptor specifically expressed on the surface of hepatocytes, enabling liver-targeted delivery of oligonucleotide drugs. Therefore, improving GalNAc can improve the delivery efficiency of oligonucleotide drugs.

[0005] Galactose (Gal) and N-acetylgalactosamine (GalNAc) are ligands that can bind to ASGPR on the liver surface. The binding affinity of GalNAc to ASGPR is approximately 50 times higher than that of Gal. Studies have shown that the affinity order of GalNAc with various antennary structures is tetraantennary > triantennary > biantennary > monoantennary galactosamine. In recent years, progress has been made in liver-targeted delivery of nucleic acid drugs by using N-acetylgalactosamine (GalNAc), a high-affinity ligand for ASGPR, as a targeting molecule. Various GalNAc conjugations have been successfully implemented to siRNAs and ASOs, and this technology has been utilized to develop drugs for the treatment of diseases such as amyloidosis, hemophilia, hypercholesterolemia, and hepatitis B.

[0006] Different GalNAc structures have significantly different nucleic acid delivery efficiencies. There is a need in the art to develop new GalNAc compounds to improve the delivery efficiency of liver-targeted drugs, such as complement inhibitor drugs. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention provides novel GalNAc compounds containing triazole structures. By incorporating triazole into the linker of the GalNAc compound, a series of novel GalNAc compounds containing triazole structures can be constructed modularly, simply, and quickly using click chemistry. The GalNAc ligand is attached to the 3' end of the oligonucleotide using phosphoramidite solid-phase synthesis. Compared with the prior art, the GalNAc compounds of the present invention significantly improve the efficiency of oligonucleotide delivery to the liver, making them useful for the development of drugs for treating liver-related diseases. [Means for solving the problem]

[0008] The present invention provides a GalNAc compound represented by formula (I), or a pharmaceutically acceptable salt thereof: [ka] (wherein L1 is -HNC(O)- or -C(O)NH-, L2 is -(CH2)n1- or -(CH2CH2O)n2CH2-, where n1 and n2 are integers from 1 to 7; Z1 is -(CH2)n3-, where n3 is an integer from 1 to 7; L3 is -HNC(O)- or -C(O)NH-; n is an integer from 0 to 10, L4 is -(CH2)n4-, where n4 is an integer from 0 to 7; A is, [ka] and R1 is hydrogen, C 1~6 Alkyl group, C 1~6R2 is 4,4'-dimethoxytrityl, monomethoxytrityl, trityl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, triisopropylsilyl, or isopropyldimethylsilyl; R3 is hydrogen, a hydroxy protecting group, a phosphorus-containing reactive group, -CO(CH2) x CONH-E, or -CO(CH2) x COOH, where x is an integer from 1 to 10, and E is controlled pore glass (CPG) or polystyrene; B is oxygen or sulfur, G is [ka] and where: X1 is -(CH2) a - or -(CH2CH2O) a CH2-, and a is an integer of 1 to 5; X2 is -HNC(O)- or -C(O)NH-; X3 is -(CH2) b and b is an integer from 1 to 6; X4 is [ka] where c and d are integers from 0 to 5; Y1 is 0 or 1, Y2 is 0 or 1, Y3 is 1, 2, or 3; When Y3 is 1, K is CH2, when Y3 is 2, K is CH, and when Y3 is 3, K is carbon.

[0009] In some embodiments of the invention, L1 is -C(O)NH-*, and the * end is linked to G.

[0010] In some embodiments of the invention, L2 is -(CH2)3-, -(CH2)4-, -(CH2)5-, -(CH2)6-, -(CH2)7-, or -(CH2CH2O)3CH2-.

[0011] In some embodiments of the invention, L2 is -(CH2)5-, or -(CH2CH2O)3CH2-.

[0012] In some embodiments of the invention, n is 0.

[0013] In some embodiments of the invention, L4 is none, -(CH2)3-, -(CH2)4-, -(CH2)5-, -(CH2)6-, or -(CH2)7-.

[0014] In some embodiments of the invention, L4 is none, -(CH2)3-, or -(CH2)5-.

[0015] In some embodiments of the invention, R1 is hydrogen or a methoxy group.

[0016] In some embodiments of the invention, R2 is a 4,4'-dimethoxytrityl group (DMTr).

[0017] In some embodiments of the present invention, R3 is -CO(CH2)2CONH-E, where E is controlled pore glass.

[0018] In some embodiments of the present invention, B is oxygen.

[0019] In some embodiments of the present invention, X1 is -(CH2)3-.

[0020] In some embodiments of the invention, X2 is -HNC(O)-**, and the ** end is linked to X3.

[0021] In some embodiments of the invention, X3 is -(CH2)2-, -(CH2)4-, or -(CH2)5-.

[0022] In some embodiments of the invention, X4 is [ka] and the *** end is linked to X3.

[0023] In some embodiments of the present invention, Y 1 is 1.

[0024] In some embodiments of the invention, Y2 is 1.

[0025] In some embodiments of the invention, Y3 is 3 and K is carbon.

[0026] In some embodiments of the invention, A is [ka] or [ka] and E is the controlled pore glass.

[0027] In some embodiments of the invention, G is: [ka] [ka] or [ka]

[0028] In some embodiments of the present invention, the compound of formula (I) has a structure of formula (IA): [ka] (wherein L1, L2, L4, B, A, X1, X2, X3, X4, Y1, and Y2 are defined as above.)

[0029] In some embodiments of the present invention, the compound represented by formula (I), or a pharmaceutically acceptable salt thereof, is capable of binding to the asialoglycoprotein receptor (ASGPR).

[0030] In some embodiments of the present invention, the compound represented by formula (I) is any of the following compounds: [ka] [ka] [ka] [ka] [ka] or [ka]

[0031] The present invention also provides a conjugate represented by formula (IIA) or (IIB), or a pharmaceutically acceptable salt thereof. [ka] or [ka] (wherein Oligo represents an oligonucleotide, X represents a hydroxy group or a mercapto group, and G1 represents [ka] and The definitions of L1, L2, Z1, L3, L4, n, R1, R2, R3, X1, X2, X3, X4, Y1, Y2, Y3, and K are as described above.) As will be understood by those skilled in the art, in the conjugates represented by formula (IIA) or (IIB), the structural unit [ka] During solid phase synthesis, the oligonucleotide is synthesized by a monomer containing a five-membered sugar ring (i.e., [ka] or [ka] ) and then linked to the hydroxy group in

[0032] In the conjugate represented by formula (IIA), R3 is preferably hydrogen.

[0033] In the conjugate represented by formula (IIB), R2 is preferably hydrogen.

[0034] In some embodiments of the present invention, the oligonucleotide is any one or a combination of at least two selected from the group consisting of small interfering nucleotides (siRNA), DNA, microRNA (miRNA), small activating RNA (saRNA), small guide RNA (sgRNA), transfer RNA (tRNA), antisense oligonucleotides (ASO), and aptamers, preferably antisense oligonucleotides (ASO) or small interfering nucleotides (siRNA).

[0035] In some embodiments of the present invention, each nucleotide of the antisense oligonucleotide (ASO) or small interfering nucleotide (siRNA) is independently a modified or unmodified nucleotide.

[0036] In some embodiments of the invention, the oligonucleotide modulates expression of a target gene.

[0037] In some embodiments of the invention, the oligonucleotide is a double-stranded RNAi agent.

[0038] In some embodiments of the present invention, the double-stranded RNAi agent is a double-stranded RNAi agent that inhibits expression of the PCSK9 gene.

[0039] In some embodiments of the invention, at least one strand of the double-stranded RNAi agent comprises a 3' overhang of at least one nucleotide.

[0040] In some embodiments of the invention, at least one strand of the double-stranded RNAi agent comprises a 3' overhang that is at least two nucleotides.

[0041] In some embodiments of the present invention, the double-stranded region of the double-stranded RNAi agent is 15 to 30 nucleotide pairs in length.

[0042] In some embodiments of the invention, the double-stranded region of the double-stranded RNAi agent is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotide pairs in length.

[0043] In some embodiments of the present invention, the double-stranded region of the double-stranded RNAi agent is 17 to 25 nucleotide pairs in length.

[0044] In some embodiments of the present invention, the double-stranded region of the double-stranded RNAi agent is 19 to 23 nucleotide pairs in length.

[0045] In some embodiments of the invention, the double-stranded region of the double-stranded RNAi agent is 21 nucleotide pairs in length.

[0046] In some embodiments of the invention, each strand of the double-stranded RNAi agent has between 15 and 30 nucleotides.

[0047] In some embodiments of the invention, the number of nucleotides in the sense strand and / or the antisense strand of the double-stranded RNAi agent is any selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30, respectively.

[0048] In some embodiments of the invention, each strand of the double-stranded RNAi agent has between 19 and 25 nucleotides.

[0049] In some embodiments of the invention, the sense strand of the double-stranded RNAi agent has 21 nucleotides and the antisense strand has 23 nucleotides.

[0050] In some embodiments of the present invention, the double-stranded RNAi agent comprises any one or a combination of at least two selected from the group consisting of oligonucleotide duplexes formed by pairing an unmodified sense strand with an unmodified antisense strand as shown in the table below (the sequences are listed in Table 8 of the specification of CN117210468A). [Table 1]

[0051] In some embodiments of the invention, the sense strand of the double-stranded RNAi agent has 1-3 nucleotide substitutions, additions, or deletions compared to any of the sequences of SEQ ID NOs: 1-12.

[0052] In some embodiments of the invention, the antisense strand of the double-stranded RNAi agent has 1 to 3 nucleotide substitutions, additions, or deletions compared to any of the sequences of SEQ ID NOs: 13-24.

[0053] In some embodiments of the present invention, the double-stranded RNAi agent comprises at least one modified nucleotide, and the modified nucleotide comprises any one or a combination of at least two selected from the group consisting of deoxynucleotides, 3'-terminal deoxy-thymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, unlocked nucleotides, conformationally fixed nucleotides, constrained ethyl nucleotides, abasic nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-hydroxy-modified nucleotides, 2'-O-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidites, nucleotides containing unnatural bases, tetrahydropyran-modified nucleotides, 1,5-anhydrohexitol-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing phosphorothioate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, and nucleotides containing 5'-phosphate mimetics.

[0054] In some embodiments of the present invention, the modified form of nucleotides in the sense and antisense strands of the double-stranded RNAi agent is a chemical modification of the 2' position of the ribose of the nucleotide.

[0055] In some embodiments of the present invention, the chemical modification at the 2'-position of the nucleotide ribose of the double-stranded RNAi agent is any one or a combination of at least two selected from the group consisting of a 2'-methoxy modification, a 2'-O-methoxyethyl modification, a 2'-fluoro modification, a 2'-benzyloxy modification, a 2'-methylcarbonylamino modification, and a 2'-pyridinemethoxy modification.

[0056] In some embodiments of the present invention, the chemical modification at the 2' position of each nucleotide ribose of the double-stranded RNAi agent is selected from a combination of a 2'-methoxy group and a 2'-fluoro.

[0057] In some embodiments of the present invention, the chemical modification of the 2'-position of each ribose of each nucleotide of the double-stranded RNAi agent is such that all odd-numbered positions of the sense strand are 2'-fluoro modified and all even-numbered positions are 2'-methoxy modified, and all odd-numbered positions of the antisense strand are 2'-methoxy modified and all even-numbered positions are 2'-fluoro modified.

[0058] In some embodiments of the invention, the nucleotide monomers of the double-stranded RNAi agent are linked via 3',5'-phosphodiester bonds.

[0059] In some embodiments of the invention, the nucleotide monomers of the double-stranded RNAi agent are linked via thio-modified 3',5'-phosphodiester bonds.

[0060] In some embodiments of the invention, the double-stranded RNAi agent has the following modifications:

[0061] The antisense strand has one of the following modified forms A, B, or C: [Table 2] The sense strand has either of the following modified forms a and b: [Table 3] In the table, 2'-OMe is a 2'-methoxy group, 2'-F is 2'-fluoro, and PS is a thiophosphate backbone.

[0062] In some embodiments of the present invention, the antisense strand of the double-stranded RNAi agent has any of the following modified forms (I) to (X): (I) In the 5'-3' direction, the 2nd, 3rd, 4th, 6th, 14th, and 16th positions are all 2'-fluoro-modified, the 7th position is GNA-modified, and the remaining positions are all 2'-methoxy-modified, and the 1st and 2nd nucleotides, the 2nd and 3rd nucleotides, the 1st and 2nd penultimate nucleotides, and the 2nd and 3rd penultimate nucleotides are all linked via a thiophosphate bond. (II) In the 5'-3' direction, the 2nd, 3rd, 4th, 6th, 14th, and 16th positions are all 2'-fluoro-modified, and the remaining positions are all 2'-methoxy-modified, and the 1st and 2nd nucleotides, the 2nd and 3rd nucleotides, the 1st and 2nd penultimate nucleotides, and the 2nd and 3rd penultimate nucleotides are all linked via a thiophosphate bond. (III) In the 5'-3' direction, positions 2, 3, 4, 6, 14, and 16 are all 2'-fluoro-modified, and the remaining positions are all 2'-methoxy-modified. The nucleotides at positions 1 and 2, the nucleotides at positions 2 and 3, the nucleotides at positions 1 and 2 from the end, and the nucleotides at positions 2 and 3 from the end are all linked via thiophosphate bonds. Position 1 is a 5'-trans vinyl phosphate nucleotide. (IV) In the 5'-3' direction, positions 2, 4, 5, 6, 14, and 16 are all 2'-fluoro-modified, position 7 is GNA-modified, and the remaining positions are all 2'-methoxy-modified. The nucleotides at positions 1 and 2, the nucleotides at positions 2 and 3, the nucleotides at positions 1 and 2 from the end, and the nucleotides at positions 2 and 3 from the end are all linked via thiophosphate bonds. Position 1 is a 5'-trans vinyl phosphate nucleotide. (V) In the 5'-3' direction, the 2nd, 4th, 5th, 6th, 14th, and 16th positions are all 2'-fluoro-modified, and the remaining positions are all 2'-methoxy-modified, and the 1st and 2nd nucleotides, the 2nd and 3rd nucleotides, the 1st and 2nd penultimate nucleotides, and the 2nd and 3rd penultimate nucleotides are all linked via a thiophosphate bond. (VI) In the 5'-3' direction, positions 2, 4, 5, 6, 14, and 16 are all 2'-fluoro-modified, and the remaining positions are all 2'-methoxy-modified. The nucleotides at positions 1 and 2, the nucleotides at positions 2 and 3, the nucleotides at positions 1 and 2 from the end, and the nucleotides at positions 2 and 3 from the end are all linked via thiophosphate bonds. Position 1 is a 5'-trans vinyl phosphate nucleotide. (VII) In the 5'-3' direction, the 2nd, 6th, 14th, and 16th positions are all 2'-fluoro-modified, the 7th position is GNA-modified, and the remaining positions are all 2'-methoxy-modified, and the 1st and 2nd nucleotides, the 2nd and 3rd nucleotides, the 1st and 2nd penultimate nucleotides, and the 2nd and 3rd penultimate nucleotides are all linked via a thiophosphate bond. (VIII) In the 5'-3' direction, the 2nd, 6th, 14th, and 16th positions are all 2'-fluoro-modified, and the remaining positions are all 2'-methoxy-modified, and the 1st and 2nd nucleotides, the 2nd and 3rd nucleotides, the 1st and 2nd penultimate nucleotides, and the 2nd and 3rd penultimate nucleotides are all linked via a thiophosphate bond. (IX) In the 5'-3' direction, positions 2, 6, 14, and 16 are all 2'-fluoro-modified, and the remaining positions are all 2'-methoxy-modified. The nucleotides at positions 1 and 2, the nucleotides at positions 2 and 3, the nucleotides at positions 1 and 2 from the end, and the nucleotides at positions 2 and 3 from the end are all linked via thiophosphate bonds. Position 1 is a 5'-trans vinyl phosphate nucleotide. (X) In the 5'-3' direction, the 2nd, 4th, 5th, 6th, 8th, 10th, 12th, 14th, 16th, and 18th positions are all 2'-fluoro-modified, and the remaining positions are all 2'-methoxy-modified, and the 1st and 2nd nucleotides, the 2nd and 3rd nucleotides, the 1st and 2nd penultimate nucleotides, and the 2nd and 3rd penultimate nucleotides are all linked via a thiophosphate bond.

[0063] In some embodiments of the present invention, the sense strand of the double-stranded RNAi agent has any of the following modified forms (1) to (3). (1) In the 5'-3' direction, the 7th, 9th, 10th, 11th, and 17th positions are all 2'-fluoro-modified, and the remaining positions are all 2'-methoxy-modified. The 1st and 2nd nucleotides and the 2nd and 3rd nucleotides are all linked via thiophosphate bonds. (2) In the 5'-3' direction, the 7th, 9th, 11th, and 17th positions are all 2'-fluoro-modified, and the remaining positions are all 2'-methoxy-modified. The 1st and 2nd nucleotides and the 2nd and 3rd nucleotides are all linked via thiophosphate bonds. (3) In the 5'-3' direction, positions 7 and 9 are all 2'-fluoro-modified, the remaining positions are all 2'-methoxy-modified, the nucleotides at positions 1 and 2 and the nucleotides at positions 2 and 3 are all linked via thiophosphate bonds, and position 11 is a thymine nucleoside.

[0064] In some embodiments of the invention, the double-stranded RNAi agent has any of the following modifications: the antisense strand has the modified form A described above, and the sense strand has the modified form a described above; the antisense strand has the modified form B described above, and the sense strand has the modified form a described above; the antisense strand has the modified form C described above, and the sense strand has the modified form a described above; the antisense strand has the modified form B described above, and the sense strand has the modified form b described above; The antisense strand has the modification form C above, and the sense strand has the modification form b above.

[0065] In some embodiments of the present invention, the antisense strand of the double-stranded RNAi agent has modified groups at positions 2 to 8 from the 5' end, and the modified groups are UNA, GNA, or DNA, and the structures of UNA and GNA are as follows: [ka] (The bases are adenine, guanine, cytosine, thymine, or uracil.)

[0066] In some embodiments of the present invention, the phosphorylation of the 5' carbon atom of the glycoside of the 5' terminal nucleotide of the modified antisense strand in the double-stranded RNAi agent includes, but is not limited to, the following 5' phosphorylated groups: 5'-vinylphosphonate group (5'-E-VP), 5'-methylphosphonate group (5'-MP), 5'-C-methylphosphate group, 5'-thiophosphate group (5'-PS), and 5'-phosphate group (5'-P), the structures of which are shown below. [ka] (where R is hydrogen, hydroxyl group, amine group, C 1~4 Alkyl group, aromatic group, C 1~4 Alkoxy group, C 1~4 alkylcarbonylamino group, or halogen; The bases are adenine, guanine, cytosine, thymine, or uracil.

[0067] In some embodiments of the present invention, the double-stranded RNAi agent comprises any of the oligonucleotide duplexes comprising a modified sense strand paired with a modified antisense strand as shown in the table below (sequences are listed in Table 38 of the CN117210468A specification). [Table 4] TIFF2026034359000031.tif245165TIFF2026034359000032.tif94164

[0068] In some embodiments of the present invention, the conjugate represented by formula (IIA) or (IIB) is a conjugate of a double-stranded RNAi agent and YK-GAL-401, YK-GAL-402, YK-GAL-403, YK-GAL-404, YK-GAL-405, or YK-GAL-406, wherein the double-stranded RNAi agent comprises any of the following oligonucleotide duplexes comprising paired sense and antisense strands: (1) the sense strand has the sequence shown in SEQ ID NO: 29, and the antisense strand has the sequence shown in SEQ ID NO: 41; (2) the sense strand has the sequence shown in SEQ ID NO: 30, and the antisense strand has the sequence shown in SEQ ID NO: 42; (3) The sense strand has the sequence shown in SEQ ID NO: 30, and the antisense strand has the sequence shown in SEQ ID NO: 41.

[0069] In some embodiments of the present invention, the conjugate is a conjugate of a double-stranded RNAi agent and YK-GAL-401, YK-GAL-402, YK-GAL-403, YK-GAL-404, YK-GAL-405, or YK-GAL-406, wherein the double-stranded RNAi agent comprises an oligonucleotide duplex comprising a sense strand set forth in SEQ ID NO: 30 paired with an antisense strand set forth in SEQ ID NO: 41.

[0070] In some embodiments of the present invention, the conjugate of formula (IIA) or (IIB) is a conjugate of a double-stranded RNAi agent and YK-GAL-401, YK-GAL-402, YK-GAL-403, YK-GAL-404, YK-GAL-405, or YK-GAL-406, wherein the sequence of the double-stranded RNAi agent is as follows: Sense strand: 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: 46) Antisense strand: 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: 47)

[0071] In some embodiments of the present invention, the conjugate of formula (IIA) is any of the following conjugates: [ka] [ka] [ka] [ka] [ka] or [ka] (However, the sense strand of the siRNA has the sequence shown in SEQ ID NO: 46, and the antisense strand of the siRNA has the sequence shown in SEQ ID NO: 47.)

[0072] The present invention also provides a pharmaceutical composition comprising the conjugate represented by formula (IIA) or (IIB) and at least one pharmaceutically acceptable excipient.

[0073] The present invention also provides use of the conjugate represented by formula (IIA) or (IIB) or the pharmaceutical composition 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 cells.

[0074] In some embodiments of the present invention, the disease caused by the expression of a particular gene in hepatocytes is selected from chronic liver disease, hepatitis, liver fibrosis, liver proliferative disease, dyslipidemia, and complement-associated disease.

[0075] The present invention also provides a method for inhibiting the expression of a specific gene in a hepatocyte, the method comprising contacting the hepatocyte with an effective amount of a conjugate represented by formula (IIA) or (IIB).

[0076] The present invention provides a kit comprising a conjugate according to formula (IIA) or (IIB).

[0077] All publications and patents mentioned in this invention are incorporated herein by reference in their entirety. If the application or terminology used in any of the incorporated by reference publications and patents conflicts with the application or terminology used in this invention, the application and terminology of this invention shall control.

[0078] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter.

[0079] Unless otherwise defined, all technical and scientific terms used herein have the common meaning within the field to which the claimed subject matter belongs. In the event that there are multiple definitions for a term, those in this specification prevail.

[0080] Other than in the examples, or where otherwise indicated, in the specification and claims, all numerical values ​​expressing quantitative properties, such as dosage, are to be understood as being modified in all instances by the term "about." It is also to be understood that the numerical ranges set forth herein are intended to include all subranges within that range, and any combination of the various endpoints of that range or subrange.

[0081] As used herein, words such as "comprise," "contain," or "include" mean that the elements before the word include the elements listed after the word and their equivalents, and do not exclude elements not listed. As used herein, the terms "contain" or "comprise" may be open, semi-closed, or closed. In other words, the terms also include "consisting essentially of" or "consisting of."

[0082] In the present invention, the term "hydroxy-protecting group" refers to a protecting group commonly used to protect the hydroxy of a ribose structure in the synthesis of RNA or its derivatives, such as an acetyl group, a phenoxyacetyl group, a pivaloyl group, a benzyl group, a 4-methoxybenzyl group, a benzoyl group, a triphenylmethyl group, a 4,4'-dimethoxytrityl group (DMr), a monomethoxytrityl group (MMTr), a 9-phenylxanthen-9-yl group, a 9-(p-tolyl)-xanthen-9-yl group, a trimethylsilyl group, a tert-butyldimethylsilyl group (TBDMS), a cyanomethoxymethyl group, a 2-(cyanoethoxy)ethyl group, a cyanoethoxymethyl group, and the like, preferably a 4,4'-dimethoxytrityl group.

[0083] In the present invention, the term "phosphorus-containing reactive group" refers to a phosphorus-containing group that can react with other molecules, particularly hydroxyl or amine groups contained in other nucleotide units or other nucleotide analogs, by nucleophilic attack. Typically, such a reaction produces an ester internucleoside linkage that links a nucleotide or nucleotide analog unit to another nucleotide or nucleotide analog unit. These phosphorus-containing reactive groups are known in the art and are generally known as P III or P V and the phosphorus-containing reactive groups include phosphoramidites, H-phosphonates, phosphate triesters, and phosphorus-containing chiral auxiliaries, such as [ka] Including, but not limited to:

[0084] In the present invention, controlled pore glass (CPG) and polystyrene (highly cross-linked polystyrene microbeads) are solid supports for oligonucleotide synthesis, insoluble particles that bind to oligonucleotides during the synthesis process, and are commercially available.

[0085] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0086] The term "alkyl group" refers to a straight or branched chain alkyl group having a specified number of carbon atoms (e.g., C1-C6). Alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like.

[0087] The term "alkoxy group" refers to R X refers to the -O- group, where R X is an alkyl group as defined above.

[0088] The term "pharmaceutically acceptable salt" refers to a salt obtained by reacting a compound with a pharmaceutically acceptable (relatively non-toxic, safe, and suitable for use by patients) acid or base. When a compound contains a relatively acidic functional group, a base addition salt can be obtained by contacting the free form of the compound with a sufficient amount of a pharmaceutically acceptable base in a suitable inert solvent. Pharmaceutically acceptable base addition salts include, but are not limited to, sodium salts, potassium salts, calcium salts, aluminum salts, magnesium salts, bismuth salts, ammonium salts, and the like. When a compound contains a relatively basic functional group, an acid addition salt can be obtained by contacting the free form of the compound with a sufficient amount of a pharmaceutically acceptable acid in a suitable inert solvent. Pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochloride salts, sulfate salts, methanesulfonate salts, and the like.

[0089] In the present invention, the term "pharmaceutically acceptable excipient" refers to all substances contained in a pharmaceutical preparation other than the active ingredient.

[0090] The term "treatment" refers to any of the following: (1) alleviating one or more biological symptoms of a disease, (2) interfering with one or more points in the biological cascade leading to a disease, or (3) slowing the progression of one or more biological symptoms of a disease.

[0091] The term "prevention" refers to reducing the risk of developing a disease.

[0092] The oligonucleotides of the present invention include single-stranded oligonucleotides (e.g., antisense oligonucleotides, simply called ASOs) and double-stranded oligonucleotides (e.g., small interfering nucleotides, simply called siRNAs).

[0093] The oligonucleotide of the present invention includes natural oligonucleotide and chemically modified oligonucleotide.Herein, chemical modification includes nucleoside modification (including sugar moiety modification and nucleic acid base modification) and internucleoside bond modification.Chemical modification of oligonucleotide does not include the case where only nucleic acid base sequence is different.Herein, "natural" refers to the situation corresponding to naturally occurring RNA or DNA.

[0094] According to common knowledge in the art, the above preferred conditions can be arbitrarily combined to obtain each embodiment of the present invention.

[0095] All reagents and raw materials used in the present invention are commercially available.

[0096] The positive and innovative effects of the present invention are as follows: the present invention designs a series of novel GalNAc compounds with completely different chemical structures compared to GalNAc compounds of the prior art. The GalNAc compounds designed according to the present invention incorporate a triazole structure into the linking arm, and by taking advantage of click chemistry, a series of novel GalNAc compounds containing a triazole structure can be rapidly constructed. GalNAc-oligonucleotide conjugates prepared with the GalNAc compounds of the present invention can achieve efficient liver-targeted delivery, and their activity is significantly improved compared to representative GalNAc compounds of the prior art.

[0097] 1. Oligonucleotide conjugates prepared with the GalNAc compounds YK-Gal-401 to YK-Gal-403 of the present invention exhibit significantly improved inhibition of PCSK9 protein expression in mouse serum compared with conventional GalNAc conjugates. For example, the inhibition rates at day 7 with inc-YK-Gal-401 are 21.5%, 14.5%, and 12.7% higher than those of inc-YK-Gal-301, inc-GalNAc1b, and inc-NAG0052, respectively, and the inhibition rates at day 14 are 17.3%, 10.4%, and 10.8%, respectively.

[0098] 1) The siRNAs conjugated with the GalNAc compounds of the present invention showed significant differences in the inhibition rates of PCSK9 protein expression in mouse serum, with the inhibition rates of YK-Gal-401 to YK-Gal-403 being significantly higher than those of YK-Gal-404 to 406. For example, the inhibition rates of inc-YK-Gal-401 were 20.7% and 16.0% higher than those of inc-YK-Gal-404 on days 7 and 14, respectively.

[0099] 2) The GalNAc compounds YK-Gal-401 to YK-Gal-403 of the present invention exhibit significantly improved inhibition of PCSK9 protein expression in mouse serum when conjugated with siRNA compared to conventional GalNAc compounds. For example, the inhibition rate of inc-YK-Gal-401 is 21.5% and 17.3% higher than that of inc-YK-Gal-301 on days 7 and 14, respectively.

[0100] 3) There is a significant difference in the inhibition rate of PCSK9 protein expression in mouse serum between GalNAc compounds and siRNA conjugates with similar structures. Therefore, GalNAc compounds with similar chemical structures do not necessarily have similar oligonucleotide delivery efficiencies, and the GalNAc oligonucleotide conjugates prepared with them do not necessarily have consistent or even significant differences in the inhibition rate of PCSK9 protein expression in mouse serum.

[0101] 2. Oligonucleotide conjugates prepared with the GalNAc compounds YK-Gal-401 to YK-Gal-403 of the present invention exhibit significantly improved inhibition of PCSK9 gene expression in mouse liver compared with conventional GalNAc conjugates. For example, the inhibition rate of inc-YK-Gal-401 was significantly improved by 21.1% and 9.8% over the inhibition rates of inc-YK-Gal-301 and inc-GalNAc1b, respectively.

[0102] 1) The GalNAc compound-conjugated siRNAs of the present invention significantly inhibit PCSK9 gene expression in mouse liver, with the inhibition rates of YK-Gal-401 to YK-Gal-403 being significantly higher than those of YK-Gal-404 to YK-Gal-406. For example, the inhibition rate of inc-YK-Gal-401 is 19.4% higher than that of inc-YK-Gal-404.

[0103] 2) The GalNAc compounds YK-Gal-401 to YK-Gal-403 of the present invention significantly improve the inhibition of PCSK9 gene expression in mouse liver by siRNA conjugated to them compared with conventional GalNAc compounds. For example, the inhibition rate of inc-YK-Gal-401 is 21.1% higher than that of inc-YK-Gal-301.

[0104] 3) There are significant differences in the inhibition rates of PCSK9 gene expression in mouse livers between structurally similar GalNAc compounds and siRNA conjugates. Therefore, GalNAc compounds with similar chemical structures do not necessarily have similar oligonucleotide delivery efficiencies, and the GalNAc oligonucleotide conjugates prepared with them have inconsistent and potentially very large differences in the inhibition rates of PCSK9 gene expression in mouse livers.

[0105] 3. Oligonucleotide conjugates prepared with GalNAc compounds YK-Gal-401 to YK-Gal-403 of the present invention significantly reduced serum LDL-C in mice compared with GalNAc prepared using conventional techniques. For example, the inc-YK-Gal-401 group showed significantly improved LDL-C reductions on day 14 compared with the inc-YK-Gal-301, inc-GalNAc1b, and NAG0052 groups, respectively, by 36.4%, 20.6%, and 17.8%, respectively.

[0106] 1) The siRNAs conjugated with the GalNAc compounds of the present invention significantly reduced serum LDL-C levels in mice, with YK-Gal-401 to YK-Gal-403 achieving significantly higher levels than YK-Gal-404 to 406. For example, the reduction in serum LDL-C by inc-YK-Gal-401 was significantly higher than that by inc-YK-Gal-404 on days 7 and 14, showing a significant improvement of 33.0% and 36.0%, respectively.

[0107] 2) The GalNAc compounds YK-Gal-401 to YK-Gal-403 of the present invention significantly improved the reduction of serum LDL-C in mice when conjugated with siRNA compared to conventional GalNAc compounds. For example, the reduction by inc-YK-Gal-401 was 33.4% and 36.4% higher than that by inc-YK-Gal-301 on days 7 and 14, respectively.

[0108] 3) There is a significant difference in the level of LDL-C reduction in mouse serum between structurally similar GalNAc compounds and siRNA conjugates. Therefore, GalNAc compounds with similar chemical structures do not necessarily have similar oligonucleotide delivery efficiencies, and the GalNAc oligonucleotide conjugates prepared with them do not necessarily have consistent or significant differences in the inhibition rates of PCSK9 protein expression in mouse serum.

[0109] 4. The compounds designed according to the present invention can efficiently deliver oligonucleotides to the liver.

[0110] For example, in the inc-YK-Gal-401, inc-YK-Gal-402, and inc-YK-Gal-403 groups, the liver fluorescence intensities reached 9.03E+09, 8.79E+09, and 8.92E+09, respectively, at 4 hours, and 9.11E+09, 8.95E+09, and 9.01E+09, respectively, at 8 hours, indicating that the GalNAc compounds designed according to the present invention can efficiently deliver oligonucleotides to the liver of animals. [Brief explanation of the drawings]

[0111] [Figure 1]This shows the percentage inhibition of PCSK9 protein expression in mouse serum 7 and 14 days after administration of each test drug group (inc-YK-Gal-401, inc-YK-Gal-402, inc-YK-Gal-403, inc-YK-Gal-404, inc-YK-Gal-405, inc-YK-Gal-406, inc-YK-Gal-301, inc-GalNAc1b, and inc-NAG0052). [Figure 2] This shows the inhibition rate of PCSK9 gene expression in mouse liver 14 days after administration of each test drug group (inc-YK-Gal-401, inc-YK-Gal-402, inc-YK-Gal-403, inc-YK-Gal-404, inc-YK-Gal-405, inc-YK-Gal-406, inc-YK-Gal-301, inc-GalNAc1b, and inc-NAG0052). [Figure 3] The reduction in LDL-C levels in the serum of mice 7 and 14 days after administration of each test drug group (inc-YK-Gal-401, inc-YK-Gal-402, inc-YK-Gal-403, inc-YK-Gal-404, inc-YK-Gal-405, inc-YK-Gal-406, inc-YK-Gal-301, inc-GalNAc1b, and inc-NAG0052). [Figure 4] Fluorescence intensity in the liver and kidney of mice 4 and 8 hours after administration of each test drug group (inc-YK-Gal-401, inc-YK-Gal-402, inc-YK-Gal-403, inc-YK-Gal-404, inc-YK-Gal-405, inc-YK-Gal-406, inc-YK-Gal-301, inc-GalNAc1b, and inc-NAG0052). DETAILED DESCRIPTION OF THE INVENTION

[0112] In order 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 clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the described embodiments of the present invention, all other embodiments obtained by those skilled in the art without any creative efforts are within the protection scope of the present invention.

[0113] The present invention may be embodied in other specific forms without departing from its essential attributes. It should be understood that, where there is no contradiction, any and all embodiments of the present invention may be combined with the technical features of any or more other embodiments to obtain additional embodiments. The present invention also includes additional embodiments obtained from such combinations.

[0114] The present invention will be further described below with reference to examples. However, the present invention is not limited to the following examples. The operating conditions used in the examples can be further adjusted according to different requirements of specific applications, and operating conditions not specified are conventional conditions in the industry. In specific examples of the present invention, the raw materials used are commercially available. Unless otherwise specified, all temperatures are in degrees Celsius. The technical features included in various embodiments of the present invention can be combined with each other unless they are mutually inconsistent.

[0115] The following abbreviations represent the following reagents: DCM: dichloromethane, PE: petroleum ether, EA: ethyl acetate, THF: tetrahydrofuran, DMF: N,N-dimethylformamide, ACN: acetonitrile, KOH: potassium hydroxide, DCE: 1,2-dichloroethane, TsOH·H2O: p-toluenesulfonic acid monohydrate, EDCI: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, HOBt: 1-hydroxybenzotriazole, THPTA: tris(3-hydroxypropyltriazolylmethyl)amine, VCNa: sodium ascorbate TFA: trifluoroacetic acid, TEAB: triethylamine ammonium bicarbonate aqueous solution, HBTU: O-benzotriazolyl-N,N,N',N'-tetramethyluronium hexafluorophosphate, DIPEA: N,N-diisopropylethylamine, DMAP: 4-dimethylaminopyridine, TMSOTf: trimethylsilyl trifluoromethanesulfonate, Py: pyridine, AcO: acetic anhydride, CPG-NH: amino-modified controlled pore glass (Controlled Pore Glass) (manufactured by Yakumei Kotoku Co., Ltd.).

[0116] Example 1: Synthesis of GalNAc Compounds 1. Synthesis of YK-GAL-401 The synthesis scheme is as follows: [ka] [ka]

[0117] Step 1: Synthesis of G1-2 Compound G1-1 (5.0 g, 22.6 mmol, 1.0 eq) was dissolved in anhydrous DMF (60.0 mL) and stirred at 0 °C. Propargyl bromide (40.3 g, 271 mmol, 29.2 mL, 12.0 eq) was then added, and finely ground KOH (15.2 g, 271 mmol, 12.0 eq) was added batchwise to the reaction mixture over 15 min. The mixture was heated to 35 °C and stirred for 24 h. A sample was taken and analyzed by LC / MS, which showed that the starting material had disappeared. Ethyl acetate (300 mL) was added to the reaction mixture and washed with water (50 mL × 3). The organic phase was dried over Na2SO4, filtered, and concentrated under reduced pressure to remove the solvent. The crude product was purified on a silica gel column (petroleum ether / ethyl acetate) to give yellow oil G1-2 (5.30 g, 15.8 mmol, 69.9% yield). MS(ESI) m / z[M−H] - :334.2; 1 HNMR: δppm4.90(s,1H),4.14(d,J=2.4,6H),3.77(s,6H),2.43-2.41(m,3H),1.41(s,9H).

[0118] Step 2: Synthesis of G1-4 Compound G1-3 (22.3 g, 57.3 mmol, 1.2 eq) was dissolved in DCE (200 mL), 3A molecular sieves (20.0 g) were added, and the mixture was cooled to 0 °C. Next, TMSOTf (21.2 g, 95.6 mmol, 17.3 mL, 2.0 eq) and 3-(benzyloxycarbonylamino)-1-propanol (10.0 g, 47.8 mmol, 1.0 eq) were added sequentially at 0 °C. The mixture was heated to 15 °C and stirred for 10 min. A sample was taken and analyzed by LC / MS, which showed that the raw materials had disappeared. Saturated sodium bicarbonate solution (200 mL) was added to the reaction mixture, and the mixture was extracted with dichloromethane (200 mL × 3). The organic phase was dried over NaSO, filtered, and concentrated under reduced pressure to remove the solvent. The crude product was purified on a silica gel column (petroleum ether / ethyl acetate) to give a white solid G1-4 (13.4 g, 24.9 mmol, 52.1% yield). MS (ESI) m / z [M+H] + :539.1; 1HNMR(400MHz,CDCl3)δppm7.38-7.29(m,5H),6.31(d,J=8.7Hz,1H),5.33-5.28(m,1H),5.14-5 .02(m,3H),4.98(dd,J=11.1,3.3Hz,1H),4.32(d,J=8.5Hz,1H),4.16-4.09(m,3H),3.98-3.93 (m,1H),3.76(t,J=6.7Hz,1H),3.57-3.48(m,1H),3.39(td,J=9.9,3.1Hz,1H),3.12-3.05(m,1 H),2.13(s,3H),2.04(s,3H),1.99(s,3H),1.93(s,3H),1.83-1.76(m,1H),1.65-1.57(m,1H).

[0119] Step 3: Synthesis of G1-5 Compound G1-4 (13.0 g, 24.1 mmol, 1.0 eq) and TsOH·HO (4.59 g, 24.1 mmol, 1.0 eq) were added to a solution of palladium on carbon (6.50 g, 26.4 mmol, 1.09 eq) in tetrahydrofuran (130 mL). The reaction mixture was stirred at 25 °C for 2 h under hydrogen gas (15 psi). A sample was taken and analyzed by LC / MS, which showed that the starting material had disappeared. The reaction mixture was filtered and concentrated under reduced pressure to remove the solvent, yielding crude product G1-5 (15.0 g) as a white solid. MS (ESI) m / z [M−H] - :403.2.

[0120] Step 4: Synthesis of G1-6 EDCI (7.47 g, 38.9 mmol, 1.05 eq) and HOBt (5.51 g, 40.8 mmol, 1.1 eq) were added to a dichloromethane solution (150 mL) of 5-azidopentanoic acid (5.31 g, 37.1 mmol, 1.0 eq), and the mixture was cooled to 0 °C. Next, DIPEA (9.59 g, 74.2 mmol, 12.9 mL, 2.0 eq) and compound G1-5 (15.0 g, 37.1 mmol, 1.0 eq) were added sequentially to the above solution. The mixture was heated to 20 °C and stirred for 1 h. A sample was taken and analyzed by LC / MS, which showed that the starting material had disappeared. The reaction mixture was diluted with dichloromethane (150 mL) and washed with saturated NaHCO3 (150 mL x 3). The organic phase was dried over Na2SO4, filtered, and then concentrated under reduced pressure to remove the solvent, yielding crude product G1-6 (18.0 g) as a colorless liquid. MS (ESI) m / z [M−H] - :528.3; 1 HNMR(400MHz,CDCl3)δppm6.30(d,J=8.8Hz,1H),6.17(dd,J=7.1,4.6Hz,1H),5.34(dd,J=3.4,1.1Hz, 1H),5.03(dd,J=11.2,3.4Hz,1H),4.44(d,J=8.4Hz,1H),4.29-4.08(m,3H),4.03-3.98(m,1H),3.88(t d,J=6.7,1.2Hz,1H),3.71-3.57(m,1H),3.45(td,J=9.2,3.5Hz,1H),3.31(t,J=6.7Hz,2H),3.14-3.02 (m,1H),2.27(t,J=7.2Hz,2H),2.16(s,3H),2.04(s,3H),2.00(s,3H),1.96(s,3H),1.84-1.58(m,6H).

[0121] Step 5: Synthesis of G1-7 G1-2 (400 mg, 1.19 mmol, 1.0 eq) was added to a DMSO solution (50.0 mL) of G1-6 (2.53 g, 4.77 mmol, 4.0 eq). Next, CuSO4 (1.71 g, 10.7 mmol, 1.65 mL, 9.0 eq) and THPTA (3.11 g, 7.16 mmol, 6.0 eq) were added to water (15.0 mL). VCNa (3.54 g, 17.89 mmol, 15.0 eq) was then added to the above DMSO solution and stirred at 25 °C for 2 h. A sample was taken and analyzed by LC / MS, which showed that the starting material had disappeared. The reaction mixture was diluted with dichloromethane (100 mL) and washed with water (100 mL x 2). The organic phase was dried over Na2SO4, filtered, and concentrated under reduced pressure to remove the solvent. The crude product was purified on a silica gel column (petroleum ether / ethyl acetate, dichloromethane / methanol) to give a white solid G1-7 (1.90 g, 987.5 μmol, 82.8% yield). MS (ESI) m / z [M-2H] 2- :960.8; 1 HNMR(400MHz,CDCl3)δppm7.62(s,3H),6.99(d,J=9.0Hz,3H),6.73(t,J=6.2Hz,3H),5.32(d,J=3.3Hz, 3H),5.28(s,3H),5.05(dd,J=11.2,3.4Hz,3H),4.55-4.49(m,10H),4.37(t,J=7.0Hz,6H),4.27-4.06( m,10H),4.00-3.95(m,3H),3.92-3.85(m,3H),3.67(s,6H),3.54-3.42(m,6H),3.13-3.08(m,3H),2.32 -2.23(m,7H),2.13(s,10H),2.01(s,10H),1.96(s,10H),1.88(s,9H),1.70-1.61(m,9H),1.38(s,10H).

[0122] Step 6: Synthesis of G1-8 Trifluoroacetic acid (9.21 g, 80.7 mmol, 6.00 mL, 81.7 eq) was added to a dichloromethane solution (18.0 mL) of G1-7 (1.90 g, 988 μmol, 1.0 eq) and stirred at 25 °C for 0.5 h. A sample was taken and analyzed by TLC (dichloromethane / methanol), which showed the disappearance of the starting material. The reaction mixture was then concentrated under reduced pressure to remove the solvent, and the crude product was rotary evaporated with toluene (50.0 mL) a total of three times to give a yellow oil, G1-8 (1.80 g, 967 μmol, 97.8% yield, 98.0% purity). LC / MS (ESI) m / z [M-2H] 2- :910.7.

[0123] Step 7: Synthesis of G1-10 HBTU (174 mg, 460 μmol, 1.5 eq) and DIPEA (79.4 mg, 614 μmol, 107 μL, 2.0 eq) were added to a DMF solution (3.0 mL) of G1-9 (preparation method: see CN116854754B) (200 mg, 307 μmol, 1.0 eq). Next, crude product G1-8 (955 mg, 491 μmol, 1.6 eq, TFA) (pH adjusted to approximately 7 with DIPEA) was added to the above reaction solution and stirred at 25 °C for 6 h. A sample was taken and detected by LC / MS, which showed that the starting material had disappeared. The reaction solution was used directly in the next step. MS (ESI) m / z [M-2H] 2- :1227.0.

[0124] Step 8: Synthesis of G1-11 G1-10 (750 mg, 305 μmol, 1.0 eq) was added to DMF (4.0 mL). DIPEA (118 mg, 915 μmol, 159 μL, 3.0 eq), succinic anhydride (244 mg, 2.44 mmol, 8.00 eq), and DMAP (37.3 mg, 305 μmol, 1.0 eq) were then added, and the reaction mixture was stirred at 25 °C for 40 h. A sample was taken and analyzed by LC / MS. The main peak was the desired product. The reaction mixture was filtered, and the filtrate was purified by preparative chromatography to give a white solid, G1-11 (120.0 mg, 46.0 μmol). MS (ESI) m / z [M-2H]2- :1276.8; 1 HNMR(400MHz,CDCl3)δ7.55(s,2H),7.43-7.36(m,2H),7.26(dd,J=9.1,2.8H z,3H),7.21-7.17(m,6H),7.11(dd,J=8.3,6.2Hz,1H),6.88(d,J=9.0Hz,2H) ,6.76-6.70(m,3H),6.68(t,J=6.0Hz,2H),5.28(d,J=3.3Hz,2H),5.09(t,J= 5.2Hz,1H),5.00(dd,J=11.2,3.4Hz,2H),4.93(d,J=2.3Hz,1H),4.57-4.41(m ,7H),4.32(t,J=6.9Hz,4H),4.24-4.00(m,8H),3.95-3.90(m,2H),3.86-3.8 2(m,3H),3.71-3.61(m,10H),3.48-3.37(m,5H),3.34-3.29(m,3H),3.18-3.0 2(m,5H),2.73(q,J=7.3Hz,7H),2.64-2.57(m,2H),2.47(t,J=6.9Hz,2H),2. 32-1.75 (m, 54H), 1.72-1.58 (m, 11H), 1.50-1.41 (m, 4H), 1.19-1.09 (m, 20H).

[0125] Step 9: Synthesis of YK-GAL-401 G1-11 (160 mg, 62.5 μmol, 1.0 eq) was added to DMF (10.0 mL). DIPEA (64.7 mg, 500 μmol, 87.20 μL, 8.0 eq), HBTU (118 mg, 312 μmol, 5.0 eq), DMAP (37.3 mg, 305 μmol, 1.0 eq), and CPG-NH (1.1 g) were then added, and the reaction mixture was stirred at 40 °C for 16 h. LC / MS analysis showed that the starting material had disappeared. The reaction mixture was filtered, and the filter cake was washed with MeOH (20 mL × 4) and DCM (20 mL × 4) successively. It was then dried under nitrogen gas to give a pale yellow solid, which was used directly in the next step.

[0126] The crude product (1.2 g) was dissolved in pyridine (10.0 mL). Acetic anhydride (2.17 g, 21.3 mmol, 2.0 mL, 45.3 eq) was added and the reaction mixture was stirred at 40 °C for 1 h. The reaction mixture was then filtered, and the filter cake was washed with MeOH (20 mL × 4) and DCM (20 mL × 4) in that order. The cake was then dried under nitrogen gas to give a pale yellow solid, YK-GAL-401 (1.0 g, loading: 37.1 μmol / g).

[0127] A typical method for measuring the carrier loading amount was as follows: YK-GAL-401 was weighed into a 1.5 mL centrifuge tube, and then 1.0 mL of PTSA (CAS: 104-15-4) (0.1 M ACN solution) was added to the centrifuge tube. The test tube was sealed and mixed uniformly. The solution turned orange and was allowed to stand for 1 hour. 100 μL of the solution was added to a 4.0 mL centrifuge tube. 3.0 mL of PTSA (0.1 M ACN solution) was added to the centrifuge tube. The test tube was sealed and mixed uniformly, and PTSA (0.1 M in ACN) was loaded into a reference cuvette. The absorbance of the blank solution at 411 nm was measured. The sample solution was loaded into a cuvette, and the absorbance of the sample solution at 411 nm was measured, and the loading amount was calculated.

[0128] 2. Synthesis of YK-GAL-402 The synthesis scheme is as follows: [ka] [ka]

[0129] Step 1: Synthesis of G2-3 Compounds G2-1 (1.30 g, 11.3 mmol, 1.04 eq) and G2-2 (2.00 g, 10.9 mmol, 1.0 eq) were dissolved in anhydrous ethyl acetate (20.0 mL) and cooled to 0 °C. EDCI (2.17 g, 11.3 mmol, 1.04 eq) was then added batchwise, and stirring was continued at 0 °C for 1 h. The mixture was then slowly heated to 25 °C and stirred for 4 h. A sample was taken and detected by LC / MS, which showed that the raw materials had disappeared. The reaction mixture was washed successively with water (20 mL × 3), 10% aqueous NaHCO3 solution (20 mL × 3), water (20 mL × 2), 5% aqueous KHSO4 solution (20 mL × 2), and water (20 mL × 3). The organic phase was dried over Na2SO4, filtered, and then concentrated under reduced pressure to remove the solvent, yielding brown oil G2-3 (2.50 g, 8.89 mmol, 81.8% yield). 1 HNMR(400MHz,CDCl3)δppm3.72(t,J=6.4Hz,2H),2.94(t,J=6.4Hz,2H); 19 FNMR (376MHz, CDCl3) δ-152.54~-152.63(m,2F),-157.46(t,J=21.8Hz,1F),-161.98~-162.13(m,2F).

[0130] Step 2: Synthesis of G2-4 DIPEA (1.54 g, 11.9 mmol, 2.08 mL, 2.0 eq) was added to a dichloromethane solution (30.0 mL) of compound G1-5 (3.55 g, 5.97 mmol, 1.0 eq) and compound G2-3 (1.68 g, 5.97 mmol, 1.0 eq). The mixture was stirred at 25 °C for 2 h. A sample was taken and analyzed by LC / MS, which showed that the starting material had disappeared. The reaction mixture was washed sequentially with aqueous NaHCO3 (30.0 mL x 1) and water (30 mL x 3). The organic phase was dried over Na2SO4, filtered, and concentrated under reduced pressure to remove the solvent. The crude product was purified by preparative chromatography to give a white solid, G2-4 (710 mg, 1.42 mmol, 23.7% yield). MS (ESI) m / z [M−H] - :500.2; 1HNMR(400MHz,CDCl3)δppm6.37-6.28(m,1H),6.14(d,J=8.8Hz,1H),5.35(d,J=3.3Hz,1H),5.0 2(dd,J=11.2,3.4Hz,1H),4.43(d,J=8.4Hz,1H),4.30-4.23(m,1H),4.20-4.10(m,2H),4.06-4 .01(m,1H),3.92-3.84(m,1H),3.71-3.57(m,3H),3.46(td,J=9.1,3.3Hz,1H),3.21-3.09(m,1 H),2.62-2.44(m,2H),2.17(s,3H),2.04(s,3H),2.01(s,3H),1.98(s,3H),1.71-1.66(m,2H).

[0131] Step 3: Synthesis of G2-5 G1-2 (110 mg, 328 μmol, 1.0 eq) was added to a DMSO solution (20.0 mL) of G2-4 (575 mg, 1.15 mmol, 3.5 eq). Next, CuSO4 (471 mg, 2.95 mmol, 453 μL, 9.0 eq) and THPTA (855 mg, 1.97 mmol, 6.0 eq) were added to water (4.0 mL). VCNa (974 mg, 4.92 mmol, 15.0 eq) was then added to the above DMSO solution and stirred at 25 °C for 2 h. A sample was taken and analyzed by LC / MS, which showed that the starting material had disappeared. The reaction mixture was added to water (2.0 mL), extracted with dichloromethane (3 mL x 3), and washed with water (2 mL x 3). The organic phase was dried over Na2SO4, filtered, and then concentrated under reduced pressure to remove the solvent. The crude product was purified by preparative chromatography to give a white solid G2-5 (350 mg, 190 μmol, 58.0% yield). MS (ESI) m / z [M-2H] 2- :918.6; 1HNMR(400MHz,CDCl3)δppm7.69(s,3H),7.09(t,J=5.7Hz,3H),6.97(d,J=9.0Hz,3H),5.35(d,J=3.2Hz,3 H),5.08(dd,J=11.3,3.4Hz,3H),4.77-4.66(m,6H),4.52(s,6H),4.47(d,J=8.5Hz,3H),4.26-4.11(m,9H) ),3.96-3.91(m,6H),3.60(s,6H),3.52-3.47(m,2H),3.42-3.36(m,3H),3.16-3.11(m,3H),3.04-2.97( m,3H),2.86-2.80(m,3H),2.15(s,9H),2.04(s,9H),2.00(s,9H),1.85(s,9H),1.78(s,8H),1.42(s,9H).

[0132] Step 4: Synthesis of G2-6 Trifluoroacetic acid (1.07 g, 9.42 mmol, 700 μL, 49.5 eq) was added to a dichloromethane solution (2.8 mL) of G2-5 (350 mg, 190 μmol, 1.0 eq) and stirred at 25 °C for 0.5 h. A sample was taken and analyzed by LC / MS, which showed the disappearance of the starting material. The reaction mixture was directly concentrated under reduced pressure to remove the solvent, and the crude product was rotary evaporated with toluene (10.0 mL) a total of three times to give a red oil, G2-6 (523 mg). MS (ESI) m / z [M-2H] 2- :868.6.

[0133] Step 5: Synthesis of G2-7 HBTU (65.6 mg, 173 μmol, 1.5 eq) and DIPEA (29.8 mg, 230 μmol, 40.2 μL, 2.0 eq) were added to a DMF solution (1.5 mL) of G1-9 (75.0 mg, 115 μmol, 1.0 eq). Next, crude product G2-6 (200 mg, 115 μmol, 1.0 eq, TFA) (pH adjusted to approximately 7 with DIPEA) was added to the above reaction mixture and stirred at 25 °C for 2 h. A sample was taken and analyzed by LC / MS, which showed that the starting material had disappeared. The reaction mixture was used directly in the next step. MS (ESI) m / z [M-2H] 2-:1184.8.

[0134] Step 6: Synthesis of G2-8 G2-7 (273 mg, 115 μmol, 1.0 eq) was added to DMF (3.0 mL). DIPEA (44.7 mg, 345 μmol, 60.2 μL, 3.0 eq), succinic anhydride (104 mg, 1.04 mmol, 9.0 eq), and DMAP (56.3 mg, 461 μmol, 4.0 eq) were then added, and the reaction mixture was stirred at 30 °C for 24 h. A sample was taken and analyzed by LC / MS, which showed that the starting material had disappeared. The reaction mixture was filtered, and the filtrate was purified by preparative chromatography to give a white solid, G2-8 (110 mg, 44.5 μmol, 38.6% yield). MS (ESI) m / z [M-2H] 2- :1234.9; 1 HNMR(400MHz,CDCl3)δppm7.66(s,3H),7.45(d,J=7.7Hz,2H),7.33(d,J=8.8Hz,4H),7.28(s,1H),7.24(s,1H),7.20-7.13(m,4H),7.02(d,J =9.0Hz,2H),6.81(d,J=8.7Hz,3H),5.35(d,J=3.3Hz,3H),5.08(dd,J=11.2,3.4Hz,3H),4.73-4.69(m,5H),4.53-4.47(m,8H),4.26-4.10(m ,11H),3.97-3.89(m,7H),3.78(s,6H),3.65(s,5H),3.56-3.44(m,4H ),3.40-3.37(m,5H),3.24-3.21(m,2H),3.16-3.09(m,4H),3.01-2.94 (m,4H),2.82-2.78(m,2H),2.69-2.64(m,2H),2.56-2.53(m,2H),2.15 (s,9H),2.04(s,9H),2.00(s,9H),1.93-1.76(m,32H),1.27(br,10H).

[0135] Step 7: Synthesis of YK-GAL-402 G2-8 (110 mg, 42.74 μmol, 1.0 eq, TEA) was added to DMF (8.0 mL), followed by DIPEA (44.2 mg, 342 μmol, 59.5 μL, 8.0 eq), HBTU (81.0 mg, 213 μmol, 5.0 eq), DMAP (5.22 mg, 42.7 μmol, 1.0 eq), and CPG-NH (713 mg, 1.21 mmol, 28.4 eq). The reaction mixture was stirred at 40 °C for 16 h. LC / MS analysis showed the disappearance of the starting material. The reaction mixture was filtered, and the filter cake was washed with MeOH (10 mL × 4) and DCM (10 mL × 4) sequentially and dried under nitrogen gas to give a pale yellow solid, which was used directly in the next step.

[0136] The crude product was dissolved in a pyridine and acetic anhydride solution (Py / AcO = 5 / 1, 9.0 mL), and the reaction mixture was stirred at 40 °C for 40 min. The reaction mixture was then filtered, and the filter cake was washed with DCM (5.0 mL x 4) and MeOH (5.0 mL x 4) in that order. It was then dried under nitrogen gas to obtain a pale yellow solid, YK-GAL-402 (726 mg, loading: 33.15 μmol / g). The loading was measured according to the general method for measuring loading on a support described above.

[0137] 3. Synthesis of YK-GAL-403 The synthesis scheme is as follows: [ka] [ka]

[0138] Step 1: Synthesis of G3-1 EDCI (1.92 g, 10.0 mmol, 1.05 eq) and HOBt (1.42 g, 10.50 mmol, 1.1 eq) were added to a solution of 6-azidohexanoic acid (1.50 g, 9.54 mmol, 1.0 eq) in dichloromethane (40.0 mL) and the mixture was cooled to 0 °C. Next, DIPEA (2.47 g, 19.1 mmol, 3.32 mL, 2.0 eq) and compound G1-5 (3.86 g, 9.54 mmol, 1.0 eq) were added sequentially to the above solution. The mixture was heated to 25 °C and stirred for 2 h. A sample was taken and analyzed by LC / MS, which showed that the starting material had disappeared. The reaction mixture was diluted with dichloromethane (20.0 mL) and washed with saturated NaHCO (40.0 mL) and then with water (40.0 mL). The organic phase was dried over Na2SO4, filtered, and then concentrated under reduced pressure to remove the solvent, yielding crude product G3-1 (1.42 g, 2.61 mmol, 32.7% yield) as a yellow oil. MS (ESI) m / z [M−H] - :542.2; 1 HNMR(400MHz,CDCl3)δppm6.32(d,J=8.8Hz,1H),6.09-6.01(m,1H),5.34(d,J=2.7Hz,1H),5.04(dd,J= 11.2,3.3Hz,1H),4.45(d,J=8.4Hz,1H),4.28-4.09(m,3H),4.03-3.98(m,1H),3.88(td,J=6.7,1.2Hz,1 H),3.71-3.62(m,1H),3.44(td,J=9.3,3.4Hz,1H),3.28(t,J=6.8Hz,2H),3.10-3.03(m,1H),2.24(t,J =7.5Hz,2H),2.16(s,3H),2.04(s,3H),2.01(s,3H),1.96(s,3H),1.76-1.63(m,6H),1.46-1.38(m,2H).

[0139] Step 2: Synthesis of G3-2 G1-2 (230 mg, 686 μmol, 1.0 eq) was added to a DMSO solution (50.0 mL) of G3-1 (1.30 g, 2.40 mmol, 3.5 eq). Next, CuSO4 (985 mg, 6.17 mmol, 947 μL, 9.0 eq) and THPTA (1.79 g, 4.11 mmol, 6.0 eq) were added to water (6.0 mL). VCNa (2.04 g, 10.3 mmol, 15.0 eq) was then added to the above DMSO solution and stirred at 25 °C for 2 h. LC / MS analysis showed that the starting material had disappeared. The reaction mixture was diluted with water (30.0 mL), extracted with dichloromethane (30.0 mL x 3), and then washed with water (30.0 mL x 3). The organic phase was dried over NaSO, filtered, and then concentrated under reduced pressure to remove the solvent. The crude product was purified by preparative chromatography to give a white solid, G3-2 (1.0 g, 508 μmol, 74.2% yield). MS (ESI) m / z [M-2H] 2- :981.8; 1 HNMR(400MHz,CDCl3)δppm7.60(s,3H),6.84(d,J=8.9Hz,3H),6.62-6.54(m,3H),5.34(d,J=3.3Hz,3H),5.06(dd,J=11. 2,3.4Hz,3H),4.57(s,6H),4.50(d,J=8.5Hz,3H),4.36(t,J=6.9Hz,6H),4.28-4.07(m,10H),4.00-3.95(dt,J=9.9,5.0H z,3H),3.90(t,J=6.7Hz,3H),3.69(s,6H),3.59-3.55(m,3H),3.51-3.41(m,3H),3.11-3.06(m,3H),2.21(t,J=7.4Hz,6 H),2.15(s,9H),2.03(s,9H),1.99(s,9H),1.93(s,9H),1.82(s,9H),1.72-1.61(m,9H),1.40(s,9H),1.37-1.29(m,6H).

[0140] Step 3: Synthesis of G3-3 Trifluoroacetic acid (3.07 g, 26.9 mmol, 2.0 mL, 52.9 eq) was added to a dichloromethane solution (10.0 mL) of G3-2 (1.00 g, 508 μmol, 1.00 eq) and stirred at 25 °C for 1 h. A sample was taken and analyzed by TLC (dichloromethane / methanol), which showed the disappearance of the starting material. The reaction mixture was then concentrated under reduced pressure to remove the solvent, and the crude product was rotary evaporated with toluene (20.0 × 3 mL) a total of three times to give a yellow oil, G3-3 (950 mg, 458.21 μmol, 90.09% yield, 90% purity). MS (ESI) m / z [M-2H] 2- :931.7.

[0141] Step 4: Synthesis of G3-4 HBTU (174 mg, 460 μmol, 1.5 eq) and DIPEA (79.4 mg, 614 μmol, 107 μL, 2.0 eq) were added to a DMF solution (3.0 mL) of G1-9 (200 mg, 307 μmol, 1.0 eq). Next, crude product G3-3 (837 mg, 430 μmol, 1.4 eq, TFA) (pH adjusted to approximately 7 with DIPEA) was added to the above reaction mixture and stirred at 25 °C for 4 h. A sample was taken and analyzed by LC / MS, which showed that the starting material had disappeared. The reaction mixture was used directly in the next step. MS (ESI) m / z [M-2H] 2- :1247.8.

[0142] Step 5: Synthesis of G3-5 G3-4 (760 mg, 304 μmol, 1.0 eq) was added to DMF (4.0 mL). DIPEA (112 mg, 912 μmol, 158 μL, 3.0 eq), succinic anhydride (243 mg, 2.43 mmol, 8.0 eq), and DMAP (37.1 mg, 304 μmol, 1.0 eq) were then added, and the reaction mixture was stirred at 35 °C for 24 h. A sample was taken and analyzed by LC / MS, which showed the disappearance of the starting material, but the desired product was detected. The reaction mixture was filtered, and the filtrate was purified by preparative chromatography to give a white solid, G3-5 (400 mg, 142 μmol, 46.7% yield, 96.0% purity, TEA). MS (ESI) m / z [M-2H]2- : 1298.2; 1 HNMR (400 MHz, CDCl3) δ ppm 7.53 (s, 3H), 7.40 - 7.36 (m, 2H), 7.31 - 7.24 (m, 4H), 7.22 - 7.07 (m, 6H), 6.84 (d, J = 8.9 Hz, 2H), 6.77 - 6.70 (m, 4H), 6.59 (t, J = 6.1 Hz, 3H), 6.08 (s, 1H), 5.28 (d, J = 3.3 Hz, 3H), 5.09 (t, J = 5.1 Hz, 1H), 5.04 - 4.90 (m, 4H), 4.50 (s, 6H), 4.45 (d, J = 8.4 Hz, 3H), 4.30 (t, J = 6.9 Hz, 6H), 4.21 - 4.02 (m, 10H), 3.93 - 3.88 (m, 3H), 3.87 - 3.81 (m, 4H), 3.70 (d, J = 9.3 Hz, 12H), 3.50 (d, J = 7.8 Hz, 3H), 3.39 (td, J = 9.2, 3.7 Hz, 3H), 3.32 (s, 3H), 3.16 (dd, J = 9.8, 4.3 Hz, 2H), 3.08 - 2.99 (m, 4H), 2.76 (q, J = 7.3 Hz, 9H), 2.63 - 2.57 (m, 2H), 2.47 - 2.44 (m, 2H), 2.15 (t, J = 7.3 Hz, 6H), 2.09 (s, 9H), 1.97 (s, 9H), 1.92 (s, 9H), 1.87 (s, 9H), 1.71 - 1.67 (m, 3H), 1.66 - 1.56 (m, 9H), 1.51 - 1.40 (m, 4H), 1.29 - 1.24 (m, 6H), 1.10 (t, J = 7.2 Hz, 11H).

[0143] Step 6: Synthesis of YK-GAL-403 G3-5 (400 mg, 154 μmol, 1.0 eq) was added to DMF (10.0 mL). DIPEA (159 mg, 1.23 mmol, 214 μL, 8.0 eq), HBTU (291 mg, 769 μmol, 5.0 eq), DMAP (18.8 mg, 154 μmol, 1.00 eq), and CPG-NH (2.25 g) were then added, and the reaction mixture was stirred at 40 °C for 16 h. LC / MS analysis showed that the starting material had disappeared. The reaction mixture was filtered, and the filter cake was washed with MeOH (20 mL × 4) and DCM (20 mL × 4) successively. It was then dried under nitrogen gas to give a pale yellow solid, which was used directly in the next step.

[0144] The crude product (2.4 g) was dissolved in pyridine (15.0 mL). Acetic anhydride (3.26 g, 31.9 mmol, 3.0 mL, 34.5 eq) was then added, and the reaction mixture was stirred at 40 °C for 1 h. The reaction mixture was then filtered, and the filter cake was washed with MeOH (30 mL x 4) and DCM (30 mL x 4) in that order. The cake was then dried under nitrogen gas to obtain a pale yellow solid, YK-GAL-403 (2.2 g, loading: 32.8 μmol / g). The loading was measured according to the general method for measuring loading on a support.

[0145] 4. Synthesis of YK-GAL-404 The synthesis scheme is as follows: [ka]

[0146] Step 1: Synthesis of G4-1 The crude product G1-8 (537 mg, 280 μmol, 1.4 eq, TFA) and G1-9A (preparation method see CN116854754B) (124 mg, 200 μmol, 1.0 eq) were used as starting materials, and the reaction was carried out according to the synthesis method for G1-10. A sample was taken and detected by LC / MS, which showed that the starting material had disappeared. The reaction solution was used directly in the next step. MS (ESI) m / z [M-2H] 2- :1212.0.

[0147] Step 2: Synthesis of G4-2 Using G4-1 (485 mg, 200 μmol, 1.0 eq) as the starting material, the reaction was carried out according to the synthesis method for G1-11 to obtain a white solid G4-2 (187 mg, 74.2 μmol, 37.1%). MS (ESI) m / z [M-2H] 2- :1262.1; 1 HNMR(400MHz,CDCl3)δ7.63(s,2H),7.53-7.45(m,2H),7.36-7.33(m,3H),7.29-7.11(m,7H),6.92(d,J=9.1Hz,2H), 6.79-6.68(m,5H),5.12-5.05(m,3H),5.06(d,J=2.3Hz,1H),5.01-4.97(m,2H),4.79-4.61(m,8H),4.53(t,J=6.0Hz, 4H),4.34-4.20(m,3H),4.14-4.09(m,4H),3.98-3.86(m,5H),3.80(s,6H),3.77-3.65(m,2H),3.47-3.32(m,10H),3 .25-3.12(m,3H),2.85-2.61(s,9H),2.53-2.44(m,2H),2.39-1.89(m,52H),1.82-1.49(m,19H),1.19-1.09(m,18H).

[0148] Step 3: Synthesis of YK-GAL-404 Using G4-2 (126 mg, 50.0 μmol, 1.0 eq) as the starting material, the reaction was carried out according to the synthesis method for YK-GAL-401 to obtain a pale yellow solid, YK-GAL-404 (783 mg, loading: 35.9 μmol / g). The loading amount on the carrier was measured according to the general method for measuring the loading amount on the carrier.

[0149] 5. Synthesis of YK-GAL-405 The synthesis scheme is as follows: [ka]

[0150] Step 1: Synthesis of G5-1 The crude product G1-8 (537 mg, 280 μmol, 1.4 eq, TFA) and G1-9B (preparation method see CN116854754B) (125 mg, 200 μmol, 1.0 eq) were used as starting materials, and the reaction was carried out according to the synthesis method for G1-10. A sample was taken and detected by LC / MS, which showed that the starting material had disappeared. The reaction solution was used directly in the next step. MS (ESI) m / z [M-2H] 2- :1215.2.

[0151] Step 2: Synthesis of G5-2 Using G5-1 (481 mg, 198 μmol, 1.0 eq) as the starting material, the reaction was carried out according to the synthesis method for G1-11 to obtain a white solid, G5-2 (173 mg, 68.4 μmol). MS (ESI) m / z [M-2H] 2- :1265.2; 1 HNMR(400MHz,CDCl3)δppm7.53(s,1H),7.50(s,1H),7.44(d,J=8.0Hz,2H),7.25-7.16(m,4H),7.05-6.91(m,4H),6.8 5-6.72(m,6H),6.85-6.72(m,2H),5.48-5.45(m,4H),5.39(d,J=4.0Hz,2H),5.12-4.99(m,3H),4.57-4.32(m,11H),4 .19-4.06(m,6H),3.96-3.87(m,4H),3.85-3.79(m,14H),3.68(s,6H),3.63-3.56(m,12H),3.37-3.25(m,11H),3.15( t,J=5.9Hz,5H),3.11-2.89(m,4H),2.48-2.34(m,12H),2.21-1.98(m,25H),1.82-1.61(m,14H),1.28-1.08(m,13H).

[0152] Step 3: Synthesis of YK-GAL-405 Using G5-2 (171 mg, 67.5 μmol, 1.0 eq) as the starting material, the reaction was carried out according to the synthesis method for YK-GAL-401 to obtain a pale yellow solid, YK-GAL-405 (1.1 g, loading: 35.9 μmol / g). The loading amount on the carrier was measured according to the general method for measuring the loading amount on the carrier.

[0153] 6. Synthesis of YK-GAL-406 The synthesis scheme is as follows: [ka]

[0154] Step 1: Synthesis of G6-1 The crude product G1-8 (402 mg, 210 μmol, 1.4 eq, TFA) and G1-9C (preparation method see CN116854754B) (88 mg, 150 μmol, 1.0 eq) were used as starting materials, and the reaction was carried out according to the synthesis method for G1-10. A sample was taken and detected by LC / MS, which showed that the starting material had disappeared. The reaction solution was used directly in the next step. MS (ESI) m / z [M-2H] 2- :1198.0.

[0155] Step 2: Synthesis of G6-2 Using G6-1 (479 mg, 150 μmol, 1.0 eq) as the starting material, the reaction was carried out according to the synthesis method for G1-11 to obtain a white solid, G6-2 (146 mg, 58.8 μmol). MS (ESI) m / z [M-2H] 2- :1247.8; 1HNMR(400MHz,CDCl3)δppm7.47(d,J=6.4Hz,2H),7.36-7.31(m,3H),7.27-7.23(m,4H),7.20(d,J=6.0Hz,1H),7.15-7.08(m,2H),7.04- 6.97(m,4H),6.90-6.83(m,3H),5.54-5.51(m,2H),5.42(d,J=3.9Hz,2H),5.36(t,J=4.0Hz,2H),4.89-4.81(m,1H),4.55-4.47(m,5H), 4.38-4.24(m,7H),4.16-4.04(m,4H),3.80(s,6H),3.74-3.65(m,11H),3.59-3.52(m,4H),3.46-3.28(m,15H),2.83-2.78(m,7H),2.64 (t,J=6.5Hz,4H),2.59-2.50(m,4H),2.47-2.41(m,4H),2.33-1.97(m,33H),1.86-1.62(m,20H),1.37-1.29(m,5H),1.16-1.13(m,13H).

[0156] Step 3: Synthesis of YK-GAL-406 Using G6-2 (146 mg, 58.8 μmol, 1.0 eq) as the starting material, the reaction was carried out according to the synthesis method for YK-GAL-401 to obtain a pale yellow solid, YK-GAL-406 (832 mg, loading: 34.1 μmol / g). The loading amount on the carrier was measured according to the general method for measuring the loading amount on the carrier.

[0157] 7. Synthesis of control product YK-GAL-301 [ka] The target product was obtained according to the synthesis method of YK-GAL-301 on page 35 of the specification of CN116854754B.

[0158] 8. Synthesis of control product GalNAc1b [ka] According to the synthesis method of GalNAc1b on page 145 of the specification of WO2023014938A1, 377.2 mg of the product (total yield was 0.58%) was obtained. MS (ESI) m / z [M+H] + =1137.9.

[0159] 9. Synthesis of control product NAG0052 [ka] According to the synthesis method of NAG0052 on page 100 of the specification of WO2023109938A1, 288 mg of the product (total yield 1.26%, loading 31.5 μmol / g) was obtained.

[0160] Example 2: Conjugation of GalNAc Compounds with Oligonucleotides In this example, the same siRNA sequence was used for synthesis, and the conjugated siRNA sequence was the sequence numbered inc, where the inc sequence is as follows: Sense strand (inc-SS): 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: 46) Antisense strand (inc-AS): 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: 47) Here, A, U, C, and G represent the base composition of the nucleotide, dT represents a deoxythymine 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 f is 2'-F modified, and s represents that the two nucleotides adjacent to the left and right of s are linked by a thiophosphate group.

[0161] This sequence was obtained from a publicly available sequence and is the oligonucleotide sequence of the siRNA drug inclisiran, which is sold overseas.

[0162] The inclisiran sequence is a chemically modified synthetic double-stranded small interfering RNA (siRNA) that targets and binds to the mRNA encoding the PCSK9 protein, inhibiting the production of PCSK9 protein through the RNA interference mechanism, thereby regulating the recycling and reuse of LDL receptors, strengthening their binding to LDL, and achieving the goal of lowering LDL levels in the blood.

[0163] 1. Preparation of GalNAc-conjugated siRNA sense strand Oligonucleotide GalNAc conjugates were synthesized on solid supports using phosphoramidite chemistry. For the synthesis of inc-YK-Gal-401, inc-YK-Gal-402, inc-YK-Gal-403, inc-YK-Gal-404, inc-YK-Gal-405, inc-YK-Gal-406, inc-YK-Gal-301, and inc-NAG0052 conjugates, the GalNAc-CPG compound synthesized in Example 1 was used as the solid support. For the synthesis of the inc-GalNAc1b conjugate, a standard CPG was used as the solid support, and the control inc-GalNAc1b monomer was attached as the first monomer. Here, GalNAc was conjugated to the 3' end of the siRNA sense strand in all cases. The specific structures of the GalNAc compounds used are shown in Table 1.

[0164] Table 1. GalNAc compounds conjugated to the siRNA sense strand [Table 5] TIFF2026034359000053.tif132163

[0165] Experimental Process (1) Preparation of reagents and monomers The corresponding CPG carrier was selected and placed in the designated reagent position on a 192P model DNA / RNA synthesizer. The monomer solution in acetonitrile (1 / 20, w / v), 0.25 M 5-benzylmercaptotetrazole in acetonitrile (1 / 4, v / v) was used as the activating agent, 0.2 M xanthan hydride in acetonitrile / pyridine (1 / 4, v / v) was used as the thiolation reagent, 0.05 M iodine in water / pyridine (1 / 9, v / v) was used as the oxidation reagent, 20% acetic anhydride (in acetonitrile (v / v)) was used as capping agent A, 20 / 30 / 50 (1-methylimidazole / pyridine / acetonitrile, v / v / v) was used as capping agent B, 20% diethylamine (in acetonitrile (v / v)) was used as the decyanoethylation reagent, and 3% dichloroacetic acid (in toluene (v / v)) was used as the DMT removal reagent.

[0166] (2) Synthesis of crude product After inputting the specified oligonucleotide sequence, setting up the synthesis program, and confirming its accuracy, the oligonucleotide synthesis was started repeatedly. The monomer binding time was approximately 1 minute, of which the oxidation time was approximately 30-45 seconds and the sulfurization time was approximately 2 minutes. When the cycle was completed, the solid-phase synthesis of the oligonucleotide was completed.

[0167] (3) Deprotection After the synthesis was completed, the solid support was transferred to a reactor, and the oligonucleotide was decomposed from the solid support using concentrated aqueous ammonia (25-28%) at 50-60°C for 16-24 hours. The system was then cooled to room temperature, filtered, rinsed with a mixture of purified water and ethanol, and the filtrate was combined and concentrated at low temperature to obtain a crude residue.

[0168] (4) Refining The crude residue after deprotection was dissolved in purified water and purified by HPLC. The peak solution of the product was collected and the content was measured using a microplate reader, and the molecular weight was confirmed by ESIMS.

[0169] 2. Preparation of siRNA antisense strand without GalNAc conjugation The siRNA antisense strands were synthesized according to the method for synthesizing the siRNA sense strand, using a general CPG solid support, and the synthesis scale of each antisense strand complementary to the sense strand was 1 μmol.

[0170] 3. Preparation of Double-stranded siRNA to be Conjugated The siRNA sense strand and complementary antisense strand were mixed in a 1:1 ratio according to UV absorption content, heated to 95°C, and cooled to room temperature for 3 minutes to form a duplex. The resulting duplex solution was characterized by HPLC. If the purity of the product passed, the content was measured using a microplate reader, and the solid powder was lyophilized and stored for later use.

[0171] In Table 1, each GalNAc compound was conjugated to the siRNA numbered inc, and the resulting conjugates are designated inc-YK-Gal-401, inc-YK-Gal-402, inc-YK-Gal-403, inc-YK-Gal-404, inc-YK-Gal-405, inc-YK-Gal-406, inc-YK-Gal-301, inc-GalNAc1b, and inc-NAG0052, respectively.

[0172] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0173] Example 3: Inhibition of PCSK9 gene expression by GalNAc-conjugated siRNA in mouse serum and liver and effects on LDL-C levels When GalNAc-conjugated inclisiran siRNA sequences enter the bloodstream, they bind to the ASGPR on the hepatocyte membrane via GalNAc and enter hepatocytes. Once in hepatocytes, the inclisiran siRNA sequences bind to the RNA-induced silencing complex (RISC) and, via the antisense strand, bind to the mRNA encoding PCSK9 protein, inhibiting its production. The reduction in PCSK9 protein in the liver promotes LDL-R circulation, increases the number of LDL-R receptors on the surface of hepatocytes, and further reduces plasma LDL-C levels. Therefore, the higher the amount of inclisiran siRNA sequences delivered to the liver, i.e., the more efficient the GalNAc delivery, the lower the amount of PCSK9 protein in the liver and serum, and the lower the LDL-C levels in the liver.

[0174] The results show that compared with conventional GalNAc oligonucleotide conjugates, the inc-YK-Gal-401, inc-YK-Gal-402, and inc-YK-Gal-403 experimental groups exhibited significantly improved inhibition rates of PCSK9 gene expression in mouse serum and liver, and significantly improved LDL-C reduction levels. For example, the inhibition rates of serum PCSK9 protein by inc-YK-Gal-401 were 21.5%, 14.5%, and 12.7%, respectively, on day 7, and 17.3%, 10.4%, and 10.8%, respectively, on day 14, compared with inc-YK-Gal-301, inc-GalNAc1b, and inc-NAG0052. The inhibition rate of the PCSK9 gene in the mouse liver was improved by 21.1%, 9.8%, and 5.1%, respectively, on the 14th day, and the LDL-C reduction level was improved by 33.4%, 19.1%, and 16.7%, respectively, on the 7th day and by 36.4%, 20.6%, and 17.8%, respectively, on the 14th day, showing a significant improvement.

[0175] Animal preparation First, hPCSK9 transgenic mice (SPF grade, purchased from Jiangsu Jixi Pharmaceutical Biotechnology Co., Ltd.) were acclimatized and randomly divided into a negative control group (no treatment) and an siRNA test drug group, with five mice per group, all male, according to the serum PSCK9 protein content.

[0176] Administration method and dosage The drug was administered by a single subcutaneous injection at a dose of 6 mg / kg, a volume of 1 mL / kg, and a concentration of 6 mg / mL, and the day of administration was designated as day 0.

[0177] 1. Inhibitory efficiency of various GalNAc-conjugated siRNAs on PCSK9 protein expression in mouse serum The oligonucleotide conjugates prepared with the GalNAc compounds of the present invention exhibited significantly improved inhibition rates of PCSK9 protein expression in mouse serum compared with conventional GalNAc compounds. For example, the inhibition rates of inc-YK-Gal-401 on day 7 were 21.5%, 14.5%, and 12.7% higher than those of inc-YK-Gal-301, inc-GalNAc1b, and inc-NAG0052 on day 7, and the inhibition rates on day 14 were 17.3%, 10.4%, and 10.8%, respectively.

[0178] Experimental Process Approximately 200 μL of blood (without anticoagulation) was collected from the orbital venous plexus of the experimental animals before administration (D0), on day 7 after administration (D7), and on day 14 after administration (D14). The whole blood samples were temporarily stored in an icebox before centrifugation, and then centrifuged at 4°C for 10 minutes at 4000 r / min to separate the serum. The PCSK9 protein level in the serum was detected using an ELISA kit (Sino Biological Co., Ltd.).

[0179] Experimental results The inhibition rates of serum PCSK9 protein in the negative control group and each test drug group on days 7 and 14 after administration are shown in Table 3 and Figure 1. Data were statistically analyzed using GraphPad Prism 9 software.

[0180] 1. Structural differences in GalNAc compounds Table 2. Comparison of GalNAc compound structures [Table 6]

[0181] 1) The chemical structures of the GalNAc compounds YK-GAL-401 to YK-Gal-406 designed according to the present invention are very similar.

[0182] From Tables 1 and 2, it can be seen that the chemical structures of the GalNAc compounds YK-GAL-401, YK-GAL-402, YK-GAL-403, YK-GAL-404, YK-GAL-405, and YK-GAL-406 designed according to the present invention are very similar, in that the 1'-position of ribose is all oxygen, the number of antennae is all three, and the antennae all contain triazole structures.

[0183] In these compounds, the individual groups are only slightly different. For example, the 2'-substituent of the ribose in YK-GAL-401, YK-GAL-402, and YK-GAL-403 is a methoxy group, while that in YK-GAL-404, YK-GAL-405, and YK-GAL-406 is hydrogen. The main chain structures of YK-GAL-401 to YK-GAL-404 are all -(CH2) 10 The structure of YK-GAL-405 is -(CH2CH2O)3CH2C(O)NH-, that of YK-GAL-406 is -(CH2)8C(O)NH-, and the length of the antenna chain is slightly different, but the rest of the structure is exactly the same.

[0184] 2) Compared with GalNAc compounds produced by the prior art, some of the GalNAc compounds YK-GAL-401 to YK-GAL-406 designed according to the present invention have very similar chemical structures, while others have very different chemical structures.

[0185] The GalNAc compounds YK-GAL-401, YK-GAL-402, YK-GAL-403, YK-GAL-404, YK-GAL-405, and YK-GAL-406 designed according to the present invention have very similar chemical structures to the prior art GalNAc compounds YK-GAL-301 and NAG0052. For example, YK-GAL-301 and NAG0052 also contain three antennae, and the main chain structure contains aliphatic chains and amide bonds, but the antennae of YK-GAL-301 and NAG0052 do not contain triazole structures.

[0186] The GalNAc compounds designed according to the present invention have a chemical structure that is significantly different from GalNAc1b, for example, GalNAc1b has only one antenna, a very short chain, and the chain does not contain a triazole structure.

[0187] 2. Inhibition rate of PCSK9 protein expression in mouse serum by various GalNAc-conjugated siRNAs Table 3. Inhibition rate of PCSK9 protein expression in mouse serum [Table 7]

[0188] 1) The siRNAs conjugated with the GalNAc compounds of the present invention showed significant differences in the inhibition rates of PCSK9 protein expression in mouse serum, with the inhibition rates of YK-GAL-401 to YK-Gal-403 being significantly higher than those of YK-GAL-404 to 406. For example, inc-YK-Gal-401 showed 20.7% and 16.0% higher inhibition rates than inc-YK-Gal-404 on days 7 and 14, respectively.

[0189] Table 3 shows that all siRNAs conjugated with GalNAc compounds of the present invention can inhibit the expression of PCSK9 protein in mouse serum. There were significant differences in the inhibition rates of PCSK9 protein expression in mouse serum between different GalNAc conjugates. The inhibition rates of inc-YK-Gal-401, inc-YK-Gal-402, inc-YK-Gal-403, inc-YK-Gal-404, inc-YK-Gal-405, and inc-YK-Gal-406 on day 7 were 94.2%, 92.4%, 93.1%, 73.5%, 84.3%, and 85.0%, respectively, and the inhibition rates on day 14 were 95.4%, 92.8%, 94.3%, 79.4%, 88.7%, and 90.2%, respectively.

[0190] The inhibition rates of inc-YK-Gal-401, inc-YK-Gal-402, and inc-YK-Gal-403 all exceeded 90%, while the inhibition rate of inc-YK-Gal-404 was between 70% and 80%. Inc-YK-Gal-401, which had the highest inhibition rate, showed significant improvements of 20.7% and 16.0%, respectively, on days 7 and 14 compared with inc-YK-Gal-404, which had the lowest inhibition rate.

[0191] 2) The GalNAc compounds YK-GAL-401 to YK-GAL-403 of the present invention exhibited significantly improved inhibition of PCSK9 protein expression in mouse serum when conjugated with siRNA compared with conventional GalNAc compounds. For example, the inhibition rates of inc-YK-Gal-401 were 21.5% and 17.3% higher than those of inc-YK-Gal-301 on days 7 and 14, respectively.

[0192] The inhibition rates of the siRNA conjugates inc-YK-Gal-301, inc-GalNAc1b, and inc-NAG0052 prepared with conventional GalNAc compounds on day 7 were 72.7%, 79.7%, and 81.5%, respectively, and the inhibition rates on day 14 were 78.1%, 85.0%, and 84.6%, respectively.

[0193] The GalNAc compounds of the present invention, compared with conventional GalNAc compounds, showed significantly improved inhibition rates of PCSK9 protein expression in mouse serum when conjugated with siRNA. For example, the inhibition rates of inc-YK-Gal-401, inc-YK-Gal-402, and inc-YK-Gal-403 on day 7 were 21.5%, 19.7%, and 20.4%, respectively, higher than those of inc-YK-Gal-301, 14.5%, 11.9%, and 13.4%, respectively, higher than those of inc-GalNAc1b, and 12.7%, 10.9%, and 11.6%, respectively, higher than those of inc-NAG0052. The inhibition rates on day 14 were significantly improved by 17.3%, 14.7%, and 16.2% compared to those by inc-YK-Gal-301, 10.4%, 7.8%, and 9.3% compared to those by inc-GalNAc1b, and 10.8%, 8.2%, and 9.7% compared to those by inc-NAG0052.

[0194] 3) Conjugates of structurally similar GalNAc compounds with siRNA show significant differences in the inhibitory rates of PCSK9 protein expression in mouse serum.

[0195] The GalNAc compounds designed according to the present invention are structurally very similar, but the activity of their siRNA conjugates is significantly different. For example, when comparing YK-GAL-401 and YK-GAL-404, the only difference is the 2'-position of the ribose ring, which is a methoxy group in YK-GAL-401 and a hydrogen atom in YK-GAL-404. The remaining structures are identical, but inc-YK-Gal-401 exhibited a significantly higher inhibition rate of PCSK9 protein expression in mouse serum than inc-YK-Gal-404, by more than 20%.

[0196] Comparing YK-GAL-406 and YK-GAL-301, only the main chain and the Athena chain were different: the main chain of YK-GAL-406 had two methylene groups less than that of YK-GAL-301, and the Athena chain of YK-GAL-406 contained a triazole structure, whereas the Athena chain of YK-GAL-301 did not. However, the inhibition rate by inc-YK-Gal-406 on day 7 was significantly improved by 12.3% compared to inc-YK-Gal-301.

[0197] Comparing YK-GAL-401 and inc-NAG0052, although the individual groups are only slightly different, the inhibition rate by inc-YK-Gal-401 on day 7 was significantly improved by 12.7% compared to the inhibition rate by inc-NAG0052 on day 7.

[0198] Therefore, GalNAc compounds with similar chemical structures do not necessarily have similar oligonucleotide delivery efficiencies, and the GalNAc oligonucleotide conjugates prepared with them may not have consistent or even significant differences in their inhibition rates of PCSK9 protein expression in mouse serum.

[0199] 3. Inhibitory efficiency of various GalNAc-conjugated siRNAs against PCSK9 in mouse liver The oligonucleotide conjugates prepared with the GalNAc compounds of the present invention exhibited significantly improved inhibition of PCSK9 gene expression in mouse liver compared with conventional GalNAc compounds. For example, the inhibition rates of inc-YK-Gal-401 were significantly improved, by 21.1% and 9.8%, compared with those of inc-YK-Gal-301 and inc-GalNAc1b, respectively.

[0200] Experimental Process On day 14 (D14) after administration, the experimental animals were anesthetized and sacrificed, and the livers were harvested by perfusion.

[0201] Depending on the tissue weight, 1 mL of 100 mg of RNA dissolving solution (trizol, Ambion) was quickly added to a 1.5 mL RNase-free EP tube containing 1 mL of RNA dissolving solution (trizol). Three 3 mm steel beads (RNA-free treated) were added to the tube, which was then placed in a tissue homogenizer and run at 50 Hz for 30 seconds, followed by a 10-second pause, three times to prepare tissue homogenate.

[0202] The homogenate was centrifuged at 12,000 × g for 3 minutes at 4°C, and 400 μL of the supernatant was transferred to a 1.5 mL RNase-free EP tube and placed on ice. 80 μL of chloroform was added to each tube, and the tube was shaken vigorously for 15 seconds and allowed to stand at room temperature for 5 minutes. The mixture was centrifuged at 12,000 × g for 15 minutes at 4°C, and 150 μL of the supernatant was transferred to a new EP tube.

[0203] An equal volume of isopropanol was added, the tube was inverted to gently mix the liquid therein uniformly, and the mixture was left at -20°C for 10 minutes, then centrifuged at 12,000 xg at 4°C for 15 minutes, and the supernatant was discarded.

[0204] The RNA precipitate was gently washed by adding 1 mL of 75% ethanol, centrifuged at 7,500 × g for 5 minutes at 4 °C, and the supernatant was aspirated. The rinse was repeated once more, centrifuged at 7,500 × g for 5 minutes at 4 °C, and the residual ethanol was removed using a micropipette tip.

[0205] Residual ethanol was removed by drying at room temperature for 10 minutes and then dissolved in 150 μL of RNase-free ddH2O. RNA concentration was detected using a micro-volume UV spectrophotometer. PCSK9 gene expression was detected by qPCR.

[0206] Experimental results The qPCR results (Mean ± SD) were statistically analyzed and plotted using GraphPad Prism 9 software. The specific results are shown in Table 4 and Figure 2.

[0207] Table 4. Inhibition rate of PCSK9 gene expression in mouse liver [Table 8]

[0208] 1) The GalNAc compound-conjugated siRNAs of the present invention significantly inhibited PCSK9 gene expression in mouse liver, with the inhibitory effects of YK-GAL-401 to YK-GAL-403 being significantly higher than those of YK-GAL-404 to YK-GAL-406. For example, the inhibitory effect of inc-YK-Gal-401 was 19.4% higher than that of inc-YK-Gal-404.

[0209] Table 4 shows that all siRNAs conjugated with the GalNAc compounds of the present invention can inhibit the expression of the PCSK9 gene in mouse liver. There were significant differences in the inhibition rates of PCSK9 gene expression in mouse liver among different GalNAc conjugates. The inhibition rates of inc-YK-Gal-401, inc-YK-Gal-402, inc-YK-Gal-403, inc-YK-Gal-404, inc-YK-Gal-405, and inc-YK-Gal-406 were 90.9%, 89.3%, 90.1%, 71.5%, 83.2%, and 81.6%, respectively.

[0210] The inhibition rates of inc-YK-Gal-401 and inc-YK-Gal-403 were all over 90%, while the inhibition rate of inc-YK-Gal-402 reached 89.3%, while the inhibition rate of inc-YK-Gal-404 was only 71.5%. Inc-YK-Gal-401, which had the highest inhibition rate, showed a significant improvement of 19.4% over inc-YK-Gal-404, which had the lowest inhibition rate.

[0211] 2) The GalNAc compounds YK-GAL-401 to YK-GAL-403 of the present invention exhibited significantly improved inhibition of PCSK9 gene expression in mouse livers when conjugated with siRNA compared to conventional GalNAc compounds. For example, the inhibition rate of inc-YK-Gal-401 was 21.1% higher than that of inc-YK-Gal-301.

[0212] The inhibition rates of inc-YK-Gal-301, inc-GalNAc1b, and inc-NAG0052 conjugated with siRNA prepared with GalNAc compounds according to the prior art were 69.8%, 81.1%, and 85.8%, respectively.

[0213] The GalNAc compounds of the present invention, compared with conventional GalNAc compounds, showed significantly improved inhibition rates of PCSK9 protein expression in mouse serum when conjugated with siRNA. For example, the inhibition rates of inc-YK-Gal-401, inc-YK-Gal-402, and inc-YK-Gal-403 were 21.1%, 19.5%, and 20.3% higher than those of inc-YK-Gal-301, respectively, and 9.8%, 8.2%, and 9.0% higher than those of inc-GalNAc1b, respectively, demonstrating significant improvements.

[0214] 3) Conjugates of structurally similar GalNAc compounds with siRNA show significant differences in the inhibitory rates of PCSK9 gene expression in mouse liver.

[0215] The GalNAc compounds designed according to the present invention are structurally very similar, but the activity of their siRNA conjugates is significantly different. For example, when comparing YK-GAL-401 and YK-GAL-404, the only difference is the 2'-position of the ribose ring, which is a methoxy group in YK-GAL-401 and a hydrogen atom in YK-GAL-404. The remaining structures are identical, but inc-YK-Gal-401 exhibited a significantly improved inhibition rate of PCSK9 gene expression in mouse liver compared to inc-YK-Gal-404, by 19.4%.

[0216] Comparing YK-GAL-406 and YK-GAL-301, only the main chain and the Athena chain were different: the main chain of YK-GAL-406 had two methylene groups less than that of YK-GAL-301, and the Athena chain of YK-GAL-406 contained a triazole structure, whereas the Athena chain of YK-GAL-301 did not. However, the inhibition rate of inc-YK-Gal-406 was significantly improved by 11.8% compared to that of inc-YK-Gal-301.

[0217] Therefore, GalNAc compounds with similar chemical structures do not necessarily have similar oligonucleotide delivery efficiencies, and the GalNAc oligonucleotide conjugates prepared with them may not be consistent in their inhibition rates of PCSK9 gene expression in mouse liver, and there may be significant differences.

[0218] 4. Effects of various GalNAc-conjugated siRNAs on serum LDL-C levels in mice The oligonucleotide conjugates prepared with the GalNAc compounds of the present invention significantly improved the reduction of LDL-C in mouse serum compared with GalNAc compounds prepared using conventional techniques. For example, the LDL-C reduction levels in mice treated with inc-YK-Gal-401 on day 14 were significantly higher than those of inc-YK-Gal-301, inc-GalNAc1b, and NAG0052, respectively, by 36.4%, 20.6%, and 17.8%, respectively.

[0219] Experimental Process Approximately 200 μL of blood (without anticoagulation) was collected from the orbital venous plexus of the experimental animals before administration (D0), on day 7 after administration (D7), and on day 14 after administration (D14). The whole blood samples were temporarily stored in an icebox before centrifugation, and then centrifuged at 4°C and 4000 r / min for 10 min to separate the serum, and the serum LDL-C level was detected.

[0220] Experimental results Serum LDL-C (Mean ± SD) was statistically analyzed and plotted using GraphPad Prism 9 software. The specific results are shown in Table 5 and Figure 3.

[0221] Table 5. Lowering levels of LDL-C in mouse serum [Table 9]

[0222] 1) The GalNAc compound-conjugated siRNAs of the present invention significantly reduced serum LDL-C levels in mice, with the reduction levels achieved by YK-GAL-401 to YK-GAL-403 being significantly higher than those achieved by YK-GAL-404 to 406. For example, the reduction levels achieved by inc-YK-Gal-401 were significantly higher than those achieved by inc-YK-Gal-404 on days 7 and 14, respectively, demonstrating a significant improvement of 33.0% and 36.0%, respectively.

[0223] Table 5 shows that all siRNAs conjugated with GalNAc compounds of the present invention can reduce LDL-C levels in mouse serum. Different GalNAc conjugates showed significant differences in the level of LDL-C reduction in mouse serum. The reduction levels of inc-YK-Gal-401, inc-YK-Gal-402, inc-YK-Gal-403, inc-YK-Gal-404, inc-YK-Gal-405, and inc-YK-Gal-406 on day 7 were 49.2%, 39.6%, 44.5%, 16.2%, 33.7%, and 30.3%, respectively, and the reduction levels on day 14 were 53.5%, 41.3%, 50.4%, 17.5%, 35.0%, and 32.2%, respectively.

[0224] In both inc-YK-Gal-401 and inc-YK-Gal-403, the reduction level on day 7 exceeded 40% and on day 14 exceeded 50%. In inc-YK-Gal-402, the reduction level on day 7 reached 39.6% and on day 14 reached 41.3%, while inc-YK-Gal-404, the reduction level on day 7 was only 16.2%. The reduction level on day 14 was only 17.5%, whereas the reduction levels on day 7 by inc-YK-Gal-401, inc-YK-Gal-402, and inc-YK-Gal-403 were 33.0%, 23.4%, and 28.3%, respectively, higher than that of inc-YK-Gal-404, and the reduction levels on day 14 were 36.0%, 23.8%, and 32.9%, respectively, showing significant improvements.

[0225] 2) The GalNAc compounds YK-GAL-401 to YK-GAL-403 of the present invention exhibited significantly improved LDL-C reduction levels in mouse serum compared with conventional GalNAc compounds. For example, the reduction levels achieved by inc-YK-Gal-401 were 33.4% and 36.4% higher than those achieved by inc-YK-Gal-301 on days 7 and 14, respectively.

[0226] For inc-YK-Gal-301, inc-GalNAc1b, and inc-NAG0052, which were conjugated with siRNA prepared with conventional GalNAc compounds, the LDL-C reduction levels at day 7 were 15.8%, 30.1%, and 32.5%, respectively, and the LDL-C reduction levels at day 14 were 17.1%, 32.9%, and 35.7%, respectively.

[0227] The GalNAc compounds of the present invention, compared with conventional GalNAc compounds, showed significantly improved reductions in serum LDL-C levels in mice with siRNA conjugated to them. For example, inc-YK-Gal-401, inc-YK-Gal-402, and inc-YK-Gal-403 showed significantly improved reductions in serum LDL-C levels on day 7 compared with inc-YK-Gal-301 by 33.4%, 23.8%, and 28.7%, respectively, compared with inc-YK-Gal-301, by 19.1%, 9.5%, and 14.4%, respectively, compared with inc-GalNAc1b, and by 16.7%, 7.1%, and 12.0% compared with inc-NAG0052. The reduction levels on day 14 were significantly improved by 36.4%, 24.2%, and 33.3% compared to inc-YK-Gal-301, by 20.6%, 8.4%, and 17.5% compared to inc-GalNAc1b, and by 17.8%, 5.6%, and 14.7% compared to inc-NAG0052.

[0228] 3) Conjugates of structurally similar GalNAc compounds with siRNA show significant differences in the level of reduction in LDL-C in mouse serum.

[0229] The GalNAc compounds designed according to the present invention are structurally very similar, but the activity of their siRNA conjugates is significantly different. For example, when comparing YK-GAL-401 and YK-GAL-404, the only difference is the 2'-position of the ribose ring, which is a methoxy group in YK-GAL-401 and a hydrogen atom in YK-GAL-404. The remaining structures are identical, but inc-YK-Gal-401 showed a significant improvement in reducing serum LDL-C in mice by more than 30% compared to inc-YK-Gal-404.

[0230] Comparing YK-GAL-406 and YK-GAL-301, only the main chain and the Athena chain were different: the main chain of YK-GAL-406 was two methylene groups shorter than that of YK-GAL-301, and the Athena chain of YK-GAL-406 contained a triazole structure, whereas the Athena chain of YK-GAL-301 did not. However, inc-YK-Gal-406 showed a significant improvement in reducing serum LDL-C in mice by 15.1% compared to inc-YK-Gal-301.

[0231] Comparing YK-GAL-401 and inc-NAG0052, although the individual groups are only slightly different, inc-YK-Gal-401 showed a significant improvement over inc-NAG0052 in reducing LDL-C in mouse serum by 17.8%.

[0232] Therefore, GalNAc compounds with similar chemical structures do not necessarily have similar oligonucleotide delivery efficiencies, and the GalNAc oligonucleotide conjugates prepared with them may not have consistent or even significant differences in their inhibition rates of PCSK9 protein expression in mouse serum.

[0233] Example 4: Distribution of various GalNAc-conjugated siRNAs in various tissues and organs in mice The results demonstrate that GalNAc compounds designed according to the present invention can efficiently deliver nucleic acids to the liver of animals.

[0234] Experimental Process Six 6- to 8-week-old wild-type C57BL / 6 mice were used per group. Each group received one of the Cy5 fluorescent siRNA conjugates: inc-YK-GAL-401, inc-YK-GAL-402, inc-YK-GAL-403, inc-YK-GAL-404, inc-YK-GAL-405, inc-YK-GAL-406, inc-YK-GAL-301, inc-GalNAc1b, and inc-NAG0052. The negative control group received no siRNA. Mice were weighed and administered a dose of 6 mg / kg subcutaneously. The injection volume was determined by the volume administered and did not exceed 0.1–0.2 mL.

[0235] After administration, the abdominal hair of the mice was shaved, and the mice were placed in a supine position in a small animal in vivo imaging system under isoflurane anesthesia. The mice were imaged in the Cy5 channel 4 and 8 hours after administration. The in vivo imaging software LivingImage was used to statistically analyze the luminescence intensity of the mice and compare the differences between each test group.

[0236] Experimental results The detection results of the fluorescence intensity (representing the relative content of oligonucleic acid) in the liver and kidney of mice are shown in Table 6 and Figure 4. The results demonstrate that the GalNAc compound designed according to the present invention can efficiently deliver nucleic acids to the liver of animals.

[0237] Table 6. Fluorescence intensity in mouse liver and kidney [Table 10]

[0238] As can be seen from Table 6, Cy5 fluorescently labeled oligonucleotides were distributed in both the liver and kidney of mice. Among them, inc-YK-Gal-401 showed the highest fluorescence intensity (i.e., the highest relative content of oligonucleotide) in the mouse liver, reaching 9.03E+09 at 4 hours and 9.11E+09 at 8 hours. The liver fluorescence intensities of inc-YK-Gal-402 and inc-YK-Gal-403 were also very high, reaching 8.79E+09 and 8.92E+09, respectively, at 4 hours and 8.95E+09 and 9.01E+09, respectively, at 8 hours. This indicates that the GalNAc compounds designed according to the present invention can efficiently deliver oligonucleotides to the liver.

[0239] As described above, the present invention designs a series of novel GalNAc compounds with completely different chemical structures compared to GalNAc compounds of the prior art. The GalNAc compounds designed according to the present invention incorporate a triazole structure into the linking arm, and by taking advantage of click chemistry, a series of novel GalNAc compounds containing a triazole structure can be rapidly constructed. GalNAc-oligonucleotide conjugates prepared with the GalNAc compounds of the present invention can achieve efficient liver-targeted delivery and have significantly improved activity compared to representative GalNAc compounds of the prior art.

[0240] 1. Oligonucleotide conjugates prepared with the GalNAc compounds YK-GAL-401 to YK-GAL-403 of the present invention exhibited significantly improved inhibition of PCSK9 protein expression in mouse serum compared with conventional GalNAc conjugates. For example, the inhibition rates of inc-YK-Gal-401 on day 7 were 21.5%, 14.5%, and 12.7% higher than those of inc-YK-Gal-301, inc-GalNAc1b, and inc-NAG0052 on day 7, and the inhibition rates on day 14 were 17.3%, 10.4%, and 10.8%, respectively.

[0241] 1) The siRNAs conjugated with the GalNAc compounds of the present invention showed significant differences in the inhibition rates of PCSK9 protein expression in mouse serum, with the inhibition rates of YK-Gal-401 to YK-Gal-403 being significantly higher than those of YK-Gal-404 to 406. For example, inc-YK-Gal-401 showed 20.7% and 16.0% higher inhibition rates than inc-YK-Gal-404 on days 7 and 14, respectively.

[0242] 2) The GalNAc compounds YK-Gal-401 to YK-Gal-403 of the present invention exhibited significantly improved inhibition of PCSK9 protein expression in mouse serum when conjugated with siRNA compared with conventional GalNAc compounds. For example, the inhibition rates of inc-YK-Gal-401 were 21.5% and 17.3% higher than those of inc-YK-Gal-301 on days 7 and 14, respectively.

[0243] 3) There is a significant difference in the inhibition rate of PCSK9 protein expression in mouse serum between GalNAc compounds and siRNA conjugates with similar structures. Therefore, GalNAc compounds with similar chemical structures do not necessarily have similar oligonucleotide delivery efficiencies, and the GalNAc oligonucleotide conjugates prepared with them do not necessarily have consistent or even significant differences in the inhibition rate of PCSK9 protein expression in mouse serum.

[0244] 2. Oligonucleotide conjugates prepared with the GalNAc compounds YK-GAL-401 to YK-GAL-403 of the present invention exhibited significantly improved inhibition of PCSK9 gene expression in mouse liver compared with conventional GalNAc conjugates. For example, the inhibition rate of inc-YK-Gal-401 was significantly improved by 21.1% and 9.8% over the inhibition rates of inc-YK-Gal-301 and inc-GalNAc1b, respectively.

[0245] 1) The GalNAc compound-conjugated siRNAs of the present invention significantly inhibited PCSK9 gene expression in mouse liver, with the inhibition rates of YK-Gal-401 to YK-Gal-403 being significantly higher than those of YK-Gal-404 to 406. For example, the inhibition rate of inc-YK-Gal-401 was 19.4% higher than that of inc-YK-Gal-404.

[0246] 2) The GalNAc compounds YK-Gal-401 to YK-Gal-403 of the present invention exhibited significantly improved inhibition of PCSK9 gene expression in mouse liver compared with conventional GalNAc compounds. For example, the inhibition rate of inc-YK-Gal-401 was 21.1% higher than that of inc-YK-Gal-301.

[0247] 3) There are significant differences in the inhibition rates of PCSK9 gene expression in mouse livers between structurally similar GalNAc compounds and siRNA conjugates. Therefore, GalNAc compounds with similar chemical structures do not necessarily have similar oligonucleotide delivery efficiencies, and the GalNAc oligonucleotide conjugates prepared with them have inconsistent and potentially very large differences in the inhibition rates of PCSK9 gene expression in mouse livers.

[0248] 3. Oligonucleotide conjugates prepared with the GalNAc compounds YK-GAL-401 to YK-GAL-403 of the present invention significantly reduced serum LDL-C in mice compared with GalNAc prepared using conventional techniques. For example, the inc-YK-Gal-401 group showed significantly improved LDL-C reductions on day 14 compared with the inc-YK-Gal-301, inc-GalNAc1b, and NAG0052 groups, respectively, by 36.4%, 20.6%, and 17.8%, respectively.

[0249] 1) The GalNAc compound-conjugated siRNAs of the present invention significantly reduced serum LDL-C levels in mice, with YK-Gal-401 to YK-Gal-403 significantly reducing LDL-C levels compared with YK-Gal-404 to YK-Gal-406. For example, the reduction in serum LDL-C by inc-YK-Gal-401 was 33.0% and 36.0%, respectively, on days 7 and 14, demonstrating a significant improvement over the reduction in serum LDL-C by inc-YK-Gal-404.

[0250] 2) The GalNAc compounds YK-Gal-401 to YK-Gal-403 of the present invention significantly improved the level of LDL-C reduction in mouse serum when conjugated with siRNA compared with conventional GalNAc compounds. For example, the reduction level achieved by inc-YK-Gal-401 was 33.4% and 36.4% higher than that achieved by inc-YK-Gal-301 on days 7 and 14, respectively.

[0251] 3) There is a significant difference in the level of LDL-C reduction in mouse serum between structurally similar GalNAc compounds and siRNA conjugates. Therefore, GalNAc compounds with similar chemical structures do not necessarily have similar oligonucleotide delivery efficiencies, and the GalNAc oligonucleotide conjugates prepared with them do not necessarily have consistent or significant differences in the inhibition rates of PCSK9 protein expression in mouse serum.

[0252] 4. The compounds designed according to the present invention can efficiently deliver oligonucleotides to the liver.

[0253] For example, in the inc-YK-Gal-401, inc-YK-Gal-402, and inc-YK-Gal-403 groups, the fluorescence intensities in the liver reached 9.03E+09, 8.79E+09, and 8.92E+09, respectively, at 4 hours, and 9.11E+09, 8.95E+09, and 9.01E+09, respectively, at 8 hours, indicating that the GalNAc compounds designed according to the present invention can efficiently deliver oligonucleotides to the liver of animals.

[0254] Although the applicant has stated that the GalNAc compound of the present invention and its use are illustrated by the above examples, the present invention is not limited to the above examples, i.e., it does not mean that the present invention cannot be carried out without relying on the above examples. Any improvements to the present invention, equivalent replacement of each raw material of the product of the present invention, addition of auxiliary ingredients, selection of specific methods, etc., are all within the protection scope and disclosure of the present invention, as will be apparent to those skilled in the art.

Claims

1. A GalNAc compound represented by formula (I), or a pharmaceutically acceptable salt thereof: 【Chemistry 1】 (however, L １ is —C(O)NH—*, and the * end is connected to G, L ２ is -(CH ２ ) n １ - or -(CH ２ CH ２ O)n ２ CH ２ -, where n １ and n ２ is an integer from 1 to 7, Z １ is -(CH ２ ) n ３ -, where n ３ is an integer from 1 to 7, L ３ is —HNC(O)— or —C(O)NH—, n is 0, L ４ is -(CH ２ ) n ４ -, where n ４ is an integer from 0 to 7, A is, 【Chemistry 2】 and R １ is hydrogen or C １～６ is an alkoxy group, and R ２ is a 4,4'-dimethoxytrityl group or a monomethoxytrityl group, and R ３ is -CO(CH ２ ) ２ CONH-E, where E is controlled pore glass; B is oxygen or sulfur; G is, 【Transformation 3】 and where: X １ is -(CH ２ ) ３ - and X ２ is -HNC(O)-**, and the ** end is X ３ is connected to X ３ is -(CH ２ ) b-, where b is an integer from 1 to 6; X ４ teeth, 【Chemistry 4】 and the *** end is X ３ is connected to Y １ is 1, Y ２ is 1, Y ３ is 3, K is carbon.

2. 2. The GalNAc compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the GalNAc compound of formula (I) satisfies one or more of the following conditions: (1) L ２ is -(CH ２ ) ３ -, -(CH ２ ) ４ -, -(CH ２ ) ５ -, -(CH ２ ) ６ - or -(CH ２ ) ７ - is; (2) L ４ Is, None, -(CH ２ ) ３ -, -(CH ２ ) ４ -, -(CH ２ ) ５ -, -(CH ２ ) ６ - or -(CH ２ ) ７ - is; (3) R １ is hydrogen or a methoxy group; (4) R ２ is a 4,4'-dimethoxytrityl group; (5) B is oxygen.

3. L ２ is -(CH ２ CH ２ O) ３ CH ２ 2. The GalNAc compound of formula (I) according to claim 1, wherein R is -, or a pharmaceutically acceptable salt thereof.

4. G is, 【Transformation 5】 【Transformation 6】 or 【Transformation 7】 2. The GalNAc compound of formula (I) according to claim 1, wherein:

5. 2. The GalNAc compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof, which is a compound having the following structure: YK-GAL-401, YK-GAL-402, YK-GAL-403, YK-GAL-404, YK-GAL-405, or YK-GAL-406, wherein E is controlled pore glass. 【Transformation 8】 【Chemistry 9】 【Chemistry 10】 【Chemistry 11】 【Chemistry 12】 or 【Chemistry 13】

6. A conjugate of formula (IIA) or (IIB) or a pharmaceutically acceptable salt thereof: 【Chemistry 14】 or 【Chemistry 15】 (wherein Oligo represents an oligonucleotide, X represents a hydroxy group or a mercapto group, and G １ teeth, 【Chemistry 16】 and R ２ is hydrogen, and R ３ is hydrogen, and L １ , L ２ , Z １ , L ３ , L ４ , n, R １ , X １ , X ２ , X ３ , X ４ , Y １ , Y ２ , Y ３ and K are defined as in claim 1; The sense strand of the oligonucleotide has the sequence shown in SEQ ID NO: 46, and the antisense strand has the sequence shown in SEQ ID NO:

47.

7. The conjugate according to claim 6, or a pharmaceutically acceptable salt thereof, is a conjugate of an oligonucleotide and YK-GAL-401, YK-GAL-402, YK-GAL-403, YK-GAL-404, YK-GAL-405, or YK-GAL-406 according to claim 5, wherein the sense strand of the oligonucleotide has the sequence shown in SEQ ID NO: 46 and the antisense strand of the oligonucleotide has the sequence shown in SEQ ID NO:

47.

8. 7. The conjugate of claim 6, or a pharmaceutically acceptable salt thereof, wherein the conjugate is any of the following conjugates: 【Chemistry 17】 [Chemistry 18] 【Chemistry 19】 【Chemistry 20】 【Chemistry 21】 or 【Chemistry 22】 (However, the sense strand of the siRNA has the sequence shown in SEQ ID NO: 46, and the antisense strand of the siRNA has the sequence shown in SEQ ID NO: 47.)

9. A pharmaceutical composition comprising the conjugate according to any one of claims 6 to 8 or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.

10. A kit comprising the conjugate according to any one of claims 6 to 8 or a pharmaceutically acceptable salt thereof.

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